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548152	Upregulated expression of human neutrophil peptides 1, 2 and 3 (HNP 1-3) in colon cancer serum and tumours: a biomarker study	Background Molecular markers for localized colon tumours and for prognosis following therapy are needed. Proteomics research is currently producing numerous biomarker studies with clinical potential. We investigate the protein composition of plasma and of tumour extracts with the aim of identifying biomarkers for colon cancer. Methods By Surface Enhanced Laser Desorption/Ionisation – Time Of Flight / Mass spectrometry (SELDI-TOF/MS) we compare the protein profiles of colon cancer serum with serum from healthy individuals and the protein profiles of colon tumours with normal colon tissue. By size exclusion chromatography, we investigate the binding of HNP 1-3 to high mass plasma proteins. By microflow we investigate the effect of HNP 1-3 on mammalian cells. Results Human Neutrophil Peptides -1, -2 and -3 (HNP 1-3), also known as alfa-defensin-1, -2 and -3, are present in elevated concentrations in serum from colon cancer patients and in protein extracts from colon tumours. A fraction of HNP 1-3 in serum is bound to unidentified high mass plasma proteins. HNP 1-3 purified from colon tumours are lethal to mammalian cells. Conclusions HNP 1-3 may serve as blood markers for colon cancer in combination with other diagnostic tools. We propose that HNP 1-3 are carried into the bloodstream by attaching to high mass plasma proteins in the tumour microenvironment. We discuss the effect of HNP 1-3 on tumour progression.	Background The diagnostic stage of colon cancer determines survival. Patients diagnosed with localized tumours have a 75% probability of 5 year survival, whereas patients diagnosed with distant metastases only have a 5–10 % probability of 5 year survival (reviewed in [ 1 ]). Recently a number of studies have been published in which Surface Enhanced Laser Desorption/Ionisation-Time Of Flight/Mass Spectrometry (SELDI-TOF/MS) has been applied to biological samples from patients with various forms of cancer [ 2 - 4 ] leading to the identification of protein markers with clinical potential. Here we present a SELDI-TOF/MS study of colon cancer serum and tumours. We show that the expression of Human Neutrophil Peptides -1, -2 and -3 (HNP 1 -3), also known as alfa-defensin-1, -2 and -3, is upregulated in the tumour microenvironment, as compared to normal colon tissue. This finding is reflected in serum. We find that HNP 1-3 is present in elevated concentrations in serum from patients diagnosed with tumours in the colon, as compared to serum from a healthy control group matched by age and gender. By size-exclusion analysis we add to the existing evidence that HNP 1-3 bind to high mass plasma proteins, explaining the presence of HNP 1-3 in serum. By microflow analysis, we show that HNP 1-3 purified from colon tumours are lethal to mammalian cells. The HNP 1-3 peptides are part of the defensin family of peptides (reviewed in [ 5 - 7 ]), which are a fundamental component of the immune system and have the capacity to kill / inactivate a broad range of pathogens. Defensins are also known to function as regulators of both the innate and the adaptive immune system. We discuss the possible effects of HNP 1-3 in the tumour microenvironment. Methods Biological samples All biological samples were obtained by trained staff at Glostrup Hospital, Denmark. Written consent was obtained from all donators. Permission was obtained from the Danish Scientific Ethical Committee and the Danish Data Protection Agency. Tissue screening Normal colon tissue samples and colon tumour samples were obtained from the removed fragment of the patient's colon after surgical treatment for colon cancer. Tissue samples were stored at -80°C until use. The protein content was extracted from the tissue: 100 mg tissue sample was thawed on ice and homogenised on a Wheaton Overhead Stirrer for 2 minutes at speed step 2, in 500 ul Lysis buffer (100 mM TRIS-HCl, pH 8.0, 9.5 M UREA, 2% CHAPS). The samples were centrifuged at 14,000 rpm for 10 minutes and the pellet was discarded (repeated twice). The tissue protein extracts were stored at -80°C until use. Pilot studies were performed on different chips (data not shown) and the NP20 (Normal Phase) (Ciphergen) chip was chosen for the tissue screening. NP20 chips were placed in Bioprocessor (Ciphergen) and pre-treated with 50 ul tissue binding buffer (50 mM TRIS-HCl, pH 8.0) for 5 minutes on shaker (250 rpm) (repeated twice). 5 ul tissue protein extract was diluted in 50 ul tissue binding buffer and incubated in Bioprocessor on NP20 chips for 40 minutes at room temperature on shaker (250 rpm). Spots were washed twice in 250 ul tissue washing buffer (50 mM TRIS-HCl, pH 8.0) for 5 minutes. The chips were air dried for 10 minutes, followed by treatment with two times 0.6 ul 100% sinapinic (SPA) matrix solution. SPA was obtained from Ciphergen in 5 mg aliquots and dissolved (150 ul MQ water, 150 ul acetonitrile, 1.5 ul Tri-Fluoro-acetic-Acid (TFA)) immediately before the screenings. Serum screening Colon cancer serum samples were obtained from patients before surgical treatment. Normal serum was obtained from a group of healthy individuals matched by age and gender to the cancer patients. All serum samples were stored at -80°C until use. Serum pilot studies were performed on different chips to monitor the presence of HNP 1-3 in serum (data not shown). The immobilised metal affinity capture (IMAC30) chip was chosen for the actual screening and was pre-treated with nickel before analysis: 5 ul 100 mM NiSO4 were added to each spot and left on shaker (250 rpm) for 5 minutes (repeated twice). The chips were placed in Bioprocessor and incubated with 100 ul MQ for 5 minutes on shaker (250 rpm). Each spot was treated with 50 ul serum binding buffer (100 mM TRIS-HCl, pH 7.5, 500 mM NaCl, 0,1% Triton X-100) and left on shaker for 5 minutes (250 rpm). Serum samples were thawed on ice and 1 ul serum was diluted in 50 ul serum binding buffer and applied to spots and left on shaker (250 rpm) at room temperature for 40 minutes. The sample solution was removed and the spots were washed twice in 200 ul serum washing buffer (100 mM PBS, pH 7.4, 700 mM NaCl), followed by one wash in 200 ul MQ water. The chips were removed from the Bioprocessor and left to air dry for 20 minutes followed by treatment with two times 0.6 ul SPA. Only freshly made matrix solutions were used and the instrument was calibrated daily. Cancer and normal samples were run side by side. The chips were analysed on a PBS II instrument (Ciphergen). All spectra in each screening were normalised based on total ion current. Purification and identification of HNP 1-3 100 ul protein extract from colon tumour tissue in tissue lysis buffer was loaded unto a RP-HPLC column (uRPC C2/C18 ST 4.6/100, Pharmacia Biotech, Flow rate: 0.5 ml/min, Fraction size: 0.5 ml) in buffer A (0.065% TFA in MQ-water) and proteins were eluted in a gradient of 0–100% buffer B (0.05% TFA in acetonitrile (ACN)). Elution of peptides was monitored by absorbance at 280 nm. All protein-containing fractions were analysed by Matrix Assisted Laser Desorption/Ionisation-Time of flight (MALDI-TOF) on the PBS II instrument: 1.5 ul fraction was incubated with 0.6 ul SPA on a Gold array (Ciphergen) and left to crystallise, followed by an additional treatment with 0.6 ul SPA and analysed in the PBSII instrument. The HNP 1-3 containing fraction (32% buffer B) was further purified on a peptide gelfiltration column (Superdex Peptide HR 10/30, Pharmacia Biotech, Flow rate 0.9 ml/min, Fraction size: 0.5 ml, Buffer: 50% ACN, 0.1 % TFA). Elution of peptides was monitored by absorbance at 280 nm and protein-containing fractions were again analysed by MALDI-TOF. Purified HNP 1-3 were identified by on-chip trypsin digestion: 10 ul of HNP 1-3 fraction was applied to an NP20 chip and left on shaker (250 rpm) at room temperature for 40 minutes. The solution was removed and the spot was washed twice with 10 ul water. In order to denature peptides prior to digestion, the chip was left on heating block (80°C) for 5 minutes. The chip was cooled on ice for 2 minutes. 10 ul trypsin digestion solution (0.01 ug/ul trypsin in 50 mM NH 4 HCO 3 , pH 8,0) was added, and the chip was left for 10 hours at 40°C in humidity chamber after which the chip was left to air dry for 20 minutes. 1 ul CHCA matrix (prepared as the SPA matrix solution) was added and the peptide map was analysed on the PBS II instrument. Peptide maps of trypsin autodiggest were used as controls. Identification was done with the PepIdent software on the Expasy server. For the reduction experiment, HNP 1-3 were first denaturation by heating (10 minutes at 80°C) followed by treatment with DTT (200 mM, 30 minutes at room temperature) and the peptides were incubated on an NP20 chip and analysed on the PBS II instrument. Size exclusion chromatography of HNP 1-3 50 ul colon cancer serum was loaded unto a peptide gelfiltration column (Superdex Peptide HR 10/30, Pharmacia Biotech, optimal separation range: 1 to 7 kDa, flow rate: 0.5 ml/min, fraction size: 0.5 ml, buffer: 10 mM Ammonium carbonate, pH: 8.0). Elution of peptides was followed by absorbance at 280 nm. All protein containing fractions were analysed by MALDI-TOF on PBS II (Ciphergen) as described above. Maximum signal intensity of 40 individual peaks was plotted as a function of elution volume and an approximate elution curve was calculated. Study of HNP 1-3 by microflow For micro flow experiments, canine kidney cells (MDCK cells) were plated onto poly-d-lysine coated cover slips at a concentration 3000 cells/well, grown in Dilbeccoo's Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) for five days with the result of confluent islands. Microflow was performed in an Eppendorf micromanipulator 5171 and transjector 5246 system mounted on a Leica DMIRBE inverted research microscope. Micro capillaries (borosilicate with filament, Sutter Instruments Company, Novato, California, USA) were pulled to an outer diameter of 0.85 nm on a Sutter P-97 Micropipette Puller. The dye-loaded cells were visualized by excitation at 470 nm and recorded at 509-nm emission using Haupage version 3.3.18038 software and Kappa CF 15/4 MC-S camera (Leica). The MDCK cells were recorded (in CO2 independent media) on the inverted DMIRBE inverted research microscope. The capillary was placed 20 nmover the confluent cells with a constant flow (1300 hPa). The MDCK cells were exposed to peptide and calcein (20 mM) fractions for 60 minutes. Results HNP 1-3 expression in tissue and serum We performed pilot studies of colon tumour and normal colon tissue on a variety of chips with different chemical properties and with different binding and washing conditions. Based on these preliminary studies, we found that the expression of three peptides with mass/charge ratio (m/z) values of 3372, 3443 and 3486 (subsequently identified as HNP 2, 1 and 3, respectively), were upregulated in the tumour samples. The three peptides were visible on different chips and under different binding conditions (data not shown). The strongest signal of HNP 1-3 in tissue extract was obtained on the NP20 (Normal Phase) chip, whereas the strongest signal of HNP 1-3 in serum was observed on the IMAC30 (Ni) (immobilised metal affinity capture chip, activated with nickel), and these conditions were chosen for the actual screenings. We emphasize that in general the protein profiles of serum and tissue were very different when using the same protein chip for serum and tissue extract. However, individual peaks were present on several types of chips, and observed in both serum and in tissue extract, for example the characteristic triplet with m/z 3372, 3443 and 3486. Protein extract from 40 colon tumour and 40 normal colon tissue samples were analysed on NP20 chips and 125 colon cancer serum samples and 100 normal serum samples were analysed on IMAC30 (Ni) chips. All spectres in each screening were pooled and normalised based on overall ion current. Each spectrum produced approximately 40 to 90 protein peaks in the range from 2 to 80 kDa (FIG. 1A–C ). Statistical analysis of the intensity values of HNP 1-3 in the tissue screening (FIG. 2A .) showed that HNP 1-3 were significantly upregulated in tumours (p < 0.0005) and statistical analysis of HNP 1-3 expression in the serum screening (FIG. 2B .) showed that HNP 1-3 were significantly upregulated in cancer serum (p < 2.2e -16 ). Compared to other peptides in the same range, HNP 1-3 showed average signal intensity in most normal colon tissue extract, whereas the HNP 1-3 signal was extremely high in most tumour samples (in some tumour spectres the HNP 1-3 peaks were the strongest of all peaks). In the normal serum samples the HNP 1-3 signals were weak and only slightly stronger in the cancer serum. Identification of HNP 1-3 The markers were purified by RP-HPLC, peptide gelfiltration and on-chip purification, after which they were identified by peptide mapping as HNP-2 (3372 Da), HNP-1 (3442 Da) and HNP-3 (3486 Da) (Table 1A ). The masses correspond to the peptides in their oxidised states with three disulfide bridges. After reduction with DTT, HNP-1 and HNP-2 increased 6 Daltons in mass, due to reduction of the six cysteines (Table 1B ). We were not able to reduce HNP-3. Size exclusion chromatography of HNP 1-3 50 ul colon tumour extract in Lysis buffer was applied to a peptide gelfiltration column. Elution of peptides was followed by absorbance at 280 nm. All fractions were analysed by MALDI-TOF on the PBS II instrument (Ciphergen). Maximum signal intensity of 40 individual peaks was plotted as a function of the elution volume and an approximate elution curve was calculated (FIG. 3 ). HNP 1-3 peptides were primarily eluted in early fraction together with high mass proteins above 20 kDa and also, but to a lesser degree, in fractions together with other peptides of similar mass interval (2 to 4 kDa) (FIG. 3 ). Cytoxic assay The cytotoxicity of HNP 1-3 purified from colon tumours was tested by exposing MDCK cells to different fractions purified from colon tumours. Calcein were added to the fractions and the solutions were left to flow over the cells for one hour. By fluorescence microscopy calcein was observed to accumulate in cells exposed to HNP 1-3/calcein fractions, whereas cells treated with fractions containing other (unidentified) tissue peptides did not uptake calcein (FIG. 4C&D ). Further, by microscopy we observed that cells exposed to HNP 1-3 appeared more diffuse and had enlarged nuclei, indicating apoptosis (FIG. 4A&B ). Discussion Elevated concentrations of HNP 1-3 in colon cancer serum Abnormal concentration of HNP 1-3 in blood has previously been demonstrated in connection with benign conditions. Elevated concentrations of HNP 1-3 following infection (bacterial- / non-bacterial- infection and pulmonary tuberculosis) has been found in plasma, blood and other body fluids [ 8 ], and plasma HNP 1-3 concentrations have been shown to be elevated in patients with septicemia or bacterial meningitis [ 9 ]. HNP 1-3 have been found in urine from patients with transitional cell carcinoma of the bladder [ 10 ] and HNP-1 has been found in salvia of patients with oral carcinomas [ 11 ]. Finally, HNP 1-3 are found in excess amounts in tears after ocular surface surgery [ 12 ]. Our study is the first that demonstrate elevated concentrations of HNP 1-3 in blood following tumour growth. Elevated concentrations of HNP 1-3 in colon tumours HNP expression has previously been linked to different types of tumours and cell lines. HNP-1 has been detected in lung tumours [ 13 ] and in the submandibular glands of patients with oral carcinomas [ 14 ]. By RT-PCR, mass spectrometry and flow cytometric analysis, HNP 1-3 have been shown to be expressed by cell lines deriving from renal cell carcinomas [ 15 ] and the expression of a specific HNP precursor peptide has been shown to be upregulated in human leukemic cells [ 16 ]. Our results suggest that HNP 1-3 are extremely abundant in colon tumours. This is in agreement with a study of HNP-1 in lung tumours, where the maximum observed level was 26 nanomoles per gram wet tissue [ 13 ]. In order for these excessive amounts of peptide to be detectable in serum, the peptides must be present extracellular in the tumour environment, such as observed in studies of HNP 1-3 expression in kidney [ 14 ] and brain [ 17 ]. Usually tumour expressed peptides are not easily detected in serum or plasma by SELDI-TOF mass spectrometry techniques; the highly concentrated plasma proteins out compete the signal of low abundance peptides. We suggest that HNP 1-3 are detectable in serum only because they are expressed in exceptionally high amounts in the tumour microenvironment. Previous studies indicate that HNP 1-3 expression in tumours primarily originate from tumour invading eosinophils [ 13 ] and neutrophils [ 14 , 18 ]. However, it has been shown that the excess amounts of HNP 1-3 observed in urine from bladder cancer patients were often produced by the actual bladder cancer cells [ 10 ], and that highly invasive bladder cancer cells produced more HNP 1-3 than less invasive ones (Holterman DA, in press, personal communication). Since our tissue screening is based on comparison of tissue samples, we cannot say whether the peptides are produced by the colon cancer cells or by tumour infiltrating neutrophils. In the case of active inflammatory bowel disease, it is not clear whether epithelial expression of HNP1-3 is induced by the inflammatory state or the whether the peptides are released by adjacent neutrophils and taken up by the epithelial cells [ 6 ]. Here it is believed that the peptides provide protection against microbial invasion when the mucosal barrier is damaged (such as is the case in inflammatory bowel diseases). HNP 1-3 are known to stimulate bronchial epithelial cells to upregulate interleukin-8 production [ 19 ], a potent neutrophil chemotactic factor. Thus, the upregulated expression of HNP 1-3 in tumours may primarily originate from invading immune cells, but could be initiated by HNP 1-3 producing cancer cells. Size exclusion chromatography of HNP 1-3 We explain the elevated concentrations of HNP 1-3 in colon cancer serum by unspecific binding between HNP 1-3 and high mass plasma proteins. We suggest that the peptides attach to plasma proteins in the tumour area and are carried into the bloodstream. The HNP 1-3 we observe in high mass fractions from size exclusion, could also be explained by multimerisation: In one study it was demonstrated that defensins form voltage dependent channels in lipid bilayer membranes and further conductance investigations suggested that the channels were formed by multimers containing 2–4 molecules [ 20 ] and a crystal structure study [ 21 ] of HNP-3 revealed an amphiphilic dimmer. We interpret the size exclusion results as evidence for interaction between HNP 1-3 and unidentified high mass proteins through unspecific interactions; the peptides are eluted in very early fractions which would probably require the presence of multimers of more than five peptides and our study does not reveal the presence of multimers containing four, three or two peptides. Further, our interpretation is in agreement with a number of previous studies that show that HNPs are bound to plasma protein in vitro and that high concentrations of HNPs causes precipitation of plasma proteins; specifically 2-macroglubulin and C1 complement [ 22 , 23 ] has been shown to bind defensin. Another study [ 24 ] showed that HNP-1 bind to various plasma proteins, notably serum albumin, and it was found that serum, or serum albumin, was able to inhibit the anti-viral activity of HNP-1. This ability to bind to plasma proteins could also explain why HNP 1-3 lysis of mammalian cells is hindered in the presence of serum [ 25 ]. Together these observations add to the growing realisation that common plasma proteins may carry disease specific peptides and therefore should not be ignored in biomarker research. Common to beta-defensin 2 (another member of the defensin family) and HNP 1-3 is an uneven distribution of surface charges. Beta-defensin 2 has been shown to bind to a chemokine receptor [ 26 ]. It has been suggested that the positively charged cluster found in defensin peptides and chemokines, may play a common role in binding to receptors, but is not important for determining receptor specificity [ 27 ]. This surface charge may also explain the binding of HNP 1-3 to plasma proteins. The observation that defensins are localised to lymphocyte nuclei [ 28 ], could similarly be explained by unspecific binding to shuttle proteins. The function of HNP 1-3 The exact concentration of HNPs in the tumour microenvironment may decide the in vivo function of HNP 1-3. One study showed that HNP 1-3 mediates lysis of tumours in a concentration dependent manner [ 25 ]. This is in agreement with another study that show that only relatively high concentrations of HNP-1 (10 -4 M) are cytotoxic for human monocytes, whereas lower concentration of HNP-1 (10 -8 to 10 -9 M) increases TNF-alpha production by monocytes [ 29 ]. In a study of renal cell carcinoma lines [ 14 ] it was shown that HNP 1-3 were cytotoxic to all tested cell lines when present in high concentrations (above 25 ug/ml), but at lower concentration HNP 1-3 stimulated growth of a subset of tumour cell lines. We add to these results by demonstrating that in vivo HNP 1-3 purified from colon tumours are capable of lysing MDCK cells. Our study was based on a 60 minutes microflow study. This screening set-up did not allow us to investigate the minimum concentration of HNP 1-3 necessary for lysis. Conclusions The high concentration of HNP 1-3 observed in tumours and the observation that HNP 1-3 are capable of lysing mammalian cells may lead to the conclusion that the peptides serve to the benefit of the host by killing tumour cells. However, in one study HNP 1-3 were found to bind to HLA-Class II molecules and were capable of reducing the proliferation of a HLA-DR-restricted T-cell line after stimulation [ 30 ] and could in this way help the cancer cells avoid local immune recognition. Defensins also regulate the systemic immune response. Through interaction with the chemokine receptor CCR6, beta-defensins recruits dendritic and T cells[ 26 ] (discussed in [ 31 ]) and HNP 1-3 are capable of recruiting leukocytes to sites of infection in mice [ 32 ]. Upregulated immune responses are known to stimulate tumour proliferation; immune cells are actively recruited by tumours to exploit their pro-angiogenic and pro-metastatic effects (reviewed in [ 33 , 34 ]). Whether the high concentrations of HNP 1-3 in the tumour limits the tumour growth or on the contrary stimulate tumour proliferation is not clarified; we emphasise that HNP 1-3 are expressed in inflammatory bowel disease and could be involved in the early stages of carcinogenesis. We suggest that the prominent surface charge on defensins, their unspecific binding to other proteins (such as high mass plasma proteins) and the observed excess amounts of peptides found in tumours, could provide the peptides with broad antagonising effects on numerous receptors in the tumour microenvironment. In this way HNP 1-3 peptides may serve to the benefit of the tumour. Our results add to the evidence that HNP 1-3 bind to high mass plasma proteins. We suggest that the peptides are released in the inflammatory site and passively diffuse into the blood stream. HNP 1-3 have been observed in primary tumours of different tissues and the peptides are known to play a fundamental role in the innate immune system. We suggest that the peptides may serve as markers for colon cancer in combination with established diagnostic tools and as prognostic markers following therapy. Competing interests This study was partly financed by Colotech ltd. and partly by Glostrup Hospital. Jakob Albrethsen, Rikke Bøgebo and Hans Raskov receive salary from Colotech A/S. Steen Gammeltoft, Jesper Olsen and Benny Winther receive salary from Glostrup Hospital. Authors' contributions JA performed the SELDI-TOF/MS screenings, the protein identification experiments and the size exclusion study. RB did the statistical analysis, BW did the microflow study, JO obtained the biological samples. HR and SG planned the project. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC548152.xml
546037	Light-Dependent Development of Circadian Gene Expression in Transgenic Zebrafish	The roles of environmental stimuli in initiation and synchronization of circadian oscillation during development appear to vary among different rhythmic processes. In zebrafish, a variety of rhythms emerge in larvae only after exposure to light-dark (LD) cycles, whereas zebrafish period3 (per3) mRNA has been reported to be rhythmic from day 1 of development in constant conditions. We generated transgenic zebrafish in which expression of the firefly luciferase (luc) gene is driven by the zebrafish per3 promoter. Live larvae from these lines are rhythmically bioluminescent, providing the first vertebrate system for high-throughput measurement of circadian gene expression in vivo. Circadian rhythmicity in constant conditions was observed only after 5–6 d of development, and only if the fish were exposed to LD signals after day 4. Regardless of light exposure, a novel developmental profile was observed, with low expression during the first few days and a rapid increase when active swimming begins. Ambient temperature affected the developmental profile and overall levels of per3 and luc mRNA, as well as the critical days in which LD cycles were needed for robust bioluminescence rhythms. In summary, per3-luc zebrafish has revealed complex interactions among developmental events, light, and temperature in the expression of a clock gene.	Introduction The circadian clock controls biological processes such as behavior, gene expression, and physiology in diverse organisms, ensuring that these processes to take place at appropriate times of the day. This is crucial for many organisms, such as plants, which must synchronize photosynthesis with day-night cycles. Animals also synchronize to environmental cycles because of more subtle but nevertheless important needs such as predator avoidance, food availability, and optimal temperatures for various processes. Circadian clocks in all species share the following properties: They persist even in constant environmental conditions with periods near 24 h; they can be reset by environmental stimuli such as light and temperature; and their periods are relatively constant at different temperatures. Recently, remarkable progress has been made in elucidation of molecular mechanisms of circadian clocks in diverse organisms. The common feature of clocks is cycling gene expression due to intracellular transcriptional feedback loops [ 1 , 2 , 3 ]. Genetic analysis in higher metazoan species, for example the fruit fly Drosophila, has been extremely valuable in identifying important players of the clockworks and their roles [ 4 , 5 , 6 ]. Identification of clock genes in Drosophila lead to molecular dissection of clock mechanisms in mammals mainly by testing whether homologs of Drosophila clock genes are involved in mammalian clocks. While this approach is informative, it harbors the risk of missing important factors that would have been found by forward genetic searches without preconceptions. Furthermore, gaps in our understanding of the clock mechanisms include factors responsible for the expression of positive transcription factors Clock and Bmal and mechanisms for clock protein turnover. In this regard, zebrafish is in a unique position as a vertebrate species in which large-scale forward genetic screens are convenient [ 7 , 8 ]. Furthermore, zebrafish circadian clocks have been shown to possess unique properties such as light entrainability of molecular rhythms in cultured organs and cells [ 9 , 10 , 11 ]. A behavioral screening for circadian mutations has been successfully carried out in zebrafish [ 12 ]. However, due to the limited capacity of this method, it is not suited to high-throughput screening. A method relying on bioluminescence rhythms mediated by luciferase reporting has been successfully used to screen for mutants affecting circadian gene expression in plants, cyanobacteria, and flies [ 13 , 14 , 15 ]. In vertebrates, however, luciferase reporting has been used mainly for recording circadian gene expression in cultured tissues and cells, because of technical difficulties in these species [ 16 , 17 , 18 , 19 , 20 ]. Bioluminescence rhythms mediated by a zper4-luc promoter fusion construct have been studied successfully in the zebrafish PAC-2 cell line [ 21 ]. While this approach was useful for promoter dissection, generation of transgenic animals is necessary for mutagenesis screening. In this study, transgenic zebrafish were made in which cycling expression of the firefly luc gene is driven by the promoter of per3 [ 22 ]. This promoter was chosen because per3 mRNA has been shown to oscillate rhythmically in embryos as well as in a cell line [ 11 , 22 ]. For mutagenesis screening, it is most convenient and economical to test the youngest possible animals and avoid raising the animals that give negative results. In this regard, per3-luc was considered ideal for mutagenesis screening, because an in situ hybridization study showed that per3 mRNA cycles starting on day 1 postfertilization with or without any entraining signals [ 22 ]. It was suggested that maternal per3 mRNA present in the oocyte can set the phase of per3 mRNA rhythms in early embryos. This result, however, is not consistent with other studies involving development of circadian rhythms: Rhythms of melatonin production require a light-dark (LD) transition later than 20 h postfertilization [ 23 ]; circadian swimming rhythms in larval fish develop during the first 4 d of development and require entraining signals late in embryonic development [ 24 ]; and rhythms of cell proliferation in larval fish develop only after exposure to several LD cycles [ 25 ]. In order to determine the earliest possible developmental time when rhythmic luc expression can be monitored in the per3-luc transgenic fish, embryos from the transgenic lines were monitored for bioluminescence from day 1 of development ( Protocol S1 ). To our surprise, very low and non-oscillating levels of bioluminescence were detected during the first 4–5 d into development. Furthermore, consistent with the rhythms of melatonin production, locomotor activity, and cell division, rhythmicity of the per3 gene expression gradually developed during the first several days postfertilization, and was observed only if fish were exposed to LD cycles during the hatching period or later. It was also found that ambient temperature affects per3-luc -mediated bioluminescence in a complex way. This study defines conditions under which the per3-luc transgenic fish can be used for mutagenesis screening and other types of studies. Results Generation of per3-luc Transgenic Fish To develop a system in which circadian gene expression in zebrafish can be monitored in vivo, transgenic fish were generated in which the expression of the firefly luc gene is driven by the promoter of the per3 gene [ 22 ]. The construct was made by modifying a bacterial artificial chromosome (BAC) originally screened for sequences in the first coding exon of per3 ( Figure 1 ) . By comparison of per3 cDNA sequence to the genomic sequence from another BAC clone (CH211–138E4) from this region, it was found that the cDNA contains another exon 5′ to the first coding exon. By comparing BAC-end sequences from the construct to genomic sequences from CH211–138E4 as well as with the Ensembl Zebrafish whole genome shotgun assembly sequence version 4 ( http://www.ensembl.org/Danio_rerio/ , the construct was found to be approximately 72 kb long, and spans from 26 kb upstream of exon 1 to intron 19 of per3, and contains part of another gene 5′ to per3 ( Figure 1 ). One canonical and two noncanonical E-boxes were found within 1 kb of the per3 promoter (unpublished data). In the modified BAC, the coding portion of the first coding exon was replaced by the luc and kanamycin resistance (Km r ) genes ( Figure 1 ). This rather long construct was made because it has been shown in zebrafish that a reporter gene is more consistently expressed in a context of a longer BAC construct than in a conventional short construct with just a few kilobases of promoter sequences [ 26 ]. Figure 1 Schematic Map of the per3-luc Construct The top graphic shows the exon-intron structures of per3 and the flanking gene. The BAC clone 8M06 screened for the first coding exon of per3 is approximately 72 kb long, and extends from about 26 kb upstream of exon 1 to intron 19 of per3 . The bottom graphic shows the magnified view of the first coding exon in the BAC 8M06 and the modified BAC construct. The white and black boxes represent noncoding and coding sequences, respectively, of the first coding exon of per3 . The coding sequence of this exon was replaced with an approximately 3-kb fragment containing luc and Km r . Arrows under the construct represent primers used for the screening of transgenic lines. After screening 147 injected founders by PCR, five independent transgenic lines were found. Each positive founder was bred to a wild-type fish, and their progeny were individually tested for bioluminescence as 5–7-d-old larval fish. Larval fish with bioluminescence above background (more than 100 counts per second [cps]) were raised as transgenic F1 fish. Three of the five lines emitted bioluminescence above background. The level of bioluminescence varied depending on the line. The strongest-glowing line (#23) was used for this study unless otherwise stated. All the animals used in this study were the progeny of crosses between a transgenic line and the AB wild-type strain. Therefore, these animals carried the transgene in hemizygous condition. Light Signals Are Necessary for per3-luc Rhythms One of the intended usages of the per3-luc transgenic zebrafish is screening for mutations that affect bioluminescence rhythms. Since it is most convenient to screen the youngest possible animals, bioluminescence from transgenic embryos was monitored first. It was also expected that per3-luc -mediated bioluminescence in embryos should cycle from day 1 of development even in constant conditions, because per3 mRNA expression detected by in situ hybridization has been demonstrated to oscillate from day 1 postfertilization in constant conditions [ 22 ]. Therefore, embryos carrying the transgene in hemizygous condition were collected and their bioluminescence monitored for 10 d starting from day 1 postfertilization. Surprisingly, when embryos were exposed to only one 14 h light: 10 h dark (14:10 LD, lights on at 8 A.M. ; lights off at 10 P.M. CST) cycle on day 1, the majority of the animals showed no circadian rhythmicity of bioluminescence ( Figure 2 A and 2 B; Table 1 ). Nevertheless, a characteristic developmental profile of luc expression was observed. Bioluminescence mediated by per3-luc stays rather low until day 4, when there is a small peak of bioluminescence, followed by a small dip on day 5 and a rapid increase that reaches the second peak on days 7–9. The slow decline of luminescence after that point may be due to substrate deprivation common in luciferase reporting [ 27 ]. Importantly, this developmental profile was also observed in two other lines of per3-luc, albeit with much lower overall luminescence counts (unpublished data). Figure 2 Bioluminescence in Embryos That Experienced Different Numbers of LD Cycles during Development Embryos hemizygous for per3-luc were collected and monitored for bioluminescence while exposed to different numbers of 14:10 LD cycles starting on day 1 postfertilization followed by DD. (A) One LD, (B) one LD, (C) two LD, (D) three LD, (E) four LD, (F) five LD, (G) six LD, and (H) six LD. In (I), embryos were exposed to two LDs, one on day 1 and the other on day 6 of development. Black and white bars on top of each plot represent the times when the lights were off and on, respectively. Since overall bioluminescence levels can vary among clutches and experiments, normalized bioluminescence was averaged and plotted in each graph. Number of animals that were averaged is given at top right corner of each plot. Error bars represent standard error of the mean (SEM) For the one-LD and six-LD groups, plots for two experiments are shown here. These experiments showed small differences in developmental profiles, possibly due to differences in room temperature (about 1 °C), therefore could not be pooled. For the two- to five-LD groups, data from two experiments were pooled. The small but abrupt increase of luminescence that occurred only during the light period of LD cycles is considered an artifact made visible by low bioluminescence counts during the first several days of development, because the same level of fluctuation was observed in empty wells under LD condition. Table 1 Rhythmicity and Periods of Larval Zebrafish: Varying Number of LD Cycles Data are included for 5.5- to 10-d-old larval fish that had experienced varying numbers of LD during development. For each group, results of two experiments were pooled. Mean ± SEM of rhythm statistic [ 54 ] and period were calculated for rhythmic individuals only. A series of G-tests showed that percent rhythmic values were significantly different ( p < 0.001) among the seven groups a Further tests showed that these percent rhythmic values are significantly higher ( p < 0.05) than the others. Except for the one- to three-LD groups that were mostly arrhythmic, differences in rhythm statistic among groups were not quite significant (Wilcoxon/Kruskal-Wallis test, p = 0.027, α = 0.017) Since the light signal on day 1 was not enough to elicit detectable rhythmicity of bioluminescence, an increasing number of LD cycles were given to embryos while they were monitored for bioluminescence (see Protocol S1 ). LD cycles on days 2 and 3 did not increase rhythmicity on subsequent days, although small fluctuations of bioluminescence were discernible on days 7–10 in the averaged plots ( Figure 2 C and 2 D; Table 1 ). The number of animals expressing significant rhythmicity during the last 4.5 d of the record increased gradually when the number of LD cycles was increased from three to six ( Figure 2 D– 2 H; Table 1 ). The circadian fluctuation was superimposed on the developmental profile also seen in embryos entrained by fewer numbers of LD cycles ( Figure 2 ). To determine whether the number of LD cycles or the developmental stage at which the last LD transition occurred is more important for robust rhythmicity of luciferase reporting, embryos were monitored for bioluminescence while experiencing two LD cycles, one on day 1 and the other on day 6 ( Protocol S1 ). Luminescence rhythms on the last 4.5 d of the record for this group of animals were as robust as those exposed to six LDs ( Figure 2 G– 2 I; Table 1 ). Thus, the developmental stage at which the last LD transition occurred, rather than the number of LD cycles, was important for light entrainment of per3-luc rhythms. There was no systematic effect of the time of the last lights-off on free-running periods ( Tables 1 and 2 ). Table 2 Rhythmicity and Periods of Larval Zebrafish: Varying LD and Temperature Data are included for 5.5- to 10-d-old larval fish that had experienced varying numbers of LD at different temperatures during development. Results from one of two experiments with similar results are shown here. Mean ± SEM of rhythm statistic and period were calculated only for rhythmic individuals. A series of G-tests showed that percent rhythmic values among the six groups were significantly different ( p < 0.001) a Further tests showed that these percent rhythmic values were significantly higher ( p < 0.01) than the others. For the six-LD groups, the rhythm statistic was not significantly different between the two temperature groups ( t -test, p = 0.61) Luciferase Reporting Reflects per3 Expression The lack of bioluminescence rhythms during the first few days of development was rather unexpected because of the previously reported per3 mRNA rhythms [ 22 ]. Therefore, mRNA cycling of per3 and luc was compared by real-time quantitative PCR (qPCR). As in the experiment shown in Figure 2 G and 2 H, embryos were exposed to six LD cycles and transferred to constant darkness (DD). Embryos were collected and their mRNA was extracted on days 3 and 8. Both per3 and luc mRNA levels were much lower on day 3 compared to day 8 ( Figure 3 ). What appears to be approximately 2-fold oscillations of per3 and luc mRNA on day 3 (see the insets on Figure 3 A and 3 C) were not statistically significant ( p = 0.47 for per3 , and p = 0.08 for luc by the Wilcoxon/Kruskal-Wallis test). In contrast, approximately 5-fold fluctuations of the transcripts were observed on day 8 ( Figure 3 B and 3 D). This increase in overall expression levels and cycling amplitudes reflect the observed bioluminescence profile, although there were qualitative differences between bioluminescence and RNA as well as between per3 and luc mRNA (see Discussion). Importantly, the peak phase of mRNA cycling on day 8 was 5–7 h advanced compared to the phase of bioluminescence cycling (compare Figure 2 G and 2 H to Figure 3 B and 3 D). Figure 3 Temporal Expression of luc mRNA Is Similar to That of per3 during Development Embryos hemizygous for per3-luc were collected from naturally breeding parents and kept in 14:10 LD cycles (lights on at 8 A.M. CST) at 22 °C for 6 d. Larval fish were shifted to DD at the end of the light phase on day 6. Total RNA was extracted from 2- to 3-d-old embryos and 7- to 8-d-old larval fish, and was subjected to real-time PCR for per3 and luc mRNA levels. (A) Expression of per3 mRNA per embryo on day 3 was determined every 6 h starting at 10 A.M. (2 h after lights-on). (B) The per3 mRNA per animal on day 8 was measured every 4 h starting at 10 A.M. (C) Levels of luc mRNA on day 3 were determined as in (A). White and black bars at the bottom represent light and dark phases, respectively, for (A) and (C). (D) Cycling of luc mRNA on day 8 was determined as in (B). The gray and black bars represent the time when the light would have been on and off, respectively, had the LD cycles continued. For each of per3 and luc , mRNA levels were normalized to the peak level on day 8 (10 A.M. time point). The y-axis scales were set at 120% maximum for all plots to allow direct comparison of mRNA extracted on days 3 and 8. The x-axis scales are given in both hours and days postfertilization to facilitate the comparison with Figure 2 . In order to show more detailed temporal profiles of mRNAs on day 3, plots with smaller y-axis scales were shown in the insets at top right corners of (A) and (C). Each plot is the average of three identical experiments, and error bars represent SEM. An identical experiment was also done at 24 °C with essentially the same results (unpublished data). Effects of Ambient Temperature on per3-luc -Mediated Bioluminescence The experiments presented above were done at 21–24 °C simply because fish survived better at these rather low temperatures (see Materials and Methods ). However, the previously documented circadian studies on zebrafish, including those involving development of rhythmicity, have been done mainly at the higher temperatures of 25–28.5 °C [ 22 , 23 , 24 , 25 ]. Since higher temperatures accelerate development in general, it was conceivable that development of bioluminescence rhythms may be faster at higher temperatures. However, it was not possible to do the same experiment at higher temperatures, because fish do not survive well in microtiter wells at temperatures higher than 25 °C. Therefore, embryos were raised in petri dishes at two different temperatures, 22 °C and 28.5 °C, while exposed to two, four, or six LD cycles. Subsequently, they were placed in microtiter wells and bioluminescence was recorded in DD at 21–24 °C ( Protocol S1 ). The majority of embryos that were raised at 22 °C were arrhythmic after they were entrained by two or four LD cycles, but they were highly rhythmic after six LD cycles ( Figure 4 A, 4 C, and 4 E; Table 2 ). This is largely consistent with the trends observed in Figure 2 and Table 1 , although more fish were rhythmic in this experiment for the two-LD and six-LD groups, and less in the four-LD group. The increased percentage of rhythmicity in this experiment for the six-LD group may be due to the fact that embryos raised in petri dishes are generally healthier than those raised in 96-well plates. Embryos raised at 28.5 °C showed significantly higher rhythmicity for the four-LD group than the same group raised at 22 °C ( p < 0.05; Figure 4 C and 4 D; Table 2 ). This result shows that embryos raised at higher temperatures can be entrained earlier than those raised at lower temperatures. Figure 4 Effects of Temperature on Development of per3-luc Expression Transgenic embryos were entrained by two, four, or six LD cycles at either 22 °C or 28.5 °C, and monitored for bioluminescence in DD at 21–24 °C. (A) Two LDs at 22 °C, (B) two LDs at 28.5 °C, (C) four LDs at 22 °C, (D) four LDs at 28.5 °C, (E) six LDs at 22 °C, and (F) six LDs at 28.5 °C. The insets in (E) and (F) show the last 3 d of the record with magnified y-axis scales. Black and white bars on top of each plot represent the times when the lights were off and on, respectively. Actual amount of bioluminescence in cps is averaged and plotted in each graph. Number of animals that were averaged is given at top right corner of each plot. Error bars represent SEM. Results of one of two identical experiments with similar results are shown here. In contrast to the effects on rhythmicity, temperature during the first several days of development seemed to have no systematic effects on periods: The two methods used for period estimation showed opposite effects of developmental temperature on periods (see the six-LD groups in Table 2 ). It should be pointed out that this may not mean that per3-luc rhythm is temperature compensated, because all the fish were monitored in the same temperature condition. Besides development of rhythmicity, the developmental profile of luc expression and baseline level of bioluminescence were also affected by prior ambient temperature. For the two-LD groups, the first and second developmental peaks came earlier in the 28.5 °C than in the 22 °C group ( Figure 4 A and 4 B). Higher temperatures also caused elevated levels of baseline bioluminescence, especially in the six-LD group ( Figure 4 E and 4 F). This higher bioluminescence cannot be caused by high specific activity of equivalent luciferase enzyme, because all of the fish were monitored at the same temperature, and the difference in bioluminescence level persisted through over 4 d of monitoring. Therefore, this difference in luminescence most likely reflects a difference in the level of luc expression. Part of this difference between the two temperature groups may be explained by the fact that animals raised at higher temperatures are more mature and therefore express more luciferase than do those raised at lower temperatures. However, 10-d-old animals raised at 22 °C, which should have reached the plateau of luminescence (see Figure 2 ), showed much lower bioluminescence than 10-d-old fish raised at 28.5 °C (compare Figure 4 E and 4 F). Therefore, maturity of animals cannot explain the difference either. It seems that animals raised at higher temperatures simply express more luciferase than do those at lower temperatures. This was confirmed by a real-time PCR experiment ( Figure 5 ). Cycling amplitudes and peak levels of per3 and luc mRNA on day 6 were much higher in fish raised at 28.5 °C than those at 22 °C. The difference was especially large for luc, for which an approximate 40-fold difference between the two temperature groups was found at Zeitgeber Time 0 (2 h after lights-on; Figure 5 C). Figure 5 Levels of per3 and luc mRNA Are Elevated by High Temperatures during Development (A) A schematic diagram showing how embryos were entrained and collected for RNA extraction. Embryos were entrained in 14:10 LD cycles (lights on at 8 A.M. ; lights off at 10 P.M. CST) at two different temperatures, 22 °C and 28.5 °C. On day 6, half of the animals were sacrificed for RNA at 10 A.M. (2 h after lights-on) and at 10 P.M. (at lights-off). The rest of the animals were transferred to DD at 22 °C at 10 P.M. on that day, and sacrificed for RNA on day 10 at 10 A.M. and 10 P.M. The white and black bars represent day and night, respectively, and the gray bars the time at which lights would have been on had the LD cycles continued. The arrowheads indicate the time at which the animals were sacrificed for RNA extraction. (B) Relative mRNA level per animal for per3 on days 6 and 10 of the experiment quantified by real-time qPCR. The levels were normalized to the value of the 10 A.M. time point on day 6 at 28.5 °C. (C) Relative RNA level per animal for luc measured from the same samples used in (B). For both (B) and (C), averages of three experiments are shown. Error bars represent SEM. Bioluminescence in the high-temperature group gradually decreased over several days after they were transferred to lower temperatures, but did not fully return to the level of the low-temperature group (see Figure 4 E and 4 F). This is consistent with per3 and luc mRNA levels determined by real-time PCR ( Figure 5 ). Taken together, high temperatures elevate the level and cycling amplitudes of per3 and luc mRNA, at least in larval fish, and this level and amplitude can gradually decrease after the animals are shifted down to lower temperatures. Optimal Condition for Recording Larval per3-luc Rhythms Fold amplitudes of bioluminescence rhythms were higher when embryos were entrained by LD cycles for 6 d at 22 °C rather than at 28.5 °C (see Figure 4 E and 4 F). Furthermore, survival of the animals was better if they were raised at 22 °C than at 28.5 °C (96.9% and 0% survival, respectively, on day 7). Therefore, embryos were entrained by six LDs at 22 °C in a petri dish, and tested for bioluminescence rhythms in DD ( Protocol S1 ). In this experiment, embryos survived better than they did when they were placed in 96-well plates from day 1 onward (89.6% survival on day 12 in this experiment, compared to 71.5% on day 10 for the experiments presented in Figure 2 ). Furthermore, 88.1% ( n = 42) of the fish were rhythmic with a 25.2 ± 0.7 h (mean ± standard deviation) period under this condition, and their rhythms persisted for 6 d, albeit with some damping ( Figure 6 A and 6 B). In addition, per3-luc rhythms were tested in LD ( Protocol S1 ). Amplitudes of bioluminescence rhythms in LD were higher than in DD, although they also damped slightly, possibly due to substrate deprivation ( Figure 6 C and 6 D). A slightly higher percentage of fish was rhythmic in LD (95.0%, n = 179) compared to DD, although the difference was not significant ( p > 0.1, G-test). The waveform of the rhythm in LD was different from that in DD: The ascending part of the wave that happens during the day was steeper in LD than in DD ( Figure 6 ), suggesting that light may induce transcription of per3 . Figure 6 Bioluminescence Rhythms Mediated by per3 - luc in DD and LD Measured for Six Days (A) Average plot of bioluminescence rhythms in DD. Animals were entrained in 14:10 LD cycles for 6 d at 22 °C and tested for approximately 6.5 d in DD. (B) Representative plot of bioluminescence rhythm in DD for an individual. (C) Average plot of bioluminescence rhythms in 14:10 LD cycles. Animals were entrained in 6 LD cycles at 22 °C prior to the monitoring. (D) Individual plot of bioluminescence rhythm in LD cycles. For each of DD and LD experiments, two experiments have been performed with essentially the same results. Only one of two experiments is shown for each of DD and LD. The first 12 h of data were deleted from each plot. Black and white bars on top of each plot represent the time when lights were off and on, respectively. Numbers of animals that were averaged is given at top right corner of (A) and (C). Error bars represent SEM in (A) and (C). Discussion The per3-luc transgenic zebrafish system presented here is unique, because it is the only vertebrate system in which circadian gene expression in the whole animal can be studied in a high-throughput manner. This property of per3-luc in combination with zebrafish genetics makes the transgenic fish suitable for mutagenesis screening for circadian mutants. Embryos were tested initially, because it is more efficient to screen embryos than older animals, and movements of older animals can cause noise in bioluminescence signals [ 28 ]. However, the current study clearly demonstrates that monitoring embryos is not an option for any circadian studies using these lines. Of the conditions tested, raising fish to 6 d of age at 22 °C under LD cycles produced the most robust free-running rhythms. Rhythms measured under LD cycles were even stronger, and this condition has been used successfully in previous screens [ 15 ]. In addition to mutagenesis screening, it will facilitate other studies such as circadian organization of central and peripheral oscillators, entrainment pathways by various environmental cues, and physiological effects on circadian rhythms as have been studied in other organisms [ 17 , 18 , 29 , 30 , 31 , 32 , 33 ]. Development of per3 RNA Cycling in Larval Zebrafish Larval fish that experienced an LD-DD transition later during development were more rhythmic than those shifted to DD earlier. This may mean that there is a critical developmental period after which per3-luc rhythms can be entrained. Alternatively, per3-luc rhythms might damp so fast that rhythms entrained a few days earlier could not be detected. We think the latter possibility unlikely for the following reasons: per3-luc -mediated bioluminescence rhythms persisted reasonably well for at least 6 d in DD if entrained properly ( Figure 6 A); at 28.5 °C, the rhythmicity of larval fish entrained for 4 d was comparable to that of fish entrained for 6 d, suggesting that bioluminescence rhythms do not damp in 2 d ( Table 2 ); and luc mRNA cycling was almost undetectable during the first few days of development (see Figure 3 C), and this low-amplitude fluctuation cannot give rise to high-amplitude oscillations unless there is a separate rhythm-amplifying mechanism operating during development, such as synchronization of cellular oscillators. The results presented here, namely the gradual development of rhythmicity and responsiveness to entraining stimuli during the first several days of development and requirement of light signal, is consistent with previous observations on rhythms of melatonin production, locomotor activity, and the cell cycle in larval zebrafish [ 23 , 24 , 25 ]. It is also consistent with several studies in other vertebrate species involving gene expression [ 34 , 35 , 36 ] and physiological rhythms [ 37 ]. In both insects and mammals, free-running behavioral rhythms can develop normally in the absence of entraining signals, but their phases are not synchronized [ 38 , 39 , 40 ]. DeLaunay et al. [ 22 ] reported that per3 RNA detected by in situ hybridization cycled synchronously from day 1 of development [ 22 ]. In our hands, however, per3 and per3 -driven luc RNA in 3-d-old larval fish detected by real-time PCR were not significantly rhythmic. Low-amplitude oscillations of both mRNAs may exist at this early developmental stage, because a similar trend (high in the morning) was observed in all three independent experiments done. However, this does not mean that the low-amplitude per3 RNA oscillations that occur earlier during development can amplify into high-amplitude ones without any entrainment by LD cycles. The increase of cycling amplitude over the course of several days of development in LD may happen within each cell that expresses per3 . Alternatively, cycling amplitude may increase because various cellular oscillations present become synchronized. However, it was not only the cycling amplitude that increased with age, but the overall levels of per3 mRNA also. Therefore, more cells may start expressing per3 with high-amplitude oscillations later during development. This rather simple scenario is in fact what happens in developing Drosophila; as soon as the central pacemaker Lateral Neurons start expressing the PERIOD protein, the molecular rhythm is entrainable, and so is the eventual behavioral rhythmicity [ 40 , 41 ]. In any case, increasing amplitudes within cells, synchronization of oscillators, and more cells with high-amplitude oscillations are not mutually exclusive. It is worth mentioning that rhythms of melatonin release starts as early as day 2 postfertilization, and this roughly corresponds to the time when the light-sensitive pineal gland is formed [ 23 ]. Another photosensitive organ, the retina, becomes photoresponsive as early as day 3 postfertilization [ 42 ]. Therefore, these organs become photosensitive, and/or develop rhythmicity prior to robust oscillations of per3 RNA. Again, clocks in some tissues may develop earlier than in other tissues. It is also possible that per3 may not be expressed in all the clock cells. The biological significance of the developmental profile of per3 -driven luc expression is not known. Most animals seemed to hatch between the first minor developmental peak and the subsequent trough (unpublished data). However, hatching itself is unlikely to induce per3 expression in early embryos, because dechorionated per3-luc embryos showed developmental profiles of bioluminescence similar to those of nondechorionated siblings, albeit with an accelerated second rise of bioluminescence (unpublished data). Larval fish after the hatching period are supposed to have completed most of their morphogenesis and start swimming actively [ 43 ]. It may be that per3 is important for rhythmic processes specific to hatched animals, such as behavioral rhythms [ 24 ]. Consistency between per3 Expression and Bioluminescence The developmental and circadian profiles of per3 -driven bioluminescence largely reflected endogenous per3 expression. However, the amplitude of bioluminescence cycling was greatly reduced compared to that of per3 or luc RNA. In general, proteins synthesized from cycling mRNAs show amplitude reduction and phase delay due to protein stability [ 44 ]. Consistently, luciferase-reporting studies in other organisms also showed dampening of cycling. For instance, in the per - luc transformants of Drosophila, approximately 6-fold cycling of luc RNA was reduced to 3- to 4-fold bioluminescence rhythms [ 27 ]. Similar reduction in cycling amplitude was observed for suprachiasmatic nuclei from the per1-luc mouse [ 18 ]. However, the reduction of amplitude was even more dramatic in per3-luc larval fish. This may mean that the luciferase protein is somehow more stable in larval fish than in flies or mice. It should be noted that the luciferase protein itself is quite stable, but the enzymatic activity of this protein is unstable [ 28 , 45 ]. Therefore, the apparent stability of the luciferase protein in larval fish may be in fact stability of luciferase activity. Besides the difference between bioluminescence and mRNA, there were differences between per3 and luc mRNA, measured by real-time qPCR. The difference between days 3 and 8 of development was much larger for luc than per3 . Furthermore, the difference between the 22 °C and 28.5 °C groups was larger for luc than for per3 . These differences may be due to positional effect of the insert in the particular transgenic line used in this study. However, differences in bioluminescence levels between the first few days and older fish were found in two other independent per3-luc lines (unpublished data). Therefore, it is more likely that these differences between the two mRNA species reflect a property of the transgene itself. Although the BAC transgene used in this study has approximately 26 kb of upstream sequences, there may be critical sequences missing from this transgene, such as the first coding exon. Alternatively, posttranscriptional modification could be responsible for the difference. Posttranscriptional control of clock gene mRNA expression has been documented elsewhere [ 18 , 28 , 46 , 47 ]. Effects of Ambient Temperature on per3 Expression Increasing ambient temperature had three effects on development of per3 expression: It increased peak levels and cycling amplitudes of mRNA, and accelerated the developmental profile and development of responsiveness to the entraining stimuli. The latter two effects presumably are due simply to the fact that development is accelerated by higher temperatures. These results are not consistent with the report that development of a cell-cycle rhythm in larval fish was not affected by ambient temperature [ 25 ]. The increase in peak level and cycling amplitudes of per3 mRNA induced by increased temperature is mostly independent of developmental speed. The peak per3 mRNA level in the high-temperature group on day 6 was higher than that in the low-temperature group on day 10, when bioluminescence levels should have reached the plateau (see Figure 5 ). Ambient temperatures are known to affect levels of clock gene expression in Drosophila and Neurospora [ 47 , 48 , 49 ], and this could be the mechanism for clock resetting by temperature shifts [ 49 ]. Since our study was done on developing animals, it remains to be seen whether the increase in per3 expression by elevated temperatures holds true in adults. Furthermore, it was not possible to test larval fish at 28.5 °C, and therefore it is not known whether the per3 mRNA rhythm is temperature-compensated. Levels of per3 and luc mRNA in fish raised at higher temperatures did not fully return to the levels in fish raised at lower temperatures, even after 4 d at lower temperatures. Therefore, there may be a mechanism for maintaining circadian periods despite change in per3 expression levels. Materials and Methods Animals Animals used in this study were derived from the University of Oregon AB strain. Adults were kept under 14:10 LD (lights on at 8 A.M. ; lights off at 10 P.M. CST) cycle, and group-housed in plastic tanks in a Z-MOD holding system (Marine Biotech) with recirculating filtered water at about 28.5 °C. They were fed commercial flake food in the morning, baby brine shrimp at midday, and adult brine shrimp in the evening. Experimental protocols were approved by the Institutional Animal Care and Use Committee. Embryos were collected from naturally breeding fish in the morning, by plastic mesh traps that prevented parents from eating their progeny [ 23 ]. For microinjection, one- to two-cell-stage embryos were required. Therefore, male and female breeders were separated by a divider when they were placed in a trap. By removing the divider, fish were allowed to breed just before microinjection was performed [ 50 ]. Construction of per3-luc transgene First, a zebrafish BAC library was screened for BACs containing 5′ coding sequences of per3 [ 22 ] using a PCR-based screening kit (Incyte Genomics, Wilmington, Delaware, United States). One of two such clones, 8M06, was used for the construction of the transgene. The primers used for the screening were: forward, 5′- CCAGTAAAACGTCGTCGTCA-3′; reverse, 5′- GTCTGGGCCTGGAGAAGAGT-3′. The per3 sequence from the initiation codon to the end of the first coding exon was replaced by a gene cassette containing the luc and Km r genes by homologous recombination in E. coli (see Figure 1 ) [ 51 , 52 ]. The luc / Km r gene cassette was constructed as follows. First, the Km r gene was cloned into NotI and SacI sites of pBluescriptSK+ (Stratagene, La Jolla, California, United States); then a HindIII-BamHI fragment containing luc and the SV40 polyA signal from pGL3-Basic (Promega, Madison, Wisconsin, United States) was cloned upstream of Km r into HindIII and BamHI sites of pBluescriptSK+; finally, a NotI site between Km r and the polyA signal was destroyed by digesting the clone with NotI and BamHI, followed by treatment with the Klenow fragment, and ligation of these blunted ends together. Using this gene cassette as a template, a PCR fragment flanked by approximately 50-bp homology arms was amplified. The primer sequences used for the PCR reaction were: forward, 5′- GGGTTGTGAATCAGATCTTCAGTAGAGGAGGACAGGAGATCTCACAGGGAATGGAAGACGCCAAAAACATAAAGAAAG-3′; reverse, 5′- GTGCAGATTAAGTCAAATTCCACATAAAAAAAGCCACATTTCAAGTGTAC CGTTAATAATTCAGAAGAACTCGTC-3′. The forward primer contains 25 bp of sequence from the 5′ end of the luc coding sequences flanked by a 53-bp overhang corresponding to sequences just upstream of the initiation codon of per3 . The reverse primer consists of a 50-bp overhang that corresponds to the intron sequences just downstream of the first coding exon, and 25 bp of sequence from the 3′ end of Km r as the primer. The PCR fragment was purified and electroporated into DH10B cells containing the BAC and the plasmid pBAD-αβγ [ 51 ] as a source of recombinase genes. Cells in which the BAC was successfully modified were selected by kanamycin. The luc sequences in the modified BAC clones were checked for PCR errors by sequencing. One PCR error that resulted in a Val 217 to Ala change in the luciferase protein sequence was found. However, this is a conservative change, and fish injected with this construct showed bioluminescence above background. Therefore, it was judged that luciferase encoded by this construct can still function. Generation of per3-luc transgenic lines The per3-luc transgene was purified, linearized by NotI digestion, and injected into one- to two-cell-stage zebrafish embryos according to [ 53 ] with minor modifications. Injected embryos were raised to adulthood and individually bred to a wild-type fish or pairwise bred to each other. The progeny were tested for the presence of the transgene by PCR. PCR primers used for the screening were: Per35F, 5′- GCACCAGTAAAACGTCGTCA-3′; Per33R, 5′- TCATTCTCACTGGCAGAGCA-3′; Luc5R2, 5′- GTTTTAGAATCCATGATAATA-3′; Kan3F2, 5′- CTTTTTGTCAGAAGACCGACC-3′. The approximate positions of these primers with respect to the construct are shown in Figure 1 . Transgenic fish were identified as those fish that gave an approximately 600-bp PCR product with the Per35F/Luc5R2 primer combination, and an approximately 800-bp product by the Kan3F2/Per33R combination, when their genomic DNA was used as templates. Nontransgenic fish gave no products with either of these primer combinations. In vivo measurement of bioluminescence rhythms Embryos or larval fish were placed individually in every other well of a white 96-well Optiplate (Perkin-Elmer, Wellesley, California, United States) with 200 μl of Holtfreter solution (7.0 g of NaCl, 0.4 g of sodium bicarbonate, 0.2 g of CaCl 2 , and 0.1 g of KCl [pH 7.0] in 2 l of ddH 2 O) aerated overnight and containing 0.5 mM D-luciferin potassium salt (Biosynth, Naperville, Illinois, United States) and 0.013% Amquel Instant Water Detoxifier (Kordon brand; Novalek, Hayward, California, United States). Once loaded with animals, four such plates were subjected to automatic monitoring of bioluminescence every 30 min by the Topcount multiplate scintillation counter (Perkin-Elmer) equipped with six detectors and plate stackers. The room temperature was set at 21–22 °C, and the machine at 24 °C. However, due to the heat created by the machine, temperature at the bottom of the stacker was 1–2 °C higher than the room temperature. In order to minimize high background counts under lighted conditions, each plate was dark-adapted for approximately 5 min before being counted for bioluminescence. Each well was counted for 4.8 s every 30 min. The plates were illuminated with two white fluorescent lamps, each facing the left or right side of the stacker. The approximate intensity of the light that reached the plates was 17–35 lux, depending on the position of the plates within the stacker. Experimental protocol The experimental protocol for each experiment involving in vivo bioluminescence measurement is described in Protocol S1 . Bioluminescence data analyses Bioluminescence data from the Topcount were imported into Microsoft Excel 2000 by the Import and Analysis macro (kindly supplied by Steve Kay, Scripps Institute). In many of the experiments performed on the Topcount, some plates were placed in the machine several days earlier than others in order to monitor fish that experienced different numbers of LD cycles ( Protocol S1 ). Therefore, at the end of the recording period that typically lasted for approximately 2 wk, many fish that were placed in the machine earlier had been dead for a few days. Since only the data up to 10-d old larval fish were analyzed for the experiments presented in Figures 2 and 4 , simple observation of fish after the recording period could overestimate the number of fish that had died while the 10-d worth of data were collected. When a per3-luc fish dies in luciferin solution, it emits a burst of high bioluminescence counts (> 2,000 cps). This burst of luminescence is typically followed by a low background level of luminescence (< 50 cps). Furthermore, intermediate levels of spikes were also found in many plots just before the burst of high bioluminescence. Therefore, in order to eliminate data from dead fish, data that exceeded 5,000 cps, or those that went down below 50 cps, at any point of the analyzed portion of the data were first discarded. Then an averaged plot of the remaining data from each clutch of embryos was examined, and the highest count on days 8–9 was determined for experiments presented in Figure 2 . For the other experiments, highest counts from the entire averaged plots except for day 1 of the record were determined. Then data that exceeded twice that value were also discarded as those with medium-sized spikes. This second round of data elimination was done in this way because overall luminescence counts varied among different clutches, possibly due to varying sizes of eggs laid. It should be noted that this procedure also eliminated records from fish that were alive, but that showed one or more transient spikes of bioluminescence. Those data with transient spikes were eliminated anyway, because the spikes can severely affect the accuracy of the data analysis program. Period and rhythmicity for each animal were determined by a macro [ 54 ] based on MatLab 6.5 (Mathworks, Natick, Massachusetts, United States). With this macro, periods were determined by the maximum entropy spectral analysis (MESA) and autocorrelation, and rhythmicity by autocorrelation. Some fish that were apparently arrhythmic by visual examination of the plot gave rhythm indexes with a confidence interval higher than 95% by autocorrelation due to spurious peaks and small confidence intervals. Autocorrelation plots of these fish, however, were almost always nonsinusoidal and/or did not have five clear peaks (one at the center and two on each side of the center peak). Therefore, each set of data was judged blindly by three people as to whether its autocorrelation plot was sinusoidal with five peaks. Fish were judged as rhythmic only if two to three people found their autocorrelation plots sinusoidal, and their rhythm statistic values exceeded 1. Real-time qPCR Total RNA was extracted from 9–42 embryos or larval fish raised in petri dishes using TRIzol reagent (Invitrogen, Carlsbad, California, United States). The number of animals used for each extraction was recorded. Once extracted, total nucleic acid concentration was determined by a spectrophotometer. In order to prevent genomic DNA contamination, RNA samples were treated with Turbo DNA-free (Ambion, Austin, Texas, United States), and the concentration determined again by a spectrophotometer. Total RNA (0.5–1 μg) was subjected to cDNA synthesis by Superscript II Reverse Transcriptase (Invitrogen) using Oligo (dT) 12–18 (Invitrogen) as the primer in a 25–40 μl reaction volume. Real-time PCR was performed in a 25 μl reaction volume containing a probe, forward and reverse primers, and qPCR Mastermix according to the manufacturer's instruction (Eurogentec, Seraing, Belgium). Each reaction was quadrupled in order to minimize pipetting errors. The primers and TaqMan MGB probes for per3 and luc were designed and synthesized by the Assays-by-Design Gene Expression service (Applied Biosystems, Foster City, California, United States): per3 forward, 5′- GCCCTGGCAGCACCA-3′; per3 reverse 5′- GAAAGCTGGAGGACGAGGAA-3′; probe, 5′-6-FAM- CTAAGAGCTCAAAATCC-NFQ-3′; luc forward, 5′- GCAGGTGTCGCAGGTCTT-3′; luc reverse, 5′- GCGACGTAATCCACGATCTCTTTT-3′; probe, 5′-6-FAM- TCACCGGCGTCATCG-NFQ-3′. The ABI Sequence Detection System 7000 (Applied Biosystems) was programmed to perform the following protocol: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. In this study, relative amount of per3 or luc cDNA per animal was calculated by the standard-curve method [ 55 ] rather than by normalizing those RNA species to a constitutive control gene, for the following reasons: Both per3 and luc were compared between two different developmental stages as well as among different times of the day. It was also important to calculate the amount of each mRNA species per animal in order to compare these data to bioluminescence data. The amount of a specific control RNA, as well as the total RNA, may differ among fish of different ages, in which case RNA per animal cannot be calculated by the relative quantification method using a constitutive control. As a concentration standard, a single-stranded DNA oligonucleotide of known concentration was used for each gene. These oligonucleotides span from the 5′ end of the forward primer to the 5′ end of the reverse primer, and including 75 bp for per3 and 110 bp for luc (Biosource, Camarillo, California, United States). The standard concentration was varied from 10 2 to 10 7 copies per reaction in 10-fold increments. For every qPCR experiment, reactions for standards were performed in four replicates along with reactions for cDNA samples. Statistics To test whether percentages of rhythmic fish among different experimental groups were equal, the G-test was performed using Microsoft Excel 2000 according to Sokal and Rohlf [ 56 ]. If multiple tests were performed for a set of data, critical value of the G-statistic was adjusted for the experimentwise error rate [ 56 ]. For all the other numerical data, JMP 3.1.5 (SAS Institute, Cary, North Carolina, United States) was used for the following tests: Each set of data were first subjected to the test for normality. If the data were normally distributed, the one-way analysis of variance or the t -test was performed. The nonparametric Wilcoxon/Kruskal-Wallis test was performed on data that were not normally distributed even after various transformations (logarithmic, square root, and inverse) were tried. Where multiple tests were performed on a set of data, the experimentwise error rate (α) was adjusted by the Dunn-Sˇidák method [ 56 ]. Supporting Information Protocol S1 Experimental Protocol for Bioluminescence Experiments (27 KB DOC). Click here for additional data file. Accession Numbers The GenBank ( http://www.ncbi.nlm.nih.gov/ ) accession numbers of the sequences discussed in this paper are per 3 cDNA (NM_131584) and BAC clone CH211–138E4 (AL929204). The Ensembl ( http://www.ensembl.org/Danio_rerio/ ) ID of the flanking gene of per 3 mentioned in Figure 1 is ENSDARG00000023492.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC546037.xml
521696	The effect of oxythioquinox exposure on normal human mammary epithelial cell gene expression: A microarray analysis study	Background Inter-individual variation in normal human mammary epithelial cells in response to oxythioquinox (OTQ) is reported. Gene expression signatures resulting from chemical exposures are generally created from analysis of exposures in rat, mouse or other genetically similar animal models, limiting information about inter-individual variations. This study focused on the effect of inter-individual variation in gene expression signatures. Methods Gene expression was studied in primary normal human mammary epithelial cells (NHMECs) derived from four women undergoing reduction mammoplasty [Cooperative Human Tissue Network (National Cancer Institute and National Disease Research Interchange)]. Gene transcription in each cell strain was analyzed using high-density oligonucleotide DNA microarrays (HuGeneFL, Affymetrix™) and changes in the expression of selected genes were verified by real-time polymerase chain reaction at extended time points (ABI). DNA microarrays were hybridized to materials prepared from total RNA that was collected after OTQ treatment for 15, 60 and 120 min. RNA was harvested from the vehicle control (DMSO) at 120 min. The gene expression profile included all genes altered by at least a signal log ratio (SLR) of ± 0.6 and p value ≤ 0.05 in three of four cell strains analyzed. Results RNA species were clustered in various patterns of expression highlighting genes with altered expression in one or more of the cell strains, including metabolic enzymes and transcription factors. Of the clustered RNA species, only 36 were found to be altered at one time point in three or more of the cell strains analyzed (13 up-regulated, 23 down-regulated). Cluster analysis examined the effects of OTQ on the cells with specific p53 polymorphisms. The two strains expressing the major variant of p53 had 83 common genes altered (35 increased, 48 decreased) at one or more time point by at least a 0.6 signal log ratio (SLR). The intermediate variant strains showed 105 common genes altered (80 increased, 25 decreased) in both strains. Conclusion Differential changes in expression of these genes may yield biomarkers that provide insight into inter-individual variation in cancer risk. Further, specific individual patterns of gene expression may help to determine more susceptible populations.	Background Oxythioquinox (Morestan™ or OTQ, Bayer Corp) is a prototypical pesticide that was first used in 1968 on crops such as apples, pears, cucumbers, and gherkins. However, its use was later confined to non-food crops, limiting exposure to nursery and greenhouse employees. OTQ is a member of the quinoxaline class of pesticides, which also includes chlorquinox and thioquinox. Principal agricultural use of OTQ was limited to the states of California, Washington, Florida, New York, Pennsylvania, Ohio and Michigan [ 1 ]. The use of OTQ in the United States was voluntarily cancelled in 1999 with stocks in use until 2001, although OTQ is still listed in many different regions for use as an insecticide [ 2 , 3 ]. Further, OTQ is still in use today in other areas of the world, including Australia and the Caribbean [ 4 , 5 ]. Scientifically acceptable toxicity studies of OTQ are sparse. Early in vivo studies in rats found alterations of a variety of metabolic enzymes following OTQ exposures, including alkaline phosphatase [ 6 ]. Although not directly analyzed, the results of this study combined with an earlier study [ 7 ] suggest a direct effect of OTQ on succinate dehydrogenase, or other enzymes with a thiol group. Further work by Carlson et al (1970) [ 7 ] showed that, although OTQ has a low acute toxicity, cumulative exposure to this pesticide is not well-tolerated by exposed animals, with the majority of damage found in the liver of these animals. Further studies looking mainly at hepatic enzyme function found that OTQ exposure inhibits some hepatic enzyme functions [ 8 ]. OTQ was shown to be a carcinogen and hepatotoxin in laboratory animals in later studies [ 9 ]. OTQ has also been classified as a probable human carcinogen [ 10 ]. However, potentially carcinogenic exposures have already occurred and OTQ is still in use outside of the US, its mechanism of action remains of interest. In addition, OTQ has been shown to have an inhibitory effect on cytochome P450s, enzymes known to have a pivotal role in carcinogen metabolism [ 8 , 11 - 13 ]. Although early studies focused on hepatic effects, carcinogenic potential may occur in other tissues as well. While there have been multiple pesticides implicated in breast cancer, no studies have been published related to OTQ exposure and human cancer incidence [ 13 ]. This study is similar to work currently being carried out to determine gene expression profiles of a variety of environmental agents, including chemicals, physical agents and physiologic stresses [ 14 - 19 ]. The National Institute of Environmental Health Systems (NIEHS) has recently funded a consortium to expand the study of gene expression profile signatures for various chemicals, with the long-term goal to use these analyses in a validated gene expression profile signature database. A recent report by Shan etal. 2002 [ 20 ] is a good example of the differences between gene expression profile signatures for two chemicals in an animal system. This report profiled gene expression in rat carcinomas induced by two carcinogens, 2-amino-1-methyl-6-phenylimidazo [4,5- b ]pyridine (PhIP) and 7,12-dimethylbenz [ a ]anthacene (DMBA), and was able to show that while both chemicals altered expression in some genes in a similar fashion, each induced unique gene expression patterns. The primary goal of this study was to look for consistent changes common to all donors that could potentially be used as biomarkers of exposure, creating a gene expression profile following OTQ exposure in normal human cells. Given previous research in hepatoxicity, ideally normal human liver cells would have been used as a model system. However, we needed a normal human tissue that was readily accessible. Therefore, gene expression was studied in primary normal human mammary epithelial cells from four different donors in response to OTQ exposure. Current microarray analysis of pesticide exposure focuses on animal studies, not allowing for analysis of inter-individual variation. With the use of microarrays including clinical diagnosis, genetic susceptibility to disease and treatment, the need to determine the potential role of inter-individual variation on gene expression patterns in all potentially exposed tissues. Genes found to be altered in multiple cell strains could be considered biomarkers for individual populations. Given the small number of cell strains used, follow-up analysis in a larger number of samples is required to confirm any potential biomarkers. Biomarkers derived from this study could potentially be used in future epidemiology studies analyzing the effects of pesticide exposure. Further, this gene expression profile could be compared to those of other pesticides and of known carcinogens to develop a more detailed mechanistic definition of these chemicals. Methods Cell culture Primary normal human mammary epithelial cells (NHMECs) were derived from tissues salvaged at reduction mammoplasty obtained though the Cooperative Human Tissue Network (National Cancer Institute and National Disease Research Interchange). Development and characterization of cell strains was achieved using standard methods [ 21 ]. Cells were grown in MEGM media (Clonetics, Cambrex, Pittsburgh, PA) at 37°C and 5% CO 2 . OTQ treatment Treatment was performed on cells in passage six at 70% confluency, as routinely performed in our laboratory. Preliminary studies analyzed a range of OTQ concentrations (0 – 12.5 μM) selected based on previous research [ 6 , 7 ] and time points (0 – 24 hours), and showed maximum effect of OTQ on p53 expression with minimal toxicity at 2 hours with a final concentration of 6.25 μM. Cells were treated by diluting the stock OTQ/DMSO mixture in media and adding this solution to aspirated cells, allowing even exposure to all cells. DMSO (0.001%) alone was used as a vehicle control. At the end of the treatment period, cells were removed for RNA isolation. Cell viability was determined by Trypan Blue exclusion assay. Indirect immunofluorescence Confluent cells in passage five were trypsinized and plated on eight-well slides (LabTek II Slide System, Nunc, Naperville, IL). These cells were grown to 70% confluency before being treated with OTQ (6.25 μM) for 15, 60 and 120 min or DMSO (0.001%) for 120 min. At the end of treatment, the media was aspirated and the cells were fixed with methanol. Slides were then stained with anti- p53 antibody (1:1000, DO-1, Santa Cruz Biotech, Santa Cruz, CA) and incubated overnight at 4°C. The next day, the media was again removed and the secondary antibody (1:1500, goat anti-mouse FITC, Santa Cruz Biotech) was incubated for one hour at room temperature. Slides were washed in triplicate with phosphate buffered saline (PBS) and cover slips were added. Slides were dried for one hour at room temperature before viewing, using the laser scanning confocal microscope BX50 (Olympus), and quantitative analysis was performed [ 22 ]. Relative p53 expression was quantified between different time points and strains by determination of the area under the integrated intensity curve (Fluoview, Olympus, B & B Microscopes, Pittsburgh, PA). Microarray analysis Microarray analysis was performed in duplicate using the HuGeneFL high-density oligonucleotide microarrays (Affymetrix™, Santa Clara, CA). Protocols from the Affymetrix Expression Analysis Technical Manual were followed RNA was isolated from cells with Trizol (Gibco, Grand Island, NY), followed by purification with RNEasy Mini Kit (Qiagen, Valencia, CA). Spectrophotometer measurements were required to give a 260/280 ratio of 1.9–2.1 for use in microarray analysis. Double-stranded cDNA was then synthesized from total RNA (Superscript Choice System, Invitrogen, Carlsbad, CA). An in vitro transcription (IVT) reaction (Enzo, Farmingdale, NY) was then performed to produce biotin-labelled cRNA from the cDNA. Excess biotinylated dUTPs were removed by RNEasy Mini Kit before being fragmented and added to a hybridization cocktail, including Eukaryotic Hybridization controls (Affymetrix), BSA and herring sperm DNA (Gibco, Grand Island, NY) and biotinylated anti-streptavidin antibody (Vector Laboratories, Burlingame, CA). Hybridization on microarrays was performed for 16 hours at 45°C in the Gene Chip Hybridization Oven with rocker (Affymetrix). Microarrays were washed and stained using the protocol, as described in the Affymetrix Manual, with the GeneChip Fluidics Station 400 (Affymetrix). Arrays were then scanned with the Affymetrix Scanner (Hewlett Packard, Palo Alto, CA). Expression profiles were analyzed using Microarray Suite 5.0, MicroDB 3.0 and Data Mining Tool 3.0 (Affymetrix). Affymetrix arrays are produced using multiple 25-mer oligonucleotides (11–20 per target gene). Each oligonucleotide is created to match the selected region of the target gene (perfect match, PM), while a similar oligonucleotide is created altered in the 13 th position to control for non-specific binding (mismatch, MM). Results are given in signal intensities with a p -value determined from perfect match/mismatch (PM/MM) intensities by Tukey's Biweight analysis. Each array was normalized to a scaling factor of 1500 to correct for array variation. All arrays for each cell strain were analyzed on the same day to minimize variation. Signal log ratio was determined by comparison of the signal intensities for the baseline (vehicle control) and the treatment array. This is computed using a one-step Tukey's Biweight method by taking a mean of the log ratios of probe pair intensities across the two arrays. This method helps to filter out differences due to different probe binding coefficients that may lead to false positives and/or negatives. A signal log ratio of zero represents no change in gene expression as a result of OTQ exposure. A signal log ratio of one is equivalent to a fold change of two between the treatment and control. The results described here are the average of both duplicates, with the average percent variability between duplicate arrays being 1.5% (the average difference found between duplicates, related to array to array variability as well as technical variability in processing the array). Only relative changes equal to or greater than 0.6 signal log ratio (SLR) were considered a significant change as a result of exposure. The biological significance of each change is determined with Wilcoxon's signed rank test with the Affymetrix software. Gene chip analysis was performed by self-organizing map (SOM) clustering, focusing on genes with a detection p value of 0.05 or less at one or more time points. Analysis was performed to comply with MIAME standards. Real-time polymerase chain reaction analysis (RT-PCR) cDNA synthesized from each sample as in the Affymetrix analysis (Invitrogen) was used in a one-step RT-PCR analysis reaction. Analysis was performed in duplicate on the ABI 7700 cycler, with the SYBR Green Master Mix (ABI) and samples were normalized using both 18S and GAPDH expression levels for each sample. Primers were designed using Primer Express ® (ABI) to yield unique fragments for each gene under study. Reactions were set up following recommended protocols using 100 pmol of each primer (Sigma-Genosys) and approximately 60 ng template per reaction. Reactions were performed in duplicate for each sample for 40 cycles (95°C/15 sec denaturing step; 60°C/1 min annealing/extension step). Fold change was determined based on average cycle threshold (C T ) values for all duplicates and converted to signal log ratio. Results Trypan blue exclusion test Trypan Blue was used to analyze toxicity by measuring cell viability for each cell strain for each treatment. The results showed a range of viability from 92–97% at all time points, except for the last time point in strain 3, which had a viability of only 65% at 120 min (results not shown). This decrease in viability at 120 min was not found to be directly correlated to p53 or p21 protein expression, or to any particular gene expression pattern. The dose of OTQ used was based on these findings. Indirect immunofluorescence Baseline p53 protein levels were visually compared to those after treatment in each cell strain. Integrated fluorescence intensities were measured on each optical slice of cells. The fluorescence was determined as the area under the curve in arbitrary units (AU) (Figure 1 ). This result was compared between time points for each cell strain. An increase was seen in p53 expression in all cell strains with increasing duration of an OTQ exposure. However, one strain (3) showed a 10-fold lower p53 expression at each time point. Figure 1 Quantitative analysis of immunofluorescence microscopy. Confocal microscopy analysis of p53 expression. Integrated intensity measures were obtained from Fluoview and graphed with GraphPad Prism (GraphPad Software, San Diego, CA) to determine area under the curve as a measure of comparative p53 protein expression for each treatment time point. Data is shown for each time point per cell strain on the ordinate and the intensity on the abscissa (arbitrary units, AU). There was an increase in p53 protein expression for all of the strains tested in direct correlation with OTQ exposure. In one intermediate strain (3), p53 expression was 10-fold lower at all time points. DNA microarray DNA microarray analysis found no change in p53 , despite the increase in p53 protein levels observed by immunofluorescence. Studies of benzo [a]pyrene exposure in our laboratory have also found similar results for p53 expression [ 23 ]. The effect of OTQ exposure on other cell cycle genes, however, was determined by DNA microarray analysis. Although inter-individual variation between donors in response to OTQ was evident, there were also some genes found to be increased consistently in all strains by microarray analysis (Table 1 ). Self-organizing map (SOM) clustering was used to group genes with similar patterns of alteration in each of the strains. SOM analysis was performed following filtering of the total genes on the array, limiting the SOM analysis to only genes found to be present on at least one array analyzed. Following suggested analysis with the Affymetrix system, the default settings of the Affymetrix software were selected, including selecting threshold filtering (min = 20, max = 20000), row variation filtering (max/min = 3 and max-min = 100), and row normalization (mean = 0, variance = 1). Working with a 3 × 3 analysis to obtain 9 clusters generally gave an optimal amount of different clusters with less than 100 genes per cluster. From the SOM clustering analysis, genes altered by a signal log ratio of ± 0.6 or greater were chosen for closer study. Table 1 Genes altered following oxythioquinox exposure. Table represents data mined from HuGeneFL microarrays (Affymetrix). All genes selected have a signal log ratio of ± 0.6 unless otherwise noted. Representative genes for each group were selected based on their function and are shown here. GenBank ID Name Peak Expression Level (SLR) Functional Class Genes increased in three or more strains (n = 13): U22028 CYP2A13 1.5 xenobiotic metabolism U20734 junB 3.42 transcription V01512 cfos 2.04 transcription S85655 prohibitin 0.75 cell proliferation S82240 RhoE GTPase 2.81 signal transduction M69043 MAD-3 mRNA encoding IkB-like activity 0.83 apoptosis M63573 cyclophilin 1.68 immune response U05861 Dihydrodiol dehydrogenase 2.52 xenobiotic metabolism Genes decreased in three or more strains (n = 23): L05624 MAP Kinase Kinase -0.67 signal transduction U18018 E1A enhancer -1.34 transcription X56681 junD -1.03 transcription X68836 S-adenosylmethionine synthetase -2.57 cell metabolism J04973 Cytochrome bc-1 -3.12 cell metabolism Genes altered in at least two of four cell strains (n = 189): X03484 raf oncogene 1.46 carcinogenesis M60974 growth arrest and DNA-damage-inducible protein (gadd45) 1.51 DNA damage M57731 Human gro-beta 1.85 immune response X66899 EWS 1.2 carcinogenesis Z29087* Cyclin D1 Promoter 1.03 cell cycle control L10910 Splicing Factor CC1.3 0.62 RNA processing M83667 NF-IL6 Permeability Factor 1.55 transcription M27281 Vascular Permeability Factor -1.09 cell proliferation L28010 HnRNP F protein -0.55 RNA processing U72649 BTF2 -1.86 carcinogenesis M19267 tropomyosin -1.16 cardiac M38258 retinoic acid receptor gamma 1 -0.94 cell metabolism U42031 Immunophilin -1.41 immune response U67122 ubiquitin-related SUMO-1 -1.03 protein metabolism X70340 Transforming growth factor alpha -0.57 cell proliferation M34458 Lamin B -1.33 cell proliferation *No accession number was used by Affymetrix. This accession number most closely matches the probe description and sequence. SOM clustering was used to show patterns of expression in each cell strain, and followed by further subclustering of those clusters of interest across all cell strains (Figure 2 ). This analysis was performed with Data Mining Tool 3.0 (Affymetrix). For comparison to earlier versions of the Affymetrix software, a signal log ratio of one is equal to a fold change of two. SOM clustering led to the selection of genes found to be altered, with the overall total of 215 genes (13 increased in 3 or more strains; 23 decreased in 3 or more strains). To be included, the genes must have a signal log ratio of ± 0.6, which is approximately a 1.5 fold change. Signal log ratio was used to linearize the data for ease of analysis. The full list of genes altered can be found at . Figure 2 SOM clustering. Self-organizing map clustering groups' genes by similar expression patterns. Each data point represents an OTQ treatment time point. Data points are plotted in order of cell strain with four points per strain (DMSO, 15 min, 60 min, and 120 min). Levels of expression are not given, as patterns of expression are based on relative expression levels. Panels 4 and 7 of this graph show genes increased at 15 minutes for one strain (3) follow similar pattern in second strain (4). Similar pattern is also seen in panels 6 and 8 of this graph. From the full list of genes that fit these criteria, selected genes were chosen due to their potential role in carcinogenesis, whether by cell cycle control, immune response or other specific functions. Functions were described as annotated by NetAffx [ 24 ]. These genes are listed in Table 1 . Some genes were selected based on their possible role in disease as a result of pesticide exposure. Table 1 contains 8 genes increased in at least three of the four cell strains analyzed by 1.5 fold, as compared to the vehicle control and a list of 5 genes decreased in three or more of the four strains analyzed. Included in this list are two genes involved in the metabolic activation of endogeneous chemicals, cytochrome P4502A13 (CYP2A13) and dihydrodiol dehydrogenase . Although expression was slightly increased at one time point, the temporal patterns were slightly different (Figures 3 and 4 ). This increase in all cell strains suggests a potential role for these genes in OTQ metabolism. These results are confirmed at these time points as well as at 12 h and 24 h by RT-PCR (Table 2 ). Genes also found to be increased in at least three of four cell strains analyzed include genes involved in transcription ( junB , cfos ), immune response ( cyclophilin ), and apoptosis ( MAD-3 ). These genes showed a consistent increase in expression following exposure to OTQ (Table 1 ). Genes that showed a consistent decrease in expression following exposure include signalling pathway genes ( MAP kinase kinase ), cell metabolism genes ( S-adenosylmethionine synthetase , cytochrome bc-1 ), and transcription factors ( E1A enhancer ). Figure 3 Expression pattern for CYP2A13. DNA microarray analysis of NHMEC strains. Analysis was performed as described on HuGeneFL high-density oligonucleotide microarrays (Affymetrix). Results are plotted as duration of exposure vs signal log ratio (SLR). Signal log ratio is a measure of comparative expression of the treatment vs. vehicle control (0.001% DMSO). Signal log ratio of one is equal to a fold change of two. A SLR of 0.6 (Fold Change ~1.5) was the arbitrary limit of our analysis. All genes given an Absent call by analysis software are shown with a SLR of zero. Asterisks indicate a statistically significant variation in expression from the control level as measured by Tukey's Biweight analysis. Figure 4 Expression pattern for dihydrodiol dehydrogenase. DNA Microarray analysis of NHMEC strains. Analysis was performed as described on HuGeneFL high-density oligonucleotide microarrays (Affymetrix). Results are plotted as duration of exposure vs signal log ratio (SLR). Signal log ratio is a measure of comparative expression of the treatment vs. vehicle control (0.001% DMSO). Signal log ratio of one is equal to a fold change of two. A SLR of 0.6 (Fold Change ~1.5) was the arbitrary limit of our analysis. All genes given an Absent call by analysis software are shown with a SLR of zero. Asterisks indicate a statistically significant variation in expression from the control level as measured by Tukey's Biweight analysis. Table 2 RT-PCR results. Table represents real-time PCR data from selected genes of interest. Time points used were 15 min, 120 min, 12 h and 24 h where shown. Results shown are results of replicate analysis with duplicate samples. Samples not analyzed represented by N/A. Strain Gene SLR 15 min SLR 120 min SLR 12 h SLR 24 h 1 BTF2 1.41 N/A 1.53 0.3 2 BTF2 -1.56 N/A -1.32 -1.74 3 BTF2 0.18 N/A -0.89 -16.61 4 BTF2 0.62 N/A -0.36 0.65 1 CYP2A13 5.58 6.01 3.84 10.6 2 CYP2A13 2.7 0.62 1.1 -0.6 3 CYP2A13 5.76 4.77 -0.6 -1.64 4 CYP2A13 5.45 1.1 0.33 -1.51 1 DDH 1.23 -0.67 1.75 1.47 2 DDH -0.47 -0.62 0.96 5.41 3 DDH 0.86 -3.47 2.01 -3.84 4 DDH 3.44 N/A 5.53 6.09 1 EWS -1.18 -1.84 0.69 -5.64 2 EWS -6.64 -0.4 1.32 1.54 3 EWS -0.42 -0.22 -1.79 -6.64 4 EWS N/A 2.92 0.78 0.86 1 GADD45 -0.09 -0.22 -1.32 -5.06 2 GADD45 -1.03 -1.69 -1.94 -1.32 3 GADD45 0.82 -2.32 -1.56 -4.32 4 GADD45 1.34 9.05 6.4 7.72 1 MAD-3 -0.71 N/A -0.15 -3.64 2 MAD-3 -1.89 N/A -1.06 -0.38 3 MAD-3 0.34 N/A -0.58 -6.64 4 MAD-3 4.4 N/A -4.38 0.51 1 PROHIBITIN 0.31 N/A -0.3 0.96 2 PROHIBITIN -0.6 N/A 0.3 -1.18 3 PROHIBITIN -1.06 N/A -1.94 -2.84 4 PROHIBITIN 2.3 N/A -1.09 0.92 1 RAF 0.01 -2.25 1.21 -5.64 2 RAF -1.51 -0.27 -1.43 -0.06 3 RAF 0.37 -3.84 -1.12 -7.67 4 RAF 1.33 7.57 4.8 7.22 1 RhoE 0.59 N/A -0.62 -0.71 2 RhoE 0.26 N/A -1.18 1.08 3 RhoE 1.55 N/A -0.94 -5.64 4 RhoE 2.35 N/A 1.06 2.05 Some genes of interest were variably altered by strain, examples of which are also listed in Table 1 . These include genes involved in carcinogenesis ( raf oncogene , GADD45 , EWS , Cyclin D1 ) as well as immune response ( immunophilin , gro-β ), cell proliferation ( TGFβ ), and RNA processing ( HnRNP F protein ). Real-time PCR Real-time PCR was used to confirm and extend results seen by microarray analysis for selected genes. Following the original microarray analysis, patterns of some genes appeared to be changing at the latest time point (120 min), so extended time points were selected (12 and 24 h) to see a more complete expression profile for these genes. Due to limited amount of cDNA, genes were analyzed at 15 min and/or 120 min, and then analyzed at 12 and 24 h by RT-PCR. Extended time points were selected to look at specific genes found altered at the earlier time points. These genes were selected due to their function and/or pattern of expression, and determining their expression pattern at later time points was of interest. Results are shown in Table 2 . In the majority of samples, RT-PCR confirmed data found by microarray analysis for the genes listed. Some discrepancies are also shown, however, in these cases it is believed that the primer sequence is more specific to the gene in question than the probe sequence used on the array. However, extended time points, in some cases, showed that the results of early time points did not always continue to extended exposures. The RT-PCR results for prohibitin , DDH , and CYP2A13 at 24 h showed a decrease in expression in some of the cell strains analyzed. Samples not available for RT-PCR analysis are listed as N/A in Table 2 . Discussion The purpose of this study was to determine if microarray analysis of four normal human mammary cell strains with a known haplotype could be used to find biomarkers related to either exposure in general or the specific haplotype in question. The use of only four cell strains was determined to have enough power to provide basic information to lead to further study if necessary. This study would be followed up for specific genes of interest in a larger number of cell strains, preferably bypassing the more expensive and time-consuming microarray analysis for RT-PCR only. DNA microarray analysis was used to profile the cellular response to OTQ, a quinoxaline pesticide. Analysis revealed genes with common response across the four human cell strains studied as well as inter-individual variation in response. Using only four normal human cell strains, our goal was to discover any distinctly altered genes in response to OTQ exposure. Future analysis with a larger number of cell strains will be used to follow-up this analysis on specific genes of interest. The majority of studies looking at gene expression profiles have used animal models, limiting any knowledge obtained to genetically similar organisms. Analysis with normal human cell strains, like those described here, will give more information on inter-individual variation in response to various chemicals. This information will yield clues to the metabolic pathways of the specific chemicals, and this increased knowledge will aid in determining potential hazards in the environment and the workplace. Given the large number of pesticides in use today, further examination of the effect of these chemicals on individuals is warranted. Following exposure to OTQ, NHMECs showed alterations in genes involved in a variety of functions. These included xenobiotic metabolism, transcription, and DNA synthesis. Genes altered as a result of OTQ exposure in all strains analyzed included transcription factors like junB and cfos (the AP1 complex). A number of genes involved in carcinogenesis were found altered after exposure to OTQ, both induced and down-regulated. For example, prohibitin expression is found to be up-regulated in most cell strains after OTQ exposure, with similar expression patterns associated with a decrease in cancer incidence [ 25 ]. Metabolism genes that were altered after OTQ exposure, like cytochome P4502A13 (CYP2A13) and dihydrodiol dehydrogenase , are involved in xenobiotic metabolism. It has been suggested that CYP2A13 is the main metabolic activator of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine [ 26 ]. The increase of CYP2A13 begins to wane at the final time point tested, suggesting this alteration to be somewhat transient. This is an unusual expression pattern for a cytochrome p450, as genes in this family tend to show a gradual increase in induction, and an equally gradual decrease in expression. Further analysis by RT-PCR showed that this p450 was increased at later time points (12 h and 24 h, Table 2 ). Another metabolic enzyme affected by OTQ exposure is dihydrodiol dehydrogenase. Dihydrodiol dehydrogenase is known to participate in activation of certain polycyclic aromatic hydrocarbons (PAHs), so its increase in the intermediate variant after OTQ exposure may result in an increase in PAH activation [ 27 , 28 ]. Alterations of genes like this may suggest an indirect role for OTQ in carcinogenesis. Of these two genes, only CYP2A13 seems specific to exposure to OTQ. DDH has been found to be increased following exposure to various chemicals, including malathion, di-n-butyl phthalate, and benzo [a]pyrene (Gwinn in preparation, 2004) [ 23 , 29 ]. Some of the early results at 15 min by microarray analysis may have been a consequence of the stress of exposure, regardless of the chemical. Extending analysis to later times determined whether results seen at these early time points were still valid after longer exposure to OTQ. RT-PCR results given in Table 2 show that in most cases, the extended time points showed a continued trend of expression (whether increased or decreased), except in some cell strains for DDH , prohibitin , and MAD-3 . These results show a reverse of the early expression patterns at the later time points. Real-time PCR is a more specific method of analysis, as it only interrogates one gene at a time with primers designed uniquely to that gene. Conflicts in results between the two methods can generally be attributed to cross-reaction between probes designed for similar genes on the array. Sequence differences between the probes on the array and those used in RT-PCR may also play role in these results. The RT-PCR primers were selected specifically for the gene in question, while the probes on the array may not have been. Genes with sequence homology but with altered patterns of expression may not have been differentiated in the array analysis, but would be with the RT-PCR analysis. Inter-individual variation as a result of genetic polymorphisms in genes of interest would focus on specific at-risk worker populations. For example, the four cell strains analyzed in this study have been genotyped for a variety of genes, in particular those involved in cell cycle control and xenobiotic metabolism. Two of the four cell strains selected for analysis are heterozygote for the minor variant haplotype of p53, a cell cycle control gene (cell strains 3 and 4). Although no biological mechanism for the role of this variant in carcinogenesis has been defined, several studies associating this haplotype with various cancers support such role [ 30 - 35 ]. Analysis of genes altered in just those strains expressing this variant, including three genes involved in cell cycle control: raf oncogene (X03484), cyclin D1 (Z29087), and BTF2 (U72649) may further support an association between OTQ exposure, p53 variant status and carcinogenesis [ 36 - 38 ]. Of these, p53 has been reported to increase GADD45 transcription in response to DNA damage, which is associated with an increase in cell cycle arrest and DNA damage repair, while increased levels of raf oncogene have been associated with lung carcinogenesis [ 39 - 41 ]. Given the small number of cell strains used, this analysis needs to be extended to additional cell strains to determine the role of the p53 variants in gene expression differences. Due to the expense of microarray analysis, this is performed in only a limited number of cell strains (4), and selected genes will be further analyzed with \|RT-PCR in a larger number of cell strains. Over 80 cell strains have been established in our laboratory to date, with half of these having been genotyped for p53 . However, given that this haplotype is found only in a limited portion of the population, this varied pattern of expression in key genes in cell cycle control may highlight a specific at-risk population. Searches for similar natural compounds to replace these potentially disruptive chemicals can also use gene expression profiles [ 42 ]. Profile comparisons to that of a natural pesticide may decrease the need for organophosphates. Comparison of OTQ's gene expression profile to that of well-defined chemicals, like benzo [a]pyrene, will yield important information about OTQ's role in both genotoxicity and potential carcinogenicity. A comparison between many expression profiles is needed to further define similarities and differences between this pesticide and known carcinogens and/or other pesticides. Conclusions The overall goal of this project was to create a gene expression profile for OTQ or related pesticide analogues with the hopes of finding genes to be used as potential biomarkers of exposure. This expression profile may also be used to determine the final role of OTQ in carcinogenesis by comparing it to profiles of known carcinogens. It is possible that the main effect of OTQ exposure is not on the direct alterations in many genes, but on alterations in genes potentially involved in carcinogenesis, among them the examples of CYP2A13 and dihydrodiol dehydrogenase . The results shown here do not suggest a direct role of OTQ in carcinogenesis. They do, however, suggest OTQ exposure leads to an increase in expression of genes that do play a direct role in carcinogen; metabolism (for example, exposure to carcinogens like NNK and benzo [a]pyrene along with exposure to OTQ may lead to an increased incidence of tobacco-related cancer) [ 27 , 43 ]. Discovery of genes altered following exposure to OTQ in human cell strains may aid in future epidemiology studies on pesticide exposures. Gene expression profiling can be used to yield genetic biomarkers of exposure that, after validation, could be used in a clinical setting for early determination of organophosphate exposure, increasing early treatment of pesticide illness and thereby increasing the recovery rate of exposed individuals. List of abbreviations used OTQ, oxythioquinox DDH, dihydrodiol dehydrogenase DMSO, dimethylsulfoxide SLR, signal log ratio DMT, Data Mining Tool™ MAS, Microarray Suite™ SOM, self-organizing map NHMEC, normal human mammary epithelial cell RT-PCR, real-time polymerase chain reaction Competing interests None declared. Authors' contributions MRG participated in the design of the study, and performed all experiments. DLW was responsible for the growth and maintenance of all cell strains used. AW conceived of the study and participated in the design and coordination. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC521696.xml
535807	Inconsistent self-reported mammography history: Findings from the National Population Health Survey longitudinal cohort	Background Self-reported information has commonly been used to monitor mammography utilization across populations and time periods. However, longitudinal investigations regarding the prevalence and determinants of inconsistent responses over time and the impact of such responses on population screening estimates are lacking. Methods Based on longitudinal panel data for a representative cohort of Canadian women aged 40+ years (n = 3,537) assessed in the 1994–95 (baseline) and 1996–97 (follow-up) National Population Health Survey (NPHS), we examined the prevalence of inconsistent self-reports of mammography utilization. Logistic regression models were used to estimate the associations between women's baseline sociodemographic and health characteristics and 2 types of inconsistent responses: (i) baseline reports of ever use which were subsequently contradicted by follow-up reports of never use; and (ii) baseline reports of never use which were contradicted by follow-up reports of use prior to 1994–95. Results Among women who reported having a mammogram at baseline, 5.9% (95% confidence interval (CI): 4.6–7.3%) reported at follow-up that they had never had one. Multivariate logistic regression analyses showed that women with such inconsistent responses were more often outside target age groups, from low income households and less likely to report hormone replacement therapy and Pap smear use. Among women reporting never use at baseline and ever use at follow-up, 17.4% (95%CI: 11.7–23.1%) reported their most recent mammogram as occurring prior to 1994–95 (baseline) and such responses were more common among women aged 70+ years and those in poorer health. Conclusions Women with inconsistent responses of type (i), i.e., ever users at baseline but never users at follow-up, appeared to exhibit characteristics typical of never users of mammography screening. Although limited by sample size, our preliminary analyses suggest that type (ii) responses are more likely to be the result of recall bias due to competing morbidity and age. Inconsistent responses, if removed from the analyses, may be a greater source of loss to follow-up than deaths/institutionalization or item non-response.	Background In the absence of organized screening, self-report is often the only means to monitor mammography utilization and to investigate trends in uptake at the population level [ 1 ]. In Canada, mammographic screening occurs both within organized programs and opportunistically through routine medical practice [ 2 ]. The validity of mammography self-report has previously been studied primarily using convenience samples. Despite differences in methodology, design and population characteristics, studies from a variety of settings have found that self-reports of mammography use are valid provided women are not required to precisely recall the timing of a previous mammogram [ 3 - 14 ]. Women generally tend to underestimate the time elapsed since their most recent mammogram by an average of three months or more, though overestimation can also occur [ 4 - 6 , 8 - 13 , 15 - 17 ]. Greater discrepancies in recall may occur when the mammogram took place longer ago [ 15 , 16 ], though contrary evidence also exists [ 11 ]. With some exceptions [ 4 , 6 , 8 , 9 , 13 , 16 , 17 ], studies have not been designed to assess false negative reporting due to the challenge of verifying historical data from multiple service providers. Those that have attempted to validate non-use have indicated that women are unlikely to deny having had a mammogram when indeed they have had one [ 4 , 6 , 8 , 9 , 13 , 17 ], though false-negative self-reports are not always negligible [ 16 ]. However, the opposite is often true; women tend to report having had mammograms which are not verified against medical records [ 3 , 4 , 6 , 7 , 9 - 11 , 13 - 16 , 18 - 21 ], a particular problem in groups with low screening prevalence [ 20 ]. Valid reporting of mammography use has been found to be unrelated to various health behaviors and perceptions, socioeconomic, demographic, and questionnaire administration factors in some studies [ 4 , 10 , 12 , 13 ]. However, others provide some evidence that age, ethnicity, education, employment status, family history of breast cancer, recency of the mammogram, and the regularity with which women receive mammograms do affect self-report accuracy [ 7 , 10 - 13 , 17 , 22 ]. Few evaluations of the reliability of mammography self-report are reported in the literature. Excellent test-retest reliability for having ever had a mammogram was reported in interviews conducted 1 week, 6–30 days, or 6–8 months after an initial interview, while reliability within the past year varied from excellent to good [ 14 , 23 , 24 ]. In a socioeconomically advantaged group of women aged 50–75 followed annually for 3 years, 98 percent provided logically consistent responses to a question on ever/never use of mammography [ 25 ]. Self-reports of ever use have been shown to be more reliable among Caucasian women and those with higher income and education [ 24 ]. However, in this study, date of last mammogram was not as reliably reported 6–8 months after initial testing [ 24 ]. Based on data from the longitudinal panel of the National Population Health Survey (NPHS), the present study examines the prevalence and determinants of inconsistent self-reports of mammography utilization among Canadian women aged 40 years and older and quantifies the extent that inconsistent self-reports of mammography use contribute to biased estimates of mammography utilization and uptake. To our knowledge, this is one of the first studies of mammography utilization to provide specific longitudinal data on the determinants of inconsistent responses over time and the impact of such responses on population screening estimates. Methods The National Population Health Survey (NPHS) is a survey of the Canadian household population. Initiated in 1994–95 and repeated biennially, it is a split panel survey, combining repeated cross-sectional components with the longitudinal follow-up of a panel of respondents. A representative sample of Canadian household residents aged 12 and older from all ten provinces was sampled using a multistage probability design with stratification and clustering at various stages. The overall response rate for the baseline 1994–95 survey was 89 percent with a 96 percent response rate for the selected panel respondent [ 26 ]. On follow-up to the baseline survey two years later, 94 percent of the panel members responded [ 26 ]. Further details of the sampling procedures, design, data collection and response rates are published elsewhere [ 26 , 27 ]. This study evaluated data from longitudinal panel respondents of the 1994–95 (baseline) and 1996–97 (follow-up) waves of the NPHS to examine inconsistencies in reported mammography utilization among women aged 40+ years at first contact. Questions about mammography use were administered to female respondents through a personal interview conducted in 1994–95 and repeated by telephone approximately two years later. In both survey years, women were asked the identical question: "Have you ever had a mammogram, that is, a breast x-ray?". Those with positive responses were further probed for the time and reason of their most recent mammogram. All women provided their own health-related information; no proxy responses were allowed. Analyses were restricted to women aged 40 and older (at baseline) who participated in the first two waves of the NPHS and consented to share their information with federal and provincial governments. Two types of inconsistent responses were assessed: (i) baseline reports of ever use which were contradicted by follow-up reports of never use; and (ii) baseline reports of never use which were contradicted by follow-up reports of use prior to 1994–95. Multivariate logistic regression techniques were used to evaluate the associations between women's baseline sociodemographic and health characteristics and type (i) inconsistent responses. Variables significant at p ≤ .05 in age-adjusted analyses were eligible for entry in the multivariate logistic models. Sample size constraints permitted only simple bivariate, rather than multivariate exploration of factors associated with reports reflecting inconsistent timing of most recent use at follow-up (type (ii) response). Estimates were weighted to reflect baseline population characteristics. To account for stratification and clustering in the NPHS sampling design, 95% confidence intervals for parameter estimates were calculated using exact standard errors generated through bootstrap re-sampling methods [ 28 ]. All statistical analyses were conducted using SAS. Results (i) Inconsistent ever/never utilization Of the 3,535 women aged 40+ years who responded to the ever/never mammography question in both survey waves (Figure 1 : 2 women with missing data regarding timing of mammogram were excluded), four percent (95% CI: 3.1–4.9) reported having had a mammogram at baseline and subsequently, on follow-up reported never having had a mammogram (Table 1 ). Among women who reported having had a mammogram at baseline, 5.9% (95%CI: 4.6–7.3) reported never use at follow-up (estimate not shown). The majority of women with inconsistent responses (64.4%, 95% CI: 54.4–74.4) reported receiving a recent (i.e., <2 years ago) mammogram at baseline and most (85.6%, 95% CI: 78.2–93.1) reported that the mammogram was done as part of a regular check up (Table 1 ). It should be noted that the percentage estimates in Table 1 have been weighted according to 1994/95 population characteristics whereas the frequency data represent actual numbers of women surveyed. Table 2 presents the estimated adjusted odds ratios (95% CIs) of inconsistent ever/never responses associated with women's baseline sociodemographic and health characteristics. Among women reporting ever use at baseline (1994–95), those reporting never use in 1996–97 were significantly more likely to be outside the target age group for screening (50–69), to have lower income, to have not used hormone replacement therapy in the past month and to have never had a Pap test, after adjusting for relevant covariates. Women with lower education levels were also more likely to report such inconsistent responses between baseline and follow-up although education failed to remain a significant predictor in the multivariate model. Other variables considered but not found to be significantly associated with this outcome were rural/urban residence, place of birth, languages spoken, marital status and other social support indices and having a regular physician. (ii) Inconsistent timing Follow-up interviews were completed, on average, 1.98 years from the baseline survey (range 1.19–3.01 years). Of the 293 women who reported never use at baseline and ever use at follow-up, 17.4 percent (95%CI: 11.7–23.1) reported a time for their most recent mammogram at follow-up that was inconsistent with never use at baseline. Despite baseline reports that they had never had a mammogram, approximately half of these women reported having had a mammogram at least 5 years ago. Although limited by small numbers, determinants of such inconsistent responses were assessed with simple bivariate analyses. Inconsistencies in timing occurred more often in older women. Compared to women aged 50–69, those 70 and older were more likely to report (at follow-up) that their most recent mammogram had occurred prior to 1994–95, despite a report of never use at baseline (OR = 6.96, 95%CI: 2.42–20.0). Women reporting fair or poor self-rated health were also more likely to report a time for their most recent mammogram at follow-up that was inconsistent with never use at baseline (OR = 2.44, 95% CI: 0.99–6.05). Impact of inconsistent reporting on uptake estimates Depending on how inconsistent responses are handled, different measures of use and uptake of mammography may be obtained. The lack of a gold standard such as a medical chart for validation makes the choice of a corrective measure unclear. If inconsistent ever/never responses are included in the analysis unchanged, 67.3 percent (95% CI: 65.1–69.5%) of women would be classified as ever having had a mammogram in 1994–95 while 71.7 percent (95% CI: 69.6–73.7%) would be classified as ever users in 1996–97. Conversely, if it is assumed that inconsistent ever/never responses represent false-positive responses at baseline (an assumption supported by our study findings), the 1994–95 prevalence estimate becomes 63.3 percent (95% CI: 61.0–65.6%), demonstrating an absolute increase in mammography use of 8.4 percent (95% CI: 7.1–9.6%) by this cohort of women by 1996–97. Discussion Although a limited number of studies have assessed the reliability of mammography self-report [ 23 - 25 ], detailed evaluations have not been conducted for population-based longitudinal surveys. In this study, reliability could not be assessed, per se, as women's status of never having had a mammogram could normally be expected to change over a two year span. However, by examining inconsistencies in responses expected to remain constant and in responses regarding logical timing of mammography use, it is possible to examine potential concerns regarding response reliability and recall bias, respectively. In longitudinal studies, inconsistent data removed during data cleaning can yield significant losses, and may lead to bias, depending on the amount of attrition at each time point and the magnitude of the differences between those retained in the panel and those lost by such attrition [ 29 ]. Longitudinal studies of health must be acutely aware of causes of attrition because losses accumulate over survey waves [ 30 ]. Although direct comparison with our sample was not possible due to the expectation that behavior might have changed in a 2-year time span, earlier findings from longitudinal studies of fairly affluent [ 25 ] and low-income [ 24 ] populations and a population-based study [ 23 ], indicated that women reliably report having ever had a mammogram with estimated reliability measured by Cohen's kappa ranging from 0.82–0.87 [ 23 , 24 ]. Our finding that 4 percent (95% CI: 3.1–4.9 percent) of the women participating in the second wave of this longitudinal study inconsistently reported ever having had a previous mammogram was surprisingly high. Previous studies have found that initial use refuted on subsequent interviews occurred in 2–2.9 percent of respondents [ 24 , 25 ]. Our analyses of factors associated with inconsistent ever/never responses indicate that women reporting ever having had a mammogram at baseline but never use at follow-up exhibited many of the sociodemographic and health behaviour characteristics (e.g., lower income, outside age groups targeted for screening, non-users of Pap screening and hormone replacement therapy) commonly observed among non-participants in mammography screening in previous studies [ 31 - 38 ]. Such findings provide support for the assumption that the 1994–95 response of ever use is more likely erroneous. Additional factors (e.g. being born in an Asian country) previously associated with non-use of mammography [ 31 , 33 ] also showed a positive association with providing an inconsistent response; however, small numbers resulted in high variability once clustering and stratification were taken into account and precluded further analysis of this variable. Validation studies also provide support for our assumption that inconsistent ever/never responses (as assessed in the present longitudinal panel) are most likely to be explained by false-positive responses at baseline. Women are more likely to falsely claim having had a mammogram, than not having one [ 4 , 6 , 8 , 9 , 13 , 17 , 39 , 40 ]. The majority of women in our study with inconsistent ever/never responses also indicated (at baseline) that their most recent mammogram had occurred within the last two years, a finding consistent with past research [ 10 ]. Imputation, suggested as a remedy for item non-response [ 30 ], may equally be used to deal with inconsistencies with evidence, in this situation, favoring treatment of women's earlier responses as false-positive. Among women who reported never use at baseline and ever use at follow-up, approximately 17.4 percent reported a time for their most recent mammogram (at follow-up) that was inconsistent with never use at baseline. If respondents truly initiated mammography use subsequent to the baseline interview, they overestimated the time elapsed since their mammogram. Such a finding is relatively inconsistent with previous studies that have generally found that women tend to underestimate the time since their last mammogram [ 4 - 6 , 8 - 13 , 15 , 17 ]. Although McGovern et al. and Caplan et al. found reverse-telescoping in approximately 9 percent of their samples [ 13 , 16 ], only 8 percent of women in this group miscalculated by more than 1 year [ 16 ]. In the present study, women reporting inconsistent timing were older and in poorer health, suggesting that competing health events may have interfered with accurate recall. Unfortunately, no gold standard was available to assess the validity of the responses women generated. Thus, the proportion of consistent responses that may actually represent invalid responses is unclear. Nor was it possible to distinguish errors in recall of timing from false reports of mammography utilization. Further, the reasons for providing inconsistent responses can only be inferred. The possibility of data entry errors is remote. Although data entry checks for consistency between survey cycles were not included among the comprehensive quality control strategies implemented by Statistics Canada, computer assisted interviewing was used by highly trained interviewers. Also, data entry errors would be expected to occur randomly and not disproportionately among women outside of the target age range or with relatively lower socioeconomic status, as observed in the present study. However, several plausible explanations for the inconsistencies exist, including survey methodology changes and deliberate or inadvertent provision of inaccurate responses by the respondent. We cannot exclude the possibility that interview changes (from a baseline personal interview to a telephone follow-up) prompted women who reported ever use in 1994/95 to alter their response in 1996–97. The NPHS used an initial personal interview to foster a good long-term relationship with the panel representative, but the cost and logistics of traveling to different regions was prohibitive. Therefore, unless the respondent objected or had no phone, future interviews (including the 1996–97 cycle) were conducted by telephone [ 26 ]. The need to maintain study procedures over time in longitudinal studies has been stressed [ 41 ], but the impact of altering the interview method on the NPHS results has not been investigated [ 26 ]. Sensitive questions may be answered more truthfully by phone. Editing of survey responses by the respondent may occur. Social desirability and a tendency to give positive responses are possible sources of over-reporting [ 3 , 11 ]. Such biases remain largely unexplored with respect to mammography use. Cognitive research has implicated comprehension as a barrier to providing valid, reliable survey responses. Several researchers employ the lead-in question 'Have you ever heard of a mammogram, that is a breast x-ray?' to identify comprehension difficulties [ 3 , 16 , 21 , 42 ] but this was not done in the NPHS where women were directly asked if they had ever had a mammogram. One study using focus group testing and in-depth interviews showed that despite some confusion between mammography and breast exams, women generally understood what mammography was [ 11 ], a finding further supported by one population-based survey [ 21 ]. However other investigations suggest this is not uniformly so [ 16 , 42 , 43 ]. One possibility cited is that confusion with other tests such as chest x-rays may lead to over-reporting of mammography [ 3 ]. There are several possible explanations for the higher rate of inconsistent responses observed in the present study. The focus of the NPHS questionnaire was not limited to preventive practices nor was it designed for reliability testing. The length and comprehensiveness of the NPHS may have contributed to greater respondent fatigue. Also, the longer time interval between surveys, relative to that observed in other studies, may have contributed to instability in responses. A more favourable level of concordance among responses may be obtained by studies that apply eligibility criteria that ensure more accurate responses (e.g. by having the respondent recall where she had her mammogram to validate her ever/never use) [ 6 , 10 , 15 ]. Finally, the overall response rate of the NPHS was higher than in comparable studies, so it potentially included a more difficult-to-reach population, less able to provide accurate responses. The inconsistent data reported here, if removed from longitudinal analyses, could yield losses to follow-up equivalent to or greater than other sources of attrition (e.g., deaths, institutionalization, non-response) over the planned 20 year course of the NPHS. The 1998–99 NPHS included probing questions to reduce inconsistencies and it should alleviate many of the problems evident here [ 44 ]. However, probes were not designed to address the larger problem of reverse-telescoping observed among some respondents in the NPHS longitudinal cohort. Incorporation of women's previous responses in subsequent interviews to avoid telescoping and stimulate recall can be used to minimize such inconsistencies [ 30 ]. A recent critical review of the accuracy of self-reported health behaviors, including mammography, provides further suggestions for enhancing the accuracy of such data [ 45 ]. Conclusions In summary, inconsistent responses represent a challenge to longitudinal, population-based evaluations of breast screening practices. Losses from inconsistent data regarding mammography participation are not negligible and may contribute to inaccurate estimates of mammography uptake. Women reporting inconsistent ever/never use in the present study displayed characteristics typical of never users, favoring treatment of women's baseline responses as false-positive. Inconsistent responses regarding the timing of recent mammography practices, however, may be primarily related to the impact of age and competing morbidity on recall. Competing interests The authors declare that they have no competing interests. Authors' contributions CB and CM contributed to the initial and revised analyses of the NPHS panel data, the interpretation of the results and the initial drafting/editing of the manuscript. CB and JS were responsible for the data linkage with Statistics Canada (for the NPHS Share File) and for the revised bootstrap analyses. JS also contributed to the interpretation of the results and editing of the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC535807.xml
549516	Prognostic indicators in peritoneal carcinomatosis from gastrointestinal cancer	Peritoneal carcinomatosis from gastrointestinal cancer has new treatment options for surgical management. The approach uses cytoreductive surgery which combines peritonectomy and visceral resection in an effort to remove all visible cancer within the abdomen and pelvis. Then the peritoneal cavity is flooded with chemotherapy solution in an attempt to eradicate residual disease. In order to select patients for this approach the quantitative prognostic indicators for carcinomatosis were reviewed, compared and contrasted. Prognostic indicators to be used to select patients for this aggressive approach at the initiation of surgery and after completion of cytoreduction were studied. Four quantitative assessments to be used at the time of abdominal exploration were the Gilly staging, Japanese gastric cancer P score, peritoneal cancer index (PCI), and the simplified peritoneal cancer index (SPCI). All have value with the PCI being the most validated and most precise. Preoperative assessments include the tumor histopathology and the prior surgical score. The completeness of cytoreduction score is an assessment of residual disease after a maximal surgical effort. An opportunity for long-term survival following treatment for carcinomatosis requires a complete cytoreduction in all reports for gastrointestinal cancer. Quantitative prognostic indicators need to be knowledgeably employed when patients with carcinomatosis are being treated. Improved patient selection with greater benefit and reduced morbidity and mortality should result.	I. Introduction Peritoneal carcinomatosis has always been regarded as a terminal condition. It is present in 10 to 30% of patients with gastrointestinal cancer at the time of their initial surgery and is a frequent finding in patients who develop recurrent cancer. Important natural history studies establish a 6-month median survival in this group of patients [ 1 - 3 ]. Recent multicenter phase II and a single phase III study evaluating the usefulness of cytoreductive surgery and perioperative intraperitoneal chemotherapy are promising [ 4 , 5 ]. Patient selection is of utmost importance in optimizing the results of treatment and excluding patients who will not benefit from a high morbidity and potentially life threatening therapy. Quantitative prognostic indicators are to serve as guidelines in the selection of treatments to maximize benefits of therapy and to exclude patients who have little or no chance to improve. They are of greatest utility in high risk and costly management protocols. Requirements of a useful quantitative prognostic indicator include reproducibility, prediction of survivorship, and assessment of morbidity and mortality. The goal is to establish management protocols that standardize the decision making process for multiple caregivers. General surgery has used quantitative prognostic indicators in the past with established benefit to patient care. Examples of quantitative prognostic indicators currently in use include Ranson's criteria, which estimates the risk of life threatening complication or death in patients with acute pancreatitis; and, the Child-Pugh score for liver cirrhosis, which evaluates the severity of liver disease correlating grades with one- and two-year survival. Currently, there are several clinical assessments at many different institutions in use for the evaluation of carcinomatosis (see Table 1 ). Our goal in this manuscript is to critically discuss these quantitative prognostic indicators. Collaborative studies between institutions would be greatly facilitated with standardized clinical tools for management of carcinomatosis from gastrointestinal cancer. Table 1 Quantitative prognostic indicators currently in use in patients with carcinomatosis. Tumor histopathology Intraoperative assessment of the extent of carcinomatosis at time of surgical exploration • Gilly peritoneal carcinomatosis staging • Carcinomatosis staging by the Japanese Research Society for Gastric Cancer • Peritoneal Cancer Index (PCI) • Dutch Simplified Peritoneal Carcinomatosis Index (SPCI) CT PCI Prior Surgical Score Completeness of Cytoreduction Score II. Histopathology In patients with carcinomatosis from gastrointestinal cancer, invasive implants are disseminated within the peritoneal cavity. However, in two conditions the biological aggressiveness of the disease will have a broad spectrum. These two diseases are mucinous appendiceal malignancies (oftentimes clinically designated pseudomyxoma peritonei syndrome) and peritoneal mesothelioma. In these diseases a non-invasive process may be widely disseminated on the peritoneal surfaces. The biological aggressiveness of the malignancy can be estimated by the pathologist in a knowledgeable histologic review of multiple specimens. For pseudomyxoma peritonei syndrome, the histologic classification described by Ronnett and colleagues has been most widely utilized [ 6 ]. Histopathologic examination categorizes the disease process into disseminated peritoneal adenomucinosis (DPAM), peritoneal mucinous carcinoma (PMCA) or a hybrid type. Disseminated Peritoneal Adenomucinosis This is a minimally invasive disease, and therefore more likely to be completely removed by cytoreduction using peritonectomy. The histology of DPAM shows a bland single layer of epithelium that surrounds lobules of mucin. There are no signet rings and there is minimal atypia. Invasion of the structures upon which tumor accumulates does not occur. The primary site for DPAM is an appendiceal adenoma which has minimally invaded the wall of the appendix. Usually, the widespread intraperitoneal dissemination of mucinous tumor is caused by a rupture of the lumen of the appendix from pressure built up by the malignant mucocele. Figure 1 shows the typical disruption of the wall of the appendix by tumor. Figure 2 presents the histologic character of DPAM. Figure 1 Right colon, terminal ileum and mucocele of the appendix. This appendix is greatly dilated; the end has ruptured releasing mucus and adenomatous epithelial cells into the free peritoneal cavity. Figure 2 Histopathology of disseminated peritoneal adenomucinosis (DPAM). (H+E × 200) Peritoneal Mucinous Adenocarcinoma This is an invasive disease in which the mucinous cancer cells show invasion into surrounding tissues. Sometimes, the signet ring morphology or lymph node metastases are present. The cancer cells will be found in multiple layers surrounding the mucinous tumor globules. There is loss of nuclear polarity and atypia is common. The quantity of mucus may be variable from one patient to the other. However, the PMCA histology may be associated with very large amounts of mucoid ascites fluid. Therefore, it is categorized as pseudomyxoma peritonei syndrome but with an aggressive tumor histology. Figure 3 provides an example of PMCA. Figure 3 Histopathology of peritoneal mucinous adenomucinosis (PMCA). (H+E × 700) Hybrid Type Disease In the hybrid type of mucinous carcinomatosis the field of view for the pathologist shows 95% or more DPAM. PMCA is present but in 5% or less of the total field of view (figure 4 ). If there is more than 5% PMCA the histology is no longer hybrid type but designated as PMCA. Figure 4 Histopathology of hybrid type mucinous appendiceal malignancy. (H+E × 100) Not surprisingly, the observation has been made by numerous groups that the non-invasive mucinous tumors (DPAM and hybrid type) are amenable to complete cytoreduction. Therefore more definitive treatment and improved survival using the combined approach is expected with DPAM and hybrid type [ 7 ]. The histologic and clinical differences between the different types of mucinous appendiceal and other gastrointestinal mucinous tumors are shown in Table 2 . Table 2 Histopathologic features of epithelial mucinous tumors of appendiceal, colonic, and small bowel origin are designated as disseminated peritoneal adenomucinosis (DPAM) and peritoneal mucinous carcinomatosis (PMCA). Features DPAM PMCA Primary site Appendix Appendix, colon, small intestine Primary diagnosis Mucinous adenoma usually in a mucocoele Mucinous adenocarcinoma Surgical appearance Mucinous tumors and mucinous ascites with redistribution Carcinomatosis with variable amounts of mucinous ascites, redistribution is prominent with large volume of ascites Peritoneal tumor • Cellularity Scant Moderate to abundant • Morphology Abundant extracellular mucin containing simple to focally proliferative mucinous epithelium. There is a single layer of cells Moderate to abundant extracellular mucin containing extensively proliferative mucinous epithelium or mucinous glands, clusters of cells, or individual cells consistent with carcinoma • Cytologic atypia Minimal Moderate to marked • Mitotic activity Rare Infrequent to frequent Lymph node involvement Almost never Moderate Liver metastases Almost never Very infrequent Parenchymal organ invasion Rare (except ovary) Frequent Hybrid type tumors show less than 5% of PMCA within DPAM. Mucinous carcinomas are divided into three grades by maintenance or loss of glandular architecture. III. Intraperitoneal Assessment of the extent of Carcinomatosis The quantitation of tumor found at the time of surgical exploration of the abdomen has proven to be of value in assessment of prognosis and treatment planning. Four different assessments have been published. They are listed in Table 1 . Gilly Peritoneal Carcinomatosis Staging The Gilly peritoneal carcinomatosis staging format was first described in Lyon in 1994 [ 8 ]. This prognostic tool takes into account the size of lesions found at operation (table 3 ). Two advantages of this system are simplicity and reproducibility. The utility of the Gilly staging device in survivorship prediction has been demonstrated in the multicentric prospective EVOCAPE study which gathered data from 370 patients with peritoneal carcinomatosis from non-gynecologic malignancies [ 2 ]. A significant difference was observed between stages 1 and 2 with a median survival of 6 months and stages 3 and 4 whose median survival was 3 months. The Gilly carcinomatosis staging has also been validated in patients having combined treatment for carcinomatosis [ 9 ]. Table 3 Gilly peritoneal carcinomatosis staging. Stage Peritoneal carcinomatosis description Stage 0 No macroscopic disease Stage 1 Malignant implants less than 5 mm in diameter Localized in one part of the abdomen Stage 2 Diffuse to the whole abdomen Stage 3 Malignant implants 5 mm to 2 cm Stage 4 Large malignant nodules (more than 2 cm) Although the Gilly system has been used for almost a decade with acceptable prognostic value, there are some criticisms regarding this system. First, it should not be designated a "staging system" because patients can only be staged once in the course of their disease at the time of diagnosis of the primary malignancy. Usually, a TNM staging system is appropriate. The system might better be called the Gilly prognostic index for carcinomatosis. A second weakness of the Gilly prognostic index concerns a failure to quantitate distribution of peritoneal surface implants in the stage 3 and 4 categories. Carcinomatosis confined to one portion of the abdomen may carry an excellent prognosis even if the localized tumor implants are of large size. If group III and group IV nodules by size are diffuse throughout the whole abdomen, certainly a much different prognosis would occur. A definitive assessment of not only the size of the nodules but also the distribution of carcinomatosis is necessary for the most accurate assessment of prognosis. The Japanese have proposed a quantitation of carcinomatosis that is very simple, has been frequently applied, and has been validated for gastric malignancy. For the original staging a "P factor" is indicated for gastric cancer patients. P-0 means that no carcinomatosis was seen by the surgeon or could be established at the time of surgery. It would currently include patients who are cytology positive for gastric cancer cells. P-1 indicates implants immediately adjacent to the stomach and above the transverse colon. P-2 indicates scattered implants within the abdomen but not of great number. P-3 indicates numerous implants throughout the abdomen and pelvis. This staging system can also be applied to patients who have carcinomatosis with recurrent gastric cancer. A major deficit of this staging system is its inability to accurately locate the carcinomatosis. Also, it has no size assessment of the cancerous implants. Although the P factor has been of great value historically in the management of primary gastric cancer as peritonectomy and intraperitoneal chemotherapy are used for treatment of carcinomatosis, a more precise prognostic assessment is needed to manage gastric cancer peritoneal seeding. Peritoneal Cancer Index The Peritoneal Cancer Index (PCI), like the other carcinomatosis assessments, is determined at the time of surgical exploration of the abdomen and pelvis. With invasive cancer it serves as an estimate of probability of complete cytoreduction and has been found to be an accurate assessment of survival when cytoreductive surgery and perioperative intraperitoneal chemotherapy are used as treatment [ 10 ]. The PCI quantitatively combines the distribution of tumor throughout 13 abdominopelvic regions with a lesion size score. Two transverse and two sagittal planes divide the abdomen into 9 regions. The upper transverse plane is located at the lowest aspect of the costal margin, and the lower transverse plane is placed at the anterior superior iliac spine. The sagittal planes divide the abdomen into three equal sectors. The lines define 9 regions, which are numbered in a clockwise direction with 0 at the umbilicus and 1 defining the space beneath the right hemidiaphragm. Regions 9 through 12 divide the small bowel into upper and lower jejunum and upper and lower ileum (Figure 5 ). To make the PCI tool more quantitative and reproducible, each region is not only defined by the surface landmarks as previously described, but can also be defined by the anatomic structures found in each region (Table 4 ). Figure 5 Peritoneal cancer index (PCI). Two transverse planes and two sagittal planes divide the abdomen into 9 regions. The upper transverse plane is located at the lowest aspect of the costal margin and the lower transverse plane is placed at the anterior superior iliac spine. The sagittal planes divide the abdomen into three equal sectors. The lines define the nine regions which are numbered in a clockwise direction with 0 at the umbilicus and 1 defining the space beneath the right hemidiaphragm. Regions 9–12 divide the small bowel. Lesion size score is determined after complete lysis of all adhesions and the complete inspection of all parietal and visceral peritoneal surfaces. It refers to the greatest diameter of tumor implants that are distributed on the peritoneal surfaces. Primary tumors or localized recurrences at the primary site that can be removed definitively are excluded from the lesion size assessment. If there is confluence of disease matting abdominal or pelvic structures together, this is automatically scored as L-3 even if it is a thin confluence of cancerous implants. Table 4 Anatomic structures involved in the 13 abdominopelvic regions of the peritoneal cancer index (PCI). Regions Anatomic structures 0 Central Midline abdominal incision – entire greater omentum – transverse colon 1 Right upper Superior surface of the right lobe of the liver – undersurface of the right hemidiaphragm – right retro hepatic space 2 Epigastrium Epigastric fat pad – left lobe of the liver – lesser omentum – falciform ligament 3 Left upper Undersurface of the left hemidiaphragm – spleen – tail of pancreas – anterior and posterior surfaces of the stomach 4 Left flank Descending colon – left abdominal gutter 5 Left lower Pelvic sidewall lateral to the sigmoid colon – sigmoid colon 6 Pelvis Female internal genitalia with ovaries, tubes and uterus – bladder, Douglas pouch – rectosigmoid colon 7 Right lower Right pelvic sidewall – cecum – appendix 8 Right flank Right abdominal gutter – ascending colon 9 Upper jejunum 10 Lower jejunum 11 Upper ileum 12 Lower ileum The lesion size (LS) score is determined after complete lysis of all adhesions and complete inspection of all parietal and visceral peritonea surfaces within the abdominopelvic regions. LS-0 indicates no implants seen. LS-1 indicates implants less than 0.25 cm. LS-2 indicates implants between 0.25 and 2.5 cm. LS-3 indicates implants greater than 2.5 cm. It refers to the greatest diameter of tumor implants that are distributed on the peritoneal surfaces. Primary tumors or localized recurrences at the primary site that can be removed definitively are excluded from the assessment. If there is a confluence of disease matting abdominal or pelvic structures together, this is automatically scored as LS-3 even if it is a thin layer of cancerous implants. The lesion sizes are then summated for all abdominopelvic regions. The extent of the disease within all regions of the abdomen and pelvis is indicated by a numerical score from 0 to 39. In 1995, Sugarbaker and Jablonski published that the PCI was a meaningful assessment for colon cancer but not for mucinous appendiceal tumors [ 11 ]. Elias et al., found survival to be more favorable in those patients with carcinomatosis from colon cancer with a PCI score of less than 16 [ 12 ]. In a larger number of patients Sugarbaker and Chang established survivorship using the PCI [ 13 ]. Five-year survival was 50% in colon cancer patients with carcinomatosis with a PCI less than 10, 20% for 11–20 and 0% in those with a PCI score greater than 20 (Figure 6 ). Tentes and colleagues validated the PCI for ovarian cancer [ 14 ]. The PCI is not only useful as a prognostic indicator but also as a guide for sequential determinations of volume of carcinomatosis over time estimating the likelihood of a complete cytoreduction at re-operative surgery [ 15 ]. Figure 6 Peritoneal carcinomatosis from colon malignancy survival by peritoneal cancer index. (Modified from Reference 13) This quantitative prognostic indicator for colon carcinomatosis established that for patients scoring greater than 20, palliation is the goal of treatment. Currently, a PCI of greater than 20 is regarded as a relative contraindication to an elective intervention for carcinomatosis from colon cancer. It is associated with a low median survival, approximately the same as median survival without surgical intervention. In patients who have a PCI greater than 20, palliative surgery is indicated in order to alleviate symptoms or to prevent symptoms that may occur in the near future. In an asymptomatic patient with colon carcinomatosis cytoreductive surgery with intraperitoneal chemotherapy with cure as a goal of treatment is probably not indicated. An exception to the utility of the PCI is found in treating patients with pseudomyxoma peritonei and minimally aggressive mesothelioma. Because the disease is non-invasive, a PCI of 39 can be converted to 0 by cytoreductive surgery. There is a low probability of recurrence after complete cytoreduction with perioperative intraperitoneal chemotherapy and therefore the PCI has no prognostic implication [ 7 ]. Another caveat that must be observed when using the PCI occurs in cases in which a low PCI score is recorded in the presence of invasive cancer at a crucial anatomic site. For example, at exploration one may find invasive tumor in and around the common bile duct with little disease elsewhere. Even thought the PCI is low, a complete cytoreduction may not be possible. In these cases, invasive cancer at a crucial anatomic site places the patient into the same category as would systemic metastasis in the lungs or bone. Only palliative surgery is indicated if residual disease post-cytoreduction will be present. Simplified Peritoneal Cancer Index The Simplified Peritoneal Cancer Index (SPCI) was established at the Netherlands Cancer Institute and has been used for colorectal and appendieal cancer staging (Table 5 ). This tool has prognostic implication for survival following cytoreductive surgery and hyperthermic intraperitoneal chemotherapy [ 16 ]. Table 5 Simplified Peritoneal Cancer Index ◆ Tumor is recorded as: indent="1" • Large (> 5 cm) indent="1" • Moderate (1–5 cm) indent="1" • Small (< 1 cm) indent="1" • None ◆ Seven abdominal regions: indent="1" • I: pelvis indent="1" • II: right lower abdomen indent="1" • III: greater omentum, transverse colon and spleen indent="1" • IV: right subdiaphragmatic area indent="1" • V: left subdiaphragmatic area indent="1" • VI: subhepatic and lesser omental area indent="1" • VII: small bowel and small bowel mesentery Verwaal and colleagues have provided important information regarding the relationship of the Simplified Peritoneal Cancer Index and the incidence of complications in patients who receive combined treatment [ 17 ]. In their review of the toxicity of combined treatment, complications increased when the cancer index recorded involvement of more than five regions (p = 0.044). Also, if the patient had recurrent colon cancer (as opposed to carcinomatosis with primary cancer) or if there was an incomplete cytoreduction, the incidence of complications was significantly higher. Verwaal et al., established that the peritoneal cancer index quantitated not only the survival outcome of these patients but also the expected morbidity and mortality of the combined treatment [ 16 ]. There are marked similarities between the SPCI and the PCI. Both the anatomic distribution of the tumor masses and the size of the tumor masses within each abdominal region are indicated. In the PCI, there are 13 anatomic sites designated by a diagram; in the Dutch SPCI, there are 7 anatomic regions designated by anatomic site. In both systems the volume of tumor in each region is to be scored quantitatively. Some shortcomings of the SPCI could be formulated. First, the epigastric region, very important in determining the completeness of cytoreduction in some diseases is not designated separately. Disease above the stomach in the lesser omental region may cause the cytoreduction to be incomplete [ 15 ]. A second major criticism of the Dutch SPCI concerns their misuse of their own tool. In their recent publications they perform a survival analysis by SPCI and a toxicity assessment by the SPCI. However, only the involvement of regions 0–7 was indicated. No tumor size in the regions was indicated [ 16 , 17 ]. Prior Surgical Score An accepted fact regarding cancer treatment is that the optimal treatment with the highest cure rate, the greatest preservation of function, and the lowest morbidity and mortality is the initial treatment. In the management of carcinomatosis the extent of prior resection before definitive cytoreduction with intraperitoneal chemotherapy has a negative impact on the survival. This occurs because of the cancer cell entrapment phenomenon. Surgery opens tissue planes whose raw surface is a favored site for cancer cell adherence, vascularization and progression. In the use of combined treatment for carcinomatosis, the non-traumatized peritoneal surface is the body's first line of defense against carcinomatosis. Cancer progression deep to peritoneal surfaces, especially disease imbedded in scar, is difficult or impossible to remove by peritonectomy or to eradicate by intraperitoneal chemotherapy. The prior surgical score (PSS) quantitates the extent of surgery prior to definitive combined treatment. It shows that the greater the surgery the poorer the results of carcinomatosis treatment. The assessment uses a diagram similar to that for PCI but excludes abdominopelvic regions 9–12. For a PSS of 0 no prior surgery or only a biopsy was performed; PSS of 1 indicates one region with prior surgery; PSS-2 indicates 2 to 5 regions previously dissected; PSS-3 indicates more than 5 regions previously dissected. This is equivalent to a prior attempt at complete cytoreduction but in the absence of perioperative intraperitoneal chemotherapy. In appendiceal cancer patients with a prior surgical score of 0–2, the survival using combined treatment was 70% at 5 years; with a prior surgical score of 3, the 5-year survival was 51% (p = 0.001) [ 18 ]. Completeness of Cytoreduction Score The Completeness of Cytoreduction Score functions as a major prognostic indicator for the survival in peritoneal mesothelima, colon cancer with carcinomatosis, gastric cancer with carcinomatosis and sarcomatosis [ 7 ]. It is to be assessed after cytoreductive surgery is completed. Complete cytoreduction (CC-0 or CC-1) or incomplete (CC-2 or CC-3) are determined. A CC-0 is apparent when there is no peritoneal seeding visualized within the operative field. CC-1 indicates nodules persisting after cytoreduction less than 2.5 cm. CC-2 has nodules between 2.5 and 5 cm, whereas a CC-3 indicates nodules greater than 5 cm or a confluence of unresectable tumor nodule at any site within the abdomen or pelvis. The CC-1 tumor nodule size is thought to be penetrable by intracavitary chemotherapy and is, therefore, designated as complete cytoreduction if perioperative intraperitoneal chemotherapy is used. Sugarbaker and colleagues found that prognosis can be estimated by completeness of cytoreduction. For colon cancer as shown in Figure 7 , there is a 40% chance of survival at 5 years in those who undergo complete cytoreduction versus 0% survival in the incomplete category [ 7 , 13 ]. Numerous other groups have confirmed the complete cytoreduction as a requirement for survival after treatment of carcinomatosis from appendiceal, colorectal and gastric cancer [ 4 , 9 , 11 , 12 , 16 - 19 ]. Figure 7 Peritoneal carcinomatosis from colon malignancy survival by cytoreduction. (Modified from Reference 13) Although no formal statement in the literature is available, it is thought that the definition of complete vs. incomplete cytoreduction varies with the histologic type of the malignancy. For example, mucinous tumors by diffusion are well penetrated with intraperitoneal chemotherapy solutions. With minimally invasive mucinous tumors such as pseudomyxoma peritonei, complete cytoreduction may occur in the combined treatment plan with tumor nodules up to a full centimeter in size. In contrast, hard fibrotic non-mucinous colon cancer is poorly penetrated by chemotherapy solution. Only cytoreduction down to no visible evidence of disease would be expected to result in long-term survival with a sclerotic malignant process. Also, some cancers may be remarkably more responsive to chemotherapy than others. This is likely the case with a majority of ovarian cancers. Their complete response to systemic chemotherapy is also frequently seen with intraperitoneal chemotherapy solutions or a bidirectional (intraperitoneal combined with intravenous chemotherapy) approach. In both these situations the definition of a complete cytoreduction scored by a CC-1 designation would vary with the clinical situation. Computerized Tomographic PCI The preoperative CT is an excellent tool in locating and quantifying mucinous adenocarcinoma within the peritoneal cavity [ 20 ]. Unfortunately, with intestinal histologic type of colon cancer the accuracy of the CT is considerably reduced [ 21 ]. However, for mucinous carcinomatosis CT scanning is an accurate prognostic indicator of the possibility of resectability. It may show segmental obstruction of the small bowel or tumor nodules greater than 5 cm on small bowel. Patients who have both of these findings have a likelihood of less than 5% of complete cytoreduction. Obstructed segments of bowel signal an invasive character of malignancy on small bowl surfaces that would be unlikely to be completely cytoreduced. Large tumor nodules on small bowel or its mesentery are unlikely to be adequately cytoreduced without visceral resection. There are some special demands on CT scanning if the radiologic examination is to be optimized. Bowel loops cut in cross section are often indistinguishable from cancer nodules. Only if maximal oral contrast using a barium sulfate compound is utilized to prepare the patient for this examination can the greatest accuracy and the greatest prognostic implications of the examination be realized. Another technical requirement is the imaging of solid tumor layered out on the peritoneal surfaces. Unless there is maximal intravenous contrast with a 60 to 120 second delay after contrast infusion will the confluence of malignancy as a thin layer on the peritoneum be imaged. In some patients, the solid tumor, or semisolid tumor may be distributed to appear as ascites on abdominal and pelvic CT. Much to the surgeon's dismay, upon opening the abdomen, a solid tumor mass filling the abdomen and pelvis and causing adherence of small bowel and small bowel mesentery will be revealed. In this situation, not even palliative surgery can be safely performed. In patients who clinically have a firm abdomen and in whom the surgeon suspects large volume of solid tumor, an ultrasound examination may be required in order to confirm an ascitic versus a solid component of the abdominal and pelvic malignancy. If ultrasound shows that there is only minimal or no ascites and that the large volume of tumor is solid or semisolid, surgical interventions are not beneficial. It is better to determine the nature of the carcinomatosis radiologically than at the time of a major surgical exploration. Conclusion Quantitative prognostic indicators are of value in management of peritoneal surface malignancy from gastrointestinal cancer. Preoperative CT PCI, intraoperative PCI and postresection CC score have all been reported valuable. As one knowledgeable applies these tests, proper selection of patients for combined treatment may increase benefit and decrease morbidity and mortality.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC549516.xml
548146	Induction of chondro-, osteo- and adipogenesis in embryonic stem cells by bone morphogenetic protein-2: Effect of cofactors on differentiating lineages	Background Recently, tissue engineering has merged with stem cell technology with interest to develop new sources of transplantable material for injury or disease treatment. Eminently interesting, are bone and joint injuries/disorders because of the low self-regenerating capacity of the matrix secreting cells, particularly chondrocytes. ES cells have the unlimited capacity to self-renew and maintain their pluripotency in culture. Upon induction of various signals they will then differentiate into distinctive cell types such as neurons, cardiomyocytes and osteoblasts. Results We present here that BMP-2 can drive ES cells to the cartilage, osteoblast or adipogenic fate depending on supplementary co-factors. TGFβ 1 , insulin and ascorbic acid were identified as signals that together with BMP-2 induce a chondrocytic phenotype that is characterized by increased expression of cartilage marker genes in a timely co-ordinated fashion. Expression of collagen type IIB and aggrecan, indicative of a fully mature state, continuously ascend until reaching a peak at day 32 of culture to approximately 80-fold over control values. Sox9 and scleraxis, cartilage specific transcription factors, are highly expressed at very early stages and show decreased expression over the time course of EB differentiation. Some smaller proteoglycans, such as decorin and biglycan, are expressed at earlier stages. Overall, proteoglycan biosynthesis is up-regulated 7-fold in response to the supplements added. BMP-2 induced chondrocytes undergo hypertrophy and begin to alter their expression profile towards osteoblasts. Supplying mineralization factors such as β-glycerophosphate and vitamin D 3 with the culture medium can facilitate this process. Moreover, gene expression studies show that adipocytes can also differentiate from BMP-2 treated ES cells. Conclusions Ultimately, we have found that ES cells can be successfully triggered to differentiate into chondrocyte-like cells, which can further alter their fate to become hypertrophic, and adipocytes. Compared with previous reports using a brief BMP-2 supplementation early in differentiation, prolonged exposure increased chondrogenic output, while supplementation with insulin and ascorbic acid prevented dedifferentiation. These results provide a foundation for the use of ES cells as a potential therapy in joint injury and disease.	Background Articular cartilage is composed of extracellular matrix (ECM), the matrix-secreting chondrocyte and water, which all account for the tissue's characteristic rigidity as well as its flexibility. These features are necessary in order to warrant life-long survival of the cartilage tissue, especially in the joint where it has to endure pressure forces caused by movement. Chondrocytes arise from a mesenchymal progenitor during development, the same progenitor that gives rise to other mesenchymal cell types including osteoblasts, adipocytes and myocytes. Bone formation can either be endochondral, when chondrocytes mature and calcify to provide a matrix for the invading osteoprogenitors, or intramembraneous involving ossification directly from a mesenchymal ancestor. All these diverse cell types may arise from the same precursor, but are distinguished by specific morphological features and with that, a certain set of characteristic proteins including transcription factors that control their differentiation. Two chondrocyte-specific transcription factors have been identified, Sox9, a member of the SOX-family of transcription factors, and Scleraxis, a member of the basic helix-loop-helix transcription factors [ 1 , 2 ]. However, most of the exclusive markers for cartilage tissue reside in the ECM. The predominant form of collagen in mature cartilage is collagen type IIB, whereas the alternatively spliced collagen IIA is found primarily during development [ 3 ]. Aggrecan is the major proteoglycan species in cartilage [ 4 ]. The transition of chondrocytes into hypertrophy is distinguished by a change in expression of Cbfa1, the osteoblast-specific transcription factor, which is also switched on during intramembraneous ossification. Distinctive to cartilage, the major collagen molecule in osseus matrix is collagen type I. In contrast, the transcription factors controlling adipogenesis are C/EBPα and PPARγ, which transactivate subsets of genes as a function of either trans-acting factor alone or requiring the co-operative effort of both [ 5 ]. C/EBPα is known to bind to and transactivate particularly the promotors of the SCD1, aP2 and the Glut4 genes [ 6 , 7 ], all highly characteristic of the adipocyte phenotype. For decades, the treatment of degenerative cartilage and bone diseases has been a challenge for orthopaedic surgeons due to the apparent inability of cartilage and bone to repair itself. Arthritis, a degenerative joint condition, is one of the most prevalent chronic health conditions in North America. Arthritis can devastate people, but to date there is no effective therapy available and patients can only be helped by surgical joint replacement. An inherent major concern is the limited availability of autografts, which significantly reduces the choice of treatable defects. However, new approaches to cell grafting are being developed in this field: increased yields of cells are achieved by the usage of bioreactors and growth factor administration, such as TGFβ 1 and BMPs [ 8 , 9 ]. Additionally, stem cells are being discovered as a new source of transplantable material. Embryonic stem cells represent a valuable source for cell transplantation since their characteristic features include an unlimited self-renewing capacity and a multilineage differentiation potential [ 10 , 11 ]. In fact, ES-derived glial precursors and cardiomyocytes have been successfully transplanted, integrated and shown to be functionally active in the transplantation site [ 12 , 13 ]. The yield of differentiation of ES cells into an intended lineage can be greatly enhanced by the addition of growth factors or induction substances. Whereas protocols for the differentiation of cardiomyocytes, neuronal cell types, insulin-producing cells or adipocytes from ES cells have been available for many years [ 14 - 17 ], only recently their differentiation into elements of the skeleton has been reported [ 18 - 20 ]. Our group has previously shown that vitamin D 3 forces ES cells to undergo osteogenesis [ 18 ]. Kramer et al. have reported in 2000 that BMP-2 pushes ES cells to the chondrogenic fate when added during days 3–5 of EB differentiation [ 21 ]. Those ES-derived chondrocytes possess a certain plasticity to undergo hypertrophy and calcify [ 22 ]. We show here, that prolonged treatment of differentiating ES cell cultures with BMP-2 in synergy with TGFβ 1 , insulin and ascorbic acid leads to improved chondrogenesis in vitro . Compared to the brief supplementation described by Kramer et al. [ 21 ], the expression of chondrocyte-specific marker genes was highly up-regulated while proteoglycan content revealed an increased chondrocytic yield from 7.26% to 57.03%. As described by other groups [ 22 ], ES-derived chondrocytes become hypertrophic and calcify. However, spontaneous calcification did not reach mineralization levels that are found in vitamin D 3 induced ES-derived osteoblasts [ 18 ]. Yet, supplementation of chondrocyte-cultures with β-glycerophosphate, ascorbic acid and vitamin D 3 starting at day 20 rescued the osteoblast phenotype. In many differentiations, we also observed an accumulation of lipid droplets and an up-regulation of adipocyte-specific genes. This direction towards adipocyte differentiation varied with the use of specific co-factors, suggesting that in the future, such spurious differentiation may be controlled, once the pathways involved in adipogenesis are better understood. Results Characterization of chondrocyte-like cells derived from ES cells Embryonic stem cell cultures supplemented with BMP-2, TGFβ 1 , insulin and ascorbic acid show typical morphological changes compared to the untreated cultures. Starting with the fourth week of culture, aggregates consisting of small round cells formed in the supplemented cultures, which stained positive with alcian blue (fig. 1A ). Little alcian blue staining was seen in control cultures (fig. 1B ). Polygonal cells, which could also be found in treated cultures, did not stain with alcian blue. Significant immunostaining for the collagen type II (COL II) protein was observed at day 32 in treated cultures corresponding to the active secretion and formation of an extracellular matrix found with chondrocytes. The COL II antibody identified the fibrillary organization of the collagen molecules in the extracellular matrix (fig. 1C ). Chondrogenic differentiation was confirmed by positive immunostaining for adult proteoglycans (fig. 1D ), which is detectable in the aggregates identified by alcian blue staining. The distribution was associated with the extracellular matrix similar to that found with the COL II antibody. Staining appeared to be diffuse as extracellular matrix and not individual cells are stained. Figure 1 Morphology and characteristics of ES-derived chondrocytes after 32 days of culture. (A, B) Determination of proteoglycans in EBs with alcian blue in chondrocyte cultures induced with TGFβ 1 [2 ng/ml] and BMP-2 [10 ng/ml] from d3–5 of culture and with BMP-2 [10 ng/ml], ascorbic acid [50 μg/ml] and insulin [1 μg/ml] from day 5 onwards (A) compared to control cultures (B). Bar = 8.3 μm. (C, D) Analysis of cartilage-specific matrix proteins in 32 day old EBs induced with the same supplements as in (A, B) by means of immunohistochemistry. Staining with anti-collagen type II (C) and anti-cartilage proteoglycan (D), respectively, both visualized by a secondary AlexaFluor 488 conjugated antibody. Bar = 106 μm. (E) Concentration-dependent effect of BMP-2 on chondrocyte-specific gene expression in 32-day old EBs. Values of cultures supplemented with 10 ng/ml BMP-2 are shown compared to 2 ng/ml as used by Kramer et al. [2000], which was set as 1. Values represent means of three independent experiments ± standard deviation, obtained by quantitative RT-PCR analysis. P < 0.01; P < 0.001. (F) Proteoglycan content of EB extracts on day 32. BMP-2 directs increased proteoglycan synthesis in both 2 ng/ml and 10 ng/ml. P < 0.1; *P < 0.01. Mean ± standard deviation, n = 3. Dependence of gene expression patterns and proteoglycan synthesis on BMP-2 Quantitative RT-PCR was used to examine the variability of RNA expression of various cartilage-specific genes in response to BMP-2. Previously, chondrogenesis was induced in ES cells using 2 ng/ml BMP-2 on days three to five of EB formation, when early mesodermal markers such as Brachyury and BMP-4 are expressed [ 21 , 23 , 24 ]. To improve chondrogenesis, we investigated whether a higher concentration of BMP-2 (10 ng/ml) could increase chondrocyte-specific gene expression. Total RNA was extracted on day 32 and quantitative real-time PCR was carried out. Expression of genes of interest in cultures supplemented with 10 ng/ml BMP-2 was normalized to GAPDH expression and compared to cultures supplemented with 2 ng/ml (fig. 1E ), which were set as 1. Expression of the small leucine-rich proteoglycans biglycan and decorin was increased 1.6 fold upon supplementation with 10 ng/ml BMP-2 (P < 0.01). Neither link protein expression nor Sox9 or scleraxis expression were affected by the higher dosage. However, expression of aggrecan and the collagen type II isoforms A and B, which are specific for mature chondrocytes, were significantly increased compared to the low BMP-2 concentration described by Kramer et al. (P < 0.001). The degree of chondrogenic differentiation under influence of BMP-2 was further quantified by metachromatic detection of proteoglycans (fig. 1F ). Secreted proteoglycans were extracted on day 32 of culture with guanidine/HCl and combined with dimethylmethylenblue. Based on our measurement of aggrecan, the synthesis of proteoglycan proteins is also increased under the influence of BMP-2. BMP-2 at 2 ng/ml initiated an induction in proteoglycan synthesis of about 2 fold (P < 0.01), whereas 10 ng/ml BMP-2 increased the proteoglycan content of EBs 2.3 fold (P < 0.1) compared to non-treated controls. This data shows, that BMP-2 causes the induced synthesis of negatively charged extracellular matrix, characterized by proteoglycans. Additive effect of TGFβ 1 , insulin and ascorbic acid on BMP-2 induced chondrogenesis Since BMP-2 is believed to play a role in late chondrogenesis [ 25 ], we studied the effect of prolonged BMP-2 supplementation beyond day 5 of culture. Additionally, the anabolic effect of insulin and ascorbic acid and the influence of the growth factor TGFβ 1 on BMP-2 induced differentiation was determined quantitatively by PCR analysis at various stages throughout EB differentiation. Table 1 shows the particular combinations of medium supplements used at different culture stages of the 'hanging drop' protocol used for differentiation, in which day 1–3 represent the hanging drop stage. During days 3–5 the resulting embryoid bodies are cultured in suspension and then plated onto tissue culture treated plastic ware on day 5. Applied concentrations were 10 ng/ml BMP-2, 2 ng/ml TGFβ 1 , 1 μg/ml insulin and 50 μg/ml ascorbic acid. Dexamethasone, which is also known to be a chondro-inducing agent [ 26 ], did not evoke a mentionable chondrogenic response in the D3 ES cell line (data not shown). Figure 2 shows changes in cartilage-specific gene expression under the influence of various differentiation co-factors throughout the 32 days of culture. TGFβ 1 (combination B) evoked a 2-fold increase in collagen II expression for both splice forms. The synergistic effect of both growth factors, TGFβ 1 and BMP-2 (combination C) began to show an increased expressions of aggrecan, link protein and COL IIA compared to cultures that were treated with one supplement alone. Additional supplementation with insulin and ascorbic acid between culture days 3 and 5 (combination D) barely increased aggrecan, link protein and COL II expression, but when given from day 5 onwards enhanced both the BMP-2 and TGFβ 1 effects (supplement combinations E and H). Addition of insulin and ascorbic acid to BMP-2 induced cultures increased collagen type IIA and aggrecan expression minimally to 1.2-fold, link protein and collagen type IIB were induced 2.7- to 2.8-fold compared to BMP-2 alone. The TGFβ 1 response was increased 3- to 4-fold. When cultures were induced with BMP-2 and TGFβ 1 in suspension (d3–5) and supplemented with insulin and ascorbic acid starting on day 5 onwards (combination F), aggrecan, link protein and COL II were up-regulated 10-fold compared to controls. Table 1 Combinations of medium supplements used at different culture stages Supplement combination Day 3–5 (hanging drops) Day 5 onwards (attached culture) A BMP-2 B TGFβ 1 C BMP-2 TGFβ 1 D BMP-2 TGFβ 1 insulin ascorbic acid E BMP-2 insulin ascorbic acid F BMP-2 TGFβ 1 insulin ascorbic acid G BMP-2 TGFβ 1 insulin ascorbic acid BMP-2 H TGFβ 1 insulin ascorbic acid Applied concentrations were 10 ng/ml BMP-2, 2 ng/v ml TGFβ 1 , 1 μg/ml insulin and 50 μg/ml ascorbic acid. The end of the culture period varied with every experiment carried out. Figure 2 Changes on cartilage markers in response to various combinations of BMP-2, TGFβ 1 , insulin and ascorbic acid as outlined in table 1. (A) Diagram of expression niveaus of cartilage-specific genes of 32 day old EBs obtained by quantitative RT-PCR and standardized to GAPDH. Untreated control values were set as 1. Mean ± standard deviation, n = 3. P < 0.1; P < 0.01; P = 0.001. (B) Proteoglycan content of extracts of 32 day old EBs treated with different combinations of BMP-2, TGFβ 1 , insulin and ascorbic acid (see table 1). Ctrl = control. Means ± standard deviation, n = 3. P < 0.1; P < 0.01; P = 0.001. n.d. = not determined. Surprisingly, when BMP-2 supplementation was maintained throughout the culture period (combination G), a dramatic increase of marker gene expression was seen. Aggrecan and COL IIB were up-regulated over 80-fold above controls and link protein and COL IIA expression was increased to 35- and 16-fold, respectively. Here, scleraxis and Sox9 expression behaved conversely. Supplement combination G showed significant decreases of scleraxis and Sox9 expression to 50 and 87% of the control, respectively (P = 0.001). Deferral of the expression of these transcription factors in favour of chondrogenic differentiation characterizes BMP-2 induced differentiation, whereupon chondrogenic processes are furthermore enhanced by TGFβ 1 , insulin and ascorbic acid. The determination of proteoglycan content was used to confirm the results obtained by quantitative RT-PCR for all supplement combinations (fig. 2B ). TGFβ 1 alone (combination B) did not alter proteoglycan levels compared to controls, and in combination with BMP-2 decreased the proteoglycan content of EBs compared to BMP-2 alone (combination C). Insulin and ascorbic acid did not enhance the proteoglycan synthesis in combination with TGFβ 1 , but caused a 4.2-fold increase in combination with BMP-2 (combination E, P = 0.01). In agreement with aggrecan gene expression, combinations F and G generated a massive 7-fold induction of proteoglycans in EBs (P = 0.001). Genetically manipulated ES cells that express GFP under the control of chondrocyte-specific aggrecan promotor were then used to quantify chondrocyte yield using fluorescence-activated cell sorting. ES cells were differentiated along the chondrocytic lineage using BMP-2 at 2 ng/ml or supplement combination G [d3–5: TGFβ 1 10 ng/ml, BMP-2 10 ng/ml; d3–32: BMP-2 10 ng/ml, ascorbic acid 50 μg/ml and insulin 1 μg/ml]. Green fluorescing chondrocytes in both cultures appeared either organized in clusters or scattered as seen in figure 3A . The Kramer protocol gave a 7.26 percent yield of chondrocytes, which was increased to 57.03% using our modified protocol (fig. 3B ). Figure 3 Quantification of chondrogenic yield by flow cytometry. (A, B) Genetically modified ES cells expressing GFP from the chondrocyte-specific aggrecan promotor. Green fluorescing chondrocytes appear as clusters of cells within the remaining cell population (A), but are also scattered in the entire population starting at day 28 of differentiation (B). (C) FACS analysis of ES-derived chondrocytes sorted by their GFP expression. Differentiation with BMP-2 only [2 ng/ml, d3–5] produced 7.26% GFP expressing cells compared to spontaneously differentiated controls, which contained very low levels of fluorescing cells (1.54%). Prolonged BMP-2 administration together with TGFβ 1 , ascorbic acid and insulin (supplement combination G, see table 1) raised chondrocyte outcome to 57.03%. Kinetic analysis of cartilage-specific gene expression during EB differentiation The degree of chondrogenic differentiation using BMP-2, TGFβ 1 , insulin and ascorbic acid supplementation was then examined over the 35-day culture period using quantitative RT-PCR of various cartilage matrix genes (fig. 4 ). Since the extent of changes in cartilage-specific genes seemed to be dependent on acute (d 3–5) or chronic (d 3–32) application of BMP-2 (supplement combination F versus supplement combination G), the stronger induction by chronic supplementation of BMP-2 was monitored throughout the culture duration. During the first three weeks of the BMP-2 induced differentiation, only minor changes in aggrecan, link protein or collagen type II expression could be detected. Starting with day 26 however, aggrecan expression was up-regulated significantly to 14-fold over control niveaus (P < 0.1). Reaching day 32, aggrecan, link protein and collagen type II A and B approached peak levels. Consistent with the aggrecan expression profile, link protein expression was found to be significantly up-regulated on days 24–27 (factor 6.3; P = 0.001). Increased values were detectable as early as day 16. On day 26, the quantification of the collagen type IIA and B showed 11- and 24-fold increases respectively. On day 14, splice form B had already reached an 8-fold increase over control values. In contrast, during the early phases of differentiation, between day 5 and 20, collagen type IIA expression was continuously increased 2-fold. Transcripts for biglycan and decorin were detectable throughout all stages of chondrogenic differentiation. With the beginning of the maturation phase in the fourth week of culture (day 21–28), both genes were up-regulated 1.5–2 fold. Transcription factors scleraxis and Sox9 (fig. 4B ) showed a similar expression profile constantly over the entire culture period. This level did not change significantly during chondrogenic differentiation, but rather decreased compared to controls. Both were already transcribed in day 5 EBs, hallmarking their participation in early differentiation events, where lineage specificity is determined. Additionally, scleraxis transcripts were engaged in later phases of development between days 16–19. Sox9 expression was also increased between days 9–11 and between days 25–27. In conclusion, cultures supplemented with BMP-2, TGFβ 1 , insulin and ascorbic acid express mRNAs of a chondrogenic phenotype, whose expression was time-dependent. Figure 4 Expression of cartilage-specific genes in the course of EB differentiation induced by BMP-2, TGFβ 1 , insulin and ascorbic acid (supplement combination G, table 1). (A) Aggrecan, collagen type II A and B and link protein, biglycan and decorin. (B) Scleraxis and Sox9. Results show induction factors of expression obtained by quantitative RT-PCR and normalized to GAPDH expression in comparison to corresponding spontaneously differentiated controls, which were set as 1 (mean ± standard deviation, n = 3 independent experiments, 20 EBs each). ES-derived chondrocytes undergo hypertrophy and mineralize During embryo development endochondral ossification occurs in two steps: chondrocytes arise after mesenchymal condensation and become hypertrophic, characterized by expression of collagen type X, and calcification. To test whether this was true for the ES-derived chondrocytes generated with our protocol, we assayed for the Ca 2+ content of the cultures, a measure for mineralization (fig. 5A ). Calcium was increased 1.2-fold in cells that were supplemented with BMP-2, TGFβ 1 , insulin and ascorbic acid compared to controls (P < 0.01). Previously, we have described the induction of mineralization in osteoblasts derived from ES cells with vitamin D 3 , which reach maturity at day 32 of culture [ 18 , 27 ]. Here, we observed that the level of mineralization was considerably higher in direct osteoblast differentiation [11.57 mg/dl] compared to indirect differentiation using our improved chondrocyte protocol [3.22 mg/dl]. We observed, however, that the level of calcium found in VD 3 induced ES-derived osteoblasts could be rescued in the chondrocytes by adding VD 3 on day 20 of differentiation, a time when chondrocytes could be morphologically identified in the cultures (11.18 mg/dl, P < 0.001). As shown by quantitative RT-PCR, VD 3 treated ES cell derived chondrocytes could alter their expression profile to that of d32 ES cell derived osteoblasts (fig. 5B ). However, expression of osteocalcin and bone sialoprotein was less than in VD 3 rescued chondrocyte cultures than in VD 3 osteoblast cultures. Interestingly, chondrocyte-specific genes could not be detected in VD 3 osteoblasts and waned in VD 3 rescued chondrocytes. As we have shown previously, ES-derived mineralized osteocalcin expressing osteoblasts can be identified as black appearing cells in phase contrast microscopy [ 18 ]. Figure 5C shows these black cells in the VD 3 treated cultures. No such cells are visible in control cultures or in ES-derived chondrocyte differentiations. However, by adding VD 3 back in at day 20 to the chondrocytes, mineralization can be detected, although the localization pattern is slightly different. These observations support the hypothesis that VD 3 induces intramembraneous bone formation directly from mesenchymal progenitors while BMP-2 controls endochondral bone formation. Figure 5 Degree of osteogenesis in cultures treated with chondroinducing medium (BMP, supplement combination G), osteoinducing medium (VD 3 as described in ref. 18) and control ES medium (ctrl). Osteoblast phenotype and mineralization could be increased in chondrocyte cultures by adding VD 3 to chondroinducing supplements at day 20 (BMP+VD 3 ). (A) Extent of mineralization as measured by Ca 2+ content. P < 0.01; P = 0.001. (B) RT-PCR for chondrocyte-specific and osteoblast-specific marker genes as well as Collagen X as a marker for the hypertrophic state of chondrocytes. (C) Morphology as seen in phase contrast. Black appearing cells were identified as being mineralized osteocalcin expressing osteoblasts previously [18]. No such cells can be seen in control (ctrl) and chondrocyte (BMP) but in osteoblast (VD 3 ) and rescued osteoblast cultures (BMP+VD 3 ). It has been shown by others that the BMP-2 induced alteration in cell fate is both concentration- and time-dependent [ 28 ]. Lower concentrations of BMP-2 support chondrogenesis whereas higher concentrations promote osteogenesis. In ES cells, BMP-2 at concentrations of 2 ng/ml, 10 ng/ml and 100 ng/ml did not increase the expression of bone markers with the exception of osteocalcin and osteopontin, which were significantly increased as shown by quantitative RT-PCR (fig. 6 ). In combination with VD 3 (given on days 5–30) however, alkaline phosphatase and Cbfa1 were also significantly up-regulated above controls (5.4- and 4-2-fold, respectively, P < 0.001). As we noted earlier, BMP-2 induced osteogenesis with or without VD 3 supplementation did not meet the levels that were attained by VD 3 alone, but the late addition of VD 3 on day 20 rescued bone-specific gene expression arguing for an involvement for BMP-2 in endochondral bone formation. Figure 6 Dependence of genes expressed in cartilage tissue on BMP-2 with and without vitamin D 3 (VD 3 ). All cultures contained β-glycerophosphate [10 mM] and ascorbic acid [50 μg/ml]. Mean ± SD of independent triplicates was quantified by qPCR and is normalized to GAPDH expression. Controls were set as 1. P < 0,01; **P = 0,001. OCN = osteocalcin, BSP = bone sialoprotein, Cbfa1 = Core binding factor alpha, ALP = alkaline phosphatase, OPN = osteopontin, ONT = osteonectin, Col1 = Collagen type I. Concentrations of BMP-2 up to 100 ng/ml do not lead to osteoblast-specific expression levels that are reached with VD 3 only. 100 ng/ml BMP-2 plus VD 3 given together early during differentiation can significantly up-regulate osteoblast genes. However, when VD 3 is administered later (d20) in combination with BMP-2, osteoblast gene expression is almost restored. Induction of adipocyte differentiation During treatment of EBs with various chondrocyte differentiation inducing factors, we noticed accumulation of lipid droplets, which could not be found in untreated controls. Those droplets could indeed be characterized as lipid-containing by means of Oil-Red-O staining (fig. 7A ). Quantitative real-time PCR analysis revealed a slight up-regulation of adipocyte-specific genes (ADD1, aP2, C/EBPα, GLUT-4, LPL, PPARγ and SCD1) on day 30 in most of the supplement combinations used for induction of chondrocyte differentiation (fig. 7B,C ). Supplement combination G, which was the most successful for inducing chondrocyte differentiation suppressed adipocyte differentiation. GLUT-4 was most up-regulated in combinations B and C, whereas combinations E and B induced a increase in LPL expression compared to our chondrocyte differentiation protocol. Figure 7 Characterization of ES-derived adipocytes. (A) Oil-Red-O staining of lipid droplets in ES-derived adipocytes. Bar = 42 μm. (B, C) Influence of BMP-2 alone and in combination with TGFβ 1 , insulin and ascorbic acid (see table 1) on expression of adipocyte-specific genes. Quantitative RT-PCR was performed on 30 day old EBs. Expression of genes of interest was normalized to GAPDH and compared to untreated controls. Values show means ± standard deviations on n = 3 independent experiments. P < 0.1; P < 0.01; *P = 0.001. Discussion In this study, we demonstrate an improved method for driving ES cells to a cartilaginous fate when stimulated with BMP-2 and TGFβ 1 . In the early phase of differentiation, BMP-2 operates by directing differentiation towards the cartilage lineage and acting on chondroprogenitors. During later differentiation adding mineralizing agents can trigger hypertrophy and mineralization of the ES-derived chondrocytes. In the embryo, maturation of chondrocytes in the process of endochondral ossification follows a timely regulated developmental program, whereby cellular stages can be delimitated molecularly. During ES cell differentiation into chondrocytes, developmental processes follow the same pattern, as judged by the gene expression patterns observed. Chondrocytes, osteoblasts and adipocytes are thought to arise from the same mesenchymal progenitor. Based on our previous observations around VD 3 -induced osteogenesis, we have already hypothesized that during the first 5 days of differentiation mesodermal progenitors develop, which then are susceptible to the VD 3 treatment [ 18 ]. Treatment of the cultures with TGFβ 1 at days 3–5 may augment the number of mesenchymal progenitors, as TGFβ 1 is thought to inhibit the proliferation of most cells, but to stimulate some mesenchymal cells such as osteoblasts and chondrocytes [ 8 ]. It was not surprising to see adipocytes develop in many of our cultures, as they represent another member of the TGFβ 1 promoted mesodermal lineage. The gain of adipose characteristics in culture is hallmarked by: a) the appearance of cytoplasmic lipid droplets, b) the acquisition of insulin sensitivity with regard to glucose uptake (GLUT4) and c) the expression and secretion of numerous bioactive molecules [ 5 ]. All of these characteristics of in vivo adipogenesis were met in the EBs treated with BMP-2, TGFβ 1 , insulin and ascorbic acid. Indeed, a future challenge for improving adipogenic cultures will be the discovery of regulatory pathways in adipogenesis. Once identified, such pathways may be antagonized in order to enhance ES differentiation into chondrocytes. During the revision of this paper, a study was published describing that the overexpression of the sox triad, namely Sox9, Sox5 and Sox6, markedly increased chondrocyte marker gene expression (collagen type 2, aggrecan) in ES cells within 3 days [ 29 ]. Here, TGFβ 1 , BMP-2, IGF-1 and FGF-2 had no affect on the immediate regulation of these genes. In our differentiation system, Sox9 is elevated very early during differentiation at day 5 (data not shown) underlining its role as an early controller of chondrogenesis. We have shown here that BMP-2 regulates later processes in cartilage development. Marker gene expression levels reached upon overexpression of the Sox trio do not meet the levels we have observed in this study, suggesting that Sox9 may be necessary but not sufficient do direct all progenitors to the chondrocytic lineage. Earlier this year, another study portrayed chondrogenesis in ES cells using encapsulation in alginate [ 26 ]. The usage of a 3D culture system led to an increase in Col II and aggrecan expression of about 1.5- to 2-fold above the regular 2D system of plating EBs. Comparing those values to the 80- to 90-fold up-regulation described here, it is clear that three-dimensional signals also need to be incorporated into chondrocyte differentiations to increase differentiation efficiency. Articular cartilage has been refractory to repair following degeneration. Despite its limited capacity to self-repair, cartilage is replete with cells capable of undergoing mitotic division [ 30 ]. Current hypotheses suggest that cells may be constrained by their ECM, thus preventing expansion and differentiation or that there is a limit in the bioactive molecules, which support chondrogenesis [ 31 , 32 ]. Engineering bone or cartilage usually requires the handling of autologous cells. Cells are released from the ECM using collagenase and hyaluronidase. However, the small number of available progenitors, within the tissue can be problematic [ 33 ]. Moreover, the ability of these harvested cells to proliferate is limited in the elderly, where most degenerative joint disorders occur. The outcome of conventional surgical treatment including joint resurfacing or biological autografts has been unsatisfactory following long-term evaluation [ 34 ]. This failure is caused by insufficient repair resulting in the formation of mechanically inadequate resident fibrocartilage. These disappointing results and the limited therapeutic opportunities have led investigators to focus on more appropriate bioregenerative tissue engineering approaches, which could be specifically tailored for a patient's needs. Pluripotent ES cells are now being contemplated as a new cell source for tissue engineering since they are the most multifaceted cells amongst all stem cells. While their pluripotency offers a huge potential for cell therapies directed against a wide spectrum of degenerative diseases that are ineffectively treated by traditional approaches, it also poses challenge for controlling developmental fate in the recipient. Understanding the developmental pathways regulating ES cell differentiation, will enable the creation of a cell source that can be manipulated to correct for a particular defect. This study was designed to improve upon the number of differentiated cells needed for the in vitro development of functional cartilage as this represents a first critical step in applying ES cells clinically. Additionally, the presented in vitro model of chondrogenesis may be useful for in vitro embryotoxicity tests [ 27 ] as also serve as a new tool in identifying molecular pathways that underlie chondrogenesis. Compared to other in vitro models of chondrogenesis, this model incorporates all steps of development beginning with a pluripotent, uncommitted cell and thus might further our understanding of normally unamendable stages of development. Conclusions This study was particularly designed to improve chondrocyte yields from ES cells by investigating the effect of higher BMP-2 concentrations and the complementary effects of TGFβ 1 , insulin and ascorbic acid. By examining gene expression responses and cell sorting for GFP-expressing chondrocytes, we demonstrate that using a higher concentration of BMP-2 in combination with appropriate co-factors, we can significantly enhance ES cell derived chondrogenesis compared to known protocols. In addition, we document that ES-derived chondrocytes behave naturally as they undergo hypertrophy. We show that EBs supplemented with BMP-2 also result in a small amount of adipogenesis in vitro . This observation is consistent with knowledge that adipocytes, chondrocytes and osteoblasts arise from the same mesenchymal ancestor. Accordingly, in the future, it will be necessary to understand how to discriminate these populations for cytotherapeutic applications. At this time, the presented in vitro model allows the study of mechanisms involved in BMP-2 induced chondrogenesis, osteogenesis and adipogenesis. Methods Cell culture and differentiation of embryonic stem cells Cells of the mouse ES cell line D3 (American Type Culture Collection, Rockville, Maryland, USA) were kept in permanent culture as described [ 35 , 18 ] with the additive Leukemia Inhibitory Factor (1000 U/ml, Gibco Life Technologies, Karlsruhe, Germany). Differentiation was initiated in hanging drops. Cells condensed to form EBs, which were transferred on day 3 into suspension culture. At day 5, EBs were plated into 24-well tissue culture primaria plates (Falcon, Heidelberg, Germany). The effects of BMP-2 [2 or 10 ng/ml], TGFβ 1 [2 ng/ml], insulin [1 μg/ml] and ascorbic acid [50 μg/ml] in various combinations on the differentiation of chondrocytes were examined. Medium was changed every second day. Alcian blue staining Proteoglycans secreted by ES-cell derived chondrocytes were stained with Alcian blue. Cultures were fixed in 2.5% glutaraldehyde, 25 mM sodium acetate, 0.4 M MgCl 2 containing 0.05% Alcian blue for 48 h. Wash steps in 3% acetic acid, 3% acetic acid/25% ethanol and 3% acetic acid/50% ethanol reduced unspecific binding of the dye. Metachromatic test with 1.9-dimethylmethylenblue Proteoglycan content of differentiated cultures was determined with the DMMB-assay [ 36 ]. Proteoglycans were extracted in 4 M guanidin-HCl/0.05 M sodium acetate (pH 5.8) containing 100 mM 6-amino-caproic acid, 10 mM EDTA, 5 mM benzamidine/HCl, 10 mM N-ethylmaleimide, 0.4 mM pepstatin, 1 mM PMSF and 1 μg/ml soy bean trypsin inhibitor for 48 h at 4°C. Non-completely digested cells were separated from the lysate by centrifugation. Lysate was mixed with DMMB reagent (0.16% w/v DMMB in 0.2% formic acid containing 2 mg/ml sodium formate, pH 3.5) and changes in absorption were detected at 535 nm in a spectrophotometer. Concentration of proteoglycans in samples was read against a standard curve of chondroitin sulfate C. Immunofluorescence Embryoid bodies were differentiated to the chondrocyte lineage with BMP-2, TGFβ 1 , insulin and ascorbic acid and fixed on day 32 with ice-cold methanol:aceton (7:3) at -20°C for 10 min. For staining with anti-collagen type II (Chemicon, MAB 8887) cultures were digested with pepsin for 15 min at 37°C. Staining with anti-adult proteoglycan (Chemicon MAB 2015) was performed after a 1 h digest with chondroitinase ABC at room temperature. Cells were overlaid with the appropriate dilution of the first antibodies in PBS, 10% FCS at 4°C over night. The corresponding secondary antibody, an AlexaFluor 488 goat anti-mouse IgG (H+L), F(ab') 2 fragment (A-11006, Molecular Probes, Leiden, The Netherlands) was incubated with the cells for 2 h at room temperature. Cultures were observed in a Leica Fluovert FU fluorescent microscope (Leitz, Wetzlar) with an excitation wavelength of 495 and an emission wavelength of 519 nm. RNA isolation, RT-PCR and real-time quantitative RT-PCR Total RNA was isolated from 20 EBs per probe using the RNeasy Midi Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions with on-column DNase I digestion. The amount of RNA was determined using the RiboGreen™ RNA quantitation reagent and kit (Molecular Probes). 275 ng total EB RNA was used as a template for cDNA synthesis with Superscript II (Invitrogen, Paisley, Scotland) as described [ 27 , 35 ] in a total reaction volume of 25 μl. 5 μl aliquots of the first strand reaction were used for amplification performed with specific primers (see table 2 ). Primer sequences for osteoblast-specific genes and PCR conditions have been described previously [ 18 ]. PCR products were visualized on 3% agarose gels containing 0.1 μg/ml ethidium bromide. Quantitative real-time PCR analysis was performed in an ABI Prism ® 7700 Sequence Detector. The accumulation of reaction products during PCR was monitored by measuring the increase in fluorescence caused by the binding of SYBR ® Green (Applied Biosystems, Perkin Elmer, Weiterstadt, Germany) to double-stranded DNA. Expression analysis of collagen type II A and B was done in the same PCR run with two differently labelled probes specific for either the A isoform (5'-CGAGATCCCCTTCGGAGAGTGCTGT-3'/VIC) or the B isoform (5'-CCAGGATGCCCGAAAATTAGGGCCAA-3'/FAM) [ 27 ]. Reaction mixtures were set up as suggested by the manufacturer containing either SYBR Green or probes for collagen type II. Following a 10 min Taq Polymerase activating step at 95°C, the reactions were cycled by denaturing at 94°C for 30 s and annealing and elongation for 30 s at the corresponding temperatures (table 2 ). Target gene C T -values were standardized against GAPDH expression and induction of expression in treated EBs was normalized to control EBs. Primer sequences for murine GAPDH were 5'-GCACAGTCAAGGCCGAGAAT-3' and 5'-GCCTTCTCCATGGTGGTGAA-3' (T a = 60°C). Table 2 Sequences and annealing temperatures for the primer sets used in RT-PCR Gene Primer sequence T m in °C Chondrocyte-specific Aggrecan 5'-GATCTGGCATGAGAGAGGCG-3' 5'-GCCACGGTGCCCTTTTTAC-3' 61 Collagen type II 5'-GCTGCTGACGCTGCTCATC-3' 5'-GGTTCTCCTTTCTGCCCCTT-3' 60 Collagen type X 5'-CAAGCCAGGCTATGGAAGTC-3' 5'-AGCTGGGCCAATATCTCCTT-3' 60 Link protein 5'-TTCTGGGCTATGACCGCTG-3' 5'-AGCGCCTTCTTGGTCGAGA-3' 60 Biglycan 5'-CATGACAACCGTATCCGCAA-3' 5'-ATTCCCGCCCATCTCAATG-3' 60 Decorin 5'-ATGACCCTGACAATCCCCTG-3' 5'-CCCAGATCAGAACACTGCACC-3' 60 Scleraxis 5'-GGACCGCAAGCTCTCCAAG-3' 5'-ACCCACCAGCAGCACATTG-3' 62 Sox9 5'-GCAGACCAGTACCCGCATCT-3' 5'-CTCGCTCTCGTTCAGCAGC-3' 62 Adipocyte-specific ADD1 5'-CAGTGACTCTGAGCCCGACA-3' 5'-ATGCCTCGGCTATGTGAAGG-3' 61 PPARγ 5'-ATCATCTACACGATGCTGGCC-3' 5'-CTCCCTGGTCATGAATCCTTG-3' 59 SCD1 5'-ACACCATGGCGTTCCAAAAT-3' 5'-CGGCGTGTGTTTCTGAGAACT-3' 61 C/EBPα 5'-CGCAAGAGCCGAGATAAAGC-3' 5'-GCGGTCATTGTCACTGGTCA-3' 60 GLUT-4 5'-ATGGCTGTCGCTGGTTTCTC-3' 5'-ACCCATAGCATCCGCAACAT-3' 59 AP2 5'-TGATGCCTTTGTGGGAACCT-3' 5'-GCAAAGCCCACTCCCACTT-3' 58 acrp30 5'-AAGAAGGACAAGGCCGTTCTC-3' 5'-GAGGAGCACAGAGCCAGAGG-3' 60 LPL 5'-CCAATGGAGGCACTTTCCAG-3' 5'-CCACGTCTCCGAGTCCTCTC-3' 60 Forward primer sequence is depicted from the 5' to the 3' end followed by the reverse primer. The appropriate annealing temperature for each set is given as T m in °C. Quantification of chondrocyte yield by FACS The GFP expressing plasmid pEGFP-1 (Clontech) under the control of the Aggrecan promotor (kind gift of John R. Matyas) was stably transfected into D3 ES cells using Invitrogen's Effectene system followed by neomycin selection. Genomic integration of the reporter was analyzed by RT-PCR with specific GFP primers (data not shown). ES cells were differentiated along the chondrocyte lineage, trypsinized into a single cell suspension and subjected to fluorescence-activated cell sorting on day 32 using a FACS Calibur instrument and the CellQuest software from Becton Dickinson (Germany). Ten thousand events were registered per sample and analysis of whole cells was performed using appropriate scatter gates to avoid cellular debris and aggregates. Oil-Red-O staining Cells were washed with PBS and without any fixation directly overlaid with Oil-red-O working solution (0.18% Oil-Red-O dye/60% propanol) After a staining period of 15 minutes, ES cell cultures were rinsed in distilled water until desired colour was achieved. Statistical analysis Data analysis was performed with ONE-WAY ANOVA on n = 3 experiments using SigmaStat version 2.03 (SPSS Inc., San Rafael, CA, USA). List of abbreviations ADD1 Adipocyte determination- and differentiation-dependent factor 1 aP2 Fatty acid-binding protein BMP Bone Morphogenetic Protein Cbfa1 Core binding factor alpha 1 C/EBP CCAAT/enhancer-binding protein Col Collagen DMMB Dimethylmethylenblue ECM extracellular matrix ES embryonic stem EB embryoid body FCS Fetal calf serum FGF Fibroblast growth factor GAPDH Glyceraldehyde-3-phosphate dehydrogenase GLUT4 Glucose transporter 4 IGF Insulin-like growth factor PPAR Peroxisome proliferator-activated receptor SCD Steroyl CoA desaturase Sox Sry-related high mobility group box TGFβ Transforming Growth Factor beta VD 3 Vitamin D 3 Authors' contributions NZN carried out cell culture, biochemical and molecular studies and drafted the manuscript. GK, DER and HJA participated in its design and coordination. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC548146.xml
514611	Interval exercise versus continuous exercise in patients with moderate to severe chronic obstructive pulmonary disease – study protocol for a randomised controlled trial [ISRCTN11611768]	Background Physical exercise has become a cornerstone of management of chronic obstructive pulmonary disease (COPD) because it leads to clinically relevant improvements of exercise capacity and health-related quality of life (HRQL). Despite the scarcity of randomised trials directly comparing exercise protocols, current guidelines recommend high intensity continuous exercise for lower extremities as the probably most effective exercise modality. However, for patients admitted to inpatient respiratory rehabilitation programmes, it is often difficult to initiate such an exercise programme because they are severely limited by dyspnoea and leg fatigue and therefore unable to perform continuous exercise at higher intensities and for periods longer than 30 minutes. Interval exercise may be an attractive alternative for these COPD patients because it allows high intensity exercise with recovery periods. The aim of this study is to assess if interval exercise compared to high intensity continuous exercise is not of inferior effectiveness in terms of HRQL and exercise capacity improvements but associated with better exercise tolerance in patients with moderate to severe COPD at the beginning of a respiratory rehabilitation. Methods/Design We will assign patients with moderately severe to severe COPD to either continuous exercise or interval exercise using a stratified randomisation. Patients will follow 12–15 exercise sessions during a comprehensive inpatient respiratory rehabilitation. Primary end point for effectiveness is HRQL as measured by the Chronic Respiratory Questionnaire (CRQ) two weeks after the end of rehabilitation and secondary endpoints include additional clinical outcomes such as functional exercise capacity, other HRQL measures, patients' experience of physical exercise as well as physiological measures of the effects of physical exercise such as cardiopulmonary exercise testing. Including expected drop-outs, we will need 52 patients per group to show differences corresponding to the minimal clinically important difference of the CRQ. Outcome assessors and investigators involved in data analysis will be blinded to group assignment until analyses have been carried out. Discussion Clinicians and the scientific community need evidence on the benefits and tolerance of exercise protocols available in clinical practice. The proposed trial will provide important and needed data on interval and continuous exercise for decision making in clinical practice.	Background Impaired exercise capacity, dyspnoea and reduced health-related quality of life (HRQL) are common complaints of patients with chronic obstructive pulmonary disease (COPD). A major exercise-limiting factor in COPD is peripheral muscle dysfunction characterised by atrophic muscles and reduced fatigue resistance due morphological and metabolic alterations of peripheral muscles[ 1 ] As much as 70% of COPD patients may be affected by peripheral muscle dysfunction.[ 2 ] Respiratory rehabilitation with physical exercise improves exercise capacity and HRQL.[ 3 ] Although physical exercise is a mandatory component of respiratory rehabilitation programmes[ 4 , 5 ], there is an ongoing debate about what type of exercise at which exercise intensity patients should perform.[ 1 , 6 ] There is substantial variation in exercise protocols used in practice[ 7 ] as well as in clinical trials[ 3 ]. Current guidelines recommend continuous exercise at high intensity for lower extremities[ 4 , 5 ] because a study indicated that high intensity may be more effective than low or moderate intensity.[ 8 ] However, data on high intensity continuous exercise come from a trial that included 19 patients with mild COPD who were able to exercise for 45 minutes five times per week during an outpatient programme.[ 8 ] For patients who need to be admitted to inpatient programmes because of more severe COPD and/or unstable health state, it is difficult to perform high intensity exercise and exercise sessions longer than 30 minutes because they are limited by dyspnoea and leg fatigue. Less than 20% may be able to sustain high intensity continuous exercise throughout the whole rehabilitation programme[ 9 ] To find a realistically tolerable exercise programme for these patients, who often initiate exercise programmes for the first time, is challenging. A solution to this dilemma may represent interval exercise[ 6 ] where patients exercise alternatively at high intensity and at low intensity, which allows short periods of recovery. Consequently, interval training may be better tolerated than high intensity continuous training. In addition, patients may be able to achieve a greater training load during the relatively short exercise sessions they can sustain. Patients and clinicians will accept interval exercise to treat peripheral muscular dysfunction in COPD only if it is not of inferior effectiveness compared with continuous exercise and if it is indeed associated with better compliance resulting from less dyspnoea and leg fatigue during exercise. There is limited evidence from three randomised controlled trials comparing interval exercise and continuous exercise.[ 10 - 12 ] A summary of these trials can be found in table 1 . Table 1 Trials on interval exercise in patients with COPD Population Exercise protocols and rehabilitation program Main results Coppoolse 1999 [10] 21 stable male COPD patients (mean age 65 years, FEV1 36.8% predicted) Group 1 : CT ergometer cycling at 60% of Wmax Group 2 : IT ergometer cycling at 90% of Wmax (1 min) and 45% of Wmax (2 min) 3 days/week plus CT ergometer cycling at 60% of Wmax 2 days/week 8 weeks inpatient rehabilitation with 5 exercise sessions per week of 30 min. No additional physical exercise. Significant increase of V O2 and decrease of minute ventilation with CT but no changes with IT. Significant increase of Wmax and decrease of leg pain during exercise with IT but not with CT. Only significant differences between CT and IT for V O2 /Wmax favouring CT. 91% of patients with CT and 90% of patients with IT completed the exercise program. Vogiatzis 2002 [11] 45 stable COPD patients (62% males, (mean age 65 years, FEV1 34.1% predicted) Group 1 : CT ergometer cycling at 50% of Wmax weeks 1–4, at 60% weeks 5–8 and at 70% weeks 9–12 Group 2 : IT ergometer cycling at 100% of Wmax (30 sec) and 45% of Wmax (30 sec) weeks 1–4, at 120% weeks 5–8 and at 140% weeks 9–12 12 weeks outpatient rehabilitation with 2 exercise sessions per week of 40 min. No additional physical exercise. Significant improvements of CRQ scores and Wmax and reductions of minute ventilation during CWRT in both groups. No significant differences between groups. Attendance rate for exercise sessions 88% for CT and 90% for IT. Kaelin 2001 [12] 19 stable COPD patients (89% males) (mean age 67 years, FEV1 26.9% predicted) Group 1 : CT walking on stepper (70 steps/minute) or treadmill (1.5 miles/hour). Increase of 1 MET every 2 weeks Group 2 : IT walking on stepper (70 steps/minute) or treadmill (1.5 miles/hour) with active rest to ratio of 2:1. Increase of 1 MET every 2 weeks 6 weeks outpatient rehabilitation with 3 exercise sessions per week of 10–30 min. Additional resistance training and flexibility training. Larger improvements of 6-minute walking distance with IT (80 meters) compared with CT (39 meters). No data on compliance. CT = Continuous training; IT = Interval training; CRQ = Chronic Respiratory Questionnaire Wmax = Maximum exercise capacity, measured by usual incremental exercise test; CWRT = Constant work rate test; V O2 = Maximum oxygen consumption MET = Metabolic equivalent The studies indicated that both interval and continuous training improved exercise capacity, dyspnoea and HRQL and showed insignificant differences between interval and continuous exercise. However, insignificant differences do not allow concluding that interval or continuous are of clinically equivalent effectiveness[ 13 ] These trials were too small to show clinical equivalence or non-inferiority and they did not provide evidence on the tolerance of these two exercise modalities. From a methodological point of view, the trial had several shortcomings because, for example, they did not provide details on concealment of random allocation or blinding of outcome assessors. In addition, in the trial with an inpatient rehabilitation.[ 10 ], patients of the interval exercise group had a mixed intervention (3 days of interval and 2 days of continuous exercise per week) so that differences can hardly be attributed to different interventions if they are detected at all. The investigators did not use steep ramp tests to determine exercise loads but normal incremental exercise tests. For interval exercise, muscle strength and anaerobic capacity is relevant because of the short high intensity intervals, but this is not measured by normal incremental exercise tests. In addition, training load tolerated during interval exercise may be underestimated when normal incremental exercise tests are used.[ 14 ] Exercise tests to establish training intensity should consider the exercise mode. Meyer et al. studied interval exercise in several studies [ 14 - 16 ] in patients with chronic heart failure who show similar patterns of physical deconditioning in terms of clinical manifestation as well as morphological and metabolic abnormalities [ 17 - 19 ] Meyer et al. used a steep ramp test to determine short time muscular maximum exercise capacity[ 14 ], which reflects muscle strength and anaerobic capacity, both relevant for interval exercise. Meyer et al. also assessed different ratios of work/recovery phases (1:2; 1:4 and 1:6) and found that with these ratios and relatively short phases of high intensity exercise (10–30 seconds), lactate did not accumulate presumably because of lactate elimination during the recovery phases.[ 14 ] Interval exercise with these ratios was therefore recommended as high intensity aerobic exercise modes for patients who do not sustain continuous exercise. Because of the scarcity of evidence on the comparative effectiveness of interval exercise for COPD patients, additional trials are needed.[ 1 , 6 ] Our primary objective is to assess if interval exercise is not of inferior effectiveness compared to continuous exercise of high intensity to improve HRQL and exercise capacity in patients with moderately severe to severe COPD and the secondary objective is to evaluate if interval exercise is better tolerated by COPD patients. Methods Study design (see figure 1 ) Figure 1 Flow of the study from screening for eligible patients to the final outcome assessment. All consecutive patients admitted to a teaching rehabilitation clinic for an inpatient respiratory rehabilitation (Klinik Barmelweid, Barmelweid, Switzerland) will be assessed for study eligibility by senior staff physicians. If patients are deemed eligible after exercise testing, which is part of the usual rehabilitation program, senior physicians will inform patients about the study orally and in writing. If patients are willing to participate and provide written informed consent they will be randomly assigned to respiratory rehabilitation with either interval or high intensity continuous exercise. Both groups will perform 12–15 exercise sessions and follow the rest of the rehabilitation programme. Follow-up assessments will be done during, at the end of the rehabilitation programme as well as two and twelve weeks afterwards when patients are back in their home environment. The Ethikkommission of the Kantonsspital Aarau, Aargau, Switzerland, has approved the study protocol. Patients We defined the following in- and exclusion criteria: Patients with COPD as defined by FEV1/FVC < 70% predicted, FEV1 < 50 % predicted after bronchodilation, with or without chronic symptoms (cough, sputum production) corresponding to a GOLD (Global Initiative for Chronic Obstructive Lung Disease) stage III-IV[ 20 ] and German as first or daily language. Exclusion criteria are arrhythmia (atrial flutter and fibrillation, ventricular tachycardia, premature beats > 8 per minute), ischemia during exercise testing, clinically decompensated Cor pulmonale or heart failure, untreated neoplasia or neoplasia that needed treatment within the previous two years, lung surgery within the previous three months, orthopedic, rheumatologic, vascular or neurological disorders that inhibit ergometer training, gymnastic or guided walking tours, and patients unable to perform or complete the six-minute walk test or the incremental cycle test Randomisation A third party not involved in the conduction of the trial will provide online central randomisation (DatInf GmbH, Tuebingen, Germany). A computerised 'minimisation' procedure will be used to avoid chance baseline imbalances in prognostically important variables[ 21 ] Minimisation variables will be exercise capacity (< 300 or ≥ 300 meters in six-minute walk test), the presence of affective disorders (Hospital Anxiety Depression Scale scores < or ≥ 8), status of COPD (unstable COPD = In- or outpatient medical care in the last eight weeks due to exacerbation of COPD versus stable COPD = no in- or outpatient medical care in the last eight weeks due to exacerbation of COPD) and the need for oxygen at rest (yes = long term home oxygen therapy or paO 2 < 55 mmHg or no = paO 2 ≥ 55 mmHg). Every time the study coordinator has been informed about an enrolled patients, he will enter the randomisation web site[ 22 ] enter the patient data required for randomisation (patient identification and stratification variables) and obtain group allocation. The study coordinator will then inform responsible physical therapists about group allocation. No other medical staff will have knowledge about group allocation. The randomisation provider will also send an e-mail to the study coordinator for each randomised patient with details on randomisation. This will ensure correct verification of group allocation after data analysis. Separating patient enrolment and baseline assessments (physicians and physical therapists) from the randomisation procedure (study coordinator not involved in patient enrolment or rehabilitation programme) will ensure concealment of random allocation. Interventions The rehabilitation programme will start one day after baseline assessments and study enrolment. Exercise sessions and group lessons will take place five days a week and will consist of daily cycle ergometer training, breathing therapies (30 minutes per day), and guided walking of 15 to 30 minutes. Relaxation therapies (technique according to Jacobson) take place twice a week, patient education (information about COPD, coping strategies, inhalation techniques) three times a week, smoking cessation advice once a week or more if needed. Apart from physical exercise, the rehabilitation programme of approximately three weeks will be identical for both groups. Group performing continuous exercise The target workload for this group will be ≥ 70% of the maximum exercise capacity expressed in Watts and heart rate achieved during the incremental cycle ergometer test. Patients are usually not able to perform high intensity continuous exercise from the beginning and have to adapt to physical exercise. Physical therapists will increase training load as soon as possible to ≥ 70% of the maximum exercise capacity or as high as each individual patient tolerates. In each session (see figure 2 ), patients will have a warm-up period of two minutes at 20% of maximum exercise capacity, increase the exercise intensity within two minutes to the target intensity, exercise for 20 minutes at high intensity and then have a decreasing period of two minutes (gradual decrease from 70% to 0%). Pulse oxymetry will be used to supervise patients during exercise. If oxygen saturation falls below 90%, oxygen supplementation will be provided to maintain ≥ 90%. Figure 2 The upper graph shows the continuous exercise protocol for a patient who achieved a maximum exercise capacity of 100 Watts during a usual incremental exercise test. The lower graph shows the interval exercise protocol for a patient who achieved a short time muscular exercise capacity of 200 Watts during a steep ramp test. If patients cannot sustain the workload because of perceived dyspnoea or leg fatigue or because the heart rate exceeds the limits determined during exercise testing, physical therapists will let patients rest for one minute and then resume exercise. If patients have to rest more than twice per session, physical therapists will lower the workload by steps of 10% of baseline maximum exercise capacity. In turn, if patients or physical therapists consider the workload to be too low or if patients do not reach their target heart rate at 70% of the maximum exercise capacity, physical therapists will increase workload by steps of 10% of maximum exercise capacity until patients and physical therapists consider the workload to be appropriate or until the target heart rate is reached. Group performing interval exercise Patients assigned to this group will perform a steep ramp test to determine the short time muscular maximum exercise capacity.[ 14 ] The steep ramp test is an incremental cycle ergometer test where patients pedal unloaded for 2 minutes and then pedal with increments of 25 Watts every 10 seconds until they cannot maintain a pedaling frequency above 50 per minute or above the heart rate limit set by the normal incremental exercise test (which all patients perform irrespective of group assignment). With the steep ramp test measurement of muscular maximum exercise capacity is possible because the tests lasts only for 30 to 120 seconds and patients are not limited by symptoms. Measuring muscular maximum exercise capacity is important to set the exercise load for interval exercise because the high intensity interval requires also muscle strength beside overall exercise endurance.[ 14 ] Patients should improve both endurance and muscle strength during interval exercise because both are required in daily activities. We therefore chose a work/recovery ratio of 1:2 that prevents from high lactate accumulation.[ 14 ] From short time maximum exercise capacity, we will derive the initial work rate for interval exercise (in Watts), which is set at 50% of the short time muscular maximum exercise capacity as measured by the steep ramp test. This workload corresponds in Watts approximately to 90–100% of the workload as measured by the normal incremental exercise test.[ 16 ]. Patients will start with exercise the day after the steep ramp test. Patients will perform interval exercise for twelve to fifteen sessions with a cycle ergometer. In each session, they will have a warm up period of two minutes at 20% of the short time maximum exercise capacity (figure 2 ). Then they exercise for 20 minutes at high intensity intervals of 20 seconds at 50% and at low intensity intervals of 40 seconds at 20% of the short time maximum exercise capacity, i.e. with a work/recovery ratio of 1:2. Then patients have a slow down period of two minutes before completion of the training session. Pulse oxymetry will be used and oxygen supplementation will be provided as described above. If patients cannot sustain exercise intensity because the heart rate exceeds the limits determined after exercise testing or because of perceived dyspnoea or leg fatigue, physical therapists will let patients rest for one minute and then resume exercise. If patients have to rest more than twice per session, physical therapists will lower the workload from 50% of the short time maximum exercise capacity by steps of 10% while the length of intervals remains constant. They will increase the training load again as possible for the patient. In turn, if patients or physical therapists consider the workload to be too low, physical therapists will increase workload of the high intensity interval by steps of 10% until patients and physical therapists consider the workload to be appropriate while the length of intervals remains constant. Clinical outcome measures Chronic Respiratory Questionnaire (CRQ) We will use the CRQ[ 23 ] to measure HRQL changes during rehabilitation. The CRQ is a widely used disease-specific instrument to assess symptoms of COPD patients [ 24 - 26 ] We will use the self-administered German CRQ.[ 27 , 28 ] with standardised dyspnoea questions.[ 29 ] that we have developed and validated in earlier studies. Patients will complete the CRQ in the Klinik Barmelweid at baseline, at the end of the rehabilitation, two and 12 weeks thereafter when they have returned to their home environment. HADS (Hospital Anxiety Depression Score) Affective disorders are common in patients with COPD and contribute to reduced HRQL[ 30 ] The HADS has been developed to assess symptoms of anxiety and depression in patients with physical impairment.[ 31 ] There are seven items for each domain (anxiety and depression) with statements on emotions and emotional situations. Patients express their agreement with the statements on a scale from 0 to 3. Domain scores are calculated by summing up the scores for the seven domains resulting in scores from 0 (no depression or anxiety) to 21 (depression or anxiety very likely to be present). Scores ≥ 8 indicate that there is an increased probability for the presence of an affective disorder. We will use the validated self-administered German version of the HADS.[ 32 ] The HADS will be completed in the Klinik Barmelweid at baseline, at the end of the rehabilitation, two and 12 weeks thereafter when they have returned to their home environment. Feeling Thermometer (FT) We will use a validated visual analogue scale, the FT[ 33 ], an increasingly used instrument for a global estimate of the effect of interventions, including respiratory rehabilitation[ 29 , 34 ]. The FT is a visual analogue scale presented as a thermometer with 100 marked intervals. The worst (dead = 0) and best (perfect health = 100) health states are defined anchors and facilitate comparisons between individuals and groups.[ 35 ] We will ask patients to reflect in their score how they felt in the last 7 days. The FT will be completed in the Klinik Barmelweid at baseline, at the end of the rehabilitation, two and 12 weeks thereafter when they have returned to their home environment. Six-minute walk test We will use the six-minute walk test to assess functional exercise capacity according to established criteria.[ 36 ] At baseline, patients will perform the six-minute walk test twice one day apart. We will use the results of the second six-minute walk test because the first test tends to underestimate exercise capacity due to unfamiliarity with the test.[ 36 ]. At the end of the rehabilitation and two weeks after completion of rehabilitation programme patients will perform additional six-minute walk tests under supervision of physical therapists blinded to group assignment. In addition, we will use a paperboard with a modified Borg scale on from 0 to 10 with verbal labels for 0 (no dyspnoea at all), 1–5, 7 and 10 (maximal dyspnoea) to assess the intensity of perceived dyspnoea at the end of the six-minute walk test. Monitoring of exercise sessions Dyspnoea and leg pain during exercise In each session, we will use modified Borg scale as described above to assess the intensity of perceived dyspnoea and leg pain after five minutes of exercise and at the end of the exercise sessions. Subjective experience of exercise There is no instrument available to assess the subjective experience of COPD patients with exercise, which is likely to influence compliance. We have developed a questionnaire using established methodology[ 37 ] to assess how patients experienced the sessions. The questionnaire (Exercise Tolerance Questionnaire) consists of five questions addressing the exercise limitation by shortness of breath and difficulties with breathing, leg fatigue, fatigue in general and too high exercise load. In addition, one question asks patients how they experienced the exercise session in general (from very enjoyable to very unpleasant). The report on questionnaire development will be published elsewhere. Adherence to and tolerance of the prescribed cycle ergometer exercise Physical therapists will record for every cycle ergometer exercise session if patients exercised at all (yes or no), the performed workload (in Watts), if patients reached the target workload (in Watts), adjustments of workload and the requirement for oxygen. We define adherence to exercise as "full adherence" if patients follow at least 12 exercise sessions. We consider the training to be fully tolerated if patients are able to follow the exercise protocol for at least 9 exercise sessions for continuous exercise (taking into consideration the first three exercise session when patients increase training load up to 70% of maximum exercise capacity) and for at least 12 exercise sessions for interval exercise. Adverse events Previous trials of respiratory rehabilitation or physical exercise did not report any adverse events. Nevertheless, we will record any adverse events such as injuries, cardiac events or increase of respiratory symptoms as they happen during the inpatient respiratory rehabilitation. Cardiopulmonary exercise testing At baseline and end of the rehabilitation, all patients will perform an incremental cycle ergometer test to the limit of tolerance (symptom based) under the supervision of a senior physician blinded to group assignment. Patients pedal unloaded for three minutes to warm up at 20 Watts. Then exercise load is increased by 7.5 Watts per minute until patients have to stop because of dyspnoea, leg pain, or criteria for stop testing (atrial or ventricular tachycardia, ischemia, hypoxemia). At the limit of tolerance, we will draw capillary blood samples to measures lactate concentrations and we will set the maximum exercise capacity expressed by Watts. The upper limit for the heart rate during exercise is set as the heart rate measured by the electrocardiogram at the maximum exercise capacity. During testing, we will record gas exchange and ventilatory variables form calibrated signals derived from rapidly responding gas analyzers and a mass flow sensor. We will record breath by breath the following variables: Pulmonary oxygen uptake, pulmonary CO 2 output, minute ventilation, tidal volume and respiratory frequency. All patients will perform a steep ramp test at the beginning and end of the rehabilitation programme as described above. Additional data to be collected In order to characterize the patient included in the study, we will record their age, gender, lung function (FEV1, FEV1/FVC, diffusion capacity DLCO/VA, weight, height, smoking status at the beginning and end of rehabilitation as well as two and 12 weeks afterwards, duration of disease (time since diagnosis), co-morbidities such as hypertension, heart diseases, endocrine disorders, chronic rheumatological disorders and psychiatric disorders. Data analysis The randomisation code will not be broken (investigators remain blinded to group assignment and will receive only codes, such as group 1 and 2) until a draft of the manuscript has been written. The authors will write two versions with alternative possible allocation patterns to avoid bias in the interpretation of the results. This approach is methodologically rigorous and limits introduction of bias during the interpretation of the data that some of the authors might have.[ 38 ] We will submit the appropriate manuscript regardless of the results of unmasking. After agreement on the final versions of the two articles, we will break the randomisation code and we will submit the correct version of the manuscript. Effectiveness Our null hypothesis is that high intensity continuous exercise is of clinically superior effectiveness compared with interval exercise to improve HRQL (δ ≥ 0.5 in CRQ domains scores). The alternative hypothesis is that interval exercise is not of clinically inferior effectiveness compared with high intensity continuous exercise. In the primary analysis, we will calculate the raw difference and 95% confidence intervals between groups in the mean follow-up score for the CRQ domains 2 weeks after completion of the respiratory rehabilitation programme. In an additional analysis, we will adjust for the base-line score and the four stratification variables using an analysis of covariance. We will use independent t-test to compare the change scores between groups. We will also use the confidence interval approach as recommended for equivalence and non-inferiority trials[ 13 ] We will establish non-inferiority of interval exercise if the point estimate and its 95% confidence interval for difference between the change scores of the continuous and interval exercise group is smaller than the a priori determined boundary of clinical equivalence (see figure 3 ). If the 95% confidence intervals lie outside the boundaries of clinical equivalence we will establish clinical superiority of one exercise protocol. Figure 3 Illustration of the confidence interval approach to interpret results from randomised trials. The horizontal line indicates the difference between CRQ change scores between study groups. ± 0.5 points represent the predefined boundaries of equivalence. If the whole confidence interval is on the right of 0.5 points, interval exercise is not inferior to continuous exercise. If the whole confidence interval is within boundaries the two exercise protocols are of clinically equivalent effectiveness (upper three confidence intervals). Note that there can be a statistically significant difference between study groups but without any clinical relevance. Most methodologists and statisticians agree that the boundaries of equivalence should be defined as the minimal important difference and below the differences observed in previous trials comparing active to control treatments.[ 13 , 39 ] The minimal important difference is "the smallest difference in score in the outcome of interest that informed patients perceive as important, either beneficial or harmful, and which would lead the clinician to consider a change in the management"[ 40 ]. Therefore, the minimal important difference of our two main outcome measures for effectiveness have been established empirically and are around 0.5 for the CRQ domains scores [ 41 - 44 ] and 53 meters for the six-minute walk test[ 45 ]. A recent meta-analysis showed that respiratory rehabilitation with physical exercise leads to improvements of 50 meters in the six-minute walk test. Therefore we lower the boundaries for equivalence to ± 45 meters in order to have boundaries that are below the differences observed in previous trials comparing active to control treatments.[ 13 , 39 ] We will repeat the analysis with calculations of raw and adjusted differences for all other clinical and physiologic outcome measures and test for significant differences between groups using independent t-test if data are distributed normally. We will use both intention to treat and per protocol analysis to show equivalence in either case as recommended by others[ 13 , 39 , 46 , 47 ] Exercise tolerance Our null hypothesis is that patients equally experience high intensity continuous exercise and interval exercise as measured by the Exercise Tolerance Questionnaire. The alternative hypothesis is that interval exercise is associated with the experience of less limiting symptoms compared with high intensity continuous exercise. We will use independent t-tests to compare the measures for exercise tolerance (Exercise Tolerance Questionnaire, Borg scales for dyspnoea and leg pain) between groups. Again, we will assess the raw differences between groups in the primary analysis and adjust for baseline scores and the four stratification variables in the additional analysis. Confounder variables and effect modifiers Factors, which interfere with outcome measures, can distort the results if unevenly distributed between study groups. We use three approaches to control for confounders: First, we use randomisation to allocate patients. Second, we strengthen the randomisation using a computerised minimisation procedure with factors that are likely to influence the outcome measures (exercise capacity, pulmonary state and presence and absence of affective disorders). Third, we will collect a number of variables at baseline (age, gender, lung function, time since diagnosis, cumulative dose of oral steroids in previous three months, medication, cardiovascular, musculoskelettal and endocrine co-morbidities) that may modify the effect of exercise. We will compare the distribution of these potential effect modifiers between groups and statistically assess their influence on the outcome measures with multiple linear regression models. Sample size For calculating the required sample size, we use the formula for comparison of 2 means: n = [A + B] 2 * 2 * SD 2 /DIFF 2 , where n = the sample size required in each group (double for total sample), SD = standard deviation of the outcome variable, DIFF = size of desired difference between groups. A and B depend on the desired significance level and desired power, respectively. We base our sample size calculations on the CRQ. We used empirical data from our previous trial.[ 27 ], where we applied similar inclusion criteria for patients with COPD undergoing respiratory rehabilitation, to estimate variability of the CRQ (standard deviation of the CRQ domain scores between 0.8 and 1.2). A clinically sensible way to determine the size of desired difference between groups (DIFF in formula above) is based on the minimal important difference (0.5 on the scale from 1 to 7 for the CRQ)[ 41 - 43 , 48 ] A sample size of 44 patients in each group will allow showing a difference of 0.5 in CRQ scores between the groups, assuming a standard deviation of 0.8, with a power of 90% at a significance level of 5% (one-sided). Assuming a drop out rate of 15%.[ 27 ], the total minimal sample size increases to 104. This sample size will also allow detecting a difference of 45 meters in the six-minute walking test scores between the two treatment groups with a power of 95% at a significance level of 5% (one-sided) assuming a drop out rate of 15%. A priori sample size calculations usually provide only rough estimates. Therefore, we will recalculate sample size during the study when we have the data for 30 patients in each group. To this end, we will re-assess the standard deviation of the outcome variables and, if necessary, adjust the sample size accordingly (without breaking the randomisation code). Data collection and quality control We will implemented a series of measures to ensure high data quality. 1. Site investigators will collect the data using standardized forms. All data will be collected and entered into a single database in the Horten Centre by one investigator. A second investigator will validate completeness and accuracy of data extraction by checking 20% of extracted patient data. 2. Checklists with all the data to be collected will be provided for physical therapists and physicians. 3. Teaching sessions will be held regularly for physical therapists and physicians involved in data collection aiming at robust data collection. 4. Investigators meetings will be held monthly, or more frequently if needed, to discuss recruitment of patients, problems in conducting the study, acquisition of data, to check for consistency and completeness of data and for interim analyses. 5. E-mail will serve as the first line of non-urgent communication between research team members. 6. Monthly reports will be prepared by the principal investigator that will include the number of patients recruited, stage of follow up for each patient, notification regarding missing patient data and queries from data received. Discussion In the last 30 years, researchers made great efforts to study the effectiveness of respiratory rehabilitation compared to usual care. The meta-analyses of a recent systematic review[ 3 ] showed that respiratory rehabilitation with physical exercise leads to clinically significant improvements of HRQL as well as to significant improvements of functional and maximum exercise capacity. Research in respiratory rehabilitation should now focus on the evaluation of different exercise protocols. When an effective treatment such as respiratory rehabilitation is available, patients and clinicians are not confronted with the decision whether to start treatment or not, but with the decision on the most appropriate treatment. Therefore, clinicians need evidence from pragmatic randomised controlled trials directly comparing different exercise protocols at issue rather than evidence from (explanatory) trials comparing exercise with no exercise or usual care.[ 49 ] The proposed pragmatic trial will therefore provide important and needed guidance for decision-making in respiratory rehabilitation, in particular for those COPD patients who are severely impaired and initiate respiratory rehabilitation programmes. The evidence generated in the proposed clinical trial will also be relevant for the scientific community. From the 1970s up to the mid-1990s most investigators conducted explanatory clinical trials in order to better understand how and why physical exercise is effective in patients with COPD. After the recognition of its effectiveness the debate on the optimal exercise modality arose[ 6 ]. However, only few pragmatic trials have been conducted so far to advance the understanding of the relative benefit and downsides of different exercise protocols. With the proposed trial, we compare two clinically relevant interventions with the use of a clinical trial design that is useful for clinical decision making. Such trials are currently needed to gain consensus on the optimal exercise protocol. There is a need for randomised controlled trials to explore which exercise protocols are most effective for COPD patients. Another topic that has received little attention in respiratory rehabilitation trials so far is compliance to and subjective experience of physical exercise[ 9 ] Exercise protocols and adherence to it have not been described in detail in published studies[ 3 ] and therefore very little is known about the tolerance of different training modalities. This is despite the fact that physical exercise has long been considered unfeasible in patients with COPD. Beside the paucity of data on the effectiveness and tolerance of different exercise protocols, there is also a need for trials that are methodologically sound and rigorous. Most of the studies on exercise or respiratory rehabilitation in patients with COPD did not report on details of exercise tests and protocols. In addition, study design related issues that introduce bias such as description of the randomisation procedure, concealment of allocation, sample size calculations or blinding have not been addressed frequently. Therefore, we try to use strong epidemiological methods to minimize bias in our trial. Our study could make an important contribution to the understanding of physical exercise in patients with COPD and have a significant impact on the structure of respiratory rehabilitation and exercise programmes. Competing interests None declared. Abbreviations COPD = Chronic obstructive pulmonary disease HRQL = Health-related quality of life CRQ = Chronic Respiratory Questionnaire HADS = Hospital Anxiety Depression Score FT = Feeling Thermometer Authors' contributions MP drafted and revised the manuscript. All authors participated in development of research protocols and in the design of the study. MP, CZ and HS resolved statistical and methodological issues. All authors read and corrected draft versions of the manuscript and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC514611.xml
516774	The aminoguanidine carboxylate BVT.12777 activates ATP-sensitive K+ channels in the rat insulinoma cell line, CRI-G1	Background 3-guanidinopropionic acid derivatives reduce body weight in obese, diabetic mice. We have assessed whether one of these analogues, the aminoguanidine carboxylate BVT.12777, opens K ATP channels in rat insulinoma cells, by the same mechanism as leptin. Results BVT.12777 hyperpolarized CRI-G1 rat insulinoma cells by activation of K ATP channels. In contrast, BVT.12777 did not activate heterologously expressed pancreatic β-cell K ATP subunits directly. Although BVT.12777 stimulated phosphorylation of MAPK and STAT3, there was no effect on enzymes downstream of PI3K. Activation of K ATP in CRI-G1 cells by BVT.12777 was not dependent on MAPK or PI3K activity. Confocal imaging showed that BVT.12777 induced a re-organization of cellular actin. Furthermore, the activation of K ATP by BVT.12777 in CRI-G1 cells was demonstrated to be dependent on actin cytoskeletal dynamics, similar to that observed for leptin. Conclusions This study shows that BVT.12777, like leptin, activates K ATP channels in insulinoma cells. Unlike leptin, BVT.12777 activates K ATP channels in a PI3K-independent manner, but, like leptin, channel activation is dependent on actin cytoskeleton remodelling. Thus, BVT.12777 appears to act as a leptin mimetic, at least with respect to K ATP channel activation, and may bypass up-stream signalling components of the leptin pathway.	Background ATP-sensitive K + (K ATP ) channels are important regulators of cell function, coupling energy metabolism with electrical activity. K ATP channels are comprised of two proteins, derived from the sulphonylurea receptor (SUR) family and an inwardly rectifying K + channel (Kir6.x family), the exact composition of these being dependent upon tissue [ 1 , 2 ]. For example, pancreatic β-cells and insulin-secreting clonal cell lines express K ATP channels consisting of Kir6.2 and SUR1 subunits [ 3 ]. K ATP channels are present in numerous tissues and are the target for drugs that inhibit or increase channel activity [ 4 , 5 ]. The archetypal inhibitors of these channels are the sulphonylurea class of drugs, which bind to the SUR subunit of the channel. Modulation of K ATP channel activity in pancreatic β-cells has profound effects on insulin secretion and glucose homeostasis [ 6 ]. Sulphonylureas such as tolbutamide and glibenclamide inhibit channel activity, resulting in β-cell depolarization, increased electrical activity, enhanced calcium entry and consequently increased insulin secretion [ 7 ]. In contrast, pancreatic β-cell K ATP channel activation induces hyperglycaemia in animals and man [ 8 ]. This latter action is caused by membrane hyperpolarization, reduction in cell excitability and decreased intracellular calcium resulting in reduced secretion of insulin. Such effects have been reported following application of the benzothiadiazine, diazoxide, which has been used on occasion to treat persistent hyperinsulinemic hypoglycaemia of infancy [ 8 ]. It has been demonstrated that diazoxide interacts with the sulphonylurea receptor subunit, SUR1, encompassing transmembrane domains 6–11 and the first nucleotide binding fold [ 9 ]. A similar conclusion has also been reached using a novel diazoxide analogue [ 10 ]. The presence of K ATP channels in many other tissues, notably muscle and central neurons, has stimulated interest in the development of novel, selective K ATP channel openers for the treatment of various diseases [ 10 , 11 ]. The ob gene product leptin has been demonstrated to activate K ATP channels in pancreatic β-cells [ 12 ] and insulin-secreting cell lines [ 13 ], consistent with a potential role in modifying insulin secretion [ 14 ]. One of the primary functions for this hormone is its role in the regulation of food intake and body weight [ 15 ]. Interestingly, leptin also activates K ATP channels of hypothalamic glucose-responsive neurones [ 16 , 17 ] indicating a possible role for this channel in the control of energy homeostasis and body weight. In addition, Kir6.2 knock-out mice have deficits in central glucose sensing leading to loss of glucose mediated feeding response and a defective hypoglycaemic compensatory response [ 18 ]. These latter findings suggest that hypothalamic K ATP channels may also be an important target for drug manipulation with respect to centrally driven control of glucose and energy homeostasis. The aminoguanidine carboxylate, BVT.12777 (Figure 1 ), is one of a series of structurally related molecules based on the anti-diabetic/anti-obesity agent 3-guanidinopropionic acid [ 19 ], which, like leptin, have been demonstrated to reduce body weight in obese diabetic ( ob/ob ) mice [ 20 ]. Here we demonstrate that BVT.12777 opens K ATP channels in the CRI-G1 insulin secreting cell line, a useful model for pancreatic β-cells [ 21 ], and for analysing the mechanism by which leptin opens K ATP channels [ 13 , 22 , 23 ]. Figure 1 Structure of BVT.12777 ([2-(hydrazinoiminomethyl)-hydrazino] acetic acid) Results BVT.12777 activates K ATP channels Under current clamp conditions with 5 mM ATP in the pipette solution to maintain K ATP channels in the closed state, the mean resting potential was -38.7 ± 1.7 mV (n = 10), similar to values reported in previous studies [ 13 , 22 ] under these recording conditions. Application of BVT.12777 (100 μM) hyperpolarized CRI-G1 cells (Figure 2A ) to -66.3 ± 2.7 mV (n = 10). Examination of the voltage-clamped macroscopic currents indicates that prior to the addition of BVT.12777 the slope conductance of the cells was 0.43 ± 0.03 nS (n = 10), and following exposure to BVT.12777 (100 μM), this increased to 3.45 ± 1.17 nS (n = 10). The reversal potential (obtained from the point of intersection of the current-voltage relationship) associated with the BVT.12777-induced conductance increase (Figure 2A ) was -78.5 ± 0.8 mV (n = 10), close to the calculated value for E k of -84 mV in this system, indicating increased K + conductance. CRI-G1 cells responded to BVT.12777 in an all or none manner, with cells undergoing full hyperpolarization and increase in conductance, at all concentrations (100 – 300 μM) examined. Such an effect has also been reported for leptin on CRI-G1 cells [ 13 ]. Removal of BVT.12777 from the bath solution did not fully recover the membrane potential and conductance to control values over the next 15–30 minutes (not shown). Application of the K ATP channel inhibitor, tolbutamide (100 μM) during BVT.12777 exposure (Figure 2A ) completely reversed the BVT.12777-induced hyperpolarization and decreased conductance, to -41.0 ± 4.8 mV (n = 5) and 0.58 ± 0.07 nS (n = 5) respectively, values indistinguishable from control (P > 0.05). These data indicate that BVT.12777 increases K ATP current in this cell line. This is demonstrated more clearly in cell-attached recordings from CRI-G1 cells, where bath application of BVT.12777 (100 μM) resulted in activation of single K ATP channel currents (Figure 2B ; n = 7). The increase in channel activity was evident within 5 minutes of drug application, was sustained over the time course of exposure (~30 minutes) and was not immediately reversed following removal of the drug. Figure 2C shows mean channel activity (N f .P o ), normalised to the control for each recording, plotted against time of exposure to BVT.12777. BVT.12777 activation of K ATP channels was demonstrated to be reversibly inhibited by 100 μM tolbutamide (n = 4; Figure 2B,2C ). Identical control experiments, in the absence of BVT.12777, resulted in no significant effect on K ATP channel activity, over a 30-minute test period (n = 8; P > 0.05). Figure 2 BVT.12777 activates a tolbutamide-sensitive K+ current A , the upper trace shows a current clamp recording of a CRI-G1 cell following dialysis with a 5 mM ATP-containing solution. In this and subsequent current clamp figures the trace begins approximately 5 min after formation of the whole-cell configuration. Application of BVT.12777 (100 μM) for the time indicated hyperpolarized the cell from -50 mV to -76 mV, an action readily reversed by tolbutamide (100 μM), which returned membrane potential to -54 mV. Washout of all drugs from the bath resulted in a membrane potential of -70 mV, indicating the lack of reversibility of BVT.12777. The lower plot is the current-voltage relationship for the voltage clamped currents. Cells were voltage clamped at -50 mV and 10 mV steps of 100 ms duration were applied every 200 ms (range -120 to -30 mV). BVT.12777 increased the membrane conductance relative to control and tolbutamide reversed this BVT.12777-induced conductance increase with a reversal potential of -78 mV. B , cell-attached recording from a CRI-G1 cell, at 10 mV applied to the recording pipette. Single channel openings are shown as downward deflections. Addition of 100 μM BVT.12777 induced an increase in channel activity (N f .P o ) from 0.17 in control to 0.31, and 1.25 at 10 and 20 minutes respectively, after BVT addition. Application of 100 μM tolbutamide induced a substantial inhibition of activity (to 0.02), which was reversed on washout of all drugs, with activity increasing to 0.74. The symbol C refers to the closed state of the channel in this and subsequent figures. C , diary plot of N f .P o against time from cell-attached experiments in the presence and absence of BVT.12777, where channel activity was calculated every 2 minutes. Each point is the mean of 4–7 separate determinations. The effect of BVT.12777 on K ATP channel activity in excised membrane patches was also examined. Recordings were made from inside-out patches in symmetrical (140 mM KCl in pipette and bath solutions) K + at a membrane potential of -40 mV. K ATP channels were identified by inhibition of channel activity following application of 100 μM MgATP to the inner membrane aspect of the patch, which reduced normalised N f P o from 1.0 to 0.23 ± 0.05 (n = 4; P < 0.05). Subsequent application of 100 μM BVT.12777, in the continued presence of MgATP, induced a gradual increase in K ATP channel activity (Figure 3 ), to levels similar to that of control (in the absence of MgATP). For example 15 minutes after 100 μM BVT.12777 application normalised mean channel activity had recovered to 1.18 ± 0.46 (n = 4). In experiments where no drug was added, K ATP channel currents, in the presence of 100 μM MgATP, did not activate spontaneously (n = 4). Figure 3 BVT.12777 activates K ATP channels in inside-out patches Continuous single channel currents recorded from an inside-out patch at a holding potential of -40 mV. Application of 100 μM MgATP reversibly inhibited channel activity by >90%, demonstrating K ATP identity. Addition of 100 μM BVT.12777, in the presence of 100 μM MgATP to the cytoplasmic aspect of the patch resulted in K ATP channel activation. N f .P o values were 2.96 (control, after first MgATP challenge), and 0.25 in the presence of MgATP, which increased to 0.72, 1.06 and 2.74 at 5, 10 and 20 minutes respectively, after BVT.12777 addition. BVT.12777 activates K ATP channels independently of PI 3-kinase activity Leptin and diazoxide hyperpolarized CRI-G1 cells, in a manner similar to that of BVT.12777 (data not shown). Leptin (10 nM) induced a hyperpolarization from a mean membrane potential of -47.6 ± 1.6 mV to -68.5 ± 1.9 mV (n = 8; P < 0.05), and application of tolbutamide (100 μM) reversed this action, returning the membrane potential to -47.5 ± 1.9 mV (n = 4). Diazoxide (200 μM) rapidly hyperpolarized CRI-G1 cells from a mean membrane potential of -49.9 ± 1.7 mV to -74.0 ± 1.5 mV (n = 6; P < 0.05), with tolbutamide (100 μM) also reversing this action, returning membrane potential to -46.9 ± 3.8 mV (n = 6). Leptin, but not diazoxide activation of CRI-G1 K ATP channels is PI3K dependent [ 22 , 23 ]. Thus, we investigated whether BVT.12777 activates K ATP channels in CRI-G1 cells by direct (like diazoxide) or indirect (like leptin) mechanisms. Pre-incubation of CRI-G1 cells (20 min) with inhibitors of PI 3-kinase, wortmannin (10 nM) or LY294002 (10 μM) had no significant effect on the mean resting membrane potential or slope conductance of CRI-G1 cells and did not prevent BVT.12777 from causing hyperpolarization and increased cell conductance (Figure 4A ). In the presence of 10 nM wortmannin, values for mean membrane potential and slope conductance were -44.3 ± 1.2 mV (n = 6) and 0.86 ± 0.10 nS (n = 5), and addition of 200 μM BVT.12777 hyperpolarized cells to -68.9 ± 0.8 mV (n = 6) with an increase in slope conductance to 3.10 ± 0.38 nS (n = 5). Identical results were obtained in the presence of 10 μM LY294002 (data not shown), with corresponding control values of -40.8 ± 2.8 mV (n = 6) and 0.79 ± 0.11 nS (n = 4), and in the presence of 200 μM BVT.12777, -67.9 ± 0.6 mV (n = 6) and 2.69 ± 0.35 nS (n = 4) for membrane potential and slope conductance respectively. In all experiments (i.e with either PI3K inhibitor) addition of tolbutamide (100 μM) recovered the membrane potential (-41.8 ± 1.5 and -34.0 ± 1.7 mV; n = 6) and slope conductance (0.89 ± 0.11 (n = 5) and 0.58 ± 0.06 (n = 4) nS) for wortmannin and LY294002 respectively, to values indistinguishable from controls (P > 0.1). Cell-attached recordings from CRI-G1 cells also show that wortmannin (10 – 100 nM) did not occlude BVT.12777 activation of K ATP channels (Figure 4B ). Mean channel activity in the presence of wortmannin (10 nM) was 0.02 ± 0.00 which increased to 0.16 ± 0.02, 20 minutes after exposure to 100 μM BVT.12777 (n = 3; P < 0.05). Control experiments where no BVT.12777 was added show no change in channel activity over a 30-minute period (N f .P o = 0.01 ± 0.00 and 0.04 ± 0.00 after 5 and 30 minutes respectively; n = 4). Figure 4 Wortmannin does not inhibit BVT.12777 activation of K ATP A , current clamp record of a CRI-G1 cell dialysed with 5 mM MgATP, following exposure of cells to 10 nM wortmannin for 15–20 minutes. Application of BVT.12777 (200 μM), in the continued presence of wortmannin hyperpolarized the cell from -46 to -77 mV. Tolbutamide (100 μM), applied after the BVT-induced hyperpolarization, recovered the membrane potential (to -40 mV). B , cell-attached recordings from CRI-G1 cells, following exposure of cells to 10 nM wortmannin for 15–20 minutes. Upper trace; in the continued presence of wortmannin, N f .P o was 0.01 and 0.03 after 5 and 30 minutes respectively. Lower trace, application of BVT.12777 (100 μM) to cell-attached recording in the presence of 10 nM wortmannin resulted in K ATP activation, with N f .P o values of 0.01, 0.12 and 0.27 prior to, and 10 and 30 minutes after, BVT.12777, respectively. Addition of 100 nM wortmannin did not inhibit channel activity. Heterologously expressed K ATP currents are not activated by BVT.12777 Oocytes injected with Kir6.2 and SUR1 cRNAs were challenged with sodium azide (3 mM) to elicit a reversible increase in current, which was completely blocked by 1 μM glibenclamide or 0.5 mM tolbutamide, indicating that the current was due to K ATP activation, as described previously [ 24 , 25 ]. In oocytes, previously exposed to sodium azide in order to verify Kir6.2-SUR1 expression, application of BVT.12777 (10 μM – 1 mM) did not produce any consistent increase in K ATP current (n = 16; data not shown). Consequently, we utilized an alternative expression system, the HEK 293 cell line [ 25 ]. Application of BVT.12777 (100 μM) to the bathing solution using the cell-attached recording configuration resulted in no significant increase in mean channel activity above control levels over a 30-minute period, although subsequent addition of sodium azide (3 mM) did cause a rapid increase in channel activity, which was reversed by the addition of 100 μM tolbutamide (n = 4, data not shown). Similarly, application of BVT.12777 in the presence of 0.1 mM MgATP to inside-out patches from HEK 293 cells transiently expressing Kir6.2-SUR1, did not cause activation of channel activity following 30 minutes exposure (n = 4; data not shown). Thus BVT.12777 does not appear to be capable of activating heterologously expressed Kir6.2-SUR1 currents. MAPK does not mediate BVT.12777-activation of K ATP Exposure of CRI-G1 cells to BVT.12777 (100 μM) for up to 30 minutes had no consistent effect on the phosphorylation of enzymes downstream of PI3K (PKB and its downstream target, GSK3), but did increase the phosphorylation of STAT3 (n = 4) and MAPK (n = 4; data not shown). These data are in agreement with the lack of BVT.12777 sensitivity to PI3K inhibitors on activation of K ATP channels. However, activation of MAPK has been implicated as a significant intermediate for both insulin and leptin signalling pathways in various cell types [ 26 - 29 ]. Thus, we examined the effect of UO126, a potent and specific inhibitor of the activation of the classical MAPK cascade [ 30 ], on BVT.12777 opening of K ATP channels. Application of UO126 (25 μM) inhibited approximately 90 % of K ATP channel activity in cell-attached or inside-out recordings, whereas 1–10 μM UO126, concentrations that suppresses activation of MAPKK [ 30 ], had no significant effect on channel activity (data not shown). Control cell attached recordings had a mean channel activity of 0.07 ± 0.02, which increased to 1.29 ± 0.82 (n = 3) in the presence of BVT.12777 (100 μM). Subsequent application of UO126 (1 μM) in the continued presence of BVT.12777 did not alter channel activity (data not shown), over a 15-minute period (N f .P o was 1.86 ± 1.45 (n = 3) and 2.44 ± 2.00 (n = 3), at 5 and 15 minutes respectively; P < 0.05). In addition, increasing UO126 to 10 μM had no effect on BVT.12777 induced K ATP channel activation. BVT.12777 activation of K ATP channels is dependent on actin cytoskeleton dynamics Leptin activation of K ATP channels in the CRI-G1 cell line is dependent upon reorganisation of the cytoskeleton, a process downstream from PI3K activation [ 31 ]. Therefore, we examined whether BVT.12777 opening of CRI-G1 K ATP channels occurs through alteration of actin filament dynamics. For this series of experiments the heptapeptide mushroom toxin phalloidin [ 32 ] was used to stabilise the polymerised form of actin (F-actin). As phalloidin is membrane-impermeant, it was directly applied to the internal aspect of the cell membrane. In whole-cell experiments, 10 μM phalloidin was added to the electrode solution and allowed to dialyse into the cell. The mean resting potential and slope conductance were -38.0 ± 0.6 mV and 0.66 ± 0.04 nS (n = 4) respectively, and following addition of 200 μM BVT.12777 no significant change in these parameters was observed (Figure 5A ), with a mean membrane potential of -41.7 ± 1.1 mV and slope conductance of 0.60 ± 0.08 nS (n = 4; P > 0.05). The presence of phalloidin (10 μM) in the bath solution also prevented K ATP channel activation by BVT.12777 in the inside-out isolated patch configuration (Figure 5B ). Application of 0.1 mM MgATP to the cytoplasmic aspect of inside-out patches caused 97.5 ± 2.1% inhibition of K ATP channel activity (n = 3; P < 0.05) and subsequent addition of 10 μM phalloidin had no further effect, as reported previously [ 29 ]. Subsequent addition of BVT.12777 (100 μM) failed to increase K ATP channel activity, with mean Nf.Po values of 0.06 ± 0.05 and 0.03 ± 0.01 in the absence and presence of BVT.12777 respectively (n = 3; P > 0.05). In contrast, the direct K ATP channel opener, diazoxide activates K ATP channels in the presence of phalloidin. In whole-cell experiments (Figure 5C ), diazoxide (200 μM) hyperpolarized CRI-G1 cells from a mean membrane potential of -42.6 ± 0.1 mV to -70.1 ± 0.8 mV (n = 4; P < 0.05), and increased slope conductance from 0.87 ± 0.23 to 7.39 ± 0.72, actions reversed by tolbutamide (100 μM). Figure 5 Phalloidin prevents BVT.12777 activation of K ATP A , current clamp record of a CRI-G1 cell dialysed with 5 mM MgATP and 10 μM phalloidin. Application of BVT.12777 (200 μM) had no effect on the membrane potential of the cell (-41 mV) in the presence of phalloidin. B , continuous single channel currents recorded from an inside-out patch at a holding potential of -40 mV. Application of 100 μM MgATP reversibly inhibited N f .P o from 1.25 to 0.02. Addition of 10 μM phalloidin and subsequently 100 μM BVT.12777, in the presence of 100 μM MgATP, to the cytoplasmic aspect of the patch resulted in no effect on K ATP , with N f .P o values of 0.01 and 0.03 respectively. C , current clamp record of a CRI-G1 cell dialysed with 5 mM MgATP and 10 μM phalloidin. Application of diazoxide (200 μM) induced rapid cell membrane hyperpolarization, from -55 to -72 mV, an action reversed (to -45 mV) by tolbutamide (100 μM). F-actin is disrupted by BVT.12777 The prevention of BVT.12777-induced K ATP activation by phalloidin mirrors the effect of this toxin on leptin activation of K ATP [ 31 ]. Thus, we visualised F-actin by staining with rhodamine-conjugated phalloidin. In untreated CRI-G1 cells there was pronounced phalloidin-positive labelling of the cell membrane, with more diffuse, granular staining within the cytoplasm (Figure 6A ). In contrast, cells treated with BVT.12777 (100 μM) or leptin (10 nM) for 40 min showed a marked reduction in phalloidin fluorescence intensity, with disjointed labelling at the cell membrane (Figure 6A ). The actin filament disrupter cytochalasin B [ 33 ] also reduced the intensity of phalloidin labelling but in a more punctate manner on visualisation of treated cells compared with controls (data not shown). Analysis of the mean fluorescence intensity at the cell membrane following the actions of BVT.12777 and leptin demonstrated that both treatments caused a significant reduction of the intensity of rhodamine-phalloidin labelling, by 43.0 ± 4.2% (n = 6; P < 0.05) and 62.2 ± 6.0% (n = 6; P < 0.05), respectively, compared to untreated cells (Figure 6B ). However, the directly acting K ATP channel opener, diazoxide did not cause disruption of the actin cytoskeleton (Figure 6A,6B ), with a relative intensity of rhodamine-phalloidin staining of 0.98 ± 0.16 (P > 0.05). Figure 6 BVT.12777 disrupts the actin cytoskeleton A , images of rhodamine-conjugated phalloidin fluorescence in CRI-G1 cells in control conditions and following incubation with leptin (10 nM), BVT.12777 (100 μM) or diazoxide (200 μM) for 30 minutes. All panels show representative X-Y images. Note the marked reduction in phalloidin staining in cells pre-treated with leptin or BVT.12777, and not diazoxide. Scale bars are 50 μm. B , histogram comparing the normalised fluorescence intensity relative to control in the membrane periphery of randomly selected CRI-G1 cells for each condition; (control (n = 13; cells = 195), 10 nM leptin (n = 6; cells = 90), BVT.12777 (n = 6; cells = 90) and 200 μM diazoxide (n = 4, cells = 60). Error bars indicate s.e.m. and * significance of P < 0.001. Discussion BVT.12777 induced hyperpolarization of CRI-G1 cells, with an associated increase in K + conductance, an action likely caused by the activation of K ATP channels, as the sulphonylurea tolbutamide completely reversed its effects. Cell-attached and inside-out single channel current recordings demonstrate directly that BVT.12777 activates K ATP channels. The increased K ATP current generated in isolated membrane patches resembles the effects of K ATP activators such as diazoxide [ 34 ] and sodium azide [ 35 ], which have also been shown to activate insulinoma or pancreatic β-cell K ATP channels in isolated patches in the presence of Mg-ATP. Thus, although not tested here, BVT.12777 as an activator of K ATP would be expected, as observed for diazoxide, to inhibit insulin release from CRI-G1 cells stimulated by metabolizable substrates or tolbutamide [ 36 ], although this would clearly be dependent on its action on other β-cell conductances, notably calcium channels. BVT.12777 activation of K ATP channels was only slowly reversed on withdrawal of the drug, unlike the actions of diazoxide or sodium azide, which are rapidly reversed on washout [ 35 , 36 ]. Indeed, following removal of BVT.12777 in the absence or presence of tolbutamide, enhanced K ATP channel activity was apparent for a considerable time. The slow reversibility on washout of BVT.12777 resembles the effects of the hormone leptin on CRI-G1 cell membrane potential and K ATP channel activation [ 13 ]. Leptin, via activation of the main signalling form of the leptin receptor (ObRb), has been shown to increase the phosphorylation of STAT3, MAPK and to stimulate PI3K pathways in various peripheral tissues, cell lines [ 37 ], and in hypothalamic neurones [ 38 ]. BVT.12777 although stimulating phosphorylation of STAT3 and MAPK did not stimulate PI3K dependent pathways as demonstrated by the lack of effect on the phosphorylation status of the PI3K output indicators, PKB and GSK3. It is unclear at present how this molecule induces STAT3 and MAPK phosphorylation. As K ATP activation by BVT.12777 is rapid and occurs in isolated membrane patches it is unlikely that any JAK-STAT pathway (which drives changes in transcription) contributes to this action. Leptin activation of K ATP channel currents in CRI-G1 cells has previously been shown to be independent of MAPK, but prevented by the inhibitors of PI3K [ 22 ]. However, BVT.12777 activation was not only insensitive to inhibition by the MAPKK inhibitor, UO126, it was also insensitive to the presence of the PI3-kinase inhibitors, wortmannin and LY294002, at concentrations sufficient to prevent leptin activation of K ATP in this cell line. These data led us to suspect that BVT.12777, irrespective of its ability to initiate various signalling cascades in this cell line, increased K ATP channel activity by a more direct effect on the channel subunits in a manner analogous to diazoxide, which is purported to interact directly with the SUR1 subunit [ 9 , 10 ]. This possibility was tested by heterologous expression of the β-cell subunits of K ATP channels, Kir6.2 and SUR1, in Xenopus oocytes, a commonly utilised expression system for electrophysiological studies of these recombinant channels [ 24 , 25 ]. However, BVT.12777 did not activate Kir6.2-SUR1 currents in oocytes, demonstrated to express functional K ATP channel currents. Thus we explored this question further by utilising a second heterologous expression system for Kir6.2-SUR1, HEK293 cells. Recordings from inside-out patches demonstrated that BVT.12777 did not activate Kir6.2-SUR1 currents in the presence of Mg-ATP, in contrast to diazoxide [ 39 ] or sodium azide [ 35 ]. Overall these data strongly suggest that expression of the K ATP channel subunits, Kir6.2 and SUR1 are insufficient per se to bring about sensitivity to BVT.12777, and indicate that this opener may activate this channel type by an indirect mechanism (which is not available in oocytes or HEK cells). Although the activation of K ATP channels by leptin in CRI-G1 cells is PI3-kinase dependent the lipid products of this enzyme system, such as PtdIns(3,4,5)P 3 also do not interact directly with K ATP channels [ 22 ]. Recent studies demonstrate that both leptin and PtdIns(3,4,5)P 3 increase K ATP channel activity indirectly, through changes in cytoskeletal dynamics [ 31 ]. It is well established that many ion channels and transporters are anchored in the membrane by either direct or indirect association with the cytoskeleton. In addition, there is growing evidence that altering the integrity of cytoskeletal elements, in particular actin filaments, can modulate the activity of a variety of ion channels [ 40 ] and receptors [ 41 ]. For example, disruption of actin filaments with cytochalasin is shown to increase K ATP channel activity in cardiac myocytes [ 42 ] and CRI-G1 cells [ 31 ]. Indeed, a number of lipid kinases, including PI 3-kinase, are also localised to the cytoskeleton and their activities are modulated by a variety of cytoskeletal proteins, especially those associated with actin [ 40 ]. Actin filament structure is controlled by reversible polymerisation of G-actin, which forms F-actin, and this process is under the dynamic control of various actin-binding proteins [ 43 ]. The heptapeptide mushroom toxin phalloidin [ 32 ] binds to filamentous F-actin with high affinity and stabilises the actin in this form. The addition of phalloidin to the intracellular aspect of CRI-G1 cells prevented BVT.12777, but not diazoxide, from activation of K ATP channel currents in whole cell and inside out recording configurations indicating that this molecule likely causes the opening of K ATP channels by a membrane delimited alteration of cytoskeletal dynamics. This mechanism of action is identical to that proposed for leptin and PtdIns(3,4,5)P 3 activation of K ATP in this cell line [ 31 ]. Fluorescence staining of CRI-G1 cells with rhodamine-conjugated phalloidin revealed disassembly of actin filaments by both BVT.12777 and leptin, but not diazoxide. These data provide direct support for an important role for cytoskeletal dynamics in the control of K ATP channel activity by both leptin and BVT.12777. The lack of effect of diazoxide on the actin filament structure is also supportive of this opener acting directly on the K ATP channel subunits. Conclusions BVT.12777 activation of K ATP channels in CRI-G1 cells was evident regardless of whether it was applied to the external or internal surface of the cell. BVT.12777 signalling to K ATP channels is not mediated by PI 3-kinase or MAPK, but does appear to depend on actin filament re-modelling. As leptin hyperpolarizes a sub-population of hypothalamic neurones by opening K ATP channels [ 16 ], it is feasible that at least part of the anti-obesity action of BVT.12777 may be through the activation of this potassium channel. Furthermore, as BVT.12777 acts downstream of PI3K, such an agent may act to overcome the putative central leptin resistance associated with the obese state [ 37 ]. Thus, although BVT.12777 and its close structural analogues are unlikely per se to be useful anti-obesity agents as they display hepatotoxicity [ 44 ], understanding the general principles underlying their mechanism of action may reveal clues for future anti-obesity drug development. Methods Cell culture and transfection Cells from the insulin secreting cell line, CRI-G1, and the human embryonic kidney cell line, HEK 293, were grown as described previously [ 25 , 35 ]. The preparation of mouse Kir6.2 (provided by Professor F. Ashcroft, University of Oxford), rat SUR 1 (provided by Dr G. Bell, University of Chicago) and CD4 cDNAs and transfection procedures were as described by [ 25 ]. Transfected cells were selected by visible binding of anti-CD4 coated beads (Dynal, Oslo) following incubation with the beads for 20 min. Oocyte collection and preparation Ovarian lobes were removed from mature female Xenopus laevis frogs (Blades Biological, UK) following killing of the animal by destruction of the brain. The use of animals was in accordance with the Home Office Animals (Scientific Procedures) Act (1986) and approved by the local ethics committee. Separation and selection of oocytes and the preparation and injection of cRNAs were performed as described by [ 25 ]. Western blotting CRI-G1 cells, in normal saline (containing in mM; NaCl 135, KCl 5, MgCl 2 1, CaCl 2 , 1, HEPES 10 with glucose 10 (pH 7.4) were treated with BVT.12777 (100 μM) for 0, 1, 5, 15 or 30 minutes and whole-cell extracts were prepared as described [ 23 ]. Proteins (10 μg) were suspended in loading buffer (Invitrogen) and after denaturation, loaded on to NuPage 4–12% Bis-Tris mini-gels (Invitrogen) and run at 200 V for 1 hr. Subsequently, proteins were transferred to Hybond-C Extra nitrocellulose membranes (Amersham) at 25 V for 80 minutes at room temperature. Membranes were incubated in blocking buffer (5% non-fat milk in TBST (20 mM Tris HCl, 150 mM NaCl, 0.5% Tween, pH 7.4)) for 1 hr at room temperature after which antibodies to phospho-MAPK, phospho-STAT3, phospho PKB, phospho-GSK3 and PKB (all at 1:1000) were applied at 4°C with gentle shaking, overnight. The membranes were washed with TBST (4 × 30 minutes) and incubated for 1 hr at room temperature with HRP conjugated ImmunoPure goat anti-rabbit IgG (1:5000). After washing with TBST (5 × 15 minutes), immunoreactive bands were visualised by the enhanced chemiluminescence (ECL) detection reagent (Amersham). Cytoskeletal fluorescence imaging and analysis CRI-G1 cells were gently washed in normal saline (containing in mM): NaCl 135, KCl 5, MgCl 2 1, CaCl 2 1, HEPES 10, pH 7.4, and incubated for 40 min with either 100 μM BVT.12777, 10 nM leptin, 200 μM diazoxide or 3 mM sodium azide for 30 min with the cytoskeletal disrupter, cytochalasin B (10 μM). Cells were then fixed, permeabilised, stained with rhodamine-conjugated phalloidin (2.66 U ml -1 ) and visualised using a BioRad Microradiance, confocal imaging system as described by [ 31 ]. The intensity of rhodamine-conjugated phalloidin staining in the plasma membrane was determined using BioRad Lasersharp processing software (Bio-Rad, CA, USA). Analysis lines were drawn along randomly selected regions of the plasma membrane and the fluorescence intensity determined. A histogram giving the mean fluorescence intensity was constructed for a minimum of 5 cells on each stimulated or control dish on at least 3 separate occasions. Within a given experimental series all conditions for capturing images were constant. In order to allow for quantification of experimental data obtained on separate days, the results were normalised relative to the mean plasma membrane fluorescence measured in the control cells for each day and presented as mean ± S.E.M. Statistical analyses were performed using Student's unpaired t test. p < 0.05 was considered significant. Electrophysiological recording and analysis Whole cell currents from Xenopus oocytes were measured using a two-electrode voltage clamp technique as described by [ 25 ]. Recordings were made in a high-potassium bath solution, KD96 containing (mM): KCl 96, NaCl 2, CaCl 2 1.8, HEPES 5 (pH 7.4 with KOH). Working concentrations of drugs were prepared in KD96 and superfused into the bath. Whole-cell current-clamp recordings with excursions to voltage clamp mode were used to monitor membrane potential and macroscopic currents from CRI-G1 cells. Cell-attached and excised inside-out recordings were made from CRI-G1 cells and HEK cells expressing Kir6.2 and SUR1 to examine single channel responses as described previously [ 25 , 35 ]. Single channel data were analysed for current amplitude and channel activity (N f .P o ; where N f is the number of functional channels in the patch and P o is the open probability) as described previously [ 45 ]. All data were normalised to control and are expressed as mean ± S.E.M. Statistical analyses were performed using Student's unpaired t test. P < 0.05 was considered significant. Recording electrodes were pulled from borosilicate glass and had resistances of 2–5 MΩ for whole cell recordings and 7–10 MΩ for cell-attached and inside-out experiments when filled with electrolyte solution. The pipette solution for whole-cell recordings comprised (in mM): KCl 140, MgCl 2 0.6, CaCl 2 2.73, Mg-ATP 5.0, EGTA 10, HEPES 10, pH 7.2 (free [Ca 2+ ] of 100 nM), whereas for single channel recordings the pipette solution contained (in mM): KCl 140, CaCl 2 1, MgCl 2 1, HEPES 10, pH 7.2. The bath solution for whole-cell and cell-attached recordings was normal saline whereas for inside-out patches the bath solution contained (in mM): KCl 140, MgCl 2 1, CaCl 2 2, EGTA 10, HEPES 10, pH 7.2 (free [Ca 2+ ] of 30 nM). All solution changes were achieved by superfusing the bath with a gravity feed system at a rate of 10 ml min -1 , which allowed complete exchange within 2 min. All experiments were performed at room temperature (22–25°C). Antibodies & drugs Anti-PKB, which recognises all three isoforms of PKB, and the phospho-specific PKB (Thr308), GSK3α/β (Ser21/9), STAT3 (Tyr705) and p44/42 MAPK (Thr202/Tyr204) antibodies were obtained from Cell Signalling Technology Inc. Recombinant human leptin, wortmannin and LY 294002 were obtained from Novachem-Calbiochem and BVT.12777 ([2-(hydrazinoiminomethyl) hydrazino] acetic acid) was a gift from Biovitrum (Stockholm, Sweden). Tolbutamide, Mg-ATP, diazoxide, sodium azide, phalloidin and cytochalasin B were obtained from Sigma. Rhodamine-conjugated phalloidin was obtained from Molecular Probes and UO126 from Promega. BVT.12777 was prepared as a 100 mM stock solution in normal saline and stored at -70°C prior to use. Leptin was prepared as a 10 μM stock solution in normal saline containing 0.2 % bovine serum albumin as carrier. Rhodamine-conjugated phalloidin (200 U ml -1 ) and LY 294002 (10 mM) were stored as stock solutions in 1% methanol at -20°C. Cytochalasin B was stored as a 10 mM stock solution, and diazoxide and tolbutamide as 100 mM solutions, all in DMSO at 2–4°C. Mg-ATP was stored at -20°C as a 100 mM solution in 10 mM HEPES (pH 7.2). Wortmannin and UO126 were stored as 10 mM stock solutions in Me 2 SO at -20°C. List of abbreviations used CRI-G1, Cambridge Rat Insulinoma-G1; GSK3, glycogen synthase kinase-3; HEK293, human embryonic kidney 293; JAK, janus kinase; K ATP , ATP-sensitive potassium; Kir6.2, potassium channel inward rectifier-6.2; MAPK, p42, p44 mitogen-activated protein kinase; MAPKK, MAPK kinase; ObRb, Obese (leptin) receptor-b; PKB, protein kinase B; PI3K, phosphatidylinositol 3-kinase; PtdIns(3,4,5)P 3 , phosphatidylinositol 3,4,5 tris-phosphate; STAT3, signal transducer and activator of transcription-3; SUR, sulphonylurea receptor Authors' contributions JK carried out the majority of the electrophysiology and cytoskeletal fluorescence studies. HL carried out the western blot experiments. TT and JH participated in the electrophysiological experiments. CS participated in the design and implementation of the western blot experiments. MA conceived of the study, participated in its design and co-ordination and drafted the manuscript. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC516774.xml
535813	Widespread presence of "bacterial-like" PPP phosphatases in eukaryotes	Background In eukaryotes, PPP ( p rotein p hosphatase P ) family is one of the two known protein phosphatase families specific for Ser and Thr. The role of PPP phosphatases in multiple signaling pathways in eukaryotic cell has been extensively studied. Unlike eukaryotic PPP phosphatases, bacterial members of the family have broad substrate specificity or may even be Tyr-specific. Moreover, one group of bacterial PPPs are diadenosine tetraphosphatases, indicating that bacterial PPP phosphatases may not necessarily function as protein phosphatases. Results We describe the presence in eukaryotes of three groups of expressed genes encoding "non-conventional" phosphatases of the PPP family. These enzymes are more closely related to bacterial PPP phosphatases than to the known eukaryotic members of the family. One group, found exclusively in land plants, is most closely related to PPP phosphatases from some α- Proteobacteria , including Rhizobiales , Rhodobacterales and Rhodospirillaceae . This group is therefore termed Rhi zobiales / Rh odobacterales / Rh odospirillaceae - l ike ph osphatases, or Rhilphs. Phosphatases of the other group are found in Viridiplantae , Rhodophyta , Trypanosomatidae , Plasmodium and some fungi. They are structurally related to phosphatases from psychrophilic bacteria Shewanella and Colwellia , and are termed She wanella - l ike ph osphatases, or Shelphs. Phosphatases of the third group are distantly related to ApaH, bacterial diadenosine tetraphosphatases, and are termed A paH- l ike ph osphatases, or Alphs. Patchy distribution of Alphs in animals, plants, fungi, diatoms and kinetoplasts suggests that these phosphatases were present in the common ancestor of eukaryotes but were independently lost in many lineages. Rhilphs, Shelphs and Alphs form PPP clades, as divergent from "conventional" eukaryotic PPP phosphatases as they are from each other and from major bacterial clades. In addition, comparison of primary structures revealed a previously unrecognised (I/L/V)D(S/T)G motif, conserved in all bacterial and "bacterial-like" eukaryotic PPPs, but not in "conventional" eukaryotic and archaeal PPPs. Conclusions Our findings demonstrate that many eukaryotes possess diverse "bacterial-like" PPP phosphatases, the enzymatic characteristics, physiological roles and precise evolutionary history of which have yet to be determined.	Background Reversible phosphorylation of proteins is a ubiquitous mechanism, indispensable for regulation of virtually any cellular function. Therefore, protein kinases and phosphatases are of paramount importance for normal functioning of all metabolic and signalling pathways. In eukaryotes, PPP family is one of the two known protein phosphatase families specific for Ser and Thr [ 1 - 4 ]. Unlike eukaryotic (and archaeal [ 5 ]) PPP phosphatases, bacterial members of the family have broad substrate specificity [ 6 ] or may even be Tyr-specific [ 7 - 9 ]. Moreover, one group of bacterial PPPs are diadenosine tetraphosphatases [ 10 , 11 ]. Unlike eukaryotes, in prokaryotes PPP phosphatases appear to be facultative, since entirely sequenced genomes of some bacteria and archaea do not encode them [ 5 , 12 ]. Nevertheless, when present, they appear to play essential roles [ 13 - 15 ]. Three motifs (GDXHG, GDXXDRG and GNH(E/D)), highly conserved in the N-terminal subdomains of the catalytic domains of all PPP phosphatases [ 10 , 11 ], contain most residues which coordinate metal ions in the active centre [ 16 ] and are considered as the signature of the PPP family. In a previous work [ 17 ] we identified an unusual cDNA fragment from a moss Physcomitrella patens , showing no similarities to the known PPP phosphatases beyond the presence of the GDXHG and GDXXDRG motifs. Detection of homologous cDNA sequences from Arabidopsis and rice suggested the presence of an unknown PPP group in plants, distinct from "conventional" eukaryotic PPP phosphatases [ 17 ]. We have now taken advantage of a much greater representation (as compared to 1999) of sequence databases for various species to further explore this initial observation. We present the evidence for the existence in eukaryotes of three "non-conventional" branches of the PPP family. We also identify a previously unrecognised conserved motif in the PPP catalytic domain, which can be used as a signature of "bacterial"-type PPP phosphatases. Results " Rh izobiales / Rh odobacterales / Rh odospirillaceae – like" PPP phosphatases in plants Two Arabidopsis sequences, At3g09960 and At3g09970, were retrieved using the P. patens fragment [ 17 ] as a query in TBlastN searches. They share 85% identity with each other at the protein level (see Figure 1 ). Both genes are transcribed, since full-length cDNAs have been detected in a large-scale transcription study [ 18 ]. They are arranged on chromosome 3 in tandem, suggesting their origin by a recent duplication. A number of ESTs from other plant species (but none from non-plant eukaryotes) were also detected by TBlastN searches, which in most cases provide no evidence for the existence of more than one isoform. Figure 1 Comparison of the primary structures of plant Rhilphs, related α-proteobacterial phosphatases and human PP1α as a prototype of "conventional" eukaryotic PPP phosphatases . Amino acid residues conserved in at least all but one Rhilphs and α-proteobacterial phosphatases are shown in white and shaded in black. Residues conserved in at least two thirds of the sequences are shown in white and shaded in dark grey. Residues conserved in at least half of the sequences are shown in black and shaded in light grey. Following substitutions were considered as conserved residues: Ile/Leu, Phe/Tyr, Asp/Glu, Asn/Gln, Arg/Lys and Ser/Thr. Catalytic site residues that interact with metal ions are indicated by asterisks according to [20]. SAPNY motif in PP1, conserved in most eukaryotic PPP phosphatases, is double underlined. Solanum tuberosum sequence is translation of the EST entries BQ516856, BQ516857 and BI435517. Physcomitrella patens sequence is translation of the EST entry BQ039171. Other accession numbers are indicated in Table 1. Among prokaryotes, related sequences were detected in some α -Proteobacteria , the closest matches being with Rhizobiales , Rhodobacterales and Rhodospirillaceae . Therefore, we designate this group as Rhi zobiales / Rh odobacterales / Rh odospirillaceae -like phosphatases, or Rhilphs (Figure 1 ; see also Table 1 and Figure 4 ), and the two Arabidopsis genes products At3g09960 and At3g09970 as " R hizobiales - l ike" p hosphatases 1 (RLP1) and 2 (RLP2), respectively. Table 1 Species, accession numbers (UniProt, EMBL, NCBI or TIGR Gene Index) and common names (where available) of PPP phosphatase sequences shown in Figure 4. For A. thaliana sequences, gene numbers are also indicated. Sequence No. 67 is available from Chlamydomonas reinhardtii draft genome [65]. No. Accession Species name Rhilphs 1 Q9SR61; At3g09960 Arabidopsis thaliana 2 Q9SR62; At3g09970 Arabidopsis thaliana 3 BE034080 Mesembryanthemum crystallinum 4 BQ995369 Lactuca sativa 5 AW034786 Lycopersicon esculentum 6 BU875146 Populus balsamifera 7 BG457803 Medicago truncatula 8 AV425727 Lotus japonicus 9 BM731295 Glycine max 10 BF261816 Hordeum vulgare 11 BQ788728 Triticum aestivum 12 AL731641 Oryza sativa 13 CF670562 Pinus taeda 14 BQ039171 Physcomitrella patens Group I (α-Proteobacteria) 15 Q987U4 Mesorhizobium loti 16 Q8UA33 Agrobacterium tumefaciens 17 ZP_00054691 Magnetospirillum magnetotacticum 18 ZP_00015226 Rhodospirillum rubrum 19 ZP_00014771 Rhodospirillum rubrum 20 CAE28794 Rhodopseudomonas palustris 21 Q9ABQ8 Caulobacter crescentus 22 ZP_00051041 Magnetospirillum magnetotacticum 23 ZP_00093979 Novosphingobium aromaticivorans 24 Q92V37 Sinorhizobium meliloti Group VII (heterogeneous) 25 Q8YZT4 Anabaena sp. 26 Q9WZK1 Thermotoga maritima 27 O34205 Fervidobacterium islandicum 28 NZ_AABE01000101 Cytophaga hutchinsonii Group III (γ-Proteobacteria and bacteriophage λ) 29 P03772 Bacteriophage λ 30 P55798 E. coli PrpA 31 Q8VPE2 Salmonella typhimurium PrpA 32 Q8Z487 Salmonella enterica 33 P55799 E. coli PrpB Group IV (Firmicutes) 34 Q81YR3 Bacillus anthracis 35 Q9FB69 Lactococcus lactis 36 Q97FF3 Clostridium acetobutylicum Alphs 37 BM291808 Amblyomma variegatum 38 TC9835 Ciona intestinalis 39 BU652795 Chlamydomonas reinhardtii 40 AC091781 Trypanosoma brucei 41 AC084046 Trypanosoma brucei 42 AL499620 Leishmania major 43 BQ143558 Metarhizium anisopliae 44 AC127427 Magnaporthe grisea 45 AA966318 Aspergillus nidulans 46 P40152 Saccharomyces cerevisiae ApaH 47 Q8Y1K9 Ralstonia solanacearum ApaH 48 Q9JVF4 Neisseria meningitidis ApaH 49 P05637 Escherichia coli ApaH Group VI (heterogeneous) 50 O31614 Bacillus subtilis 51 O69213 Anabaena sp. PrpA 52 Q93JF4 Streptomyces coelicolor 53 Q9RS78 Deinococcus radiodurans Group II (Cyanobacteria) 54 O54390 Microcystis aeruginosa PP1-Cyano 1 55 P74150 Synechocystis sp. 56 Q8YP31 Anabaena sp. 57 ZP_00072257 Trichodesmium erythraeum 58 Q8DGA2 Thermosynechococcus elongatus Shelphs 59 AC119500* Leishmania major 60 Q8EBN0 Shewanella oneidensis 61 Q9S427 Shewanella sp. 62 TIGR_167879 Contig1731 Colwellia psychrerythraea 63 CF394707 Pinus taeda 64 TC31593 Solanum tuberosum 65 Q944L7; At1g18480 Arabidopsis thaliana 66 BF645180 Medicago truncatula 67 Scaffold_45 Chlamydomonas reinhardtii 68 Q9LMJ5; At1g07010 Arabidopsis thaliana 69 AW266595 Mesembryanthemum crystallinum 70 BG644111 Lycopersicon esculentum 71 BG450922 Medicago truncatula 72 BI787505 Glycine max 73 Q8L676 Oryza sativa 74 TC21958 Hordeum vulgare 75 AC007863 Trypanosoma brucei 76 AL499621 Leishmania major 77 TIGR_246197 Contig433 Myxococcus xanthus 78 EAK84303 Ustilago maydis 79 O74480 Schizosaccharomyces pombe 80 EAK87480 Cryptosporidium parvum 81 Q7RIH8 Plasmodium yoelii 82 Q8IKE5 Plasmodium falciparum 83 Q8I5Y5 Plasmodium falciparum 84 Q7RR22 Plasmodium yoelii Group V (heterogeneous) 85 O87639 Streptomyces coelicolor 86 Q9RVT7 Deinococcus radiodurans Archaea 87 O28453 Archaeoglobus fulgidus PPA 88 Q8ZW26 Pyrobaculum aerophilum 89 O34200 Methanosarcina thermophila PP1-arch2 90 Y12396 Pyrodictium abyssi "Conventional" eukaryotic PPP 91 Q9U493 Plasmodium falciparum PPJ 92 Q8I728 Trypanosoma cruzi PPEF 93 BH900132 Ostreococcus tauri 94 O14829 Homo sapiens PPEF1 (PP7) 95 Q8IDE7 Plasmodium falciparum PP5 96 P53041 Homo sapiens PP5 97 P53043 Saccharomyces cerevisiae PPT 98 P32838 Saccharomyces cerevisiae PPG 99 P05323 Homo sapiens PP2A 100 O00743 Homo sapiens PP6 101 Q08209 Homo sapiens Calcineurin (PP2B) 102 P08129 Homo sapiens PP1 103 P32945 Saccharomyces cerevisiae PPQ 104 O49346 Arabidopsis thaliana PP7 * This phosphatase is unlikely to be catalytically active due to replacements of essential residues in the active centre. Figure 2 Comparison of the primary structures of " Shewanella -like" phosphatases (Shelphs) and human PP1α as a prototype of eukaryotic PPP phosphatases . Designations for conserved amino acid residues are as in Figure 1. For Oryza sativa , dashed underlined C-terminal sequence has been corrected by comparison with ESTs. Accession numbers: Plasmodium falciparum 1, Q8I5Y5; 2, Q8IKE5; Trypanosoma brucei 1, AC007863; 2, AC084046.12. Chlamydomonas reinhardtii sequence is translation of the EST entries BG855683 and BI995255. Other accession numbers are indicated in Table 1. Figure 3 Characteristic modifications (shaded in black) in the conserved PPP signature motif GDXXDRG in bacterial diadenosine tetraphosphatases and eukaryotic Alphs . Eukaryotic species are shown in bold. Plus signs indicate that gene expression is confirmed by the presence of ESTs. Figure 4 Neighbor-Net analysis of the conserved N-terminal subdomains (starting 5 amino acid residues before conserved GDXHG and ending 25 residues after GNH(E/D) of 104 bacterial, archaeal and eukaryotic PPP phosphatases . Bootstrap values exceeding 50% (out of 1000 resamplings) were obtained in a separate neihbour-joining analysis and are shown in brackets. Species and accession numbers are listed in Table 1. Note that groups designated as I, IV and VII did not receive significant bootstrap support; corresponding sequences are grouped together for convenience of their representation in Table 1. This image (and bootstrap values for the alternative splits) can be viewed at a higher resolution as the Additional File 1. Figure 5 Distinct conserved motifs in the C-termini of bacterial and "bacterial-like" PPP phosphatases from eukaryotes as opposed to archaeal and eukaryotic PPP phosphatases . A His residue directly binding a metal ion in the catalytic centre (marked with asterisk), and the elements of secondary structure are shown for bacteriophage λ phosphatase and for human PP1 according to ref. [20] and [19], respectively. The (I/L/V)D(S/T)G motif, highly conserved in bacterial and "bacterial-like" phosphatases, is highlighted. An expanded version of this alignment can be viewed as the Additional File 2. Structural features of Rhilphs All residues that are expected to bind metal ions in the catalytic centre are conserved in Rhilphs (Figure 1 ). Rhilphs do not have N- or C-terminal extensions beyond their catalytic domains, which in many PPP phosphatases have regulatory function and / or interact with regulatory proteins / subunits. Instead, they have characteristic inserts between the conserved motifs GNH(E/D) and HAG (corresponding to HGG in "conventional" eukaryotic PPPs, see Figure 1 ). Notably, much shorter inserts are found at a similar position in α-proteobacterial phosphatases (group I in Figure 1 ). Inserts in both plant Rhilphs and α-proteobacterial phosphatases contain a conserved motif LXXAXPXXH (Figure 1 ). Similarly to bacteriophage λphosphatase (λPP [ 19 ]), Rhilphs lack a region corresponding to β8, β9 and α9 of eukaryotic PPPs [ 20 - 22 ]. Like bacterial PPP phosphatases, Rhilphs do not have a SAPNY motif, conserved in the β12-β13 loop of eukaryotic PPPs. Analysis of Rhilph sequences did not reveal targeting or signal peptides. While this work was in progress, a phosphatase encoded by an Arabidopsis gene At1g07010 was reported in an independent study [ 4 ]. " Shewanella -like" PPP phosphatases in plants, red algae, fungi and unicellular parasites We undertook further TBlastN searches using full-length Arabidopsis RLP2 as a query to see whether Arabidopsis genome encodes additional "bacterial-like" PPPs. These searches identified two more genes for putative PPP phosphatases, only distantly related to Rhilphs and to any other members of the family (Figure 2 ). One of these genes is At1g07010 1 . At least three different predicted products of this gene could be found in protein databases. On the basis of comparison with EST sequences, we consider as the correct structure that of Q8RY10 with Asp and Gly at positions 109 and 208, respectively (see Figure 2 ). The other detected gene, At1g18480 , is also represented in the databases by three distinct deduced proteins. Comparison with A. thaliana ESTs confirms Q944L7 as the correct structure. Genomic and EST database searches provided ample evidence for the presence of related phosphatases in a number of green plants, including multiple angiosperm species, pine and a unicellular green alga Chlamydomonas reinhardtii (Figure 2 ; see also Table 1 and Figure 4 ). Related sequences were also identified in some fungi (several basidiomycetes and an ascomycete Schizosaccharomyces pombe , but not other ascomycetes), in Apicomplexa, Trypanosomatidae , and in a red alga Porphyra yezoensis (for available sequence from the latter species, see Figure 5 ). The most closely related prokaryotic phosphatases were detected in Myxococcus xanthus (δ- Proteobacteria ) and psychrophilic bacteria Alteromonadales (γ- Proteobacteria ): uncharacterised phosphatases from Shewanella oneidensis and Colwellia psychrerythraea and a Tyr-specific phosphatase PPI from Shewanella sp . [ 8 ]. Therefore, we designate this phosphatase group as " She wanella - l ike" ph osphatases, or Shelphs, and the products of the two prototype Arabidopsis genes At1g07010 and At1g18480 as " S hewanella - l ike" p hosphatases 1 (SLP1) and 2 (SLP2), respectively. Structural features of Shelphs Like in Rhilphs, all residues that are expected to bind metal ions in the catalytic centre are conserved in Shelphs (see Figure 1 ). Another feature common with Rhilphs is the presence of inserts (as compared to "conventional" eukaryotic PPPs) between the GNHE and H(A/G)G motifs (Figure 1 ), which are especially long in plant Shelphs. However, these inserts share no sequence similarity between Rhilphs and Shelphs and probably appeared in the two phosphatase groups independently. Like in Rhilphs, a region corresponding to β8, β9 and α9 of eukaryotic PPPs is absent in Shelphs, and the primary structure of the region corresponding to the β12-β13 loop is similar to that of typical bacterial PPPs. A.thaliana SLP1 and corresponding Shelph isoform from rice have chloroplast targeting sequence, which could not be detected in A.thaliana SLP2 and corresponding isoform from Medicago truncatula . Eukaryotic PPPs distantly related to bacterial diadenosine tetraphosphatases Identification of Rhilphs and Shelphs prompted us to perform extensive searches of eukaryotic sequence databases. These searches revealed the existence of other "bacterial-like" PPP phosphatases throughout eukaryotes. Sequences only distantly related to Rhilphs, Shelphs or any other PPP phosphatases were detected in several fungi (including a putative S. cerevisiae phosphatase reported previously [ 23 ]), in Trypanosomatidae , a tick Amblyomma , an ascidian Ciona , Chlamydomonas , pine and diatoms Fragilariopsis cylindrus and Phaeodactylum tricornutum . Blast searches using these sequences revealed that all of them share higher similarity to bacterial diadenosine tetraphosphatases (ApaH) than to other PPP groups. Therefore, we tentatively designate them as A paH- l ike p hosphatases, or Alphs (Figures 3 and 4 ; Table 1 ; partial sequences available for pine and diatoms are shown in Figure 5 ). Alphs share a distinctive common structural feature. In the GDXXDRG motif, absolutely conserved in other PPPs, the second Asp (which stabilises the protonation of a His that directly participates in catalysis [ 20 ]) is replaced by a neutral amino acid, and the Arg residue (which coordinates phosphate [ 24 ]) is replaced, with one exception, by Lys (Figure 3 ). The former of these replacements is also found in ApaH, while the latter is unique to Alphs. While higher overall sequence similarity and a common alteration in the GDXXDRG motif are compatible with closer relatedness of Alphs to bacterial diadenosine tetraphosphatases, phylogenetic analysis using full length sequences failed to produce a robust tree due to high sequence diversity (not shown). Relationship of novel eukaryotic PPP groups to known PPP phosphatases In order to better understand the relationship of "bacterial-like" PPP phosphatases in eukaryotes to each other and to bacterial PPPs, we attempted to extend our previous phylogenetic analysis of eukaryotic PPP phosphatases [ 25 ] by including PPP sequences from a number of bacteria and archaea. Primary structures of bacterial PPP phosphatases are extremely diverse and, outside the relatively conserved N-terminal subdomain of about 100 amino acids containing the GDXHG, GDXXDRG and GNH(E/D) motifs, they share only a few conserved residues. Moreover, many of the sequences have long insertions at different positions. This leads to the failure to produce informative alignments of full-length catalytic domains. Therefore, we aligned more conserved N-terminal subdomains only, an approach applied previously by Kennelly [ 6 ] to a much smaller set of PPP sequences available at that time. Phylogenetic reconstruction was attempted with either neighbor-joining (as implemented in PHYLIP [ 26 ] or SplitsTree [ 27 ]) or maximum likelihood analysis using quartet puzzling (TreePuzzle [ 28 ]); in the latter case a smaller dataset consisting of with consisting of 32 representative sequences was analyzed due to the inability of the algorithm to handle large datasets. Due to the relatively short length of the sequences and their high diversity, some of the major clades did not receive significant bootstrap support and were different depending on the method used, although most major clades, including Rhilphs and Shelphs, were recovered by both methods. Alphs tended to be grouped together by neighbor-joining but were split into smaller clades when maximum likelihood analysis was used. However, we still tentatively consider Alphs as a single group due to the characteristic replacements in their catalytic centre. To circumvent the ambiguity of the results, we used Neighbor-Net [ 29 ], a neighbor-joining based method that constructs phylogenetic networks rather than trees and thus represents conflicting signals and visualises feasible trees in a single plot (Figure 4 ; for a high-resolution image, see Additional file 1 ). The Neighbor-Net analysis accurately identified the major clades such as eukaryotic and archaeal phosphatases, as well as their closer relationship to each other than to bacterial PPPs [ 6 ]. Separation of "conventional" eukaryotic PPPs into two branches, suggested previously from the analysis of the full-length catalytic domains [ 25 ], was also recovered. As it was suggested by initial sequence similarity searches, Rhilphs, Shelphs and Alphs represent distinct major clades of the PPP family, as divergent from "conventional" eukaryotic and archaeal PPP phosphatases as they are from major bacterial clades (Figure 4 ; Additional file 1 ). Common structural elements in all "bacterial-like" PPP phosphatases from eukaryotes and bacterial phosphatases C-terminal regions of the catalytic domain of all "conventional" eukaryotic PPP phosphatases share a highly conserved (with minor variations) SAPNY motif, located in the β12-β13 loop. This loop and the Tyr residue of the SAPNY motif in particular are implicated in interaction with regulators and inhibitors [ 21 , 30 - 32 ]. β strands (β9 and β10) corresponding to β12 and β13 are conserved in λPP [ 19 ]. However, the sequence on the C-terminal side of β9 is dissimilar to SAPNY in λPP and in bacterial PPPs. A conservative replacement of the first Ser of SAPNY by Thr is found in many bacterial sequences (this Thr is however only moderately conserved and is replaced by Glu or Gln in all Rhilphs and by Val or Phe in most Shelphs). The two adjacent positions are occupied by highly conserved Asp and Gly residues, respectively, thus defining a previously unrecognised motif (I/L/V)D(S/T)G. This motif is present in all examined bacterial PPPs, as well as in all "bacterial-like" phosphatases from eukaryotes described above (Figure 5 ; see also Additional file 2 for a more complete alignment). In addition, we note the presence of another characteristic feature of bacterial and "bacterial-like" PPPs: the His residue (H248 in PP1; H186 in λPP) coordinating one of the metal ions in the catalytic centre is preceded by an absolutely conserved Gly; this residue is conserved in some archaeal but not in "conventional" eukaryotic PPPs (Figure 5 ; Additional file 2 ). Discussion In this report, we have documented the presence in different eukaryotic lineages of the genes that encode PPP phosphatases resembling those of bacterial origin, rather than "conventional" eukaryotic members of the family. Catalytic domains of these "bacterial-like" phosphatases are characterised by relatively conserved structure of the N-terminal subdomains, but very diverse organisation of the C-terminal subdomains, where conserved motifs and residues forming the active centre are separated by sequences of various length that share little or no similarity between different clades (Figure 6 ). In most cases, corresponding EST sequences could be detected, which confirms that these genes are expressed. Figure 6 Schematic diagram depicting organisation of the catalytic domains of the phosphatase groups discussed in this study . N-terminal subdomains (used in the alignment for the Neighbor-Net analysis, Figure 4) and C-terminal subdomains are shown in red and yellow, respectively. Positions of the conserved motifs in PPλ and the residues forming the active centre (underlined; shown according to ref. [19]) are shown. Positions of the LXXAXPXXH motif in plant Rhilphs and related phosphatases from Rhizobiales are indicated (green boxes). For more detailed information on the position of inserts in Rhilphs and Shelphs relative to the elements of the secondary structure, see Figures 1 and 2, respectively. The most conspicuous presence of "bacterial-like" PPPs has been detected in plants. Plants possess phosphatases from all three novel groups described in this work. Rhilphs are most closely related to PPP phosphatases from a number of α-proteobacteria, including purple photosynthetic bacteria and Rhizobiales . The absence of related sequences in eukaryotes other than land plants suggests that Rhilphs may have been acquired after plants started colonising land. Although bacterial lineage that could be the source for plant Rhilphs could not be unambiguously identified by phylogenetic reconstruction, Rhizobiales (or purple photosynthetic bacteria from which Rhizobiales are thought to have originated [ 33 , 34 ]) appear to be likely candidates. Indeed, Rhizobiales have transmissible chromosomal elements and some, like Agrobacterium , are able to integrate their plasmid genes into plant genome [ 35 ] or even transform animal cells [ 36 ], a situation that would be ideally suited for a horizontal gene transfer to occur. Interestingly, the presence of genes of rhizobial origin has been detected in plant parasitic nematodes [ 37 ]. Possible origin of plant Rhilphs from α-proteobacterial phosphatases is also supported by the presence in the enzymes of both groups of characteristic inserts in similar positions, which share some sequence similarity (see Results). Phosphatases of another group, designated as Shelphs, are found in green plants, in a red alga, in Apicomplexa , Trypanosomatidae , as well as in some fungi. The similarity between proteins from Apicomplexa and Trypanosomatidae and those from plants is well documented. Trypanosomatidae are related to photosynthetic euglenoids and are thought to have lost plastids secondarily [ 38 ]. Apicomplexan parasites have a relict plastid, originated from the engulfment of a red alga [ 39 ]. Thus, the presence of phosphatases shared by plants, red algae, Apicomplexa and Trypanosomatidae is not surprising and probably reflects the presence of Shelphs in a common ancestor of photosynthetic eukaryotes. The presence of chloroplast targeting sequence in SLP1 suggests a possible origin of Shelphs from a bacterial precursor of the chloroplast (however it should be noted that Shelphs are absent from cyanobacteria); alternatively, this sequence may have appeared secondarily. Protein Ser/Thr phosphorylation / dephosphorylation is essential for regulation of photosynthesis, and unidentified okadaic acid-insensitive protein phosphatases in chloroplasts have been reported [ 40 , 41 ]. SLP1 appears to be a good candidate for such a phosphatase. The origin of fungal Shelphs is unclear. Curiously, they are found in basidiomycetes and in an ascomycete S. pombe , but not in a number of other ascomycetes, whose genomes have been completed. Current data do not permit to discriminate between (i) the presence of Shelphs in a common ancestor of eukaryotes and their loss in such lineages as animals and many fungi, and (ii) independent acquisition of Shelphs from bacteria by an ancestor of photosynthetic eukaryotes and by fungi. Further sequencing of eukaryotic genomes may shed light on the evolutionary history of this PPP group. The third group of "bacterial-like" phosphatases detected in eukaryotes, designated here as Alphs, appears to be distantly related to bacterial diadenosine tetraphosphatases ApaH. Patchy distribution in several eukaryotic kingdoms suggests that Alphs were probably present in the common ancestor of eukaryotes, but were independently lost in many lineages, including insects, vertebrates and flowering plants. A characteristic modification of the conserved GDXXDRG motif shared only with ApaH further supports a suggestion that Alphs may represent a divergent branch of diadenosine tetraphosphatases, rather than protein phosphatases. However, relatedness of eukaryotic Alphs to bacterial diadenosine tetraphosphatases remains hypothetical, since Alph sequences are too divergent from ApaH, as well as from each other, to permit a reliable phylogenetic reconstruction. Diadenosine oligophosphates are considered as emerging signalling molecules in both intra- and intercellular signalling in eukaryotes [ 42 , 43 ]. In particular, human diadenosine oligophosphate hydrolase FHIT has been identified as a tumor suppressor [ 44 ]. It seems plausible that appearance of eukaryotic diadenosine oligophosphate hydrolases (structurally unrelated to the PPP phosphatases) may have made bacterial-type diadenosine tetraphosphatases redundant, leading to their loss in many eukaryotic lineages. It would be interesting to test experimentally whether Alphs are indeed diadenosine oligophosphatases. More generally, an important implication of our findings is that many eukaryotes possess PPP phosphatases with yet undetermined substrate specificity. Eukaryotic PPP phosphatases are generally considered as Ser/Thr specific in vivo , although they may be able to dephosphorylate phosphoTyr-containing substrates in vitro ( e.g . [ 45 , 46 ]). This is probably true for archaeal PPPs as well [ 5 ]. However, Ser/Thr specificity is not a feature of bacterial PPP phosphatases [ 7 - 9 , 13 , 47 - 49 ]. Thus, it would not be possible to predict substrate specificity of uncharacterised "bacterial-like" PPP phosphatases without experimental evidence. In particular, since Shewanella PPI is Tyr-specific [ 8 ], it would be interesting to determine substrate specificity of eukaryotic Shelphs. It is also worth noting that interest in tyrosine phosphorylation in plants has recently been stimulated by identification of plant Tyr phosphatase genes and by the finding that Tyr phosphorylation is involved in the regulation of stomatal movement (reviewed by Luan [ 50 ]). Three motifs, GDXHG, GDXXDRG and GNH(E/D) form the diagnostic signature of all PPP phosphatases [ 10 , 11 ]. We detected a (I/L/V)D(S/T)G motif, which appears to be a characteristic signature of "bacterial"-type PPPs. The existence of such a motif is striking per se , taking into account extreme structural diversity of bacterial PPP phosphatases. It indicates that (I/L/V)D(S/T)G was probably present as the fourth "universal" signature motif in the common ancestor of PPP phosphatases, and was lost in the common lineage of archaeal and "conventional" eukaryotic PPPs. An alternative possibility could be that the (I/L/V)D(S/T)G motif was acquired by a bacterium and propagated by lateral gene transfer, replacing the ancestral SAPNY-related motif. However this scenario seems less likely, since the (I/L/V)D(S/T)G motif is present, with minor variations, in virtually all bacterial phosphatases, despite their great diversity, and is replaced by SAPNY-related sequences only in archaeal and "conventional" eukaryotic PPPs. The Asp residue in the 2 nd position of (I/L/V)D(S/T)G is highly conserved and can only be replaced by Glu, indicating that the negative charge is essential. The presence of a highly conserved Gly in the 4 th position indicates that flexibility of the polypeptide chain is likely to be important. The crystal structure of bacteriophage λ phosphatase (PPλ; [ 19 ]) shows that the Asp residue of (I/L/V)D(S/T)G (Asp202) is just downstream of the β9 strand, which corresponds to the β12 strand in mammalian PP1. In PPλ, Asp202 is hydrogen bonded to a water molecule coordinated to one of the metal ions in the catalytic centre, which probably accounts for its conservation. In eukaryotic or archaeal PPPs, corresponding position is occupied by neutral residues (see Figure 5 ). It would be tempting to speculate that this difference in the region just downstream of the β9 (β12) may be responsible for a feature that is common to all "bacterial"-type but not to eukaryotic / archaeal PPPs. One such feature is the Ser/Thr specificity of the latter group. The Tyr residue of the SAPNY motif has been suggested to provide a bulky phenol ring in the β12-β13 loop, sufficient to sterically block access of phosphoTyr-containing substrates to the active site [ 32 ]. However this is unlikely to be the sole determinant of Ser/Thr specificity, since residues containing bulky aromatic rings (Tyr, Phe or Trp) are found in the same or adjacent positions in many bacterial phosphatases (Figure 5 ). Since the (I/L/V)D(S/T)G motif, absent in eukaryotic and archaeal PPPs, is involved in organisation of the catalytic centre [ 19 ], it is possible that this difference in the catalytic centre organisation may be one of the determinants of broad substrate specificity vs . Ser/Thr specificity. Conclusions So far, eukaryotic PPP phosphatases were considered as a well-defined monophyletic group of enzymes, specifically dephosphorylating phosphoSer and phosphoThr, while a much more structurally and enzymatically diverse PPP phosphatases were known to be present in prokaryotes. Our findings demonstrate that, in addition to "conventional" eukaryotic PPP Ser/Thr-specific protein phosphatases, many eukaryotes possess very diverse "bacterial-like" PPP phosphatases. Enzymatic characteristics, physiological roles and evolutionary history of these phosphatases have yet to be revealed. Methods Detection of PPP phosphatase-coding sequences Sequence similarity searches were conducted using BlastP or TBlastN [ 51 ] at NCBI [ 52 ] in the following databases: "non-redundant" (NR), "expressed sequence tags" (EST), "genomic sequence survey" (GSS) and "high-throughput genomic sequences" (HTGS). Additional Blast searches of the following databases were performed: finished and unfinished genomes of eukaryotes at the NCBI [ 53 ]; fungal genomes at the Broad Institute [ 54 ]; plant genomes at The Arabidopsis Information Resource (TAIR) [ 55 ]; Gene Index databases of tentative consensus sequences (EST assemblies) at The Institute for Genomic Research (TIGR) [ 56 ]; Chlamydomonas reinhardtii draft genome [ 57 ]. In all cases, reciprocal searches were used, i.e. hits retrieved by Blast searches were in their turn used as queries in the following Blast searches. Accuracy of gene prediction was examined by comparison of the retrieved sequences with translations of corresponding EST entries. In the absence of available ESTs, closely related sequences from other species were used. Taxonomy of the species from which the phosphatase sequences is given according to the NCBI taxonomy web site [ 58 ]. Phylogenetic analysis Multiple alignments were generated using CLUSTAL W [ 59 ] at Kyoto University Bioinformatics Centre [ 60 ] and edited manually. During manual editing, particular attention was paid to correct alignment of the PPP family signature motifs and other conserved residues known to constitute the catalytic site of PPP phosphatases. Phylogenetic tree construction by the neighbor-joining method [ 61 ] and bootstrap analysis were performed using the PHYLIP package, version 3.573 [ 26 ]. Maximum likelihood analysis was performed using TreePuzzle [ 28 ]. Possible alternative neighbor-joining based phylogenies were visualised using Neighbor-Net [ 29 ] as implemented in SplitsTree, version 4.β10 [ 62 ] Analysis of the primary structure The presence of signal peptides and targeting sequences was analyzed using TargetP [ 63 ] at the the Centre for Biological Sequence Analysis, Technical University of Denmark [ 64 ]. Abbreviations EST, expressed sequence tag, Alph, ApaH – like phosphatase; PPP, protein phosphatases of the P family; Rhilph (RLP), Rhizobiales / Rhodobacterales / Rhodospirillaceae – like phosphatase; Shelph (SLP), Shewanella – like phosphatase. Authors' contributions Both authors contributed equally to this work. Supplementary Material Additional File 1 Neighbor-Net analysis of the conserved N-terminal subdomains (starting 5 amino acid residues before conserved GDXHG and ending 25 residues after GNH(E/D) of 104 bacterial, archaeal and eukaryotic PPP phosphatases . This version of Figure 4 is the original SplitsTree file that can be viewed using SplitsTree, freely available for download (see Methods). Bootstrap values (out of 100 resamplings) are shown and can be highlighted by selecting corresponding alternative splits. The file also contains the alignment used for the analysis (Input). Click here for file Additional File 2 Distinct conserved motifs in the C-termini of bacterial and "bacterial-like" PPP phosphatases from eukaryotes as opposed to archaeal and eukaryotic PPP phosphatases . This is an expanded version of Figure 5 . Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC535813.xml
514605	A study comparing the actions of gabapentin and pregabalin on the electrophysiological properties of cultured DRG neurones from neonatal rats	Background Gabapentin and pregabalin have wide-ranging therapeutic actions, and are structurally related to the inhibitory neurotransmitter GABA. Gabapentin, pregablin and GABA can all modulate voltage-activated Ca 2+ channels. In this study we have used whole cell patch clamp recording and fura-2 Ca 2+ imaging to characterise the actions of pregabalin on the electrophysiological properties of cultured dorsal root ganglion (DRG) neurones from neonatal rats. The aims of this study were to determine whether pregabalin and gabapentin had additive inhibitory effects on high voltage-activated Ca 2+ channels, evaluate whether the actions of pregabalin were dependent on GABA receptors and characterise the actions of pregabalin on voltage-activated potassium currents. Results Pregabalin (25 nM – 2.5 μM) inhibited 20–30% of the high voltage-activated Ca 2+ current in cultured DRG neurones. The residual Ca 2+ current recorded in the presence of pregabalin was sensitive to the L-type Ca 2+ channel modulator, Bay K8644. Saturating concentrations of gabapentin failed to have additive effects when applied with pregabalin, indicating that these two compounds act on the same type(s) of voltage-activated Ca 2+ channels but the majority of Ca 2+ current was resistant to both drugs. The continual application of GABA, the GABA B receptor antagonist CGP52432, or intracellular photorelease of GTP-γ-S had no effect on pregabalin-induced inhibition of Ca 2+ currents. Although clear inhibition of Ca 2+ influx was produced by pregabalin in a population of small neurones, a significant population of larger neurones showed enhanced Ca 2+ influx in response to pregabalin. The enhanced Ca 2+ influx evoked by pregabalin was mimicked by partial block of K + conductances with tetraethylammonium. Pregabalin produced biphasic effects on voltage-activated K + currents, the inhibitory effect of pregabalin was prevented with apamin. The delayed enhancement of K + currents was attenuated by pertussis toxin and by intracellular application of a (Rp)-analogue of cAMP. Conclusions Pregabalin reduces excitatory properties of cultured DRG neurones by modulating voltage-activated Ca 2+ and K + channels. The pharmacological activity of pregabalin is similar but not identical to that of gabapentin. The actions of pregabalin may involve both extracellular and intracellular drug target sites and modulation of a variety of neuronal conductances, by direct interactions, and through intracellular signalling involving protein kinase A.	Background Gabapentin (Neurotonin ® ) and pregabalin (S(+)-3-isobutyl GABA) were both originally designed as GABA mimetics (Figure 1A ), with the intention that they would be able to cross the blood-brain barrier and interact with GABAergic systems and enhance GABA mediated inhibition. Although gabapentin appears to have diverse therapeutic utility in the treatment of pain disorders [ 1 ], psychiatric illnesses [ 2 ] and epilepsy [ 3 ], there is controversy regarding its molecular mechanisms of action. Whether the actions of gabapentin and pregabalin are mediated through GABAergic mechanisms or GABA receptors remains particularly contentious [ 4 - 6 ]. However, high affinity binding sites for gabapentin and pregabalin on distinct α 2 δ subunits of voltage-activated calcium channels have been identified and characterised [ 7 ]. For this reason voltage-activated Ca 2+ channels remain primary candidate sites of action for these novel anticonvulsant and antihyperalgesic drugs. Functional data from studies on gabapentin and pregabalin also support this contention. Specifically, both gabapentin and pregabalin inhibited hyperalgesia [ 8 , 9 ], attenuated evoked Ca 2+ influx into brain slices and reduced evoked transmitter release [ 10 ]. Additionally, gabapentin and pregabalin inhibited multiple firing of action potentials evoked by 300 ms depolarising current commands in cultured sensory DRG neurones [ 5 ]. Figure 1 Pregabalin inhibits Ca 2+ currents. A) Structure of GABA (γ-aminobutyric acid), gabapentin (1-(aminoethyl)cyclohexane acetic acid) and pregabalin (S(+)-3-isobutyl GABA). B & C) Traces of high voltage-activated Ca 2+ currents evoked from a holding potential of -90 mV by a depolarising step command to 0 mV showing inhibition by pregabalin (2.5 μM). B) Shows that pressure ejection of choline chloride extracellular solution does not induce inhibition of the Ca 2+ current but in the same neurone pregabalin does produce a response. Traces show a control Ca 2+ current (Ctrl), the current unaffected by application of choline chloride recording solution (ChCl), inhibition of current by 3 minutes application of pregabalin (PGB) and the current at 5 minutes recovery (Rec). C) Traces show a control Ca 2+ current (Ctrl), the inhibition of current after 3 minutes application of pregabalin (PGB) and full recovery of the current 5 minutes after removal of the drug pipette (Rec). D) Graph showing the distribution of inhibitory responses produced by 0.025 – 2.5 μM pregabalin. Each symbol represents a result from a different experiment. Our previous studies have focused on the inhibitory effects of gabapentin (0.25–25 μM) on whole cell voltage-activated Ca 2+ currents and K + stimulated Ca 2+ entry measured with fura-2. The cellular model systems used were primary cultures of dorsal root ganglion (DRG) neurones from 1–4 day old rats and differentiated F-11 cells (embryonic rat DRG × neuroblastoma hybrid cell line) [ 11 , 12 ]. In this present study the effects of pregabalin and gabapentin on the electrophysiological properties of cultured neonatal rat DRG neurones were measured with particular reference to Ca 2+ entry through high voltage-activated channels and enhancement of K + conductances. We had two specific aims for this project. The first was to determine whether gabapentin and pregabalin have the same mechanisms of action, by examining whether saturating concentrations of gabapentin and pregabalin act in an additive manner to attenuate Ca 2+ influx. The second aim was to further evaluate GABA receptors as target sites for these drugs in sensory neurones. This latter element to the study was conducted because it has been proposed that specific GABA B receptors with a gb1a-gb2 heterodimer composition are the sites of agonist activity of gabapentin. These specific receptors can be coupled to an inwardly rectifying K + channel and / or voltage-activated Ca 2+ channels to dampen neuronal electrical excitability [ 13 - 15 ]. However, in certain situations gabapentin and the GABA B receptor agonist, baclofen, have different actions. Weaver mutant mice (wv/wv) are insensitive to both gabapentin and baclofen, however in control littermates, Weaver control mice (+/+, wv/+), only baclofen evoked a K + current [ 16 ]. Results and discussion Actions of pregabalin on voltage-activated calcium currents The firing of multiple action potentials in response to a sustained depolarising current command is a property of a sub-population (under 20 %) of cultured DRG neurones. Application for 3 minutes, of either pregabalin (PGB; 2.5 μM) or gabapentin (GBP; 2.5 μM) reduced the frequency of action potential spikes evoked by 300 ms depolarising current step commands applied every 30 s [ 5 ]. Gabapentin attenuated repetitive action potential firing as measured by a reduction in the mean number of evoked action potentials during 300 ms depolarisations from 7 to 2 (n = 3, p < 0.01 ). Pregabalin (2.5 μM) reversibly reduced the number of action potentials during 300 ms depolarisations from 8 to 3 (n = 5, p < 0.01 ). However, pregabalin and gabapentin did not significantly alter the properties (amplitude, duration & threshold) of single action potentials evoked by 5 ms depolarising current commands. Consistent with our previous work [ 11 , 12 ], whole cell voltage-activated Ca 2+ currents (I Ca ) recorded from DRG neurones were reversibly attenuated (22 ± 11%; n = 7) by 2.5 μM gabapentin. Similarly, 3 minutes application of pregabalin (25 nM – 2.5 μM; Figure 1B & 1C ; table 1 ) inhibited the mean Ca 2+ current amplitude, measured at the peak of the inward current, and at the end of a 100 ms voltage step command to 0 mV. The inhibitory actions of pregabalin were not accompanied by any shift in the voltage-dependence of activation for I Ca or by any change in holding or leak currents. At least partial recovery of I Ca was observed 5 minutes after removal of the pregabalin-containing perfusion pipette (Figure 1B & 1C ). A very low concentration of pregabalin (0.25 nM) failed to produce significant inhibition of I Ca . However, no clear dose-dependent relationship was established for the inhibitory actions of pregabalin, and a major part of the voltage-activated Ca 2+ current was insensitive to the drug. It was also apparent that there was considerable variability in the sensitivity of the DRG neurones to any given dose of pregabalin. Some neurones did not respond to 2.5 μM pregabalin while in a few neurones this same concentration produced 60 % inhibition of I Ca (Figure 1D ). Table 1 Inhibitory actions of pregabalin on voltage-activated calcium currents. Peak control current amplitude (nA) Pregabalin Concentration (μM) Peak current in the presence of pregabalin (nA) Percentage inhibition and n value -1.52 ± 0.13 0.025 -0.98 ± 0.12 ** 31 ± 4 % (n = 8) -1.2 ± 0.14 0.25 -0.93 ± 0.18 * 22 ± 9 % (n = 6) -1.19 ± 0.11 2.5 -0.94 ± 0.09* 21 ± 3 % (n = 26) P < 0.001; P < 0.01 and P < 0.05. Each value is given ± S.E.M. Data shows inhibition but a lack of dose-dependent actions on I Ca of a wide range of pregabalin concentrations. Although varied responses were observed it was clear that a major component of I Ca was resistant to inhibition by pregabalin. Distinct voltage-activated Ca 2+ channels, defined by their alpha 1 subunit, have been suggested to be selective target sites for gabapentin and related drugs like pregabalin. With this in mind, the 1,4-dihydropyridine L-type Ca 2+ channel agonist Bay K8644 was used to determine whether pregabalin was inhibiting L-type channels. Previously, we found that in the continual presence of gabapentin, Bay K8644 enhanced I Ca [ 12 ]. In this present study similar results were obtained. After inhibiting part of the current with pregabalin (2.5 μM), Bay K8644 (1 μM) was applied with pregabalin and the enhanced I Ca was measured at its peak and at the end of a 100 ms voltage step command to 0 mV (Figure 2 ). The percentage increases in current seen with Bay K8644 in the presence of either gabapentin or pregabalin were slightly less than those seen under control conditions in cultured DRG neurones [ 17 ]. This is consistent with some L-type Ca 2+ current modulation by pregabalin and gabapentin in DRG neurones. Taken together these data suggest that neither gabapentin nor pregabalin are selectively inhibiting L-type I Ca in DRG neurones. This contrasts with the effects of gabapentin on cortical pyramidal neurones where inhibition of L-type Ca 2+ channels appears to be the predominant mechanism of action [ 18 ]. Figure 2 Bay K8644 enhanced pregabalin-insensitive current suggesting that L-type Ca 2+ channels are still available for modulation by the 1,4-dihydropyridine agonist. A) Bar chart shows data for mean calcium current amplitudes measured at the peak of the inward current (open bars) and at the end of a 100 ms voltage step command to 0 mV (solid bars, n = 6). Data under control condition (Con) in the presence of 2.5 μM pregabalin (PGB) and in the presence of both pregabalin (2.5 μM) and Bay K8644 (1 μM) are shown. B) The inset traces show voltage and Ca 2+ current records under control conditions, inhibition in the presence of 2.5 μM pregabalin (PGB) and enhancement of the Ca 2+ current during continued application of pregabalin with Bay K8644 present (Bay K & PGB). C) Shows the current inhibited by pregabalin, (obtained by subtracting the net current recorded in the presence of pregabalin from the net control current). D) Shows the additional current produced by Bay K8644, (obtained by subtracting the net current recorded in the presence of pregabalin from the net current recorded in the presence of both pregabalin and Bay K8644). Actions of pregabalin on calcium influx through voltage-activated channels, measured using fura-2 imaging Given the variable and rather modest but reversible inhibitory actions of pregabalin on I Ca we tested the actions of pregabalin on K + -evoked Ca 2+ influx using fura-2 imaging. An extracellular solution containing 30 mM K + was used to depolarise the DRG neurones and activate three consistent Ca 2+ transients [ 11 , 12 ]. Pregabalin (2.5 μM) was applied during the second K + -evoked depolarisation and it produced a mixture of reversible effects on the Ca 2+ transients. In 4 of 24 neurones the Ca 2+ flux was decreased by 54 ± 13 % ( p <0.05) but in 20 neurones from the same cultures pregabalin evoked a mean increase in Ca 2+ flux to 185 ± 20 % ( p <0.05) of the control. Raising the pregabalin concentration to 25 μM increased the proportion of inhibitory responses (n = 21 out of 49 neurones) but enhancement of Ca 2+ transients was still observed in the remaining 28 neurones (Figure 3 ). However, 250 μM pregabalin caused an increase in K + -evoked Ca 2+ influx in all neurones studied (n = 31). Although in some cells good recovery from pregabalin actions was observed (figure 3B ) the inhibitory effect was often not associated with recovery. This may reflect long lasting effects of pregabalin. Run-down in some cells can not be completely ruled out but under control conditions the mean level of run-down of three K + -evoked Ca 2+ transients was only 8.4 ± 1.5 % (n = 39). Similarly, the mean control level of increase in K + -evoked Ca 2+ transients was only 7.2 ± 1.3 % (n = 35). Figure 3 Pregabalin produced mixed actions on Ca 2+ influx evoked by 30 mM K + . A) Bar chart showing inhibition of Ca 2+ influx by pregabalin (25 μM). Data for the total Ca 2+ fluxes was normalised with respect to the first control response to K + (Ctrl). The second and third responses were obtained in the presence of pregabalin (PGB) and after washing away the pregabalin (Rec). B) Record of K + -evoked Ca 2+ transients showing partially reversible inhibition produced by 25 μM pregabalin (PGB). The period of stimulation is shown with open bars and PGB application with the filled bar. C) Bar chart showing enhancement of Ca 2+ influx by pregabalin (25 μM). Data for the total Ca 2+ fluxes were normalised with respect to the first control response to K + (Ctrl). The second and third responses were obtained in the presence of pregabalin (PGB) and after washing away the pregabalin (Rec). D) Record of K + -evoked Ca 2+ transients showing reversible enhancement produced by 25 μM pregabalin (PGB). To investigate the different responses to pregabalin, the sizes of cell somas were measured and compared. Although there is overlap, figure 4 shows that enhancement of K + -evoked Ca 2+ transients by pregabalin was mainly seen in neurones with larger cell somas and that in smaller neurones pregabalin produced inhibitory effects. Figure 4 Different responses to pregabalin were observed in different populations of cultured DRG neurones. Bar chart showing the distribution of neurones with different cell soma areas and the response to pregabalin. The distributions for intermediate and larger neurones where pregabalin (25 μM) increased K + -evoked Ca 2+ influx are shown in black bars. The distributions for small and some intermediate neurones where pregabalin (25 μM) attenuated K + -evoked Ca 2+ influx are shown in open bars. Ca 2+ -dependent conductances of DRG neurones are sensitive to ryanodine and Ca 2+ -induced Ca 2+ release has been reported in these neurones. Modulation of either Ca 2+ -induced Ca 2+ release and / or Ca 2+ homeostatic mechanisms might provide a mechanism by which pregabalin enhanced Ca 2+ transients in neurones with intermediate and large sized cell somas. This was investigated in two ways. Firstly, the actions of pregabalin on caffeine-evoked Ca 2+ transients were evaluated in nominally Ca 2+ -free extracellular conditions (NaCl-based solution with no added CaCl 2 ). Single caffeine (1 mM) responses were obtained from DRG neurones in either the absence or presence of 25 μM pregabalin. No differences in either amplitudes or durations of Ca 2+ transients were seen when 8 control caffeine responses were compared with 4 caffeine responses obtained in the presence of pregabalin (Figure 5A,5B ). The second approach was to explore a possible role of Na + / Ca 2+ exchange by bathing cells in choline chloride-based medium containing only 1 mM Na + . Under these conditions the contribution of the Na + / Ca 2+ exchanger to handling of intracellular Ca 2+ loads will be minimal. In choline chloride-based medium pregabalin produced both enhancement and inhibition of total Ca 2+ flux in 7 and 3 neurones respectively. Enhancement in Ca 2+ flux by pregabalin under these experimental conditions, suggest that inhibition of Na + / Ca 2+ exchange is not the main mechanism by which pregabalin enhances K + -evoked Ca 2+ flux (Figure 5C ). However, detailed analysis was made difficult by poor recovery of even the first Ca 2+ transient evoked in low extracellular Na + which does suggest that Na + / Ca 2+ exchange is an important homeostatic mechanism in DRG neurones. In conclusion these results indicate that modulation of Ca 2+ -induced Ca 2+ release and Ca 2+ homeostatic mechanisms do not account for pregabalin-induced enhancement of K + -evoked Ca 2+ flux in a population of DRG neurones. Figure 5 Pregabalin does not appear to modulate caffeine-evoked Ca 2+ release or Ca 2+ homeostatic mechanisms. A & B) Show example records of Ca 2+ transient evoked by caffeine (1 mM) applied to DRG neurones bathed in nominally Ca 2+ -free medium and measured using fura-2. Under these conditions the Ca 2+ transients are only due to mobilisation of Ca 2+ from intracellular stores. A) Illustrates a control caffeine response and B) shows a similar response to caffeine recorded in the presence of 25 μM pregabalin (PGB). C) Example trace showing an increase in K + -evoked Ca 2+ flux by 25 μM pregabalin (PGB) in a DRG neurone bathed with choline chloride-based extracellular medium. The periods of stimulation with 30 mM KCl are shown with open bars and pregabalin application is shown with a filled bar. D) Example trace showing an increase in K + -evoked Ca 2+ flux induced by 5 mM TEA in a DRG neurone bathed with standard NaCl-based extracellular medium. The periods of stimulation with 30 mM KCl are shown with open bars and TEA application is shown with a filled bar. Inhibitory modulation of potassium conductances could result in increased K + -evoked Ca 2+ transients. To test this alternative mechanism, a relatively low concentration, 5 mM, of tetraethylammonium (TEA) was applied with NaCl-based extracellular medium and DRG neurones were stimulated with 30 mM KCl. Attenuation of potassium conductances by TEA markedly increased the K + -evoked Ca 2+ flux in all eight DRG neurones studied, mimicking in part the action of pregabalin (Figure 5D ). Do pregabalin and gabapentin have additive effects on cultured dorsal root ganglion neurones? Pregabalin and gabapentin have related chemical structures and appear to have similar but usually modest inhibitory effects on Ca 2+ currents. Both pregabalin and gabapentin at a concentration of 2.5 μM produced a maximum level of current inhibition. Simultaneous application of pregabalin (2.5 μM) and gabapentin (2.5 μM) produced modest but significant inhibition of I Ca at the peak of the current and at the end of the stimulus (Figure 6A ). However, the percentage inhibition of I Ca produced was not significantly different for 2.5 μM gabapentin alone (22 ± 11%; n = 7), 2.5 μM pregabalin alone (21 ± 3%; n = 26) and 2.5 μM gabapentin and 2.5 μM pregabalin applied together (19 ± 2%; n = 9). Figure 6 Pregabalin and gabapentin do not have additive actions on cultured DRG neurones. A) Bar chart showing the mean Ca 2+ current amplitude recorded at the peak of the inward current (Peak) and end of a 100 ms voltage step command (End). Data is shown for measurements made under control conditions (Control), after 3 to 5 minutes application of both pregabalin (2.5 μM) and gabapentin (2.5 μM) (PGB + GBP) and after 5 minutes recovery (Recovery). B) Bar chart showing normalised data from Ca 2+ imaging experiments in which simultaneous application of both pregabalin (25 μM) and gabapentin (25 μM) (PGB & GBP) reversibly attenuated the total Ca 2+ flux. C) Record of K + -evoked Ca 2+ transients, showing the reversible inhibition produced by 25 μM pregabalin and 25 μM gabapentin (PGB + GBP, filled bar). In spite of the added complexity of mixed responses with pregabalin, experiments were carried out using fura-2 to measure K + evoked Ca 2+ flux and the effects of both ligands. Our previous experiments with gabapentin (25 μM) had only identified inhibitory effects using this experimental approach [ 11 , 12 ]. Simultaneous application of pregabalin and gabapentin (both at 25 μM) reversibly reduced the K + evoked Ca 2+ influx (Figure 6B & 6C ). Pregabalin (25 μM) and gabapentin (25 μM) together reduced the total Ca 2+ flux to 75 ± 5 % (n = 14) of the control response to 30 mM K + . Consistent with the electrophysiological experiments, this level of inhibition was similar to the inhibitory effects of pregabalin and gabapentin applied separately. The electrophysiological and fura-2 Ca 2+ imaging data show that no additive inhibitory effects were found during simultaneous application of saturating concentrations of gabapentin and pregabalin. The data therefore support the contention that both drugs have a closely related or a common site of inhibitory action. The data also indicate that in the modulation of Ca 2+ channels these drugs do not act in an additive manner even though a substantial proportion of current is resistant to both drugs. It should be emphasized however that pregabalin can reversibly enhance K + -evoked Ca 2+ transients, an effect not seen with gabapentin. Therefore there may be other additional actions of pregabalin that can be identified in Ca 2+ imaging experiments when all membrane conductances are intact. Does GABA receptor modulation alter responses to pregabalin in cultured dorsal root ganglion neurones? In this section of the study two strategies were used to evaluate the possible roles of GABA receptors in pregabalin responses in cultured DRG neurones. Firstly, pregabalin actions were studied in the presence of a saturating concentration of GABA; this initially activated all types of GABA receptor and then caused desensitization of these receptors. Secondly, the effect of a potent and selective GABA B receptor antagonist, CGP52432 [ 19 ] on pregabalin responses was evaluated. GABA (100 μM) evoked inward currents in a sub-population of cultured DRG neurones and induced a transient rise in intracellular Ca 2+ in 18 of 41 DRG neurones studied (Figure 7A & 7B ). The inward currents were due to the activation of GABA A receptor Cl - channels; with the equilibrium potential for Cl - under our recording conditions being close to 0 mV the chloride conductance results in an inward current. Although in vivo the equilibrium potential for Cl - in DRG neurones is variable, it is predicted to be between -40 mV and -20 mV because of Cl - loading into the intracellular environment (for review see [ 20 ]). Therefore GABA A receptor Cl - channel activation can produce a depolarisation of the resting membrane potential. Our data indicated that the GABA-evoked depolarisation was sufficient to result in voltage-activated Ca 2+ channel activity and produce a transient rise in intracellular Ca 2+ . In both neurones that responded to GABA and those that did not, subsequent application of pregabalin, produced either enhancement or inhibition of K + -evoked Ca 2+ influx (Figure 7A,7B,7C & 7D ; Table 2 ). These responses to pregabalin in the presence of GABA were no different in character to the pregabalin responses obtained in the absence of GABA. In neurones that showed a Ca 2+ transient in response to a GABA-evoked depolarisation, there was an apparent increase (to 83%) in the proportion of neurones that were inhibited by pregabalin. It is not clear why this is but it may reflect the sensitivity of Ca 2+ transients evoked in DRG neurones, which can only be evoked consistently in most DRG neurones by three depolarising stimuli. Thus the GABA-evoked responses may influence Ca 2+ homeostatic mechanisms and subsequent stimulated Ca 2+ entry. However, the critical observation from these experiments is that GABA receptor desensitisation does not prevent either Ca 2+ transient enhancement or inhibitory actions of pregabalin in neurones that were sensitive or insensitive to GABA. Figure 7 Activation and subsequent desensitisation of GABA receptors fails to prevent the modulation of voltage-activated Ca 2+ channels in cultured DRG neurones by pregabalin. A) Bar chart showing data obtained from neurones that responded to GABA (100 μM). All data are normalised with respect to the first Ca 2+ transient evoked by 30 mM KCl. Open bars show the relative responses to 100 μM GABA in cells (n = 3) where 25 μM pregabalin enhanced the Ca 2+ flux. Filled bars show responses in cells (n = 15) where 25 μM pregabalin inhibited the Ca 2+ flux. Data are shown for GABA responses (GABA), K + -evoked Ca 2+ transients under control conditions with GABA present (Ctrl), K + evoked Ca 2+ transients in the presence of GABA and 25 μM pregabalin (PGB) and on recovery in the continued presence of GABA (Rec). B) An example trace, showing a response to 100 μM GABA and Ca 2+ transients evoked by K + in the presence and absence 25 μM pregabalin. The long open bar shows the period of GABA application, the short open bars show K + stimulation and the filled bar the period of pregabalin application. C) Bar chart showing data obtained from neurones that did not responded to GABA (100 μM) but were continually bathed with GABA for the duration of the experiment. All data are normalised with respect to the first Ca 2+ transient evoked by 30 mM KCl. Data are shown for K + -evoked Ca 2+ transients under control conditions with GABA present (Ctrl), increased (n = 10; open bars) and decreased (n = 13; filled bars) K + -evoked Ca 2+ transients in the presence of GABA and 25 μM pregabalin (PGB) and on recovery (Rec). D) An example trace, showing no response to 100 μM GABA but responses to K + in the presence and absence of 25 μM pregabalin. The long open bar shows the period of GABA application, the short open bars show K + stimulation and the filled bar the period of pregabalin application. Table 2 Actions of pregabalin (25 μM) on K + -evoked Ca 2+ influx in the continual presence or absence of 100 μM GABA. GABA sensitivity Mean percentage of control Ca 2+ influx Number of neurones Proportion of Neurones Responders 110 ± 2 % 3 of 18 17 % Responders 77 ± 2 % 15 of 18 83 % Non-responders 121 ± 4 % 10 of 23 43 % Non-responders 81 ± 3 % 13 of 23 57 % Control 169 ± 24 % 28 of 49 57 % Control 85 ± 2 % 21 of 49 43 % Responders were those neurones in which GABA evoked a transient rise in intracellular Ca 2+ . Non-responder showed no change in fura-2 fluorescence ratio in response to GABA. Controls where neurones that pregabalin was applied to without GABA being present. The amplitudes of the GABA responses did not vary with the inhibitory or enhancing responses to pregabalin. The possibility of pregabalin-evoked inhibition of I Ca taking place through activation of G-protein coupled GABA B receptors, was then assessed. CGP52432 (10 μM) when applied alone had no effect on I Ca . In the presence of the GABA B receptor antagonist CGP52432 (10 μM), pregabalin (2.5 μM) evoked a 27 ± 3 % (n = 9) inhibition of the voltage-activated Ca 2+ current and complete recovery was observed 5 minutes after removal of the drug-containing pipette (Figure 8A ). In Ca 2+ imaging experiments 10 μM CGP52432 had no effect on the responses to 25 μM pregabalin. In the presence of CGP52432, 4 out of 16 neurones showed enhanced K + -evoked Ca 2+ influx (to 161 ± 24 % (n = 4) of control) in response to pregabalin and 12 neurones showed inhibition of Ca 2+ influx (64 ± 8 % (n = 12) of control) in response to pregabalin (Figure 8B & 8C ). Figure 8 The GABA B receptor antagonist CGP52432 had no effect on the actions of pregabalin. A) Bar chart showing the inhibitory effects of 2.5 μM pregabalin in the presence of 10 μM CGP52432 (PGB & CGP) on Ca 2+ current amplitude measured at the peak inward current (Peak) and at the end of a 100 ms voltage step command to 0 mV (End). The inset traces show the inhibition of the Ca 2+ current produced by 3 minutes application of pregabalin and CGP52432 (PGB + CGP) and partial recovery 5 minutes after removal of the drug perfusion pipette. B & C) Show example records of 25 μM pregabalin in the presence of 10 μM CGP52432 (PGB + CGP) modulating Ca 2+ flux evoked by 30 mM KCl. The open bars show the period of stimulation with K + and the filled bar the application of pregabalin in the presence of CGP52432 (PGB +CGP). Is there a role for G-proteins or G-protein coupled receptors in the responses to pregabalin in cultured dorsal root ganglion neurones? Previously, we found that the actions of gabapentin on I Ca were attenuated by pre-treating DRG neurones with pertussis toxin, an effect that did not appear to involve metabotropic GABA B receptors [ 12 ]. In this study several different approaches were taken to examine the influence of receptor and G-protein function in pregabalin actions. Firstly, the effects of a novel thiadiazole compound, SCH-202676, which inhibits ligand binding to a variety of G-protein coupled receptors (opioid, adrenergic, muscarinic and dopaminergic) were investigated. The selective and reversible action of SCH-202676 appears to involve allosteric modulation of both agonist and antagonist binding to G-protein coupled receptors [ 21 ]. Secondly, the potential influences of intracellular flash photolysis of caged GTP-γ-S and subsequent G-protein activation on pregabalin responses were also examined. SCH-202676 (10 μM) was applied to the intracellular environment via the patch pipette solution. After 5 minutes equilibration the whole cell Ca 2+ current had a mean amplitude of -0.54 ± 0.08 nA (n= 7). During this period there was a clear reduction in the inward current, although a steady state current level was reached. However, subsequent application of pregabalin (2.5 μM) resulted in a further significant reduction in Ca 2+ current amplitude to -0.43 ± 0.07 nA (n = 7, p <0.05). This represents a mean inhibition of 22 ± 5 % by pregabalin, a value very similar to the percentage inhibition produced by pregabalin in the absence of SCH-202676 (21 ± 3%). Therefore SCH-202676 had no effect on the pregabalin responses, indicating that pregabalin was not acting through an SCH-202676-sensitive G-protein coupled receptor (data not shown). Preliminary Ca 2+ imaging experiments were also conducted to determine whether extracellular SCH-202676 (10 μM, continually applied throughout the experiment) altered the effects of pregabalin (2.5 μM) on K + -evoked Ca 2+ transients. Under these experimental conditions pregabalin was found to markedly enhance or inhibit K + -evoked Ca 2+ transients (data not shown). This data again suggests that pregabalin was not acting through mechanisms that were sensitive to SCH-202676. Caged GTP-γ-S (100 μM) was applied to the intracellular environment via the patch pipette solution and after entering the whole cell recording configuration DRG neurones were left for 5 minutes to equilibrate. Once a stable control Ca 2+ current was obtained, three 200 V flashes of intense near UV light were applied to the neurone to achieve intracellular flash photolysis of the caged GTP-γ-S. We estimate that approximately 15 μM GTP-γ-S was photoreleased by the three flashes. As previously observed intracellular flash photolysis of caged GTP-γ-S reduced the amplitude and slowed the activation of I Ca [ 22 ]. The effects of photoreleased GTP-γ-S were attenuated by applying a large depolarising pre-pulse [ 23 ], which is consistent with voltage-dependent G-protein modulation of Ca 2+ channels (Figure 9A ). No recovery from photoreleased GTP-γ-S was observed during the period of the experiment. GTP-γ-S and pregabalin (2.5 μM) had additive inhibitory effects on I Ca . These additive actions of GTP-γ-S and pregabalin were apparent regardless of the order of application. One set of experiments was performed in which GTP-γ-S was photoreleased in the intracellular environment and then after stabilisation of the response pregabalin was applied (Figure 9B ). In another set of experiments pregabalin was applied first and after equilibration GTP-γ-S was photoreleased in the continual presence of pregabalin (Figure 9C ). When pregabalin (2.5 μM) was applied first it produced a mean inhibition in I Ca by 25 ± 5 % (n = 4), when applied after photorelease of GTP-γ-S, pregabalin produced a 20 ± 10 % (n = 5) inhibition of I Ca . Figure 9 Intracellular photorelease of GTP-γ-S did not alter the sensitivity of DRG neurones to pregabalin. A) Traces showing a Ca 2+ current attenuated and slowed by intracellular flash photolysis of caged GTP-γ-S (GTP-γ-S) and a Ca 2+ current showing voltage-dependent partial recovery of GTP-γ-S-evoked inhibition (GTP-γ-S + PP {pre-pulse to +120 mV}). B) Bar chart showing control (Ctrl) data, the inhibitory effects of both intracellular photorelease of GTP-γ-S (GTP-γ-S) and subsequent application of 2.5 μM pregabalin (GTP-γ-S & PGB) on the mean peak Ca 2+ current amplitude. Inset records show individual Ca 2+ currents recorded under control conditions, after intracellular photorelease of GTP-γ-S (GTP-γ-S) and after subsequent extracellular application of pregabalin for 3 minutes (GTP-γ-S + PGB). C) Bar chart showing control (Ctrl) data, the inhibitory effects of both 3 minutes extracellular application of 2.5 μM pregabalin (PGB) and subsequent intracellular photorelease of GTP-γ-S in the continued presence of pregabalin (PGB & GTP-γ-S) on the mean peak Ca 2+ current amplitude. Inset records show individual Ca 2+ currents recorded under control conditions, after application of pregabalin for 3 minutes (PGB) and after intracellular photorelease of GTP-γ-S (PGB & GTP-γ-S). None of the currents in this figure have been leak subtracted. However, neither pregabalin nor GTP-γ-S altered the leak current. Actions of pregabalin on voltage-activated potassium currents It was clear from the Ca 2+ imaging experiments that not all the cellular actions of pregabalin could be explained by the inhibition of voltage-activated Ca 2+ channels because both inhibition and enhancement in K + evoked Ca 2+ flux was observed. To investigate the actions of pregabalin further, its' effects on voltage-activated K + currents in DRG neurones were studied. Previously, Stefani and colleagues found that gabapentin modulated neuronal steady state non-inactivating K + currents [ 18 ]. Three minutes application of pregabalin (2.5 μM) had no significant effect on the voltage-activated K + current activated from a holding potential of -90 mV by a 100 ms voltage step command to 0 mV (Figure 10A ). However, raising the pregabalin concentration to 250 μM resulted in significant modulation of K + currents in DRG neurones. Pregabalin (250 μM) applied for 3 to 5 minutes produced an enhanced K + current in 11 out of 21 neurones but a modest inhibition of the outward current in 10 out of 21 neurones (Figure 10B and 10C ). DRG neurones are a heterogenous population of neurones and there is evidence that they express at least 6 diverse voltage-activated K + channels and that this expression is in part determined by the type of DRG neurone [ 24 ]. To distinguish between the two responses experiments were carried out on DRG neurones held at -30 mV to inactivate a proportion of the outward current. Under these conditions pregabalin inhibited the outward current in 9 out of 11 DRG neurones but still produced enhancement in 2 neurones. The level of inhibition increased at a holding potential of -30 mV to 30 ± 7% (n = 9) compared to 15 ± 4 % (n = 10) at -90 mV, (data not shown). Figure 10 Pregabalin modulates voltage-activated potassium currents in cultured DRG neurones. A) Traces showing that 2.5 μM pregabalin failed to significantly alter the outward K + current evoked by a 100 ms voltage step command from -90 mV to 40 mV. B) Current / voltage relationship showing enhancement of outward current following 3 minutes application of 250 μM pregabalin (filled circles = control data and open circles currents recorded in the presence of pregabalin (PGB); * = P < 0.05 & ** = P < 0.01). Inset traces show the control outward K + current activated at +40 mV and the enhanced K + current recorded in the presence of pregabalin (250 μM). C) Current / voltage relationship showing inhibition of outward K + current following 3 minutes application of 250 μM pregabalin (filled circles = control data and open circles K + currents recorded in the presence of pregabalin (PGB); * = P < 0.05). Inset traces show the control outward K + current activated at +40 mV and the attenuated K + current recorded in the presence of pregabalin (250 μM). Interestingly, in apparent contrast to pregabalin, 250 μM gabapentin was initially only found to inhibit K + currents in DRG neurones (Figure 11A,11B ). However, when studies into long-term (10–15 minutes) actions of gabapentin were investigated slowly developing outward current enhancement was identified. So similar to actions of pregabalin, biphasic responses to gabapentin were found with initial inhibition of K + currents and then a delayed enhancement of the outward current (Figure 11C ), [ 5 ]. Figure 11 Acute application of gabapentin produced modest inhibition of voltage-activated K + currents in cultured DRG neurones but long-term measurement of K + currents shows a delayed enhancement in outward current. A) Bar chart showing data for the acute (3–5 minutes) reversible inhibition of the mean K + current by 250 μM gabapentin (GBP). B) Traces showing a control outward K + current activated at 0 mV and the inhibited current activated at the same voltage after 3 minutes application of 250 μM gabapentin (GBP). C) Traces showing the biphasic response to 250 μM gabapentin. Illustrated are the control current, the inhibited current recorded after 5 minutes application of gabapentin (GBP) and the enhanced outward current measured 5 minutes after removal of the perfusion pipette containing gabapentin (Enhanced Current). Long-term modulation of K + current by pregabalin was then investigated. Dramatic increases in outward current were observed after a delay. This effect of pregabalin persisted even as pregabalin was removed from the extracellular environment, which may implicate a metabolic or intracellular signalling event in this response. The mean K + current amplitude at +40 mV increased from 3.37 ± 0.73 nA to 7.56 ± 1.10 nA (n = 11; p <0.01) 15 minutes after perfusion of 250 μM pregabalin. Figures 12A and 12B show an individual example record and trace of the delayed response to pregabalin. These responses did reverse but this took about 40 minutes with the response developing 3–10 minutes after the start of pregabalin application. No change in holding current or in the leak conductances were associated with the long-term effect of pregabalin and in the absence of pregabalin stable K + currents were recorded from DRG neurones for 16 minutes (n = 10). Figure 12 Pregabalin produced delayed enhancement of K + currents in cultured DRG neurones. A) Line graph showing a time course for the delayed action of pregabalin in a single neurone, the open bar shows the period of application of pregabalin (250 μM). Little effect of pregabalin was seen in this neurone until 5 minutes after removal of the pressure ejection pipette containing pregabalin. B) Inset traces show a control K + current activated at +40 mV, modest enhancement of the K + current after 5 minutes application of 250 μM pregabalin and the enhanced outward K + current recorded 9 minutes after application of pregabalin. Apamin mimicked the inhibitory action of pregabalin on K + current but did not prevent enhancement of the K + current by acute application of 250 μM pregabalin. C) Bar chart showing the modest inhibitory effect of apamin (1 μM) on K + current and the enhancement in K + current when pregabalin was applied in the continued presence of apamin (Apamin & PGB). D) Traces showing a control K + current, the inhibited K + current in the presence of 1 μM apamin and the enhanced K + current observed when apamin and pregabalin were applied together. Apamin prevented the inhibitory effect of pregabalin but not the enhancement of K + current. Several pharmacological experimental approaches were taken to characterise the biphasic actions of pregabalin and to determine the possible mechanism of action associated with the long-term K + current modulation. Apamin, a toxin from the honeybee, was used to block small conductance Ca 2+ -activated K + currents. Apamin (1 μM) caused a reduction in outward current at +40 mV from 2.64 ± 0.33 nA to 2.37 ± 0.39 nA, subsequent application of 250 μM pregabalin enhanced the current to 3.02 ± 0.39 nA (n = 11; p <0.01). No inhibitory effects were seen with pregabalin after treatment with apamin, suggesting that it is the apamin-sensitive Ca 2+ -activated K + channels that are inhibited by pregabalin (Figure 12C & 12D ). Gabapentin can be transported into cells via the L α-amino acid transporter and may achieve intracellular concentrations 10–20 times the levels in the extracellular environment [ 25 ]. Furthermore, actions through intracellular signalling pathways and specifically protein kinase A, have been proposed for gabapentin [ 12 ]. To examine whether the long-term enhancement of K + currents by pregabalin might involve intracellular sites of action, we applied pregabalin to the intracellular environments of DRG neurones via the patch pipette solution. Intracellular pregabalin (250 μM) evoked an increase in the K + current that started to develop within the first minute of entering the whole cell recording configuration. After an initial increase that could reflect equilibration with the KCl-based patch pipette solution containing pregabalin, a slow sustained significant increase in the outward K + current continued to develop over a 15 minute period (n = 10; p < 0.01; Figure 13A,13B ). No similar change in K + current was observed in control studies carried out in the absence of pregabalin (Figure 13C,13D ). Figure 13 Intracellular application of pregabalin enhanced K + currents in cultured DRG neurones. For these experiments pregabalin was included in the KCl-based patch pipette solution at a concentration of 250 μM. A) Bar chart showing the mean amplitudes of the K + current recorded immediately after entering the whole cell recording configuration (Initial I K ) and the maximum outward current recorded within 16 minutes of entering the whole cell recording configuration (Max I K ). B) Traces of the first K + current recorded using a patch pipette solution containing 250 μM pregabalin (Initial) and from the same cell the maximum outward K + current recorded with intracellular pregabalin (Max). C) Bar chart showing control data recorded from neurones not exposed to pregabalin. Illustrated are the mean amplitudes of the K + current recorded immediately after entering the whole cell recording configuration (Initial I K ) and the maximum K + outward current recorded within 16 minutes of entering the whole cell recording configuration (Max I K ). D) Traces of the first K + current recorded using the standard KCl-based patch pipette solution (Initial) and from the same cell the maximum outward current recorded under control conditions, within 16 minutes of entering the whole cell recording configuration (Max). Pertussis toxin pre-treatment results in the uncoupling of sensitive G-proteins from effector mechanisms, including voltage-activated Ca 2+ and K + channels. Additionally, pertussis toxin pre-treatment has previously been found to influence gabapentin actions on I Ca . Pertussis toxin pre-treatment (500 ng/ml; 16–18 hours) prevented pregabalin-induced long-term K + current enhancement in cultured DRG neurones (Figure 14A & 14B ). Figure 14 Pertussis toxin pre-treatment and intracellular (Rp)-cAMP prevented enhancement of K + current by pregabalin. A) Bar chart showing the mean amplitude of K + current recorded from DRG neurones pre-treated with pertussis toxin for 16–18 hours with 500 ng/ml (PTX Control) and after application of 250 μM pregabalin (PTX PGB), long term, up to 15 minutes monitoring of the current. B) Traces showing outward K + currents recorded from a DRG neurone pre-treated with pertussis toxin, prior to pregabalin application (PTX Control) and 10 minutes after application of 250 μM pregabalin. C) Bar chart showing mean data obtained from neurones containing (Rp)-cAMP (30 μM), which was applied to the intracellular environment via the patch pipette solution. Data shows the mean K + current amplitude recorded 1 minute and 5 minutes after entering the whole cell recording configuration (1 min; 5 min), after 5 minutes application of 250 μM pregabalin (PGB) and 10 minutes after removal of the pressure ejection pipette containing pregabalin. D) Traces from a single experiment showing the outward K + currents at 1 and 5 minutes after entering the whole cell recording configuration and allowing entry of 30 μM (Rp)-cAMP in the DRG neurones. Also shown are the K + current inhibited by 5 minutes application of 250 μM pregabalin (PGB) and the recovery of the K + current after the pressure ejection pipette containing pregabalin was removed. Intracellular (Rp)-cAMP prevented the delayed long-term enhancement of the K + current evoked by pregabalin. The possible role of cAMP-dependent protein kinase A (PKA) in the pregabalin-induced enhancement of K + current was then assessed using the inhibitor (Rp)-cAMP [ 26 ]. Intracellular application of 30 μM (Rp)-cAMP applied via the KCl-based patch pipette solution had no effect over a 5 minute equilibration period on voltage-activated K + current (n = 9). This may indicate that PKA has little or no basal or tonic activity on K + currents in DRG neurones in culture. However, when pregabalin was applied to DRG neurones loaded with (Rp)-cAMP no long-term enhancement of the outward current was observed. Under these recording conditions pregabalin still produced some inhibition of the K + current (Figure 14C & 14D ). These data provide evidence that the long-term modulation of voltage-activated K + channels by pregabalin is dependent on PKA-mediated phosphorylation. Conclusions In conclusion, these results indicate that pregabalin acts via the same basic mechanisms as gabapentin to inhibit voltage-activated Ca 2+ channels and that these inhibitory actions are independent of GABA receptor activation. Some features of the actions of pregabalin on intermediate size and large DRG neurones appear not to be seen with gabapentin. However, these distinct responses involve enhanced K + -evoked Ca 2+ transients by pregabalin rather than inhibition of Ca 2+ channels. Alpha 2 δ subunits of voltage-activated Ca 2+ channels remain a possible site of action for both pregabalin and gabapentin. Dooley and colleagues showed that both gabapentin and pregabalin attenuated K + -evoked norepinephrine release from rat neocortical slices by inhibiting P/Q-type Ca 2+ channels [ 10 ]. In cortical pyramidal neurones gabapentin predominantly works through L-type Ca 2+ channels [ 27 ]. Our work with Bay K8644 indicates that in cultured DRG neurones gabapentin [ 5 ] and pregabalin act predominantly independently of L-type channels. A recent study has also indicated that acting via G-protein coupled GABA B receptors, gabapentin selectively inhibited N-type Ca 2+ channels in hippocampal pyramidal neurones [ 15 ]. Interestingly, this selectivity of gabapentin seen in the hippocampus is different from the Ca 2+ channel modulation seen with the GABA B receptor agonist, baclofen. Our previous investigation using "toxityping" showed that gabapentin inhibited a variety of Ca 2+ channels in DRG neurones [ 11 ]. We conclude from all this work that gabapentin and pregabalin may have allosteric interactions with promiscuous α 2 δ Ca 2+ channel subunits. These α 2 δ subunits are not specifically combined with distinct pore forming Ca 2+ channel subunits (α 1 ) in all neurones and may therefore inhibit pharmacologically diverse Ca 2+ channels depending on the expression of Ca 2+ channel subunits in different neurones. However, transfection studies have shown that oocytes expressing Ca 2+ channels (Ca v 2.2) containing β1b and α 2 δ-1 or α 2 δ-2 subunits are insensitive to acute application of 50 μM gabapentin [ 28 ]. This work raises the possibility that other mechanisms independent of α 2 δ – Ca 2+ channel subunits are involved in the modulation of neuronal excitability by gabapentin. Additionally, the modulation of voltage-activated Ca 2+ channels by gabapentin and pregabalin acting through indirect mechanisms has been suggested. Candidates for such indirect mechanisms include the control of Ca 2+ channel functional expression [ 29 , 30 ] and metabotropic mechanisms linked to pertussis toxin sensitivity and PKA activation [ 12 ]. In hippocampal pyramidal neurones, uncoupling G-proteins from metabotropic receptors with N-ethylmaleimide prevents modulation of K + and Ca 2+ channels by gabapentin [ 15 ]. However, in DRG neurones it is not clear how pertussis toxin influences the actions of gabapentin and pregabalin but it may involve disruption of multi-protein complexes of G-proteins and ion channel subunits after ADP-ribosylation of Gα. The role of any G-protein coupled receptors in either gabapentin or pregabalin responses in DRG neurones are not supported by our investigations with SCH-202676. Furthermore, receptor and direct G-protein involvement in pregabalin effects on Ca 2+ channels also appears unlikely in DRG neurones. This is because in this study we found that intracellular flash photolysis of GTP-γ-S had no influence on pregabalin-evoked current inhibition and pregabalin did not alter responses to GTP-γ-S. Our findings are in agreement with those made in a previous study on cells expressing GABA B1a/B2 or GABA B1b/B2 receptor subunits. In this study GABA and the GABA B receptor agonist baclofen evoked [ 35 S]-GTP-γ-S binding responses but even at high concentrations both gabapentin and pregabalin did not [ 31 ]. The experiments designed to assess potential roles of GABA receptors in the responses to pregabalin showed that GABA receptor desensitization or the blockade of GABA B receptors did not attenuate pregabalin actions. These findings add to the published studies that indicate that gabapentin and pregabalin effects are independent of GABA receptor activation at least in some preparations [ 31 , 32 ]. Specifically, in cultured DRG neurones gabapentin was previously found not to activate a Cl - conductance to alter membrane potential and input resistance and the GABA B receptor antagonist saclofen did not influence the inhibitory action of gabapentin on I Ca [ 12 ]. Pregabalin has a higher affinity for α 2 δ subunits than gabapentin and in a number of studies is more effective. When applied alone pregabalin produced enhancement in Ca 2+ flux in some neurones, an effect not seen with gabapentin. So, it was surprising to see in the imaging experiments that when gabapentin and pregabalin were applied together they produced only inhibitory effects. This may reflect the allosteric interactions between these drugs and α 2 δ subunits of Ca 2+ channels as well as indirect modulation of Ca 2+ dependent conductances and interactions with components of cell signalling. There appear to be some inconsistencies in the pregabalin data when its actions on voltage-activated Ca 2+ currents are compared with effects on K + -evoked Ca 2+ transients. This may in part be due to effects of pregabalin on membrane conductances in addition to voltage-activated Ca 2+ currents. These effects may not be detected under voltage clamp recording conditions where Na + and K + currents are blocked to isolate Ca 2+ currents. In the imaging experiments all voltage-activated conductances are intact and may be modulated by pregabalin. Alternatively, pregabalin could potentially have a direct or indirect influence on Ca 2+ -induced Ca 2+ release from intracellular stores and alter Ca 2+ homeostatic mechanisms. These effects may be modulated differently in different sub-populations of DRG neurones and so produce the mixed responses recorded in small, intermediate and large neurones in this study. Thus different actions of pregabalin may be detected using fura-2 fluorescence imaging but these additional mechanisms may not influence the measurements of Ca 2+ currents. Although mixed responses to gabapentin were not previously identified, neither were clear effects on the amplitude of K + -evoked Ca 2+ transients apparent in DRG neurones [ 11 ], although they were seen in differentiated F-11 cells [ 12 ]. These apparent anomalies seen with pregabalin do not appear to be due to drug effects on Ca 2+ -induced Ca 2+ release or Ca 2+ homeostatic mechanisms such as Na + /Ca 2+ exchange. This conclusion was reached because pregabalin did not influence Ca 2+ transients evoked by caffeine and still enhanced K + -evoked Ca 2+ transients in choline chloride-based (low Na + ) extracellular solution. The mixed responses to pregabalin seen in the Ca 2+ imaging experiments prompted us to investigate the actions of pregabalin on voltage-activated K + currents, which could be modulated to cause an increase in Ca 2+ influx. This speculation was supported by the finding that application of the K + channel inhibitor, TEA, enhanced K + -evoked Ca 2+ transients. Stefani and colleagues have reported inhibition of outward K + currents by gabapentin [ 18 ]. Additionally, in rat hippocampal and human neocortical brain slices gabapentin has been shown to inhibit K + -evoked [ 3 H]-noradrenaline release. The activation of K ATP channels is implicated in these responses because not only does glibenclamide, a K ATP channel antagonist, attenuate the gabapentin response but also pinacidil, a K ATP channel agonist, mimics the response and did not have additive effects with gabapentin [ 33 ]. Mixed or biphasic responses to gabapentin and pregabalin were seen, with both inhibition and enhancement of K + currents observed. The inhibitory actions of both gabapentin and pregabalin on K + currents recorded from DRG neurones may not reflect a direct action of these drugs on K + channels. The effects of apamin, which prevented the inhibition of outward current by pregabalin indicated that small conductance Ca 2+ -activated K + channels were involved in the response. This is consistent with the effects of gabapentin seen in isolated cortical neurones [ 18 ]. The pregabalin inhibitory action on voltage-activated Ca 2+ channels may underlie the inhibition of outward K + current. A reduction in Ca 2+ influx would lead to reduced activation of Ca 2+ -activated K + channels and therefore a smaller outward current. If this indirect effect of pregabalin resulted in a disproportionate reduction in Ca 2+ -activated K + conductances this may result in prolonged depolarisation. In turn a prolonged depolarisation may maintain activation of Ca 2+ channels that are insensitive to pregabalin and an increase in Ca 2+ influx as seen in a sub-population of larger DRG neurones in the Ca 2+ imaging experiments. It is not clear why gabapentin, which produced modest inhibition of K + currents, did not also produce enhancement of K + -evoked Ca 2+ transients. It may be explained by a balance between inhibition of Ca 2+ influx through voltage-activated channels and modulation of Ca 2+ flux through inhibition of K + conductance. Consistent with this hypothesis, gabapentin appears to be less effective than pregabalin at modulating K + -evoked Ca 2+ transients in DRG neurones. This is supported by our previous study that showed that gabapentin did not significantly alter the peak Ca 2+ transients [ 11 ]. The delayed enhancement of voltage-activated K + currents by gabapentin and pregabalin is a powerful mechanism for reducing cell excitability. The slow development of this response and its gradual decline suggested that intracellular signalling events were involved. Pregabalin produces enhancement of K + currents when applied either inside or outside DRG neurones. The delay in the development of these responses is less with intracellular application of pregabalin, suggesting that it may have an intracellular site of action. This effect may also depend on drug uptake into cells via L α-amino acid transporters and the presence and activity of these transporters may greatly influence cell sensitivity to gabapentin and pregabalin. Although it is not clear how gabapentin and pregabalin might activate PKA, it is a candidate target site. Our previous work showed that inhibition of Ca 2+ currents by gabapentin was sensitive to cAMP analogues that activated or inhibited PKA [ 12 ]. Similarly, in this present study (Rp)-cAMP blocked enhancement of K + currents by pregabalin. In the literature there are a number of reports of PKA altering neuronal excitability and modulating K + conductances. PKA is involved in pain signalling, playing a role in prostaglandin-induced activation and sensitization of DRG neurones [ 34 ]. PKA activity attenuates KV3.2 channel currents [ 35 ] and A-type current [ 36 ] and is involved in the inhibition of K + conductances by prostaglandin E-2 [ 37 ]. Our results are not explained by these findings but PKA has also been implicated in presynaptic inhibition of GABA release [ 38 ], large conductance calcium – and voltage-activated K + channel activity [ 39 ] and cannabinoid receptor mediated modulation of ion channels [ 40 ]. In the context of our project, of particular interest is the biphasic modulation in retinal cones of voltage dependent K + and Ca 2+ channels by a synthetic cannabinoid receptor agonist and the sensitivity of these responses to Wiptide, a PKA inhibitor [ 40 ]. However, the G-protein pharmacology is different in the cone cells suggesting different PKA activation pathways in DRG neurones exposed to pregabalin. There appears to be a considerable number of apparently conflicting effects mediated by PKA, although a number of different preparations have been studied. The concept of conditional protein phosphorylation such that initial phosphorylation state affects the sensitivity of different effector targets to subsequent activation [ 39 ] could determine responses to gabapentin and pregabalin acting through PKA. A future challenge will be to determine how PKA plays roles in activation and sensitization of DRG neurones and also plays a role in gabapentin and pregabalin responses to dampen down electrical excitability in the same neurones. Perhaps answers will come when we have a better understanding of the changes in neuronal phenotype that develop with pain disorders? An interesting feature of some actions of gabapentin and pregabalin is that their effects often outlast the period of drug delivery. Maintained intracellular signalling effects of these drugs and the delayed responses involving PKA modulation of Ca 2+ and K + conductances may underlie this characteristic. Gabapentin has previously been shown to attenuate high frequency action potential firing both in CNS neurones [ 41 ] and in cultured DRG neurones [ 5 , 12 ]. Pregabalin produces the same effect, dramatically but reversibly reducing the number of action potentials fired in response to 300 ms depolarising current commands. The underlying mechanisms involved in these reductions in electrical excitability could involve inhibition of voltage-activated Ca 2+ channels with resulting reduced activation of Ca 2+ -activated K+ currents and effects on voltage-dependent ion channel availability. Although the delayed enhancement of voltage-activated K + channels could contribute, this effect appears to develop over a longer period. These actions may contribute both to anticonvulsant effects of gabapentin and pregabalin as well as their therapeutic actions in pain disorders. It is particularly worth noting that the inhibition of Ca 2+ influx is most consistently seen in small diameter cultured DRG neurones that are likely to be the pain fibres. Our data adds to previous work that has assessed the actions of gabapentin and pregabalin on K + -evoked neurotransmitter release in CNS neurones [ 10 ] and the pharmacology of pain in whole animal models [ 8 , 9 , 42 - 44 ]. The present work and these previous studies suggest that gabapentin and pregabalin mostly act through the same basic mechanisms. These mechanisms in cultured DRG neurones appear to involve allosteric inhibitory modulation of Ca 2+ channels through drug interactions with α 2 δ subunits, reduced activation of Ca 2+ -dependent conductances and modulation of both Ca 2+ and K + channels through metabotropic mechanisms that involve PKA. Interestingly, both gabapentin and pregabalin inhibit release of sensory peptides (substance P and CGRP) this may provide a mechanism of action down-stream of ion channel modulation. This effect is only observed after inflammation and may contribute to their analgesic properties of gabapentin and pregabalin [ 45 ]. Ca 2+ channels in DRG neurones are targets for antinociceptive agents but the pharmacology of these channels remains to be fully exploited in pain therapy [ 46 , 47 ]. Further studies on changes in ion channel subunit expression and alterations in intracellular signalling pathways in pain disorders may in the future open up new therapeutic opportunities. Methods Cell culture One to four-day old Sprague-Dawley rats were decapitated and dorsal root ganglia removed. DRG neurones were dissociated enzymatically (0.125% collagenase for 13 minutes and 0.25% trypsin for 6 minutes) and mechanically (trituration). Primary cultures of DRG neurones were plated on lamin-polyornithine coated coverslips and bathed in Ham's F-14 culture medium (Imperial Laboratories) containing 10% horse serum (Gibco), low NGF (20 ng/ml; Sigma), NaHCO 3 (14 mM), streptomycin (50 μg/ml) and penicillin (50 IU/ml). The cultures were maintained for up to two weeks at 37°C in humidified air with 5% CO 2 . Cultures were re-fed with fresh media after 5 days. In some experiments DRG neurones were pre-treated with pertussis toxin (500 ng/ml; for 18 hours) to ADP-ribosylate the α subunits of certain G-proteins. This prevents pertussis toxin-sensitive G-protein being activated through a range of G-protein coupled receptors and inhibits coupling to voltage-activated Ca 2+ channels and other potential effectors [ 12 , 48 ]. Electrophysiology and calcium imaging The whole cell patch clamp recording method and fura-2 Ca 2+ imaging were used to measure the inhibitory actions of pregabalin and gabapentin on Ca 2+ entry through voltage-activated channels. Initially, multiple firing properties of a sub-population of DRG neurones were studied using a patch pipette solution containing in mM: KCl, 140; EGTA, 5; CaCl 2 , 0.1; MgCl2, 2.0; HEPES, 10.0; ATP, 2.0. The extracellular solution containing in mM: NaCl, 130; KCl, 3.0; CaCl 2 2.0; MgCl 2 , 0.6; NaHCO 3 1.0, HEPES 10.0 and glucose 5.0. For recording Ca 2+ channel currents the patch pipettes were filled with CsCl-based solution containing in mM: 140 CsCl, 0.1 CaCl2, 5 EGTA, 2 MgCl2, 2 ATP, 10 Hepes. The pH and osmolarity of the patch pipette solutions were corrected to 7.2 and 310–320 mOsm.l -1 with Tris and sucrose. The extracellular bathing solution used to study Ca 2+ currents contained in mM: 130 choline chloride, 2 CaCl2, 3 KCl, 0.6 MgCl2, 1 NaHCO3, 10 HEPES, 5 glucose, 25 tetrethylammonium chloride, 0.0025 tetrodotoxin (Sigma). The pH and osmolarity of this extracellular bathing solution was corrected to 7.4 and 320 mOsml -1 with NaOH and sucrose respectively. The recording solutions used in these experiments were designed to attenuate voltage-activated Na + and K + currents and isolate voltage-activated Ca 2+ currents. The range of values for the series resistance under our recording conditions was from ~8 to 15 MΩ. Pregabalin and other drugs were applied to the extracellular environment by low-pressure ejection from a blunt pipette positioned about 50–100 μm away from the cell being recorded. Voltage-activated Ca 2+ currents were evoked by 100 ms voltage step commands applied every 30 s. Similar protocols were used to study the actions of pregabalin on voltage-activated K + currents but NaCl-based extracellular bathing solution and KCl-based patch pipette solutions were used. In some experiments GTP-γ-S was photoreleased inside DRG neurones. Caged GTP-γ-S (100 μM; Molecular Probes) was included in the CsCl-based patch pipette solution. After obtaining control Ca 2+ currents intracellular flash photolysis was achieved by flashing the neurone being studied (three 200 V flashes to produce ~15μM GTP-γ-S) using a XF-10 xenon flash lamp with a UG11 bandpass filter (Hi-Tech Scientific; [ 49 ]) All voltage-activated Ca 2+ and K + currents had scaled linear leakage and capacitance currents subtracted to obtain values for the net inward Ca 2+ current or net outward K + current. Data are given as mean ± standard error of the mean (s.e.m.) values and statistical significance was determined using a paired or independent Student's t test as appropriate. For Ca 2+ imaging in cultured DRG neurones the cultures were incubated for 1 hour in NaCl-based extracellular solution contained (in mM): NaCl, 130; KCl, 3.0; MgCl 2 , 0.6; CaCl 2 , 2.0; NaHCO 3 , 1.0; HEPES, 10.0; glucose, 5.0 and fura-2AM, 0.01; (Sigma, 1 mM stock in dimethylformamide). The pH was adjusted with NaOH to 7.4 and the osmolarity to 310–320 mOsm with sucrose. The cells were then washed for 10–20 minutes with NaCl-based extracellular solution to remove the extracellular fura-2AM and this period allowed cytoplasmic de-esterification of the Ca 2+ sensitive fluorescent dye. The cells were constantly perfused with NaCl-based extracellular solution (1–2 ml/min) and viewed under an inverted Olympus BX50WI microscope with a KAI-1001 S/N 5B7890-4201 Olympus camera attached. Some Ca 2+ imaging experiments were carried out using a Ca 2+ -free NaCl-based solution (as standard NaCl-based extracellular solution but with no added CaCl 2 ) or choline chloride-based solution (as standard NaCl-based extracellular solution but with choline chloride in place of NaCl). The fluorescence ratiometric images from data obtained at excitation wavelengths of 340 nm and 380 nm were viewed and analysed using OraCal pro, Merlin morphometry temporal mode (Life Sciences resources, version 1.20). The DRG neurones were stimulated with NaCl-based extracellular solution containing high K + (30 mM), which produced depolarisation, activation of voltage-gated Ca 2+ channels and large transient increases in intracellular Ca 2+ . Three consistent transient increases in intracellular Ca 2+ could be obtained in a single experiment on cultured DRG neurones [ 11 ]. The actions of pregabalin and gabapentin (2.5–250 μM) were investigated on the response to the second stimulus in DRG neurones. The actions of pregabalin and gabapentin on the Ca 2+ transient amplitude, duration at 1/2 peak amplitude and total Ca 2+ flux were measured. All experiments were conducted at room temperature and data are expressed as means ± s.e.m. List of abbreviations DRG, Dorsal root ganglion. cAMP, Cyclic adenosine monophosphate GABA, Gamma aminobutyric acid GBP, Gabapentin GTP-γ-S, Guanosine 5'-o(3-thio)triphosphate I Ca , Calcium current NGF, Nerve growth factor PGB, Pregabalin PKA, Protein kinase A PP, Pre-pulse (Rp)-cAMP, (R)-adenosine, cyclic 3', 5'-(hydrogenphosphorothioate) triethylammonium Authors' contributions All the authors of this manuscript contributed to electrophysiological and Ca 2+ imaging experiments. DM and RHS designed and conducted the experiments on K + currents and wrote the first draft of this manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC514605.xml
439783	Genomic Analysis of Mouse Retinal Development	The vertebrate retina is comprised of seven major cell types that are generated in overlapping but well-defined intervals. To identify genes that might regulate retinal development, gene expression in the developing retina was profiled at multiple time points using serial analysis of gene expression (SAGE). The expression patterns of 1,051 genes that showed developmentally dynamic expression by SAGE were investigated using in situ hybridization. A molecular atlas of gene expression in the developing and mature retina was thereby constructed, along with a taxonomic classification of developmental gene expression patterns. Genes were identified that label both temporal and spatial subsets of mitotic progenitor cells. For each developing and mature major retinal cell type, genes selectively expressed in that cell type were identified. The gene expression profiles of retinal Müller glia and mitotic progenitor cells were found to be highly similar, suggesting that Müller glia might serve to produce multiple retinal cell types under the right conditions. In addition, multiple transcripts that were evolutionarily conserved that did not appear to encode open reading frames of more than 100 amino acids in length (“noncoding RNAs”) were found to be dynamically and specifically expressed in developing and mature retinal cell types. Finally, many photoreceptor-enriched genes that mapped to chromosomal intervals containing retinal disease genes were identified. These data serve as a starting point for functional investigations of the roles of these genes in retinal development and physiology.	Introduction The vertebrate retina is a model system for studying both the development and function of the central nervous system (CNS). Only six major types of neurons develop within the retina, along with a single type of glial cell ( Rodieck 1998 ). These cells are readily distinguished from one another by morphology and laminar position within the retina. Birthdating studies have shown that retinal cell types are generated in overlapping intervals, with ganglion cells, cone photoreceptors, amacrine cells, and horizontal cells generated prior to birth, and bipolar neurons and Müller glia generated after birth in mice ( Sidman 1961 ; Young 1985 a, 1985 b). Rod photoreceptors, the most abundant retinal cell type in the retina, are born both pre- and postnatally, with a peak of genesis coincident with the day of birth in the mouse. These birthdating studies, together with heterochronic coculture experiments ( Belliveau and Cepko 1999 ; Belliveau et al. 2000 ; Rappaport et al. 2001 ), heterochronic transplantation ( Rappaport et al. 2001 ), and lineage analysis ( Turner and Cepko 1987 ; Holt et al. 1988 ; Wetts and Fraser 1988 ; Turner et al. 1990 ), have given rise to the competence model of retinal cell fate specification ( Cepko et al. 1996 ). The competence model states that the intrinsic ability of mitotic retinal progenitor cells to produce a particular cell fate changes continually through development. A cell produces only a single fate, or a subset of fates, at any one time even though lineage analysis has shown that most retinal progenitors have the potential to produce many or all fates over the entire period of retinal development. Interestingly, even at one time in development, retinal progenitor cells show heterogeneity in their developmental competence ( Alexiades and Cepko 1997 ; Belliveau and Cepko 1999 ; Belliveau et al. 2000 ; Rapaport et al. 2001 ). In addition to the contribution of intrinsic determinants of cell fate specification, the fates chosen by the daughters of a retinal progenitor may be influenced by extrinsic factors ( Watanabe and Raff 1990 ; Altschuler et al. 1993 ; Kelley et al. 1994 ; Levine et al. 1997 , 2000 ; Belliveau and Cepko 1999 ; Young and Cepko 2004 ). Finally, certain aspects of retinal cell fate choice, such as the specification of at least some rod and bipolar cells, appear to occur in postmitotic cells ( Ezzeddine et al. 1997 ). Although the competence model was formulated to explain cell fate choice in the retina, it is clear that cell specification in many other regions of the developing nervous system—including neural crest ( Selleck and Bronner-Fraser 1996 ), spinal cord ( Ericson et al. 1996 ), and cerebral cortex ( McConnell 1988 ; Qian et al. 2000 )—involve changes in progenitor competence over time, frequently resulting in altered sensitivity to extrinsic factors. The model of temporal changes in competence is strongly supported by recent elegant studies of Drosophila CNS development ( Isshiki et al. 2001 ; Pearson and Doe 2003 ), where a temporal order of transcription factor expression was found to set the context of cell fate determination. The fundamental similarity among these systems nonetheless accommodates mechanistic differences. The situation in the retina, where early progenitor cells cannot be induced to adopt late fates and vice versa (although see James et al. [2003] for a possible exception to this rule), is distinct from the progressive developmental restriction that is seen in the cerebral cortex, where early cortical progenitor cells are competent to generate cells of upper (late-born) and lower (early-born) layers of the cortex, but become restricted to generating only late-born fates as development proceeds ( Desai and McConnell 2000 ). It is not known what genes mediate changes in progenitor competence during retinal development. Likewise, it is not known to what extent individual retinal progenitor cells from a single time point differ in their developmental competence from one another, although a few genes that are expressed in distinct subsets of progenitor cells have been found ( Austin et al. 1995 ; Matter et al. 1995 ; Alexiades and Cepko 1997 ; Dyer and Cepko 2000 a; Brown et al. 2001 ; Wang et al. 2001 ). Moreover, the genes that regulate the differentiation of any retinal cell type following commitment to a specific fate are generally poorly understood, although a number of transcription factors such as Crx, Nrl, and NR2E3 ( Chen et al. 1997 ; Furukawa et al. 1997 a; Haider et al. 2001 ; Mears et al. 2001 ) are clearly important in rod development. Unbiased, comprehensive expression profiling studies offer the possibility of identifying the molecular components and networks underlying these processes, as well as revealing target genes involved in intermediate and terminal differentiation of individual retinal cell types. We have used serial analysis of gene expression (SAGE) to profile gene expression during the development of the mouse retina ( Blackshaw et al. 2001 ). SAGE, which provides an unbiased and nearly comprehensive readout of gene expression, is conceptually very much like expressed sequence tag (EST) sequencing, with the difference being that concatenated libraries of short sequence tags derived from each cDNA found in the sample of interest are sequenced ( Velculescu et al. 1995 ). By identifying genes that show dynamic expression via SAGE and testing the cellular expression of these genes via in situ hybridization (ISH), we can identify genes that potentially regulate proliferation, cell fate determination, and cell differentiation. Furthermore, by examining SAGE libraries made from adult tissue, genes that are specifically expressed in mature cell types can be identified. By employing both SAGE-based expression profiling and large-scale ISH analysis to determine cellular expression of developmentally dynamic transcripts, we aim to combine the strengths of these two approaches and obtain a detailed picture of molecular events taking place during development of the retina. The laminar structure of the retina, which allows identification of the major cell types expressing a transcript under examination, makes large-scale ISH particularly informative relative to many other regions of the nervous system. Results/Discussion Summary of SAGE Data SAGE was conducted on mouse retinal tissue taken at 2-d intervals from near the start of neurogenesis at embryonic day 12.5 (E12.5) to nearly the end of neurogenesis at postnatal day 6.5 (P6.5). In addition, libraries were made from P10 wild-type mice and the adult retina. Previously generated SAGE data from the microdissected outer nuclear layer (ONL) of the retina, which comprises roughly 97% rod photoreceptors, from retinal tissue from mice that were deficient for Crx (littermates of the wild-type P10 mice), and from adult hypothalamus were also incorporated into the analysis ( Blackshaw et al. 2001 ). All of these libraries were sequenced to a depth of 50,000–60,000 SAGE tags each 14 bp long. Table S1 lists the number of distinct tags found in the 12 retina1 libraries and their abundance levels, along with the number of tags that do not match any known transcript. While 10% of all unique tags found twice or more in the 12 libraries did not correspond to an identified transcript, only 3% of the tags found five times or more did not match a known transcript ( Table S1 ). Table S2 lists all individual tag levels in each of these retinal libraries, along with data from a number of other publicly available nonretinal mouse libraries. We have also created a database, accessible at http://134.174.53.82/Cepko/ , that is searchable by gene name, SAGE tag sequence, accession number, genome location, or UniGene number. It displays all SAGE tags and their levels, as well as ISH images (see below). The accuracy of the SAGE data was assessed by comparing the 15,268 SAGE tags from E14.5 retina to an unnormalized and unsubtracted set of 15,268 ESTs generated by another research group from E14.5 mouse retina of a different strain ( Mu et al. 2001 ). An r-value of 0.65 (see Figure S1 ) was obtained that compares well with SAGE expression profiles obtained in similar tissues but from different individuals that were not strain-matched ( Blackshaw et al. 2003 ). Analysis of SAGE Tag Expression Patterns in Developing Retina Using Cluster Analysis In order to determine whether the temporal pattern of a gene's expression during retinal development might predict its cellular site of expression or its molecular function, clusters of coexpressed genes were assembled. The ten libraries obtained from wild-type total retina were analyzed by cluster analysis using a new Poisson model–based k -means algorithm designed specifically for SAGE data ( Cai et al. 2004 ) (see Materials and Methods for a full description of the algorithm and the protocols used). The results for a 24-cluster analysis are shown graphically in Figure 1 . Table 1 provides a list of previously characterized genes corresponding to tags within these clusters, the number of genes associated with tags within each cluster that were tested via ISH, and select functional categories of genes that were enriched in specific clusters. Table S3 lists all SAGE tags used in the analysis and their corresponding cluster assignments. Figure 1 Median Plot of SAGE Tag K- Means Cluster Analysis Using 24 Clusters Tags present at greater than 0.1% in one or more of the ten wild-type total retina libraries are considered. SAGE libraries are plotted on the x-axis, and tag abundance, plotted as a fraction of the total tags for a gene in the library in question, is shown on the y-axis. A full list of tags and their abundance levels used for the analysis is detailed in Table S3 . Table 1 Summary of SAGE Tag K- Means Cluster Data Tags present at greater than 0.1% in one or more of the ten wild-type total retina libraries were considered. The number of SAGE tags in each cluster is shown, along with the number and percentage of SAGE tags in each cluster that match genes whose expression was examined by ISH in developing retina. Selected genes that were previously examined in the context of retinal development are indicated. P -values for GO categories that are overrepresented in individual clusters were calculated using EASE ( Hosack et al. 2003 ) and represent raw EASE scores for the categories in question Virtually every gene previously reported to regulate retinal development was detected in this analysis and showed dynamic expression during development. Several of these transcripts were found at high levels during their period of peak expression. For instance, NeuroD1 —which regulates rod photoreceptor survival, as well as possibly rod differentiation ( Morrow et al. 1999 ; Wang et al. 2001 )—makes up 0.34% of all retinal mRNA at P4.5. In the case of genes previously shown to be required for production of certain cell types in the developing retina, such as Ath5 and Chx10 —which are required for ganglion cell and bipolar neurons , respectively ( Burmeister et al. 1996 ; Morrow et al. 1999 ; Brown et al. 2001 ; Wang et al. 2001 )—peak expression typically occurred around or just after the peak time of exit from mitosis for that cell type. Certain functional categories of genes were highly overrepresented in a number of SAGE tag clusters. Ribosomal proteins, which typically showed higher expression early in development, were highly enriched in clusters 5, 9, 10, 15, and 23 ( Table 1 )—clusters that also were enriched for cell cycle regulators (particularly clusters 10 and 23). Mitochondrial proteins, by contrast, were concentrated in clusters 4 and 5. Cluster 2 consisted entirely of crystallins, which may be due to contamination by lens tissue in the E12.5 and P0.5 libraries. Phototransduction genes, on the other hand, were found to be concentrated in the late-onset clusters 1, 21, 22, and 24. Genes representing a number of other functional categories also were enriched in specific clusters, although the reasons in these cases are not clear. Examples of this include the concentration of genes involved in RNA processing in clusters 6 and 7, genes coding membrane transporters in cluster 10, and genes that are involved in vesicle-mediated transport in cluster 20. Large-Scale ISH of Dynamically Expressed Genes Genes identified by SAGE were chosen for analysis via ISH by focusing on genes that showed dynamic expression by k- means cluster analysis using Euclidean distance, and some degree of retinal enrichment (i.e., genes were expressed at lower levels in nonretinal SAGE libraries—see Table S2 ). Within this data set, genes whose presumptive function suggested that they might regulate cell fate choice (e.g., transcription factors, growth factors and their receptors, etc.) received highest priority for testing, although many genes of unknown function with developmentally dynamic expression also were tested. See Table S4 for the Gene Ontology Consortium (GO) classification of each probe tested. The analysis was restricted to genes represented by at least 0.1% of total SAGE TAGS in at least one of the retinal libraries, so as to control for sampling variability and to allow for ready detection via ISH. (Exceptions were made for a number of transcription factors and other genes of potentially major functional interest.) This abundance threshold was met by 4,133 tags. Probes corresponding to 1,051 of these tags were tested via ISH. This total included the 346 candidate photoreceptor-enriched genes tested in our previous work ( Blackshaw et al. 2001 ), as well as 37 previously characterized retinal genes that served as positive controls for ISH and to allow clarification of cellular expression patterns. Retinal expression was examined at every time point used for SAGE (see Materials and Methods for details). See Table S5 for a full list of the cellular expression data for each probe in the retina, along with the accession number of the cDNA used to generate each probe used for ISH. See also http://134.174.53.82/cepko/ for images of all of the ISH data. Classification of Cellular Gene Expression Patterns in the Developing Retina The laminar structure of the retina makes it relatively straightforward to assign a tentative identity to cells expressing a given gene. During early stages of retinal development, the outer neuroblastic layer (ONBL) consists almost entirely of mitotic progenitor cells, while newborn neurons (mostly consisting of amacrine and ganglion cells) reside in the inner neuroblastic layer (INBL). The position of mitotic progenitors within the ONBL varies depending upon their progress through the cell cycle, with S phase cells being found on the vitreal side of the ONBL near the border with the INBL and M-phase cells being found on the scleral side of the ONBL abutting the retinal pigment epithelium ( Young 1985 a, 1985 b). Around the time of birth, immature photoreceptors occupy the outer portion of the ONBL. They are comingled with mitotic cells of the G2, M, and G1 phases of the cell cycle, while the S phase mitotic progenitors are in the vitreal side of the ONBL. Finally, by P6, most retinal cells occupy their final positions within the retina. Rod and cone photoreceptors occupy the ONL. Bipolar neuron cell bodies occupy the scleral portion of the inner nuclear layer (INL); the cell bodies of Müller glia occupy a strip in the center of the INL; and amacrine cell bodies are found in the vitreal portion of the INL. The ganglion cell layer (GCL) contains both ganglion cells and a displaced amacrine cells. In the developing retina, expression in the scleral and vitreal portions of both the ONBL and INBL were scored separately, along with whether the gene in question was expressed in all or only a subset of cells in the layer in question. In the case of the adult retina, cell identity in wild-type animals could be scored readily by laminar position of the cells expressing the gene of interest ( Rodiek 1998 ), and thus the identity of expressing cells was scored directly. Extracting order from the diversity of gene expression patterns observed in the developing nervous system can be a daunting task. It is not obvious how best to generate a useful taxonomy of these expression patterns. In tackling this problem, we found it useful to classify cellular expression patterns of genes both by eye and by clustering software. Both methods have specific advantages—user classification more readily identifies rare but distinct patterns, while machine-based clustering allows more flexibility with respect to cluster number and appears to better accommodate classification of intermediate patterns. All classifications were based on the location of the ISH signal within the retinal layers over time during development. Table S6 contains the full list of expression patterns generated by visual inspection, and Table S7 has the full list of cellular expression clusters generated by clustering software. See Materials and Methods for more details on how these data were generated. Comparison of the user-annotated and machine-generated clusters demonstrated fairly strong similarities between the two sets of clusters ( Table S8 ), although genes placed in a single category by user annotation were invariably grouped into larger clusters by clustering software. On the other hand, genes in certain large clusters generated by user annotation—such as panretinal, TRAP2 -like, and Nlk -like (see Table S6 )—were dispersed among many clusters in the machine-generated data sets, with placement within particular clusters varying with replicate program runs. Genes in these categories were expressed at some level in most cells of the developing and mature retina. This variability likely reflects the relative lack of specificity of the expression pattern in these clusters. The finding that most of the highly cell-specific clusters identified by user annotation were readily distinguished by the clustering software supports this hypothesis ( Table S8 ). Using SAGE Data to Predict Cellular Expression Patterns in Developing Retina Temporal changes in gene expression as measured by SAGE turn out to be a useful but inexact method of predicting cellular expression patterns of genes within the retina. While no SAGE cluster was invariably associated with a given cellular expression pattern, genes in certain late-onset SAGE clusters (e.g., clusters 1 and 22) were highly likely to be expressed in developing rods. In the case of early-onset gene expression patterns, which would likely be expressed in retinal progenitor cells, comparison to a microarray-based study could be made. Microarray profiling data of 4N progenitor enriched versus 2N cells has led to the identification of a number of these genes as being enriched in 4N progenitor cells ( Livesey et al. 2004 ). These genes were concentrated in a limited number of SAGE tag clusters (particularly clusters 5, 15, and 23), but were largely absent from clusters that showed a perinatal peak in expression (such as cluster 6), which were enriched for genes expressed in developing rods, bipolars, and amacrine cells (see Table S9 for a full breakdown of 4N-enriched genes by SAGE tag cluster). In general, the temporal expression pattern observed in a given SAGE tag cluster was accurately reflected by the ISH data, although precise prediction of cellular expression patterns based on cluster data were not achieved. Clusters that showed postnatal peaks in expression, such as cluster 6, could contain a great diversity of cellular expression patterns, yet still be enriched for genes that showed strong expression in specific cell types that were differentiating. Table S10 , which details the percentage of tags in a given cluster that represent each specific user-annotated expression pattern, can serve as a starting point for predicting the probability that a gene matching a given SAGE tag will show a given cellular expression pattern in the developing retina. The expression clusters—whether generated by user annotation or clustering software—at best represent a lower limit to the number of distinct expression patterns within the developing retina. Although the number of distinct types of cells in the developing retina is not known, it is undoubtedly high ( MacNeil and Masland 1998 ). Particularly when considering genes expressed in subsets of cells in the ONBL, or subsets of developing amacrine cells, the level of resolution of our ISH-based screen does not allow one to distinguish many of the more complex patterns. Techniques such as multiple-probe fluorescence-based ISH ( Levsky et al. 2002 ) and single-cell microarray analysis ( Tietjen et al. 2003 ) will be required to resolve such questions as whether individual cells coexpress genes that display complex expression patterns. One interesting and potentially useful finding from the SAGE cluster data is that genes known to have highly selective cell-specific expression within a single retinal cell type could show different times of onset of expression. For instance, there is heterogeneity in the time of onset of expression among the genes that mediate rod phototransduction, a feature that has previously been reported in ferret retina ( Johnson et al. 2001 ). Phototransduction genes were found in four different clusters (see Table 1 ), with genes such as RPGRIP showing comparatively early onset of expression, followed by the progressively later onset timesof rod arrestin, rhodopsin, and, finally, Gα1 and GCAP1 (see Table S11 for a full list of tags corresponding to these genes). ISH confirmed the accuracy of the SAGE data for these onset times (see Figure S2 ). This heterogeneity of the time of onset of expression is observed for terminal differentiation markers of every cell type studied in the retina, as well as for markers of subsets of mitotic progenitor cells (see http://134.174.53.82/cepko/ for the full set of ISH data). Such profiles could be explored for the possibility of control by cascades of transcription factors. Gene Expression Patterns Define Subsets of Retinal Progenitor Cells Recent studies in systems as diverse as Drosophila neuroblast specification and the specification of neural-crest-derived cells ( Anderson 1999 ; Isshiki et al. 2001 ; Pearson and Doe 2003 ) have demonstrated the role of temporal changes in gene expression in the specification of neural cells. With respect to the retina, the competence model as originally proposed predicted that mitotic progenitor cells would show both temporal changes in gene expression across broad sets of retinal progenitors, and expression of selected genes in specific subsets of progenitor cells at a given time ( Cepko et al. 1996 ). We have identified a number of genes that show temporally restricted expression in early ONBL. By analyzing the expression of a large number of genes that were highly expressed early in development (particularly in SAGE tag clusters 5, 11, and 15), a number of genes that are expressed in broad but temporally restricted subsets of mitotic progenitor cells were identified ( Figure 2 A). sFrp2 RNA was found to be broadly expressed in the ONBL until E16, after which it rapidly decreased, a pattern that corresponded well with its SAGE tag levels. Expression of Fgf15 and Edr RNA was seen to persist longer, but neither was easily detected after P0, at which time both cyclin D1 mRNA—a recognized marker of mitotic progenitor cells in the retina ( Sicinski et al. 1995 ; Ma et al. 1998 )—and BrdU labeling were still readily detectable in the central retina. Edr RNA showed an unusual patchy distribution in the ONBL at P0—a pattern that was not detected for any other gene tested and has not been previously reported. Lhx2, by contrast, was weakly expressed in subsets of cells in the ONBL until P0, when it was dramatically and transiently upregulated throughout the ONBL. Microarray analysis of 4N versus 2N retinal cells at E16 indicates that both sFrp2 and Lhx2 are enriched in 4N mitotic progenitor cells ( Livesey et al. 2004 ). Figure 2 Genes Expressed in Subsets of Mitotic Progenitors (A) Genes expressed in temporally distinct subsets of progenitors. The first column shows relative SAGE tag levels for each gene under consideration. The UniGene identities and common names of the genes in question are Mm.19155/sFrp2, Mm.3904/Fgf15, Mm.142856/Lhx2, Mm.35829/Edr, and Mm.22288/cyclin D1 . The sections for ISH and BrdU shown here were taken from near the center of the retina at the developmental times shown. Mice were albino Swiss Websters except in the case of the adults, which were pigmented C57B/6. See Table S5 for a full list of probes used. Cellular laminae of both the developing and mature retina are indicated with colored bars. All pictures were taken at 200x. The graph plotting the fraction of mitotic cells in the retina adjacent to the BrdU staining is an estimate based on data from both rat and mouse ( Young 1985 a, 1985 b; Alexiades and Cepko. 1996 ). (B) Spatially heterogeneous ONBL. Genes that were expressed in spatial subsets of cells in the prenatal ONBL are shown. The genes shown are Mm.4541/Sox2, Mm.18789/Sox4, Mm.4605/Tbx2, Mm.29067/Mbtd1, Mm.2229/Eya2, Mm.34701/Pum1, Mm.29924/Arl6ip1, Mm.11738/Ark-1, Mm.40321/Pgrmc2, and Mm.22288/cyclin D1 . Sections were from central retina. Cellular laminae of both the developing and mature retina are indicated with colored bars. All pictures were taken at 200x. See Table S5 for a full list of probes used. To further investigate the expression of these genes in mitotic progenitor cells, ISH was performed on dissociated retinal cells in conjunction with 3 H thymidine labeling at E14, E16, and P0 ( Table 2 ). A substantially lower fraction of double-labeled cells for Fgf15 at P0 relative to earlier time points was observed, while sFrp2 labeling was absent at birth and substantially lower at E16 than at E14. Table 2 Fraction of Progenitor Cells Expressing sFRP2 and FGF15 Decreased as Development Proceeded Retinal explants were labeled with 3 H-thymidine for 1 h, and then disociated and placed on slides. ISH was performed and the fraction of cells expressing sFRP2 and FGF15 is indicated, along with the fraction of cells labeled with 3 H-thymidine, and the fraction of 3 H-thymidine-positive cells that were labeled with probe A limited number of genes have previously been reported as expressed in subsets of mitotic retinal progenitor cells, including genes such as Ath5, and have been shown to be required for retinal ganglion cell development ( Brown et al. 2001 ; Wang et al. 2001 ). We identified a large number of genes that showed selective expression at certain times during development in relatively small subsets of cells in the ONBL ( Figure 2 B). These include a large number of known and putative transcription factors, such as Sox2, Sox4, Tbx2, Eya2 and Mbtd1 (a novel polycomb family member), along with many genes of other functional classes. Particularly intriguing is the early and transient expression of Pum1, a mammalian homolog of the pumilio gene, which has been shown to mediate asymmetric mRNA distribution in Drosophila ( Micklem 1995 ). Many of these genes showed highly dynamic expression during development—rapidly shifting their cellular expression patterns in the course of a few days, as in the case of Pum1 and Sox2, or being expressed for only a few days, as in the case of Eya2 and Pgrmc2 . In some cases, these subsets were scattered throughout the ONBL, such as Eya2 at E14, while for other genes, such as Pum1 and Pgrmc2, expression was in only the scleral portion of the ONBL, suggesting that these genes may show strongest expression near M phase in retinal progenitor cells. From these data, it is difficult to determine whether most of these genes were expressed in cycling progenitor cells or cells that have newly exited from mitosis, as these two populations are intermingled in the ONBL. However, microarray analysis of 4N versus 2N cells of the early retina ( Livesey et al. 2004 ) has indicated that a number of these genes, such as Sox2, are enriched in 4N progenitor cells. See Figure S3 for more examples of genes expressed in subsets of ONBL cells and contrast with Figure S4 , which shows genes with broad but selective expression in the ONBL. The genes that are expressed in subsets of presumptive retinal progenitors include a large number of transcription factors (e.g., Sox2, Lhx2, and Eya2 ) as well as signal transduction components. These intrinsically acting factors represent potential candidates for regulating developmental competence and, by analogy with the Drosophila retina, may act combinatorially to help specify cell fate ( Flores et al. 2000 ). Furthermore, a number of genes that are expressed in temporal subsets of progenitor cells encode secreted differentiation factors such as FGF15 and sFRP2 . Since cell fate choice is determined by the interaction of intrinsic properties and extrinsic factors, these genes are good candidate regulators of cell fate determination. Strikingly, the temporal expression profile of very few progenitor-enriched cell cycle genes tracked precisely with the fraction of mitotic cells in the retina. Even many well-established markers of mitotic progenitor cells, such as cyclinD1 and cdk4 were highly expressed until P2.5 and detectably expressed as late as P6.5—long after the fraction of mitotic cells in the retina had decreased drastically ( Figure 2 A). These data imply that expression of these genes frequently persists after the end of mitosis. In addition, one might have predicted that the levels of cell cycle regulators would be highest at the earliest time point analyzed (E12.5), when the percentage of mitotic cells was highest. However, we found that progenitor-enriched genes such as cyclinD1 and cdk4 often had RNA levels that peaked around P0.5. This observation suggests that the number of mRNA molecules per cell for many of the genes that mediate mitotic activity increases as development proceeds. The functional significance of these findings is unclear, although a number of features of retinal progenitor cells change over the course of development, including the length of the cell cycle ( Young 1985 a; Alexiades and Cepko 1996 ) and the probability of producing progeny that are no longer mitotic ( Livesey and Cepko 2001 ). Genes Expressed in Immature Differentiating Retinal Cell Subtypes One characteristic expression pattern of genes likely to be involved in cell fate specification and/or the early steps of the differentiation process is restriction to newly postmitotic cells and cells actively undergoing differentiation. Many of the genes demonstrated to show such expression in developing retina, such as Crx, Nrl, and NR2E3 ( Furukawa et al. 1997 a, 1997 b; Chen et al. 1997 ; Haider et al. 2001 ; Mears et al. 2001 ) have been shown to play an active role in regulating cell differentiation. We have identified genes that are selectively expressed in immature postmitotic retinal cells of every major class, with the exception of cone photoreceptors, greatly expanding the set of genes known to be selectively expressed in immature retinal precursor cells ( Figure 3 ). KIAA0013, an uncharacterized RhoGAP, was found to be expressed exclusively in immature ganglion cells, and only expressed detectably outside in limited subsets of developing neurons, such as Cajal-Retzius cells of the developing cerebral cortex, and the developing thymus. Cdc42GAP was found to be strongly and transiently expressed in newly postmitotic rods, while the leucine zipper transcription factor Zf-1 was expressed in presumptive bipolar cells. Septin 4 was found to be selectively and persistently expressed in developing horizontal cells, while Mm.23916, a novel dual-specificity protein phosphatase, was found to be expressed selectively in immature amacrine cells. Finally Tweety1, an unconventional chloride channel ( Suzuki and Mizuno 2004 ) was strongly expressed in newly postmitotic Müller glia. Along with genes whose cellular expression could be clearly identified visually, a number of genes with strong but transient expression in undefined subsets of cells of the neonatal retina were observed. Expression of these genes persisted after the end of mitosis in the central retina (see Figure 2 A), so at least some of the cells that express them must be postmitotic. Genes in this category include inhibin βB, brain fatty acid binding protein 7, BMP7, the transcription factor Sal3, and the orphan neurotransmitter transporter NTT7 (see Figure S5 ). Figure 3 Precursor Patterns for Major Retinal Cell Types Genes that are selectively expressed in immature subtypes of retinal cells. From the top, the differentiating cell types that express the genes in question are ganglion cells (Mm.45753/KIAA0013), rod photoreceptors (Mm.103742/Cdc42GAP), bipolar cells (Mm.29496/Zf-1), horizontal cells (Mm.2214 /septin 4), amacrine cells (Mm.23916), and Müller glia (Mm.29729/Tweety1). Sections were from central retina. Cellular laminae of both the developing and mature retina are indicated with colored bars. All pictures were taken at 200x. See Table S5 for a full list of probes used. Genes Expressed in Developing Photoreceptor Cells Rod photoreceptors make up 70% of cells in the retina ( Young et al. 1985 b; Jeon et al. 1998 ). The SAGE-derived expression profile of genes selectively expressed in developing rods is thus more comprehensive than that of other cell types. Based on the ISH data and aided by our SAGE study of mature tissue ( Blackshaw et al. 2001 ), as well as previous reports of mutant mice lacking transcription factors known to be important for rod development, a model of a temporal order of transcription factor expression during rod development was made ( Figure 4 ). Transcription factors known to be involved in cell fate specification sometimes show broad expression in mitotic progenitor cells and persistent expression in mature cell types (e.g., Liu et al. 1994 ; Belecky-Adams et al. 1997 ; Livesey and Cepko 2001 ). We observed a number of genes that were expressed in early ONBL from E16 on, with expression persisting in mature photoreceptors, such as Yboxbp4 . A similar pattern were seen for the mouse ortholog of the Drosophila castor gene, though this gene was observed in a more restricted subset of cells in the ONBL at E16, and for the orphan nuclear receptor ERRβ, although this gene had relatively lower expression prenatally and had pronounced expression in an undefined subset of cells in the immature photoreceptor layer during the first postnatal week. Figure 4 Transcription Factor Cascade in Photoreceptor Development Transcription factors that are selectively expressed in developing rods (and possibly cones as well) are shown. The schematic diagram integrates gene expression data from previously identified photoreceptor-enriched transcription factors and from genes explored in this study. The genes shown are Mm.193526/Yboxbp4, Mm.3499/Rax, Mm. 89623/mCas, Mm.1635/PIAS3, and Mm.235550/ERRβ . See Figure S6 for images of the developmental expression patterns of previously characterized transcription factors. Sections were from central retina. Cellular laminae of both the developing and mature retina are indicated with colored bars. All pictures were taken at 200x. See Table S5 for a full list of probes used. In contrast to being expressed in mitotic cells as well as differentiating photoreceptor cells, a number of transcription factors were selectively expressed in postmitotic but immature photoreceptors. The Rax homeodomain factor showed, as has been previously reported ( Furukawa et al. 1997 a; Mathers et al. 1997 ), strong expression in mitotic progenitor cells in the ONBL that vanished with the end of mitosis. However, expression transiently reappeared in immature photoreceptors at P8. This situation is analogous to that seen in a number of other vertebrates, in which a duplication of the ancestral Rax gene has resulted in Rax genes with distinct expression in photoreceptor and progenitor cells ( Chuang et al. 1999 ; Chen and Cepko 2002 ). PIAS3, which encodes a SUMO lyase that directly regulates the activity of a broad subset of transcription factors ( Kotaja et al. 2002 ; Haider et al. 2001 ), was strongly and selectively expressed only in developing photoreceptors, with expression beginning at E18, peaking at P8, and largely fading away in the adult, a pattern that in many respects is reminiscent of Crx (see Figure S6 ). In contrast to these patterns, Nrl and NR2E3 showed no detectable expression prenatally, and showed peak expression around P6. Somewhat surprisingly, the RNAs for many of these transcription factors is enriched in the inner segments of photoreceptors, as are a large fraction of the other photoreceptor-enriched genes characterized in this study, a finding that is in line with our earlier work ( Blackshaw, et al. 2001 ). The functional significance of this remains unclear. In addition to transcription factors, other functional classes of genes, including genes of unknown function, were expressed in developing photoreceptors, with strongest expression typically found in the first postnatal week ( Figure S7 ). In some cases, these genes fall into pathways known to regulate rod differentiation. Both PIAS3 and the multifunctional protein Hrs ( Chung et al. 1997 ; Scoles et al. 2002 ) selectively inhibit STAT3, and thus possibly inhibit the action of ciliary-derived neurotrophic factor, a factor that has been shown to inhibit rod differentiation in rodents ( Ezzeddine et al. 1997 ; Kirsch et al. 1998 ; Schulz-Key et al. 2002 ). Cdc42GAP expression (see Figure 2 ) may mediate the polarization and initiation of outer segment formation taking place in photoreceptors at this time ( Nobes and Hall 1999 ). In other cases, genes newly identified as selectively expressed in developing photoreceptors imply the existence of novel facets of photoreceptor development. The expression of synaptic vesicle protein Cpx2 suggests that developing photoreceptors may be actively secreting some developmentally relevant signal, while the expression of Hrs also potentially suggests high levels of regulated endocytosis and destruction of unknown extracellular proteins ( Lu et al. 2003 ). The expression of the previously uncharacterized tumor necrosis factor family member Tnfsf13 and A20-like signal transduction components such as TRABID and Fln29 suggest an unexplored role for this pathway in normal photoreceptor development. Genes Expressed in Developing Interneurons of the INL Many genes were selectively expressed in the other, nonphotoreceptor retinal cell types during development. A temporal sequence of transcription factors was observed in bipolar cells as they differentiated ( Figure S8 ). The homeodomain factor Lhx4, and the uncharacterized leucine-zipper protein Zf-1 (see Figure 2 ), showed expression at E16 in the ONBL, with expression continuing postnatally and persisting in adult bipolar cells. Zfh4 was expressed in developing amacrine cells and in subsets of cells in the ONBL prior to P4, and was robustly and transiently expressed in bipolar cells, with peak expression at P6. The relatively late-onset Dbp was first seen in the second postnatal week across the INL. Chx10, as has been previously reported ( Liu et al. 1994 ), and Gli5 were broadly expressed across the ONBL prior to P4, at which point they both showed elevated expression in developing bipolar cells. Microarray analysis confirmed that both of these genes are expressed in mitotic progenitor cells ( Livesey et al. 2004 ). Possible downstream targets of these transcription factors include previously uncharacterized cell adhesion molecules such as the Ig-superfamily member Mm.41284, kinases such as Prkcl, and the putative growth factor receptor SEZ-6 . Furthermore, despite the fact that they comprise only 0.3% of the cells in the adult retina, genes that are highly enriched in both developing and mature horizontal cells ( Figure S9 ), such as the GTPase regulator Borg4, were found. Many genes tested by ISH were selectively expressed in developing amacrine cells ( Figure S10 ). The expression patterns were tremendously diverse, a fact that may reflect the reported extensive heterogeneity among amacrine cell subtypes ( MacNeil and Masland 1998 ). Certain genes, such as the kinase Unc51-like-1, ArfGAP, and the orphan G-protein-coupled receptor Mm.6393, were found to be expressed both in immature amacrine cells and in subsets of cells in the ONBL, particularly in the region of the ONBL that comprises the outer or scleral surface, where M phase mitotic progenitor cells are localized. Cytoskeletal-associated kinases such as Unc51-like-1, and small GTPases such as ArfGAP, may play a role in neurite extension or process formation. Additionally, the expression of neuropeptide receptors such as Mm.6393 in the ONBL before mature neural circuits have formed fits with data from other parts of the developing CNS showing early expression of neurotransmitter receptors and suggesting that neurotransmitters may act on mitotic progenitor cells to regulate cell cycle or cell fate specification ( Rueda et al. 2002 ; Ohtani et al. 2003 ). Similarly, recent work from our laboratory on the role of glycine receptors in the formation of rod photoreceptors ( Young and Cepko 2004 ) confirms such predictions for at least one such receptor. Other genes, such as syntrophin-associated kinase and the novel dual-specificity phosphatase Mm.23916, were confined to immature amacrines only. Syntrophin-associated kinase, in particular, may regulate maturation of synaptic connections ( Lumeng et al. 1999 ). Others genes, such as necdin, the basic helix-loop-helix transcription factor Nhlh2, and the novel PLC isoform Mm.215653, showed complex and often biphasic patterns. The Slit receptor robo3 was strongly and transiently expressed in the first postnatal week in a single sublamina within the INBL, perhaps corresponding to a single subtype of developing amacrine cells. A role for Slit-Robo signaling in regulating cortical dendrite maturation has been demonstrated ( Whitford et al. 2002 ), and these data suggest such a mechanism may be at work in regulating subtype-specific amacrine cell laminae formation in the retina. Neuropeptide Y was strongly and transiently expressed in a subset of amacrine and horizontal cells towards the end of the first postnatal week, with expression dropping dramatically in the adult—suggesting a possible role for this factor in the formation of mature retinal circuitry. Finally Mm.41638, which is weakly homologous to a lysosomal membrane protein, was expressed solely in postnatal amacrine cells, though expression remained in a more restricted subset of amacrine cells in the adult. Müller Glia Are Highly Similar to Retinal Progenitor Cells Genes selectively expressed in Müller glia share a number of defining features. Mitotic retinal progenitor cells and Müller glia showed a great degree of transcriptional overlap—far more so than other retinal cells that differentiate postnatally. Of the genes identified as being specifically expressed in Müller glia after the first postnatal week, 68% were found to be enriched in mitotic progenitor cells based on their ISH pattern, in contrast to only 14% of photoreceptor-specific genes ( Figure 5 A). Of the genes identified as enriched in 4N progenitor cells by micorarray analysis ( Livesey et al. 2004 ) that were tested by ISH in adult retina, 43% were enriched in Müller glia, compared to 11% that were enriched in photoreceptors. Figure 5 Müller-Glia-Enriched Genes (A) Müller-glia-enriched genes show stronger expression in retinal progenitors than do genes enriched in other postnatally born cell types. See Materials and Methods for details of how progenitor-enriched and cell-specific expression patterns were determined, and p -values for progenitor-enrichment of genes that are cell type–specific in the mature retina were calculated. Data on 4N-enriched transcripts were obtained from Livesey et al. (2004) . Numbers for each value are as follows. For N, the number of cell-enriched genes, N MG = 86, N Pr = 112, N BC = 21, and N AC = 57. For I, the number of genes that show retinal progenitor-enriched patterns by ISH, I total = 180, I MG = 66, I PR = 15, I BC = 4, and I AC = 8. For M, the number of genes enriched in 4N retinal progenitor cells that were tested by ISH in adult retina, M total = 28, M MG = 12, M PR = 3, M BC = 3, and M AC = 1. , p < 10 −13 ; *, p < 0.0001. (B) Müller-glia-enriched genes show strong expression in mitotic progenitors. The genes shown are: Mm.26062/ADO24, Mm.55143/Dkk3, Mm.5021/DDR1, Mm.35817, Mm.20465/GPCR37, Mm.200608/clusterin, and Mm.22288/cyclin D1 . Sections were from central retina. Cellular laminae of both the developing and mature retina are indicated with colored bars. All pictures were taken at 200x. See Table S5 for a full list of probes used. (C) Dynamic expression of metabolic genes in developing retina. Metabolic enzymes are often selectively expressed in mitotic progenitors and developing Müller glia. The genes shown are Mm.27953/glycine decarboxylase, Mm.9114/mu-crystallin, and Mm.213213/HK-R . Cellular laminae of both the developing and mature retina are indicated with colored bars. Sections were from central retina. All pictures were taken at 200x. See Table S5 for a full list of probes used. Typical expression patterns for Müller-glia-enriched genes are shown Figure 5 B. Genes in this category, such as the negative regulator of Wnt signaling Dkk3, the collagen receptor DDR1, and the endosomal protein AD024, were observed to be strongly and broadly expressed across the ONBL throughout development, though expression in the adult was restricted to Müller glia. Microarray analysis suggests that a number of these genes, including Dkk3 and DDR1, are enriched in 4N mitotic progenitor cells ( Livesey et al. 2004 ). A smaller set of genes, such as Mm.35817, GPCR37, and Tweety1 (see Figure 2 ) were found to be expressed across the ONBL early in development, but showed dramatically and transiently upregulated expression at the end of the first postnatal week as Müller glia began to differentiate. While over two-thirds of Müller-glia-enriched genes showed enriched expression in retinal progenitors relative to other cell types in the developing retina, virtually all Müller-glia-enriched genes were expressed at detectable levels in retinal progenitors (without necessarily being enriched in progenitors). In fact, only two genes that are Müller-specific in the adult— clusterin and carbonic anhydrase 2 —were expressed in mature Müller glia but not detected in mitotic progenitors. However, previous work suggests that carbonic anhydrase 2 may be expressed in retinal progenitors at levels below our ability to detect ( Vardimon et al. 1986 ), and this may be the case for clusterin as well. Additional Müller-glia-enriched genes are shown in Figure S11 . The extensive overlap in gene expression between Müller glia and mitotic progenitor cells raises the question of how closely these two cell types resemble each other at the functional level. Müller glia morphologically resemble mitotic progenitor cells in having apical and basal processes that span the radial dimension of the retina ( Rodiek 1998 )—a feature that is shared with retinal progenitor cells as well as radial glia of the developing brain, a cell type known to be the cortical progenitor cell ( Doetsch 2003 ). Müller glia are one of the last cell types to exit mitosis ( Young 1985 b; Reh and Levine 1998 ), and they are the only cell type in the mature retina that can reenter mitosis following retinal injury ( Dyer and Cepko 2000b ; Vetter and Moore 2001 ). Finally, data from chicken suggest that, at least in some birds, Müller glia can be induced to divide and give rise to some types of retinal neurons for a short period of time near the end of retinal development ( Fischer and Reh 2001 ). The question arises, then, as to whether Müller glia are fundamentally multipotent progenitor cells that are quiescent regarding cell division and the production of neurons ( Morest and Silver 2003 ; Walcott and Provis 2003 ). If they are progenitor cells, they are progenitor cells that have acquired the specialized properties needed for a support role in the mature retina, e.g., neurotransmitter reuptake and structural roles. The few genes that are specifically expressed in mature Müller glia, such as clusterin, may be emblematic of such roles. Misexpression in mature Müller glia of genes that are candidates for regulating neuronal production in the postnatal retina, followed by injury-induced division, offers a potential approach for future therapies that might lead to photoreceptor or ganglion cell replacement in diseased retinas by cells derived from Müller glia. Prominent Expression of Metabolic Enzymes in Developing Müller Glia A second notable feature of genes expressed nearly specifically in developing Müller glia is the highly dynamic and cell-specific expression of a number of metabolic enzymes ( Figure 5 ). The novel hexokinase-related gene HK-R was selectively expressed in developing Müller glia cells, but not in any other cell in the body examined. Mu-crystallin, which does not encode a crystallin in placental mammals but rather an uncharacterized homolog of the bacterial enzyme ornithine cyclodeaminase ( Segovia et al. 1997 ), showed a similar expression pattern in the retina but also was expressed in other developing sensory organs. Glycine decarboxylase was strongly and selectively expressed in retinal progenitor cells, differentiating Müller glia, and to a lesser extent, developing photoreceptors. The reasons for such high enzymatic activity in development is unclear, although some of these genes may have regulatory functions unconnected to their metabolic roles. For instance, mu-crystallin is also a thyroid hormone binding protein ( Vie et al. 1997 ). Such proteins also may regulate the abundance of small molecules that can act as signals that may be relevant for development. For example, glycine levels may be kept low by glycine decarboxylase so that taurine can bind to and activate the glycine receptor to promote rod differentiation ( Young and Cepko 2004 ). These data point to future directions of research examining the intersection of metabolism and development and suggest the usefulness of supplementing gene expression profiling with metabolomic analysis ( Watkins and German 2002 ). Dynamic Expression of Putative Noncoding RNAs in Developing Retina A number of RNA transcripts that do not appear to encode proteins were strongly expressed in the developing retina ( Figure 6 ). These transcripts are typically spliced and polyadenylated, but do not encode evolutionarily conserved open reading frames (ORFs), or any ORFs encoding proteins longer than 100 amino acids, while often showing high similarity at the nucleotide level between mouse and human ( Numata et al. 2003 ). Table S12 provides a list of these transcripts. Putative noncoding transcripts that showed developmentally dynamic expression include retinal noncoding RNA 1 (RNCR1), which was expressed throughout the ONBL during early development and which was later restricted to Müller glia. It was transcribed in a head-to-head fashion, and largely coexpressed, with Six3. This transcript showed extensive alternative splicing, and while one splice form contained a potential ORF of greater than 100 amino acids, no mouse/human conservation of this putative protein was observed, while high similarity was observed at the nucleotide level in other regions of the transcript. RNCR2 , on the other hand, was expressed in a large subset of cells in both the ONBL and INBL prenatally, with expression restricted to the INL and GCL postnatally. ISH signal for RNCR2 was strongly concentrated in what appeared to be nuclear or perinuclear regions of expressing cells. RNCR3 was expressed in a steadily increasing subset of cells in the ONBL from E14 and gradually resolved to an adult pattern that was photoreceptor-enriched but present in the inner retina at lower levels. Figure 6 Noncoding RNAs in Retinal Development A number of presumptive noncoding RNAs are strongly expressed in dynamic subsets of retinal progenitor and precursor cells. The transcripts shown are Mm.150838/ RNCR1, Mm.44854/ RNCR2, and Mm.194050/RNCR3 . Sections were from central retina. Cellular laminae of both the developing and mature retina are indicated with colored bars. All pictures were taken at 200x. See Table S5 for a full list of probes used. Although additional assays are required to conclusively demonstrate that these RNAs do not encode functional proteins, there is precedent for this conclusion from recent genomic work. Large-scale EST sequencing efforts from mouse have uncovered up to several thousand putative spliced transcripts that do not appear to encode for proteins ( Numata et al. 2003 ). Likewise, oligonucleotide array experiments using probes that tile individual human chromosomes at high density report substantial transcription from many regions not predicted to have protein-coding genes ( Kapranov et al. 2002 ; Cawley et al. 2004 ), and suggest that microarray-based expression profiling that uses probes designed only against predicted protein-coding genes may miss a significant fraction of the transcriptome. The functional role of these transcripts is obscure, although noncoding spliced RNAs such as Xist and H19 in mammals and Rox1 and Rox2 in Drosophila have been implicated in a variety of epigenetic processes ( Mattick 2003 ). The possibility that RNCR1 might somehow regulate expression of Six3 or other progenitor-specific transcripts awaits further investigation. Both Xist and Tsix, noncoding RNAs that play a crucial role in X-inactivation, were expressed in subsets of cells in the ONBL and INBL early in development, but were expressed strongly and selectively in the INL around the end of the first postnatal week ( Figure S12 ). This finding is quite surprising, given that photoreceptors and ganglion cells do not express these transcripts and would thus appear to escape X-inactivation. Since genetic evidence suggests that this is not the case for either cell type ( Reese et al. 1999 ), our findings implicate the existence of possibilities such as alternate cell-specific pathways of X-inactivation or dramatic cell-specific variations in Xist levels required to mediate X-inactivation. Expression Profiling and Candidate Gene Analysis Although we have identified a plethora of transcription factors, growth factors, and signal transduction components, the data do not clearly implicate a known signaling pathway as selectively involved in the differentiation of a given cell type within the retina. For example, negative regulators of Wnt signaling were identified, but these genes display a diversity of cellular expression patterns that cloud a simple model for their action. Dkk3 and Nkd1 are expressed broadly in progenitor cells and Müller glia, together with beta-catenin, while sFRP-2 is expressed exclusively in early progenitor cells, and Nlk is expressed strongly in postmitotic but immature cells of the postnatal retina. Another approach to the creation of models of pathways that control retinal development is to combine the ISH analysis of genes identified via SAGE with a candidate gene approach, even for genes not identified by SAGE. For example, we examined the expression of all known regulators of Wnt signaling, all fibroblast growth factor receptors, and all Slit and Robo genes whether or not SAGE tags corresponding to these genes were identified. See Table S5 and http://134.174.53.82/cepko/ for a full list of genes and their expression patterns. Cell-Specific Gene Expression in the Mature Retina Identifies Candidate Retinal Disease Genes A molecular catalog of gene expression in the adult retina was assembled with molecular markers for every major class of retinal cell ( Figure 7 ). The catalog of photoreceptor-enriched genes reported in previous work ( Blackshaw et al. 2001 ) was expanded, and a large number of genes expressed in the inner retina were identified. Some of these include genes that mark subsets of amacrine and ganglion cells. Knowledge of which genes show cell-specific expression in the retina can aid in identifying retinal disease genes. The expression of nearly half of all cloned photoreceptor dystrophy genes is selectively enriched in photoreceptors ( Blackshaw et al. 2001 ), while hereditary optic neuropathies have been suggested to be partially mediated by mutations in ganglion-cell-enriched genes ( Votruba et al. 1998 ). Furthermore, a number of other retinal and anterior segment abnormalities result from mutations in genes that are broadly expressed in retinal progenitor cells ( Hanson et al. 1999 ; Ferda Percin et al. 2000 ). See Table S13 for a full list of the chromosomal locations of the human orthologs of genes examined in this work. This list also contains a full list of mapped but unidentified Mendelian human retinal disease genes and orthologs of photoreceptor-enriched genes identified in this work that lie within those chromosomal intervals. A total of 164 photoreceptor-enriched genes not previously linked to retinal disease were found in chromosomal intervals containing retinal disease loci, representing a total of 42 distinct loci. While photoreceptor-enriched transcripts make up roughly half of all cloned retinal disease genes ( Blackshaw et al. 2001 ), roughly one-third of retinal disease genes are expressed in all cells of the retina, suggesting that it is fruitful to consider such genes when screening candidate disease genes. We find that 22 panretinally expressed genes map within intervals containing unidentified disease genes, representing 16 distinct loci. Figure 7 Catalog of Gene Expression in Adult Retina The most commonly observed patterns of gene expression in the adult retina are indicated. Data are taken from Table S5 and cover all genes examined in the adult retina. Genes are placed in a category corresponding to a single cell type if expression is substantially greater in that cell type than in any of the other cell types examined. Genes are placed in categories corresponding to multiple cell types if expression is approximately equal in more than one cell type. The number of genes expressed in photoreceptors and Müller glia differs somewhat from those used in the analysis shown in Figure 5 A, since the expression of a large number of photoreceptor-enriched genes was not examined prenatally, and a number of Müller-enriched genes were detectable in Müller glia through the end of the second postnatal week, but not in adult retina. AC, amacrine cells; BC, bipolar cells; GC,ganglion cells; HC, horizontal cells; MG, Müller glia; sAC, subset of amacrine cells; sBC, subset of bipolar cells; sGC, subset of ganglion cells Genomic Approaches to Development The retina consists of a number of distinct cell types that are relatively well defined morphologically, as well as molecularly. They undergo differentiation in defined intervals and are found in stereotypical locations within the retina. These characteristics allow a fairly straightforward evaluation of the cell-specific expression of genes within the retina. We have coupled SAGE-based expression profiling with large-scale ISH analysis to obtain an atlas of gene expression for the developing and mature retina. This atlas is useful for many purposes—in particular, providing many candidate genes for studies of retinal development and function. SAGE analysis can be nearly comprehensive ( Velculescu et al. 1995 ), but its sensitivity is limited by the number of tags sequenced, the level of expression of a transcript within a given cell, and the abundance of given cell subtypes within a tissue sample. Thus this analysis detected relatively rare cell-specific transcripts primarily for the abundant rod photoreceptors and their precursors, and for genes broadly expressed in retinal progenitor cells. Nonetheless, the catalog does include some genes selectively expressed even in the rarest cell types, such as the horizontal cells (0.3% of all retinal cells; Jeon et al. 1998 ) and subtypes of ganglion cells, as well as genes expressed selectively in small subsets of cells in the early ONBL. A recent microarray-based study in developing neural crest screened over 90 candidate genes via ISH ( Gammill and Bronner-Fraser 2002 ), and a recent study using serial stages of embryonic Drosophila has analyzed hundreds of genes by such methods ( Tomancak et al. 2002 ). However, while a number of recent studies have used microarray analysis to profile developing neural tissue, large-scale ISH-based validation of genes identified as being expressed in developing CNS by such expression profiling has not yet been conducted. Large-scale ISH studies enhance our ability to interpret expression profiling data, as the precise cellular expression of a gene in heterogeneous tissues of the developing nervous system cannot be inferred reliably from the profiling of bulk tissue. Other considerations underscore the benefits of verifying primary expression data from expression profiling methods by using other approaches. For instance, several studies describing microarray-based expression profiling of similar starting material have obtained contrasting results for sets of differentially regulated genes ( Claridge-Chang et al. 2001 ; McDonald and Rosbash 2001 ; Lin et al. 2002 ; Ivanova et al. 2002 ; Ramalho-Santos et al. 2002 ). These may result from either experimental variation among labs or biological variation in gene expression among the samples and individuals tested ( Pritchard et al. 2001 ; Blackshaw et al. 2003 ), but nonetheless suggest that large-scale verification of expression differences by techniques such as quantitative RT-PCR or ISH would aid interpretation of such differences. Studies that rely on large-scale ISH as an initial screen generate vast amounts of data, but typically have been conducted using sets of identified or random cDNAs without using expression screening to preselect genes that show high or dynamic expression in the tissue of interest ( Gawantka et al. 1998 ; Neidhardt et al. 2000 ; Kudoh et al. 2001 ; Thut et al. 2001 ). Using expression profiling to generate a set of candidate genes for large-scale ISH analysis will increase the probability of testing genes that show enriched or dynamic expression in a tissue of interest. Towards a Functional Genomics of Neural Development The data presented here provide the starting point for medium-throughput functional analysis of the role of many genes in retinal development. The use of in vivo electroporation ( Matsuda and Cepko 2004 ) and plasmid constructs encoding small inhibitory RNAs delivered by electroporation or retroviruses will make possible medium-throughput gain- and loss-of-function studies of gene function in the retina. The identification of a variety of progenitor subtypes and stage-specific precursor markers will enable a deeper interpretation of such studies. Construction of appropriate Cre lines will allow lineage analysis to determine with precision the mature cell types to which subsets of mitotic progenitor cells or posmitotic precursors give rise. Combining the knowledge of cell-specific transcription factors and cell-specific target genes, together with bioinformatic approaches that take advantage of mammalian genome sequence information in a manner like recent efforts in Drosophila ( Stathopoulos et al. 2002 ), may allow the characterization of the combinatorial code of cis - and trans -acting elements that specify mature neuronal identity. We anticipate that similar approaches are likely to be useful in any region of a developing tissue where birthdating studies have been conducted and cell subtypes can be readily identified based on their spatial localization. Materials and Methods Generation of SAGE libraries Isolation of mouse brain and retinal tissue, as well as construction of all SAGE libraries derived from retinal and hypothalamic tissue, was conducted as previously described ( Blackshaw et al. 2001 ). Publicly available mouse libraries used in the analysis include 3T3 fibroblasts (obtained from http://www.sagenet.org ), P8 cerebellar granule precursor cells maintained in culture for 24 h (GCPcntr; obtained from http://www.ncbi.nlm.nih.gov/SAGE ), P8 cerebellar granule precursor cells maintained in culture and treated with Shh for 24 h (GCP+SHH; obtained from http://www.ncbi.nlm.nih.gov/SAGE ), freshly harvested P8 cerebellar granule precursor cells (GC_P8; obtained from http://www.ncbi.nlm.nih.gov/SAGE ). Libraries from E15 and P1 cerebral cortex were obtained from Gunnersen, et al. 2002 . ). All retinal and hypothalamic SAGE data have been submitted to NCBI, and will be available for download at http://www.ncbi.nlm.nih.gov/SAGE . SAGE data analysis The SAGE 3.0.1 program (courtesy of Victor Velculescu and Ken Kinzler, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States) was used to extract SAGE tags and eliminate duplicate ditags. Identity of SAGE tags was obtained from the National Center for Biotechnology Information (NCBI) “reliable” tag map set for UniGene (available at http://www.ncbi.nlm.nih.gov/SAGE ). UniGene Build 131 of Mus musculus ( http://www.ncbi.nlm.nih.gov/UniGene ) was used for the mappings. In cases where ISH results for genes matching a “reliable” tag did not match the temporal expression profile for the tag in question, along with all cases of unknown tags (i.e., tags which had no “reliable” tag to gene assignment) that were present at greater than 0.1% of total tags in any one SAGE library, the genes were tested via NCBI BLASTN searching ( http://www.ncbi.nlm.nih.gov/BLAST/ ) against the nr and dbest databases, with Expect threshold set to 100 ( Karlin and Altschul 1990 ). A tag was considered to match a specific transcript if it corresponded to the 3′-most NlaIII site in a given polyadenylated transcript ( Velculescu et al. 1995 ). If no such match was found, tags matching the 3′-most NlaII sites in 5′ reads of retinal-derived MGC cDNAs ( Strausberg et al. 2002 ) were considered to match those transcripts, in cases where no further 3′ sequence information was available for those ESTs. Each tag representing a gene tested by ISH, moreover, was checked by BLASTN using these parameters to verify the accuracy of the NCBI tag-to-gene matches. Human orthologs of mouse genes were identified through the use of the Homologene data set and verified by BLASTN and/or BLASTX analysis using the NCBI server, or BLAT analysis using the University of California at Santa Cruz genome server ( http://genome.ucsc.edu ). In cases where no curated ortholog was present in the database, BLASTN analysis against nr, dbest, and htgs databases was used to identify transcripts that showed over 85% sequence conservation over 100 bp and did not match any repeat sequence. The University of California at Santa Cruz genome browser using the October 2003 freeze ( http://genome.ucsc.edu/cgi-bin/hgGateway ) was used to determine if any transcripts with no obvious coding sequence mapped within 5 kb of the 3′ end of an identified gene and were transcribed in the sense orientation relative to that gene. If so, these were considered to represent novel 3′ ends of that gene. All other data analysis and curation was conducted with Microsoft Excel and Microsoft Access. Tissue section, ISH, and BrdU staining ISH was conducted as previously described ( Blackshaw et al. 2001 ). For BrdU staining, mice were given a single interperitoneal injection of 37.5 mg/kg BrdU and killed 1 h later. Fresh-frozen sections were used following 15 min fixation in 4% paraformaldehyde. The protocol of BrdU staining was carried out using an anti-BrdU monoclonal antibody (Roche, Basel, Switzerland) and detected using an AP-conjugated secondary antibody, using recommended blocking and washing conditions. Dissociated cell ISH Retinas were dissected from E14.5, E16.5, and P0 mice and cultured for 1 h in DMEM/10% fetal calf serum containing 5 μCi/ml 3 H-thymidine. The labeled retinas were dissociated into single cells by incubating for 30 min at 37 °C in 100 units/ml of papain (Worthington Biochemical, Lakewood, New Jersey, United States) in Hank's balanced salt solution (HBSS) containing 10 mM HEPES (pH 7.6), 2.5 mM cysteine, and 0.5 mM EDTA. The suspensions were then gently triturated and incubated with 0.1 mg/ml DNase I for 10 min at 37 °C. The cells were pelleted, washed twice in HBSS, and plated on polyD-lysine-coated glass slides for 15 min at room temperature. Cells were fixed to the slides in 4% paraformaldehyde for 5 min at room temperature, washed twice in PBS, and dehydrated in 100% methanol. For acetylation, probe incubation, and subsequent washings, the in situ protocol detailed herein for tissue sections was used. A tyramide signal amplification system (TSA Plus, PerkinElmer, Wellesey, Massachusetts, United States) combined with an anti-digoxigenin-HRP antibody (Roche) was used according to the manufacturer's instructions to detect the signal. Autoradiographic processing was performed in emulsion (NTB2, Eastman Kodak, Rochester, New York, United States) exactly as previously described ( Alexiades and Cepko 1996 ). Classification of cellular expression data in retina by user-based classification and cluster analysis Two classification schemes of the patterns of expression over time were developed: human and machine-aided. In the first case, a single observer (S.B.) generated a presumptive minimal classification of expression patterns following visual inspection of each hybridization pattern (see Table S6 for a full list). This subjective classification took into account a relatively informal assessment of signal intensity. This approach yielded a total of 72 distinct patterns, of which 19 contained only a single member. In the second case, laminar expression within the retina was scored on a 0–5 point scale based upon visual inspection for each defined cell type in the prenatal, perinatal, and mature retina, and cluster analysis software was used to perform k- means clustering (using Euclidean distance) of cellular expression patterns (see Table S7 for the full data set). As with the cluster analysis of the SAGE data, in order to determine an optimal minimal number of clusters, the total distance among data points within the clusters of cellular expression data (within cluster dispersion) were plotted for cluster sizes from 10 to 65 over 100 simulations ( Table S14 ) using Euclidean distance measure ( De Hoon et al. 2004 ). Algorithms used for this analysis are available at http://bonsai.ims.u-tokyo.ac.jp/mdehoon/software/cluster/index.html . It was found that at approximately 45 clusters there was a pronounced discontinuity in the rate of change in the distance among points within the cluster, and this was adopted as a tentative minimal number of clusters. Determination of cell-enriched expression in adult retina and retinal progenitor cells For the data presented in Figure 5 A, numerical cellular expression data from Table S7 was used. Transcripts were assayed as enriched in a specific cell type if they showed highest (but not necessarily exclusive) expression in the cell type in question after the first postnatal week of life. Genes enriched in subsets of bipolars or amacrines were treated as bipolar- and amacrine-enriched, respectively. Whether or not a gene showed retinal-progenitor-enriched expression was determined from Table S7 by the following empirical set of criteria, which were found to cover virtually all known retinal-progenitor-enriched genes: early vO/svO or scO/sscO greater than 1, early (scO + sscO + vO + svO) greater than early (scI + sscI + vI + svI), early (vO + svO) greater than or equal to early (scO + sscO), and mid (vO + svO) greater than mid (scO + sscO). (See legend of Table S5 for a key to these abbreviations.) To determine whether genes that are cell type–specific in the adult retina are disproportionately enriched in retinal progenitors (see Figure 5 A), we have used the hypergeometric distribution statistical analysis to compute the probability that a subset of genes of a given size will have a given number of occurrences of the pattern we examine, when chosen randomly from the group of all known genes ( Johnson et al. 1992 ). Cluster analysis of SAGE data Considering the numerous types of transcripts present in a cell or tissue and the small probability of sampling a particular type of transcript at each draw, the number of sampled transcripts of each type is assumed to be approximately Poisson distributed. Statistically, when this actual sampling process is random enough, Poisson would be the most practical and reasonable assumption compared to other probability models. This assumption, with the assumption that each tag is uniquely mapped to a transcript, leads to the probability model used for clustering analysis of SAGE data (below). First, all SAGE tags were assigned at random to k groups. Second, a cluster center, which led to the expected expression pattern of each tag, was calculated for each cluster. Chi-square test statistics were used to measure the distance between the observed expression pattern and the expected expression pattern of a tag in a cluster. Third, using an iterative method, tags were moved between clusters, and intra- and intercluster distances were measured with each move. Tags were allowed to remain in the new cluster only if they were closer to it than to their previous cluster. Fourth, after each move, the expression vectors for each cluster were recalculated. Last, the shuffling proceeded until moving any more tags made the clusters more variable, increasing intracluster distances and decreasing intercluster dissimilarity (see Protocol S1 for full details of the algorithms used, as well as Cai, et al. 2004 for a more detailed discussion of applications of the protocol). To compute optimal values for the number of clusters k, the within-cluster dispersion was computed for increasing values of k . This within-cluster dispersion declined as new clusters were added. We thus looked for the reduction at each step, and observed the rate of change. Discontinuities in the rate of change were taken to indicate that a meaningful cluster number had been obtained, with the lowest number of clusters that showed such a discontinuity being used for analysis ( Hartigan 1975 ; Yeung et al. 2001 ). In order to determine the optimal number of clusters to use in the analysis of the SAGE data, the within-cluster dispersion was determined for a range of ten to 65 clusters over 100 iterations. If certain numbers of clusters gave a better fit to the data, they should show discontinuities in the rate of decrease ( Hartigan 1975 ). It was found that setting the number of k -means clusters at around 25, 40, and 55 showed these features (see Table S15 ) Database construction Data from 21 SAGE libraries and ISH images were gathered and stored in a MySQL relational database ( http://www.mysql.com ). Information on the measurement values for the SAGE libraries and ISH images can be accessed at http://134.174.53.82/cepko/ . The database was developed to provide up-to-date mapping of SAGE tags to UniGene clusters. Since a single sequence tag can represent different genes and, conversely, an individual UniGene cluster can be represented by more than one tag, both “full” and “reliable” tag-to-UniGene mappings ( Lash et al. 2000 ) have been created and can be selected by the user. The cluster assignments and their reliability were obtained from NCBI SAGEmap ( http://www.ncbi.nlm.nih.gov/SAGE ). For the database reported herein, UniGene Build 131 of Mus musculus and Build 164 of Homo sapiens ( http://www.ncbi.nlm.nih.gov/UniGene ) were used for the mappings. However, the database at http://134.174.53.82/cepko/ includes up-to-date mapping data. For each UniGene cluster, all measurement values and ISH images of associated tags are provided. Measurement values can also be segregated and summed up for each library if more than one SAGE tag is mapped to a given UniGene cluster. A plot of measurement values was also created to visualize patterns across the SAGE libraries. Additionally, for each UniGene cluster, links to gene functions using GO, accession numbers for annotated human orthologs, and LocusLink IDs have been provided. Supporting Information Figures S2–S12 show ISH data for genes that show dynamic expression in developing retina. All pictures were obtained from central retina. Cellular laminae of both the developing and mature retina are indicated with colored bars. All pictures were taken at 200x. See Table S5 for a full list of probes used. Figure S1 Comparison of E14.5 EST Versus E14.5 SAGE Data The number of times a gene was observed in a set of 15,268 individual ESTs obtained from E14.5 mouse retina (data obtained from Mu et al. [2001] ) compared to a set of 15,268 individual E14.5 retinal SAGE tags generated in this study. Only genes present at least ten times in the EST data set were considered. (1.7 MB TIF). Click here for additional data file. Figure S2 Heterogeneous Developmental Onset of Phototransduction Gene Expression The genes shown are rod arrestin, PrCdh, Gγ1, rod PDEγ, rhodopsin, peripherin 2, Gα1, and GCAP1 . (26.9 MB TIF). Click here for additional data file. Figure S3 Genes Expressed in Subsets of Cells in Developing ONBL Sections were from central retina. The genes shown are Otx2, RORβ, Yboxbp1, Mm.38347, Mm.11660, BTF3, H2Ax, Ppp1r14b, Grb10, Mm.158631, HMG-AT1, Mm.24141, KIAA1411, Mm.25018, IAP5, and Chaf1b . (25.7 MB TIF). Click here for additional data file. Figure S4 Genes Expressed Broadly in Mitotic Progenitors The genes shown are PDK3, Giα2, β-catenin, LRC8, Nrarp, Foxn4, and HMG-17 . (27.4 MB TIF). Click here for additional data file. Figure S5 Genes Expressed in Undefined Subsets of Progenitors/Precursors The genes shown are FABP7, BMP7, NTT7, Inhibin βB, and Sal3. (20.8 MB TIF). Click here for additional data file. Figure S6 Known Transcription Factors Expressed in Developing Rods These data are shown to allow direct comparison with the data in Figures 4 and S7 . The genes shown are NeuroD1, Crx, Nrl, and NR2E3. (24.9 MB TIF). Click here for additional data file. Figure S7 Genes Expressed in Developing Rods The genes shown are Cpx2, TRABID, Fln29, Mak, Mm.24642, Nlk, Hrs, Tnfsf13, and Arip2. (18.3 MB TIF). Click here for additional data file. Figure S8 Genes Expressed in Developing Bipolar Cells The genes shown are Chx10, Gli5, Dbp, Lhx4, Mm.41284, Prkcl, SEZ-6, and Zfh4. (21.4 MB TIF). Click here for additional data file. Figure S9 Genes Expressed in Developing Horizontal Cells The gene shown is Borg4. (11.0 MB TIF). Click here for additional data file. Figure S10 Genes Expressed in Developing Amacrine Cells The genes shown are Unc-51-like-1, ArfGAP, robo3, necdin, SAK, Mm.6393, Mm.34130, Nhlh2, NPY, Mm.21657, Mm.215653, and Mm.41638 . (19.1 MB TIF). Click here for additional data file. Figure S11 Genes Expressed in Developing Müller Glia The genes shown are KIAA0937, Mm.157502, Slc38a3, Nkd1, Dsp8, carbonic anhydrase 2, and cyclin D1. (40.1 MB TIF). Click here for additional data file. Figure S12 Additional Noncoding RNAs Expressed in Developing Retina The genes shown are MEG3, Xist, and Tsix. (13.6 MB TIF). Click here for additional data file. Protocol S1 Description of Methodology Used for Cluster Analysis of SAGE Tags (52 KB DOC). Click here for additional data file. Table S1 Summary of SAGE Tag Distribution The total cumulative number of tags found at each abundance level in all 12 retinal libraries (i.e., the ten libraries from total retinal of wild-type animals, the library from P10.5 crx −/− animals, and the library from microdissected ONL of adult animals) is shown. The number of tags, and the fraction of total tags, that do not show any reliable match for any gene (data from NCBI) are also shown. (14 KB XLS). Click here for additional data file. Table S2 Full List of Tag Counts in All SAGE Libraries Considered This list includes not only all libraries made from retinal tissue, but also nonretinal SAGE libraries made by this group, and other mouse libraries that are publicly available. Raw, unnormalized tag counts are shown. See Materials and Methods for more details on the SAGE libraries analyzed. (17.9 MB TXT). Click here for additional data file. Table S3 Twenty-Four-Cluster Analysis for SAGE Tags All tag abundance levels were normalized to 100,000. Tags present at greater than 0.1% in one or more of the ten wild-type total retina libraries were considered. The single most probable “reliable” tag-to-gene match ( http://www.ncbi.nlm.nih.gov/SAGE ) is shown, along with the confidence level of that assignment. Mouse UniGene number is shown for each tag-to-gene match, along with LocusLink ID, where available. In each case where a gene was analyzed by ISH in developing retina, that fact is indicated in the final column. In some cases, a gene that matched the tag with a lower confidence level was tested. In these cases, the UniGene number of the gene tested by ISH differs from that of the most probable tag match. (1.0 MB XLS). Click here for additional data file. Table S4 Molecular Function, Biological Process, and Subcellular Compartment GO Data Are Shown for Each Gene Analyzed by ISH in the Retina Gene names and LocusLink IDs for these genes are also shown (225 KB XLS). Click here for additional data file. Table S5 Complete List of Cellular Expression Patterns for Each Probe Tested The SAGE tag matching each gene tested is given, as well as the accession number of the cDNA used to generate each probe used for ISH. Cellular expression is scored on a 0–5 point scale for each time point tested, as well as for E16 embryo and P6 head cut in horizontal section. A, amacrine cells; Ast, astrocytes; B, bipolar cells; Bv, blood vessels; Cb, cerebellum; CM, ciliary margin; CP, cortical plate; Ctx, cerebral cortex; DG, dentate gyrus of hippocampus; DRG, dorsal root ganglia; EGL, external granule layer of developing cerebellum; EOM, extraocular muscles; G, ganglion cells; H, horizontal cells; Hippo, hippocampus; I, inner neuroblastic layer; In, inner nuclear layer; MG, Müller glia; MGE, medial ganglionic eminence; ND, not determined; O, outer neuroblastic layer; OB, olfactory bulb; OE, olfactory epithelium; ORN, olfactory receptor neurons; P, panretinal; PC, Purkinje cells; PNS, peripheral nervous system; Pr, photoreceptors; Pr(is), inner segments of photoreceptors; sA, subset of amacrine cells; sB, subset of bipolar cells; SC, spinal cord; sG, subset of ganglion cells; sI, subset of cells in INBL; sIn, subset of cells in INL; scI, scleral INBL; sscI, subset of cells in scleral INBL; svI, subset of cells in vitreal INBL; sO, subset of cells in outer neuroblastic layer; scO, scleral ONBL; sscO, subsets of cells in scleral ONBL; svO, subset of cells in vitreal ONBL; sPr, subset of photoreceptors; SVZ, subventricular zone; vI, vitreal INBL; vO, vitreal ONBL; VRN, vomeronasal receptor neurons; VZ, ventricular zone. (381 KB XLS). Click here for additional data file. Table S6 User-Curated Cellular Expression Clusters for Genes Tested by ISH in Retina Here, data from Table S5 are summarized such that the predominant cellular expression pattern from early (E12–E18), mid (P0–P4), and late (P6–adult) developing retina is recorded, and genes are grouped into coexpressed clusters by user annotation. The main cell types expressing the gene in the retina over the interval in question are listed, with weaker expression in other cell types being noted in parentheses. Clusters are given a name (after a representative gene) and a unique cluster number, and the presumptive cell types that show greatest expression are listed. Genes for which the full developmental expression profile was not determined are tentatively assigned to clusters that showed the best fit based on two out of three criteria, with tentative assignments being indicated as such (261 KB XLS). Click here for additional data file. Table S7 Numerical Cellular Expression Data Used for Machine-Aided Cluster Analysis of Cellular Expression Patterns of Genes Tested by ISH in Retina To obtain these numbers, data from Table S5 were modified. As in Figure S6 , expression data were summarized for early (E12–E18), mid (P0–P4), and late (P6–adult) developing retina. In cases where cellular expression changed dramatically within one of these three intervals (e.g., expression shifted from INBL to ONBL), these cellular expressions were both entered in the category in question. Genes that were not examined in all three of these time intervals were not considered in this analysis. Cellular expression data, scored on a 0–5 point scale, were then entered for each time point separately in each of the categories used to score retinal cellular expression in Table S5 . (266 KB XLS). Click here for additional data file. Table S8 Comparison of User-Curated Cellular Expression Clusters from Table S6 and a 45-Cluster Machine-Aided Analysis of the Cellular Expression Data from Table S7 The fraction listed notes the fraction of genes in the machine-generated cluster that were found in a given user-curated cellular expression cluster. The presumptive cellular expression pattern of each user-curated cellular expression cluster is also listed (following Table S6 ). (86 KB XLS). Click here for additional data file. Table S9 Comparison of 4N-Enriched Genes from Livesey et al. (2004) and SAGE Cluster Data from Table S3 Shown is the percentage of tags that matched genes enriched in 4N retinal progenitor cells found in a given SAGE tag cluster. (14 KB XLS). Click here for additional data file. Table S10 Comparison of the SAGE Tag Cluster Data from Table S3 and the 72-Cluster Analysis of the User-Curated Cellular Expression Data from Table S6 Values indicate the fraction of all tags found in a given SAGE tag cluster that were found in a specific user-curated cellular expression cluster. The presumptive cellular expression pattern of each cellular expression cluster is also listed (following Table S6 ). (209 KB XLS). Click here for additional data file. Table S11 SAGE Tags Representing the Known Photoreceptor-Specific Genes Analyzed in Figure S2 Tags in each library are expressed as the fraction of all tags that match the gene in question that were found in the ten libraries considered. (15 KB XLS). Click here for additional data file. Table S12 Candidate Noncoding RNAs Analyzed by ISH in This Study The SAGE tag corresponding to the transcript in question is listed, along with UniGene numbers, and accession numbers of the probes used for ISH for each candidate noncoding RNA. P -values for BLASTN and BLASTX mouse/human comparisons are shown. Transcripts that show high BLASTN, but low BLASTX, matches to human may represent the best candidates for noncoding mRNAs of functional importance and are indicated as likely to be genuine noncoding RNAs. NS, not significant. (17 KB XLS). Click here for additional data file. Table S13 Accession Numbers for Full-Length Transcripts for Genes Tested by ISH in This Study, Along with Their Human Orthologs Chromosomal localizations are shown for both the mouse genes and their human orthologs. Genes located within chromosomal intervals containing mapped but uncloned retinal disease genes are indicated by the name of the disease (terminology from Retnet; http://www.sph.uth.tmc.edu/Retnet/disease.htm ). User-curated cellular expression data of the genes in question (derived from Table S6 ) are shown to aid in prioritizing candidate disease genes for further investigation. ND, not determined. (291 KB XLS). Click here for additional data file. Table S14 Average Distance Analysis of Cellular Expression Data from Table S7 The values shown here are the average sum-of-squares within k- means clusters over all variables. Euclidian mean distance–directed clustering is used ( Hartigan 1975 ). The proportional reduction of error (PRE) for each number of clusters is also shown. This measures the ratio of reduction in within-cluster dispersion to the previous within-cluster dispersion ( Hartigan 1975 ). For this analysis, PRE is given by ( Ni − N ( i – 5))/ Ni, where N is the average within-cluster distance and i is cluster number. (14 KB XLS). Click here for additional data file. Table S15 Average Distance Analysis of SAGE Tag Clusters Tags present at greater than 0.1% in one or more of the ten wild-type total retina libraries were considered and were normalized to 100,000 for this analysis. The average sum-of-squares within k- means clusters for each number of clusters is shown. The PRE, given by ( Ni − N ( i – 5))/ Ni, is also shown. (14 KB XLS). Click here for additional data file. Accession Numbers The GenBank ( www.ncbi.nlm.nih.gov ) accession numbers for the genes discussed in this paper are β-catenin (NM_007614), ArfGAP (BC030682), Arip2 (NM_025292), BMP7 (NM_007557), Borg4 (NM_012121), brain fatty acid binding protein 7 (NM_021272), BTF3 (NM_145455), carbonic anhydrase 2 (NM_009801), cdk4 (NM_009870), Chaf1b (NM_028083), Chx10 (NM_007701), Cpx2 (NM_007756), Crx (NM_007770), Dbp (NM_016974), Drosophila castor gene (BC035954), Dsp8 (XM_181424), FABP7 (NM_021272), Fln29 (NM_172275), Foxn4 (NM_148935), GCAP1 (NM_008189), Giα2 (NM_008138), Gli5 (NM_031184), Grb10 (NM_010345), Gα1 (NM_008140), Gγ1 (NM_010314), H2Ax (NM_010436), HMG-17 (NM_016957), HMG-AT1 (NM_016660), Hrs (NM_008244), IAP5 (NM_009689), inhibin βB (BC048845), KIAA0937 (NM_172442), KIAA1411 (NM_026604), Lhx4 (NM_010712), LRC8 (NM_172736), Mak (NM_008547), MEG3 (NM_144513), Mm.103742/Cdc42GAP (NM_020260), Mm.11660 (AK034313) , Mm.11738/Ark-1 (BC005425) , Mm.142856/Lhx2 (NM_010710), Mm.150838/RNCR1 (AK044330), Mm.157502 (NM_026592) , Mm.158631 (XM_132295) , Mm.1635/PIAS3 (NM_018812), Mm.18789/Sox4 (NM_009238), Mm.19155/sFrp2 (NM_009144), Mm.193526/Yboxbp4 (NM_007705), Mm.194050/RNCR3 (AK044422), Mm.200608/clusterin (NM_013492), Mm.20465/GPCR37 (NM_010338), Mm.213213/HK-R (NM_145419), Mm.215653 (NM_183191), Mm.21657 (BC038057), Mm.2214/septin 4 (NM_011129), Mm.22288/cyclin D1 (NM_007631), Mm.2229/Eya2 (NM_010165) , Mm.235550/ERRβ (NM_011934), Mm.23916 (AK009781), Mm.24141 (NM_025615), Mm.24642 (NM_146168), Mm.25018 (BC010304), Mm.26062/AD024 (NM_025565), Mm.27953/glycine decarboxylase (NM_138595), Mm.29067/Mbtd1 (NM_134012), Mm.29496/Zf-1 (AK004085), Mm.29729/Tweety1 (NM_021324), Mm.29924/Arl6ip1 (BC010196) , Mm.34130 (AK012601), Mm.34701/Pum1 (NM_030722), Mm.3499/Rax homeodomain factor (NM_013833), Mm.35817 (NM_145940), Mm.35829/Edr (NM_133362), Mm.38347 (XM_126644), Mm.3904/Fgf15 (NM_008003), Mm.40321/Pgrmc2 (XM_130859), Mm.41284 (NM_153137), Mm.41638 (NM_029530), Mm.44854/RNCR2 (AK028326), Mm.4541/Sox2 (NM_011443), Mm.45753/KIAA0013 (NM_181416), Mm.4605/Tbx2 (NM_009324), Mm.5021/DDR1 (NM_007584), Mm.55143/Dkk3 (NM_015814), Mm.6393 (NM_010045), Mm.89623/mCas ( BC035954) , Mm.9114/mu-crystallin (NM_016669), necdin (NM_010882), NeuroD1 (NM_010894 , Neuropeptide Y (NM_023456), Nhlh2 (NM_178777), Nkd1 (NM_027280), Nlk (NM_008702), NPY (NM_023456), NR2E3 (NM_013708), Nrarp (NM_025980), Nrl (NM_008736), NTT7 (NM_175328), Otx2 (NM_144841), PDK3 (NM_005391), peripherin 2 (NM_008938), Ppp1r14b (NM_008889), PrCdh (NM_130878), Prkcl (NM_008857), RGPRIP (NM_023879), rhodopsin (NM_145383), robo3 (NM_011248), rod arrestin (NM_009118), rod PDEγ (NM_012065), RORβ (NM_146095), SAK (NM_019945), Sal3 (NM_026528), SEZ-6 (NM_021286), Slc38a3 (NM_023805), syntrophin-associated kinase (NM_019945), Tnfsf13 (NM_023517), TRABID (AK005926), Tsix (AF138745), Unc-51-like-1 (NM_009469), Xist (AK011511), Yboxbp1 (NM_011732), and Zfh4 (NM_030708).	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC439783.xml
539230	Unique Double-Barreled Enzyme Makes Methionine the Hard Way	null	If a cell is a complex symphony of chemical reactions, its enzymes are the instruments through which this elemental music is played. Each reaction is catalyzed by a specific enzyme, whose uniquely shaped active site not only binds reactants, but, by forming weak and temporary bonds, coaxes them into new orientations with new partners, thus creating the products. Determining exactly how any individual enzyme accomplishes this task—which amino acids make up the active site, which bonds form where when enzyme meets substrate, which electrons switch partners as new bonds form—is the work of the structural biochemist. In this issue, Martha Ludwig and Robert Pejchal elucidate the structure of cobalamin-independent methionine synthase (MetE) from the bacterium Thermotoga maritima , and describe how it catalyzes the formation of the amino acid methionine. Methionine synthases actually come in two forms, which use somewhat different mechanisms to accomplish the same task: transfer of a methyl group (CH3) from methyltetrahydrofolate to the terminal sulfur of homocysteine. The cobalamin-dependent form, MetH, relies on the cofactor cobalamin (vitamin B12), which pulls the methyl away at one active site, and then donates it at a second active site. Here, a central zinc atom binds and activates homocysteine, enabling it to attack the incoming methyl group that is attached to cobalamin. MetE, on the other hand, has no cofactor and only one active site, which sits at the junction of two eight-stranded barrels. The structure and sequence of these barrels indicate they arose through duplication of a primordial zinc-bearing, homocysteine-binding protein. This unique duplex now bears only one zinc atom, deep within the cleft separating the two barrels. As in MetH, the role of the zinc is to bind homocysteine, but in MetE, this event also induces a conformation change around the zinc. The zinc and its coordinating partners form an umbrella; entering from the handle end, the homocysteine sulfur pulls the zinc toward it and turns the umbrella inside out. Methyltetrahydrofolate initially binds along the edge of the cleft, with the methyl group on the folate oriented far from the sulfur on the homocysteine, as can be seen in the research article's Video S1 (DOI: 10.1371/journal.pbio.0030031.sv001 ). There must be subsequent conformational changes within the active site that serve to bring the two substrates together and promote transfer of the methyl group. Exactly how methyltetrahydrofolate reorients within the cleft to complete the reaction is not yet clear. The reaction catalyzed by MetE proceeds slowly, at only 1%–2% of the speed of that catalyzed by MetH. One reason for this rather sluggish activity is that homocysteine, even when activated by binding to zinc, is much poorer than cobalamin at displacing the methyl group of methyltetrahydrofolate. While MetE's unique active-site structure was made possible by gene duplication, the two barrels are no longer identical. Through evolution, the second, N-terminal, barrel has lost the ability to bind zinc or homocysteine, and indeed appears to contribute little to the active function of the enzyme. Nonetheless, this barrel may be necessary to temporarily isolate the substrates from solvent and to form the hydrophobic environment in which the reaction is more favorable. Further research may indicate more about the function of this unequal partner, and provide more detail on the exact atomic movements within the cleft at the moment of reaction.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC539230.xml
554976	Comparing strategies for United States veterans' mortality ascertainment	Background We aimed to determine optimal strategies for complete mortality ascertainment comparing death certificates and United States (US) Veterans Administration (VA) records. Methods We constructed a cohort of California veterans who died in fiscal year (FY) 2000 and used VA services the year before death. We determined decedent status using California death certificates linked to VA utilization data and the VA Beneficiary Identification and Records Locator System (BIRLS) death file. We compared the characteristics of decedents who would not have been identified by either single source (e.g., VA BIRLS alone or California death certificates alone) with the rest of the cohort. Results A total of 8,813 veteran decedents were identified from both VA decedent files and death certificates. Of all decedents, 5,698 / 8,813 (65%) veterans were identified in both source files, but 2,426 / 8,813 (28%) decedents were not identified in VA BIRLS, and 689 / 8,813 (8%) were not identified in death certificates. Compared to the rest of the cohort, decedents whose mortality status was ascertained through either single source differed by race / ethnicity, marital status, and California residence. Clinically, veterans identified from either single source had less comorbidity and were less likely to have been users of VA inpatient or long term care, but equally or more likely to have been users of VA outpatient services. Conclusion As single sources, VA decedent files and death certificates each provided an incomplete record, and death ascertainment was improved by using both source files. Potential bias may vary depending on analytic interest.	Introduction Clinicians, healthcare administrators, researchers, regulators and policymakers are concerned with optimizing mortality ascertainment using administrative data. In addition to its clinical importance, mortality informs program planning, quality assessment and improvement, and public reporting [ 1 - 8 ]. Veterans are an important, vulnerable population in which mortality has been examined as a function of race / ethnicity, service characteristics, access, and quality of care. Valid, complete reporting is critical to the success of such endeavors, and limitations in using death certificates have been acknowledged [ 9 , 10 ], although VA mortality data is generally regarded as accurate [ 11 - 15 ]. To understand the limitations of single source ascertainment, we described decedents who would not have been identified by a strategy using either VA decedent files alone or death certificates alone. We compared cases that would have been missed using either single source with the rest of the cohort based on their demographic and clinical attributes and the settings in which they received care. Methods In order to evaluate the implications for improving veterans' end-of-life care, we constructed a population-based decedent cohort [ 16 ]. For such purposes, it is particularly important to understand whether death was recorded elsewhere for veterans who were under VA care since the VA system may be responsible for much of their end-of-life care even if they do not die while receiving health care in a VA facility. Data Sources The VA Beneficiary Identification and Records Locator System (BIRLS) contains records of all beneficiaries including veterans whose survivors applied for burial benefits. It includes records of discharged military veterans post-1973 and recipients of Medals of Honor and VA education benefits. After submission to the Veterans Benefits Administration (VBA), deaths are recorded in the BIRLS Death File. A submission to the VBA is typically triggered by a family claim for death benefits (e.g. burial assistance, pension) [ 17 - 19 ]. The VA maintains a National Patient Care Database (NPCD) that contains a record of Social Security Number (SSN) linked VA and contracted health services provided to all veterans [ 17 - 19 ]. Death certificates are required for burial in California and are available for public use [ 20 ]. We first identified 345,380 decedent veterans who died during FY2000 (30 September 1999 – 1 October 2000) from the BIRLS Death File. We used SSNs to link cases to VA NPCD outpatient, inpatient, or long term care records restricted to recipients of any VA services in California within 12 months of death. We extracted records including any inpatient or long term care admission, or outpatient encounters. Veterans who entered the cohort on the basis of using outpatient services were required to have at least one clinical encounter (e.g., other than laboratory, radiology, or administrative). In addition, we used California death certificates as second source to identify decedent veterans by linking SSNs from death certificates directly to VA utilization files. California death certificates contained 462,561 records for calendar years 1999 and 2000, and we primarily matched decedents identified through death certificates to BIRLS by SSN. We manually inspected matches on SSN only and we also examined matches on criteria other than SSN (e.g. last name, first name, date of birth, date of death). Additional cases we accepted after manual inspection involved transpositions of one and rarely more than one SSN digit but agreement in other fields. Thus, the cohort included recipients of VA clinical services verified as deceased based on either BIRLS or death certificates, and all cases were linked to VA utilization files by SSN. In the final decedent cohort, we excluded cases of non-veterans receiving care at VA facilities by examining indicators of veteran status associated with visits. The VA assigns specific codes to non-veterans rendered care for various reasons (e.g., emergency or charitable care). We also considered the possibility of erroneous decedent status by looking for evidence of healthcare utilization during the 12 months after death. We excluded cases with evidence of utilization more than one month after the date of death. Variables and Analysis We used VA encounters and ICD-9-CM codes to demographically (e.g., age, gender, marital status, state of residence, and race / ethnicity) and clinically characterize decedents [ 21 - 26 ]. We identified veterans with any visit or admission for congestive heart failure (CHF), ICD-9-CM 398.91, 402.x1, 404.x1, 404.x3 428.x excluding procedures, chronic obstructive lung disease (COPD), ICD-9-CM 491–492.x, 494.x, 496, end-stage liver disease (ESLD), ICD-9-CM 571.2–571.9,572.2–572.8, dementia, ICD-9-CM 046.1, 290.0–290.43, 331.0–331.7, 333.4, 438.0, and malignant neoplasia, ICD-9-CM 140.0–208.9 [ 25 ]. To identify end-stage renal disease (ESRD), we used procedure and clinical stop codes that identify the type of care received (e.g., dialysis) [ 26 ]. We developed a complexity index of co-morbidity based on a simple count of advanced illnesses. To understand the limitations of single source mortality ascertainment, we described decedents who would not have been identified by a strategy using either death certificates alone or VA decedent files alone. We compared these cases with the rest of the cohort based on their demographic and clinical attributes and the settings in which they received care. Based on distributions, we used Wilcoxon tests for continuous and chi-square tests for categorical variables. Results From 345,380 deaths during the period 30 September 1999 to 1 October 2000 identified in BIRLS, we distinguished 6,071 decedents who were users of VA inpatient, outpatient, or long term care services in California. California death certificates included 227,308 deaths during the same period, including 3,580 additional users of VA inpatient, outpatient, or long term care services in California. Using SSN and other identifiers to match decedent cases to VA utilization data, we excluded non-veterans (n = 365), users of only non-clinical care such as laboratory tests (n = 251), those possibly alive based on subsequent VA encounter data (n = 229), and 3 cases for other reasons. Of the final cohort of 8,813 veteran decedents, 5,698 (65%) cases were identified in both source files, while 689 (8%) were only identified in VA decedent files, and 2,426 (28%) additional cases were only identified through death certificates (Figure 1 ). Figure 1 Cohort Development We examined potential biases associated with veteran decedents missed by either single source of mortality ascertainment (e.g., VA BIRLS or California death certificates). Ninety-nine percent of decedents missed by using VA data alone were California residents (vs. 92% of the remainder cohort, p < 0.001); whereas, 62% of those missed by using death certificates alone were out-of-state residents (vs. 1% of the remainder cohort, p < 0.001). Relatively fewer veterans of white or black ethnicity and relatively more veterans of missing ethnicity were represented among decedents missed by either single source strategy. The proportion of married or previously married veterans was higher and single or missing marital status lower among those decedents missed using only BIRLS, and relative proportions were reversed for a strategy using only death certificates. Decedents missed by either single source approach were less likely to have been diagnosed with an advanced chronic illness than the identified cohort. Veteran decedents missed by using only BIRLS were less likely to be diagnosed with any condition except HIV and dementia, and those missed by using death certificates alone were less likely to be diagnosed with any condition except HIV. With a BIRLS only approach, 37% of missing cases vs. 35% of the remainder cohort (p < 0.001) had no diagnosed chronic illness (death certificate only approach; 69% vs. 32%, p < 0.001). Veteran decedents missed by either single source approach were equally or more likely to have been users of the outpatient setting, but missed cases were less likely to have been users of inpatient healthcare settings (Table 1 ). Table 1 Potential Bias Associated with Alternative Strategies For Veterans' Mortality Ascertainment * BIRLS Only Strategy Death Certificate Only Strategy Cases identified by BIRLS Additional cases identified by death certificates P-value Cases identified by death certificates Additional cases identified by BIRLS P-value Number of cases 6,387 2,426 8,124 689 Age (years) 70.86 71.15 0.8253 70.95 70.79 0.6891 Gender Male 98 97 0.2733 98 98 0.8662 Race / Ethnicity White 57 54 58 31 <0.001 Black 12 8 11 6 Hispanic 5 5 5 1 Other 2 2 2 1 Missing 24 31 <0.001 23 61 Marital Status Married 46 49 47 45 0.0028 Single 16 13 15 16 Divorced 23 24 23 23 Widowed 11 12 11 11 Missing 4 2 0.0002 3 6 State of Residence California 92 99 99 38 Non-California 8 1 <0.001 1 62 <0.001 Diagnosis Cancer 35 32 0.0426 35 17 <0.001 CHF 22 19 0.0175 22 7 <0.001 COPD 28 24 0.0002 28 11 <0.001 ESLD 6 4 0.0327 6 3 0.0010 ESRD 3 1 <0.0001 3 0 0.001 Dementia 11 11 0.9656 11 3 <0.001 HIV 1 1 0.1586 1 0 0.0621 Complexity Index 0 35 37 32 69 1 35 39 38 22 2 22 18 22 6 3 7 5 7 2 4 1 1 <0.0001 1 0 <0.001 Site of Utilization Any inpatient Any long term 45 29 <0.0001 42 21 <0.001 care 20 12 <0.0001 19 7 <0.001 Any outpatient 95 96 0.0225 95 96 0.1218 *Findings are expressed as proportions unless otherwise identified. P-values reflect Wilcoxon two-sided probabilities for continuous variables and chi-square for categorical variables. Categorical tests reflect tests for differences including missing. Discussion Veterans' mortality ascertainment was significantly improved by using both VA and death certificates as source files. Our findings indicate that either single source approach for mortality ascertainment may misrepresent veteran mortality based on comparisons of race / ethnicity, marital status, severity of illness, and settings of care. Diagnoses associated with serious medical co-morbidity and the likelihood of receiving any inpatient services (e.g. hospital or long term care) were both significantly lower among veterans missed by either single source approach. Our findings are consistent with Washington State where the deaths of 25% of 533 veterans who only used outpatient services were only identified with death certificates, and 5% were only identified in BIRLS. [ 9 ] Using BIRLS only for mortality determination, it is unclear why generally healthier, primarily outpatient users are less likely to be noted. Death notification is typically triggered by benefit claims (e.g., burial assistance, pension and related benefits). Affluent veterans whose families might be less likely to file benefit claims were drawn to the VA recently [ 27 ]. However, poverty or low social support might also make it harder to file claims. On the other hand, a death certificate only approach to ascertainment misses relatively fewer non-resident veterans. Such veterans may be homeless or mobile, retired veterans, and they may seek care transiently in California, or their deaths may be recorded elsewhere. One limitation of our study is that we did not identify cases that were only decedents by virtue of VA utilization files alone rather than BIRLS, although Dominitz, et. al., identified only 2.7% of deaths this way [ 11 ]. We did not compare VA files or death certificates to the National Death Index (NDI), as have previous studies that have used the NDI as a gold standard. The NDI is a central data repository of state vital statistics that is often used as a gold standard in US mortality studies [ 28 ]. We report findings for only one state, but given similar findings in Washington State, it would be helpful to determine if this is a national issue or there are particular state issues related to BIRLS death file agreement, or concerns related to veteran morality ascertainment with California death certificates. Conclusion Researchers, managers, and policy makers should understand the limitations of sources of mortality ascertainment. The relationship of missing data to bias is related somewhat to how "missingness" is distributed by the outcome of interest. Our findings suggest these concerns may be relatively more important for studies involving veterans and racial-ethnic disparities, co-morbidity, certain disease comparisons, or settings of care. Additional study is needed to compare BIRLS, death certificates, and the NDI for mortality ascertainment in veterans. If our findings are confirmed, the VA may need to consider improving its system for mortality ascertainment through routine linkages to national mortality data. Studies of end-of-life care using decedent cohorts need to pay particular attention to the incompleteness of VA data as the sole source of mortality information. List of Abbreviations Used VA, Veterans Administration; FY, fiscal year; BIRLS, Beneficiary Identification and Records Locator System; NPCD, National Patient Care Database; SSN, Social Security Number; CHF, congestive heart failure; COPD, chronic obstructive lung disease; ESLD, end-stage liver disease; ESRD, end-stage renal disease. Competing interests The author(s) declare that they have no competing interests. Authors' contributions KL originated and oversaw all aspects of the conception, design, analysis, and publication of the study. SA, LR, and EY contributed to conception, design, and analysis. MW contributed to analysis and is responsible for programming. All authors reviewed and approved of the manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC554976.xml
524261	One Brain, One Vision	null	Not all devices that measure the same property do it in the same way—a clock might use a spring system or it might be digitally synchronized to a transmitted signal. Although both have the same goal of reporting accurate time, each is subject to different errors. Sometimes even the same device uses different systems to measure the same property. A relatively simple device like a camera will use one sensor system to capture light intensity for an image and a second sensor to capture light intensity for making automatic adjustments of aperture and flash. It does not seem outlandish, therefore, that the brain might also have developed multiple sensory systems to achieve different goals. Indeed, an influential hypothesis has argued that people use two separate visual processing systems in much the same way as a camera—one for creating our perception of the world and another for guiding our actions within it. One line of evidence supporting this dual hypothesis comes from an illusion known as Roelofs effect. Usually, people are pretty good at judging the location of even a small object. But if the small object is surrounded by a large frame and the frame itself is not centered in front of the person who is judging it, the viewer will perceive the object as shifted in a direction opposite that of the frame. This may not in itself be surprising, but the same person who perceives an offset of the object where none exists is nonetheless able to grasp it without difficulties. Figure 1 In this issue of PLoS Biology , Paul Dassonville and his colleagues reexamine the seeming dissociation of visual analysis for perception and action, and call it into question. Through a careful quantitative analysis of the conditions under which the Roelofs effect occurs, they find that it traces not to an illusory perception of the object location but to an illusory perception of self. The large frame, presented under experimental conditions in which subjects sit in darkness without access to a normal rich sensory environment, actually causes people to incorrectly perceive their own centers as rotated towards the frame and therefore to conclude that the small object is offset with respect to themselves. This may seem like a subtle distinction, and yet, since it is the observer's frame of reference that is altered, that same distorted frame of reference will be used to guide movement. Thus, the error in movement planning should cancel the error in perception, and people should have no trouble reaching for the object despite their misperception, which is indeed what is observed. Others have questioned the hypothesis that two separable neural systems process the visual world for perception and action, but this study removes one of the strongest pieces of evidence in its favor with a precise alternative explanation. No two brains may see the world identically, but the authors suggest that it may be time to concede that a single brain, at least, has the same world view.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC524261.xml
554786	Using equilibrium frequencies in models of sequence evolution	Background The f factor is a new parameter for accommodating the influence of both the starting and ending states in the rate matrices of "generalized weighted frequencies" (+gwF) models for sequence evolution. In this study, we derive an expected value for f , starting from a nearly neutral model of weak selection, and then assess the biological interpretation of this factor with evolutionary simulations. Results An expected value of f = 0.5 (i.e., equal dependency on the starting and ending states) is derived for sequences that are evolving under the nearly neutral model of this study. However, this expectation is sensitive to violations of its underlying assumptions as illustrated with the evolutionary simulations. Conclusion This study illustrates how selection, drift, and mutation at the population level can be linked to the rate matrices of models for sequence evolution to derive an expected value of f . However, as f is affected by a number of factors that limit its biological interpretation, this factor should normally be estimated as a free parameter rather than fixed a priori in a +gwF analysis.	Background Felsenstein [ 1 ] was the first to introduce an evolutionary model for DNA sequences, which allows for unequal nucleotide frequencies (see also [ 2 ]). His F81 model allows for substitutions at a rate proportional to the frequencies of the ending nucleotides. It is considered the simplest rate matrix for accommodating variable nucleotide frequencies and is therefore the starting point for the consideration of more complex models with frequency variation (e.g., the HKY model of Hasegawa et al . [ 3 ]). Goldman and Whelan [ 4 ] described new variants of these F81-based models (their +gwF (generalized weighted frequencies) models; e.g., JC+gwF for Jukes and Cantor [ 5 ], and K2P+gwF for Kimura [ 6 ]). At the heart of their +gwF variants was a new free parameter ( f ) to accommodate the frequencies of the starting, as well as ending, nucleotides in the evolutionary process: where q ij refers to the substitution rate from nucleotide i to j , π i and π j correspond to their equilibrium base frequencies, and s ij is the exchangeability between the two. In the +gwF variants, the substitution rate becomes more dependent on the ending nucleotide as f decreases from 1 to 0, with f = 0 for the classic F81-type models. This study starts with a population genetics model to derive equations that link weak selection, genetic drift, and mutation to the f parameter and evolutionary rate matrices of the +gwF variants. These theoretical derivations lead to an expected value of f = 0.5. However, as illustrated with simulations, the f parameter is complex and thus its biological interpretation must be considered with caution. Results Derivation of the rate matrix for the weak selection model The nearly neutral model of molecular evolution states that most DNA mutations of longer-term evolutionary consequence are under weak selection and are therefore prone to drift [ 7 , 8 ]. For a diploid population of size N , a neutral mutation has a probability of 1/2 N of becoming fixed in the population. However, because of drift, even slightly deleterious mutations can become fixed, but at a probability of less than 1/2 N . Advantageous mutations have higher fixation probabilities than neutral mutations. In the nearly neutral model, the distribution of alleles is determined by an equilibrium of selection, drift, and mutation. Consider a number of sites under identical evolutionary constraints and with a bias in nucleotide distribution. Assume that weak selection and drift are the causes of this bias; e.g., as for the codon usage biases in micro-organisms and Drosophila [ 9 , 10 ]. In our model, some nucleotides confer a slightly higher fitness onto the organism than do others, regardless of their position, and these can become fixed in the population through drift and/or selection. Here, we also assume that selective advantages are additive for the two alleles of the diploid organism [ 11 , 12 ]. Let the selective advantages of the four nucleotides be given by s A , s C , s G , and s T . The differences between these selection coefficients will be very close to zero, since no strong selection is expected. Consider a mutation from nucleotide i to j , with a selective advantage of s = s j - s i (a selective disadvantage exists when s is negative). For a population of size N and an effective size of N e , Kimura [ 11 ] showed that the fixation probability in this population is given by: when s ≠ 0. For s = 0, we have P ( s ) = 1/2 N . This approximation is valid for small values of s , which is the case here. The substitution rate from nucleotide i to j is proportional to P ( s j - s i ): q ij = 2 N μ ij P ( s j - s i ), (3) where μ ij is the mutation rate from i to j . For different i and j , μ ij can vary because of unequal transition versus transversion rates (for example). Furthermore, let us assume that the mutation rate is the same for either direction of substitutions between i and j . This assumption is necessary to maintain the widely used condition of time reversibility in the evolutionary process, which thereby keeps the following derivations tractable [ 1 , 13 ]. We then have: Since q ij / q ji can be written as a function evaluated at s j divided by the same function evaluated at s i , evolution is time reversible according to this model with: Here, c and c ' are constants with c ' = -l/4 N e log c , which will be chosen to make the equilibrium frequencies sum to one. The substitution rates can now be approximated as: Given an exchangeability of s ij = μ ij , this equation reduces to equation (1) with f = 0.5 and an adjustment factor of: This adjustment factor is close to one for moderate ratios of π , with a horizontal tangent around π j / π i = 1 and a slight bending downwards when deviating from this value (Fig. 1 ). Thus, a value of f = 0.5 is suggested for the +gwF variants according to these derivations of the weak selection model. Figure 1 Adjustment factor as a function of the ratio of π 's . The adjustment factor is given by (equation (7)). Evolutionary simulations Evolutionary simulations were conducted to examine the effects of violating certain assumptions in the above model of weak selection. Unless otherwise noted, these simulations were based on the K2P+gwF model with f = 0.5 and k = 2 (for the transition/transversion ratio). Simulations consisted of four sequences of length 10,000 and relied on a symmetric rooted phylogeny with all branch lengths equal to 0.10 expected substitutions per site under the model in question [i.e., ((seq1:0.10, seq2:0.10):0.10, (seq3:0.10, seq4:0.10):0.10)]. Violations of the weak selection model were incorporated in the simulations by: (1) heterogeneous sequences with sites drawn from different equilibrium base frequencies; (2) populations in disequilibrium due to changing N e ; and (3) an accelerated C to T substitution rate. Estimates of f for the simulated sequences were made with the K2P+gwF model. Forty simulations were run for each test condition, with the results for the f estimates summarized as their means and twice their standard errors. In the first set of simulations, six categories of sites with different equilibrium distributions were considered (Table 1 ). The f estimates for the simulations with each category alone were not significantly different from 0.5 (i.e., the value under which the sequences were generated). In contrast, for the simulated heterogeneous sequences (i.e., those composed of equal numbers of sites from two or three different categories), their values of f varied significantly in either direction from 0.5. Analogous results were obtained for the simulations of homogeneous and heterogeneous sequences under the HKY model (with f = 0.0 instead of 0.5). Thus, the value of f can vary considerably when heterogeneous sequences are analyzed with a +gwF model. Here, such deviations are a consequence of using a single rate matrix to analyze sequences that were derived from two or three different ones. Table 1 Starting equilibrium base frequencies and results for the simulations with either homogeneous or heterogeneous sequences (i.e., those with sites from single versus multiple categories, respectively). Categories π C π G π T Bias b f c A 0.10 0.40 0.30 0.20 0.154 0.50 ± 0.01 0.00 ± 0.01 B 0.30 0.30 0.30 0.10 0.105 0.50 ± 0.01 0.00 ± 0.01 C 0.30 0.20 0.20 0.30 0.029 0.50 ± 0.02 0.00 ± 0.03 D 0.40 0.20 0.20 0.20 0.078 0.49 ± 0.01 0.01 ± 0.01 E 0.20 0.40 0.20 0.20 0.078 0.51 ± 0.01 -0.01 ± 0.02 F 0.20 0.20 0.40 0.20 0.078 0.50 ± 0.02 -0.01 ± 0.01 A+B 0.20 0.35 0.30 0.15 0.074 0.43 ± 0.01 -0.11 ± 0.02 A+C 0.20 0.30 0.25 0.25 0.015 0.34 ± 0.03 -0.16 ± 0.03 B+C 0.30 0.25 0.25 0.20 0.015 0.24 ± 0.02 -0.33 ± 0.03 A+B+C e 0.23 0.30 0.27 0.20 0.016 0.39 ± 0.04 -0.13 ± 0.04 D+E+F e 0.27 0.27 0.27 0.20 0.010 0.68 ± 0.03 0.29 ± 0.04 a Expected nucleotide distribution. b Nucleotide bias, as information content measured in bits: . c Mean ± twice the standard error of the estimate. d f = 0:0 for these simulations with the HKY model. With f = 0:0, the HKY+gwF variant is reduced in these simulations to its more standard F81 based model. e The heterogeneous sequences in these simulations were of length 9,999, rather than 10,000, since the latter is not a multiple of 3. In the second set of simulations, N e was kept constant until the time of the most recent common ancestor for the four simulated sequences. Then, N e was either left unchanged or was suddenly changed by a certain factor. The latter was done by replacing the rate matrix derived from equation (4), resulting in new equilibrium frequencies of the nucleotides. When N e was kept constant, the selective pressures and drift were left unchanged, thereby maintaining the same starting equilibrium frequencies throughout the phylogeny. Thus, the corresponding f estimates did not significantly differ from 0.5 (Fig. 2 ). In contrast, increases in N e lowered the value of f as the efficiency of selection was increased relative to drift [ 4 ]. Correspondingly, the evolutionary process became more dominated by the ending nucleotide. This increasing dominance can be expected to continue until a new equilibrium is restored (which occurs on a longer time scale than that in these simulations). Figure 2 Two situations where f is affected by deviations from the model . (A) The effect of a change in N e on the value of f . This change in N e occurs in the most recent common ancestor of the four simulated sequences. Population ratio refers to its N e after versus before this change. (B) The effect of an increased C to T substitution rate. Categories A, B, and C are defined in Table 1. In the third set of simulations, an acceleration in the C to T substitution rate was incorporated, thereby modeling an increase in their mutation rate due to the deamination of methylated C's in CpG pairs [ 14 ]. The introduction of this bias resulted in significant deviations of f in either direction from 0.5, even though their sequences were simulated in equilibrium (Fig. 2 ). Thus, the value of f can vary considerably when the rates for reciprocal mutations are unequal. Discussion This study illustrates how selection, drift, and mutation within a population can be linked to the f parameter and rate matrices of the +gwF variants for sequence evolution. Our weak selection model relies on the fixation probabilities of mutant alleles with additive genie selection and equal mutation rates for reciprocal substitutions. What is now needed are additional studies that link other population genetics models to the +gwF variants [ 9 ]. For example, the population genetics models of Li [ 15 ], which focus on allele frequency distributions and different modes of selection and mutation, could be studied for their connections to the f parameter and +gwF rate matrices. Collectively, the three sets of simulations highlight that the f parameter is complex and can be influenced by a number of different factors [ 4 ]. This complexity limits its biological interpretation and the use of its expected value of 0.5 as derived for the weak selection model. Correspondingly, in many +gwF analyses, f ill need to be estimated as a free parameter rather than fixed beforehand. Goldman and Whelan [ 4 ] focused on amino acid sequences, where they found that the +gwF models provided better fits to the majority of their protein data sets. They also analyzed two rather small nucleotide data sets for which the general reversible model (REV) outperformed the +gwF variants. As noted by them, the REV model provides enough free parameters to cover the effects of a +gwF analysis. Thus, given sufficient data, this model will consistently outperform the simpler +gwF variants, since it can always accommodate more of the evolutionary process by virtue of its extra parameters. Nevertheless, as widely acknowledged, simpler models have their place, since they allow one to maximize analytical power for more limited data, while minimizing the risk of over-parameterization [ 13 , 16 ]. Thus, as for the JC, K2P, and HKY models, we expect their +gwF variants to remain of interest as part of the hierarchy of simple to complex models for sequence evolution. Authors' contributions Both authors contributed to the conception and design of this study and to the writing, reviewing, and final approval of this article. B.K. performed the simulations and parameter estimations.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC554786.xml
544581	Metallopanstimulin as a marker for head and neck cancer	Background Metallopanstimulin (MPS-1) is a ribosomal protein that is found in elevated amounts in the sera of patients with head and neck squamous cell carcinoma (HNSCC). We used a test, denoted MPS-H, which detects MPS-1 and MPS-1-like proteins, to determine the relationship between MPS-H serum levels and clinical status of patients with, or at risk for, HNSCC. Patients and methods A total of 125 patients were prospectively enrolled from a university head and neck oncology clinic. Participants included only newly diagnosed HNSCC patients. Two control groups, including 25 non-smokers and 64 smokers, were studied for comparison. A total of 821 serum samples collected over a twenty-four month period were analyzed by the MPS-H radioimmunoassay. Results HNSCC, non-smokers, and smokers had average MPS-H values of 41.5 ng/mL, 10.2 ng/mL, and 12.8 ng/mL, respectively (p = 0.0001). Conclusion We conclude that MPS-1 and MPS-1-like proteins are elevated in patients with HNSCC, and that MPS-H appears to be a promising marker of presence of disease and response to treatment in HNSCC patients.	Background Effective therapy for head and neck squamous cell carcinoma (HNSCC), which constitutes approximately 95% of head and neck malignancies, is dependent upon early diagnosis and intervention. Despite the obvious advantage to earlier diagnosis of head and neck malignancies, no strategy has proven to effectively detect these tumors at early stages. Most head and neck neoplasms are detected when the patient has become symptomatic from the effects of the primary disease or when lymphatic metastases are palpable. These tumors are infrequently found incidentally on physical exam, and in these cases are often discovered at an earlier stage. Stage of disease at time of diagnosis is the primary metric used for determination of therapy and prognostication of life expectancy [ 1 ]. As tumor stage advances, the morbidity of surgical resection worsens due to an increased loss of tissue volume and involvement of vital structures. Organ-sparing approaches to head and neck malignancies have been developed in an attempt to treat advanced stage lesions while avoiding conventional surgical morbidities. They have, however, not produced universally superior results to surgery both in terms of local-regional control and function. Surveillance in the post-treatment head and neck cancer patients has traditionally centered on regular physical examination [ 2 , 3 ]. Office flexible fiberoptic exams of these patients have provided an excellent means of diagnosis for early mucosal recurrences, but are dependent upon the patient's compliance with regular follow-up and often cannot detect submucosal recurrence. Anatomic imaging is used as an adjunct to regular physical exam when recurrence is suspected, when findings are suggestive of cervical lymphatic involvement, or when a patient's symptoms are out of proportion or unexplained by physical exam findings. Imaging of anatomic structures is complicated by alterations in anatomy due to previous surgery or irradiation. Furthermore, despite many promising early reports, no tumor marker has yet been adopted for clinical use which shows high specificity or sensitivity for primary or recurrent HNSCC [ 4 - 8 ]. Metallopanstimulin-1 (MPS-1) was identified, cloned and characterized in the laboratory of Dr. Fernandez-Pol from a cDNA library constructed from a human mammary carcinoma cell line (MDA-468) that was stimulated by the growth factors TGF-β1 and EGF in the presence of cyclohexamide [ 9 ]. MPS-1, a multifunctional S27 ribosomal protein, is an 84 amino acid 9.5 kD ribosomal subunit, "zinc finger" protein that is present in all tissues and expressed in large quantities in a wide spectrum of proliferating tissues and oncogenic processes [ 10 - 16 ]. When MPS-1 is over-expressed, it is either secreted or passively released down a concentration gradient into the extra-cellular space. Conventionally, ribosomal proteins are thought to be confined in their function to intracellular protein synthesis. Many recent reports have drawn attention to "extraribosomal functions" of ribosomal proteins [ 17 - 20 ]. Moreover, these extraribosomal functions have been observed in relation to oncogenesis in various models [ 21 , 22 ]. The zinc finger motif of MPS-1 and other ribosomal proteins may allow binding to nucleic acids which may result in interference with transcription and translation [ 10 , 17 , 18 ]. Practical applications of this include: 1) DNA repair, 2) gene suppression, 3) cell-cycle control, or 4) control of oncogenesis. Another related ribosomal protein, S27a, is ubiquitinilated and over expressed in human colon cancer. Like MPS-1, it is involved in cell-cycle control and DNA replication [ 23 ]. The physiology of MPS-1 expression and our initial experience with this protein in HNSCC has led us to conclude that MPS-1 and MPS-1-like proteins may be useful markers in the effort to screen for and analyze the extent of HNSCC [ 24 ]. The purpose of this study was to use an empirical MPS-H test, which measures both MPS-1 and MPS-1-like proteins, to 1) compare average MPS-H levels between HNSCC patients and normal controls and 2) to illustrate how the MPS-H test may be useful for surveillance and evaluating response to treatment for HNSCC. Patients and methods Patients A total of 125 volunteers with newly diagnosed HNSCC were prospectively enrolled from a university head and neck oncology clinic. Serum collection consisted of a pre-treatment specimen followed by collections every six weeks during the first year and quarterly during the second year. At the time of specimen collection, presence of HNSCC was judged by all available data including: physical examination (that included an office endoscopy when indicated), biopsy, and radiology [computerized tomography (CT), magnetic resonance imaging (MRI), or positron emission tomography (PET)]. A clinical assessment was rendered based on all available data as "no evidence of disease (NED)", "alive with disease", or an indeterminate status. Serum MPS-H levels in these patients (N = 709) were compared to two control groups. The first control group was comprised of 25 normal, healthy, non-smoking volunteers. The second control group included 64 actively smoking volunteers who were screened for HNSCC and were found to be free of disease during the 1999 Yul Brynner Head and Neck Screening day in St. Louis, Missouri. A total of 821 serum samples collected over a twenty-four month period were analyzed by the MPS-H radioimmuno assay (follow-up days, mean: 217, median: 166.). All patients gave informed consent under an IRB-approved protocol. MPS-H Serum Assay Technical details for the preparation of reagents for MPS-H antigen determinations, RIA procedure, and patient sample preparation are published elsewhere [ 9 , 10 ]. Each serum sample was run in duplicate by a single technician who was blinded to specimen identity. The targets of this assay are the MPS-N-terminus of both MPS-1 and MPS-1-like proteins. These proteins are activated or released from the precursor or carrier proteins by heat-denaturing of the serum under controlled conditions. The resulting proteins are collectively designated as MPS-H. Immunoreactive substances detected by the MPS-H test do not reflect the true levels of authentic immunoreactive MPS-1/S27 ribosomal protein in the circulation under non-denatured conditions. Statistical Analysis Average MPS-H levels were compared between HNSCC patients and the two control groups and across American Joint Committee on Cancer (AJCC) stages and subsites using analysis of variance (ANOVA). Based on the results from the clinical assessment we computed the receiver operating characteristic (ROC) curve for MPS-H. The ROC curve plots the trade-off between sensitivity and specificity for a range of threshold values for defining a positive result. All ANOVA and contingency table analysis were performed using SAS (version 8.1, Cary, NC) statistical software. ROC curve analysis was performed using R software, an open source implementation of the S language (version 1.4.1, ). Statistical significance was defined as p < 0.05. Results Of the 125 HNSCC patients studied, 90 were male (Table 1 ). Table 2 presents the distribution of stage and site of the primary tumor. Most patients presented in stage III (24.0%) or IV (50.4%) with primary tumors of the oral cavity (26.4%) and larynx (37.6%). Table 1 Demographic and treatment characteristics of cases Gender Radiation/Chemo Surgery Total Female N 7 28 35 % 5.6% 22.4% Male N 19 71 90 % 15.2% 56.8% Total N 26 99 125 % 20.8% 79.2% 100.0% Table 2 Summary of primary tumor stage and location. Primary Stage Tumor Site I II III IV Total Percent Hypopharynx 0 1 0 1 2 1.6% Larynx 7 4 18 18 47 37.6% Nasopharynx 0 0 0 4 4 3.2% Neck 0 1 1 3 5 4.0% Oral Cavity 5 8 4 16 33 26.4% Oropharynx 0 0 2 18 20 16.0% Parotid 0 0 2 0 2 1.6% Skin 4 2 3 3 12 9.6% Total 16 16 30 63 125 Percent 12.8% 12.8% 24.0% 50.4% 100.0% MPS-H levels in this group were compared to a control group of healthy volunteers and to a control group who volunteered for screening for head and neck cancer (Figure 1 ). Mean MPS-H was 10.2 ng/mL for the healthy control group, and 12.8 ng/mL for the smoking control group. Mean MPS-H for the HNSCC group was 41.5 ng/mL, which was significantly higher than both control groups (p < 0.0001). Furthermore, Figure 2 illustrates that among HNSCC patients, those who were successfully treated and clinically free of disease had consistently lower MPS-H levels over time than patients living with active head and neck cancer. Figure 1 Mean serum MPS-H level for patients with SCC (n = 125, all stages and sites within the head and neck), healthy control group (n = 51) and actively smoking volunteers who were screened for HNSCC (N = 64). Mean of SCC group is 41.5 ng/mL, mean of healthy control group is 10.2 ng/mL, and mean of screening controls is 12.8 ng/mL (p < 0.0001). Error bars denote 1 standard deviation. Figure 2 Serial MPS-H levels in HNSCC patients treated and without clinical disease (No Disease) and patients with unresectable disease or receiving palliative therapy with persistent clinical disease (Alive with Disease). Error bars denote 1 standard deviation and vary widely in the AWD group due to its small size and patient attrition over time from death. We next computed the receiver operating characteristic (ROC) curve for the MPS-H levels. Figure 3 shows that the area under the ROC curve (0.73; 95% CI: 0.71–0.75; p = 0.001), is significantly different from 0.5, which suggests that there is moderate diagnostic accuracy associated with MPS-H. Analysis of MPS-H levels as a function of AJCC stage or head and neck sub site were performed and were not significant. Larger numbers of earlier stage (I and II) tumors and greater numbers among the various sub sites might result in significance. Figure 3 Receiver operating characteristic (ROC) curve for MPS-H test. Area under the curve is 0.73 (CI 95 : 0.69 – 0.76, p = 0.001) We have observed several instances where elevated MPS-H levels in patients presenting with head and neck neoplasms dropped to normal levels following successful therapy. We have also noted examples of persistent elevations or increases in MPS-H levels in patients with failure to respond to therapy or with recurrence of tumor respectively. Several cases of patients presented in Figures 4 , 5 , 6 illustrate these phenomenons. Patient 1 (Figure 4 ) was a female with a T 4 N 0 M 0 SCC of the floor of mouth with positive tumor margins on the cut edge of the mandible, having failed a recent limited surgery by another surgeon. Her presenting MPS-H value was borderline positive when she had little clinical disease [1 on the x axis] and rose immediately postoperatively. A transient rise after surgical ablation or induction chemotherapy is a documented phenomenon observed with numerous tumor markers (personal communication J.A. Fernandez-Pol). The elevation likely results from a large initial disruption of cells within the tumor resulting in a dumping of intracellular MPS-1 into the circulation. This usually returns to baseline in 4 to 6 weeks if the tumor is successfully treated. The patient was clinically NED in the immediate postoperative period and during radiation treatment. Approximately 6 months post operatively, the patient developed a neck mass and by the next visit skin metastasis were seen. On last follow-up, the patient had progression of skin and neck metastasis at which time she decided to pursue hospice care. She expired 6 weeks later. Figure 4 Patient without evidence of disease from surgery through radiation therapy (draws 1–5). The patient suffered a recurrence of clinical disease following radiation therapy (draws 6–8), which progressed until death. Figure 5 Patient followed for 24 months without evidence of recurrence on physical exam, endoscopy, or FDG-PET. Figure 6 Patient with unresectable tumor. Note MPS-H level response to chemotherapy. Patient subsequently expired after refusing further therapy. Patient 2 (Figure 5 ) is a male who was diagnosed with a T 4 N 2c M 0 SCC of the larynx, which required total laryngectomy, bilateral neck dissections, and postoperative radiation therapy. The patient joined this study 3 months following his surgery, while clinically NED, and has remained free of clinical recurrence for 24 months. The patient had three FDG-PET scans at six-month intervals following his surgery that were all negative for recurrent disease or metastasis. His persistently low MPS-H levels over time (range from 7.7–19.2 ng/mL), was suggestive of ongoing disease-free status. Patient 3 (Figure 6 ) was a female who presented with a T 4 N 0 M 0 SCC of the hypopharynx. She was severely malnourished with multiple medical problems and thought to be a poor surgical candidate. She decided to pursue induction chemotherapy (Carboplatin and Taxol) to be followed by radiation therapy. She underwent three rounds of chemotherapy at 21-day intervals and had a 50% decrease in her tumor size as judged by office endoscopy. She suffered severe GI problems during her therapy, opted not to continue on to radiation, and enrolled in hospice care. The patient did not present for additional follow-up after entering hospice and died three months later. This case illustrates the potential utility of MPS-H as a marker for tumor response to chemotherapy and/or irradiation. Discussion Histological examination of the tumor, surgical margins, and cervical nodes are the current means of determining extent of disease. When surgical extirpation is not undertaken, staging is performed based on a radiological assessment, biopsy, and physical examination. These methods are used either to determine adequacy of resection and the need for adjuvant therapy or to select an alternative primary (non-surgical) therapy, respectively. Limitations of the histologic method include microscopic disease that escapes diagnosis due to a small number of malignant cells, subtle histological changes not classified as cancer, previous treatment effect upon tissues, or pathologic sampling error. CT and physical examination both suffer from modest sensitivity and specificity in detecting many early head and neck neoplasms. As a result, local and regional treatment failures are not uncommon in both surgical and non-surgical treatments of head and neck cancer. This may be due to an underestimation of the tumor burden. This may also be explained by the current diagnostic emphasis upon analysis of structure (microscopic) or anatomic extent of disease, both of which are imperfect, rather than its biologic activity as might be measured by a tumor marker or a functional scanning technique such as FDG-PET. The MPS-H test has been used in conjunction with conventional tumor-specific markers to improve sensitivity and specificity of tumor serodiagnosis [ 12 ]. Of all malignancies in which MPS-H has been studied to date, epithelial malignancies possess no alternative tumor markers in clinical use that have been effective for diagnosis or surveillance [ 4 - 8 , 23 ]. This observation in conjunction with the current dependence on anatomic evaluation for diagnosis of epithelial malignancies has led us to preliminary investigations of the utility of MPS-H serologic diagnosis for the detection of head and neck epithelial neoplasms. Control groups of healthy volunteers and those with systemic, non-malignant diseases have been studied using the MPS-H test. Statistical analysis has revealed that those without malignancy have MPS-H serum levels less than 10 ng/mL, those with malignancy have levels greater than 20 ng/mL, and those with bony metastasis have levels in excess of 100 ng/mL [ 12 ]. Additionally, MPS-H levels have been documented to decline with successful treatment of malignant disease whereas non-responders to therapy persisted with high levels of MPS-H [ 12 ]. Serum samples from prostate carcinoma patients with high levels of MPS-H (>500 ng/mL) have demonstrated the authentic 9.4 kDa MPS-1 protein, at least one protein with sequence homology to the N-terminus of MPS-1, and a high molecular weight precipitation interfering protein. More accurate detection of recurrent SCC of the head and neck by the MPS-H test may provide a new way to improve survival. Physical exam, conventional imaging, and biopsy are the current gold standard to determine recurrence. Currently, FDG-PET is the most promising way to assess early tumor recurrence of the head and neck but is quite expensive [ 25 , 26 ]. Its sensitivity and specificity have been reported to be approximately 90% [ 25 , 26 ]. No standard serum tumor marker is routinely used for head and neck cancer surveillance, which limits alternatives to conventional exams or frequent FDG-PET imaging [ 4 - 8 , 25 ]. In the present study we compared FGD-PET to MPS-H levels in head and neck cancer patients. FDG-PET interpretation and MPS-H level determination were performed independently and blinded from the results of the other test. FDG-PET positive scans were not all confirmed by biopsy in our study. A statistically significant correlation was noted between FDG-PET positive cases and high MPS-H serum levels in head and neck cancer patients. MPS-H and FDG-PET agreed in 103 out of 183 cases. In 12 cases MPS-H was elevated but no cancer was found by FDG-PET, suggesting that the patients may have had an early recurrence detectable by MPS-H but not yet by FDG-PET. The 68 FDG-PET positive cases that show low MPS-H levels suggest that some tumors were unable to produce high levels of MPS-H, perhaps due to previous chemotherapy, or that some results represent false positive PET scans. Further study with larger patient groups is ongoing to assess the optimal cutoff levels of MPS-H and the correlation between FDG-PET and MPS-H. Shortcomings of this study include a limited time span, a large proportion of advanced stage cancers, limited controls (both size, smoking status, and age/gender match), and ability to define an absolute cut off value for normal vs. abnormal. We recognize that other factors such as age and other malignancies may effect MPS levels. Future studies will attempt to address and/or control for these issues. Considering the data presented in this paper, which agrees with previous results with other tumor types, and the compelling need to expedite the early diagnosis of primary and recurrent epithelial malignancies of the head and neck, we are further evaluating the MPS-H tests as a tool for diagnosis in a larger group of HNSCC patients. Additionally, we are working to improve the diagnostic technology used to detect MPS-H [ 27 - 29 ]. Since there is a reasonable correlation between detection of MPS-H in the sera and FDG-PET positivity for SSC, these results raises the potential of the MPS-H test for becoming a test for HNSCC, followed by selective confirmatory FDG-PET imaging. These preliminary results will be verified in a larger population. The role for using MPS-H as a general screening tool among at-risk populations without the diagnosis of HNSCC is also currently being evaluated. Competing interests The authors declare that they have no competing interests. Authors' contributions BCS: Principal Investigator, Principal Editor CSH: Data analysis and manuscript preparation CL: Medical student research assistant for project FD: Member of multidisciplinary head and neck oncology team, recruiter of subjects VJL: Member of multidisciplinary head and neck oncology team, supplier of PET data, data analysis. PDH: Laboratory technical support with MPS assay Funding sources This study was funded by the clinical salaries of BCS, FRD, and VJL. CML and CSH were students at the time of this study. MPS assays were performed at the Department of Veterans Affairs James A. Cochran Medical Center, Molecular Oncology Laboratory, St. Louis, MO. PDH was an employee of the US Government at the time of this research.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544581.xml
524513	An unusual case of chronic meningitis	Background Chronic meningitis is defined as symptoms and signs of meningeal inflammation and persisting cerebrospinal fluid abnormalities such as elevated protein level and pleocytosis for at least one month. Case presentation A 62-year-old woman, of unremarkable past medical history, was admitted to hospital for investigation of a four-week history of vomiting, malaise an associated hyponatraemia. She had a low-grade pyrexia with normal inflammatory markers. A CT brain was unremarkable and a contrast MRI brain revealed sub-acute infarction of the right frontal cortex but with no evidence of meningeal enhancement. Due to increasing confusion and patient clinical deterioration a lumbar puncture was performed at 17 days post admission. This revealed gram-negative coccobacilli in the CSF, which was identified as Neisseria meningitidis group B. The patient made a dramatic recovery with high-dose intravenous ceftriaxone antibiotic therapy for meningococcal meningitis. Conclusions 1) Chronic bacterial meningitis may present highly atypically, particularly in the older adult. 2) There may be an absent or reduced febrile response, without a rise in inflammatory markers, despite a very unwell patient. 3) Early lumbar puncture is to be encouraged as it is essential to confirm the diagnosis.4) Despite a delayed diagnosis appropriate antibiotic therapy can still lead to a good outcome.	Background Bacterial Meningitis usually presents as an acute illness, predominantly affecting children and young adults. It typically presents with the classical clinical triad of fever, neck stiffness and an altered mental state. However, it may rarely present as a chronic illness, without the classic clinical features noted in acute meningitis. Case presentation A 62-year-old retired woman was admitted to hospital via her GP for investigation of a four-week history of vomiting and malaise associated with hyponatraemia. She was initially diagnosed as suffering from viral gastroenteritis. However, the vomiting had persisted and had become associated with a mild frontal headache. She had an unremarkable past medical history and was not taking any regular medication. She had never smoked and there was no recent antecedent foreign travel. On examination she appeared clinically dehydrated but otherwise looked well, and was alert and orientated. She was apyrexial and had no rash, photophobia, neck stiffness or stigmata of endocarditis. She had a sinus tachycardia of 104/minute, with normal heart sounds, and a blood pressure of 130/76 mmHg. Chest, abdominal and neurological examinations were unremarkable. She had a plasma sodium of 127 mmol/L (135–145 mmol/L), potassium of 3.4 mmol/L (3.5–5.0 mmol/l), urea 4.8 mmol/L (3.0–6.5 mmol/L) and creatinine of 68 mmol/L (60–125 mmol/L). There was no biochemical evidence of the syndrome of inappropriate antidiuretic hormone (SIADH) production (serum osmolality 261 mmols/kg; urine osmolality 71 mmols/Kg; urine sodium < 10 mmol/L). Serum complement and plasma immunoglobulin levels were unremarkable with no evidence of immunosuppression. In addition, she had a normal full autoimmune profile and thyroid function. Random cortisol level was mildly elevated at 799 nmol/L (normal 140 – 700 nmol/L) consistent with a stress response. Her initial white cell count (WCC) was mildly elevated at 13.0 × 10 9 /L (normal 4–11 × 10 9 /L) with a neutrophilia of 10 × 10 9 /L (normal 2–7.5 × 10 9 /L). Her ECG and chest X-ray were normal. Her C-reactive protein (CRP) was slightly elevated at 10 mg/L (normal <5 mg/L) and erythrocyte sedimentation rate (ESR) was normal at 5 mm/hour. Her Chest X-ray and electrocardiogram were normal. Initial microbiological investigations (blood cultures, urine microscopy and culture) were normal. Initial management consisted of slow intravenous rehydration with normal saline and antiemetic therapy, which led to a mild symptomatic improvement. Upper gastrointestinal endoscopy revealed mild oesophagitis. During the ensuing two weeks her laboratory investigations remained stable (CRP normal; ESR normal; sodium 127–131 mmol/L; WCC 11–13 × 10 9 /L). However, on day 4 of admission she developed a low-grade pyrexia of 37.5°C, which persisted (<38°C). A CT scan of the head revealed periventricular patchy white matter changes but no features of raised intracranial pressure or space occupying lesion. Unfortunately the patient had become slowly more lethargic, withdrawn, and depressed. By day 17 of admission, although alert, she was uncooperative with intermittent confusion. Her symptoms of intermittent nausea and vomiting with occasional frontal headache continued. On day 18 she underwent a lumbar puncture (LP) as she still had a low-grade pyrexia (temperature 37.5°C) and neutrophilia of 9.3 × 10 9 /L). In addition, her nausea and vomiting had failed to fully settle with supportive treatment. The LP results were as follows: cerebrospinal fluid (CSF) appearance was pale yellow and clear; protein = 5.69 g/L (0.15–0.4 g/L); CSF glucose 1.7 mmol/L versus plasma glucose 5.7 mmol/L (ratio = 30%, normal > 50%); CSF WCC = 106/mL (normal <5 WCC/mL) – 99% lymphocytes. Gram's stain revealed gram-negative coccobacilli; acid-fast bacilli were not seen. She was commenced on intravenous ceftriaxone. Contrast MRI brain revealed sub-acute infarction of the right frontal cortex but with no evidence of meningeal enhancement. EEG demonstrated slow wave activity, which was consistent with a meningo-encephalitis. Within 48 hours of intravenous antibiotics she was more alert, orientated, and sitting out of bed. CSF culture grew gram-negative cocci, which was identified as Neisseria meningitidis group B, type NT, subtype NT P1.16/nt. She underwent contact tracing and completed a 10-day course of intravenous ceftriaxone. She continued to make a slow but progressive recovery. After a period of rehabilitation and intense physiotherapy she was discharged home 40 days after admission, with mild residual gait ataxia. Conclusions This case report presents two important clinical concepts: firstly, the presentation of chronic meningitis and secondly, the clinical presentation of bacterial meningitis in the older adult (defined as > 60 years old). The diagnosis was delayed due to the highly atypical clinical presentation [ 1 ]. Chronic meningitis is defined as symptoms and signs of meningeal inflammation and persisting cerebrospinal fluid (CSF) abnormalities such as elevated protein level and pleocytosis for at least one month [ 2 , 3 ]. It affects less than 10% of meningitis sufferers and is linked to a large variety of both infective and non-infective causes [ 4 ]. However, whilst there are numerous published individual case reports on chronic meningitis, there is a definite paucity of large case series in the literature. The most common cause of chronic meningitis is Mycobacterium tuberculosis, which accounts for 40–60% of cases [ 3 , 5 ]. Other relatively frequent causes include malignancy (8–13%) and crytococcal infection 7–11%) [ 3 , 5 ]. In up to 33% of cases no underlying cause is identified [ 3 , 5 ]. Chronic meningococcal meningitis is rare and is limited to a few isolated case reports in the literature [ 6 - 8 ]. There are several distinguishing features that may help to differentiate chronic meningitis from adult acute bacterial meningitis (table 1 ). The classic triad of clinical features of meningitis (fever, neck stiffness, altered mental state), whilst seen in up to 85% of patients presenting with acute bacterial meningitis is far less commonly seen in chronic meningitis [ 3 , 5 , 9 ]. Focal neurological signs with cranial nerve palsies and abnormal CT brain findings are also far more commonly seen in chronic meningitis [ 5 , 10 ]. Table 1 Features distinguishing chronic meningitis (bacterial and non bacterial) compared with acute bacterial meningitis Description Acute bacterial meningitis Chronic meningitis Aetiology Variable Neisseria meningitides 13–56% [10,17, 39] Streptococcus pneumoniae 24–37% [10,17] Variable TB- 40–60% [3,5] Malignancy 8–13% [3,5] Cryptococcus 7–11% [3,5] Unknown 30–33% [3,5] Clinical features - Classic triad of fever, headache and neck stiffness 85% [9] 10% [4] - Fever 78–91% [39] 44% [4] - Headache 32–68% [39] 79% [4] - Neck Stiffness 58–82% [39] 75% [5] - Altered Mental state 52–82% [39] 41% [4] - Focal neurology 23% [39] 32% [5] - Papilloedema <1–4% [9,10] 30% [5] - Cranial Nerve Palsies 4% [10] 24% [5] Mortality Variable – aetiology dependent 19.7–25% overall [10,17] 37–44% ≥ 60 years old [1,10] 10–25% < 60 years old [10,16–20] Variable – aetiology dependent 29%- overall [5] Elevated WCC, CRP and ESR Elevated Normal or only mildly elevated [5] Hyponatraemia <10% >90% [5] Cerebrospinal fluid analysis 10% – lymphocytic [9,17] 90% – neutrophilic [9,17] Gram stain positive 57–90% [9,10,17] >90% lymphocytic [5] <10% neutrophilic [5] Gram stain positive <10% [5] Abnormal CT 2.7 – 13% [10,40] 60% [5] WCC, white cell count; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate. Hyponatraemia (as in our patient), whilst very uncommon in acute bacterial meningitis, is seen in the vast majority of cases of chronic meningitis [ 5 , 11 ]. Although there was a persistent mild neutrophilia, both the CRP and ESR were normal throughout the course of the disease, which, whilst being highly unusual for acute meningitis, has been reported, in chronic meningitis [ 12 , 13 ]. Acute bacterial meningitis is usually a rapidly progressive and highly lethal disease in older adults [ 1 ]. Rapid diagnosis is vital as the prognosis worsens with treatment delay leading to a high rate of sustained neurological deficit in this age group [ 14 , 15 ]. Despite the widespread use of antibiotics the overall case mortality rate remains unchanged and is far higher (37–44%) in the older adult compared with that seen in younger adults (10–25%) with significant long-term morbidity (up to 70% of infected patients) in survivors [ 16 - 20 ]. Given the success of childhood immunization, and an increasingly aging population, the proportion of older adults presenting with bacterial meningitis is increasing [ 16 ]. There are several additional factors, which make the older adult more prone to bacterial meningitis. Older adults often have underlying acute and chronic diseases (e.g. diabetes, renal or hepatic failure) with immunosenescence (age related decline in immune function) [ 1 , 21 ]. This can lead to symptoms, which can be confused with those of meningitis and at the same time increase the propensity to infection [ 1 , 21 ]. The role of immunosenescence in predisposing patients to bacterial meningitis is not clearly defined, but appears to relate to defects in innate, specific cellular and humoral immunity leading to an attenuated immune response [ 1 , 22 - 24 ]. Persons who lack or are deficient of antibody-dependent, complement-mediated lysis (bacteriocidal activity) are most susceptible to meningococcal disease [ 25 ]. Our patient had an unremarkable past medical history with normal complement and immunoglobulin levels with no evidence of immunosuppresion [ 26 - 28 ]. The clinical presentation of bacterial meningitis is more variable in the older as compared with the younger adult, with fewer patients manifesting with the classic symptoms of fever, neck stiffness and altered mental state than among younger adults [ 1 ]. It has been suggested that 1 of 3 findings (fever, neck stiffness, altered mental state) is present in virtually all patients with meningitis and that the absence of these features virtually excludes meningitis with a high negative predictive value (table 1 ) [ 1 ]. Our patient had none of these features on presentation and had been unwell for four weeks prior to presentation, but did develop a mild fever (<38°C) and cognitive dysfunction during her inpatient stay. The blunted febrile response is well recognised in older adults in general [ 29 ]. Our patient's CSF showed a lymphocytosis, raised protein, and low glucose ratio, which are seen in only 10% of bacterial meningitis cases. This CSF profile would normally suggest infection with Listeria monocytogenes meningitis or alternative causes such as tuberculous and fungal infection [ 30 , 31 ]. Neisseria (N) meningitidis is a leading cause of bacterial meningitis in the Western World and tends to predominate in young adults [ 19 , 20 , 25 ]. N. meningitidis is a gram-negative, aerobic diplococcus. It is classified into serogroups (e.g. A,B,C etc) according to the immunological reactivity of their polysaccharides [ 25 ]. The most prevalent serogroups implicated in clinical meningococcal meningitis are serogroup B (62%, as in our patient) and the more virulent serogroup C (22%) [ 19 , 20 ]. The relatively reduced virulence of serogroup B may partly explain the chronicity of presentation and reduced inflammatory response seen in our patient. Serogroups B and C have a seasonal variation occurring more commonly in the first quarter of the year (our patient presented in February) [ 19 ]. Meningococcal meningitis is also more common among the following groups: persons of black race; lower socioeconomic classes; those exposed actively or passively to tobacco smoke; persons exposed to overcrowding and amongst binge drinkers [ 32 - 37 ]. This case highlights the diagnostic challenge associated with bacterial meningitis presenting in an older patient. The presentation was made even more difficult owing to the blunted febrile response, the lack of inflammatory response observed in laboratory tests and the chronicity of the patient's symptoms. The diagnosis required thorough investigation during the inpatient stay. Early lumbar puncture is to be encouraged as it is essential to confirm the diagnosis. Despite a delayed diagnosis appropriate antibiotic therapy can still lead to a good outcome. Competing interests The authors declare that they have no competing interests. Authors' contributions MD generated the idea of writing the case report and was the consultant in charge of the patient. CD reviewed the case notes of the patient and wrote the original draft of the case presentation. CB significantly revised the original draft and added the conclusions, references and figures. AH offered considerable help with the manuscript revisions. All authors contributed to the final version of the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC524513.xml
544595	The migration of physicians from sub-Saharan Africa to the United States of America: measures of the African brain drain	Background The objective of this paper is to describe the numbers, characteristics, and trends in the migration to the United States of physicians trained in sub-Saharan Africa. Methods We used the American Medical Association 2002 Masterfile to identify and describe physicians who received their medical training in sub-Saharan Africa and are currently practicing in the USA. Results More than 23% of America's 771 491 physicians received their medical training outside the USA, the majority (64%) in low-income or lower middle-income countries. A total of 5334 physicians from sub-Saharan Africa are in that group, a number that represents more than 6% of the physicians practicing in sub-Saharan Africa now. Nearly 86% of these Africans practicing in the USA originate from only three countries: Nigeria, South Africa and Ghana. Furthermore, 79% were trained at only 10 medical schools. Conclusions Physician migration from poor countries to rich ones contributes to worldwide health workforce imbalances that may be detrimental to the health systems of source countries. The migration of over 5000 doctors from sub-Saharan Africa to the USA has had a significantly negative effect on the doctor-to-population ratio of Africa. The finding that the bulk of migration occurs from only a few countries and medical schools suggests policy interventions in only a few locations could be effective in stemming the brain drain.	Background Doctors migrate from developing countries to wealthier countries in order to further their careers, or improve their economic or social situation. The World Health Organization (WHO) has long recognized that migration of health personnel from developing to developed countries creates unfortunate imbalances in the global health workforce [ 1 ]. America's physician workforce has been significantly infused with foreign-trained international medical graduates (IMGs) since World War II. The purpose of this paper is to describe a sub-population of IMGs in the USA, those who have trained in one of the 47 African subcontinent nations. African governments have been very clear about their objections to the wholesale migration of their physicians to rich countries. In 1996, South Africa's then-Deputy President Thabo Mbeki implored the World Health Assembly to take measures to stop the flow of physicians from poor countries to rich ones. In 1995, South Africa itself banned the recruitment of doctors from other Organization of African Unity countries [ 2 ]. Nonetheless, large numbers of African-trained physicians leave home upon completion of their medical school training in search of careers in higher-income countries. They leave behind health systems in sub-Saharan Africa that are severely stressed: life expectancy is only 50 years, 162 children in 1000 die before reaching their fifth birthdays, and only half have access to clean water sources [ 3 ]. Further, AIDS prevalence among those 15 to 49 years old is estimated to be 8.4% [ 4 ], and in four countries, adult HIV prevalence exceeds 30% [ 5 ]. While health improvements in Africa will require a broad agenda of development activities, access to an educated workforce of health professionals is also essential [ 6 ]. African country health systems and workforce data are poor, making it difficult to estimate the effects of physician migration on sending countries. The World Bank has documented this data gap, noting "Quantitative data on the health workforce is notoriously unreliable in most countries...In poor countries, government and professional information systems are weak, when they exist at all, and are rarely comprehensive (often there is no information on the private sector) and up-to-date" [ 7 ]. Indeed, the way many African country ministries of health learn about the extent of their own emigration is through gleaning data presented by destination countries [ 8 ]. This paucity of sending-country data makes it difficult to fully describe the impact of migration on countries of origin. The 47 nations of sub-Saharan Africa have a total of 87 medical schools, although 11 countries have no medical school at all and 24 have only one each (see Table 1 ). The population of the subcontinent totals over 660 million people, with a ratio of fewer than 13 physicians per 100 000 population, or a total of 82 949 doctors [ 9 ]. By comparison, the United Kingdom (UK) has 164 physicians per 100 000 and the USA has over 279 physicians per 100 000 (or almost 800 000 doctors for a population of 284 million). Table 1 Physician workforce distribution and number of medical schools by African country Country Population (in 1000s) Physicians per 100 000 population Total number of physicians FAIMER number of medical schools Angola 10 132 7.7 780 1 Benin 6428 5.7 366 1 Botswana 1578 23.8 376 0 Burkina Faso 12 217 3.4 415 1 Burundi* 5714 6 343 1 Cameroon 14 792 7.4 1095 1 Cape Verde 0.04 17.1 68 0 Central African Republic 3501 3.5 123 1 Chad 8419 3.3 278 1 Comoros 0.578 7.4 43 0 Congo 2809 25.1 705 1 Congo (DR) 51 810 6.9 3575 3 Côte d'Ivoire 15 866 9 1428 1 Equatorial Guinea 0.474 24.6 117 0 Eritrea 4232 3 127 0 Ethiopia* 62 651 2 1253 3 Gabon* 1223 20 245 1 Ghana 19 509 6.2 1210 3 Guinea 8642 13 1123 1 Guinea-Bissau 1278 16.6 212 1 Kenya 30 310 13.2 4001 2 Lesotho 1847 5.4 100 0 Liberia 3149 2.3 72 2 Madagascar 15 506 10.7 1659 3 Malawi* 10 874 2.3 250 1 Mali 10 665 4.7 501 1 Mauritania 2668 13.8 368 0 Mauritius 1179 85 1002 1 Mozambique# 16 934 2.57 435 1 Namibia 11 826 29.5 3489 0 Niger 10 174 3.5 356 1 Nigeria 123 750 18.5 22 894 16 Rwanda* 7405 4 296 1 Sao Tome & Principe 0.16 46.7 75 0 Senegal 9784 7.5 734 2 Seychelles 0.08 132.4 106 1 Sierra Leone 5203 7.3 380 1 So. Africa 42 351 56.3 23 844 8 Somalia 7253 4 290 1 Sudan 35 080 9 3157 14 Swaziland 1,120 15.1 169 0 Tanzania 33 768 4.1 1384 4 The Gambia 1367 3.5 48 0 Togo 5033 7.6 383 1 Uganda* 23 496 3 705 3 Zambia 9799 6.9 676 1 Zimbabwe 12 186 13.9 1694 1 TOTAL/AVG 663 529 12.5 82,949 1.8 avg Avg. physicians per country 1765 Indicates countries for which physicians/100 000 data came from World Bank instead of World Health Organization # Mozambique data come from Stephen S. Gloyd, as no data are available from WHO or World Bank Note on calculations: Average physicians per 100 000 population in Africa overall = total number of doctors (82 949)/total population (663 529) Sources: Population data from United States Census Bureau IDB Summary international Data Base (United Nations and National Statistics Offices) Number of physicians from the World Health Organization Number of FAIMER (Foundation for the Advancement of International Medical Education and Research) medical schools comes from The dependence of the United States on IMGs is encoded in various policies, most specifically Medicare's financial support for significantly more residency positions than we have domestic medical school graduates [ 10 ]. Additionally, the USA will waive the exchange visitor requirement that would otherwise return IMGs to their home countries after residency training in exchange for agreements to practice in underserved USA settings. Further, the USA will grant permanent residency status to IMGs under a variety of conditions [ 11 ]. The UK has initiated efforts to meet its own health workforce planning needs while paying attention to global equity considerations by adopting a formal "code of practice" that prohibits its National Health Service employers from recruiting health professionals from a long list of developing countries [ 12 ]. While this code has not resulted in a reduction in nurse recruitment, the number of physicians migrating to the UK has declined for a brief period (but is now back up) [ 8 , 13 ]. Recently, two prominent medical journals in the UK, the Lancet and the British Medical Journal , have editorialized on the effects of the brain drain in poor countries, recommending an international code of ethics prohibiting the recruitment of developing world health professionals by rich countries [ 14 , 15 ]. While the UK has a centralized health system well positioned to address these issues, both within its health care system and with representatives of other nations, the USA, in contrast, has a fractured health system that is less able to engage these issues. Agencies of the USA government have been reluctant, unable or unwilling to impede free-market driven physician migration. United States policies have always been quite friendly to physician migration, even taking into account toughened medical licensing examinations and tightened immigration rules over the past four or five decades. Furthermore, even though some types of immigration have been more restricted since September 11, 2001, Congress subsequently expanded the number of foreign physicians who will be granted favorable immigration status (HR 2215, passed 10/3/02 increases the number of J-1 visa waivers allocated to state health departments from 20 to 30; further, the Department of Health and Human Services took over the role formerly played by the USA Department of Agriculture in handling applications of J-1 waivers, thereby ensuring additional foreign physicians will have access to waivers.). One of the most common initial points of entry for IMG physicians into the USA medical workforce is residency training program enrollment, even if physicians have already completed postgraduate training in their home countries. The reliance of many inner-city hospitals on IMGs has thwarted calls by medical policy organizations, such as the Council on Graduate Medical Education, to reduce the number of IMGs admitted to residency programs as a means of narrowing the IMG pipeline to the USA There is little debate within the USA government or other institutions about the social justice implications of obtaining health professionals from poor countries [ 16 ]. Typically, research on the issues surrounding the role(s) of IMGs in the USA has focused on 1)whether IMGs practicing here contribute to a surplus of physician labor (which could tend to lower physician salaries and/or drive up health care costs) [ 17 - 19 ]; 2) the quality of care delivered by IMGs [ 20 ]; and 3) the contribution of IMGs to the "health safety net" in rural or underserved areas [ 21 ]. The ethics of health professional migration from poor countries to rich ones is complicated by the competition of legitimate interests – each country's need for an adequate health workforce as opposed to each individual's human right to travel. When health professionals travel to receive training and then return to apply their skills, there are advantages to the home country. Additionally, emigrants of all social classes from poor countries typically send funds home to relatives, although sub-Saharan African remittances, at less than USD 5 billion, comprise the lowest dollar amounts of any other poor world region [ 8 ]. Further, it must be noted that individuals having benefit of public funds for their medical training are sending their remittances home to private parties with no direct gain for the health or education systems. Immigration theory informs us that "push factors" prompt professionals to leave poor countries in favor of settling in higher income countries [ 22 ]. Negative factors in the sending countries include insufficient suitable employment, lower pay, unsatisfactory working conditions, poor infrastructure and technology, lower social status and recognition, and repressive governments. Simultaneously, "pull factors" in wealthier countries systematically attract physicians. These include training opportunities, higher living standards, better practice conditions and more sophisticated research conditions. The "world systems framework theory" stresses the more permeable barriers between and among countries created by the standardized curriculum and English language used in world medical schools, the use of common research methods and shared scientific knowledge, the easy articulation of requirements of practice across countries, and the weakened nationalism that occurs as a result of professional training [ 23 ]. Other theories characterize migration as a decision of family units, rather than individuals, emphasizing the insurance nature of establishing what are, in effect, "branch offices" in multiple locations [ 24 ]. Given the enduring migration from poor countries to rich ones, only likely to increase with the international liberalization of trade in health services [ 25 ], concerns for global health require the maintenance of an adequate health workforce in poor countries. Methods To describe the numbers and types of physicians practicing in the USA who earned medical degrees in Africa, we performed a cross-sectional study using the 2002 American Medical Association Physician Masterfile [ 26 ]. This data set contains detailed information on all 771 491 active physicians who were licensed to practice medicine in the year 2002 (excluding those physicians employed by federal entities such as the Veterans Administration, federal prisons or the military). We reviewed these data for all physicians in the USA who received their training in sub-Saharan Africa (those 47 countries south of the Saharan desert on the African continent). These data included year of birth, gender, year of medical school graduation, name of medical school, current practice location, specialty of practice, and practice activity (office-based, hospital-based, in residency, or conducting teaching or research). Birth country information is missing for 68% of those who graduated from a sub-Saharan African medical school, so we did not analyze birth country data. To detect changes in migrant waves over time, we analyzed the data by cohorts, categorizing physicians who had graduated from medical school during four periods: before 1970, during the 1970s, during the 1980s, and 1990 and beyond. We linked geographic data about practice locations to a four-category, rural-to-urban status and taxonomy, a condensed version of the Rural-Urban Commuting Area (RUCA) codes, to determine whether these physicians are practicing in rural or urban areas. RUCAs are a census tract-based classification scheme, that have also been adapted for zip codes, combining USA Census population data with work commuting information to characterize the types of rural and urban status [ 27 ]. Research colleagues in Canada and the UK provided some data on sub-Saharan African physicians in their countries, as well. Results A total of 179 978 (23.3%) of the 771 491 active non-federal physicians in the USA in the year 2002 received their medical qualification in another country. The largest portion of these, or 115 835 physicians, originate from low and lower-middle income nations, as defined by the World Bank. Indeed, the most frequent countries of origin of IMGs in the USA include India (36 634), the Philippines (17 755), Mexico (10 404), and Pakistan (8563). Canadian physicians conventionally are not included in the IMG count because the body that accredits USA medical schools (the Liaison Committee on Medical Education (LCME)) offers reciprocal accreditation to Canadian medical schools (accredited by the Committee on Accreditation of Canadian Medical Schools). Canadians are, however, still subject to relevant immigration requirements. Sub-Saharan African medical schools in 22 countries have trained approximately 5334 physicians currently practicing in the USA. Only nine nations, however, have lost more than 40 physicians each (see Table 2 ). Some 86% are from three countries (Nigeria, South Africa and Ghana). Nigeria, with more than twice the population of any other country in the region and 16 medical schools, has lost 2158 physicians who are now practicing in the USA; South Africa, with eight medical schools, has lost 1943 physicians; and Ghana, with three medical schools, has lost 478 physicians to the USA. By region, West Africa lost 2697 physicians and Southern Africa 1943. It is also suspected there are many more physicians from these countries working in the USA, although they are not licensed as physicians. Table 2 Country of medical school of sub-Saharan African international medical graduates (IMGs) in the United States and Canada Country of training Number of African-trained IMGs in USA 1 Number of African-trained IMGs in Canada 2 Number of physicians remaining in home country 3 % of total African-trained now in USA or Canada 4 Nigeria 2158 123 22 894 9 South Africa 1943 1845 23 844 14 Ghana 478 37 1210 30 Ethiopia 257 9 1564 15 Uganda 133 42 722 20 Kenya 93 19 4001 3 Zimbabwe 75 26 1694 6 Zambia 67 7 676 10 Liberia 47 8 72 43 Other 12 countries 83 35 12 912 1 Total/Average 5334 2151 69 589 10 1. American Medical Association: Physicians' professional record (AMA-PPD). 2002 2. Buske, Lynda. Associate director of research, Canadian Medical Association. Personal communication. February 3, 2003. 3. Number of physicians from the World Health Organization. Available at: Calculation: [(Col. 1 + Col. 2)/ (Col. 1 + Col. 2 + Col. 3)]100 = percent Other 12 countries with at least one graduate in the United States. An analysis by school indicates only ten medical schools produced 79.4% of the sub-continent's graduates who are practicing in the USA. The medical schools most frequently attended by Sub-Saharan African IMGs in the USA include the University of the Witwatersrand (South Africa, 1053 physicians), the University of Cape Town (South Africa, 655), the University of Ibadan (Nigeria, 643), the University of Lagos (Nigeria, 429), the University of Nigeria (Nigeria, 394), the University of Ghana (Ghana, 389), Addis Ababa University (Ethiopia, 200), the University of Benin (Nigeria, 183), the University of Ife (Nigeria, 156), and the University of Pretoria (South Africa, 132), for a total of 4234 physicians. An analysis of the numbers of sub-Saharan African IMGs coming to the USA in each of the last decades illustrates that it takes some time between graduation and emigration. The number of recent graduates currently in a USA residency program is higher than those in previous decades because of the obvious correlation between age and career stage. Among sub-Saharan physicians in the USA, 78.3% are male. The picture is changing over time, however. Of the cohort who were trained in 1969 or earlier, 90% were male, but now only 66.3% of those who graduated from medical school in 1990 or later are male (see Table 3 ). Table 3 Characteristics of sub-Saharan African international medical graduates in the United States by graduation year cohort 1969 or earlier 1970–1979 1980–1989 1990–2000 OVERALL Number 720 1167 2268 1179 5334 Currently in residency (%) 0 2.3 17.6 59.7 21.2 Gender (% male) 90.0 86.5 76.7 66.3 78.3 Generalists (%) 19.0 28.3 47.8 57.9 41.9 current practice location: Urban (%) 95.3 94.1 93.7 95.7 94.4 Large rural (%) 2.4 3.3 3.6 2.6 3.1 Small rural (%) 1.7 2.1 1.8 1.1 1.7 Isolated rural (%) 0.7 0.6 0.9 0.6 0.7 Note: includes residents. Source of data: American Medical Association: Physicians' professional record (AMA-PPD) 2002. The average age of sub-Saharan African physicians in the USA is 43 years, compared to 46 years for all USA physicians. Forty two percent of sub-Saharan African physicians in the USA are under 40 years, and another 32% are between 40 and 50. Among the large contributing countries, Nigerian physicians are the youngest cohort (63% are under 40), and South Africans are the oldest (only 20% are under 40). A higher proportion of sub-Saharan physicians were in residency training programs (21.2%) than were USA physicians (14.1%), because many emigrate specifically for that reason. While 41.9% are in generalist specialty areas, compared to 34.7% of USA-trained physicians, the number has been rising with each new cohort. Table 3 illustrates that 57.9% of those trained in the 1990s selected a generalist practice specialty, compared to 28.3% of those trained in the 1970s. This apparently rising interest in generalist practice may be an artifact, however, as a prerequisite to internal medicine specialization is training in general internal medicine. While 31.6% of all sub-Saharan African physicians in the USA are identified as family practitioners or general internists, it may be that this ratio of generalists will increase, as 45.4% of those in residency programs are in those two specialties. The next largest specialty groups are pediatrics (9.7%), psychiatry (5.5%), anesthesiology (5.4%), obstetrics and gynecology (3.3%) and general surgery (3.0%). Urban areas attracted 93% of sub-Saharan African physicians (compared to 90.9% of other IMGs), even after excluding residents, who are typically based in urban teaching hospitals. Graduates of USA medical schools distribute themselves similarly, with 87% of USA-trained physicians in urban areas, even though a smaller 81% of the population lives in urban areas [ 27 ]. The states attracting the largest numbers of sub-Saharan Africa physicians include New York, California, Texas, Maryland, Illinois, Georgia, Pennsylvania, and New Jersey (see Figure 1 ). These are the same states that draw the largest portion of immigrant physicians generally. Figure 1 Origin and distribution of African-trained physicians in the 11 US states with the most such physicians The 1943 physicians trained in South African medical schools are somewhat different from their fellow African trainees from the subcontinent. They are older, more of them are male, they are typically white (94%) and they are more often in a subspecialty practice. This may reflect a particular wave of physicians seeking subspecialty training and practice opportunities in the USA during the political turmoil South Africa experienced in the 1970s and 1980s. It is unlikely they are seeking training abroad that is unavailable in their home country, as the medical training opportunities in South Africa are quite comprehensive. Discussion The 5000-plus physicians trained in sub-Saharan Africa who have migrated to the USA comprise only a small proportion of the total number of IMGs practicing in the USA. However, their relatively small numeric addition to the USA medical workforce contrasts markedly with the impact of their migration on the medical workforce in sub-Saharan Africa. Moreover, in absolute terms, the USA has drawn more of the medical workforce of Africa than either Canada, with 2151 African graduates (Lynda Buske, Canadian Medical Association, personal communication, 2/3/03), or the UK, with 3451 (Bonnie Sibbald, University of Manchester, personal communication, 1/24/03), largely because of the relative size of the health care system in the USA. Including the USA, the UK, and Canada, then, 10 936 physicians trained in sub-Saharan Africa are practicing in the three countries, a number that represents 12% of all African physicians. We expect there are many more African physicians who are in the UK, as well, as our figures from there include only those who arrived post-1992. While some of the physicians in residency training will return home, there are unknown others who could be practicing medicine at home but were not able to get licenses abroad and therefore are engaged in other occupations. Almost all the medical schools sending graduates to the USA provide their instruction in English. Our study provides some measures of sub-Saharan Africa physician migration to the USA. A next step is to collect data from other developed countries and begin to create a physician migration data set from multiple countries. Migration from country to country within the subcontinent is also worthy of further examination, and it would be useful to identify the relationship between country of birth and country of training. There are likely to be some medical schools that draw students from several African countries. It should be noted, as well, that there are physicians practicing in Africa who did not train there, most prominently Cubans. Some sub-Saharan countries lose a larger proportion of their physicians to the USA than others. For example, while Ghana has a reported 1210 practicing physicians in its country, 478 graduates of Ghanaian medical schools are practicing in the USA. Without even considering those who have migrated to other countries, these 478 Ghanaian graduates in the USA represent 30% of Ghana's potential medical workforce (see Table 2 ). If none of those had come to the USA, the physician-to-population ratio in Ghana would rise from 6.2 to 8.7 per 100 000, or a 40% increase. By comparison, South Africa has lost 14% of its potential workforce to the USA and Canada. The migration of physicians from sub-Saharan Africa represents a lost investment of significant training costs, since graduates of medical schools in Africa are likely to have contributed financially to only a small portion of the costs of their medical education [ 28 ]. Medical education is estimated to cost Ghana about USD 9 million per year and Nigeria USD 20 million (Hagopian & Ofosu, et al. The flight of physicians from West Africa: views of African physicians and implications for policy , unpublished 2003). The United Nations Commission for Trade and Development has estimated that each professional leaving Africa costs the continent USD 184 000, or USD 4 billion a year – one third of official development funds to Africa [ 29 ]. The loss of trained health personnel also contributes to a general decline in average incomes, as physicians generate skilled health system jobs beyond their own. Lost tax revenues from absent physicians represent significant losses as well. Ostensibly, the USA welcomes IMGs for two purposes. First, as a form of foreign aid, it provides specialty training that physicians can take back to their home countries for the benefit of residents of those nations. Second, IMGs fill positions in specialties and locations that are less attractive to their USA counterparts, and may help to correct physician maldistribution in some rural or underserved areas of the USA. (There are several federal agencies, along with state health departments who "sponsor" physicians who have completed their residency training in the USA on J-1 exchange – or "student"- visas. These sponsorships allow foreign national physicians to gain approval from the State Department and the USA Citizenship and Immigration Services to waive J-1 visa requirements that would otherwise require them to return to home countries for at least two years. In exchange for this waiver, physicians find employment with a health agency or private physician in a health professional shortage area.) Longitudinal tracking of physicians entering the USA has indicated, however, that few IMGs ever leave the USA after arriving for residency training [ 30 ], and there is conflicting evidence about whether IMGs are more likely to practice in safety net practices for low-income and underinsured people [ 21 , 31 , 32 ]. Attracting physicians to rural practice in most countries is difficult, and is accomplished only through a careful set of policies designed to provide incentives for rural service. In most poor as well as rich countries, physicians are concentrated around urban hospitals that offer tertiary care, even though more rational service delivery systems might focus on a geographically decentralized system of primary and preventive care. Poor countries that offer medical training to produce too many physicians with highly technical skills, some of whom who cannot find satisfying jobs, may further contribute to physician migration [ 33 ]. India and the Philippines, for example, clearly over-produce physicians who are intended for an international market. Our findings show African physicians are unlikely to select small or remote rural practice opportunities in either their home countries or in the USA, but the preponderance of African physicians in American inner-city underserved areas may to some extent be helpful to USA needs by boosting the number of minority physicians in the urban health workforce. The growing number of African immigrant female physicians follows the trend for increasing numbers of female physicians trained in the USA, and probably has similar implications. Some researchers have found, for instance, that female physicians are less likely to practice in rural areas [ 34 ]. While the sub-Saharan Africa region as a whole loses many of its physicians, it is apparent that a small handful of medical schools is the sources of the majority of this migration. Ten medical schools in four countries – South Africa, Nigeria, Ghana and Ethiopia – produce 79.4% of the émigré physicians to the USA, out of a total of 87 medical schools in the region. This suggests policy approaches to reducing the "brain drain" from Africa could be targeted at only these few countries or medical schools, a less daunting task than addressing the problem in 47 different countries. Medical migration results from the complex interaction of myriad social, legal and economic forces. Single country policies are unlikely to alter the flows significantly. Even if the USA acknowledges that that it benefits from luring medical professionals here for whose medical school training we do not pay, solutions that would be compatible with social justice principles are not clear. Furthermore, if all African doctors returned to their home countries today, they would not necessarily find satisfactory employment opportunities in cash-strapped health systems. Conclusions The 57th World Health Assembly, in 2004, adopted a resolution to urge member countries to develop strategies to mitigate the adverse effects of migration of health workers; to develop policies that could provide incentive for health workers to remain in their countries; and, among other issues, requests WHO to help countries set up information systems to monitor the movement of health resources for health, and to include human resources for health development as a top-priority program at WHO from 2006 to 2015 [ 35 ]. In an ideal world, freedom of movement is a universal right for individuals, as there is ostensibly no rational reason why anyone would have a stronger right to be in any place more than anyone else [ 36 ]. Today, however, differences in wealth between countries create flows of educated people seeking better opportunities far from home. One result is that resource-strapped African (and other poor) countries have invested significant resources in educating health professionals who will never serve the populations that were taxed (or took out high-interest loans from international lenders) to pay for their training. Importing health professionals from poor countries to provide care in rich countries is not consistent with a rational workforce policy rooted in social justice principles. In the short run, Mullan [ 37 ] and others are right to recommend that the USA expand its incentives to USA graduates to practice in rural and underserved areas through the National Health Service Corps and other programs. Grumbach [ 18 ] recommends reducing the number of excess residency training positions by limiting the Medicare subsidy. In response to a recent report by the USA Physicians for Human Rights [ 38 ], The New York Times editorialized that the "obvious long-term solution to the medical brain drain is for wealthier countries to reimburse Africa's health and educational systems for the cost of poaching their professionals, and to greatly increase the financing and technical help for Africa's health systems" [ 39 ]. This unprecedented attention to the issue of the African medical brain drain in a major USA publication, coupled with a radical call for reparations, suggests USA policy makers may be called to address this issue. The same Physicians for Human Rights report that prompted The New York Times response made a strong recommendation that the International Monetary Fund, World Bank, and other donors refrain from withholding loans or grants from countries that increase their spending on "health, education and other sectors and activities needed to promote human development, including to enhance salaries to health staff or to hire new health personnel." One of the major limitations African nations face in addressing their health workforce problems is the lack of reliable data on how many health workers have graduated from their schools, how many are working in the country and in what locations, and how many have emigrated. It is urgent that poor countries put together the information systems required to track these data, as a basis for workforce policy and investment decisions. And, finally, the fact that so few medical schools generate the vast majority of emigrants creates an opportunity to focus attention in a strategic way. These schools might be enticed to redirect their missions towards producing graduates who intend to serve their own countries. This would likely require curriculum changes, admissions policy changes, and a change in faculty culture to ensure that emigration is not promoted as a mark of prestige. Competing interests The author(s) declare that they have no competing interests. Authors' contributions AH conceived the project, designed the research and wrote the paper. MJT and KEJ actively participated in the conceptualization and re-writing of the paper. MF conducted the data management and analysis. LGH provided guidance, advice and editing assistance. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544595.xml
534099	Comparison of frozen and RNALater solid tissue storage methods for use in RNA expression microarrays	Background Primary human tissues are an invaluable widely used tool for discovery of gene expression patterns which characterize disease states. Tissue processing methods remain unstandardized, leading to unanswered concerns of how to best store collected tissues and maintain reproducibility between laboratories. We subdivided uterine myometrial tissue specimens and stored split aliquots using the most common tissue processing methods (fresh, frozen, RNALater) before comparing quantitative RNA expression profiles on the Affymetrix U133 human expression array. Split samples and inclusion of duplicates within each processing group allowed us to undertake a formal genome-wide analysis comparing the magnitude of result variation contributed by sample source (different patients), processing protocol (fresh vs. frozen vs. 24 or 72 hours RNALater), and random background (duplicates). The dataset was randomly permuted to define a baseline pattern of ANOVA test statistic values against which the observed results could be interpreted. Results 14,639 of 22,283 genes were expressed in at least one sample. Patient subjects provided the greatest sources of variation in the mixed model ANOVA, with replicates and processing method the least. The magnitude of variation conferred by processing method (24 hours RNALater vs 72 hours RNALater vs. fresh vs frozen) was similar to the variability seen within replicates. Subset analysis of the test statistic according to gene functional class showed that the frequency of "outlier" ANOVA results within each functional class is overall no greater than expected by chance. Conclusions Ambient storage of tissues for 24 or 72 hours in RNALater did not contribute any systematic shift in quantitative RNA expression results relative to the alternatives of fresh or frozen tissue. This nontoxic preservative enables decentralized tissue collection for expression array analysis without a requirement for specialized equipment.	Background Many of the hopes for achieving clinical benefits of genomic medicine will hinge on the ability to develop an efficient specimen conduit from clinic to laboratory. Quantitative gene expression studies have created unprecedented tissue collection and handling challenges. In particular, the rapid degeneration of RNA, and possible perturbation of expression following excision place a high premium on prompt stabilization of tissue samples intended for expression analysis. This can be accomplished by sending a dedicated trained technologist outfitted with the necessary specialized equipment, such as liquid nitrogen, into the clinical environment. Alternatively, clinicians can be enabled to process the specimens directly in the course of patient care and send them in some stable form by unrushed and routine means for centralized processing. The latter is greatly preferred when patients are physically dispersed, and becomes essential in a multi-institutional setting. High throughput quantification of RNA expression in solid tissues has become a commonplace modality for genome-wide discovery of mechanisms of disease. Typically, groups of samples classified into comparison groups are used as a training set for expression pattern discovery, followed by validation in a fresh challenge set of annotated cases. The likelihood of success is highly dependent on the accuracy of classification within the training set, and ability to control random variables introduced during tissue processing and analytical measurement of RNA abundance. Efforts to standardize RNA quantification include sharing of information regarding probe design and use [ 1 ], or centralized design and production of analytical reagents and platforms by commercial entities using good manufacturing procedures (GMP). Flash freezing, either by immersion in liquid nitrogen or on dry ice, is the most common means of stabilizing tissue samples intended for RNA analysis. Local access to the necessary materials and expense of cold shipping and/or storage limit these collection capabilities in most clinical settings. An additional disadvantage of frozen storage is that homogenization of frozen tissue must be accomplished rapidly to avoid the rapid RNA degeneration that occurs during thawing of a previously frozen sample. Room temperature immersion of fresh tissue samples in aqueous sulfate salt solutions (such as ammonium sulfate) at controlled pH precipitates degenerative RNAses [ 2 ] and other solubilized proteins, thereby preserving the tissue with intact RNA [ 3 ]. Tissues preserved in this manner are compatible with most RNA isolation protocols, and may be archivally stored for extended periods at -60°C. A commercial preparation of this preservative, RNALater (Ambion), is increasingly being used by individual investigators and cooperative groups [ 4 ] for collection of human tissues. There have been promising reports of microarray-based RNA expression studies using RNALater-preserved tissues [ 5 - 10 ]. Solid tissues stored for a week in RNALater at room temperature give comparable RNA yields, and specific gene RNA abundance as with frozen tissue[ 8 ]. RNA yields are not affected substantially by storage at room temperature compared to 4°C, for storage intervals up to 3 months [ 11 ]. RNALater preserved tissues and cell suspensions are suitable starting points for RNA quantification by quantitative RT-PCR [ 11 ] and expression microarray hybridization [ 12 ]. One shortcoming of the prior work is that the potential changes contributed by RNALater use have not been precisely measured relative to random processing effects. We studied the effects of differences between storage conditions on gene expression as measured by expression array. Duplicate uterine myometrial tissue samples from three women were processed under each of 4 fixed storage conditions – fresh, frozen, 24 hours RNA-later and 72 hours RNA-later. The 24 labeled cRNA samples (Figure 1 ) were hybridized to HG-U133A Affymetrix microarrays. Then, for each microarray a data matrix was generated of 22,283 probe sets (genes) by quantitative expression levels in each RNA sample, and the effect of subject source, tissue processing, and replicates (Table 1 ) determined by ANOVA. Subset analysis by gene functional class was then performed to determine if storage condition has a specific effect on particular groups of genes. Figure 1 Experimental design. Tissue aliquots from 3 women were aliquoted, in duplicate, into four storage groups before RNA isolation and microarray hybridization. ANOVA design elements including fixed (storage group), random (woman, duplicate processing), and random interactive (woman × storage) effects as listed in Table 1. Table 1 Variability Sources in ANOVA Model (See Figure 1). Mixed ANOVA Model:Xij = u + ai + Bj + Eij where Xij is the observation (LN intensity), ai is the tissue storage effect, Bj is the individual variability effect and Eij is the noise term. Variability Source Type df Tissue Storage Fixed 3 Woman, Individual Variation Random 2 Interaction (Woman × Storage) Random 6 Replication (RNA isolation, chip Processing) Random 12 We found no systematic bias in measured quantitative level of gene expression by processing method, indicating that short term storage in RNALater is a valid alternative to traditional frozen storage. Results Of the 22,283 genes, 14,639 did not have absolutely null expression across all 24 samples. We fit the mixed model ANOVA from their log values and recorded this F statistic. The permutation distribution was used to assess the significance of F statistics calculated for each gene in the dataset. In this approach all 13,824 or (4!) 3 possible ways of permuting 4 pairs of replicate samples within each subject were considered. For each of these, the F statistics were computed for each gene. To control the overall error rate, the distributions of the maximum F statistics over the genes were used. That is, for each gene, the p-value is the proportion of permutations with the maximum F statistics over all genes greater or equal to the observed value for a particular gene. A test declaring as significant any genes with p < 0.05 then guarantees that the chance of any false positives being selected is < 5%. Similar analyses were performed replacing the distribution of the maximum F statistic with the distribution of the F statistics at the 95 th percentile and then at the 90 th percentile. After closer examination of the 387 genes in the 5% tail, we noted that most were exhibiting expression values below 100 for all 24 samples. In fact, within a storage condition, 2 out of 3 patients exhibited null expression while the third patient showed expression values other than null but less than 100 for at least one of their replicates. Therefore, as an additional analysis, any expression values less than 100 were recoded as 100. Genes that showed expression levels of 100 across all 24 samples, and therefore lacked variability, were then removed from the analysis. This resulted in 7,853 genes for which there was at least one sample with expression level greater than 100 across all 24 samples. Patient subjects provided the greatest sources of variation in the mixed model ANOVA, with replicates and processing method the least (Figure 2 ). The magnitude of variation conferred by processing method (24 hours RNALater vs 72 hours RNALater vs. fresh vs frozen) was similar to the variability seen within replicates. This is strong evidence that those individual expression profiles characteristic of the source tissue (woman) are unlikely to be obscured by the small amount of variation introduced by the processing method chosen. Figure 2 Sources of variation in the mixed ANOVA model . Distribution of Mean Squares Errors by variation source are plotted for those genes with at least one tissue showing expression at a level above LN(100) (7853 genes). Note that individual women emerge as the dominant source of variation. Variation contributed by tissue storage is of approximately the same magnitude as that seen between duplicate samples within the same storage group. Boxes encompass inner quartiles, horizontal line represents the median or the second quartile, and whiskers delimit 1.5 times the interquartile range. Because of the very large number of data points, outliers were suppressed in this summary plot. The distribution of ANOVA test statistics on those 7,853 genes where at least one sample of 24 had expression at a level exceeding 100 is compared in Figure 3 to those seen in the randomly permuted dataset. In the actual dataset, the maximum observed F statistic was 25.52; the observed F statistic at the 95th percentile was 3.51; and the observed F statistic at the 90 th percentile was 2.58. Corresponding p-values were 0.94, 0.55 and 0.51, respectively. The values of test statistics seen at the 95% level in a randomly permuted dataset (Figure 3 , thin solid line) are greater than those of the observed dataset (Figure 3 , thick solid line). This indicates that the model variation contributed by processing method is of the same magnitude as that seen randomly. Figure 3 Distribution of actual test statistics vs. randomly permuted background. Distribution of ANOVA F-statistics from the model shown in Figure 1 were calculated for the observed (FStat Observed) and permuted datasets. The maximum, 95th percentile, and 90th percentile F-Statistics in the permuted dataset provide an index of the distribution of test results expected for a random sample. The values of test statistics seen at the 95% level in a randomly permuted dataset are greater than those of the observed dataset. Genes were included if at least one tissue showed expression above LN(100) (7853 genes). Subset analysis of the test statistic according to gene functional class (Table 2 ) showed that the frequency of "outlier" ANOVA results within each functional class is overall no greater than expected by chance. Test statistic distribution was compiled by functional annotation for those 7853 genes which had at least one sample with detectable expression above a level of 100. Nine separate classification schemas containing a total of 127 functional classes were studied. Probe sets were rank ordered by decreasing ANOVA test statistic, and enrichment in the nominal p = 0.05 tail (F>3.51, containing top 387 of 7853 expressed) plotted by functional class (circles) and schema (box plot) (Figure 4 ). Table 2 Test statistic results by functional class within 9 annotation schema. Amongst 9 schema, a total of 127 functional classes ("Classes") contained expressed genes ("Total Genes"), and of these, 102 classes had a sufficient number of expressed genes (>50, "Class>50") to enumerate ("Genes F>3.51") and calculate fractional representation ("%>3.51", also Figure 3) in the nominal p = 0.05 tail at a test statistic cutoff of 3.51. 7853 genes with expression in at least one tissue >LN(100) were included. Code Schema Classes Class>50 TotalGenes Genes F>3.51 %>3.51 1 Biological Process (GO) 22 14 3591 197 5.5 2 Cellular Role (Proteome) 14 13 1775 92 5.2 3 Cellular Component (GO) 15 12 2845 154 5.4 4 Molecular Localization (Proteome) 9 10 1788 93 5.2 5 Organismal Role (Proteome) 17 12 1524 64 4.2 6 Biochemical Function (Proteome) 16 14 2727 150 5.5 7 Subcellular localization (Proteome) 8 7 2047 116 5.7 8 Molecular Function (GO) 21 15 4473 239 5.3 9 Pathways (GenMAPP) 6 5 756 33 4.4 Figure 4 Proportion of functional gene classes in which the test statistic falls within the nominal p < 0.05 tail. Nine publically available gene functional annotation schemas (listed in Table 2), each composed of multiple functional gene classes, were used to determine if specific functional subsets of expressed genes were more likely to show significant change in RNA detection between tissue treatments. Percentage of individual gene classes with test statistics above the nominal p = 0.05 level (F Statistic >3.51 in the dataset of 7853 expressed genes) are plotted on the Y axis for each of 9 classification schema (X axis). Results for functional classes of genes with a minimum of 50 available expressed genes are shown as individual data points (circles) the distribution of which is summarized for each schema by the superimposed notch plot. There are only two high outliers (arrows) amongst the 113 gene classes shown. These are a messenger RNA splicing factors in the Biological Process (GO) schema (Schema 1), and translation factor in the Pathways (GenMAPP) schema (Schema 9). This frequency of 2/113 outliers is no greater than expected by chance, (>5). Discussion Storage of fresh whole human tissues for up to 72 hours at room temperature in RNALater does not introduce quantitative bias into RNA expression determinations with the Affymetrix U133A array. Several differing standards justify this conclusion. First, by construction of a test model (Figure 1 ) incorporating both random reproducibility estimates (replicate determinations) and between-sample differences it has been possible to demonstrate that the magnitude of variation introduced by RNALater processing is equivalent to that seen within routine repeat specimens in a common processing group (Figure 2 ). Second, the extent of result variation conferred by RNALater processing is not statistically significant when measured against the randomly permuted dataset (Figure 3 ). This is an important element in evaluation of large datasets in which small numbers of individual variables may randomly demonstrate extreme values of the test statistic. Lastly, there is no evidence that specific functional subgroups of genes have aberrant behavior in this regard (Figure 4 ). Uterine myometrium was selected for these experiments because its components (myocytes, fibroblasts, vascular elements) are evenly intermingled throughout the myometrial compartment, lending itself to physical subdivision into equivalent aliquots. This would not be possible with more complex tissues in which differing cell types are distributed asymmetrically within the specimen. Despite the equivalency of subdivided fractions that underwent varying storage treatments, it must be noted that this is a hormonally responsive tissue whose expression patterns would be expected to differ between individual women as a function of monthly changes in circulating sex hormones. We did not control for hormonal factors or indication for hysterectomy (prolapse or fibroids) but selected patients randomly. It comes as no surprise that expression differences between women, irrespective of processing method, emerged as the dominant source of inter-sample variation. This was anticipated in constructing the model, by assigning the subject source of specimens as a random variable which could be measured against the fixed processing effects. It is likely that if a larger number of women had been included in the study, the observed biologic variation attributable to subject would have been even greater. Since our goal was to compare magnitude of variation contributed by subjects to that conferred by processing method, we achieved a balanced design by having comparable degrees of freedom for those two variables. There are several critical procedural elements that must be highlighted for successful preservation of solid tissues in aqueous sulfate salt solutions such as RNALater. These reagents enter the tissue through passive diffusion, a process which follows simple physical principles. The distance between the tissue surface, which is exposed to preservative, and the innermost regions of the fragment should be minimized. We did this by cutting the tissues into 2 mm thick slices, thereby reducing the diffusion distance to 1 mm or less. Clumping of multiple fragments into a mass that excludes preservative may obviate the benefits of fine division. This can be avoided either by gentle agitation or placement in a sufficiently large container that individual pieces are likely to disperse. Results reported here are for tissues stored at room temperature (23–25°C). Storage under cooler conditions (4°C) as recommend by the manufacturer of RNALater were not directly evaluated in this experiment because it was our intent to mimic storage interval and conditions commonly encountered when sending a specimen by express courier to a centralized processing facility. Storage at temperatures substantially higher than 25°C, especially before the preservative has had an opportunity to penetrate the tissue, should be avoided. Conclusions Split samples of fresh human tissue yield quantitatively similar RNA expression profiles whether processed fresh, frozen, or following 24–72 hour storage in RNALater. Formal statistical analysis shows patient source is the predominant source of variation between samples, with processing method contributing a random level of variation comparable to that seen in split duplicates (replicates). Subset analysis by functional gene category did not identify a specific class of genes which responded differently by processing method. Use of nontoxic ambient environment tissue preservatives makes it practical to engage practicing clinicians directly in decentralized sample collection for high throughput expression analysis in a central location. Tissue handling closely resembles that used by clinicians to prepare specimens for routine pathology analysis. Upon receipt in a centralized facility, the samples can either be immediately homogenized or archived at -60°C. Methods Tissue handling and storage Normal fresh uterine myometrial tissues were collected randomly from three women undergoing hysterectomy for benign uterine disease. For each hysterectomy, a single 4 to 8 gram tissue fragment was subdivided into eight aliquots composed of thin slices measuring no more than 2 mm in thickness. Replicate aliquots were immediately triaged into one of four storage conditions prior to homogenization: 1)immediate homogenization; 2)flash frozen in liquid nitrogen and storage for 48 hours at -80°C; 3)24 hour immersion in RNALater at room temperature with gentle agitation; or 4)72 hours immersion in RNALater at room temperature with gentle agitation. RNA isolation Tissue was solubilized in Trizol reagent (Gibco BRL, Grand Island, NY), and RNA isolated according to the manufacturers instructions. In brief, the aqueous phase was resolved by addition of chloroform, and RNA precipitated from the aqueous phase by addition of isopropyl alcohol. Pelleted RNA was washed with 70% ethanol, dried, and resuspended in water. Quality of total RNA was assessed by running a non-denaturing 1% agarose tris-acetate buffer which confirmed the integrity of 18S and 28S ribosomal bands for all 24 total RNA preparations. Microarray chip hybridization and data normalization Double-stranded cDNA was generated from 8 μg total RNA using the Superscript Choice System (Life Technologies) with T7-(dT)24 oligomer. cDNA was purified by phenol/chloroform extraction and ethanol precipitation. Biotin-labeled cRNA was prepared using the Enzo BioArray HighYield RNA Transcript labeling kit (Affymetrix). Unincorporated NTPs were removed from the biotinylated cRNA using an RNeasy kit (Qiagen). 10 μg of quality, fragmented cRNA was hybridized to each Affymetrix HG-U133A arrays containing probe sets representing approximately 22,000 genes. Array hybridization, washing was done according to the manufacturer's protocol (Affymetrix, GeneChip ® Expression Analysis Technical Manual) and all arrays were scanned under a low PMT (Photo Multiplier Tube) of 570 nm. Global scaling to a target value of 75 was applied to normalize all the arrays so they were comparable (Affymetrix Microarray Analysis Suite MAS5.0). The Affymetrix average-difference expression data and the P/A calls were used in the analysis. Those probe sets determined to have no detectible signal above background mismatch hybridization (Call of "Absent") were assigned a nominal value of 1 to facilitate future log transformations. Probesets having at least one tissue with detectable expression (call of "present") and an average difference above either 1 or 100 were selected to define subsets of 14639 permissively or 7853 stringently expressed genes, respectively. Further analysis was performed using the natural log transformed data of these probe subsets. Data files for all specimens processed are deposited online at the Gene Expression Omnibus at the National Center for Biotechnology Information [ 13 ]. Biostatistical analysis For this two factor study, a mixed model analysis of variance (ANOVA)was used, regarding storage condition as a fixed factor with four levels and subject as a random factor with three levels. The analysis of variance calculations for sums of squares in the mixed model ANOVA are identical to those for the fixed ANOVA model. Similarly, the degrees of freedom and mean squares are exactly the same. The mixed ANOVA model departs from the fixed ANOVA model only in the expected mean squares and the consequence choice of the appropriate test statistic. The mixed model also included a random storage by subject interaction. Replicate samples enabled us to estimate the replication error in the model. To test for the presence of storage main effects for each gene we divided the mean square for storage by the mean square for the interaction effect between storage and subject [ 14 ]. The ANOVA test statistic was calculated using C++. Functional annotation of probesets on the U133A chip, were downloaded from the Netaffx tm download center [ 1 ]. The March, 2003 version matches individual probesets with functional annotations (Table 2 ) from public domain databases including: the Gene Microarray Pathway Profiler, Gene Ontology Consortium, Proteome BioKnowledge Library, and Kyoto Encyclopedia of Genes and Chromosomes. Within each schema (comprised of many functional classes of genes), each gene is assigned to a primary functional class. Each probe set may be represented in several different schemas. Individual functional classes with at least 50 probesets represented within the U133A array were plotted by schema to show fractional representation within the nominal 0.05 tail (Figure 4 ). This provides a rapid and intuitive manner to identify functional classes of genes biased towards high test statistics in the ANOVA model. Authors' contributions GM and JW conceived and designed the research plan and participated in all aspects of data collection and analysis. DF participated in data analysis and interpretation. DN and DZ performed the statistical analysis. CL and HB performed the RNA isolations, chip hybridizations, and data collation.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC534099.xml
535344	Bi-directional modulation of AMPA receptor unitary conductance by synaptic activity	Background Knowledge of how synapses alter their efficiency of communication is central to the understanding of learning and memory. The most extensively studied forms of synaptic plasticity are long-term potentiation (LTP) and its counterpart long-term depression (LTD) of AMPA receptor-mediated synaptic transmission. In the CA1 region of the hippocampus, it has been shown that LTP often involves a rapid increase in the unitary conductance of AMPA receptor channels. However, LTP can also occur in the absence of any alteration in AMPA receptor unitary conductance. In the present study we have used whole-cell dendritic recording, failures analysis and non-stationary fluctuation analysis to investigate the mechanism of depotentiation of LTP. Results We find that when LTP involves an increase in unitary conductance, subsequent depotentiation invariably involves the return of unitary conductance to pre-LTP values. In contrast, when LTP does not involve a change in unitary conductance then depotentiation also occurs in the absence of any change in unitary conductance, indicating a reduction in the number of activated receptors as the most likely mechanism. Conclusions These data show that unitary conductance can be bi-directionally modified by synaptic activity. Furthermore, there are at least two distinct mechanisms to restore synaptic strength from a potentiated state, which depend upon the mechanism of the previous potentiation.	Background Fast excitatory synaptic transmission in the central nervous system, which is mediated predominantly by the AMPA subtype of glutamate receptors, can undergo long-term bi-directional modifications in strength [ 1 - 3 ]. These persistent changes have been proposed to be key synaptic processes involved in learning and memory. The best characterised form of bi-directional synaptic plasticity is LTP / LTD of glutamatergic transmission in the CA1 region of the hippocampus. Although there has been intensive investigation of the induction and expression of LTP and LTD (see [ 4 ]), the precise molecular mechanisms by which these alterations in synaptic strength occur remain unclear. Possible mechanisms could be presynaptic such as changes in the release process (probability of neurotransmitter release or the amount of L-glutamate released from vesicles (e.g., [ 5 - 9 ]), and / or postsynaptic (e.g., [ 10 - 12 ]), such as a change in the number of AMPA receptors (AMPARs) available to bind transmitter, or in the properties of existing receptors (P open , activation or de-activation kinetics, desensitisation or single-channel conductance, γ). Recent studies have provided information on the possible mechanisms underlying postsynaptic alterations in synaptic strength. There is evidence that AMPA receptors are inserted into the postsynaptic membrane during LTP [ 13 - 15 ] and removed from the synapse upon induction of LTD [ 16 , 17 ]. However, it has also been shown that LTP can involve a rapid increase in γ of existing AMPA receptors [ 18 ]. This could be caused by a Ca 2+ /calmodulin-kinase II (CaM-KII)-mediated phosphorylation of GluR1 which occurs during LTP [ 19 ], and which causes an increase in γ of GluR1 homomers in transfected cells [ 20 ]. Therefore, another potential mechanism for LTD could be a decrease in γ caused by a dephosphorylation of AMPARs. We have recently used peak-scaled non-stationary fluctuation analysis (non-SFA; [ 21 ]) of synaptic currents recorded from CA1 pyramidal cell dendrites [ 18 ], to investigate the molecular basis of de novo LTD (LTD at naïve pathways; [ 22 , 23 ]). In these studies, LTD was never associated with a change in γ [ 17 ]. Indeed, evidence was presented that the underlying mechanism involved a reduction in the number of surface expressed AMPA receptors (LTD N ). In the present study we have investigated a second form of LTD known as depotentiation (DP), which is a reversal of pre-established LTP [ 24 - 27 ]. LTP can be associated with either an increase in γ (LTPγ) or no change in γ (LTP N ) [ 18 ]. We were, therefore, interested to determine whether DP of LTPγ involved a decrease in γ and hence whether γ is a bi-directionally modifiable parameter. We find a reciprocal relationship between LTP and DP, such that DP of LTPγ invariably involves a restoration of the pre-LTP γ (DPγ) whereas DP of LTP N never involves a change in γ (DP N ). These data show, firstly, that there are two distinct molecular mechanisms for the reduction of synaptic strength that are dependent on the nature of the preceding LTP and, secondly, that γ can indeed be bi-directionally modified in response to synaptic activity. Results Using whole-cell recordings from the proximal apical dendrites of hippocampal CA1 pyramidal cells, minimal stimulation of nearby afferents evoked EPSCs that could be reliably resolved from failures (trials in which stimulation produced no synaptic response; Figure 1 , see also Figure 3 ). These high resolution recordings enabled both a failures analysis to be performed [ 28 ] and, using non-SFA, an estimate of γ of synaptically-activated AMPARs to be obtained [ 18 ]. LTP To investigate the mechanism of DP, LTP was first induced in 18 cells by pairing afferent stimulation (baseline frequency) with a holding potential of 0 mV. This resulted in stable LTP (EPSC amplitude = 186 ± 16 % of baseline, n = 18). In agreement with a previous study [ 18 ], cells fell into two groups with respect to changes (> 20%) in γ of AMPA receptor channels during LTP. In the majority of cases (11/18 cells), LTP was associated with an increase in γ (LTPγ; 246 ± 26% of baseline; range 136 – 363%). In the other 7 cells there was no increase in γ during LTP (100 ± 4 % of baseline; range 85 – 118 %), indicating that there was an increase in the functional number of channels activated (LTP N ). As noted previously [ 18 ], there were no differences between the two groups of neurons with respect to a variety of baseline parameters. Depotentiation of LTPγ Figure 1 shows two examples from the group of cells that exhibited LTPγ, as indicated by the change in the current-variance plot obtained from non-SFA (Figure 1B ). As previously reported [ 18 ], the increase in γ was not associated with any change in EPSC kinetics (Figure 1C ) indicating that AMPA receptor channel kinetics were not affected [ 29 ]. Failures analysis (Figure 1C ) of this group of cells (Figure 2 ) revealed that LTP was associated with changes in success rate (1 – failure rate) in some cells (Figure 2B ), and potency (mean EPSC amplitude excluding failures) in all cells (Figure 2C ), as previously reported under these recording conditions [ 18 , 28 ]. For this group of cells, DP, induced by pairing stimulation (baseline frequency) with a holding potential of -40 mV, always resulted in a reversal of the γ increase, as indicated by the current-variance plot (Figure 1B ; Figure 2D ). Similar to the preceding LTPγ, this form of DP (DPγ) was also associated with no change in EPSC kinetics (τ rise : baseline = 1.6 ± 0.2 ms, LTPγ = 1.7 ± 0.3 ms, DPγ = 1.7 ± 0.2 ms, n = 11; τ decay : baseline = 8.3 ± 0.6 ms, LTPγ = 8.2 ± 0.6 ms, DPγ = 8.9 ± 0.7, n = 11; Figure 1C ). Failures analysis showed that DPγ was associated with a decrease in success rate in most cells (Figure 2B ) and a full reversal of the potency increase (Figure 2C ). These data show that the primary mechanism for DP is the reversal of any increase in γ caused by LTP. Indeed, the changes in γ were sufficient to account for the potency changes during both LTPγ and DPγ. (In most cells the alterations in γ actually exceeded the potency changes. This is most likely due to an underestimate of the potency change due to dendritic filtering, which affects measurements at the peak of EPSCs greater than during the tail, from where the non-SFA estimates are obtained; see [ 18 ]). Depotentiation of LTP N Figure 3 shows an example from the group of cells that exhibited no change in γ during LTP (LTP N ), as indicated by the current-variance plot (Figure 3B ). The change in EPSC amplitude for LTP N neurons (Figure 4A ) was similar to that for LTPγ neurons (Figure 2A ). Failures analysis (Figure 3D ) of this group of cells (Figure 4 ) revealed that LTP was associated with changes in success rate in some cells (Figure 4B ), and potency in most cells (Figure 4C ; P < 0.01), as previously reported under these recording conditions [ 18 , 28 ]. In contrast to DPγ, DP in these cells was never associated with a change in γ (DP N ; Figure 3B , Figure 4D ). There was also no change in EPSC kinetics with LTP or DP (τ rise : baseline = 1.9 ± 0.4 ms, LTP N = 2.0 ± 0.3 ms, DP N = 1.8 ± 0.3 ms, n = 7; τ decay : baseline = 9.7 ± 0.7 ms, LTP N = 9.7 ± 0.6 ms, DP N = 10.1 ± 0.9, n = 7; Figure 3C ). Failures analysis of LTP N and DP N (Figure 3D , Figure 4 ) showed similar changes to the LTPγ group of cells in EPSC amplitude (Figure 4A ), success rate (Figure 4B ) and potency (Figure 4C ). These data suggest that there is a second mechanism for DP, not involving a decrease in γ, which co-exists at CA1 synapses. Therefore, there are two mechanisms for the expression of DP that depend upon the expression mechanism of the previous potentiation. Role of NMDA receptors in depotentiation It has been shown previously that DP at CA1 synapses may be blocked either by NMDA receptor antagonists [ 26 ] or by mGlu receptor antagonists [ 27 ] and that this may depend on previous history [ 30 ]. In the present study we wished to focus on NMDA receptor-dependent synaptic plasticity and therefore used pairing protocols designed to activate NMDA receptors sufficiently to, firstly, induce NMDA receptor-dependent LTP and, secondly, to induce NMDA receptor-dependent DP. To verify that we were indeed investigating NMDA receptor-dependent DP we performed a series of experiments using the NMDA receptor antagonist D-AP5 interleaved with control experiments. Following the induction of LTP, D-AP5 (50 μM) was bath applied for 15 minutes before delivering the DP induction stimulus. Whilst DP was induced in the control experiments (25 ± 11% of baseline, n = 5; p < 0.05) it was blocked by D-AP5 (89 ± 21%, n = 4; Figure 5 ). Relationship of changes in success rate, potency and γ to the magnitude of DP To gain further insights into the underlying mechanisms of DP we compared changes in EPSC amplitude, success rate, potency and γ for the individual experiments (Figure 6 ). A decrease in success rate indicates a reduction in probability of transmitter release (Pr) and/or a reduction in the number of functional synapses (n). A decrease in potency indicates a reduction in quantal amplitude (postsynaptic response to the release of a single quantum of transmitter, q) or, if multiple synapses are activated, a reduction in Pr or n. In both types of neuron (i.e., LTPγ and LTP N ), a small depression of less than 50 % (to 71 ± 7% of baseline; n = 4) was associated with no change in success rate (Figure 6A ; success rate ratio = 1.00 ± 0.01) but an equivalent decrease in potency (Figure 6B ; potency ratio = 0.70 ± 0.08), as is also observed for de novo LTD [ 17 ]). This indicates that the depression in these cells was associated primarily with a decrease in q. For larger depressions (to 26 ± 4 % of baseline; n = 14) there was also a marked decrease in success rate (success rate ratio = 0.52 ± 0.08; P < 0.01 vs success rate ratio for the group with DP < 50%) as well as a decrease in potency (potency ratio = 0.54 ± 0.04). This suggests that in these cells there was an additional decrease in Pr or n. Therefore, for DPγ, the change in γ did not fully account for the amplitude change in every cell (Figure 6C ) but did account for the potency change (Figure 6D ). Discussion In this study we have shown that there are two mechanisms for NMDA receptor-dependent DP, a reduction in γ (DPγ) and a decrease in the number of activated AMPA receptors (DP N ). As reported previously for different data sets from this age of rats [ 9 , 18 ] there are two forms of LTP; in approximately two-thirds of neurons LTP was associated with an increase in γ (LTPγ) whilst in the remainder there was no change in γ (LTP N ). In the present study we saw a similar proportion of LTP expressed by changes in γ versus N. Strikingly, we observed a precise relationship between the mechanism of DP and the form of preceding LTP; LTPγ was always reversed by DPγ, and LTP N was always reversed by DP N . In some experiments, induction of DP caused a decrease in EPSC amplitude below the initial baseline. This is most likely due to the simultaneous induction of DP and de novo LTD since in slices taken from juvenile animals, de novo LTD is readily induced by this [ 17 ] and other [ 22 , 23 ] induction protocols. This is in contrast to previous experiments using adult tissue in which DP induction depressed synaptic responses only as far as the initial baseline and where the same induction protocol was unable to induce de novo LTD [ 27 ]. Analysis of the mechanism of de novo LTD under the present experimental conditions demonstrated that it was associated with no change in γ [ 17 ]. Therefore the coexistence of DP and de novo LTD does not interfere with the analysis of DPγ. In addition, there is sometimes a small, gradual run-down of synaptic responses observed in minimal stimulation experiments using two-week-old animals [ 17 ] see also [ 31 ]. Whilst this effect tends to exaggerate changes in amplitude and success rate during DP in some neurons, it does not significantly interfere with estimates of potency or γ (see [ 17 ]). Mechanisms underlying DP N What might be the mechanism underlying DP that is not associated with a decrease in γ (i.e., DP N )? It is unlikely that this type of depression is due to a change in channel kinetics because there was no change in EPSC kinetics (see [ 18 , 29 ]). Therefore the mechanism is most likely a reduction in the number of activated AMPARs. This could be due to a presynaptic mechanism such as a reduction in release probability, the L-glutamate content of vesicles or the amount of L-glutamate discharged during fusion. Indeed there is evidence that some forms of LTD are expressed presynaptically [ 5 , 35 ]. Postsynaptic mechanisms for a reduction in the number of activated AMPA receptors include a reduction in their P open [ 36 ]or in the physical number of receptors present in the postsynaptic membrane [ 37 ]. The present observations for DP N are indistinguishable from those that we and others have reported recently for de novo LTD [ 17 , 38 ]. For example, we showed that similar effects were obtained using the postsynaptic injection of a peptide (pep2m) that disrupts the interaction between NSF and GluR2 [ 39 - 42 ]. The effects of pep2m and those of de novo LTD were mutually occlusive, indicating a convergence of mechanisms. These data argue strongly for a postsynaptic mechanism of expression. Furthermore, since pep2m causes the removal of AMPA receptors from the membrane surface, as determined immunocytochemically [ 38 , 42 ], it is most likely that de novo LTD is due to the physical elimination of synaptic AMPA receptors. Other evidence for a postsynaptic mechanism for de novo LTD includes a reduction in the postsynaptic sensitivity to glutamate [ 43 , 44 ], the dephosphorylation of serine 845 of the GluR1 subunit [ 45 , 46 ] and a rapid internalisation of AMPA receptors [ 47 ] associated with LTD. Therefore, by analogy, we feel that the postsynaptic removal of AMPA receptors is also the most likely explanation for DP N (Figure 7A ). Accordingly, a reduction in AMPA receptor number would account for the changes in potency without changes in success rate observed with modest DP N . The removal of an entire synaptic complement of AMPA receptors would explain the additional change in success rate seen with large depressions associated with DP N in some cells. Mechanisms underlying DP γ A number of possible underlying mechanisms could account for the change in γ during LTPγ and DPγ. Non-SFA cannot distinguish between 1) a change from a single low conductance state to a single high conductance state, 2) changes in open times within a burst and, 3) changes in the proportion of time spent in different conductance states. Since AMPA receptors are known to have multiple conductance states [ 32 , 33 ] we have postulated that the proportion of time spent in different conductance states is the modifiable parameter [ 18 ]. Such a change is detectable with the type of analysis that we have used, which provides a weighted mean of the various sub-conductance states [ 48 ]. Independent support for this hypothesis is provided by a study which shows that phosphorylation of GluR1 at serine 831 increases the proportion of time AMPA receptors spend in high conductance states, as determined by single channel recording [ 20 ]. Indeed, phosphorylation of this residue occurs during LTP [ 19 ]. Thus a possible mechanism of DPγ is the dephosphorylation of serine 831 [ 45 ], perhaps involving calcineurin [ 49 ], resulting in a lower proportion of time AMPA receptors spend in the higher conductance states (Figure 7B ). Other theoretical possibilities exist to explain DPγ. For example, the silencing of synapses close to the patch electrode leaving more distant synapses contributing a lower net γ due to electrotonic filtering, or a decrease in trial-to-trial asynchrony of transmitter release. We believe that these possibilities are unlikely because in every cell in which de novo LTD was induced there was never a change in γ [ 17 ]. If such possibilities were likely, statistically one would expect similar changes to occur for both de novo LTD and DP. Moreover, we have also investigated these possibilities using our standard compartmental model [ 29 ]. This shows that 1) the electrotonic filtering required to achieve an artefactual decrease in γ would have a pronounced effect on τ decay which was never observed experimentally, and 2) pronounced changes in asynchrony necessary to cause an artefactual change in γ cause large deviations from the parabolic relationship in the current-variance plot (unpublished observations) and alterations in τ rise [ 29 ], also never observed experimentally. Another possibility is that alterations in vesicle fusion pore dynamics leading to substantial changes in the peak and time-course of cleft glutamate are caused by synaptic plasticity [ 7 ]. Such changes could differentially affect estimates of γ [ 50 ], however they would be associated with substantial changes in τ rise and τ decay [ 7 ], which were never observed experimentally. Conclusions In summary, we have shown that there are two distinct molecular mechanisms for the reduction of synaptic strength. Although previous studies have provided evidence that LTD is associated with dephosphorylation of serine 845 on GluR1 [ 45 ] and internalisation of AMPA receptors [ 16 , 17 ] it is not known whether this represents two separate mechanisms or two components of the same process. For example, dephosphorylation of GluR1 could drive the internalisation of AMPA receptors. Here we show, for the first time, the co-existence of two distinct mechanisms for the expression of DP, using functional criteria under identical experimental conditions. The relationship between the two mechanisms is critically dependent upon the recent experience of the synapse, which may be governed by the phosphorylation state of the AMPA receptor complement [ 45 ]. Further work is now required to elucidate the relationship between these two fundamental mechanisms for modulating synaptic strength and the precise molecular mechanisms involved in each form of plasticity. Methods Electrophysiology Hippocampal slices (400 μm) were obtained from 12–15 day old rats and perfused with an extracellular solution containing (in mM): 124 NaCl, 3 KCl, 1.25 NaHPO 4 , 26 NaHCO 3 , 2 CaCl 2 , 1 MgSO 4 , 15 glucose, 2 ascorbic acid, 0.05 picrotoxin, saturated with 95% O 2 / 5% CO 2 , at room temperature (23–25°C). Individual dendrites were visualised using infrared illumination and DIC optics and approached under visual control. Whole-cell dendritic recordings of synaptic currents were obtained at a holding potential of -70 mV using patch electrodes (6–10 MΩ) filled with a solution containing (in mM): 135 CsMeSO 4 , 8 NaCl, 10 HEPES, 0.5 EGTA, 4 Mg-ATP, 0.3 Na-GTP, 5 QX-314, pH 7.25, 285 mOsm. Schaffer collateral-commissural fibers were stimulated at 0.5 Hz using a platinum monopolar or concentric bipolar electrode, which was positioned 20–40 μm from the dendrite parallel to the input pathway. The stimulus intensity was set to evoke some failures to enable a failures analysis to be performed and to ensure that the majority of EPSCs were, for any given trial, evoked by release from a single site. However, to elicit sufficient EPSCs to obtain a baseline estimate of γ before "washout" of LTP it was usually necessary to set the success rate fairly high (usually above 50%). As a result it is likely that multiple release sites contributed to the recordings. LTP was induced by pairing 40–60 stimuli (baseline frequency) with a holding potential of 0 mV. DP was induced following the induction of stable LTP by 100–200 stimuli (baseline frequency) at -40 mV holding potential. All recordings were made using an Axopatch 1B amplifier, signals were filtered at 5 kHz (8 pole Bessel filter), digitised at 10 kHz and stored on computer. EPSC amplitude and input resistance were analysed and displayed on-line using the 'LTP' program [ 51 ]). Series resistance was estimated by measuring the peak amplitude of the fast whole-cell capacitance current in response to a -1 mV step applied to the cell during each sweep. The amplitude was estimated by fitting the capacitance transient with a double exponential (from 0.5 ms after the peak) and determining the current at the beginning of the step. Series resistance was stable throughout recordings (series resistance values [MΩ]: baseline = 42 ± 3, LTP = 40 ± 3, DP = 43 ± 3, n = 17). Analysis Non-SFA was performed as described previously [ 18 ]. Briefly, synaptic currents were aligned by their point of maximal rise, and averaged. The average response waveform was scaled to the peak, subtracted from individual responses and the variance of the decays calculated. The variance was plotted vs . the mean current amplitude and the single channel current was estimated by fitting the data to: σ 2 = iI - I 2 /N + b l , where σ 2 is the variance, I is the mean current, N is the number of channels activated at the peak, i is the single channel current and b l is the background variance. The single channel conductance (γ) is then γ = i/V, where V is the driving force (holding potential – assumed reversal potential of 0 mV). Response amplitude was measured in two ways. For failures, amplitude was estimated by measuring the difference between the average current over two time windows of equal length, one immediately before the stimulus artefact and the other centred on the peak of the mean EPSC. For estimation of the amplitude of successes the peak amplitude over a set time window was determined. Failures were identified visually, and potency was calculated as the mean EPSC amplitude excluding failures. For analysis of EPSC kinetics, rise time was estimated by the time-constant of a single exponential fit of the rising phase of the mean EPSC waveform. For decay, the time-constant of the single exponential fit to the decay phase was used. For display of individual EPSC traces in the figures, the stimulus artefact was digitally subtracted using an average of identified failures. All histograms are represented as smoothed line plots (SigmaPlot ver 5.0). Data are expressed as % of baseline (i.e., 100 % = no change). Statistical significance was assessed using the Student's t -test (one or two-tailed, paired or unpaired as appropriate; P < 0.05 as significant). All values are expressed as mean ± s.e.m. Authors' contributions AL performed most of the electrophysiology. MJP and MAW also contributed some recordings. PM provided some information that guided the work. TB performed the modelling that provided the theoretical frame-work. JI helped with the design and analysis of experiments. GLC coordinated the work and contributed to its design. All authors read and approved the manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC535344.xml
521494	Open Access gains attention in scholar communication	null	Open Access is one of the attempts to maximize the exchange of information, and therefore benefits the scholar communication [ 1 ]. Molecular Cancer offers Open Access to all of its content, thereby providing a platform to present information to specialists and the public in order to further promote free exchange of ideas, concepts and findings in all fields of cancer-related biomedical science. All the published articles in the journal are determined by the peer review process. Open Access has following broad benefits for science and the general public: • All articles become freely and universally accessible online; so an author's work can be read by anyone at no cost. • The authors hold copyright for their work and grant anyone the right to reproduce and disseminate the article, provided that it is correctly cited. • A copy of the full text of each Open Access article is permanently archived in an online repository separate from the journal, such as PubMed Central, the US National Library of Medicine's full-text repository of life science literature, the repositories at the University of Potsdam in Germany, at INIST in France and in e-Depot, the National Library of the Netherlands' digital archive of all electronic publications. • Authors are assured that their work is disseminated to the widest possible audience. This is accentuated by the authors being free to reproduce and distribute their work, for example by placing it on their institution's website. It has been suggested that free online articles are more highly cited because of their easier availability [ 2 ]. • The information available to researchers will not be limited by their library's budget, and the widespread availability of articles will enhance literature searching. • The results of publicly funded research will be accessible to all interested readers and not just those with access to a library with a subscription. As such, Open Access could help to increase public interest in, and support of, research. Please note that this public accessibility may become a legal requirement in the USA if the proposed Public Access to Science Act is made law [ 3 ]. • A country's economy will not influence its scientists' ability to access articles because resource-poor countries (and institutions) will be able to read the same material as wealthier ones, although creating access to the internet is another matter. Molecular Cancer published a number of interesting papers, and the list of the top ten most accessed articles is available at . All papers accepted by Molecular Cancer appear as 'accepted manuscript' on the web pages and are subsequently included in PubMed. A fully formatted portable document file is available approximately two to three weeks after acceptance along with a web-version of the article. The on-line publication, to the exclusion of print, has many advantages: Coloured pictures can be presented along with large sets of supporting data (movies, tables, pictures, et cetera) without additional charges. In addition, the on-line submission process allows a fast and effective handling of papers and allows authors to check the status of their submitted manuscript(s). There is no limitation in space, but concise papers are more likely to be read. The peer review policy, described in [ 4 ], ensures a fair evaluation of the work. We wish to thank our authors for sending their work to Molecular Cancer , all members of the editorial board and the reviewers for their ongoing support for Open Access publishing and for aiming higher standards for Molecular Cancer . The acceptance rate of Molecular Cancer did not change significantly, compared to the last report [ 4 ]. One out of three incoming articles are accepted for publication at Molecular Cance r after revisions. In addition to indexing in PubMed, PubMed Central and other search engines, Molecular Cancer is working closely with the Institute for Scientific Information to ensure that citation analysis of our articles will be available. Competing interests PJC is Editor-in-Chief and CS is Deputy Editor of this journal. Both do not receive any remuneration for their efforts but they are exempted from the article processing fee for this journal.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC521494.xml
522823	Spatial dependency of Buruli ulcer prevalence on arsenic-enriched domains in Amansie West District, Ghana: implications for arsenic mediation in Mycobacterium ulcerans infection	Background In 1998, the World Health Organization recognized Buruli ulcer (BU), a human skin disease caused by Mycobacterium ulcerans (MU), as the third most prevalent mycobacterial disease. In Ghana, there have been more than 2000 reported cases in the last ten years; outbreaks have occurred in at least 90 of its 110 administrative districts. In one of the worst affected districts, Amansie West, there are arsenic-enriched surface environments resulting from the oxidation of arsenic-bearing minerals, occurring naturally in mineral deposits. Results Proximity analysis, carried out to determine spatial relationships between BU-affected areas and arsenic-enriched farmlands and arsenic-enriched drainage channels in the Amansie West District, showed that mean BU prevalence in settlements along arsenic-enriched drainages and within arsenic-enriched farmlands is greater than elsewhere. Furthermore, mean BU prevalence is greater along arsenic-enriched drainages than within arsenic-enriched farmlands. Conclusion The results suggest that arsenic in the environment may play a contributory role in MU infection.	Background Buruli ulcer (BU) is a skin disease, which usually begins as a painless nodule or papule and may progress to massive skin ulceration. If untreated BU may lead to extensive soft tissue destruction, with inflammation extending to deep fascia. The parts of the body most affected are the extremities. Subsequent complications may include contractural deformities. The main form of treatment is wide excisional surgery, including amputation of limbs, which requires prolonged hospitalization and is thus a significant burden on hospital resources and budgets. In recent years, there has been increased incidence of BU in West Africa (including Benin, Burkina Faso, Cote d'Ivoire, Ghana, Guinea, Liberia and Togo), Mexico, French Guyana, Papua New Guinea and Australia. The disease seems to affect mostly impoverished inhabitants in remote and rural areas; children are the most vulnerable, accounting for about 70% of the cases [ 1 ]. The World Health Organization (WHO) has recognized BU as the third most prevalent mycobacterial disease after tuberculosis and leprosy and has called for urgent action to control it [ 2 ]. The causative agent of BU is Mycobacterium ulcerans (MU), which was first described in Bainsdale, Australia, in 1948 [ 3 ]. From the medical point of view, MU is among the group of mycobacteria that are potentially pathogenic in humans and animals under special circumstances [ 4 ]. It is suggested MU enters through a small break or trauma in the skin because it is not known to penetrate through intact or healthy skin [ 5 , 6 ]. Portaels et al. [ 7 ] have suggested that insects may be involved in the transmission of the disease because insects found in the roots of trees tested positive with the mycobacterium. Marsolliers et al. [ 8 ] found through an experimental study that the bite of MU-infected waterborne insects transmitted infection to mice. In terms of human infection, however, the reservoir of MU and the mode of transmission of BU are still unclear [ 4 , 9 - 12 ]. Epidemiological data suggest that environmental factors such as climate, soil, geology, geochemistry, etc. may indirectly influence or contribute to MU infection [ 4 ]. In addition, the frequencies of some diseases caused by mycobacteria indicate that species are distributed geographically [ 13 ]. For example, MU has been observed mainly in the tropics and [ 4 ] especially in anthropogenically-polluted areas [ 14 ]. Since MU is known to be present in nature although its reservoir is not known and since the epidemiology of BU is still unclear, there is a need to have a better understanding of environmental, ecological, and behavioural factors that predispose to infection. Spatial analysis potentially contributes important information leading to the understanding of the epidemiology and etiology of BU. The main objective of this paper is to explore relationships between some spatial environmental factors and the prevalence of BU. Results For the buffers tested, P values range from 0.09 to 0.46. The buffers with the highest P values (i.e., 0.09) are 100 m for drainage channels and 400 m for farmlands. With these buffers 24 of the 61 settlements (i.e., 39%) fall within 100 m of arsenic-enriched drainage channels (Figure 4 ) and 41 of the 61 settlements (i.e., 67%) fall within 400 m of As-enriched farmlands (Figure 5 ). The mean BU prevalence within the drainage buffer is 0.7% whereas the mean BU prevalence inside the farmland buffer is 0.6%. Thus the naturally smaller number of settlements within the drainage buffer (i.e., 24) has a slightly higher BU prevalence than the relatively larger number of settlements within the farmland buffer (i.e., 41). Figure 4 Map of As-enriched farmlands. Distances to As-enriched farmlands (red; with > 15 ppm As in stream sediments) and locations of villages with BU cases. Figure 5 Map of As-enriched streams. Distances to As-enriched streams (black; with > 15 ppm As in stream sediments) and locations of villages with BU cases. Discussion Siting of rural settlements in the study area is based primarily on proximity to and availability of water for drinking and other domestic purposes. Consequently, many settlements are located within the optimum buffer distance of 100 m from drainage channels. Where water is abstracted from drainage channels enriched in arsenic, chronic ingestion of arsenic-enriched water through drinking and cooking is likely. This renders the inhabitants susceptible to several kinds of diseases [ 15 , 16 ] including BU. Amofah et al. [ 17 ] studied 90 BU patients and found that 52 used surface water as the source of their drinking water. The result of the statistical analysis corroborates this observation in that BU prevalence is highest where the inhabitants have ready access to domestic water supplies from arsenic-enriched surface drainage. Subsistence farmlands, especially those that depend partially on irrigation, tend to be located along stream floodplains. Soils in these floodplains have a high cation exchange capacity [ 18 ] so that, where streams carry high concentration of arsenic, there is accumulation of arsenic in the soils of the floodplains. These high concentrations of arsenic are in part taken up by the foodcrops grown there [ 19 - 21 ]. The results of the statistical analysis suggest that a high proportion of settlements with high BU prevalence exploit such floodplain farmlands enriched in arsenic. Through consumption of arsenic-enriched drinking water and arsenic-enriched foodcrops, inhabitants in some settlements in the Amansie West District are prone to chronic ingestion of higher-than-average (but sub-toxic) levels of arsenic. Arsenic interacts with and inhibits several enzymes in the body [ 15 ] leading to several multisystemic non-cancer effects [ 16 ], which could predispose to defect the immune system [ 22 ]. Subjects exposed to high levels of arsenic concentrations were known to have impaired immune response [ 23 ]. Immunosuppression due to arsenic has been found to defect antigen processing of splenic macrophages with consequent defective mechanism of helper T-cells [ 24 , 25 ]. Down-regulation of the immune system is known to be a risk factor for the development of BU [ 26 , 27 ]. Several studies [e.g., [ 28 - 31 ]] have reported of impaired resistance to viral/bacterial infection via arsenic ingestion. Conclusions The results of this study reveal spatial dependency of BU prevalence upon proximity to drainage channels and farmlands containing > 15 ppm arsenic. Proximity implies chronic exposure to and/or ingestion of elevated concentrations of arsenic, which influences susceptibility to infection. Methods Research hypotheses It has been consistently theorized that BU is acquired when MU enters the body through a skin rupture [ 32 , 27 ]. However, several people who were affected by the disease do not recall having any break or trauma in their skin prior to being infected [ 33 ]. A possible alternative is entry through non-ruptured but unusually unhealthy or thin skin. Several dermatological diseases (e.g., Bowen's disease, hyperkeratosis, hyperpigmentation) are related to arsenic ingestion and exposure [ 34 ]. Bioaccumulation of arsenic in the fatty tissues of the skin [ 35 ], due to its high lipid solubility [ 36 , 37 ], may provide a favourable environment for MU in the skin because arsenic is known to help microorganisms grow [ 38 ]. It can be hypothesized, therefore, that (a) arsenic induces MU adhesion to human tissues and (b) arsenic influences the ability of MU to establish BU. In a case study, Amofah et al. [ 17 ] reported that about 44% of the BU patients were farmers whilst about 54% were school children. In Ghana many children help their parents on farms. Not only do farmers and children come in contact with natural drainage areas on their journeys to and from their farmlands, but also the farms are located near water bodies or drainage systems for obvious irrigation purposes [ 39 ]. If farmlands and surface drainage channels are contributory factors to BU, farmlands and surface drainage channels enriched in arsenic may contribute to still higher prevalence of BU. Research methodology Spatial analysis of data provides opportunities for epidemiologists to study associations between environmental factors and spatial distribution of diseases [ 40 ]. A geographic information system (GIS) is capable of analyzing and integrating large quantities of geographically distributed data as well as linking geographic data to non-geographic data to generate information useful in further scientific (or medical) research and in decision-making. In this study, topographic map data, stream sediment geochemical data for arsenic, ASTER satellite imagery and locations of settlements with BU cases were the basic data inputs into the GIS. Spatial data processing was carried out (a) to delineate arsenic-enriched catchment basins based on arsenic concentrations in stream sediment samples, (b) to delineate farmlands from ASTER satellite imagery and determine arsenic-enriched farmlands based on catchment basin data and (c) to extract drainage channels from the topographic map and determine arsenic-enriched drainage channels based on arsenic-enriched catchment basins. Proximity analysis was undertaken to determine spatial relationships between BU-affected areas and the arsenic-enriched areas determined from the data inputs. The study area History of BU in Ghana The study area is in Ghana, where the first case of BU was reported in 1971 and, between 1991 and 1997, more than 2000 cases have been reported [ 41 ]. The disease has affected all of the ten regions and at least 90 of the 110 districts in Ghana [ 42 ]. The Ashanti Region is the worst affected, accounting for about 60% of all reported cases, of which the greatest percentage is in the Amansie West District (Figure 1 ). Figure 1 The study area. Amansie West District, Ghana, showing the study area (box) and villages with BU cases (black dots). Location of study area The Amansie West District lies between latitudes 6°N and 6°45'N and longitudes 1°30'W and 2°15'W. It covers an area of about 1,136 km 2 . The district capital, Manso Nkwanta, is about 40 km south of Kumasi. The district is drained by the Offin and Oda rivers. Vegetation in the district is composed mainly of secondary forests, thicket, forb regrowth (i.e., soft-stemmed leafy herbs, mostly the weeds, which appear on farms and have to be cut regularly) and swamp vegetation. Vegetation thrives in ferric fluvisols, which are the major soil types in the district. These soils have been developed through yearly rainfall ranging from 125 to 200 cm with temperatures of 22 to 30°C. The landscape of the district varies from gentle to broken. The district is underlain by Lower Proterozoic Birimian and, to a lesser extent, Tarkwaian rocks. Throughout Ghana, Birimian rocks of West Africa are mainly volcanic greenstones with intervening sedimentary rocks and granitoid intrusions, in places containing deposits composed of pyrite, arsenopyrite, minor chalcopyrite, sphalerite, galena, native gold and secondary hematite [ 43 ]. The district has about 310 settlements (though not all these settlements are mapped) with a population in 2000 of 108,726. There are approximately equal percentages of males and females (49% and 51%, respectively), of whom 70% are farmers and 22% are engaged in legal and 'galamsey' (or illegal) mining. The study area is the east-central part of the Amansie West District (covered by a single topographic map sheet, 0602C1), with an area of about 623 km 2 , comprising 61 settlements and including the Bilpraw goldmine (formerly a treasure mine of the Ashanti Kings). The BU cases per settlement range from 1–29. Materials The following are the sources of spatial data input to the GIS. • Incidence of BU per settlement in 1999, obtained from Korle-BU Teaching Hospital, Accra, Ghana. • Settlement population estimates for 2000, projected by the Ministry of local government and rural development. • Topographic map (Sheet 0602C1, 1974, at a scale of 1: 50,000), a single sheet covering the study area, obtained from the Survey Department, Accra, Ghana. • Location map (at scale of 1: 62,500) of stream sediment samples collected in part of the Amansie West District in 1992 and list of arsenic concentrations determined in these samples, obtained from the Geological Survey Department, Accra, Ghana. • Boundary map (at scale of 1: 250,000, surveyed in 1991) of the district, obtained from the Amansie West District Administration. • ASTER imagery (level 1B) acquired on 15/01/2002, obtained from the US Geological Survey. • Landuse/landcover map of Ghana (traced on Landsat TM data of 1998 and published in the same year), obtained from the Remote Sensing Application Unit (RSAU), University of Ghana, Legon. The GIS operations were carried out in three principal steps: (1) spatial data capture; (2) generation of spatial factor maps; and (c) spatial data analysis. The GIS operations were carried out using ILWIS (Integrated Land and Water Information Systems), a GIS software package developed by the International Institute for Geo-information Science and Earth Observation (ITC) in the Netherlands. Spatial data capture The different analog maps were scanned then georeferenced (by defining the x and y coordinates of the corner points of the maps) into a UTM coordinate system. From the scanned map, spatial data were captured by screen digitizing. From the topographic map, rivers, streams and gullies were digitised as line segments as were elevation contours. The boundaries of the district were digitised as line segments and then polygonized. The locations of centres of 61 settlements (identifiable on the topographic map) were digitised as points and the BU incidence in 1999 was recorded as spatial attribute of each settlement. From the stream sediment sample location map, the locations of the samples were digitised as points and the arsenic concentrations (in ppm) were recorded as a spatial attribute of each sample. The ASTER imagery was also georeferenced to the same coordinate system using eight reference points (tie points), which were selected in the image and which could be identified in the topographic map. Using an affine transformation, a root mean square error (RMSE) of 0.58 pixel was obtained in georeferencing the ASTER imagery. For each of the settlements with incidence of BU the percentage prevalence of BU was calculated. Prevalence expresses cases of a disease in terms of the proportion of the population afflicted at a specified time [ 44 ]. It is expressed here as the number of BU cases in a settlement in 1999 divided by the estimated population in 2000 multiplied by 100 to yield a percentage. Spatial factor maps The spatial factor maps generated from the stream sediment geochemistry data for use in the spatial analysis were: (a) map of arsenic-enriched catchment basins; (b) map of arsenic-enriched farmlands; (c) map of arsenic-enriched drainage channels. Arsenic-enriched catchment basins The stream sediment geochemical data for arsenic were initially analysed statistically to determine a threshold value that divides the data into background (normal) classes and anomalous (abnormally high) classes of arsenic concentrations. The data are lognormally distributed and, after removing obvious outliers in the data, a geometric mean of 8.9 ppm As and standard deviation of 2.8 ppm As were obtained. The threshold value was therefore set at 15 ppm As (i.e., approximately the mean plus two standard deviations). The spatial distribution of arsenic was then mapped through the generation of a catchment basin anomaly map in which a sample catchment basin is assigned the geochemical attribute of the corresponding sample [ 45 , 46 ]. Generation of sample catchment basins involved the following steps (using ILWIS): • creation of a raster digital elevation model (DEM) through interpolation of elevation contours; • generation of raster map of drainage lines; and • calculation of sample catchment basin boundaries via an iterative calculation procedure involving the DEM and the raster map of drainage lines. The catchment basin map of arsenic concentrations was then classified into a binary map showing arsenic-normal areas (with ≤ 15 ppm As) and arsenic-enriched areas (with > 15 ppm As) as shown in Figure 2 . About 24% of the study area is occupied by arsenic-enriched catchment basins. Figure 2 Binary map of the catchment basin. Binary map (of the catchment basin) showing arsenic-normal and arsenic-enriched areas. Arsenic-enriched farmlands A supervised classification of ASTER imagery was carried out to distinguish between the major landcover/landuse classes known in the area. These landcover classes are (a) forest areas, (b) residential areas or settlements (bare of vegetation), and (c) farmlands. Using the available landuse/landcover map and topographic map as references, training pixels of known landuse/landcover classes were selected using a colour composite of ASTER bands 2, 3 and 4. These three bands gave the highest optimal index factor (OIF), which indicates the combination of three spectral bands that provide optimum information about landcover [ 47 ]. The box classifier [ 48 ] was chosen for the image classification. The classified image (Figure 3 ), which was also validated in the field, has an overall accuracy of at least 91% with reference to the landcover/landuse map. The classified image indicates that about 91% of the area is farmland. Figure 3 Landcover/landuse map. Landcover/landuse map based on supervised classification of ASTER data. To determine arsenic-enriched farmlands, a Boolean AND operation was performed by crossing the catchment basin anomaly map and the classified landcover/landuse image. About 21% of the total area of farmlands in the classified image is arsenic-enriched. Arsenic-enriched portions of the drainage systems A Boolean AND operation was performed by crossing the catchment basin anomaly map and the raster map of drainage lines. About 22% of the total length of drainage lines is indicated to be arsenic-enriched. Spatial data analysis The inhabitants of a settlement earn their livelihoods by exploiting the resources of the surrounding land. This land influences their exposure to infections and to environmental factors that dispose to infections. Proximity analysis was therefore used to determine spatial relationships between BU prevalence per settlement and (i) arsenic-enriched farmlands and (ii) arsenic-enriched portions of the drainage system. The proximity analysis was carried in two principal steps. First, maps of distances from arsenic-enriched farmlands and arsenic-enriched portions of the drainage system were generated. Second, the point map of BU prevalence per settlement was overlaid on (or crossed with) each of these maps. A buffer is a zone of specified distance around a selected map feature. A GIS creates buffer zones around selected map features such as arsenic-enriched farmlands and arsenic-enriched portions of drainage systems. Around each of these, buffers were set at intervals of 100 m up to 1000 m. Each buffer zone map was crossed with BU prevalence data of settlement to determine how many of these fall within and outside of the buffer. At each increasing interval of 100 m, a test of the significance of the difference of the mean BU prevalence within the buffer and outside of the buffer is made using the t-statistic: where , are the sample means, is the pooled sample variance, n i and n j are the sample sizes from population i and j . Using t ij and degrees of freedom given by n i + n j -2, a t distribution look-up table provides the probability, P that the means are significantly different. Authors' contributions AAD carried out the research and drafted the manuscript. EJMC guided parts of the research and both EJMC and MH reviewed the manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC522823.xml
509318	A Red-Blooded Transcription Factor	null	Every multicellular organism depends on the coordinated actions of a multitude of cell types, each designed to carry out specific jobs. In vertebrates, for example, the task of ferrying oxygen to organs, tissues, and cells throughout the body is shouldered by red blood cells. These cells must develop early in the embryo to nourish the body and must be maintained at the right levels throughout adulthood to keep the organism healthy. Specialized red blood cells arise from undifferentiated stem cells in a developmental process called hematopoiesis. All vertebrates have the same basic plan for hematopoiesis during their development, with one group of stem cells producing embryonic blood cells and, later, another group in a different part of the body making adult blood cells. Such developmental programs are tightly orchestrated by suites of transcription factors—proteins that turn genes on and off. But which transcription factors act—and how and where they act—is still largely unknown. Scientists and doctors alike want to better understand differentiation in order to grasp not only how it proceeds normally but also how it can go wrong and lead to diseases, such as leukemia, in which white blood cells become cancerous, and aplastic anemia, in which bone marrow stem cells make too few red blood cells. To discover the genes involved in hematopoiesis, Leonard Zon's group at Children's Hospital, Boston, and their collaborators exposed zebrafish to mutagens and then studied the individuals that developed relevant disorders. In 1996, the group produced three zebrafish lines with embryos lacking fully differentiated red blood cells; since these fish also had especially shimmery tails, the lines were named moonshine. Now Zon and his colleagues have identified the mutant gene responsible for the moonshine zebrafish lines' peculiar traits. The moonshine (mon) gene, it turns out, encodes a transcription factor with wide effects on the embryo's mesoderm, the set of cells that eventually form the circulatory system, muscles, and skeleton. The researchers found that all three lines of mutant zebrafish had mutations in the mon gene. In the mutant fish, stem cells that produce red blood cells were present initially, but the defective moonshine protein was unable to keep the blood cells alive. These blood cells underwent apoptosis, or programmed cell death, leaving the fish unable to make red blood cells, and they died at about two weeks old. Hemoglobin staining shows that hematopoiesis is defective in a moonshine mutant (bottom) compared to a wild-type zebrafish embryo (top) The researchers found that the protein encoded by the mon gene is most similar to the human and mouse Tif1-gamma, one of a family of proteins known to link DNAbinding proteins with other factors that activate or repress gene activity. Using a DNA analysis technique in mouse cell cultures, the researchers found Tif1-gamma in nuclear bodies, multi-protein complexes in cell nuclei that help regulate gene expression. But the Tif1-gamma nuclear bodies didn't fit into any known class of such complexes, so it's an open question how Tif1-gamma acts and what other proteins help regulate it. Finding a transcription factor such as Tif1-gamma involved in early cell specialization opens a door to a suite of studies on the targets of the transcription factor, as well as other genes that act along with mon to affect red blood cell development.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC509318.xml
543577	Thermodynamics of weight loss diets	Background It is commonly held that "a calorie is a calorie", i.e. that diets of equal caloric content will result in identical weight change independent of macronutrient composition, and appeal is frequently made to the laws of thermodynamics. We have previously shown that thermodynamics does not support such a view and that diets of different macronutrient content may be expected to induce different changes in body mass. Low carbohydrate diets in particular have claimed a "metabolic advantage" meaning more weight loss than in isocaloric diets of higher carbohydrate content. In this review, for pedagogic clarity, we reframe the theoretical discussion to directly link thermodynamic inefficiency to weight change. The problem in outline: Is metabolic advantage theoretically possible? If so, what biochemical mechanisms might plausibly explain it? Finally, what experimental evidence exists to determine whether it does or does not occur? Results Reduced thermodynamic efficiency will result in increased weight loss. The laws of thermodynamics are silent on the existence of variable thermodynamic efficiency in metabolic processes. Therefore such variability is permitted and can be related to differences in weight lost. The existence of variable efficiency and metabolic advantage is therefore an empiric question rather than a theoretical one, confirmed by many experimental isocaloric studies, pending a properly performed meta-analysis. Mechanisms are as yet unknown, but plausible mechanisms at the metabolic level are proposed. Conclusions Variable thermodynamic efficiency due to dietary manipulation is permitted by physical laws, is supported by much experimental data, and may be reasonably explained by plausible mechanisms.	Background Carbohydrate restriction as a general strategy for weight loss continues to gain in popularity and its utility and generally protective effect in lipid profile and glycemic control continues to be demonstrated, at least in an experimental setting [ 1 - 4 ]. The subject nonetheless remains controversial. Those critics who grant efficacy of low carbohydrate diets nonetheless contend that they act strictly by caloric restriction and there is no special effect of carbohydrate reduction. Beyond caloric restriction, several studies have shown increased weight loss on low carbohydrate diets compared to isocaloric low fat diets, the so-called metabolic advantage (see table 2 ). Although no clear experimental error has been demonstrated, critics continue to maintain that something must be wrong because the laws of thermodynamics would be violated [ 5 ], that "a calorie is a calorie" [ 6 ] We have previously shown [ 2 , 7 ] that this is not correct and it is our intention here to review the fundamental physics underlying the phenomenon of metabolic advantage. An outline may be described: Can metabolic advantage happen? If so, what mechanisms might account for such a phenomenon? Does it, in fact, occur? Table 2 Isocaloric low carbohydrate (CHO) vs. higher carbohydrate investigations Reference %CHO %CHO Wt. loss(kg) ± SEM p Low High Low CHO arm (no. subjects) High CHO arm Rabast et al (1978) [31] 10 68 14.0 ± 1.4 (25) 9.8 ± 1.0 (20) 0.10 Rabast et al (1981) [32] 12 70 12.5 ± 0.9 (7) 9.5 ± 0.7 (7) <0.01 Golay, Allaz et al (1996) [33] 15 45 8.9 ± 0.6 (22) 7.5 ± 0.5 (21) 0.1 Golay, Eigenheer et al (1996) [34] 25 45 10.2 ± 0.7 (31) 8.6 ± 0.8 (37) 0.13 Piatti et al (1994) [35] 35 60 4.5 ± 0.4 (10) 6.4 ± 0.9 (15) 0.3 Layman et al (2003) [36] 44 59 7.5 ± 1.4 (12) 7.0 ± 1.4 (12) 0.8 Baba et al (1999) [38] 25 68 8.3 ± 0.7 (7) 6.0 ± 0.6 (6) <0.05 Lean et al (1997) [37] 35 58 6.8 ± 0.8 (40) 5.6 ± 0.8 (42) 0.1 Young et al (1971) [39] 7 23 16.2 ± 0.9 (3) 11.9 ± 0.8 (3) <0.05 Greene et al (2003) [40] 5 55 10.4 ± 2.1 (21) 7.7 ± 1.1 (21) 0.25 Metabolic advantage: can it happen? We have previously presented arguments that there is no violation of physical principles [ 2 , 7 ] and, ironically, that suggesting a change in body mass to be independent of macronutrient composition would itself be a violation of the second law of thermodynamics [ 7 ]. Here, we reframe these arguments in a more pedagogically direct way and we provide simple examples. The misunderstanding that continues to be repeated in the expression "a calorie is a calorie" appears to be exclusive reference to the first law of thermodynamics. The difficulty with this theoretical approach is that it is only part of the relevant physics and its relationship to biologic systems. The first law says that in any transformation the total energy in the system can be accounted for by the heat added to the system, the work done by the system on its environment and the change in energy content of all the components of the system. It is important to understand, however, that the first law does not say what the relative distribution between these effects will be for any process. In fact, the first law does not even allow us to say whether the process will occur at all. To understand the progress of a physical change it is necessary to understand the second law which introduces an entity known as the entropy, S, a measure of disorder in all processes. In all real (irreversible) processes, entropy increases, usually written ΔS > 0. The most common marker of increasing entropy is heat, although it is by no means the only evidence for increased entropy. In systems at constant temperature and pressure (i.e. biologic systems)), the first and second law are combined in the Gibbs Free Energy, ΔG, which represents the maximum useful work that can be performed by the process. The actual process however, in general derives less useful work than permitted by the theoretically available ΔG due to inefficiency in energy capture. A proper accounting of entropy and efficiency must be included if we are to understand energy utilization in biological and biochemical systems. Biological systems and thermodynamics It is also important in the discussion of biological systems to understand that they are open systems, i.e. they take in nutrients and oxygen and excrete carbon dioxide, water, urea and other waste products, as well as heat. The importance with respect to weight considerations is that mass and energy are conserved (the more general statement of the first law of thermodynamics), but they are not conserved entirely within the organism. To illustrate the proper interpretation of the first law of thermodynamics consider a subject whose resting energy expenditure is met by the production of 95 moles of ATP. Since oxidation of a single mole of glucose provides 38 moles of ATP, 2.5 moles of glucose will be needed to meet this individual's resting energy requirements. It is important to note that the resultant carbon dioxide, water, and heat are not retained within the organism. The useful retained energy is in the 95 moles of ATP (Figure 1B ). (Similar equations could be written for lipid or protein but we restrict our discussion to glucose for simplicity). Figure 1 A: Oxidation of glucose in a calorimeter is completely inefficient. The products of oxidation are carbon dioxide and water, and all of the energy produced is released as heat. 1B: To illustrate the proper interpretation of the first law of thermodynamics in living organisms we must consider that conservation of matter and energy includes excretion of products into the external environment. None of the products of oxidation (CO 2 and H 2 O) remain within the organism. There is stoichiometric balance and no net weight change. Only the ATP, representing the useful energy, is retained. The wasted heat constitutes 60% of the energy of oxidation, while the efficiency is reflected in the retained ATP, available for reactions in the organism. Body fat stores are signified as TAG (triacylglycerol) 1C . A common way of thinking of weight loss is from reduction of caloric intake. If our subject ingests 2.3 moles of glucose (or equivalent lipid and/or protein) and produces only 90 moles of ATP, then homeostasis will enlist body stores of fat (and/or lean body mass) to yield the additionally required 5 moles ATP. The additional resultant CO 2 and H 2 O (and heat) will be excreted (and radiated) leading to weight loss. 1D : If efficiency is reduced then our subject would have to eat more (e.g. 2.9 moles of glucose, or equivalent lipid/protein) to produce 95 moles of ATP and remain at the same weight. The additional CO 2 and H 2 O produced will be excreted maintaining constant weight. 1E : Under conditions of reduced metabolic efficiency (from 40% to about 38% in this example), 90 moles of ATP will be produced from oxidation of 2.5 moles glucose (or equivalent lipid/protein). The remaining 5 moles ATP needed for homeostasis must be made up from oxidation of body stores of lipid or lean mass. This results in weight loss, exactly as it does for the example of reduced caloric intake (Figure 1C). The illustration above can be compared to the oxidation of glucose in a calorimeter in which no useful energy is obtained and the total energy of oxidation is measured as the heat produced. This process is completely inefficient. A traditional (Atwater) value for glucose obtained in the calorimeter is approximately 4 kilocalories of energy per gram (Figure 1A ). By contrast, the living organism above metabolizes and oxidizes glucose so that approximately forty percent of the energy of oxidation is retained as useful ATP (38 moles per mole of glucose)) whereas sixty percent is released as heat, the inefficiency in this mode of oxidation. The entropy (i.e. the second law of thermodynamics) shows up in this inefficiency . The calorimeter heat can no longer be interpreted in a simple way. The energy stored in useful ATP represents the efficiency of 40% (neglecting the difference in entropy between the structures of the products and reactants). This value approximates the efficiency for oxidation of carbohydrate as well as lipid, whereas proteins are generally oxidized at a lower value of approximately 30–35% (Figure 1B ). Summary of thermodynamics in living organism 1. The second law of thermodynamics dictates that there is an inevitable metabolic inefficiency in all biological and biochemical processes with heat and high entropy molecules (carbon dioxide, water, urea) as the most common products. 2. The first law of thermodynamics is satisfied in living (open) systems by properly accounting for the mass excreted and the heat radiated and exported in high entropy molecules. Weight loss due to reduced caloric intake The most common example of weight loss is reduction of caloric intake. At the risk of oversimplification, if our subject ingests fewer than 2.5 moles of glucose and produces, for example, only 90 moles of ATP from food, then homeostasis would require enlisting endogenous body stores for further oxidation. This oxidation would then provide the additional 5 moles of ATP required. Oxidation of body stores (lipid or lean body mass) will result in production of additional carbon dioxide, urea, water and heat. The excretion of these products will result in weight loss. (Figure 1C ). Weight loss due to increased metabolic inefficiency The implication of the first and second laws of thermodynamics is that reduced efficiency has precisely the same result as reduced caloric intake. One conceptually simple means of reducing efficiency involves the process of uncoupling in mitochondria. ATP is produced in a variety of cellular locations. Glycolysis produces a net of two ATP's per molecule of glucose, in the cell cytoplasm. On the other hand, we recall that 36 additional molecules of ATP are produced from glucose as a result of the mitochondrial TCA cycle and electron transport. A critical part of the process involves the development of a hydrogen ion gradient across the mitochondrial membrane. This concentration gradient provides the energy that is converted into ATP as hydrogen ions pass down the gradient through the ATP synthase particle, entirely analogous to the energy in a high-pressure gas in a cylinder with a movable piston. (The expansion of the gas is like diffusion down a gradient: It does work against the piston). In the mitochondrion the energy of moving down the gradient is captured in ATP, the medium of exchange for the performance of work within cells. This capture of energy, referred to as coupling the energy to the formation of ATP, is the essential process permitting work to be done by living systems. There are known endogenous and pharmacologic agents, which result in uncoupling the formation of ATP from the dissipation of the gradient. Uncouplers such as 2, 4-dinitrophenol bypass ATP synthase and cause hydrogen ion gradient dissipation without ATP formation that can result in organ dysfunction causing death. More modest degrees of uncoupling may be caused by the class of endogenous compounds we know as uncoupling proteins (UCP's). Three different isoforms, UCP1, UCP2 and UCP3 have been identified thus far in mammalian tissues. While the overall and relative physiologic importance of these proteins remains incompletely understood in human tissues, UCP1 has been shown in mice [ 8 ] to result in modest degrees of uncoupling in brown fat. Elevation of fatty acid concentration has been associated with induction of UCP3 and even with pathologic reductions of myocardial efficiency in rat heart [ 9 ]. For purposes of illustration, then, we may consider that there may be physiologic triggers that result in oxidative uncoupling, reducing the overall efficiency of glucose metabolism. For example if efficiency is reduced from 40% to 35%, the result will be the production of only 34 moles of ATP instead of the usual 38. While this represents a mechanism better demonstrated in rats than humans, our subject would require more glucose to make 95 moles of ATP. Now 2.9 moles of glucose would be required to produce 95 moles ATP. Our subject would either eat more and stay at the same weight (Figure 1D ) or would eat 2.5 moles of glucose, the same amount as previously, but would produce less ATP. By eating only 2.5 moles of glucose our subject's metabolism would enlist oxidation of body stores to make up the additional ATP needed for homeostasis. This would result in weight loss exactly as it did for reduced caloric intake. (Figure 1D ). The essence of the second law of thermodynamics is that it guarantees inefficiency in all metabolic processes . However, variation of efficiency is not excluded. In fact, the laws of thermodynamics are silent on the existence of variable efficiency. If efficiency can vary (as in the example of oxidative uncoupling) then "a calorie is a calorie" is no longer a true statement. The role of uncoupling proteins in humans, as indicated, is as yet incompletely defined [ 10 ]. However, thermodynamic principles permit variable efficiency, and its existence must be determined empirically. Metabolic advantage: how could it happen? It is possible that metabolic efficiency may be decreased by oxidative uncoupling as described above. Polymorphisms connecting uncoupling proteins with obesity or propensity to gain weight have been identified in humans [ 11 , 12 ] although these are not firmly established and the effect of dietary intervention is unknown. Other mechanisms are better understood and are described below. Substrate cycling and protein turnover Substrate or "futile" cycles refer to the dynamic process that must accompany the thermodynamic steady state [ 13 ]. In particular, increased cycling of metabolic intermediates utilizes ATP and generates heat. The simplest examples are the numerous kinase-phosphatase pairs that regulate metabolism. In addition, although not generally considered in the category of substrate cycling, inefficiency results from the repeated breakdown and re-synthesis of proteins, lipids, and carbohydrates in cycles that use ATP for no apparent net gain. Such mechanisms, however, far from futile, allow for precision in the regulation of metabolism and constitute one of the uses of ATP. Protein turnover, in particular, provides for error correction or removal of "old" or damaged proteins. The effect of metabolic path on the energetics of oxidation is illustrated in Table 1 which summarizes the analysis from our earlier paper [ 2 ]. In this example, a mole of glucose directly oxidized to CO 2 and water generates 38 moles of ATP with an overall efficiency of about 38.5%. On the other hand, if glucose is first incorporated into glycogen, followed by hydrolysis of the glucose and subsequent oxidation, 2 moles of ATP are lost per mole in this cycle with overall efficiency reduced to 35%. Similarly an amino acid from an "average" protein, when directly oxidized to CO 2 , produces ATP with an efficiency of about 33%. If the amino acid is first incorporated into a protein and later hydrolyzed and oxidized, four ATP's per molecule are used for synthesis of the peptide bond. This reduces the efficiency to 27%. Smaller degrees of inefficiency are seen for lipid cycles (Table 1 ) but multiple cycles may have a cumulative effect. It is estimated, for example, that half of depot fatty acids in triacylglycerol have been through at least one cycle [ 14 ]. It should be apparent that variation in efficiency is not a thermodynamic issue but an empiric question to be determined by the requirements of metabolism. Table 1 Effect of Path on energetics of oxidation Macronutrient & path Mass ATP/mole Kcal/gm Efficiency (%) Glucose → CO 2 180 38 1.54 38.5 Glucose → glycogen → glucose → CO 2 180 36 1.40 35 "Average" AA → CO 2 1.32 33 AA → Protein → AA → CO 2 -4 1.08 27 Palmitate → CO 2 256 129 3.68 40.9 Palmitate → Ketone → CO 2 256 121 3.45 38.3 *Adapted from Feinman, Fine: 2003 Metabolic Syndrome and Related Disorders (1): 209–219 [2] Thyrotoxicosis Thyroid hormone decreases efficiency possibly by mechanisms involving both uncoupling and cycling described above: oxidative uncoupling as well as increased futile cycling of intermediates [ 15 ]. It is observed in thyrotoxic mice that UCP1 decreases efficiency in brown fat at the mitochondrial level [ 8 ]. In humans, the role of UCP1 in thyrotoxicosis is less certain due to the relative paucity of brown fat. On the other hand, activation of the adrenergic system via phosphoenolpyruvate carboxykinase ultimately increases "futile" metabolic cycling of intermediates ([ 15 ]). Thyrotoxicosis is well known to result in weight loss, often with increased food intake and increased generation of heat, indicative of metabolic inefficiency. The use of thyroid hormone has even been suggested therapeutically to induce weight loss in obese individuals, although its toxicity has limited this application. Inefficiency in metabolic processes with weight loss and increased heat generation, therefore, is known to occur on clinical grounds. Even without a complete understanding of the relative importance of different underlying cellular mechanisms in humans, the potential for biochemical processes to reduce their efficiency must be considered established as a feature of mammalian metabolism . Protein induced protein turnover There is abundant evidence that dietary protein stimulates protein breakdown and re-synthesis. In particular, branched chain amino acids, and especially leucine, are documented to act as nutritional signals acting via both the insulin and mTOR signaling pathways [ 16 - 18 ]. On the macroscopic level, the energetic cost of protein turnover is demonstrable as excess heat generated during a high protein meal. Thermogenesis (thermogenic effect of feeding; old name: specific dynamic action) has been defined as the extra heat generated during a meal due to digestion or metabolism. Johnston et al [ 19 ] compared the energy expended during 9 hour intravenous feedings of a high protein meal, vs. an isocaloric high carbohydrate meal; both contrasted with a 9 hour fast. The protein meal, with 70% of its caloric value due to protein, had significantly greater thermogenesis than the high carbohydrate meal (70% of calories from carbohydrate). These data have been reproduced in numerous studies [ 19 - 22 ]. The overall energy costs of protein turnover and synthesis have been estimated in various animal species, including man, and tabulated by Vernon Young ([ 23 ]), based on data from other investigators [ 24 - 26 ]. Despite the substantial experimental difficulties involved, the cost of protein synthesis clusters at around 4–5 kcal/gram in 8 species of birds, marsupials and mammals, including man. The high energetic cost is understandable in view of the multiple ATP-requiring processes involved. The cost of protein turnover can reduce efficiency from 33% to 27%, merely in the formation and hydrolysis of a single peptide bond (requiring 4 ATP's per bond formed: Table 1 ). In addition, protein processes that are ATP-dependent include formation of the ribosomal initiation complex, translation and folding of the protein, and protein degradation (both ubiquitin-dependent and -independent pathways) [ 23 ]. The energy costs of protein turnover could therefore account for a metabolic advantage in high protein diets, independent of carbohydrate content. This mechanism may also contribute to inefficiency in low carbohydrate diets, often high in protein. Gluconeogenesis-stimulated protein turnover in carbohydrate restriction The following hypothesis is suggested from classic studies of starvation done in chronically fasted obese individuals [ 27 , 28 ]. The brain's metabolism requires 100 grams of glucose per day. In the early phase of starvation, glycogen stores are rapidly reduced, so the requirement for glucose, is met by gluconeogenesis. Approximately 15–20 grams are available from glycerol production due to lipolysis, but fatty acid oxidation generally cannot be used to produce glucose. Therefore, protein breakdown must supply the rest of substrate for conversion to glucose in the early phases of starvation. By 6 weeks of starvation, ketone bodies plus glycerol can replace 85% of the brain's metabolic needs, the remainder still arising from gluconeogenesis due to protein. It should be mentioned that, since the fundamental role of ketones is to spare protein, it might be expected that the reliance on protein would actually decrease with time, perhaps relating to the anecdotal observation of "hitting the wall" on weight loss diets. Very low carbohydrate diets, in their early phases, also must supply substantial glucose to the brain from gluconeogenesis. For example, the early phase of the popular Atkins or Protein Power diet restricts dieters to about 20–30 grams of carbohydrate per day, leaving 60–65 grams to be made up from protein-originated gluconeogenesis. One hundred grams of an "average" protein can supply about 57 grams of glucose so 110 grams protein would be needed to provide 60–65 grams glucose. Increased gluconeogenesis has been directly confirmed using tracer studies on day 11 of a very low carbohydrate diet (approx 8 grams/day) [ 29 ]. If indeed, 110 grams of endogenous protein is broken down for gluconeogenesis and re-synthesized, the energy cost, at 4–5 kcal/gram could amount to as much as 400–600 kcal/day. This is a sizable metabolic advantage. Of course, the source of protein for gluconeogenesis may be dietary rather than endogenous. Whereas endogenous protein breakdown is likely to evoke energetically costly re-synthesis in an organism in homeostasis, dietary protein may conserve energy. The source of protein for the observed gluconeogenesis [ 29 ] remains an open question, but there is no a priori reason to exclude endogenous rather than dietary sources. This is therefore a hypothesis that would need to be tested. The extent to which the protein for gluconeogenesis is supplied by endogenous protein would explain very high-energy costs. It should be noted, however, that even if limited to breakdown of dietary protein sources, there would be some energy cost associated with gluconeogenesis. Metabolic advantage: does it happen? Having established that there is no theoretical barrier to metabolic advantage and that there are plausible mechanisms that could account for such an effect, we must ask whether it can be demonstrated experimentally, that is, whether the proposed effects are of sufficient magnitude to be a practical feature of weight reduction strategies, in particular very low carbohydrate diets. If so there will be increased weight loss for the same caloric intake, or metabolic advantage. A recent animal model provides support for greater metabolic inefficiency in rats fed carbohydrate restricted diets compared with higher carbohydrate, leading to excess weight loss [ 30 ]. Human data in Table 2 illustrates 10 clinical trials of isocaloric diets with a lower versus higher carbohydrate arm in each trial [ 31 - 40 ]. It can be seen that the lower carbohydrate arm in 9 of 10 studies demonstrates increased weight reduction in comparison with the higher carbohydrate arm. Three of the studies show statistical significance (p < 0.05 or better). Even without statistical significance of individual studies, however, the likelihood that the lower carbohydrate arm would have an advantage in 9 of 10 studies is equivalent to the likelihood of 9 coin toss experiments having excess heads in comparison to excess tails. The 9 th binomial coefficient shows this probability to be p < 0.01. While the above suggests the possibility of metabolic advantage, it does not prove it, nor do we know the magnitude of the effect, or the factors that control it. The studies above were chosen from among those quoted by many of the authors who have disputed the existence of metabolic advantage. Nonetheless, a formal meta-analysis would be necessary to avoid the possibility of conscious or unconscious bias in their selection. Further, it would be necessary to establish evidence that energetically costly metabolic processes are more prevalent in low carbohydrate diets than in diets of higher carbohydrate content. Whereas the proposed mechanisms are plausible, they need to be proven. Conclusions Thermodynamics is not the limiting factor behind the concept of metabolic advantage. On the contrary, thermodynamics guarantees inefficiency in all metabolic processes and is silent on the possibility that inefficiency may be augmented in some instances. A familiar example of inefficiency is thyrotoxicosis, with attendant weight loss and heat generation despite unchanged or increased caloric consumption. The theoretical possibility of inefficiency and metabolic advantage due to macronutrient compositional change exists, but demonstration of the phenomenon can only be resolved experimentally. Isocaloric dietary studies with a low vs. a higher carbohydrate arm support the experimental possibility of metabolic advantage. A formal meta-analysis would be required to evaluate this more objectively. Further studies, including tracer methods, would be required to establish mechanisms. The presence of high quantities of dietary protein (often a feature of low carbohydrate diets) is known to stimulate protein turnover, an energetically costly process. However, it is unclear whether this is the only factor, or whether it is necessary for metabolic advantage to occur. In particular, obligate gluconeogenesis from endogenous sources may also contribute to induction of protein turnover. Competing interests The author(s) declare that they have no competing interests.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC543577.xml
535350	Distinct gene expression profiles in different B-cell compartments in human peripheral lymphoid organs	Background There are three major B-cell compartments in peripheral lymphoid organs: the germinal center (GC), the mantle zone (MNZ) and the marginal zone (MGZ). Unique sets of B-cells reside in these compartments, and they have specific functional roles in humoral immune response. MNZ B cells are naïve cells in a quiescent state and may participate in GC reactions upon proper stimulation. The adult splenic MGZ contains mostly memory B cells and is also known to provide a rapid response to particulate antigens. The GC B-cells proliferate rapidly and undergo selection and affinity maturation. The B-cell maturational process is accompanied by changes in the expression of cell-surface and intracellular proteins and requires signals from the specialized microenvironments. Results We performed laser microdissection of the three compartments for gene expression profiling by cDNA microarray. The transcriptional program of the GC was dominated by upregulation of genes associated with proliferation and DNA repair or recombination. The MNZ and MGZ showed increased expression of genes promoting cellular quiescence. The three compartments also revealed distinct repertoires of apoptosis-associated genes, chemokines and chemokine receptors. The MNZ and GC showed upregulation of CCL20 and CCL18 respectively. The MGZ was characterized by high expression of many chemokines genes e.g. CXCL12 , CCL3 , CCL14 and IFN-associated genes, consistent with its role in rapid response to infections. A stromal signature was identified including genes associated with macrophages or with synthesis of extracellular matrix and genes that influenced lymphocyte migration and survival. Differentially expressed genes that did not belong to the above categories include the well characterized BCL6 and CD10 and many others whose function is not known. Conclusions Transcriptional profiling of B-cell compartments has identified groups of genes involved in critical molecular and cellular events that affect proliferation, survival migration, and differentiation of the cells. The gene expression study of normal B-cell compartments may additionally contribute to our understanding of the molecular abnormalities of the corresponding lymphoid tumors.	Background Appropriate T- and B-cell migration and timely interaction with antigen presenting cells (APC) are essential for the development of humoral immune responses [ 1 , 2 ]. Specialized compartments within lymphoid tissues facilitate these interactions [ 3 ]. Distinct populations of B-cells reside in these microenvironments, and, upon antigen stimulation, cells with appropriate antigen receptors differentiate and migrate among these compartments for a proper immunological reaction [ 4 - 7 ]. The initiation of a T-dependent B-cell response results from cognate interaction between a T-helper cell and a B-cell that primes the B-cell into two developmental pathways. An extrafollicular reaction takes place in the T zone and leads to the production of plasma cells with unmutated immunoglobulin (Ig) genes. The other pathway initiates a germinal center (GC) reaction, whereby activated B lymphocytes originating from extrafollicular foci enter the GC and undergo a stringent process of positive selection and affinity maturation. The selected cells differentiate into either memory B-cells or plasma cells with mutated Ig genes. The GC provides the important microenvironment for this crucial B-cell maturational process [ 8 , 9 ]. In follicles with developing GCs, the resting B-cells that are not the part of the GC response are pushed outward to form the mantle zone (MNZ) or corona around the GC B-cells. The mantle cell is a pre-GC, immunologically naïve B-cell that is also the putative cell of origin of mantle cell lymphoma [ 10 ]. These B-cells express unmutated immunoglobulin genes, sIgD high , CD27 - [ 11 ] and are mostly restricted to the follicular mantle zone [ 12 ]. In the human spleen, there is a well defined zone between the follicular B-cells and the red pulp called the marginal zone (MGZ) containing marginal-zone macrophages, granulocytes and dendritic cells that are specialized to capture blood-borne antigens and present them to the resident marginal zone B-cells [ 13 ]. Unlike primary lymphoid follicles in spleen and lymph nodes, which contain mostly mature recirculating B-cells, non-recirculating B-cells are enriched in the splenic MGZ. These cells are specially adapted to respond rapidly to T-independent (TI) antigens and have a lower threshold than recirculating or immature B-cells for activation, proliferation and differentiation into antibody-secreting cells [ 7 ]. They may therefore provide the early rapid humoral response prior to the more refined but delayed response from the GC reaction. Most adult human MGZ B-cells have the IgM high , IgD low and CD27 + phenotype, suggesting that this zone contains mainly memory B-cells [ 14 ]. Many previous studies [ 15 , 16 ] have provided important information concerning the biology of the GC. While morphology and immunophenotype are useful in defining the various B-cell compartments of peripheral lymphoid organs, the molecular signals that affect the life span, survival, retention, migration and functions of the cells in these compartments have not been widely investigated. We used the Lymphochip cDNA microarray [ 17 ] to investigate the differences in gene expression profiles in the three different B-cell compartments, the MNZ with mostly naïve B-cells, the MGZ containing memory B-cells [ 18 ] and specialized non-recirculating B-cells and the GC with a mixture of highly proliferative centroblasts and more differentiated and non-dividing centrocytes. For this study, we used both tissue compartments isolated by laser capture microdissection (LCM) and naïve and memory B-cells isolated by fluorescence-activated cell sorting (FACS). The microdissected samples contained the dominant B-cell population in each compartment as well as other cell populations in the physiological microenvironment, whereas the FACS-sorted cells contained more uniform B-cell subsets. By comparing FACS-sorted cells with the corresponding compartment from LCM, we have identified a stromal cell gene expression signature that may provide insight into stromal/B-cell interaction. Results and discussion Isolation of naïve and memory B-cells and different anatomic B-cell compartments GC and MNZ could be readily dissected from tonsillar frozen sections, but MGZ could only be reliably obtained from the spleen (Figure 1 ). Immunostaining was not applied on the sections to be microdissected because it was difficult to obtain cells from sections on charged slides and because immunostaining also led to a marked loss of amplifiable RNA from the sections, even when a rapid procedure was used [ 19 ]. Hence, immunostaining was performed on a consecutive section to guide the dissection. Immunostaining by us and others has shown that the MNZ contained over 90% B-cells, which are the IgD + CD27 -, similar to FACS-sorted naïve B-cells. The GC was easily recognizable and generally contained a higher percentage of non B-cells, including T-cells, macrophages and follicular dendritic cells (FDC). The MGZ was obtained from a spleen with a morphologically clearly defined MGZ containing mostly IgM + CD27 + B-cells, corresponding to the phenotype of FACS-sorted memory B-cells [ 14 ]. The MGZ also contained scattered T-cells and has been shown by others to contain specialized macrophages and fibroblasts [ 20 , 21 ]. The FACS-sorted populations were over 90% pure, according to post-sort immunophenotyping (Figure 2 ). Figure 1 Three different B-cell compartments isolated by LCM. Frozen sections of reactive tonsils or spleens were fixed, and a consecutive section was immunostained for CD3 to guide the dissection. The three B-cell compartments (GC, MNZ and MGZ) were isolated using LCM with the Arcturus PixCell II system (Arcturus Engineering, Mountain View, CA). Cells were captured at the 15-μm with laser set to pulse at 60 mW for 200 ms. The GC and MNZ were clearly recognizable, and the MGZ was obtained from a spleen with a morphologically clearly defined MGZ. Only well-defined GC, MNZ and MGZ were dissected to avoid contamination. Figure 2 FACS-sorting of naïve and memory B-cells from splenocytes. A single B-cell suspension prepared from a fresh spleen was isolated using the Human B-cell Isolation Kit ( See methods ) and subjected to 3-color cell sorting. Memory B-cells from the splenic B-cells were gated on the IgM high IgD low CD27 + fraction, while naïve B-cells were gated at IgM low IgD high CD27 - . FACS-sorted populations were over 90 % pure, judging from post sort immunophenotyping. Gene expression profiling analytical approach Fifteen data sets corresponding to the five sample groups were generated. Different hybridizations were correlated through a correlation matrix plot, and replicated hybridizations were shown to be closely related (R ≥ 0.85). The plots allowed us to check reproducibility of the microarray assay among different samples of each tissue (Figure 3 ). The number of genes showing differential expression between two compartments and the magnitude of difference calculated by t-statistics were further filtered by Significance Analysis of Microarrays (SAM) approach, as described previously [ 22 ]. On the Lymphochip, over 20% of the genes are represented by multiple clones, and, generally, several clones of same genes are selected by our analytical algorithm. The differentially expressed genes among the three compartments identified by SAM were grouped according to their major functional attributes and then viewed through Tree View. Figure 3 Correlation Coefficient Mapping. Reproducibility of the different hybridization experiments was checked through correlation coefficient mapping programmed in MATLAB. High correlation is seen among samples from same compartment or FACS-sorted population. Confirmation of the microarray analysis with semi-quantitative RT-PCR and with real time quantitative PCR The differential expression of some of the transcripts that had no previously reported association with any of the compartments was further validated by a semi-quantitative RT-PCR. No discrepancies were found with any of the selected genes. By PCR analysis, some of the transcripts had almost exclusive expression in one compartment: ARK2 in GC, CCL2 0 in MNZ and CMRF-35H in MGZ. Other transcripts were expressed in all compartments with a relatively high differential expression in one, such as SET and FAIM in GC, Cyclin G2 in MNZ, and NM23-H1 and CARD11 in MGZ (Figure 4 ). Figure 4 Confirmation of the Microarray analysis by semi quantitative RT-PCR. aRNA amplified from GC, MNZ and MGZ -cells was reversely transcribed and amplified by PCR. The human HPRT gene was used as the comparative standard. Five fold serially dilutions (4 dilutions) of each cDNA amplified with gene specific primers and analyzed by electrophoresis in 2% agarose gel. The transcripts had either an almost exclusive expression in one compartment ( ARK2 in GC, CCL20 in MNZ and CMRF-35H in MGZ), or they were expressed in all compartments with a significant differential expression in one – for example, SET and FAIM in GC, Cyclin G2 in MNZ and NM23-H1 , CARD11 and GAS2 in MGZ. Some of the results of the semi-quantitative RT-PCR were further validated by the SYBR ® Green real-time quantitative PCR (data not shown). The results corresponded well with both microarray and semi-quantitative RT-PCR. Gene expression characteristics in anatomic B-cell compartments Genes controlling cell proliferation and quiescence (Figure 5 , 6 ) Comparing the gene expression profiles of LCM GC and FACS-sorted GC B-cells [ 17 ] revealed that the GC B-cell signature was largely represented in the microdissected GC profile. The GC gene expression profile was dominated by the increased expression of genes associated with proliferation (e.g., CCNB1 , PCNA , Ki67 ), kinetochore association (e.g., CENPF , BUB1 and BUB3 ), functional components of mitotic checkpoints (e.g., CENPE and TTK ) and regulators of cell-cycle related events, including centrosome separation/segregation and cytokinesis (e.g. KNSL5 , ARK2 ), as expected from the known high proliferation rate of centroblasts. GC also highly expressed genes involved in DNA repair (e.g., RAD54 , BRCA2 , RAD51 , ERCC5 and MSH2 ), as expected from the frequent physiological double-strand DNA breaks associated with somatic hypermutation and isotype switching. The GC profile also showed increased levels of transcripts involved in DNA replication (e.g., DNAJ , DNA2L , DNMT1 , TOP , RFC4 and RPA1 ) and in transcription and translation (e.g., EIF2 , TAF , TCF , UHRF1 , UBD and UBE2 ). The low expression of the cyclins CCNA , CCNB1 and CCNF and of CDC2 (also known as CDK1 ) is consistent with the resting state of the MNZ and MGZ B-cells. Characteristically CCNA expression is very low in G o phase and begins to increase in early G 1 . For the cell to enter the G2/M phase, an association with CDC2 is required [ 23 ]. The transition requires CCNB1 to form a complex with CDC2 and relocate to the nucleus. This nuclear localization is mediated by CCNF [ 24 ]. However, MNZ and MGZ B-cells may also employ different mechanisms in maintaining quiescence. Cyclin G2 ( CCNG2 ) was highly expressed in MNZ cells as compared to either GC or MGZ. The function of CCNG2 differs from the conventional cyclins in negatively regulating the cell cycle [ 25 ]. Studies on HeLa cells have showed that DNA damage induces the production of cyclin G2, which then arrests the cell cycle at the G1/S boundary, and this function is independent of p53. Cyclin G2 can directly interact with the catalytic subunit of protein phosphatase 2A (PP2A) and prevent cell cycle progression. The low expression of CARD11 in MNZ may also be part of the program in maintaining the quiescent state. CARD11 has been shown to be critical for immune receptor signaling of both T and B-cells through the activation of JNK and NF-κB [ 26 ]. The increased transcriptional level of CD72 may be involved in maintaining the quiescent state in MNZ B-cells. CD72 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain and functions as a negative regulator of B-cell signaling [ 27 ]. Interestingly, many genes associated with proliferation were expressed at even lower levels in MGZ than in MNZ cells. These cells highly expressed growth inhibitory genes such as CMRF-35H [ 28 ], CBL-B [ 29 ], and GAS2 [ 30 ], which may contribute to the quiescent state in MGZ B-cells. Figure 5 Cell proliferation and quiescence. Differential gene expression among B-cell compartments. Genes identified in SAM analyses were merged according to functional or operational categories and visualized in Tree View. Color changes within a row indicate expression levels relative to the median of the sample population. Only transcripts differing 2-fold or more in their magnitude than the median or mean of the other two compartments are shown. Apoptosis (Figure 7A ) The markedly decreased BCL2 expression in GC B-cells makes them vulnerable to undergo apoptosis unless rescued by survival signals [ 31 ]. An increase in the expression of proapoptotic genes e.g. BIK , FASL and PDCD8 ( AIF ) suggests a further increase in susceptibility to apoptosis in the GC. However, FAIM is overexpressed in the GC and may represent a protective mechanism in GC B-cells that have appropriate BCR signaling and CD40L stimulation with resultant upregulation of FAIM and increased resistance to FASL-mediated apoptosis [ 32 ]. Presumably, GC B-cells with sIg having poor antigen affinity will be ineffective in activating FAIM. In addition, a TNF receptor family member (TNFRSF17/BCMA) which promotes the B-cell survival [ 33 ] showed increased transcription in the GC. Programmed Cell Death 4 (PDCD4), which functions mainly as an inhibitor of translation by inhibiting the activity of eIF4A helicase, which helps to unwind the 5' end of mRNAs, was markedly repressed in the GC B-cells [ 34 ]. This suggests that PDCD4 repression facilitates the rapid proliferation of centroblasts, which requires a high rate of protein synthesis. Both MNZ and MGZ had higher expression of BCL2 , but have different profiles of other apoptosis/survival genes that may represent specific adaptation of these cells to their unique physiological states and microenvironment. The expression of BNIP3 , encoding a proapoptotic protein of the BCL2 family[ 35 ], is markedly down regulated in MGZ cells, perhaps providing additional protection against apoptosis in memory B-cells. On the other hand, TCL1 was upregulated in MNZ only and may have an antiapoptotic role in that population. There was increased expression of Suppressor of Death Domains (SODD) in MGZ cells, suggesting a complex regulation of signaling through the TNFR superfamily. SODD is associated with TNFR1 in vivo, maintaining the receptor in an inactive monomeric state. The release of SODD from TNFR1 permits the recruitment of proteins such as TRADD and TRAF2 to the activated TNFR1 signaling complex [ 36 ]. It has been demonstrated that TNF-induced activation of NF-κB is accelerated in SODD-deficient cells. The high expression of SODD may be a major mechanism to dampen TNFR1 signaling in MGZ B-cells in the resting state. The higher expression of CARD11 in MGZ may have a pro-survival function, but it may also have a role in MGZ organization. It has been shown that loss of CARD11 in mice resulted in the complete loss of CD5 + peritoneal cells and reduced number of IgD high IgM low mature splenic B-cells, indicating its role in B-cell development [ 37 ]. Two closely related genes, NM23-H1 and NM23-H2 , which share an amino acid identity of 88%, were highly expressed in MGZ. NM23 H1 is a granzyme A-activated DNase (GAAD) that is inhibited by SET [ 38 ]. The high expression of NM23-H1 and the low expression of its inhibitor SET was opposite in their expression profile in the GC, suggesting that this expression may influence apoptosis in opposite directions in these two B-cell compartments. Figure 6 DNA repair, replication and protein synthesis. Figure 7 (A) Apoptosis and cell survival and (B) Cytokines and chemokines and their receptors. Chemokines, cytokines and their receptors (Figure 7B ) Chemokines attract primary B-cells and play an important role in the homing and localization of B-cell subsets at different stages of antigen-independent and dependent reaction [ 39 ]. Our microarray data revealed that CCL18 , encoding a chemokine secreted by immature dendritic cells (DC), is specifically upregulated in the GC compartment. Our finding was supported by a recent report showing that CCL18 is produced by GC DC and can attract MNZ B-cells towards GC [ 40 ]. The higher expression of CCL18 may be especially important during the onset of a GC reaction, the time point to recruit antigen primed pre-GC B-cells, which then interact with GC DC to initiate and maintain the GC reaction. The GC compartment showed increased expression of CXCL10 ( IP-10 ). which has pleiotropic effects, including stimulation of monocytes and T cell migration [ 41 ]. The GC also showed increased transcriptional level of genes that may suppress or control inflammatory responses; e.g., SOCS1 limits cellular response to IFNγ, IL-2 and IL-6[ 42 , 43 ]. Macrophage inhibiting cytokine 1 ( MIC1 , also known as PLAB ) [ 44 ], is only expressed by activated macrophages, but not by resting macrophages. Its higher expression in the GC may reflect the presence of moderate numbers of macrophages and its possible role in suppressing the inflammatory response in the GC. Increased expression of the chemokine CCL20 was observed in MNZ cells. Human naïve and memory B-cells express the cognate receptor for CCL20, CC-chemokine receptor 6 (CCR6) [ 45 ]. The high expression of CCL20 may play a vital role in naïve B-cell migration and localization in the MNZ. The chemokine gene CXCL12 (also know as stromal cell-derived factor 1, SDF1 ) is highly expressed in MGZ cells. The receptor for CXCL12 is CXCR4, which is present on CD34 + cells, myeloid cells, CD4 + T cells, B-cells, epithelial cells, endothelial cells, and dendritic cells. In the bone marrow, stromal cells secrete CXCL12, which is involved in the emigration of hematopoietic precursors to the marrow during embryogenesis [ 46 ]. In peripheral lymphoid organs CXCL12 may be involved in the migration of B-cells and possibly other cells, such as T cells and plasma cells, to the MGZ. CCL14 (also known as HCC1 ) and CCL3 (also known as MIP-Iα ) were more highly expressed in the MGZ. CCL14 can activate human monocytes via receptors that also recognize CCL3 [ 47 ]. CCL3 is a proinflammatory cytokine important in the clearance of viral infections and the stimulation of the innate immune response [ 48 ]. Thus, the expression of this gene may be important in innate immunity in the MGZ of the spleen. CXCL13 and CCL5 were upregulated in both microdissected MGZ and MNZ compared with the FACS-sorted B-cell populations. Previous studies have established an important role for CXCL13 (BLC) in the development of Peyer's patches (PP) and many peripheral lymph nodes. It also controls B-cell migration and thus the organization of B-cell areas [ 49 ]. CCL5 (RANTES), a stromal related chemokine, elaborated by activated T, NK and macrophages has been shown to interact with CD44 to activate the MAPK pathway [ 50 ]. It is possible that CCL5, under appropriate conditions, contributes to cellular activation that may be particularly relevant to MGZ B-cells, in which rapid response on recognition of antigen stress signal is important. A number of IFN-induced genes ( AIM2, IFNGR1 , and IFNAR -1 and - 2 ) were also preferentially expressed and may reflect the unique function of the MGZ to provide the first rapid response to particulate or T cell-independent antigens. The MGZ also showed increased expression of many members of G protein pathways consistent with more active chemotaxis, cell motility and secretory functions. In addition, MGZ cells showed higher expression of IL-7Rα , consistent with the role of the IL-7R in MGZ organization [ 51 ]. Aside from its role in B-cell differentiation and proliferation IL-7Rα expression is also required for the recruitment of precursor cells to develop in secondary lymphoid organs and for the proper structural organization of these organs [ 52 ]. Figure 8 Stromal signature. Extracellular matrix and stromal signature (Figure 8 ) Cells function within the context of a three-dimensional (3D) extracellular matrix (ECM) that participates in regulating cellular motility, proliferation and survival. In the GC, COL9A3 , which encodes collagen IX [ 53 ], and COL2A1 , which encodes collagen XI [ 54 ], were uniquely overexpressed. In the marginal zone COL14A1 , COL16A1 , COL3A1 and COL6A3 were expressed at higher level, which suggests a role for these genes in the synthesis of specific extracellular matrix. There was also a marked overexpression of macrophage metalloelastase 12 ( MMP12 ), encoding a metalloproteinase that preferentially degrades elastin and takes part in the remodeling of extracellular matrix. No collagen-specific gene was up-regulated in the MNZ. Microdissected compartments contained a minor component of stromal T cells, macrophages, dendritic cells and fibroblasts whereas FACS-sorted cells from lymphoid tissues comprise almost exclusively B-cells. Thus, an insight into the gene expression profile of the stromal elements can be obtained by comparing the expression profile of FACS-sorted and microdissected cells. We found a set of genes that likely represent the stromal signature. Osteonectin (SPARC) , upregulated in LCM samples, encodes a matrix-associated protein that elicits changes in cell shape, inhibits cell-cycle progression, and influences the synthesis of extracellular matrix [ 55 ]. It regulates endothelial barrier function through F-actin-dependent changes in cell shape [ 56 ]. Two members of the Maf family ( MafB , and c-Maf ) were also part of the stromal signature. The Maf family of genes encode bZip nuclear transcription factors and play an important role in morphogenesis and cellular differentiation [ 57 ]. These genes are expressed in a variety of organs, including the spleen, in agreement with our finding. The MGZ expressed elevated levels of ICAM1 and VCAM1 . MGZ B cells express the integrin LFA1 which binds to its ligands ICAM1 and VCAM1, and this interaction may control the localization of these B cells [ 58 ] in this compartment. Our results also showed elevated expression of VCAM1 , ITGAL (LFA-1) and ITGA6 in the MNZ, suggesting a role for these adhesion molecules in mantle cell localization as well. The kruppel-like transcription factor BCL11a (also called Evi9), which is essential for normal B-cell lymphopoiesis, was upregulated in LCM cells only. Interestingly, bone marrow from BCL11a -/- mouse can induce thymic lymphoma in wild type mice. Thus, the increased expression of BCL11a in the MNZ and MGZ may be physiologically relevant to the function of lymphocytes in these regions [ 59 ]. Figure 9 Other unique compartment markers. Other differentially expressed genes (Figure 9 ) Many genes know to be specifically expressed in GC B-cells are found to be upregulated: e. g., BCL6 , CD10 , GCET1 , GCET2 , JAW1 and CD38 . A number of genes were clearly upregulated in the MNZ or MGZ but their functional significance is largely unknown. Some of these would be interesting targets for further investigation. Among the genes encoding surface molecules, CD59 was highly expressed in the GC. CD59 antigen is a small protein that inhibits complement-mediated pore formation or lysis by preventing the formation of membrane-embedded C9 multimers [ 60 ]. It is likely that the over expression of CD59 in GC can prevent complement-mediated damage to FDCs with entrapped immune complex. CD10 and CD38 are well established markers of GC B-cell and over expression of the corresponding mRNA in the GC is expected [ 61 - 63 ]. Notably, CIITA was markedly down regulated in GC cells, associated with a general low expression of MHC transcripts. Conclusions The gene expression profiles of the three B-cell compartments reflect distinctive functional attributes of the resident B-cell populations. They also showed different molecular microenvironments that allow the different B-cell populations to differentiate and function properly. GC B-cells have a high proliferation gene signature, whereas MNZ and MGZ cells are characterized by signals that help to maintain the quiescent state. Genes involved in the apoptosis pathway are differentially expressed in the three B-cell compartments, reflecting different adaptations for survival in different B-cell populations. Expression of different chemokines, their receptors, and stromal molecules have been detected. Many of these have been implicated in the establishment of the normal lymphoid architecture in peripheral lymphoid organs and in attracting distinct immune-cell populations to specific lymphoid areas. The expression of unique sets of genes may also reflect the functional adaptation of cells in a specific location, such as genes involved in DNA repair in the GC and genes that are active in innate immune response to infection in the MGZ. Gene expression profiling of B-cell compartments has allowed us to obtain a global survey of the molecular signals that are functionally important in B-cell subpopulations as well as the respective microenvironments. One of the major challenges is to delineate the functions of the uncharacterized genes that are unique to each of the compartments. Another challenge is to exploit these normal transcriptional profiles to further our understanding of the normal immune response and the derangements resulting in the corresponding lymphoid tumors. Methods Laser capture microdissection (LCM) Tissue blocks of tonsils and spleens were snap frozen in O.C.T immediately after surgery. Four micrometer thick frozen sections of reactive tonsils or spleens on plain glass slides were fixed with 70% ethanol for 30 seconds, rinsed in DEPC water and stained with hematoxylin for 30 seconds, followed by another water rinse. The sections were then dehydrated with 70%, 95% and 100% ethanol for 10 seconds each. Finally, the slides were passed through xylene twice, each for 30 seconds. A consecutive section was immunostained for CD3 to guide the dissection. The three B-cell compartments (GC, MNZ and MGZ) were isolated using LCM with the Arcturus PixCell II system (Arcturus Engineering, Mountain View, CA). To avoid contamination, only well-defined GC, MNZ and MGZ were dissected. Cells were captured at the 15-μm laser setting on CapSure LCM Caps (Arcturus). The laser was set to pulse at 60 mW for 200 ms. The Institutional Review Board of the University of Nebraska approved the usage of tissues for this study. Cell preparation and FACS sorting Tissue from fresh spleens or tonsils was cut into small pieces in cold RPMI-1640, and cells released by grinding with a glass tissue homogenizer. The crude cell suspension was passed through a nylon mesh (Spectrum Laboratories, Inc) to generate a single-cell suspension. B-cells were firstly isolated using the Human B-cell Isolation Kit (a cocktail of hapten-modified antibodies to CD2, IgE, CD4, CD11b, CD16 and CD36) and the Midi Macs system (Miltenyi Biotec, Auburn, CA). The highly enriched B-cell population (negative fraction) was subjected to 3-color cell sorting. Briefly, 1 × 10 7 B-cells were stained with IgM-Cy-chrome, IgD-FITC and CD27-PE (BD Pharmingen, SanDiego, CA) at 4°C for 30 min. MGZ B-cells were isolated from the splenic B-cells gated on the IgM high IgD low CD27 + fraction, whereas MNZ B-cells were selected based on IgM low IgD high CD27 - using the BD FACSVantage™ SE high-speed cell sorter (Becton-Dickinson, SanJose, CA) RNA extraction and T7 RNA amplification Total RNA was extracted from each sample of microdissected cells with Trizol™ (Gibco BRL, Carlsbad, CA) and further purified with the RNeasy Mini Kit (Qiagen, Valencia, CA). RNA amplification was performed using a modified Eberwine protocol [ 64 ]. Briefly, first-strand cDNA was synthesized by reverse transcription using oligo dT(25)-T7 anchoring primer and superscript II at 42°C for 1 hour. Second-strand synthesis was performed with 40 units E. coli DNA polymerase I, 2 units RNase H, 10 units E. coli DNA ligase (Life Technologies, Carlsbad, CA) in 150 μl volume. Antisense RNA (aRNA) was generated by in vitro transcription (IVT) using the Ampliscribe™ High yield Transcription Kit (Epicentre Technologies, Madison, WI) containing 1000 units AmpliScribe T7 enzyme at 37°C for 8–12 hours, as per the manufacture's instruction. Second-round amplification and IVT were performed as described previously [ 65 ]. The quality and quantity of aRNA were monitored on agarose gel electrophoresis and by spectrophotometer. Typically, 30–50 μg of aRNA were generated from each 10 ng of total RNA by two rounds of amplification. Gene expression profiling using the Lymphochip Analysis of gene expression was performed using the Lymphochip cDNA microarray, which contained 15,132 cDNA clones representing 7399 known or uncharacterized genes [ 66 ]. Labeled cDNA from each compartment was first hybridized with a test cDNA microarray to assess the quality and quantity of the amplified aRNA before using them on the Lymphochip. In each experiment, reverse transcription was carried out on 8–9 μg of aRNA, and aminoallyl-dUTP was incorporated into the cDNA using a dUTP: dTTP ratio of 4:1 . The aminoallyl group on the dUTP reacts with the ester group on the cyanine dyes. Cy3 dye was used to label the standard cDNA and Cy5 dye the test probe, and hybridization was performed as previously described [ 17 ]. Data and statistical analysis procedure Each tissue type was independently isolated, amplified and profiled in three separate experiments to enhance the reliability of the gene expression data. Images of hybridized microarrays were obtained and processed using GenePix 4000B microarray scanner (Axon Instruments, Union City, CA). Spots or areas of an array with obvious blemishes were flagged and excluded from subsequent analyses. Fluorescence ratios were normalized for each array by applying a single scaling factor to all fluorescent ratios from the array [ 17 ]. The correlation coefficients among 15 hybridized cDNA microarrays were calculated and expressed in Correlation Coefficients Mapping (CCM), programmed in MATLAB © (Mathworks, Inc, Natick, MA), which provided an overview of the similarity of expression profiles between multiple samples. Only genes with at least two values out of the triplicate experiments showing similar behavior were included for analysis. The expression data for each gene from an individual compartment was median/mean centered with weighted variance across the two or three replicates showing similar behavior. The initial data reduction was performed using the two-tailed student t-test to compare the differences in gene expression levels between individual compartments. Genes differentially expressed between the two compartments with a p-value of less than 0.05 were selected for further analysis using the Significance Analysis of Microarrays (SAM) approach, as described previously [ 22 ]. SAM assigns each gene a score based on its change in average expression between two groups, relative to the gene's standard deviation of permutated measurements. The scatter plots for observed relative difference vs expected relative difference between two compartments were used to find the potentially significant genes and plotted in the T-distance histogram correlating with the p-values. The genes selected from the common set of the analysis result from both t-statistics and SAM were grouped according to their functional characteristics after analyzing through OMIM or Gene Ontology database ( or ) and viewed by TreeView. Semi-quantitative and real-time quantitative PCR To confirm the differential mRNA expression of the genes identified by the Lymphochip in different B-cell compartments, a semi-quantitative RT-PCR was employed. In brief, 200 ng aRNA was reversely transcribed into cDNA with 200 ng random primer using MMLV-RNase H - reverse transcriptase as per the manufacturer's instructions (Invitrogen, Carlsbad, CA). Five-fold serially diluted cDNAs from GC, MNB and MZB were amplified with gene-specific primers for 30 cycles with the following cycling conditions: A denaturation step at 94°C for 2.5 minutes and then each PCR cycle at 94°C for15 sec, 52°C for 30 sec, and 72°C for 30 sec followed by a final extension at 72°C for 5 min. The human HPRT transcript was used as the comparative standard. The products were analyzed by electrophoresis in 2% agarose gel. The primers were designed to amplify the cDNA close to the 3' end of the transcript, and all the PCR products were less than 200 bp in length. Some of the results from the semi-quantitative RT-PCR were also validated by the real-time quantitative PCR with DyNAmo™ HS SYBR ® Green qPCR Kit (MJ Research, Reno, NV) on DNA Engine OPTICON2 (MJ Research, Reno, NV) as per the manufacturer's instructions. The PCR protocol used an initial denaturation of 95°C for 15 minutes followed by 35 cycles (95°C for 10 sec, 52°C for 15 sec and 70°C for 20 sec). The plate was read at 70°C according to the melting point of the amplicon. Serial dilutions of cDNA from the lymphoid standard [ 67 ] were used to construct standard curves for the target genes ( FAIM , CCL3 , SODD , NM23-H1 , CARD11 , Cyclin G2 and CIITA-8 ) and the endogenous reference genes (HPRT). For each unknown sample, the relative amounts of target cDNAs and reference cDNAs applied to the PCR reaction system were calculated using linear regression analysis from the corresponding standard curves [ 68 ]. Then the normalized expression level of the target gene in each sample was calculated by dividing the quantity of the target transcript with the quantity of corresponding reference transcript. The normalized values of the target transcript were used to compare its relative expression levels in different samples. Authors' contributions YS carried out the LCM, in vitro RNA amplification and semi-quantitative PCR. JI participated in the design of the study, microarray procedure, data analysis, and drafted the manuscript. LX, RL and SS participated in data analysis. JE provided the microarray facility and various technical assistance and advice. LS and AR provided the reference standard, the Lymphochip and helpful discussions. KD, GZ, TM participated in helpful discussions and interpretation of the data. WCC conceived, organized and supervised the study, and participated in the analysis and interpretation of the data. All authors have read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC535350.xml
551615	Familial hypercholesterolemia in St.-Petersburg: the known and novel mutations found in the low density lipoprotein receptor gene in Russia	Background Familial hypercholesterolemia is a human monogenic disease caused by population-specific mutations in the low density lipoprotein (LDL) receptor gene. Despite thirteen different mutations of the LDL receptor gene were reported from Russia prior to 2003, the whole spectrum of disease-causing gene alterations in this country is poorly known and requires further investigation provided by the current study. Methods Forty-five patients with clinical diagnosis of FH were tested for the apolipoprotein B (apoB) mutation R3500Q by restriction fragment length analysis. After exclusion of R3500Q mutation high-sensitive fluorescent single-strand conformation polymorphism (SSCP) analysis and automatic DNA sequencing were used to search for mutations in the LDL receptor gene. Results We found twenty one rare sequence variations of the LDL receptor gene. Nineteen were probably pathogenic mutations, and two (P518P, T705I) were considered as neutral ones. Among the mutations likely to be pathogenic, eight were novel (c.670-671insG, C249X, c.936-940del5, c.1291-1331del41, W422X, c.1855-1856insA, D601N, C646S), and eleven (Q12X, IVS3+1G>A, c.651-653del3, E207X, c.925-931del7, C308Y, L380H, c.1302delG, IVS9+1G>A, V776M, V806I) have already been described in other populations. None of the patients had the R3500Q mutation in the apoB gene. Conclusions Nineteen pathogenic mutations in the LDL receptor gene in 23 probands were identified. Two mutations c.925-931del7 and L380H are shared by St.-Petersburg population with neighbouring Finland and several other mutations with Norway, Sweden or Denmark, i.e. countries from the Baltic Sea region. Only four mutations (c.313+1G>A, c.651-653del3, C308Y and W422X) were recurrent as all those were found in two unrelated families. By this study the number of known mutations in the LDL receptor gene in St.-Petersburg area was increased nearly threefold. Analysis of all 34 low density lipoprotein receptor gene mutations found in St.-Petersburg argues against strong founder effect in Russian familial hypercholesterolemia.	Background Familial hypercholesterolemia (FH) (OMIM #143890) is one of the most common monogenic human diseases. It is inherited as an autosomal dominant trait with the prevalence of the heterozygous form conventionally considered to be about 1 of 500 in most populations [ 1 ]. Elevated blood serum cholesterol is due to impaired removal of low-density lipoproteins (LDL) from blood by LDL receptors, and it is associated with early onset coronary artery disease and myocardial infarction. Impairment of LDL receptor function usually results either from the absence or deficiency of the LDL receptor (OMIM 606945) itself or from a common mutation (R3500Q) in the gene of the receptor's ligand, apolipoprotein B (OMIM +107730) causing type B of the autosomal dominant hypercholesterolemia (OMIM #144010) [ 2 ]. Furthermore, a third type of monogenic autosomal dominant hypercholesterolemia (OMIM #603776) is due to the recently discovered defects in the proprotein convertase PCSK9 gene (OMIM 607786) [ 3 ]. A form of recessively inherited hypercholesterolemia (OMIM #603813) has a prevalence of less than 1:10 000000 and is due to defects in the LDL receptor adaptive protein ARH (OMIM *605747) [ 4 , 5 ]. Even though many forms of monogenic hypercholesterolemia are known, only apoB gene and LDL receptor gene variations seem to contribute significantly to the CHD morbidity in most populations [ 6 ]. The spectrum of LDL receptor mutations varies between different human populations and more than 900 mutations in the LDL receptor gene have been characterized worldwide [ 7 - 9 ]. A recent review [ 9 ] shows a clear difference in the LDL receptor gene mutations spectra for Western European countries, but this review gives nearly no data on the genetics of FH in the Eastern European countries and Russia. However, many mutations were described from Poland [ 10 ] and Bulgaria [ 11 ] and already thirteen different LDL receptor gene mutations have been published from the Russian population prior to 2003 [ 12 ]. In the present research we expand the study of the molecular genetic basis for FH in St.-Petersburg known to be the most well studied region of Russia in respect to FH-causing mutations and report 21 mutations, previously unknown in Russia. Methods Patients Patients with FH were recruited from two lipid clinics of St.-Petersburg, namely Institute of Human Brain and Institute of Experimental Medicine. The clinical diagnosis of familial hypercholesterolemia was based on the following criteria: highly elevated plasma total cholesterol and LDL-cholesterol, presence of tendon xanthomata, corneal arcus or both, and positive family history of myocardial infarction and hypercholesterolemia with at least one first-degree relative affected. Forty-five probands fulfilling at least two of three criteria listed above were selected for the study. Full data of patients including their lipid data are given in the Discussion section (see Table 2 ). Informed consent was obtained in each case for DNA testing procedures. Biochemical procedures Genomic DNA was extracted from blood white cells using a standard method [ 13 ]. The patients were initially tested for the apoB mutation R3500Q by restriction fragment length analysis [ 14 ]. Exons of the LDL receptor gene were then amplified [ 15 ], and PCR products were subjected to gel-electrophoresis followed by ethidium bromide or silver staining of DNA to exclude non-specific amplification. Single-strand conformation polymorphism (SSCP) analysis was performed in an ABI 377 DNA Sequencer (PE-Applied Biosystems) sequencing device using 4,25% non-denaturing MDE polyacrylamide gels (Cambrex) at 20°C. The gels were run under two different conditions, i.e. standard MDE gel or with the addition of 5% glycerol to the MDE gel. Automated DNA sequencing was performed in part in the ABI 377 DNA Sequencer (PE-Applied Biosystems), in part in an ALFExpress-2 DNA sequencer (Amersham Life Sciences), using primers for routine PCR DNA amplification. In the case of samples bearing the deletions, PCR products were cloned into a commercially available vector (TACLONE, Medigen, Novosibirsk) and sequenced using universal and reverse primers. Results None of the 45 patients had the apoB R3500Q mutation, whereas 25 patients had mutations in the LDL receptor gene. Large genomic rearrangements in the LDL receptor gene were previously shown to be an uncommon cause of FH in St.-Petersburg [ 16 ] and had been excluded in most patients of the current group. Therefore we restricted our search to point mutations and to minor deletions and insertions. We identified 21 sequence variations (Table 1 ) of which two probably were not pathogenic (in the following considerations we give numbering of aminoacids in the LDL receptor according to Yamamoto's nomenclature, [ 17 ] ). The T705I mutation (known also as FH Paris-9) is not associated with elevated serum cholesterol [ 18 ], and the transition c.1617C>T (P518P) is synonymous. We consider the remaining 19 variations to be pathogenic. Eleven mutations have been described in other populations, but to our knowledge the remaining 8 mutations have not been described so far [ 7 , 8 ]. From two of these nucleotide substitutions (C249X and W422X) the stop codon arises, four are frame-shift mutations leading to premature stop codons (FsK202:S205X; FsK290:N309X; FsV409:S423X; FsV597:A622X), and two result in amino acid substitutions (D601N and C646S). Sequencing of the cloned mutant allele bearing the five-nucleotide deletion (c.936-940del5 or FsE291) is demonstrated on Fig. 1 . Mutations c.313+1G>A, c.651-653del3, C308Y and W422X were found in two probands each. Rapid tests were developed for most of the mutations and all of those were confirmed by using these methods (Table 1 ). Various detection methods, including heteroduplex analysis, restriction enzyme tests and SSCP for several mutations from the list are illustrated by Figures 2 , 3 . Cosegregation of the mutations and elevated blood serum cholesterol was demonstrated in seventeen families (see Fig. 2 , 3 and Table 1 ). Totally the mutations were confirmed in 9 relatives and excluded in 27 members of the proband's families. Most important the diagnosis of FH was excluded in 9 and set in 2 children of probands before adulthood, i.e. prior to age 18. In these children mutation detection was of crucial importance to set the diagnosis and to suggest further life style. In case of the Q12X mutation, the c.97C>T transition leads to occurrence of a new Mae I restriction site (CTAG) in exon 2 that allows a simple detection of this mutation. Restriction enzyme test was performed for the proband and her 4 descendants (Fig. 2 ). The presence of the mutation was confirmed in the son of the proband and excluded in the daughter and two grandchildren. In some other mutations no restriction enzyme tests can be developed for their rapid detection. For example, it is true for the recurrent mutation IVS3+1G>A (c.313+1G>A) that was identified by means of SSCP and verified by sequencing in probands from two unrelated families. SSCP patterns on silver-stained gels differ strikingly in patients with and without the mutation. The presence of the mutation was confirmed in the son of the proband by SSCP. In heterozygotes a deletion of 41 nucleotides at c.1291-1331 in exon 9 (mutation FsV409:S423X) results in occurrence of specific heteroduplexes. Besides, a PCR fragment of smaller size as compared to the normal allele is revealed in silver-stained polyacrylamide gels (Fig. 3 ). This mutation was identified in the son and daughter of the proband and excluded in the second daughter and in her child (Fig. 3 ). Discussion Previously 13 mutations in the LDL receptor gene have been reported in FH patients residing in St.-Petersburg [ 12 ] (see Table 3 ). Eight of those have not been reported in other populations. Our study revealed 21 mutations in the LDL receptor gene, out of which only T705I was previously reported from St.-Petersburg [ 12 ]. Nineteen of those 21 mutations are likely to be pathogenic. We exclude P518P (CCC>CCT; c.1617 C>T) from the list of pathogenic mutations since the transition c.1617 C>T results neither in an amino acid substitution nor in appearance of the new consensus splicing sequences. P518P mutation was found in the proband with mutation C646S, but it was not clarified if the mutations were in cis- or trans- position. Also we consider the T705I variant not to be a primary cause of FH, since the I705 allele itself is not associated with elevated cholesterol level [ 18 ] and the probable hypercholesterolemic effect of this mutation may be due to its linkage with other pathogenic LDL receptor gene mutations. Indeed, a variation in intron 7 (c.1061-8T) of unclear functional significance was shown to be very tightly linked to the I705 allele [ 19 , 20 ]. We have not searched for this intronic variant in the LDL receptor in the patient with the T705I substitution. Among pathogenic mutations reported here, eleven have been described in other populations (Table 1 ) [ 7 , 8 ], and eight are novel. Six of the novel mutations lead to premature stop codons and result in truncated protein chains that probably lose their function. One out of these truncating mutations (c.936-940del5 or FsE291) also changes invariant nucleotides nearby exon-intron junction and thus may affect splicing. The D601N missense mutation, causing substitution of aspartic acid by asparagine has not been reported before. It seems likely that such a substitution might cause the loss of function since one other mutation in the same codon (D601Y) was described in familial hypercholesterolemia subjects [ 7 ]. The C646S also has not been reported before, but 5 other mutations affecting this codon have been described, one of which, C646Y (FH French Canadian-2), results in a transport defective protein (mutation class 2A) [ 21 ]. We, therefore, find it very likely that the C646S mutation is also pathogenic, but expression studies are needed to justify the effect of both missense mutations D601N and C646S. Previously described mutations were considered to be pathogenic due mostly to their listing in FH mutation databases even though functional studies were not systematically performed. Indeed, V806I mutation (known as variant FH New York -5) [ 22 ] occurs in the LDL receptor internalization signal NPVY, for which the consensus sequence NPxY is given (where X is not a conserved aminoacid) [ 23 ] and thus the substitution of isoleucin for valine may be not crucial for the LDL receptor function. The V776M mutation may have effect on LDL receptor mRNA splicing rather than to be realized on the protein level since the V776M mutation changes the invariant G at the 3' end of exon 16. However, this mutation is likely to be pathogenic since it was reported already in patients from La Habana, Cuba [ 24 ]. The apoB R3500Q mutation was not detected in any of our patients. This finding is in agreement with the previous observation that the R3500Q mutation had not been found in St.-Petersburg [ 25 ]. The mutation has been detected in another part of Russia only in 2 of 71 patients with symptoms of familial hypercholesterolemia [ 26 ]. The apoB R3500Q mutation is almost exclusively found in Caucasian individuals, and almost all subjects with the mutation carry the same haplotype. One of the highest frequencies of the mutation has been found in the Swiss population (approximately 1/200) [ 27 ], and Miserez and Muller [ 27 ] hypothesized that the mutation may have arisen in Switzerland 10,000 – 6,000 years ago. The prevalence of the mutation declines with increasing distance from the Central Europe [ 27 , 28 ], and the prevalence of the apoB R3500Q mutation is therefore expected to be low in the St.-Petersburg area (< 1/1000). A precise estimate would require a random sampling of the general population. Mutations in LDL receptor gene typical for various ethnic groups were revealed in St.-Petersburg (Table 1 ). In particular, several mutations found in Denmark, Finland, Norway and Sweden are present in the St.-Petersburg population. The ethnic origin of these families is unclear and according to questioning and family names no evidence of a Scandinavian origin of probands was obtained. Only exception from our previous FH group was the proband with the E207X mutation who stated his German roots and indeed this mutation was previously reported from several families from Germany [ 29 ]. Interestingly, no specific Slavic or Eastern-European founder mutations were found in St.-Petersburg when comparing the LDL receptor mutation spectrum of Russia to those of Poland [ 10 ], Bulgaria [ 11 ] or Czech Republic [ 30 , 31 ]. G571E was found in Russia, Czech Republic and Poland but this mutation was also reported from many other countries worldwide. The C188Y mutation found in Russia [ 32 ] and in Czech Republic [ 30 , 31 ] cannot be considered a founder Slavic mutation, since it was reported from unique families in each country. Only one candidate for a Russian founder mutation is C139G identified in four unrelated families in different regions of the country, but this mutation is absent in related nations of the Eastern Europe as well as in other countries in the world. In our study we were able to find LDL receptor gene pathogenic mutations in 23 of 45 patients. Many methods are considered to be superior to routine isotopic SSCP when screening for mutations in DNA (see e.g. [ 33 ]for comparison of SSCP and DHPLC). However, we believe that mutations could have been overlooked due to intrinsic limitations of SSCP method in our hands only in few cases. Fluorescent SSCP, used in the current study, can be superior even to DHPLC and is a sensitive method for mutation screening, especially when different gel electrophoresis conditions are applied [ 34 ]. Recent studies indicate, that SSCP run under two different conditions detect up to 96% of heterozygous variations (P.H. Nissen, unpublished results). In our study LDL receptor gene mutations were found in 56% (14 out of 26) patients selected by SSCP and in 53% (10 out of 19) patients which DNA was subjected to direct sequencing of all gene exons (Table 2 ). We do not find it likely that many patients are underdiagnosed due to presence of large genomic rearrangements. In a study conducted in St.-Petersburg, in the partly overlapping sample of FH patients, only one case of a large deletion was found in the sample of 50 probands, giving the rough estimate of genomic rearrangements 2% [ 16 ]. In fact in mixed populations (such as that of that of St. Petersburg) the contribution of large rearrangements to the spectrum of pathogenic mutations seems to vary from 6% in Great Britain [ 35 ] to 2.5% in English-speaking Canadians [ 36 ]. More important, some of intronic mutations leading to defects of splicing could be missed, since the design of primers [ 15 ] mostly following recommendations of Hobbs et al. [ 22 ] with the only exclusion for primers to amplify exon 3 allowed analyzing only 14 out of 34 intron-exon boundaries in the LDL receptor gene. Recent investigation [ 37 ] demonstrated that a high percent of previously missed LDL receptor mutations may be localized in introns. In the cited study [ 37 ] in the patient sample after modifying the gene analysis procedure nearly 27% of patients turned out to have intronic mutations, despite the functional significance of these mutations have still to be validated. In the apoB gene we only looked for the R3500Q mutation, and the possibility that other mutations in that gene are responsible for hyperlipidemia cannot be ruled out. This possibility is unlikely, since the apoB gene has been studied extensively in other laboratories and no other pathogenic variants have been determined. To conclude we cannot definitely rule out the possibility that some undiscovered mutations in the LDL receptor gene have not been found. However, the possibility that the patients have mutations in a different gene involved in FH still remains since the mutation in the PCSK9 gene [ 3 ] were not tested in this study. With the two exceptions, mutations previously reported from Russia [ 12 , 38 ] have been confined to a single family. The exceptions were the mutations G197del (FH-Lithuania), found in high percent of Ashkenazi Jewish families with FH from St.-Petersburg [ 39 ], and the C139G mutation found in two Slavic families from St.-Petersburg [ 40 , 12 ], one family in Novosibirsk [ 38 ] and one family in Moscow [ 41 ]. In this study 4 mutations, namely c.313+1G>A, c.651-653del3, C308Y and W422X were found in two unrelated families. Together with the previous findings we discovered 34 mutations in the LDL receptor gene in St. Petersburg of which only six were detected in more than one family. Fourteen LDL receptor gene mutations were discovered by the other Russian FH team from Moscow, Russia out of which only C139G is shared with St.-Petersburg population [ 12 , 41 ]. To date a total of 47 different FH mutations are known from Russia. The data presented here enlarge the spectrum of mutations found in the Russian population and make us regard it as genetically heterogeneous. Conclusions We identified nineteen pathogenic mutations in the LDL receptor gene in 23 probands and two probably neutral mutations. In our study only four mutations in the LDL receptor gene (c.313+1G>A, c.651-653del3, C308Y and W422X) were found to be recurrent, i.e. all of those were found in two apparently unrelated families. Together with the data obtained earlier [ 12 , 38 ] our results present an evidence against the strong founder effect among Russian FH patients, and it is likely that in the St.-Petersburg area there is as much genetic heterogeneity as in most other areas of the world. Competing interests The author(s) declare that they have no competing interests. Authors' contributions FMZ, MYM, VIG, PHN, GGN, AS performed cloning, SSCP, sequencing and familial analysis, BML, VOK, DD, ADD selected patients, VBV and OF participated in the study design and coordination. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC551615.xml
554779	General practitioner attitudes to the care of people with epilepsy: an examination of clustering within practices and prediction of patient-rated quality of care	Background There is wide variation in the quality of care provided by primary care practices to individuals with chronic illnesses. Individual doctor attitudes and interest have been demonstrated to influence patient outcomes in some instances. Given the trend towards larger practices and part-time working, continuity of care is likely to fall and thus practice-based rather than individual general practitioner attributes and attitudes are likely to become increasingly important. The aim in this paper was to examine the extent to which individual general practitioner (G.P.) attitudes to the care of people with epilepsy cluster within practices and predict patient-rated quality of care. Methods The sample consisted of 1255 people with active epilepsy (a recent seizure or on anti-convulsant medication for epilepsy) and 199 GPs from 82 general practices. Measures of GP attitudes (a 17-item GP attitudes questionnaire) and patient-rated quality of epilepsy care were obtained. 1210 individuals completed initial questionnaires and 975 patients filled in final questionnaires one year later. Responses were achieved from 64 practices (83% of total) and 115 GPs (60% of total). Results 2 main factors were found to underlie GP attitudes to the care of people with epilepsy and these demonstrated clustering within practices "epilepsy viewed as a primary care responsibility" (Eigenvalue 3.98, intra-class correlation coefficient (ICC) 0.40), and "medication skills"(Eigenvalue 2.74, ICC 0.35). GP-rated scores on "epilepsy care being a primary care responsibility" were a significant predictor of patient-rated quality of GP care (p = 0.031). Other contributory factors were seizure frequency (p = 0.044), and patient-rated "shared decision making" (p = 0.022). Conclusion Specific general practitioner attitudes to the care of people with epilepsy cluster within practices and are significantly associated with patient-rated quality of epilepsy care. It is important to take these findings into consideration when planning primary care interventions to ensure people with epilepsy receive the benefits of available medical and surgical expertise.	Background There is wide variation in the quality of care provided by primary care practices to individuals with chronic illnesses [ 1 ]. Individual doctor attitudes and interest have been demonstrated to influence patient outcomes in some instances [ 2 , 3 ] and it has been argued that specific attitudes are more predictive of behaviour than general attitudes[ 4 ]. For chronic diseases, in line with the distinction proposed by Katz [ 5 ], these doctor attitudes may be separated into perceptions of knowledge, skills and personal preferences. The importance of these specific doctor attitudes on patient outcomes is however largely unknown. There is a marked trend to larger partnerships in primary care practices and more flexible working practices. It is likely, therefore, that continuity of care will continue to fall, and that patient experience of care of a particular condition will be based on contact with more than one general practitioner [ 6 ]. Thus practice-based rather than individual general practitioner (GP) attributes and attitudes are likely to become increasingly important. The extent to which GP attitudes to specific chronic conditions cluster within practices, is however currently unknown. There is evidence that where attitudes within a group are shared this enhances the influence of individual attitudes on behaviour [ 7 ]. Thus, on the basis of this observation, if attitudes are shared by general practitioners within practices, these group-based attitudes are then more likely to influence GP behaviour and the quality of care provided by the clinician. In the next few years there is likely to be considerable reorganisation of the way in which epilepsy care within primary care is delivered, with GPs taking on a more active role in providing care. Information on how individual general practitioners view and value their role in providing epilepsy care is considered as important [ 8 ]. However, what may be more important is whether or not these views are shared within the practice and if these attitudes influence the quality of care provided by the practice. If this is the case, then taking GP attitudes in a given practice into account will be crucial in deciding how primary care services for people with epilepsy are best organised and improved. In this paper, data from a completed community-based study on people with epilepsy are used to examine the following questions: 1. To what extent do individual general practitioner attitudes to the care of people with epilepsy cluster within practices? 2. Do general practitioner attitudes predict how people with epilepsy rate the quality of the general practitioner care of their epilepsy? Methods General practitioners and adults with epilepsy taking part in an intervention study in Greater Manchester provided information for this study. The results of the intervention study (a prompt and reminder card for general practitioners to complete, held by patients or placed in their medical records and used opportunistically over the course of a year) have been reported [ 9 ]. No group differences in patient rated outcome measures were found for the intervention study [ 9 ]. Ethical approval was obtained in the 4 areas of Greater Manchester from where patients were approached. The patients who consented to participate in the study had their medical records examined to extract data on recording of clinical information about epilepsy and other markers of quality of care. Patients were also sent questionnaires for self-completion. These included both a generic quality of life measure- the EUROQOL 5D[ 10 ] and a disease specific quality of life and quality of care measure, the "Living with Epilepsy" questionnaire which has been psychometrically tested and shows good reliability and validity [ 11 ]. Self-rated seizure frequency (included in the "Living with Epilepsy" questionnaire) was based on the response to a question "How many epileptic attacks have you had in the past year" with the 3 response categories being "None", " Less than one a month" and "One or more a month". General practitioners completed a 17-item GP epilepsy attitudes questionnaire at the end of the study. Responses to items such as "I feel comfortable changing the type of anti epileptic drugs in my patients" were scored using a Likert scale. The attitudes scale was developed and validated for a previous study and results reported in an earlier paper [ 12 ]. Statistical analysis Mean practice scores for each item on the GP attitudes questionnaire were computed and significant factors underlying the grouping identified (using eigenvalues >1.2 and the Scree test.). Clustering of responses on the attitudes questionnaire were examined using the intra-class correlation coefficient both for individual items as well as for a mean score of responses to items loading on each of the main factors. Finally linear regression analysis with the patient-rated quality of care provided by the practice as the dependent variable and GP attitudes and other patient derived measures (such as seizure frequency, age, gender, other long term illness) as independent variables was carried out (using aggregated GP attitude and patient scores). Intervention group was adjusted for. P values of 0.05 and 95% confidence intervals were used to assess significance. Results 1255 patients consented to participate in the study and 975 patients filled in final questionnaires. 199 GPs from 82 practices consented to participate in the study. Responses were obtained from 115 GPs (60% of total) from 64 practices (83% of total). 29 practices had a single respondent. These practices were excluded from the analysis of attitude clustering. In this study 54% of individuals were seizure-free in the previous year ("controlled" seizures) and 46% had reported a seizure in the previous year ("uncontrolled" seizures). Factor analysis and clustering of GP attitudes Factor analysis with varimax rotation was undertaken on aggregate GP responses. Four factors had an eigenvalue of above 1.2. Three of these factors were selected after the scree test. Both the Kaiser-Meyer-Olkin measure of Sampling adequacy test (0.778) and Bartletts test for sphericity (Chi-square 413.7, Df 120, p < 0.0001) suggested that factor analysis was appropriate for this data set. Using guidelines for identifying significant factor loadings based on sample size from Hair et al.[ 13 ] a cut-off of 0.65 was used. Individual items within each factor were used to generate mean factor scores. These mean factor scores were normally distributed. Responses to the 11 questions that were included in the first three factors were further examined. The aim was to detect if significant clustering of responses to these items occurred within practices. The average cluster size was 2.74. The results of the factor analysis and clustering of attitudes for the two main factors are listed in Table 1 . The other two factors were excluded. The first excluded factor was not clinically meaningful with only two disparate items loading on it ("epilepsy care straightforward", "epilepsy patients viewed as being well-informed"). The second excluded factor explained less than 10% of variance and only had one item loading on it ("self-perceived knowledge of epilepsy"). Table 1 Two main extracted factors, significant factor loadings and intraclass correlation coefficients (ICC) for general practitioner attitudes within practices Factor loading 1 ICC 2 Factor 1: "Primary care responsibility"(Eigenvalue 3.98, 24.9% of variance explained)-mean scores 0.40 "Not too time pressured to take on epilepsy care" 0.785 0.37** "GP has primary responsibility for organising follow up care" 0.769 0.13 "Epilepsy care not too difficult to organise" 0.767 0.19* "Epilepsy care not a specialist responsibility" 0.732 0.34 "Epilepsy care should be based in general practice" 0.684 0.44 "Annual structured review should be carried out in primary care" 0.657 0.10 Factor 2: "Medication skills"(Eigenvalue 2.74, 17.1% of variance explained) 0.35 "Comfortable adjusting dose of medication" 0.724 0.31** "GP responsible for adjusting treatment if more fits" 0.718 0.25* "Comfortable adjusting type of medication" 0.655 0.17 p < 0.05, ** p < 0.01 1 (based on mean GP scores per practice) 2 (based on individual GP scores in practices with >1 respondent) Do GP attitudes predict how patients rate the quality of GP care of their epilepsy? Data from 60 practices where both patient and general practitioner data were available were used in the linear regression analysis. As the data were obtained at the end of an intervention study, the intervention group was also included as an independent variable. The results of this analysis are given in Table 2 . Significant predictors of patient-rated quality of GP care were patient seizure frequency and patient -rated "shared decision making" and GP -rated score on "epilepsy care being a primary care responsibility" (Factor 1). Recording of clinical information about epilepsy was not a significant predictor of patient-rated quality of GP care. Table 2 Linear regression analysis: GP and patient predictors of patient rated satisfaction with GP care of epilepsy Unstandardized Coefficient Standardized Coefficient t value Sig. 95% Confidence intervals for B B Std Error Beta Lower bound Upper bound Patient measures Age -.012 .006 -.19 -1.80 .079 -.025 .001 Gender -.4 .258 -.19 -1.55 .128 -.921 .120 Long term health problems other than epilepsy -.141 .255 -.05 -.55 .583 -.655 .373 Anxiety score .051 .028 .24 1.80 .078 -.006 .108 Depression scores -.048 .041 -.17 -1.16 .255 -.131 .036 Ease of talking to GP about epilepsy .392 .348 .13 1.128 .266 -.309 1.109 GP takes views of epilepsy into account ("shared decision making")* .931 .390 .31 2.38 .022 .144 1.719 Seizure frequency* .306 .148 .26 2.07 .044 .008 .604 GP measures Epilepsy as primary care responsibility* (factor 1) .154 .069 .29 2.23 .031 .015 .294 Medication skills (factor 2) -.033 .059 -.07 -.564 .576 -.152 .086 Data adjusted for intervention group r 2 = 0.635, adjusted r 2 = 0.525, standard error = 0.224 * p value <0.05 Some further bivariate analyses were also undertaken. Recording of clinical information about epilepsy by GPs was not significantly associated with the GP-rated score on epilepsy care being a primary care responsibility but was associated with seizure frequency. Discussion In this study two main factors ("epilepsy viewed as a primary care responsibility" and "medication skills") were found to underlie GP attitudes to the care of people with epilepsy. Responses to questions constituting these factors demonstrated a high and significant level of clustering within practices. The main factor that accounted for the largest proportion of variance, general practitioner-rated "epilepsy viewed as a primary care responsibility", significantly predicted patient-rated quality of care. Patient-rated shared decision-making and seizure frequency were other significant predictors of patient-rated quality of GP epilepsy care. Recording of clinical information by GPs about epilepsy was not associated with GP attitudes to epilepsy care but was related to patient seizure frequency. In this study general practitioner attitudes to the care of people with epilepsy were found to cluster within practices to a considerable extent. This has not previously been shown in the U.K. A recent Dutch study [ 14 ] showed that GPs working in the same partnership showed more resemblance in overall attitudes to patient care and behaviour than GPs not working in the same partnership and hypothesised that social processes in partnerships and local circumstances may be particularly relevant. The present study has quantified these intra-practice GP similarities in terms of attitudes to one specific chronic condition. Moreover the results of this study also demonstrate that certain general practitioner attitudes predict patient-rated quality of care provided by the practice. The high level of clustering of GP attitudes and the effect of these attitudes on patient-rated outcomes in terms of quality of care, may have important implications in determining the effectiveness of practice level interventions in primary care. These results suggest, firstly, that when planning educational interventions, changing GP attitudes within practices should also be a key aim and, secondly, to focus on changing attitudes for the practice as a whole rather than simply for individual general practitioners. In addition, for practice level intervention studies (especially those using patient-rated quality of care as an outcome measure) an estimate of clustering of doctor attitudes as well as estimates of clustering of patient responses when carrying out power calculations should be incorporated to avoid making a Type 1 error. There is relatively little information of the relationship of GP attitudes to patient ratings of the quality of GP epilepsy care. Existing evidence suggests that GPs with a special interest in a particular condition improves outcomes [ 2 ]. The results of the present study extend these findings by highlighting the importance of specific attitudes (accepting a key role in management) rather than perceptions of specific skills (skills in medication management) in predicting patient rated quality of care. The results of a multilevel analysis examining patient and doctor predictors of patient satisfaction from the Netherlands [ 15 ] suggested that most of the variance in "patient satisfaction" scores was at the patient level (age, morbidity and previous negative experience with the GP being the main predictors) with only 5–10% of the variance in "patient satisfaction" being at the doctor level. However in that Dutch study [ 15 ], specific GP attitudes were not included as predictors and the "patient satisfaction" score was a composite score incorporating measures of accessibility, availability, humaneness of the GP and information provision. Patient-centred communication skills are known to be associated with improved patient satisfaction [ 16 ] and our analysis indeed found that patient-rated shared decision making ("GP took my views into account") was another significant predictor. Patient ratings of the quality of care do vary according to whether individuals have controlled or uncontrolled seizures. Individuals with controlled seizures rate the quality of care provided higher than individuals with uncontrolled seizures. However why the ratings of care provided are higher is not clear as individuals with controlled and uncontrolled epilepsy differ from each other in other characteristics that may influence quality ratings apart from seizure frequency (e.g. depression scores, social functioning). At practice level GP attitudes are not related to mean practice patient seizure frequency. Although it is likely that individual GP attitudes will be influenced by whether an individual patient has controlled or uncontrolled seizures it is not possible to empirically demonstrate this relationship, as nearly all general practitioners will see a mix of individuals with "controlled" and "uncontrolled" seizures in a given year. Their attitudes to the care of people with epilepsy will be influenced by this spectrum of epilepsy severity (and often to a greater extent by other factors including significant events with individual patients). In terms of limitations of the results, some practices did not consent to take part in this intervention study and not all GPs who participated completed questionnaires. However there were no significant differences between practices that participated and did not participate in terms of size, average deprivation or training status [ 9 ]. Moreover responses to the GP questionnaire were received from over 80% of practices that participated and 60% of the doctors that participated. Aggregate scores were used when doctor and patient views were analysed. This will reduce variability and may result in a loss of statistical power. However given that many different patient-doctor encounters are likely within a given year this approach was the most pragmatic. Although the results were obtained at the end of an intervention study that may have influenced attitudes one of the groups in the study was a control group and no significant differences in GP attitudes between groups was found. Furthermore, results on GP attitudes in the present study were very similar to those found in a previous survey using this scale [ 12 ]. Conclusion Specific general practitioner attitudes to the care of people with epilepsy are significantly associated with patient-rated quality of epilepsy care and cluster within practices. It is important to take these findings into consideration when planning interventions and services. General practitioners need to have good knowledge and skills in the management of epilepsy and should be aware of and utilise current guidelines for good clinical epilepsy care [ 17 - 19 ] to fully utilize medical and surgical expertise in managing epilepsy. Recognising and addressing general practitioner attitudes to the care of people with epilepsy may be important in ensuring these goals of good epilepsy care are met. Competing interests The author(s) declare that they have no competing interests. Authors' contributions AT designed and ran the study, undertook the analysis and wrote the manuscript. MR was involved in the design and running of the study and edited the paper. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC554779.xml
516238	We still fail to account for Mendel's observations	Background The present article corrects common textbook accounts of Mendel's experiments by re-establishing what he wrote and how he accounted for his observations. It notes the long-established tests for the validity of any explanations that purport to explain observations obtained by experiment. Application of these tests to Mendel's paper shows that the arguments he used to explain his observations were internally consistent but were, on one crucial issue, implausible. The same tests are applied to the currently accepted explanation for Mendel's observations. Conclusions The currently favoured explanation for Mendel's observations is untenable. It misrepresents Mendel, fails to distinguish between the parameters and the variables of any system of interacting components, its arguments are inconsistent, it repeats the implausibility in Mendel's paper, fails to give a rational explanation for his observed 3:1 trait ratio and cannot explain why this ratio is not always observed in experimental practice. A rational explanation for Mendel's observations is initiated. Readers are challenged to complete the process before a further article appears.	1. Background We all talk, more or less knowingly, about Mendelian genetics. But four questions need to be asked and answered. 1. Do we understand Mendel's work? To judge from nearly all modern accounts of genetics, we do not. Mendel's paper of 1866 has been persistently misrepresented ever since it was rescued from obscurity in 1900. 2. Do we teach our students a rational description of the inheritance of traits? The answer is again no. Why? Because our current depiction of the inheritance of traits or characteristics is based on false statements, inconsistent arguments and an implausible assertion. 3. Does the current description of Mendelian genetics account for his observations of dominant and recessive traits? No, for the reasons given in answering question 2. 4. Do we account rationally for Mendel's observation of a 3(dominant):1(recessive) trait ratio in some but not all of his experiments? The answer is again no. The reasons will become clear in this article and its successor. A survey of the relevant literature for the period from 1900 to 2003 shows that the various misrepresentations of Mendel's first paper [ 1 ] are of long standing. This is not the place to review all the accumulated historical evidence. The present article concentrates on demonstrating that the currently favoured depiction of elementary Mendelian genetics is untenable; it fails to achieve its intended purpose. A change in the concepts and notation for the interpretation (and teaching) of elementary genetics is suggested. There are two long-established tests of the validity of any hypothesis or proposed explanation for the results observed by experiment. The first test asks: Are all the arguments employed consistent, one with all the others? The second test asks: Are all the proposed mechanisms plausible? Could they be confirmed by experiment, i.e. by a "real"experiment or by a logical "thought experiment". Both tests must be passed if the proposed explanations for the observations are to be accepted. If judgement is being passed on work carried out in the distant past, allowance must be made for the availability or lack of availability of tests of plausibility at that time. On the other hand, we should not hesitate to criticise a current explanation that fails tests of plausibility that are now available but were not available in the past. These two tests of validity (consistency and plausibility) will be applied to Mendel's explanation for his observations and to the currently favoured explanation for his observations. We must first re-establish what experiments Mendel performed and what he wrote in his published accounts of these experiments in order to correct the various false textbook descriptions of Mendel's work. For this purpose it is necessary to study authentic reprints of his two papers [ 1 , 2 ]. The first paper is the one we are concerned with here; it was reprinted [ 3 ] and in a version [ 4 ] correcting several type-setting errors that occurred when Mendel's manuscript was set in typescript. The translation into English by Sherwood [ 5 ] avoided several errors in earlier attempts to translate Mendel's Versuche paper [ 1 ]. There may be other sound translations, but Sherwood's version is strongly recommended. It is accurate and also captures Mendel's literary style. 2. Mendel's experiments and his conclusions 2.1. Why did Mendel carry out his experiments? Many earlier biologists had noted the appearance of hybrid plants but their findings did not show how hybrids arose, whether there was any regularity in their occurrence, or how their properties were related to those of their parents. Mendel showed that there was a general rule for the appearance of hybrid plants and that an exact relationship existed between the traits displayed by hybrids and those displayed by their parents. Hence the title of his first paper: Versuche über Pflanzen-hybriden (Investigations on plant hybrids). 2.2. Mendel's preliminary work and his conditions for successful experimentation Mendel recognised five preconditions for success in his experiments on the origin of hybrids: (i) He needed suitable plants for his experiments. He chose Pisum sativum (the edible pea plant) for most of his work because many established varieties were readily available; and because the flowers enclose the reproductive organs, so minimising accidental cross-fertilisation by insect-or air-borne pollen. (ii) Pisum sativum , like all leguminosae, is androgynous. The flowers contain both male (pollen or sperm) and female (germinal or ova) cells and are therefore normally self-fertilising. This provided experimental advantages, as we shall see. (iii) It was necessary to have stocks of true breeding plants for his cross-fertilisation experiments. He therefore spent much time establishing that 22 varieties of edible pea plants were in fact true breeding. He discarded those plants that were not true breeding before starting his experiments on hybridisation. (iv) He had to ensure that any cross-fertilisations were strictly under his control. To achieve this control, he removed all the immature pollen-bearing stamens from a true-breeding pea plant that displayed a particular trait, e.g. green seeds, then transferred pollen to these emasculated flowers from another true breeding pea plant that displayed an alternative form of the same trait (e.g. yellow seeds). (v) Success depended on meticulous enumeration of the occurrence of hybrids, and of alternative traits, in the populations of plants that arose from his cross-and self-fertilisation experiments; and on repetition of each cross-fertilisation and self-fertilisation experiment in order to obtain reliable, average, results. Table 1 reveals the magnitude of Mendel's undertaking and records his observations on the occurrence of hybrids, and of plants displaying either dominant or recessive traits (see further descriptions in the following section). Reciprocal crosses gave the same results; Mendel thus established that male and female sex cells contributed equally to the final outcomes. Table 1 Mendel's novel observations summarised. Mendel demonstrated that crossing parental plants bearing alternative forms ( A ) and ( a ) of any one of seven traits generated a F1 population of plants (not shown) all of which were hybrids ( Aa ). Each of these F1 hybrid plants displayed only one of the two alternative parental traits, defined as the dominating trait ( A ). When these F1 hybrid plants were allowed to self-fertilise, the ratio of dominant to recessive traits in the F2 population was always close to 3:1. Pairs of parental plants Their F2 progeny Dominant traits ( A ) Recessive traits ( a ) Number of F2 plants examined Dominant:recessive trait ratio in the F2 population Green pods Yellow pods 580 2.82:1 Axial flowers Terminal flowers 858 3.14:1 Red flowers White flowers 929 3.15:1 Long stems Short stems 1064 2.84:1 Inflated pods Constricted pods 1181 2.95:1 Round seeds Wrinkled seeds 7324 2.96:1 Yellow seeds Green seeds 8023 3.01:1 2.3. Bateson's notation for successive stages in breeding experiments The following account uses the notation proposed by Bateson [ 6 ] for successive generations arising from sexual reproduction:- P = the original male and female parental generations; F1 = the first filial progeny population arising from crosses between plants of the P generation; F2 = the second filial generation that arises from sexual reproduction by members of the F1 generation – and so on. The advantage of Bateson's notation is that it does not depend on any preconceived ideas about the mechanisms of inheritance of traits during sexual reproduction. It can therefore be used to describe the stages in Mendel's experiments without misrepresenting any of his observations, arguments or conclusions. 2.4. Mendel's initial observations summarised Table 1 shows the results of seven different cross-fertilisations between parental (P) plants displaying alternative forms of the same trait, e.g. red rather than white forms of the trait "flower colour"; all individual plants in the F1 population displayed only one of the two parental trait forms. Also shown are the results observed by Mendel when he allowed these F1 plants to self-fertilise; the ratio of ( A ) form to ( a ) form plants was, in every case, close to 3:1. Mendel also carried out experiments in which he cross-fertilised plants displaying concurrently two or three trait differences, and then recorded the occurrence of each trait in the F1 and F2 generations. These results are not shown here but they were consistent with the findings exemplified in Table 1 . These initial findings led Mendel to a remarkable generalisation and a definition. (i) All plants in the F1 population displayed only one of any two differing trait forms ( A ) and ( a ) displayed by the parental (P) plants. (ii) He defined the trait form that was displayed in the F1 plants as das dominirende Merkmal ( A ) – the dominating trait (A) . He defined the alternative trait form, which did not appear in any of the F1 plants, as das recessivem Merkmal (a) – the recessive trait ( a ). 2.5. Further experiments Mendel now faced the problem of explaining how the 3(dominant):1(recessive) trait ratio arose in the F2 population of plants (Table 1 ). In further experiments on each of the seven crosses shown in Table 1 , he was able to show that those F2 plants he had identified by the symbol ( a ) were 'constant form' (true-breeding) plants; i.e., when they were allowed to self-fertilise, all their F3 progeny displayed the same parental trait ( a ). On the other hand, when F2 plants initially identified by the symbol ( A ) were allowed to self-fertilise some proved to be 'constant form' plants because, when they were allowed to self-fertilise, they produced F3 progeny that again displayed this same parental trait ( A ). But other plants initially identified by the symbol ( A ) in the F2 population were not 'constant form' plants. Some of their F3 progeny did display the original parental trait ( A ). Other plants in the same F3 population displayed the alternative parental trait ( a ). Yet other plants in this F3 population were again not 'constant form' plants. They were like the F1 plants (their "grandparents") and like the F2 parents from which they were immediately derived. When they were allowed to self-fertilise, some of their progeny displayed the ( A ) form, some the ( a ) form of trait and some were again like the F1 plants. The experimental procedures Mendel used to make these distinctions are readily understood by reading a reprint of the original paper or a reliable translation. Given this ability to distinguish, by experiment, between those plants initially designated ( A ) and those now designated ( Aa ), Mendel was able to state the average distribution of trait forms among the plants of the F2 population as (one dominant: two hybrid: one recessive) or, in his notation, (A + 2 Aa + a) ; i.e. the 3:1 trait ratio factored into the proportions 1:2:1. Mendel was now able to add a further generalisation: When F1 plants were allowed to self-fertilise, 1/4 of the F2 population displayed the 'constant form' parental trait (A) that was displayed by the F1 plants, 1/4 displayed the 'constant form' parental trait (a) that did not appear in any of the F1 plants (Table 1 ), and 1/2 were hybrids ( Aa ) that displayed only the dominant trait ( A ) but were not 'constant form' plants. 2.6. Mendel's notation Mendel used upper case and lower case italicised letters throughout his paper to denote, by definition, dominant and recessive traits . Examples have already been given of the use of letters ( A ) and ( a ) when only one trait difference between parental plants was tested (Table 1 ). Mendel made similar use of the letters ( B ) and ( b ), ( C ) and ( c ) when he described experiments in which two or three trait differences were displayed concurrently. For reasons given in Section 2.5 these single letters also designated what Mendel called 'constant forms' of traits. Plants displaying these traits were 'true-breeders'; they were the parental plants he used in cross-fertilisations (Table 1 ). There is one further crucial feature of Mendel's single letter notation for 'constant form' traits. These letters ( A, a, B, b, C, c ) did not represent the structure or composition of the traits. All the traits shown in Table 1 obviously had complex compositions. But, irrespective of such complexity, each dominant trait was denoted by ( A ) and each recessive trait by ( a ) in Table 1 . The traits were what Mendel could see with his own eyes. He distinguished a dominant trait from a recessive trait by qualitative observations. He was not concerned with and did not analyse the structural composition of the traits. The letters ( A, a, B, b, C, c ) represented classes of traits – a dominant class represented by an upper case letter, and a recessive class of trait represented by the corresponding lower case letter (Table 1 ). It is necessary to recognise these facts if a rational explanation for Mendel's observation is to be obtained; and if gross misrepresentations of Mendel's paper are to be detected. Why then did Mendel use a combination of letters (e.g. Aa ) to represent hybrid plants? This will become clear in section 2.7. 2.7. Postulates and arguments; Mendel's explanations of his observations Mendel accounted for the two generalisations (section 2.4) by the following postulates and arguments; they were based on his further experiments (section 2.5):- (1) All the F1 plants were hybrids ( Aa ) in welcher beide Merkmale vereinigt sind – in which both (parental) traits ( A and a ) were united; trait ( a ) was not displayed by these hybrids, so that these hybrids displayed what he had defined as the dominating trait ( A ) only. (2) The traits ( A ) and ( a ) in the F1 hybrids ( Aa ) segregated into traits ( A ) and ( a ) during formation of the male pollen (sperm) cells and also during formation of the female germinal cells (ova). Thus, each pollen cell and each germinal cell carried only one trait – either ( A ) or ( a ) but not both. (3) Fertilisation of one germinal cell by one pollen cell was a random event. (4) When a pollen cell bearing trait ( A ) fertilised a germinal cell bearing the same trait ( A ), all their progeny displayed the trait ( A ). Likewise, when a pollen cell bearing a trait ( a ) fertilised a germinal cell bearing the same trait ( a ), all their progeny displayed the trait ( a ). But when a pollen cell bearing trait ( A ) fertilised a germinal cell bearing the alternative trait ( a ), the resulting plant was the hybrid ( Aa ); if the pollen cell displaying a trait ( a ) fertilised a germinal cell displaying the alternative trait ( A ), the outcome was again a hybrid ( Aa ). In either event, the hybrid ( Aa ) displayed only the dominant trait ( A ). (5) Mendel illustrated these postulates and explanations in a diagram (Figure 1 ) showing the consequences of self-fertilisation of F1 hybrids ( Aa ), given that traits ( A ) and ( a ) in the hybrid ( Aa ) first segregated into individual pollen cells (sperm) and individual germinal cells (ova) before recombining, in random fashion, during formation of the F2 population. The arrows in Figure 1 represent the fertilising event. Figure 1 Mendel's diagrammatic explanation for the formation of the F2 population of plants produced by self-fertilisation of his F1 hybrids. Mendel proposed that F1 hybrids ( Aa ) contained a dominant trait ( A ) that was displayed and a recessive trait ( a ) that was not displayed. Self-fertilisation of F1 hybrids ( Aa ) then involved segregation of the component traits ( A ) and ( a ) into individual male pollen and female germinal cells, as shown in his diagram. Mendel proposed that if a male pollen cell carrying a trait ( A ) fertilised a female germinal cell carrying the same trait ( A ), the progeny would display trait ( A ). He used the analogous argument for the generation of progeny bearing trait ( a ). Only if male and female sex cells carried differing forms of a given trait ( A or a but not both) would the progeny be hybrids ( Aa ). Thus random recombination of the segregated traits during self-fertilisation of hybrids would yield (on average) the F2 population of plants represented by the trait series ( A + 2 Aa + a ) shown below Mendel's original diagram. Note two crucial points:- (i) Mendel observed and recorded the occurrence of traits ( die Merkmale ) or the characters (die Charaktere) in his plants and their seeds, not the mechanisms underpinning these occurrences. These mechanisms could not have been investigated in 1866. (ii) All Mendel's explanations were based solely on observations of the changes in the occurrence of alternative traits in successive populations that arose from cross-or self-fertilisations and back-crosses. 2.8. Comment Mendel was a well-trained scientist [ 7 ], an astute thinker, a careful and systematic experimentalist, an expert hybridiser and an exemplary writer but he was not the first geneticist. That title should go, possibly, to Bateson [ 6 , 8 , 9 ] for advocating Mendel's experimental methods, for showing that Mendel's findings could be repeated in animals, and for emphasising that combination , segregation and recombination of traits during gametogenesis was the most important feature of Mendel's work. Moreover, Bateson realised [[ 6 ]; in a footnote on page 133] that the occurrence of alkaptonuria, one of the "Inborn Errors of Metabolism" first reported by Garrod [ 10 - 12 ], was an example of Mendelian recessivity of a trait or character. Bateson, incidentally, coined the word "genetics". Another leading contender for the title "the first geneticist" was the Danish biologist, Johannsen [ 13 , 14 ], an equally enterprising experimentalist and astute thinker. Johannsen [ 14 ] was the first to define the term "das gen ; (plural) die gene" as the determinant of a trait; he was also the first to make a clear distinction between the genotype (der Anlagetypus) and the phenotype (der Erscheinungstypus ) on the basis of his experiments with self-fertilising bean plants. In Johannsen's experiments the weights of individual beans were the characteristics or traits. He had, in effect, repeated Mendel's experiments but by measuring a trait (individual bean weights in successive populations of plants) he was able to introduce three new concepts (gene, genotype and phenotype) that were the most significant, after Mendel's concepts of combination, segregation and recombination of traits during gametogenesis, in understanding the origin of genetic phenomena (the origin of changing traits). Failures to recognise the significance of Johannsen's work [ 13 , 14 ] prevented the development of rational concepts in genetics for at least the first two decades of the 20 th century. This failure is, surprisingly, still evident in current depictions of elementary Mendelian genetics (Section 3). 2.9. The tests of validity applied to Mendel's explanation for his observations It is clear that Mendel's experimental procedures (sections 2.2, 2.5) were sound; his notation was simple, unambiguous and consistently applied (section 2.6). His arguments (section 2.7) for a combination of traits in forming the F1 hybrids ( Aa ) are consistent with his arguments for the segregation of the component traits of the hybrid into separate gametes, and their random recombination in generating the F2 population ( A + 2 Aa + a ). Mendel's arguments pass the test of consistency. It is equally clear (but hitherto not noticed) that Mendel's explanations failed the test of plausibility. Mendel postulated that a F1 hybrid ( Aa ) was formed by combining the two differing traits ( A ) and ( a ) of their parents. He did not explain how a F1 hybrid ( Aa ) displayed only trait ( A ) and how it did not display trait ( a ), even when some F2 plants, like one of the two original parental (P) plants, did display trait ( a ). What explanation could we now give for this selective display of only one of two traits that are said to be combined in a hybrid? It may be (and has been) argued by some that trait ( A ) was displayed by the hybrids ( Aa ) because ( A ) was a dominant trait and ( a ) was a recessive trait. Such statements do not even qualify as a circular argument. They are illogical. Such statements fail to distinguish between an arbitrary definition and a plausible explanation. Mendel's definition of a dominant trait should be seen as an arbitrary device that accounts for his observation (by experiment) that his hybrids ( Aa ) in the F2 populations ( A + 2 Aa + a ) displayed trait ( A ) but not trait ( a ). A word of caution is necessary. Mendel's formulation ( Aa ) for a hybrid was crucial in establishing his consistent arguments; it was also the basis for Bateson's recognition that the essential features of Mendel's work were the concepts of combination, segregation and recombination of alternative traits (i.e., components of the phenotype). If we now wish to replace Mendel's implausible formulation ( Aa ) for a hybrid by a plausible formulation, we face the prospect of abandoning the rest of Mendel's arguments. That is not to say that we abandon admiration for Mendel's work. For its time, it was unsurpassed and should be recognised as one of the important steps in the development of experimental procedures in what became known as genetics. We should take care not to misrepresent Mendel's experiments and his arguments. It will become clear that misrepresentations of Mendel's paper have served only to sustain untenable concepts in current biology. In the post-Mendel era we assert that it is not components of the phenotype that segregate and recombine. It is the alleles (i.e., components of the genotype) that combine, segregate and recombine. May we then anticipate that modern explanations of Mendel's observations will pass the tests of consistency and plausibility? 3. Current accounts of elementary Mendelian genetics 3.1. Explanations of Mendel's observations The currently favoured explanation for Mendelian heredity in general, and in particular for the occurrence of Mendel's 3(dominant):1(recessive) trait ratio, is shown in Figure 2 . Figure 2 The currently favoured depiction of Mendelian inheritance following self-fertilisation of F1 hybrids represented by the allele pair ( Aa ). Section 3.1 of the text records the arguments commonly used in attempts to account for the alleged F2 trait series ( AA + 2 Aa + aa ) and for Mendel's 3(dominant):1(recessive) trait ratio. Sections 3.2 and 3.3 discuss the faults in these arguments. The assertions and descriptions generally attached to Figure 2 are as follows. (i) Mendel explained his experimental results by assuming that particles or factors (now called alleles) determined or specified the observed traits. (ii) ( A ) is a dominant allele; (a) is a recessive allele. (iii) The alleles in the male and female heterozygous somatic cells (Aa) segregate into separate gametes. Each gamete then contains only one dominant allele (A) or only one recessive allele (a). (iv) Fertilisation is a completely random event. Given a large number of fertilisation events, the possible recombinations of alleles are those displayed in the four squares. (v) Therefore the average distribution of the alleles at one diploid locus in the resulting progeny population of individual plants will be ( AA + 2 Aa + aa ). It is then argued that: (vi) The dominant allele pair ( AA ) will give rise to a dominant trait ( AA ). (vii) The recessive allele pair ( aa ) will give rise to a recessive trait ( aa ). (viii) In the heterozygote ( Aa ), the recessive allele is ineffective, or is suppressed by the dominant allele ( A ), so that only the dominant allele ( A ) is expressed in the heterozygote. Expression from one ( A ) is as effective as that from two dominant alleles ( AA ). Thus the heterozygote ( Aa ) expresses a dominant trait. (ix) Therefore the allele series ( AA + 2 Aa + aa ) is expressed (in a population of the progeny plants, animals or cells) as the trait series ( AA + 2 Aa + aa ). (x) This trait series gives rise to Mendel's 3(dominant):1(recessive) trait ratio (by the arguments in vi, vii, viii). 3.2. Faults in these currently favoured descriptions of Mendelian genetics There are seven faults in the descriptions and arguments attached to Figure 2 . (i) Mendel is misrepresented; he did not assume that particles or factors specified the observed traits. It is historically inaccurate and scientifically misleading to suppose that he made any such assumption. (ii) The letters ( A ) and ( a ) are Mendel's notation for dominant and recessive traits (Figure 1 , Table 1 ). If we are to continue to discuss Mendelian genetics, these notations (and the nomenclature dominant and recessive ) should refer to traits alone. (iii) Figure 2 fails to distinguish between the components of the genotype and the components of the phenotype (Johannsen, Section 2.8) because it asserts that alleles are dominant or recessive; and uses the same notation ( A and a ) and the same nomenclature ( dominant and recessive ) for both. (iv) Because we must not confuse alleles with traits, we could reasonably write an allele series as ( UU + 2 Uu + uu ); this states that a given locus, in three genetically related diploid cells, comprises a pair of two normal alleles ( UU ), or one normal and one mutant allele ( Uu ), or a pair of two mutant alleles ( uu ). Mutations change the allele constitution or composition at a locus. The modern (non-Mendelian) notation ( AA + 2 Aa + aa ) in Section 3.1 (items vi, vii) then states explicitly that a dominant trait ( AA ) comprises two aliquots ( A + A ) of some material substance or of two doses of dominance ( A + A ); likewise that a recessive trait ( aa ) is composed of two entitities ( a + a ) or two doses of recessivity. This is simply not true. It was not true in Mendel's time and it is not true today. Furthermore, it is not what Mendel's notation meant. It was pointed out (Sections 2.5, 2.6) that Mendel's notation ( A ) and ( a ) distinguished classes of traits, specifically 'constant form' classes of traits (Table 1 ). To substitute ( AA ) for ( A ) and ( aa ) for ( a ) in a trait series is illogical and indicates a regrettable failure to read Mendel's paper with the care that should be given to one of the classic papers in biology. (v) If the arguments attached to the homozygotes in Figure 2 are sound, they should also apply to the heterozygote. It is argued in Figure 2 that two dominant alleles ( AA ) generate a dominant trait ( AA ); and that two recessive alleles ( aa ) generate a recessive trait ( aa ). In other words, it is asserted that there is a direct, positive, linearly proportional (or additive) relationship between the allele constitution at a gene locus and the constitution of the trait expressed from that locus. If we are to be consistent, the same arguments should apply to the heterozygote ( Aa ). On the contrary, the arguments in section 3.1 (item viii) state that one dominant allele ( A ) in a heterozygote ( Aa ) is as effective as two dominant alleles ( AA ) in the homozygote. The arguments in item (viii) are therefore inconsistent with arguments in items (vi) and (vii). Item (viii) also transfers Mendel's implausible assertion that a hybrid ( Aa ) displays only trait ( A ) to the equally implausible assertion that one allele ( A ) in a heterozygote ( Aa ) is as good as two such alleles in a homozygote ( AA ). The argument in item (viii) that allele ( a ) is ineffective is an extreme case; it is therefore not generally applicable. The alternative argument, that allele ( a ) in a heterozygote is suppressed by the dominant allele ( A ), lacks any experimental support or rational theoretical justification. Items vi, vii and viii attached to Figure 2 are arbitrary, irrational and implausible devices applied to the heterozygote alone; they seem to have been introduced solely in order to arrive at the desired result. (vi) Figure 2 and the attached arguments thus fail to give rational explanations for the occurrence of dominant and recessive traits and for Mendel's 3(dominant):1(recessive) trait ratio. (vii) Figure 2 does not and cannot account for the observation that dominance and recessivity are not observed for all traits. The assertion in Figure 2 that the alleles are themselves "dominant" or "recessive" (and thus determine that traits are dominant or recessive) conflicts with inability of Figure 2 to explain why dominant and recessive traits are not always observed; nor does Figure 2 account for the observation that, when dominance and recessivity do occur, they do not always exhibit a 3:1 trait ratio. 3.3. Comments on these faults It is necessary to restate fault (iii) in section 3.2 in more widely applicable terms. It is illegitimate to use the same notation and nomenclature for a parameter and a variable in the same system. Parameters are those components of any system that are directly accessible to the experimentalist; they can be changed and maintained by the experimentalist at the new value, at least for the duration of an experiment. Variables are those components of the same system that are not directly accessible to the experimentalist; they can be changed and maintained at a new value only by making a finite change in at least one parameter of the system or of its immediate environment. The magnitudes of individual variables, in any system, respond to changes in the magnitude of one or more parameters of the system or of the immediate environment. In the case under discussion, the alleles are parameters (and part of the genotype); the traits are variables (and part of the phenotype). If the parameters and variables of any system of interacting components are represented by the same notation and the same nomenclature, confusion will inevitably result – as illustrated by Figure 2 , by the assertions (i) and (ii) and by the false arguments (vi) to (x). Traits may be dominant or recessive [ 1 ]; alleles cannot also be dominant or recessive. Figure 2 , and the arguments attached to it, fail all tests of consistency and plausibility (Section 2.9); they also fail the test of historical accuracy. 3.4. Another example of the improper transfer of dominance/recessivity from traits to alleles The primary error in Figure 2 is the illegitimate transfer of Mendel's terms "dominant" and "recessive" from traits (variables) to alleles (parameters), followed immediately by the reverse (and perverse) argument that the traits specified by the alleles must be dominant or recessive because the alleles are dominant or recessive . This habit is unscientific. It also occurs in discussion of mutations of non-catalytic proteins. When haemoglobin A (HbA) is mutated to the sickle cell haemoglobin (HbS), the three possible trait forms are correctly depicted as follows: (A/A) – the homologous, normal/normal protein, condition; (A/S) – the heterologous, normal/mutant protein (sickle cell), condition; (S/S) – the homologous, mutant/mutant protein, condition. Contrast these depictions with those sometimes found: (A/A) – the dominant condition; (A/S) – the sickle cell condition; (S/S) – the recessive condition. These latter statements depend solely on the illegitimate transfer of Mendel's terms dominant and recessive from traits (variables) to alleles (parameters) and the contention that, if alleles are themselves dominant or recessive, their expressed traits must always be dominant or recessive. If changes in the composition of non-catalytic proteins do explain the occurrence of Mendel's dominant and recessive traits, we require a demonstration that does not depend on these illogical notions. The sickle cell trait (A/S) in humans is significantly different from the normal trait (A/A). Those carrying the sickle cell (A/S) condition enjoy an advantage in areas where malaria is endemic . They do not die from malaria as frequently as those in the population with the (A/A) condition. The sickle cell condition (A/S) is debilitating but, provided it is not too debilitating, the frequency in the local population of those carrying the (A/S) protein pair is greater than it would be in malaria-free areas. This higher frequency of the sickle-cell (A/S) condition in areas where malaria is endemic is often said to be an example of "over-dominance". The term "over-dominance" is inappropriate. It presumably arose from the illegitimate transfer of the terms dominant and recessive noted above. The appropriate term is "heterozygous superiority". The "superiority" indicates the better chance of surviving in regions where malaria is endemic. 4. Conclusions: beginning a rational explanation for Mendel's observations The illegitimate use in Figure 2 of the same notation ( A and a ), and the same nomenclature (dominant and recessive), to describe an allele series and a trait series can be traced to Sutton [ 15 ]. Sutton asserted that the proportions of the chromosome pairs in the F2 population "would be expressed by the formula AA :2 Aa : aa which is the same as that given for any character in the Mendelian case." Mendel's expression ( A + 2 Aa + a ) gave the proportions of characters in his F2 population as A :2 Aa : a . Sutton gave no justification for rewriting these proportions in the form AA :2 Aa : aa . By writing the expression for chromosome pairs as AA :2 Aa : aa and the expression for the proportions of F2 characters as AA :2 Aa : aa , Sutton established a direct, one-for-one, relationship between pairs of chromosomes and the traits arising from them. This false relationship also persists in the currently favoured depiction of Mendelian genetics (Figure 2 ). Sutton's notation for pairs of chromosomes ( AA :2 Aa : aa ) was later transferred to pairs of alleles (what Sutton described as subunits of the chromosomes). It would be easy to blame Sutton for our present confusions. We should remember that Sutton, and those in the early years of the 20 th Century who copied his error, were struggling to understand the hereditary origin of traits. We may more reasonably ask: Why, one hundred years later, are these obvious errors still one of the features of Figure 2 ? Have these errors not been noticed before or, if they have been noticed, why they have not been corrected? Why also has the inconsistency and the implausibility of the arguments attached to Figure 2 not been noticed or corrected? Why (in both of the examples given in sections 3.3 and 3.4) are alleles (components of the genotype) not distinguished, as they surely should be in genetics, from traits (components of the phenotype) by using different notations and nomenclatures for each? Traits (variables) may be dominant or recessive, as defined by Mendel. Alleles (parameters) are, always have been, and can only be normal or abnormal (mutant). Harris (pages 143–157 in reference [ 16 ]), for example, referred consistently to normal and abnormal alleles (not to dominant and recessive alleles), whereas, as noted above, alkaptonuria was a Mendelian recessive trait or character (page 133 in reference [ 6 ]; page 19 in reference [ 16 ]). A review of 13 textbooks of genetics showed that in 12 instances, dominance and recessivity were defined specifically as properties of genes or alleles. These texts, published between 1982 and 2002, were intended for student use; their definitions of dominance and recessivity ignore Mendel's definition of dominance and recessivity as properties of the traits (sections 2.4, 2.5, 2.6, 2.7); they take no account of the need to distinguish between the parameters and variables of a system of interacting components (section 3.2). In one of these 12 texts, it was further claimed that: "Mendel proposed the existence of what he called particulate unit factors for each trait". In another, that: "Mendel realised that some genes (dominant genes) expressed themselves when present in only one copy". In a third that: "Mendel imagined that during the formation of pollen and egg cells, the two copies of each gene in parents segregate". Of these three quoted texts: The first misrepresented Mendel; he did not "propose the existence of particulate unit factors for each trait". The second misrepresented Mendel by transferring his term dominirende ("dominating") from traits to genes; the second and the third quoted texts ignored the fact that the term " das gen " (plural " die gene ") was first used and its role as the determinant of traits postulated by Johannsen, 43 years after Mendel's paper was published (Section 2.8); Mendel did not mention the word gene (Section 3.2). Of the 13 texts examined, only one gave a definition of dominance and recessivity that would have been recognised by Mendel. Even so, this author contradicted his correct definition of dominance and recessivity as properties of components of the phenotype by giving an explanation of elementary Mendelian genetics that employed Figure 2 and its associated arguments. All 13 of the texts examined ignored or contradicted the verifiable historical evidence (sections 2.2–2.7) and failed to make the obligatory distinction between the functions of alleles and the properties of traits. The correct nomenclature for alleles used by Harris (pages 143–157 in reference [ 16 ]) is, unfortunately, rarely if ever employed by other authors. Pasternak [ 17 ], for example, accepted that "in strict genetic terms, dominance and recessivity are descriptions of the phenotype and not of the genes." but then continued: "However, few textbooks bother to make the distinction, because it was both convenient and highly ingrained for geneticists and others to refer to dominant and recessive alleles." Ingrained it may be, convenient (and scientifically legitimate) it is not. If we continue to propose Figure 2 and the attached arguments as an explanation of Mendel's work, we deceive ourselves and encourage irrational thinking in our students at a time in their education when they are most vulnerable. It is extraordinary that an "explanation", like Figure 2 , should still be found in textbooks intended for student instruction; it exposes our own confusion but explains nothing of scientific value in genetics. Any student who criticised Figure 2 and the attached arguments in an answer to an examination question would have shown commendable scientific insight but, according to current teaching, would be deemed to have failed that question. Barker [ 18 ], writing on another topic, suggested that it might take 50 rather than 25 years for textbooks "to get it right". On the evidence presented here, Barker was too optimistic. The four errors introduced by Sutton [ 15 ] remain uncorrected (Figure 2 ) 100 years later. To be fair to authors of textbooks of genetics, every author inevitably relies on what has been written by preceding authors. However that may be, we are faced with an uncomfortable question. Are we content to continue to deceive ourselves, to give our students a false picture of what Mendel achieved, and to provide them with untenable 'explanation' of his remarkable observations (Figure 2 )? Presumably not, especially when we can very easily begin, in this article, to construct a rational explanation for Mendel's observations and for other observations of current interest in genetics. A fresh approach to the origins of dominant and recessive traits is needed. As a first step, we need to represent normal and mutant alleles by symbols that differ from those ( A, a, B, b, C, c ) used by Mendel to represent traits . We must replace symbols ( A and a ) for alleles in Figure 2 by quite different symbols; e.g. ( U ) to represent a normal allele, not a "dominant allele"; and ( u ) to represent a mutant or abnormal allele, not a "recessive allele" . The F2 allele series in Figure 2 would then be, on average, UU + 2 Uu + uu. Similarly, the trait series in Figure 2 must be replaced by Mendel's notation (A + 2 Aa + a) because, as explained earlier, Mendel was concerned (as we are, first and foremost) only with understanding the origin of two classes of trait – the dominant class ( A ) and recessive class ( a ). We will later be concerned with the quantitative composition of traits. We have, however, already identified an implausibility in Mendel's notation ( Aa ) for a hybrid that, allegedly, displayed the trait ( A ). An implausibility, like an inconsistency, must be eliminated if we are to arrive at an internally consistent and plausible account of Mendel's observations. The implausible notation ( Aa ) can be eliminated by replacing it by the single symbol ( H ) for a hybrid. We have now adopted a stance that, in sharp contrast to Figure 2 , distinguishes clearly between determinants and that which is determined. We have allocated a nomenclature and notation for alleles that is distinct from that allocated to traits. We have differentiated clearly between the parameters of the system (in this particular case, the components of the genotype) and the variables of the system (in this particular case, the components of the phenotype). Mendel found, by experiment, that the proportions of different plant forms in his F2 populations were 1(dominant trait):2(hybrids):1(recessive trait) or, in his notation, ( A + 2 Aa + a ). Replacing Mendel's notation ( Aa ) for a hybrid by the single symbol ( H ) does not alter Mendel's experimental observation of the proportions of trait forms in the F2 populations (section 2.5). It does mean that we can avoid Mendel's implausible postulate that, although recessive trait plants did display trait ( a ), his hybrids ( Aa ) did not. We have, of course, to discover an experimentally verifiable mechanism that would explain why hybrids ( H ) display a trait that is sometimes indistinguishable and sometimes distinguishable from trait ( A ). Our remaining task is to explain rationally how this series of normal and mutant alleles (UU + 2 Uu + uu) in the F2 population is expressed as the trait classes ( A + 2 H + a ) in that population, where all that we have done is to replace Mendel's implausible ( Aa ) by a plausible ( H ). Note also that we have now also eliminated the illegitimate use of paired symbols for Mendel's dominant ( A ) and recessive ( a ) traits. Most of the clues that facilitate this task are present in this article. One clue is missing, but it can be inferred by asking how one allegedly dominant allele ( U ) in a heterozygote ( Uu ) could be as effective as two such alleles ( UU ) in a homozygote. A further article will provide the answers, but in the interval readers may like to rise to the challenge of explaining: (1) how dominant and recessive traits arise from normal and mutant alleles , and (2) why Mendel's 3:1 trait ratio, though not uncommon, does not always occur.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC516238.xml
497040	Integrated web service for improving alignment quality based on segments comparison	Background Defining blocks forming the global protein structure on the basis of local structural regularity is a very fruitful idea, extensively used in description, and prediction of structure from only sequence information. Over many years the secondary structure elements were used as available building blocks with great success. Specially prepared sets of possible structural motifs can be used to describe similarity between very distant, non-homologous proteins. The reason for utilizing the structural information in the description of proteins is straightforward. Structural comparison is able to detect approximately twice as many distant relationships as sequence comparison at the same error rate. Results Here we provide a new fragment library for Local Structure Segment (LSS) prediction called FRAGlib which is integrated with a previously described segment alignment algorithm SEA. A joined FRAGlib/SEA server provides easy access to both algorithms, allowing a one stop alignment service using a novel approach to protein sequence alignment based on a network matching approach. The FRAGlib used as secondary structure prediction achieves only 73% accuracy in Q3 measure, but when combined with the SEA alignment, it achieves a significant improvement in pairwise sequence alignment quality, as compared to previous SEA implementation and other public alignment algorithms. The FRAGlib algorithm takes ~2 min. to search over FRAGlib database for a typical query protein with 500 residues. The SEA service align two typical proteins within circa ~5 min. All supplementary materials (detailed results of all the benchmarks, the list of test proteins and the whole fragments library) are available for download on-line at . Conclusions The joined FRAGlib/SEA server will be a valuable tool both for molecular biologists working on protein sequence analysis and for bioinformaticians developing computational methods of structure prediction and alignment of proteins.	Background Protein structure is obviously modular, with similar structural segments, such as alpha helices and beta strands found in unrelated proteins. Such segments, identified from structure, are used extensively in description and analysis of protein structures [ 1 , 2 ]. Several groups have demonstrated that only a small library of segments is sufficient to rebuild experimental protein structures with high accuracy [ 3 ]. Predicted local structure segments (PLSS) are also used in structural prediction, starting from the nearest neighbor approach to secondary structure prediction [ 4 - 6 ]. This idea was later extended and lead to even more successful applications of PLSSs in ab initio structure prediction by Baker and colleagues, who developed a library of sequence-structure motifs called I-sites [ 7 ]. Those motifs are later assembled in a complete protein structure by a program ROSETTA [ 8 ]. Predicted local structure segments are also used in a novel protein alignment algorithm, based on the comparison of PLSSs for two proteins treated as networks and finding a common path through networks describing the two proteins [ 9 ]. The underlying idea in all those approaches is that because global folding constraints can override local preferences, the prediction of structure segments from local sequence is by necessity uncertain. Therefore, instead of trying to predict a correct local structure, all possible local solutions are identified and other constraints (folded structure in Rosetta, or compatible alignment in SEA) are used to identify a globally consistent solution. Prediction of local structure segments can be approached in two different ways. A first possibility, used in most nearest neighbor secondary structure algorithms, is to use a representative set of proteins with known structure as source of structure segments, but without any restrictions on a number or type of segments. In this approach, we don't make any assumptions about the compositions and distributions of segments in the library and this approach can be compared to unsupervised learning approach. In a second approach, used for instance in the I-site method, only segments from a specifically constructed fragment library are used in prediction, thus this approach is similar to supervised learning. Interestingly, some limited tests suggest that the former approach leads to lower prediction accuracy [ 10 ]. The same tests suggested the possibility that different segment libraries could lead to different prediction, and likely, some segment libraries would be better suited to some tasks. Following this observation, we have developed the FRAGlib – a fragment library specifically designed to complement a segment alignment SEA. SEA alignment algorithm was developed previously in our group [ 9 ] and originally used in conjunction with the I-site library. I-site library [ 7 ] was originally developed to be used in ab initio folding predictions and anecdotal evidence suggested that it may not be ideally suited for alignment purposes. In this note we describe a combined FRAGlib/SEA server and first benchmarking results of this method. Implementation Database of Short Fragments FRAGlib is based on the idea of developing a uniform coverage of all known types of local structural regularity with the distribution based on that observed in natural proteins. The collection of segments is constructed using representative set of proteins from the ASTRAL database [ 11 , 12 ]. For each protein in this set, each continuous segment with regular secondary structure, including the flanking residues on both sides, is added to the FRAGlib (see below for details). We do not utilize any further clustering algorithm so our database contains no-unique entries and it is redundant both in terms of structure and sequence information. Local structure is described by the SLSR (Symbolized Local Stuctures Representation) codes consisting of 11 symbols { HGEeBdbLlxc }, each representing a certain backbone dihedral (phi and psi) region [ 7 , 13 ]. Protein local structure is described as a string of local-structure symbols and a local structure segment is defined as a 5–17 amino acid fragment with constant local structural codes. Segments are then extended by two additional residues offset at the beginning, and at the end of a segment. We store all such segments with their sequence, SLSR style local structures representation codes and the homology profile [ 14 , 15 ], derived from that of their parent protein. The library is highly redundant, i.e. there are many segments with the same structural description, but each of the redundant fragments is coming from a different parent protein (or a different part of the same parent protein), therefore it has a different sequence and a different profile associated with it. FRAGlib prediction In a next step, FRAGlib segment library is used to assign local structure segments for a new protein (query) based only on sequence information using a variant of the FFAS profile-profile alignment algorithm [ 16 ]. A profile for the query protein is calculated following the FFAS protocol, then for all possible overlapping segments of length from 7 to 19 amino acids, their profiles are compared to those of the segments from the FRAGlib database and the score of each alignment is calculated using a FFAS-like scalar product of composition vectors at each position. Since the segments being compared have the same length, no dynamic programming alignment is necessary and the score calculation can be highly optimized. As the result of this procedure, each position in the query protein can be assigned to all of the possible LSSs in the database, each with a specific score (see Figure 1 ). Only reduced sets of predicted LSSs, rather arbitrarily limited to the first 20 highest scoring segments are kept for further analysis. This cut-off is the only free parameter of the method, and can be set by user using the Web interface of the server. The Q3 quality of the FRAGlib used as a secondary structure prediction algorithm (data not shown), with the prediction based on the single best scoring segment for each position is 73% on a standard secondary structure prediction benchmark. The Q3 gives percentage of residues predicted correctly as helix, strand, and coil or for all three conformational states. SEA Segment Alignment Approach to Protein Comparison The principal motivation to develop the FRAGlib segment prediction was to further improve the alignment quality for comparing distantly related proteins, which is one of the most important problems in practical application of comparative modeling and fold recognition [ 17 ]. To address this problem, we have previously developed a SEA algorithm, which compares the network of predicted local structure segments (PLSSs) for two proteins using the network matching approach. In a previous paper we have demonstrated that the SEA algorithm, using I-site server for PLSSs prediction and a simple sequence-sequence scoring for segment comparison resulted in alignments better than the FFAS profile-profile alignment algorithm and several other alignment tools. A full description of the SEA algorithm is available in the previous manuscript [ 9 ], so only a brief summary is presented here. Every residue in each of the proteins being aligned is described as a vertex in the graph. Two artificial vertices are added to the very beginning of each protein as a source vertex, and also at the end as a sink vertex. For each PLSS is described as an edge between the vertices representing its first and last positions. For some PLSS protocols, some parts of the protein may not be covered by any predicted segments, so virtual edges are added to all neighbor residues to form a complete, continuous network. Each assembly of connected PLSSs corresponds to a path in this network. In a next step, PLSSs networks of two proteins are compared by the SEA algorithm. For each pair of positions i and j , with position i coming form the first protein and position j from the second protein, all possible segments covering each of the positions must be considered in a combinatorial way and compared to get the optimal similarity score. It is not the sequences or secondary structures at two positions that are compared, but all segments that cover these two positions. This is the main feature of SEA that makes it different from standard sequence pair-wise alignments. The computational complexity of SEA is about O ( NMC 1 C 2), where C 1 and C 2 are the average numbers of segments that cover a position in each protein (the segment coverage). Detailed description of the SEA mathematical algorithm together with benchmarks results obtained using the I-site server calculated PLSSs network can be found elsewhere [ 9 ]. The integrated FRAGlib and SEA server is available at [ 18 ]. The FRAGlib database and segment prediction provides the PLSSs network for each aligned protein, and the SEA algorithm aligns the two networks. On Figure 2 we present the flowchart of the integrated web service. Preliminary benchmarks for the FRAGlib/SEA server and presented below. A full paper on the FRAGlib algorithm is in preparation. Results and Discussion We use here as a benchmark the database of 409 family-level similar pairs [ 19 ]. Each protein pair shares at least one similar domain as identified by SCOP [ 20 ]. Segments coming from the proteins of the same SCOP family as the proteins being compared were removed from the FRAGlib calculated PLSSs network. Further analysis of the SEA results also confirmed that the memorization is not a problem here, as all the SEA alignment are build predominantly from segments that are not locally optimal. To evaluate the improvement we use two measures of alignment quality: the classical root mean square deviation (RMSD) and the shift score [ 1 ]. The shift score measures misalignment between a predicted alignment of two proteins and the reference alignment. The shift score measure ranges from -ε(default as -0.2) to 1.0, where 1.0 means an identical alignment. RMSD is dependent on alignment length and the shift score is dependent on the reference alignment, so both measures are less than perfect in comparing alignments. In our case we use as the reference alignment provided by the CE structural method [ 21 ]. We chose the CE, which is available as a single file executable for various operating systems, as an example of purely structural alignment tool. It is a method for fast calculation of pairwise structure alignments, which aligns two proteins chains using characteristics of their local geometry as defined by vectors between Cα positions. Heuristics are used there in defining a set of optimal paths joining termed aligned fragment pairs with gaps as needed. The path with the best RMSD is subject to dynamic programming in order to achieve an optimal alignment. For specific families of proteins additional characteristics are used to weight the alignment. 'Table 1 [see Additional file 5]' compared the quality of the FRAGlib/SEA (identified as SEA F in the Table) alignment with that of the structural alignment prepared with the CE algorithm [ 21 ] and the SEA algorithm used with I-site segment prediction (SEA I ), SEA algorithm used with the actual (not predicted) local structure segments (SEA T ), local single predicted structures (SEA loc ) and few other publicly available alignment tools. All the results other than the FRAGlib/SEA alignments, as well as alignment quality evaluation, were adopted from the original SEA manuscript [ 9 ]. The results presented in 'Table 1 [see Additional file 5]' show that SEA F significantly improves the alignment quality as compared to all other methods, including SEA I (SEA using I-site prediction), bringing it close to (and in the shift based quality measure actually improving on) the SEA algorithm using the actual structure segments. Conclusions The benchmarks show that SEA with FRAGlib (SEA F ) integrated prediction service better incorporate diversities of local structure predictions over known methods. It produces also more accurate alignments in comparison to SEA I (based on the I-site library), or the SEA with single predicted structures (SEA loc ). Comparing those sequence pairwise alignments we can observe that predicted local structure information seems to improve the alignment qualities. Alignments from SEA using FRAGlib method of describing diversities of local structure prediction have the same quality as alignments using true local structures derived from their known 3D structures SEA T . Availability and requirements An integrated SEA/FRAGlib server is available at [ 18 ]. Both components can be used separately, SEA alignment with arbitrary PLSSs and FRAGlib for other purposes than segment alignment, but the integrated server provides the complete alignment method for comparing pairs of protein sequences using a network matching algorithm. The fragments library prediction method (FRAGlib) is also available as the separate http server at [ 22 ]. The software is freely available to academics. Contact Dariusz Plewczynski darman@bioinfo.pl or Adam Godzik adam@burnham.org for information on obtaining the local copy of a software. Authors' contributions DP designed, implemented, and evaluated the FRAGlib program. The benchmark dataset and programme for aligning two short sequence profiles were provided by LJ. The integration of FRAGlib predictions within SEA network alignment software together with benchmark evaluation of the SEA method was done by YY. AG was responsible for the overall project coordination. All authors have read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC497040.xml
520749	Methylation profiles of thirty four promoter-CpG islands and concordant methylation behaviours of sixteen genes that may contribute to carcinogenesis of astrocytoma	Background Astrocytoma is a common aggressive intracranial tumor and presents a formidable challenge in the clinic. Association of altered DNA methylation patterns of the promoter CpG islands with the expression profile of cancer-related genes, has been found in many human tumors. Therefore, DNA methylation status as such may serve as an epigenetic biomarker for both diagnosis and prognosis of human tumors, including astrocytoma. Methods We used the methylation specific PCR in conjunction with sequencing verification to establish the methylation profile of the promoter CpG island of thirty four genes in astrocytoma tissues from fifty three patients (The WHO grading:. I: 14, II: 15, III: 12 and IV: 12 cases, respectively). In addition, compatible tissues (normal tissues distant from lesion) from three non-astrocytoma patients were included as the control. Results Seventeen genes ( ABL, APC, APAF1, BRCA1, CSPG2, DAPK1, hMLH1, LKB1, PTEN, p14 ARF , p15 INK4b , p27 KIP1 , p57 KIP2 , RASSF1C, RB1, SURVIVIN , and VHL ) displayed a uniformly unmethylated pattern in all the astrocytoma and non-astrocytoma tissues examined. However, the MAGEA1 gene that was inactivated and hypermethylated in non-astrocytoma tissues, was partially demethylated in 24.5% of the astrocytoma tissues (co-existence of the hypermethylated and demethylated alleles). Of the astrocytoma associated hypermethylated genes, the methylation pattern of the CDH13, cyclin a1, DBCCR1, EPO, MYOD1 , and p16 INK4a genes changed in no more than 5.66% (3/53) of astrocytoma tissues compared to non-astrocytoma controls, while the RASSF1A, p73, AR, MGMT, CDH1, OCT6,, MT1A, WT1 , and IRF7 genes were more frequently hypermethylated in 69.8%, 47.2%, 41.5%, 35.8%, 32%, 30.2%, 30.2%, 30.2% and 26.4% of astrocytoma tissues, respectively. Demethylation mediated inducible expression of the CDH13, MAGEA1, MGMT, p73 and RASSF1A genes was established in an astrocytoma cell line (U251), demonstrating that expression of these genes is likely regulated by DNA methylation. AR gene hypermethylation was found exclusively in female patients (22/27, 81%, 0/26, 0%, P < 0.001), while the IRF7 gene hypermethylation preferentially occurred in the male counterparts (11/26, 42.3% to 3/27, 11%, P < 0.05). Applying the mathematic method "the Discovery of Association Rules", we have identified groups consisting of up to three genes that more likely display the altered methylation patterns in concert in astrocytoma. Conclusions Of the thirty four genes examined, sixteen genes exhibited astrocytoma associated changes in the methylation profile. In addition to the possible pathological significance, the established concordant methylation profiles of the subsets consisting of two to three target genes may provide useful clues to the development of the useful prognostic as well as diagnostic assays for astrocytoma.	Background Diffusely infiltrating astrocytoma is a leading group of the primary central nervous system tumors, accounting for more than 60% of all primary brain tumors [ 1 , 2 ]. It may arise aggressively from the normal astrocytes, or evolve stepwise from the less its benign precursors. Owing to the difficulties with its early diagnosis and surgical removal of all residue diseased tissues, rapid progression, and frequent reoccurrence, the most advanced form of astrocytoma, glioblastoma (WHO grading IV) represents an extremely life-threatening intracranial malignant tumor both inside and outside of China [ 1 , 2 ]. Molecular genetic analyses have demonstrated multiple genetic lesions implicating to pathogenesis of astrocytoma, glioblastoma in particular. In addition to the frequent amplification and deletion of the EGF receptor gene ( EGFR ) [ 3 ], the main genetic events affecting the following tumor suppressor genes: the members of the INK4A initiated cell-cycle arrest pathway (the p16 INK4a ) [ 4 ], the p14 ARF [ 5 ], the RB1 [ 6 ] and the p53 [ 7 ]), a wide spectrum of the cell surface receptor genes (i.e., CD44 , integrin , and receptors for various growth factors), and the PTEN genes [ 8 ]. Transcription in eukaryotes is regulated at multiple levels and inversely correlated with the hypermethylated state as well as the chromatin condensation. It has been well established that the methylation status of CpG islands directly affects the DNA-protein interactions by eliminating the otherwise occurring sequence specific binding of the transcription factors whereas inducing the DNA-bindings of members of the methyl-CpG binding protein family (MBD). Histone modifications (deacetylation and methylation) may occur subsequently leading to chromatin condensation and a long-term transcriptional silencing status of the affected DNA segments. Over 40% of the protein coding genes have at least one CpG island within or near to their promoter, an strong indication for transcription of which is likely to be under the control of DNA methylation status. Three DNA methyl transferases are involved in the control of the methylation state of the CpGs in genome. DNA methyl transferase I is mainly responsible for the maintenance of the methylation status of the genome after DNA replication, while IIIA and IIIB act principally in the de novo DNA methylation in the early development of high eukaryotes. DNA methylation patterns in somatic cells are established during the early development and contribute to the allele-specific transcription silencing of the imprinted genes, including the silenced alleles in the X-chromosome and other chromosomes. The epigenetic pattern (the DNA methylation profiles of the genome) in high eukaryotes is integral to the normal execution of the biological activities in cells and needs to be actively maintained. In addition to the changes linked to the cell lineage specific pattern of gene expression, both global hypomethylation and local hypermethylation of the CpG islands occur progressively as cell ages. Aberrant DNA methylation pattern changes gene transcription that has been etiologically linked to cancer formation [ 9 , 10 ]. The genome-wide hypomethylation has been believed to activate transcription of the otherwise silenced transposon like repetitive sequences (such as the Alu and LINE repeats in mammals). As a result, the transposition occurs more prevalently so that the genomic instability in cancer cells will be significantly increased [ 11 - 13 ]. The hypermethylated state of the promoter CpG islands has been etiologically associated with transcription inactivation of a number of tumor suppressor genes in tumors, which are hypomethylated and transcribed in their normal counterparts. Therefore, the hypermethylated CpG island(s) of those genes have been regarded as a defect, reminiscent to the loss of heterozygosity or other types of genetic deletion for total inactivation of the tumor suppressor genes in cancer. The most noticeable example is the p16 INK4a gene that has been frequently hypermethylated in almost all types of the tumors examined [ 14 - 17 ] including hepatocellular carcinoma [ 18 ]. The loss of the genetic imprinting (changes in DNA methylation status) has been found to reactivate transcription of the otherwise silenced allele of the genes such as the insulin like growth factor 2 gene, which has been well documented in human tumors [ 19 ]. On the other hand, the reverse process, i.e., demethylation of the promoter CpG island, has also been found instrumental to the transcription activation of the otherwise inert genes in tumor cells [ 20 ]. A prominent example is the gene encoding the melanoma antigen, MAGEA1 that was hypermethylated and transcriptionally silenced in the normal liver tissues, and demethylated prevalently in the hepatocellular carcinoma tissues [ 18 ], correlating well with the elevated level of its expression in HCC [ 21 , 22 ]. The over-expressed gene, SURVIVIN , has also been reported to be demethylated in human ovarian cancer [ 23 ]. Despite of the fact that the elevated levels of expression of three DNA methyl transferase genes were detected in virtually all cancers, the profiles of the hypermethylated genes vary with both the types and stages of cancers. Therefore, the undefined defects in the epigenetic homeostasis during carcinogenesis, rather than the aberrant expression of any given DNA methyl transferase, are more likely to account for the cancer type specific pattern of DNA methylation at both global and local levels. Methylation profiling of the promoter CpG islands has been an important information gathering process for new insights into our understanding of the role of DNA methylation in both initiation and progression of human carcinogenesis. It would result in development of the DNA methylation based assays for cancer diagnosis as well as identification of the cancer genes suffering from the epigenetic defects . However, as the majority of studies had only targeted one or a few genes in rather small patient groups, the concurrent hypermethylation behavior of multiple genes has only been addressed in a limited number of tumor types, such as colorectal cancer. The majority, if not all, of the previous studies on the astrocytoma associated changes in methylation profiles have only examined a small number of genes for methylation status at the promoter CpG island [ 25 , 26 ]. In this study, we determined the methylation profiles of as many as thirty four genes in a cohort consisting of 53 astrocytoma patients and established the concordant methylation behavior of up to three targets. Our observations should provide new insights into the DNA methylation epigenetic defects in human astrocytoma. Methods All the experiments were performed according to protocols described previously [ 18 ]. The primer pairs for the methylation specific PCR were either adopted ( APC, BRCA1, CDH1, DAPK1, hMLH1, p14 ARF , p15 INK4b , RASSF1A, RB1 and VHL ) or designed according to the same principle with assistance of the software packages for the CpG islands identification and the primer design [ Additional file 1 ]. Tissue samples Tissue samples and DNA preparation With the informed consent of all patients and approval of the ethics committee, the tumor samples were collected from astrocytoma patients (n = 53) during operation at the Tiantan Neurosurgical Hospital in Beijing. The pathological classification of tumor tissues was carried out and the stage of each astrocytoma patients was determined according to the WHO classification [ 1 ]. No significant geographic impart was observed as patients came from different places in China and went to Beijing for treatment. In addition, the compatible tissues (normal tissues distant from the lesions) were surgically obtained from three non-astrocytoma patients [gangliocytoma (21 years old, male), angiocavernoma (49 years old, male) and meningioma (54 years old, female)] as the normal controls, which have been subjected to the proper pathological evaluation. Total genomic DNA was extracted from frozen tissue specimens (50 – 100 mg) according to a standard protocol with some modifications [ 18 , 27 ]. Frozen pulverized powders of the specimens were re-suspended with 2 ml lysis buffer: 50 mM Tris-HCl pH 8.0, 50 mM EDTA, 1% SDS, 10 mM NaCl plus 100 μg/ml boiling-treated RNase A (Sigma). Following one hour of incubation at 37°C, Proteinase K (Roche, USA) was added to the cellular lysates for a final concentration of 100 μg/ml and the digestion was carried out at 55°C for 2 hours. Organic extractions with a half volume of Phenol/Chloroform/Isoamyl alcohol (1:1:0.04) were repeatedly carried out until no visible interphase remained after centrifugation. DNA was precipitated from the aqueous phase in the presence of 0.3 M NaOAc pH 7.0 and two and a half volumes of ethanol and followed by one 70% ethanol-washing and dissolved at 65°C for 30 minutes with 0.2 – 0.4 ml TE (10 mM Tris-HCl pH 7.4 and 1 mM EDTA)and stored at 4°C till use. The DNA concentrations were calculated according to the OD 260 nm readings. Bisulphate treatment of DNA and Methylation specific PCR (MSP) The methylation status of the promoter CpG islands of thirty four genes in all DNA samples was analyzed by MSP on the sodium-bisulfite converted DNA [ 18 ]. In detail, 10 μg DNA in 50 μl TE was incubated with 5.5 μl of 3 M NaOH at 37°C for 10 minutes, followed by a 16 hour treatment at 50°C after adding 30 μl of freshly prepared 10 mM hydroquinone and 520 μl of freshly prepared 3.6 M sodium-bisulfite at pH 5.0. The DNA was desalted using a home-made dialysis system with 1% agarose (detailed protocol will be provided upon request). The DNA in the desalted sample (approximately 100 μl in volume) was denatured at 37°C for 15 minutes with 5.5 μl of 3 M NaOH followed by ethanol precipitation with 33 μl 8 M NH 4 OAC and 300 μl ethanol. After washing with 70% ethanol, the gently dried DNA pellet was dissolved with 30 μl TE at 65°C for 10 min. The DNA sample was finally stored at -20°C until further use. PCR reaction was carried out in a volume of 15 μl with 50 ng or less template DNA with FastStart Taq polymerase (Roche, Germany) as follows. After an initial heat denaturing step 4 minutes treatment at 94°C, 30 cycles of 92°C for 15 sec, varying temperatures with primer pairs ( Additional file 1 ) for 15 sec and 72°C for 20 sec, was carried out. The PCR products were separated by 1.2% ethidium bromide containing agarose gel electrophoresis with 1 × TAE and visualized under UV illumination. To verify the PCR results, representative bands from each target were gel-purified and cloned into T-vector (Promega, USA) followed by automatic DNA sequencing provided by BuoCai (Shanghai, China). Only verified results are presented in this report. To optimize the MSP procedure, the M. Sss I treated DNA was used as the methylated control template. In detail, the DNA from a normal liver tissue of the healthy liver donor [ 18 , 24 ] was batch cleaved with EcoR I, followed by M. Sss I treatment according to the manufacture's instruction (New England Biol., Boston, USA) for over night. The purified DNA was bisulphate treated as usual and subjected to MSP with the primer pairs for each of thirty three genes (except for the MAGEA1 gene), and only the verified targets were included for the study of the astrocytoma tissues. Statistical analysis The methylation data were dichotomized as 1 for the co-existence of the methylated and unmethylated alleles, 2 for methylated allele only and 0 for the unmethylated for both alleles to facilitate statistical analysis using contingency tables. The methylation profiles of each individual gene (in percentage) classified by the genders and grading of the patients were presented both in table and in plot. The statistic analyses for the association between the methylation profile of the gene and each of the clinical-pathological parameters were carried out with the statistics package , where both Pearsong's Chi-square test with Upton's adjustment and Fisher exact test were used to examine the tissue samples with the low expected values. The relative frequency with a 95% confidence interval (P < 0.05) for a binomial distribution was calculated for the whole as well as each subtype of astrocytoma patients. The concordant methylation behavior of the genes was established by comparing frequency of co-occurrence of 2 to 3 target subsets with a mathematic method, namely Discovery of Association Rules [ 28 ], which is frequently utilized for association analysis. Demethylation of U251 cells with 5-Aza-2'-deoxycytidine U251 cells (an established glioma cell line) were cultured in DEME plus 10% new born calf serum at 37°C in a 5% CO 2 atmosphere. When cell culture reached 50% confluence, they were treated with 5-Aza-2'-deoxycytidine (Sigma A3656) at the final concentration 10 and 20 nM, respectively for 3 days. The primer pairs for the RT-PCR (Table 1 ) was either adopted from published papers or designed with an assistance of the software . The total RNA was extracted with Trizol solution according to manufacturer's instruction (Invitrogen, USA), and cDNA was obtained using the Supertranscript plus reverse transcriptase with the oligo-dT as primers. PCR with single pair of the target primers run for 15 cycles, followed by another 15 cycle PCR reactions in the presence of the beta-actin primers (Table 1 ) (the parameter of each cycle is 94°C 20", 60°C for 20" and 72°C for 30"). The resulted PCR products were visualized under UV illumination after an electrophoretic separation on a 1.2% agarose. The methylation status of the target was analyzed by MSP. Table 1 The primers for RT-PCR analysis Primer Name sequence PCR Product Length (bp) Accession Number beta-actin L AAGTACTCCGTGTGGATCGG 616 NM_001101 beta-actin R TCAAGTTGGGGGACAAAAAG cdh13f GCTGGACTGGATGTTGGATT 246 NM_001257 cdh13t TTGAGGGTTGGTGTGGATTT magea1rf ACCTGACCCAGGCTCTGT 401 NM_004988 magea1rt CTCACTGGGTTGCCTCTG mgmtrf AAACGCACCACACTGGAC 404 NM_002412 imgmtrt AGGATGGGGACAGGATTG p73f AGATGAGCAGCAGCCACAG 218 NM_005427 p73t GTACTGCTCGGGGATCTTCA rassf1arf GTCTGCCTGGACTGTTGC 401 NM_007182 rassf1art AGCAGGGCCTCAATGACT Results and discussion Clinical-pathological classification To establish the methylation profile of thirty four genes during the process of astrocytoma development, we recruited 53 astrocytoma patients (27 female and 26 male; 49 primary and 4 recurrent) for this study. 14 cases were pathologically classified at the Grade I pilocytic astrocytoma (10–62 years old, mean: 39.1; 9 female, 5 male), 15 cases at the Grade II diffuse astrocytoma (4–50 years old, mean: 33.1; 10 female, 5 male), 12 cases at the Grade III anaplastic astrocytoma (1–72 years old; mean: 40.4; 4 female, 8 male), and 12 cases (including 4 recurrent cases) at the Grade IV glioblastoma (22–66 years old, mean: 44.6; SD = 22–66, 4 female, 8 male) (Table 2 ). The normal brain tissues distant from the lesions were also obtained from three non-astrocytoma patients who underwent brain surgery as normal controls in this study. Table 2 The clinical and pathological profiles of the patients Astrocytoma Non astrocytoma Gender female 27 1 male 26 2 Age, y <40 27 1 40–60 23 2 >60 3 0 Grade Age Mean Range I 39.1 10 to 62 14 II 33.1 4 to 50 15 III 40.4 1 to 72 12 IV 44.6 22 to 66 12 Recurrent 4 Primary 8 Aberrant Methylation profiling in astrocytoma The technical considerations The methylation-specific PCR (MSP) is widely used for methylation profiling of the genes in human cancers for both its easiness and sensitivity. However, the necessary steps have to be taken to eliminate both false positive and negative results. Comparing the MSP-data with the non-PCR data by Southern analysis of the methylation sensitive restriction enzyme is a valuable choice, as our previous work where the hypomethylated status of both p14 ARF and p15 INK4b genes shown by MSP was confirmed by Southern analysis [ 18 ]. Alternatively, the PCR reaction with the in vitro methylated genomic DNA (by M. Sss I) as template would be an ideal positive control for the absence of methylated targets in tumor tissue samples. By taking extra caution, we carried out MSP of all the targets with the M. Sss I treated normal liver DNA as positive control templates, except for the MAGEA1 gene was unmethylated in the normal liver tissue. While only the PCR reaction designated to the unmethylated template gave rise to the detectable bands with the parental DNA, the PCR bands were evident in both reactions with the M. Sss I treated DNA ( Additional File 2 ). Therefore, failure to detect the methylated alleles with the tissue samples should genuinely reflect the lack of methylated targets. To control the false positive with either pair of primers, the representative PCR products, were T-cloned and sequenced. Only the positive PCR results with the expected sequence profiles were scored and analyzed further. The methylation profiling of thirty four targets in astrocytoma Eleven of the thirty four target genes were previously studied either in astrocytoma or other types of tumors. The published PCR conditions for these genes: APC, BRCA1, CDH1, DAPK1, hMLH1, p14 ARF , p15 INK4b , p16 INK4a RASSF1A, RB1 and VHL ( Additional file 1 ) were adopted to enable the relevant inter-study comparisons if necessary. The remaining twenty three targets were selected from a list of genes displaying the altered pattern of the promoter CpG island in various biological settings including cancers. Their CpG islands were identified via bioinformatical tools and the primer pairs were designed accordingly [ 18 , 24 ]. Some of these thirty four genes have been shown to play a role in carcinogenesis, whereas the others have no obvious association with human carcinogenesis. Since it is still disputed whether DNA methylation mediated the gene silencing is causative in the malignant transformation of cell, we specifically selected both sets of genes in this study. The "cancer unrelated" genes selected encode erythropoiesis ( EPO ) [ 29 ], a ubiquitously expressed transcription factor ( OCT6 ) [ 30 ], and the myogenesis lineage-specific transcription factor ( MYOD1 ) [ 31 ]. The majority of the cancer associated genes examined were tumor suppressor genes including genes operating in the RB1/p16 INK4a pathway ( p14 ARF , p15 INK4b , p16 INK4a , and RB1 ) [ 32 ], and two cyclin-dependent kinase inhibitors ( p27 KIP1 [ 33 ] and p57 KIP2 ) [ 34 ]. Other genes in this subset were a p53 analogue:( p73 ) [ 33 , 35 ], two alternative forms of a tumor suppressors in the Ras mediated signal transduction pathway ( RASSF1A , and RASSF1C [ 36 ]), VHL [ 37 ], APC [ 38 ], PTEN [ 6 ], the deleted in bladder cancer chromosome region candidate 1 ( DBCCR1 ) [ 39 ], and the Wilms tumor 1 gene( WT1 ) [ 40 ]. We included the genes encoding the cell membrane proteins or nuclear receptors which act actively in the intercellular interactions: melanoma specific antigen A1 ( MAGEA1 ) [ 41 ], caveolin 1 ( CAV ) [ 42 ], chondroitin sulfate proteoglycan 2 ( CSPG2 ) [ 43 ], androgen receptor ( AR ) [ 44 ], and cadherins ( CDH1 [ 45 ] and CDH13 ) [ 46 ]. Three genes implicated in signal transduction were also selected: cyclin a1 [ 47 ], the interferon regulatory factor 7 ( IRF7 ), and a serine/threonine kinase 1 (Peutz-Jeghers syndrome) gene ( LKB1 ) [ 14 ]. There were the genes encoding the O-6-methylguanine-DNA methyltransferase ( MGMT ) [ 14 ]and metallothionein 1 A gene ( MT1A ) [ 48 ] which play a key role in the cellular response to alkalyting agents and heavy metal stress. The genes acting in DNA repair process were hMLH1 [ 49 ], and BRCA1 [ 50 ], while four genes are involved in apoptosis ( APAF1 [ 51 ], DAPK1 [ 15 ], and SURVIVIN [ 23 ]). Finally, the proto-oncogenes in this group were represented by v-abl homologue 1 ( ABL ) [ 52 ] ( Additional files 3 , 4 , 5 , 6 , 7 , 8 , 9 ). The genes displayed the uniformly unmethylated profiles in astrocytoma Of the unmethylated genes in all samples tested, EPO was a cancer unrelated gene, while "cancer associated" genes included ABL (1), APAF1 (2), APC (3), BRCA1 (5), CAV (6), CDH13 (8), DAPK1 (11), hMLH1 (14), LKB1 (16), p14 ARF (22), p15 INK4b (23), p27 KIP1 (25), p57 KIP2 (26), PTEN (28), RASSF1C (30), RB1 (31), SURVIVIN (32), and VHL (33) genes ( Additional files 3 , 4 , 5 , 6 , 7 , 8 , 9 ). Lack of hypermethylation of the RB1 gene in our observation was inconsistent with a recent report that the hypermethylated RB1 gene was detected in 19% of astrocytoma patients (26/136 cases analyzed) [ 53 ]. Since the same region was looked at in this work, the discrepancy noticed may simply reflect the inherent difference in the patient cohorts between our work and the published [ 53 ]. The genetic defects affecting the PTEN gene contributed to the pathogenesis of astrocytoma [ 54 ]. Lack of the hypermethylation of its promoter CpG island in both normal and astrocytoma tissues indicates that the DNA hypermethylation mediated silencing mechanism unlikely plays a significant role in the PTEN inactivation that occurs frequently in astrocytoma. This explanation might also be applicable to the no change type of methylation behavior for both the tumor associated genes ( ABL (1), APAF1 (2), APC (3), BRCA1 (5), CAV(6), CDH13 (8), DAPK1(11), hMLH1(14), LKB1(16), p14 ARF (22), p15 INK4b (23), p27 KIP1 (25), p57 KIP2 (26), PTEN (28), RASSF1C (30), RB1 (31), SURVIVIN (32), and VHL (33) genes) and the "cancer unrelated" genes ( EPO (14)) ( Additional files 3 , 4 , 5 , 6 , 7 , 8 , 9 ). The genes with the astrocytoma specific alteration in methylation As shown in Additional files 3 , 4 , 5 , 6 , 7 , 8 , 9 , thirteen genes ( CDH1 (7), CSPG2 (9), cyclin a1 (10), DBCCR1 (12), IRF7 (15), MGMT (18), MT1A (19), MYOD1 (20), OCT6 (21), p16 INK4a (24), p73 (27), RASSF1A (39) and WT1 (34)) were unmethylated in all three normal controls. In contrast, these genes were hypermethylated to various extents in the astrocytoma samples. The following six genes were marginally hypermethylated: p16 INK4a , EPO , DBCCR1 and MYOD1 genes were hypermethylated in 1.9% (1/53) of astrocytoma tissues, while both CDH13 and cyclin a1 genes were hypermethylated in 5.7% (3/53) of astrocytoma cases. No significant changes of these six genes shown in here acted against the notion that DNA methylation related mechanisms underline potential inactivation of this set of genes in the pathogenesis of astrocytoma. The infrequent hypermethylation of the p16 INK4a gene in astrocytoma was a total surprise, as it was frequently reported hypermethylated in various human tumors tested, including in HCC where we have previously found that the p16 INK4a , MYOD1 , CDH13 and cyclin a1 genes were frequently methylated [ 18 , 24 ]. To further verify this unexpected observation, we repeated the MSP analysis on five astrocytoma samples (shown unmethylated) along with one HCC sample (previously shown heterozygously methylated). As shown in panel 1, Fig. 1 , MSP patterns of the astrocytoma as well as HCC tissues remained the same. The identities of which were also confirmed by sequencing (panel 2, Fig. 1 ), showing that while the MSP products with the primers specific to the methylated targets in the HCC sample (Z92K) contained CpGs, the unmethylated targets in all the five astrocytoma tissues (21, 22, 26, A11 and B6) contained TpGs. Therefore, lack of hypermethylation of the p16 INK4a gene in astrocytoma was unlikely incorrect, which is consistent with a recent report that inactivation of the p16 INK4a gene in 48% of astrocytoma cases was genetic [ 55 ]. Figure 1 MSP/sequencing analyses of the p16 INK4a gene in astrocytoma and hepatocellular carcinoma Both electrophoretic patterns of the PCR products of the p16 INK4a in each of five astrocytoma cases (21, 22, 26, A11 and B6) and one HCC case (Z92K) (indicated respectively, at the top of figures) were presented. To indicate the methylation status, the sequenced data are aligned with the wild-type sequence. The remaining 7 targets were hypermethylated more frequently, occurring in 26.4% to 69.8% (14 to 37/53) of astrocytoma cases. The OCT6 gene was hypermethylated in 30.2% of the astrocytoma cases (16/53). Despite of the association of the OCT6 methylation with the aging process reported previously, we found no significant correlation/association of the OCT6 methylation to any clinical-pathological features, including age, gender and clinical grading of the patients. The significance of such a prevalent occurrence of the hypermethylated OCT6 gene remains to be determined. The RASSF1A (hypermethylated in 37/53 cases, 69.8%) is a variant of the recently identified tumor suppressor, the RASSF1 gene that acts at downstream of the Ras mediated apoptotic pathway and is capable of binding to Ras in a GTP dependent manner [ 36 ]. The RASSF1A gene has a more extended 5' part and its promoter CpG island displays a tumor specific hypermethylated profile in a variety of tumors, HCC in particular. Furthermore, lack of the RASSF1A expression in nineteen established tumor cell lines correlates with the hypermethylated state of its promoter CpG island [ 36 ]. The RASSF1C gene has its own promoter CpG island, but is not methylated in any tumors. The methylation behavior of these two genes was very similar to our previous observation in hepatocellular carcinoma, where 22/29 cases (79%) had the fully methylated 1A along with the unmethylated 1C variants [ 18 ]. As shown in Additional file 4 , 5 , 6 , 7 , 8 , 9 , the RASSF1A promoter-CpG island was methylated in 69.8% (37/53) of astrocytoma tissues, while the C variant was not methylated in any astrocytoma tissues. The hypermethylated state of the RASSF1A promoter CpG island was not correlated with gender, age and clinical grading. Consistent with the hypermethylated status of the RASSF1A gene in U251 cells, no expression at the mRNA level was detected. Partial demethylation of its promoter by the treatment with 5-Aza-2'-deoxycytidine indeed resulted in its transcription (Fig. 2 ). Figure 2 The methylation state and expression profiles of the CDH13, p73, MAGEA1, MGMT and RASSF1A genes in U251 astrocytoma cells before and after the demethylation treatment with 5-Aza-2'-deoxycytidine U251 cells were subjected to the 10 and 20 nM 5-Aza-2'-deoxycytidine (5-Aza) treatment for 3 days before both DNA and RNA were prepared for either MSP analyses or RT-PCR assessments. Panels; A, the methylation status of the CDH13, p73, MAGEA1, MGMT and RASSF1A genes and B, the expression profiles of each of these five genes, respectively in U251 cells. The p73 gene encodes a homologue to TP53, and loss of its heterozygosity has been observed in up to 90% of oligodendrogliomas and in 10–25% of diffuse astrocytoma [ 56 , 57 ]. In this study, we found that the p73 gene was prevalently methylated (25/53, 47.2%) with no significant association with any clinical-pathological parameters, such as gender and the WHO grading. The occurrence of the hypermethylated p73 gene was more prevalent in our results than a recent report which detected the hypermethylated p73 gene in 18% (5 /28) of the WHO grade IV but not in grade III astrocytoma [ 35 ]. Again, even the partially elevated demethylated status of its promoter CpG island in U251 cells resulted in reactivation of p73 transcription (Fig. 2 ). Both genetic defects and epigenetic abnormalities of the WT1 gene have been etiologically implicated in the formation of the Wilm's tumor [ 58 ]. In this study, we also found that the WT1 gene was hypermethylated in 30% (16/53) of cases, implying its possible involvement in the formation of astrocytoma. Tumor resistance to the cytotoxic chemotherapies may result from the disrupted apoptosis programs and remains a major obstacle in cancer treatment. In this study, the interferon regulatory factor 7 ( IRF7 ) gene was analyzed. The analogue ( IRF1 ) of IRF7 has been implicated in the IFN gamma mediated apoptosis with a profound effect on the chemo-sensitivity of tumor cells [ 59 , 60 ]. In consistence with the recent report that the IRF7 expression was negatively regulated by the promoter methylation [ 61 ], we found that the IRF7 gene was hypermethylated in astrocytoma (14/53, 26.5%) ( Additional file 4 , 5 , 6 , 7 , 8 , 9 ), with a strong male inclination (11/26, 42.3% verse the female group: 3/27, 11%, χ 2 = 6.632, P = 0.014). Although the gender difference remains to be understood, such a strong male association with IRF7 hypermethylation may have prognostic value. O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme, removes alkylating adducts from the O(6) position of guanine and protects cells from cytotoxic and mutagenic stress. Silencing of the MGMT gene has been suggested to predispose the neoplastic clones to acquisition of the guanine to adenine point mutations in K- ras and p53 [ 62 ] and is associated with low-levels of micro-satellite instability in colorectal cancer [ 63 ]. We found that the MGMT gene was prevalently hypermethylated in astrocytoma (35%, 19/53), and its transcription could be reactivated by demethylation with 5-Aza-2'-deoxycytidine in U251 cells (Fig. 2 ). Hence, the MGMT hypermethylation in astrocytoma may indeed have the pathological significance. In this connection, a recent report suggested that the astrocytoma sensitivity to the alkylating type of chemotherapeutics might be contributed by the hypermethylated MGMT gene [ 64 ]. Expression of the metallothionein I A ( MT1A ) is inducible by a number of adversary agents such as heavy metals and oxidative stress. Both basal and inducible expression of this gene has been impaired in various tumor cell lines and attributed to the hypermethylated state of this gene [ 48 ]. In this study, we found that the MT1A gene was hypermethylated in 30% (16/53) of cases, with no significant gender and grading difference. The functional and pathological implications of the MT1A hypermethylation in astrocytoma remain to be established. Cadherins, the calcium-dependent proteins, contribute to various biological processes such as differentiation, migration and extra-cellular signal transduction of cell. Loss of expression of both E-cadherin ( CDH1 ) and H-cadherin ( CDH13 ) has been found in parallel with the hypermethylated promoter CpG islands in various cancers [ 65 , 66 ]. In this study, we found that the CDH1 gene was hypermethylated in 32.8% (17/53) of astrocytoma tissues, while the CDH13 gene was not methylated in all the astrocytoma tissues examined ( Additional files 4 , 5 , 6 , 7 , 8 , 9 ). In contrast, in human hepatocellular carcinoma [ 18 ], the CDH1 gene was unmethylated, while the CDH13 gene was frequently hypermethylated. Obviously, the molecular basis for tumor type specific methylation patterns of these two genes remains to be determined. Although the hypermethylation mediated gene silencing of the tumor suppressor genes is at the focal point of the epigenetic studies, demethylated status of the promoter CpG islands has been linked to the tumor associated activation of the normally silenced genes [ 19 - 23 ]. Therefore, we also studied both MAGEA1 and SURVIVIN genes. The promoter CpG islands were hypermethylated in normal tissues (for MAGEA1 in HCC [ 18 ] and for SURVIVIN in ovarian cancer [ 23 ]) and demethylated in parallel with the transcriptional activation in tumor cells. The unmethylated status of the SURVIVIN gene in astrocytoma is consistent with the over-expression of this gene (unpublished observations). However, its unmethylated status in all the non-astrocytoma tissues acts odd with the notion that its demethylation is associated with pathogenesis in human ovarian cancer reported previously [ 23 ]. Our previous studies indicated that demethylation of the promoter CpG island was correlated well with the over-expression profile of the MAGEA1 gene [ 18 , 21 ] in HCC. The MAGEA1 gene was fully hypermethylated in all four cases of the normal liver tissues but significantly demethylated in HCC tissues (21/28, 75%). It was found fully hypermethylated in all the three control tissues and in 74.5% (40/53) of the astrocytoma tissues and partially hypermethylated (13/53, 25.5%) in the other astrocytoma tissues. The occurrence of the MAGEA1 demethylation in HCC differed significantly from astrocytoma (75% verse 25.5%, P < 0.001). As it was fully methylated in the normal tissue, the partial hypermethylation (both hypermethylated and demethylated alleles existed) would imply that the event resulting in the loss of the hypermethylation state of the MAGEA1 gene indeed occurred in astrocytoma and should be scored positive for the changes in the methylation pattern in this study. The same principle has been applied for the opposite changes from the unmethylated pattern in the normal control to the partial or full hypermethylated status of all the other genes in astrocytoma tissues. It was also found that the partial demethylated status of the MAGEA1 gene in U251 cells induced by 5-Aza-2'-deoxycytidine occurred co-currently with activation of its transcription (Fig. 2 ). The gender association of the methylation profiles of the AR and IRF7 gene in astrocytoma By statistic analysis with both Pearson Chi-Square and Fisher's Exact tests, associations of the DNA methylation profiles of the targets displaying no less than 24.5% changes (the RASSF1A, p73, MGMT, CDH1, OCT6, WT1 as well as MAGEA1 genes) with the clinical pathological parameters (age, grading and gender) were assessed. The methylation profiles of the AR and IRF7 genes were found gender-oriented. The AR gene encodes the androgen receptor that plays a key role in the signal transduction pathways in response to the male steroid hormone, androgen and has been reported to be inactivated via the epigenetic mechanism in prostate cancers [ 67 ]. Physiologically, the AR gene should express exclusively in the somatic cells in males, while lacking of its expression in females is likely mediated by DNA methylation based mechanisms. Indeed, the hypermethylated along with the unmethylated AR genes were only found in the normal female brain tissue, but not from two male non-astrocytoma samples. The hypermethylation of the AR gene occurred frequently in the female group (81.5%, 22/27) but not in any males (0%, 0/26, χ 2 = 36.22, P = 0.000). It may simply be gender associated and do not have any significant relevance to carcinogenesis of astrocytoma. It was also noticed that hypermethylation of the IRF7 gene displayed an opposite gender inclination, detected in 11% of the female patients (3/27), and 42% of male patients (11/26, χ 2 = ?6.632, P = 0.014). Despite of the difficulty to offer a mechanistic interpretation, the potential prognostic value of such a gender-associated phenomenon might be worthwhile exploring in future. Demethylation by 5-Aza-2'-deoxycytidine treatment of the astrocytoma cells in culture resulted in partial demethylation and reactivated expression of the genes The hypermethylated status of the promoter CpG island has been linked to gene transcription silencing in a number of biological settings. The effect of the astrocytoma associated changes in the methylated state of the promoter CpG islands detected in this study on gene expression was assessed in U251 astrocytoma cells treated with the a demethylating agent, 5-Aza-2'-deoxycytidine. We used MSP to establish the methylation status of the promoter CpG island of all the genes with the astrocytoma associated methylation changes ( Additional files 3 , 4 , 5 , 6 , 7 , 8 , 9 ) in U251 astrocytoma cells, and analyzed the ability of 5-Aza-2'-deoxycytidine to demethylate five genes, as measured by MSP, and reactivate their expression, as detected by RT-PCR. As shown in panel 1 of Fig. 2 , while the CDH13 , MAGEA1 and p73 genes were heterozygously methylated, both MGMT and RASSF1A genes were fully hypermethylated in U251 cells. The CDH13 gene was found expressed, while the rest transcriptionally inert as measured by the RT-PCR. Although both methylated and unmethylated alleles for p73 and MGMT genes were evident in U251 cells, no expression was detected, indicating that the unmethylated allele may remain silent by the other mechanisms, including the genetic defects at critical control region. By the 5-Aza-2'-deoxycytidine treatment, both demethylation of the promoter CpG island and activation of transcription of these five genes were achieved (Fig. 2 ). Despite of the fact that demethylation of the promoter CpG islands was incomplete in samples treated with 20 nM 5-Aza-2'-deoxycytidine (Fig. 2 ), the expression of this five genes was either induced (the MAGEA1 , MGMT , p73 and RASSF1A genes) or elevated (the CDH13 gene). The concordant methylation behavior of the promoter CpG islands of the genes in Astrocytoma The DNA methylation mediated epigenetic changes also display the tumor type specific patterns, which seem to reflect the differentiation and maturation histories of the cell lineages as well as the aging process during which both global hypo- and local hyper-methylation occur. Hypermethylation of the promoter CpG islands in accord with the transcriptional silencing of the tumor suppressor genes, such as the p16 INK4a , and RASSF1A genes, has been well established in human tumors [ 16 , 68 ]. However, it remains unclear whether there is a common mechanism for the concurrent methylation changes of multiple tumor suppressor genes in tumors. To address this matter, it is necessary to examine a large number of genes for frequent changes in methylation in any type of human tumors. The concordant methylation behavior of multiple genes was firstly detected in colon cancer [ 69 ], based upon a comprehensive methylation profiling of over thirty genes. In this study, we have profiled the methylation status of thirty four genes in a cohort of 53 astrocytoma and 3 non-astrocytoma patients. Twenty three of these genes had not been studied previously in astrocytoma. As far as the number of the genes is concerned, this study is the most extensive in the astrocytoma field to our knowledge. Among thirty four genes, sixteen genes exhibited the astrocytoma associated changes in methylation profiles of the promoter CpG islands and nine genes displayed rather frequent changes (the occurrence ≥ 13/53, frequency ≥ 24.5%) ( Additional file 8 ). Four of 53 cases (7.5%) maintained the same methylation profile as the normal control. The rest 49 cases (92.5%) suffered from the methylation changes as much as no less than one target, an occurrence was significantly lower than in HCC, where all the cases displayed methylation changes affecting no less than three targets in the studies involved with twenty or twenty four targets [ 18 , 24 ], indicating that alterations in DNA methylation \occur more frequently in HCC than in astrocytoma. This may be contributed by the apparent anatomic inaccessibility of the brain to environmental adverse factors in comparison to the liver. The size of the subsets containing various number of the target affected (from one to nine) ranged from 1 to 11 cases, and peaked with 10 cases at three and 11 cases at five target subsets ( Additional file 9 ). To identify the most frequent changes of the target sets (one to three), a mathematic method called "the Discovery of Association Rules" [ 28 ] was used. The co-occurrence (case number/the total) and frequency (% of the total) of any subset of the targets that changed in methylation together in astrocytoma were counted and compared. In the entire cohort of patients in this study, the most altered target was the RASSF1A gene, 69.8% (37/53). The two genes that most altered together were the RASSF1A and p73 genes, hypermethylation of which was found in 20 (37.7%). Three genes that changed together were the former two plus CDH1 or OCT6 , hypermethylation of which occurred in 20.8% cases (11/53) (Column 2, a, Additional file 10 ). Furthermore, the occurrence in methylation change in any target in the two gene subsets was 79.3% (42/530 and in three gene subsets was 81.1–83% (43–44/53) (Column 3, a, Additional file 10 ). Since the hypermethylated AR is associated closely with the female gender of the astrocytoma patients and devoid of any association with the formation of astrocytoma, it was taken out from this analysis. Hypermethylation of the RASSF1A gene occurred in 21 female cases (77.8%, 21/27). Both RASSF1A and WT1 were hypermethylated in 13 (13/27, 48.1%); and the former two plus the hypermethylated p73 or CDH1 or OCT6 were found in 9 female cases (9/27, 33.3%), respectively (Column 2, b, Additional file 10 ). The subsets in the male patient group showed very different patterns. The single to three target subsets were the RASSF1A (16/26, 61.5%); the RASSF1A and IRF7 (10/26, 38.5%); and the former two plus the p73 or MGMT or MT1A (5/26, 19.2%), respectively (c, Additional file 10 ). In Grade I astrocytoma, the subsets for one, two and three targets were RASSF1A (10/14, 71.4%), RASSF1A plus p73 (6/14, 42.9%), and the former two plus either WT1 or IRF7 or MAGEA1 as well as RASSF1A plus CDH1 and WT1 (3/14, 21.4%). For Grade II astrocytoma, the corresponding sets consisted of the RASSF1A (12/15, 80%), the RASSF1A and MGMT or IRF7 (5/15, 33.3%), and the RASSF1A and MGMT plus p73 or OCT6 , or MT1A , or WT1 as well as the RASSF1A and IRF7 and MT1A (3/15, 20%), respectively. For Grading III astrocytoma, those subsets were composed of the RASSF1A (8/12, 66.7%), the RASSF1A and CDH1 (5/12, 41.7%), and the formal two plus MGMT (4/12, 33.3%), respectively. For Grading IV astrocytoma, the comparative subsets contained the RASSF1A or p73 (7/12, 58.3%), the RASSF1A and p73 (6/12, 50%), and the former two plus MGMT or OCT6 (4/12, 33.3%), respectively. (d-g, Additional file 10 ). Our methylation profiling efforts described in this report provided the following informative targets: the RASSF1A , p73 , WT1 , MGMT , CDH1 , OCT6 , and IRF7 genes. The established concordant methylation profiles of these eight genes ( Additional file 10 ) may provide useful clues for the epigenetic biomarker selection to for the novel diagnostic and prognostic assays of astrocytoma. The hypermethylated status of this lest of genes in the serum, and biopsies of the suspected astrocytoma patients may serve as good diagnostic indicators, if they can be reliably detected. With the death/survival profiles of this cohort of astrocytoma patients available in the future, the methylation profile established in this study may have certain prognostic value. Abbreviations HCC: Hepatocellular carcinoma; PCR: polymerase chain reaction; MSP: methylation specific PCR; ABL : v-abl Abelson murine leukemia viral oncogene homolog 1; APAF1 : apoptotic protease activating factor; APC : adenomatosis polyposis coli; AR : androgen receptor; BRCA1 : breast cancer 1; CAV : caveolin 1, caveolae protein; CDH1 : cadherin type 1, E-cadherin; CDH13 : cadherin 13, H-cadherin; CSPG2 : chondroitin sulfate proteoglycan 2 (versican); cyclin a1 : cyclin A1; DAPK1 : death-associated protein kinase 1; DBCCR1 : deleted in bladder cancer chromosome region candidate 1; EPO : erythropoietin; hMLH1 : mutL homolog 1, colon cancer, nonpolyposis type 2; IRF7 : interferon regulatory factor 7; LKB1 : serine/threonine kinase 11 (Peutz-Jeghers syndrome); MAGEA1 : melanoma antigen, family A, 1 (directs expression of antigen MZ2-E); MGMT : O-6-methylguanine-DNA methyltransferase; MT1A : metallothionein 1A (functional); MYOD1 : myogenic factor 3; OCT6 : POU domain, class 3, transcription factor 1; p14 ARF : the alternative reading frame of the cyclin-dependent kinase inhibitor 4a; p15 INK4b : cyclin-dependent kinase inhibitor 4b; p16 INK4a : cyclin-dependent kinase inhibitor 4a; p27 KIP1 : cyclin-dependent kinase inhibitor 1B (p27, KIP1); p57 KIP 2 : cyclin-dependent kinase inhibitor 1C (p57, KIP2); p73 : tumor protein p73; PTEN : phosphatase and tensin homolog; RASSF1A : ras association (RalGDS/AF-6) domain family 1 protein isoform 1a; RASSF1C : ras association (RalGDS/AF-6) domain family 1 protein isoform 1c; RB1: retinoblastoma 1; VHL : von Hippel-Lindau syndrome; WT1 : Wilms tumor 1. Competing interests None declared. Authors' contributions JY, HYZ, JG, executing the experiments; SL and JHL, providing the patient samples; WL and YFW, carrying out the mathematic analyses of the data JDZ: designing and organizing experiments as well as completing manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 The target promoter CpG islands and the primer pairs for methylation specific PCR. This file contains his study. Click here for file Additional File 2 Methylation profiles of thirty three genes on the in vitro methylated genomic DNA by M. Sss I methyl transferase. The Eco RI restricted genomic DNA from the liver tissue of a healthy donor was in vitro methylated overnight with M. Sss I methyl transferase and subjected to the MSP analysis, followed by electrophoresis in a 1.3% agarose gel. , the DNA size markers, NL, the untreated sample, U and M, MSP with the pair of primers specific to the unmethylated and methylated targets, respectively. Panels: 1, ABL; 2, APAF1; 3, APC; 4, AR; 5, BRCA1; 6, CAV; 7, CDH1; 8, CDH13; 9, CSPG2; 10, cyclin a1; 11, DAPK1; 12, DBCCR1; 13, EPO; 14, hMLH1; 15, IRF7; 16, LKB1; 17, MGMT; 18, MT1A; 19, MYOD1; 20, OCT6; 21, p14 ARF ; 22, p15 INK4b ; 23, p16 INK4a ; 24, p27 KIP1 ; 25, p57 KIP2 ; 26, p73; 27, PTEN; 28, RASSF1A; 29, RASSF1C; 30, RB1; 31, SURVIVIN; 32, VHL and 33, WT1. Click here for file Additional File 3 Methylation profiles of thirty four genes in astrocytoma (part I). Both electrophoretic patterns of the representative PCR products of each of thirty four targets (indicated respectively, at the top of figures) and the sequencing verification of the one representative PCR product were presented. To indicate the methylation status, the sequenced data are aligned with the wild-type sequence. , size markers, the bands of 250 bp and 100 bp were shown. U, the unmethylated; M, the hypermethylated. Panels: 1, ABL; 2, APAF1; 3, APC; 4, AR; 5, BRCA1; 6, CAV; 7, CDH1; 8, CDH13; 9, CSPG2; 10, cyclin a1; 11, DAPK1 and 12, DBCCR1. Click here for file Additional File 4 Methylation profiles of the promoter CpG islands of thirty four genes in astrocytoma (part II). Both electrophoretic patterns of the representative PCR products of each of thirty four targets (indicated respectively, at the top of figures) and the sequencing verification of the one representative PCR product were presented. To indicate the methylation status, the sequenced data are aligned with the wild-type sequence. , size markers, the bands of 250 bp and 100 bp were shown. U, the unmethylated; M, the hypermethylated. Panels: 13, EPO; 14, hMLH1; 15, IRF7; 16, LKB1; 17, MAGEA1; 18, MGMT; 19, MT1A; 20, MYOD1; 21, OCT6 and 22, p14 ARF> . Click here for file Additional File 5 Methylation profiles of the promoter CpG islands of thirty four genes in astrocytoma (part III). Both electrophoretic patterns of the representative PCR products of each of thirty four targets (indicated respectively, at the top of figures) and the sequencing verification of the one representative PCR product were presented. To indicate the methylation status, the sequenced data are aligned with the wild-type sequence. , size markers, the bands of 250 bp and 100 bp were shown. U, the unmethylated; M, the hypermethylated. Panels: 23, p15 INK4b ; 24, p16 INK4a ; 25, p27 KIP1 ; 26, p57 KIP2 ; 27, p73; 28, PTEN; 29, RASSF1A; 30, RASSF1C; 31, RB1; 32, SURVIVIN; 33, VHL and 34, WT1. Click here for file Additional File 6 The summary of the astrocytoma cases displaying no or changes in the methylation profiles (part I). The frequency (%) of the astrocytoma displaying no or the changes in the methylation profile of each target from the normal control were counted and presented in table as well as plotted in the figure below. The filled, shading and empty boxes indicate the cases where only hypermethylated allele, both hypermethylated and unmethylated alleles and only unmethylated alleles were detected, respectively. The frequency (%) of the hypermethylated targets (except for the MAGEA1 gene) among the total cases was scored for positive changes in astrocytoma. The MAGEA1 was fully methylated (3/3, 100%) in the control, and become partially demethylated in some astrocytoma, therefore, demethylation of the MAGEA1 in astrocytoma was scored as positive changes. Sub-tables: a, the female patient group, b, the male patient group, and c, the control. Click here for file Additional File 7 The summary of the astrocytoma cases displaying no or changes in the methylation profiles (part II). The frequency (%) of the astrocytoma displaying no or the changes in the methylation profile of each target from the normal control were counted and presented in table as well as plotted in the figure below. Sub-tables d-h, the WHO grading I to IV, respectively; The filled, shading and empty boxes indicate the cases where only hypermethylated allele, both hypermethylated and unmethylated alleles and only unmethylated alleles were detected, respectively. The frequency (%) of the hypermethylated targets (except for the MAGE A 1, where the heterozygously hypermethylated) among the total cases was presented in the plot. Click here for file Additional File 8 The occurrences and frequency of changes in methylation. , One of three cases was methylated; *, The MAGEA1 gene was fully methylated in the normal tissues and partially demethylated in astrocytoma patients as indicated in the relevant cells. Therefore, the astrocytoma associated changes in methylation of this gene is opposite to the rest, i.e., demethylation rather than hypermethylation. Figure is each cells are the frequency in % and occurrence (case number). Click here for file Additional File 9 The summary of changes in the methylation pattern in subsets. Both occurrence (case number) and frequency (%) for the subsets having no change in methylation and changes in one to nine genes are presented in % and (case number) in the top half of table, which was also plotted. Both occurrence (case number) and frequency (%) for the subsets having no change in methylation and changes in, at least, one to nine genes are presented in % and (case number) in the bottom half of table. Click here for file Additional File 10 The summary of the concordant methylation behavior of the hypermethylated targets in astrocytoma. The co-occurrence (/total case) and frequency (%) of a panel subsets consisting of one to three targets were treated with method "Discovery Association Rules" and presented. Sub-tables: a, the total, b, the female, c, the male, and d-g, the grade I to IV, respectively. Column 1 is the number of target in each subset. Column 2 is the co-occurrence (case number/total) (frequency in %). Column 3 is the occurrence of any single target in each subsets, presented in case number (frequency %). The column 4 is the gene(s) in subset. N.B., In view of the strong female inclination of the AR methylation and lacking of any association with astrocytoma, AR has been taken off from this analyses. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC520749.xml
524507	Classification between normal and tumor tissues based on the pair-wise gene expression ratio	Background Precise classification of cancer types is critically important for early cancer diagnosis and treatment. Numerous efforts have been made to use gene expression profiles to improve precision of tumor classification. However, reliable cancer-related signals are generally lacking. Method Using recent datasets on colon and prostate cancer, a data transformation procedure from single gene expression to pair-wise gene expression ratio is proposed. Making use of the internal consistency of each expression profiling dataset this transformation improves the signal to noise ratio of the dataset and uncovers new relevant cancer-related signals (features). The efficiency in using the transformed dataset to perform normal/tumor classification was investigated using feature partitioning with informative features (gene annotation) as discriminating axes (single gene expression or pair-wise gene expression ratio). Classification results were compared to the original datasets for up to 10-feature model classifiers. Results 82 and 262 genes that have high correlation to tissue phenotype were selected from the colon and prostate datasets respectively. Remarkably, data transformation of the highly noisy expression data successfully led to lower the coefficient of variation (CV) for the within-class samples as well as improved the correlation with tissue phenotypes. The transformed dataset exhibited lower CV when compared to that of single gene expression. In the colon cancer set, the minimum CV decreased from 45.3% to 16.5%. In prostate cancer, comparable CV was achieved with and without transformation. This improvement in CV, coupled with the improved correlation between the pair-wise gene expression ratio and tissue phenotypes, yielded higher classification efficiency, especially with the colon dataset – from 87.1% to 93.5%. Over 90% of the top ten discriminating axes in both datasets showed significant improvement after data transformation. The high classification efficiency achieved suggested that there exist some cancer-related signals in the form of pair-wise gene expression ratio. Conclusion The results from this study indicated that: 1) in the case when the pair-wise expression ratio transformation achieves lower CV and higher correlation to tissue phenotypes, a better classification of tissue type will follow. 2) the comparable classification accuracy achieved after data transformation suggested that pair-wise gene expression ratio between some pairs of genes can identify reliable markers for cancer.	Background Tumor development is a process in which gene expression is modified, causing abnormal cell behaviour [ 1 ]. Many techniques have been developed to identify abnormalities of gene expression, as reflected by abundance of mRNA transcripts between normal and tumor. The completion of the Human Genome Project and advances in DNA-array technology have allowed highly parallel genetic analyses to take place on a genome-wide scale. They have revolutionized the way tumors are studied, and promised to provide a better and more thorough understanding of the underlying mechanisms for tumorigenesis. Eventually, they will lead to more comprehensive diagnosis/prognosis of tumor with more effective therapeutic interventions. Despite its advantages, the DNA-array technology poses three major challenges that render the interpretation of expression data less efficient than expected. Firstly, the gene expression data is inherently variable due to various factors that either depend on biological factors that remain difficult to control (cross-contaminated samples of tumor and normal cells), or depend on difficulties in setting up of the experiment (RNA extraction) [ 2 ]. These drawbacks interfere with the subsequent array analysis aimed to identify reliable markers that best correlate with the tissue phenotypes. Efforts have been devoted to address these drawbacks by incorporating various raw data scaling, data filtering, normalization and improvement of the classifier algorithm [ 3 ]. Promising results have been reported claiming near-perfect classification accuracy [ 4 ]. However, the usually small number of samples per class in most studies and the highly biased cross validation procedures cast doubt on the classification accuracy in terms of their statistical significance [ 5 ]. This statistical constraint creates a further challenge for DNA-array technology where the number of features in arrays is in thousands while tissue samples are available in limited number. This causes high probability for any classification to be correct by chance alone. Thirdly, although it has been recently established that genes segregate into clusters of interacting networks [ 6 ] instead of acting as one single entity, most cancer DNA-array studies have only investigated single gene aberration (up/down-regulated) when comparing tumor expression profiles to their corresponding normal tissue controls. In an interesting study, B∅ and Jonassen tried to circumvent some of these difficulties by investigating genes in pairs. They demonstrated that gene pairs can be used to improve discrimination between different tissue classes [ 7 ]. This idea of studying genes in pairs, or even in higher order clusters, should be explored further to reveal new features of complex expression profiling datasets. In this study, we introduced a novel data transformation meant to investigate relationships between pair-wise gene expression ratios and tissue phenotype within a given experiment. With this procedure, we aimed to discover strong cancer-related signals (features) that exist in the form of pair-wise ratios (or higher order relationship when we extend to N-feature model classifier for N>2) in a given sample, while improving the signal to noise ratio of the dataset by minimizing its coefficient of variation (CV). The underlying concept for adopting pair-wise gene expression ratios as the discriminating axes for tissue type classification is that an experiment is self-consistent (in terms of factors affected either by the biology of the phenomenon of interest, or of the experimental setting, or both). With this approach we could "subtract" correlated variations by considering the sample as a whole, without making inferences such as those needed for normalization. Basically, we avoided studying gene expression in an absolute term because this requires robust normalization method to account for arrays from different experiments, different platforms and different profiling technologies. By resorting to analyze features in the form of ratios, we attempted to minimize the effect of normalization and look for co-varying signals in each experiment. Methods Colon and prostate cancer datasets The 62 colon cancer sample dataset is composed of measurements for 1,988 gene probes, of which 40 were labelled as tumor and 22 were labelled as normal. The samples were collected from patients, their RNAs were extracted and hybridised to Affymetrix Hum6000 arrays. Please refer to paper [ 8 ]. The normalized dataset can be downloaded at . The 102 prostate cancer sample dataset is composed of measurements for 12,600 gene probes, of which 52 were labelled as tumor and 50 were labelled as normal. The samples were collected from patients, their RNAs were extracted and hybridised to Affymetrix U95Av2 arrays. Please refer to paper [ 9 ]. The normalised dataset can be downloaded at . Both datasets were pre-processed to eliminate those probe pairs that showed significant fluctuation in their hybridisation signals (those greater than 3 standard deviation away from the mean for their ESTs, and the probes pairs that showed an overall higher intensity in their mismatch probe cells (MM) than their corresponding perfect match probe cells (PM); these probe pairs indicate non-specific hybridisation by background RNAs). Both datasets used average intensity as quantitative measurements of the level of gene expression. Base-10 logarithmic transformations were performed for each dataset. Initial gene selection For downstream classification analysis, we extracted only the genes whose expression pattern correlated strongly to the tissue phenotype. To achieve this, we first calculated the correlation coefficient r i (Equation 1) for each gene i using the full dataset, and ranked the genes according to their correlation coefficient r i . For the calculation of r , we assigned a number to each tissue phenotype: 1 for normal tissue and 10 for cancer tissue. After obtaining the correlation coefficients for all genes, we used a simple threshold value (\| r \|>0.4) to select the set of cancer-related genes. There were two reasons for set the threshold value at 0.4. When lower thresholds were used, we incorporated many genes that were not known to be cancer-related (data not shown). Furthermore, too many genes will later cause computer tractability problem when we calculate their pair-wise gene expression ratio for each tissue sample and later the N-feature model classifier. At \| r \|>0.4, we were able to account for most of previously known cancer related genes. where V 1 is a vector representing the gene expression pattern for gene #1; V sample is the dichotomous representation of tissues; S V1 and S sample standard deviation of V 1 , V sample ; , are the mean of V 1 , V sample . Transforming the gene expression data to investigate the expression equilibrium between genes pairs The raw expression data within a sample tissue was transformed into measurement of the pair-wise gene expression ratio for any combinatorial pairs of genes. For the 1,988 gene expression intensities for each sample ( e 1 , e 2 ... e 1988 ), there are 1988 C 2 combinations ( e 1 / e 2 , e 1 / e 3 ...) of pair-wise gene expression ratios (Figure 1 ). This transformed matrix is referred to as M . Each row/column corresponds to a specific gene and the entry at the intersection of row X and column Y corresponds to the expression equilibrium between gene X and gene Y. Such matrix has a diagonal entry of value 1 because e 1 /e 1 equals to unity. Feature partitioning method [ 4 ] for classification of normal/tumor tissues using single gene expression Regarding the F eature P artitioning M ethod (FPM), in order to discriminate between the normal/tumor tissues based on specific feature i (single gene expression), the first step is to determine the threshold value, T i , that can optimally splits all the tissue samples into tumor and normal tissue. The FPM algorithm has a recursive version [ 4 ], in which a decision tree depicting the classification rules for tissue samples was generated recursively. Both methods differ in the way T i s are derived. Nonetheless, they are very intuitive and non-parametric in nature. Also, they restrict no priori distribution patterns for features used. We adopted the simple FPM for tissue classification where each feature was treated individually. There are two criteria for deriving a valid threshold value T i for each feature. First, it has to delineate correctly (discriminating efficiency = 100%) the one-dimensional region (R feature_i ) for either all the normal/tumor tissues using all tissue samples. Secondly, it has to minimize the percentage of false prediction for the other tissue type. Take gene #1659 for example. To fulfill the two aforementioned criteria, it was determined that the region greater than 63.7 (R #1659 ) incorporates all the tumor samples (Figure 2 ). It classified correctly all tumors (discriminating efficiency = 100%) with an overall false prediction of 13.9% in the normal set. This was performed repeatedly for all features until all the threshold values ( T i...all features ) were determined. Now, to classify an unknown sample using 2-feature model classifier, a combination of any two features and their corresponding pre-determined threshold values T i s (selected from T i...all features for each dataset) were recruited. The outcome of the tissue class will be determined depending on whether one/both the expression values of the unknown sample fall completely in either the normal/cancer region (R feature_i ). This is to say that if any of the two features from the unknown sample meets the criteria (R feature_i ) to be either normal/tumor tissue type (based on our definition, R feature_i is a region with 100% discriminating efficiency for a specific tissue type), the unknown sample will be assigned to be normal/tumor respectively. This is repeated exhaustively for all possible combinations constituting of any two features. The procedure will be repeated for all tissue samples to evaluate the overall classification accuracy for 2-feature model classifier. In total, we evaluated the classification of tissue samples based on different combinations of N genes and investigated the classifiers up to 10-feature model classifier. Classification of normal/tumor tissues using transformed datasets The classification procedures and the two criteria for determining the threshold value were the same as explained in previous paragraph. The only difference here is that the definition of "feature" refers to pair-wise gene expression ratio derived from lower/upper triangular matrix of M . Take the ratio #1537/#1831 for example. To fulfill the two aforementioned criteria, it was determined that the region greater than 0.755 (R #1537/#1831 ) incorporates all the tumor tissue samples (Figure 2 ). It classifies correctly all tumor tissue samples with a false prediction of 6.4%. This is performed repeatedly for all entries in M until all the threshold values are determined. Now, to classify an unknown sample using 2-feature model classifier, a combination of any two features (pair-wise gene expression ratio) and their corresponding pre-determined threshold values T i s (selected from T i...all features for each dataset) were recruited. The outcome of the tissue class will be determined depending on whether one/both the expression values of the unknown sample fall completely in either the normal or cancer region (R feature_i ). This is to say that if any of the two features (pair-wise gene expression ratio) from the unknown sample meets the criteria (R feature_i ) to be either normal/tumor (based on our definition, R feature_i is a region with 100% discriminating efficiency for a specific tissue type), the unknown sample will be assigned to be normal/tumor respectively. This is repeated exhaustively for all possible combinations constituting of two features. The procedure will be repeated for all tissue samples to evaluate the overall classification accuracy for 2-feature model classifier. In total, we evaluated the classification of tissue samples based on different combinations of N genes and investigated the classifiers up to 10-feature model classifier. Constructing the relationship tree for the top 25 genes We calculated the cross correlation coefficient r (Equation 1) for all pair combinations of the top 25 genes listed in Table 6 and Table 7 . Prior to the construction of a relationship tree for the top 25 genes for colon and prostate cancer, the cross-correlation coefficient was used to construct the pair-wise distance matrix D . Each entry in the pair-wise distance matrix was measured by the value of (1- r ). Each row/column corresponds to a specific gene and an entry at the intersection of row X and column Y corresponds to the distance of gene expression between gene #X and gene #Y. Such matrix has a diagonal entry of value 0. Only the lower/upper triangular matrix of D is required to construct the relationship tree. After obtaining lower/upper triangular matrix of D , the neighbor-joining method (NJ) algorithm was used to construct the relationship tree [ 10 ]. Computer hardware and software A Sun Fire 6800 Server with 24 CPUs (each running with a clock speed of 900 MHz) was employed throughout this study. The computation of correlation coefficient and classification procedures were implemented using the Matlab Technical Programming language (Matlab programs can be downloaded at . Results After initial gene selection, respectively 82 and 262 genes (\| r \| > 0.4) were selected from the colon and prostate dataset for downstream analysis (Table 1 and Table 2 ). Topping the list in both tables were genes that have been found to be either over-expressed/under-expressed in tumors [ 11 ]. The first three genes most correlated to cancer in the colon dataset were heavy chain of non-muscle myosin, human monocyte-derived neutrophil-activating protein (MONAP) and human desmin genes. This agrees with the findings from [ 12 , 13 ] that used other statistical tests ( z -score, t -test) in a comparable analysis. The heavy chain of non-muscle myosin, denoted as the embryonic smooth muscle myosin heavy chain (SMemb), was found to be down-regulated in cancer. It was also determined experimentally to be a target for the protein encoded by the metastasis-related mts-1 gene [ 14 ]. Furthermore, it was demonstrated recently by 5'RACE analysis that heavy chain of non-muscle myosin interacts with ALK genes that have tyrosine kinase activity and oncogenic properties [ 15 ]. The human monocyte-derived neutrophil-activating protein (MONAP, interleukin-8), was second on the list. It was significantly up-regulated in the tumor compared to the normal samples. This protein has been linked to the progression of several human cancer types [ 16 ]. It was believed that over-expression of MONAP plays an important role in tumor angiogenesis and tumor aggression. The human desmin gene is the third on the list, and it was found to be down-regulated in tumor. Interestingly, this gene also showed significantly reduced expression in other cancer types such as the melanoma cell line [ 17 ]. From the prostate dataset, the most cancer-correlated gene is the human hepatoma gene coding for serine protease hepsin. Brief literature search in PubMed showed that hepsin is a well-characterized transmembrane protease that is expressed at high level in tumor. Three separate studies identified hepsin as a significant cancer biomarker that can be used for cancer diagnosis [ 18 ]. The second gene on the list was the human mitochondrial matrix protein P1. This gene has been correlated to different cancer types with consistent up-regulation in tumor [ 13 ]. The third gene is the carcinoma-associated antigen GA733-2, which was among the 216 cancer markers identified by Ernst's group in Germany [ 19 ]. Effect of data transformation on coefficient of variation To date, reliable markers with low coefficient of variation (CV) are generally lacking. Discovering robust cancer marker is crucial for the purpose of successful cancer diagnosis. We investigated the CV between samples after data transformation: the lowest CVs decreased to 16.5% in the colon dataset while it increased to 25.8% for the prostate dataset (Table 3 and Table 4 ). Topping the list for both dataset were the pair-wise gene expression ratio for genes #119/#54 (elongation factor 1-delta and 40S ribosomal protein S24) and #10614/#5871 (zq58b03.r1 Homo sapiens cDNA and nuclear matrix protein NXP2), which revealed informative pair-wise gene interaction in relation with their corresponding tissue phenotypes. They reflected how cell adjusts to their pair-wise product in response to physiological changes. Based on these observations, we found that the relative abundance between the numerator and denominator exhibited a strong mutual dependency, and had strong correlation to tissue phenotype. For pair-wise gene expression ratio #119/#54, the elongation factor 1-delta is involved in a sequence of events during the decoding of mRNA on the ribosome [ 20 ]. For the ratio of #10614/#5871, it corresponds to novel genes that do not yet have known function. A search in the DNA non-redundant (nr) database for gene #10614 yielded 83% DNA identity to a segment on chromosome 9. On the other hand, a search in non-redundant (nr) database for #5871 revealed 72.3% DNA identity to the cDNA of mouse that incorporates proteins involved in chromosome partitioning and cell decision [ 21 ]. Prior to data transformation the lowest coefficients of variations for single gene expression were 45.3% and 24.5% for colon and prostate datasets respectively. When using the data transformation we proposed, significant improvement was achieved in the colon dataset. Interestingly, this was followed by an improved data correlation to the tissue phenotype as well as to the classification efficiency. We did not observe a similar improvement of the CV, data correlation to tissue classes or classification efficiency in the prostate dataset. Correlations of the single gene expression and pair-wise gene expression ratio The distribution of correlation coefficients between genes and tissue phenotypes for the colon and prostate datasets is shown in Figure 3 . The distributions are positively and negatively skewed for both datasets. The two red lines separate genes with \| r \| >0.4 from the bulk (Table 1 and 2 ). They retained respectively 82 and 262 genes from the colon and prostate datasets. To study the possible interaction between pair-wise genes, we estimated the statistical correlation of gene expressions. Both the distributions for the correlation coefficient and the extreme cases are shown in Figures 4 and 5 . Both figures emphasize the true nature of gene-gene co-regulations – a complex biological mechanism, that most often has been over-simplified when we treat the gene expression as an independent variables [ 22 ]. For example, Figure 4 and Figure 5 suggested that the expressions of genes belonging to a common subset are most likely correlated to each other (e.g.: Gene #31 vs #119 in colon cancer ( r = 0.95306) and gene #7775 vs #10749 in prostate cancer ( r = 0.92922)). It should be pointed out that the two humps in the probability density function are not zero-centered, but concentrated at non-zero correlation r . For colon dataset, positive correlation was the dominant type. For prostate dataset, a balanced distribution in their gene correlation was observed. We determined that some improvement in tissue classification is achieved when pair-wise gene expression ratio was used as discriminating axes instead of using a single gene expression (Figure 2 ). The reason is that pair-wise gene expression ratio has higher correlation to tissue phenotype with lower CV (Table 5 ). Gene expression and tissue type correlation Several previous studies have already endeavored to identify correlations between specific gene expression and cancerous transformation [ 4 , 13 , 23 ]. In the present study, we identified several novel target genes that clearly distinguish the two different tissue phenotypes with high discriminating efficiency (>74%) (Table 6 and Table 8 ). Some of those have previously been documented in studies that did not involve expression profiling as cancer related genes (Human monocyte-derived neutrophil-activating protein (MONAP) and Human hepatoma mRNA for serine protease hepsin), others (Human gene for heterogeneous nuclear ribonucleoprotein (hnRNP), P24480 CALGIZZARIN, Human mitochondrial matrix protein P1, Human mRNA for aldose reductase and human adipsin) have not been identified from in-silico studies of tissue DNA-array expression data. The cancer related genes for colon and prostate cancer were ranked according to their discriminating predictive power. The list should provide hints for researchers during selection of molecular target for diagnostic, prognostic or attempts to cure the disease. Overall classification results and accuracies for each N-feature model classifier across two datasets were reported in Table 6 , 7 and 8 . In the following section, we will discuss a few important genes or pair-wise gene expression ratios from Table 6 and Table 7 that resulted in the optimum classification accuracy (Table 8B ). They are the most efficient combination of discriminating axes for classifying tissue types because they delineate correctly all the normal/tumor tissues with the lowest percentage of false prediction. For the sake of brevity, we will discuss three single gene expressions and two pair-wise gene expression ratios from colon cancer. For prostate cancer, two single gene expressions and two pair-wise gene expression ratios will be discussed. For colon cancer single gene expression, three axes for discriminating tissue types are: 1) Human monocyte-derived neutrophil-activating protein (MONAP); 2) Human desmin gene and 3) Human cysteine-rich protein (CRP) gene. Their threshold values were determined to be 62.73, 2787.0 and 749.4 respectively. For colon cancer pair-wise gene expression ratio, the two axes for discriminating tissue types are: 1) #1831/#1537 and 2) #753/#768. Their threshold values were reported to be 1.32 and 1.85 respectively. For prostate cancer individual gene expression, the two axes for discriminating tissue types are: 1) Human hepatoma mRNA for serine protease hepsin and 2) Human adipsin. Their threshold values were reported to be 115.0 and 182.0 respectively. For prostate cancer pair-wise gene expression ratio, the two axes for discriminating tissue types are: 1) #6185/#5840 and 2) #6185/#6749. Their threshold values were reported to be 2.69 and 2.55 respectively. To illustrate graphically the result of tissue classification, two examples, each based on three genes or pair-wise gene expression ratios that altogether yielded the optimum classification efficiency for the prostate cancer are shown (Figure 6 , Figure 7 ). Constructing the relationship tree for top 25 gene for colon and prostate cancer The relationship tree for top 25 genes listed in Table 6 and Table 7 were constructed based on the cross-correlation between gene expressions (Figure 8 ). We employed the established 'neighbor-joining' clustering method [ 10 ] to group different genes based on their correlated expression patterns across all tissue samples (meaning that genes expression that are correlated will appear in the same branch of the clustering tree), using a novel distance measurement to quantify how change in the expression for one gene interfered with that of another gene. The principle of this method is to cluster pairs of operational taxonomic units (OTUs [=neighbors of similar gene expression]) that minimize the total branch length at each stage of clustering of OTUs starting with a star-like tree. Figure 8 revealed two major clusters of genes. The first cluster corresponded to down-regulated genes, the second cluster represented up-regulated genes. Also, the most efficient discriminating axes (feature genes) reside at the basal position for each cluster. In bacteria many genes are co-expressed as single transcription units. This was used as a control study to validate the methodology of grouping genes, we implemented this distance measurement on bacteria gene arrays ( B. subtilis and E. coli ) and successfully determined the co-regulated operon gene structures (supplementary file #1). Discussion Data transformation to investigate pair-wise gene expression ratios As the expression profiling technologies mature, the identification of significant cancer-related signals from noisy datasets (characterized by a high CV) remains a major challenge. In particular, a robust normalization method is critical to ascertain that arrays from two experiments are comparable with minimum noise prior downstream analysis. However, the existing normalization methods pose limitations due to the lack of good models to account for sources of experimental and biological variations [ 24 ]. Hoffmann et al. [ 25 ] employed different normalization methods to analyse the same dataset, and demonstrated that the numbers of genes detected as differentially expressed differed by a huge factor depending on which normalization methods used. The problem is exacerbated further by the presence of different array formats, experimental designs and methods. Here, instead of resolving to single gene expression, that depends heavily on normalization, for tissue classification, we presented a transformation method that uses pair-wise gene expression ratios within the same experiment as the discriminating axes. By doing so, we aimed to minimize the influence of different normalization methods considering that an experiment is self-consistent with the same factors affecting all genes in the same fashion. The rationale is that even when the normalization methods differ between two array experiments, their pair-wise gene expression ratios within the same experiment will remain relatively stable. If reliable cancer-related signal, exist in the form of pair-wise gene expression ratio, were indeed discovered successfully, they will be relatively independent from the normalization method used on a dataset. The improvement in CV (Table 3 ) and overall classification accuracy (Table 7 ) for colon dataset after introduction of data transformation signifies two implications: First, the transformation is able to increase the signal to noise ratio (SNR) of the cancer related signal because the resulted pair-wise gene expression ratios correlate stronger to tissue phenotype. Second, because the pair-wise gene expression ratios are less dispersed than single gene expression, using the pair-wise gene expression ratios to classify tissue types will be much more reliable and accurate (Table 8 ). Despite the benefits mentioned, this data transformation introduced a computational limitation due to the enormous amount of feature combinations to be processed, especially when N-feature model classifiers for N>4 are considered (If 100 features are selected, and 10-feature model classifier is investigated, the search space will be 100 C 10 = 1.731030945644000 × 10 13 different combination of features). As a result, more computation time will be required to search all possibilities. As an example, the discriminating axes that accounted for the optimum accuracy in 1 to 3-feature model classifier are reported in Table 9 . Regarding the high classification accuracy reported in Table 8 , it should be stressed that this was achieved by involving all tissue samples during the derivation of the threshold value, T i , in the feature selection procedure. In other word, instead of adopting the more conservative classification accuracy test where only a subset of tissue samples are used to derive a set of classification criteria (threshold values), we adjusted our methodology to use all tissue samples so that our results are unbiased (when comparing the outcome from single gene and pair-wise gene ratio) and in-line with our objective that is to compare the classification efficiency between single gene and pair-wise gene ratio. Admittedly, we have a noisy dataset whereby selecting a subset of tissue samples that are a representable population for the entire dataset remains a challenge [ 5 ] (given that we have a small and unbalanced dataset, particularly the colon dataset). Eventually, we might run into ambiguous/contradicting results using a different population subset of tissue samples. Furthermore, we might miss important features (single gene expression/ pair-wise gene expression ratio) because of the biased training dataset. By including all tissue samples for both studies (single gene and pair-wise gene ratio), we aimed to derive the most reliable threshold values and classified tissue samples based on them. Since the same methodology was applied for both studies, the comparison of classification efficiency is valid and will reflect how well each feature (single gene and pair-wise gene ratio) can be used to delineate tissue samples. The implication derived from the classification results For colon dataset, three axes for discriminating tissues are: 1) Human monocyte-derived neutrophil-activating protein (MONAP); 2) Human desmin gene and 3) Human cysteine-rich protein (CRP) gene. The association of the first two genes and cancer biology had been discussed earlier. We will discuss the Human cysteine-rich protein gene. The expression and induction of this protein has been associated with protection against DNA damage, oxidative stress and apoptosis [ 26 ]. In the colon dataset, we observed down-regulation of this protein in tumor. This suggested lack of protection against DNA damage. For colon cancer pair-wise gene expression ratio, the two axes for discriminating tissues are: 1) #1831/#1537 and 2) #753/#768. Using these two axes, 98.4% of the tissue samples can be classified correctly. The expression ratio between #1831 (gelsolin precursor) and #1537 (vascular endothelial growth factor) was able to discriminate 93.6% of the total tissue data. The vascular endothelial growth factor was determined recently to be a plausible biomarker for colon cancer [ 27 ]. Gelsolin had been found to suppress tumorigenicity in different cancer samples, including lung, bladder and breast [ 28 ]. When they were used individually as a discriminating axis, they were only able to classify correctly 66.1% and 67.7% of all tissue samples. Furthermore, the expression ratio between #753 (Human cysteine-rich protein) and #768 (the macrophage migration inhibitory factor) was able to discriminate 90.3% of total tissue type. The human cysteine-rich protein was discussed in the previous section. The macrophage migration inhibitory factor (MIF) functions as a pluripotent cytokine involved in broad-spectrum pathophysiological events in association with inflammation and immune responses. Several reports, including ours, have suggested that MIF is also involved in tumorigenesis [ 29 ]. When they were used individually as single discriminating axis, they were only able to classify correctly 83.9% and 66.1% of all tissues. For prostate cancer single gene expression, the two axes for discriminating tissues are: 1) Human hepatoma mRNA for serine protease hepsin, and 2) Human adipsin. The first gene was discussed in the previous paragraph. For the second gene, adipsin had also been suggested by Chow et al. [ 30 ] as a good cancer marker for studying the basic biology of cancer. For prostate cancer pair-wise gene expression ratio, the two axes for discriminating tissues are: 1) #6185/#5840 and 2) #6185/#6749. Using these two axes, all tissue samples can be classified correctly. The expression ratio between #6185 (Human hepatoma mRNA for serine protease hepsin) and #5840 ( Homo sapiens mRNA for KIAA1109 protein) was able to discriminate 92.2% of total tissues. The human hepatoma mRNA for serine protease hepsin had been determined to be an important marker for cancer cell development [ 11 , 18 ]. The KIAA1109 protein is an unknown protein in human chromosome four [ 31 ]. A homology search against the non-redundant databases yielded no significant hit to known genes. When they were used individually as a discriminating axis, they were only able to classify correctly 86.3% and 61.8% of all tissues. On the other hand, the expression ratio between #6185 (Human hepatoma mRNA for serine protease hepsin) and #6749 ( Homo sapiens mRNA for KIAA1055 protein) was able to discriminate 90.10% of total tissues. The human hepatoma mRNA for serine protease hepsin was discussed in the previous section. The KIAA1055 protein is an unknown protein in human chromosome 15 [ 21 , 31 ]. A homology search against the non-redundant databases yielded 40.7% DNA identity to a novel human cDNA that had been found to function as a cancer inhibiting protein [ 21 ]. When they were used individually as a discriminating axis, they were only able to classify correctly 86.3% and 62.8% of all tissues. Conclusion By comparing the tissue classification methods based on the single gene expression and the pair-wise gene expression ratio in two microarray datasets, we reached the following conclusions: 1. The minimum coefficient of variation decreased from 45.33% to 16.53% for colon dataset but increased marginally from 24.54% to 25.78% in prostate dataset. 2. The correlation coefficient, r , of the discriminating axis that correlates maximally to the tissue phenotype improves from 0.63 to 0.79 and 0.71 to 0.75 in colon and prostate dataset respectively. 3. The optimum accuracy for 1-feature model classifier (using single gene or pair-wise gene expression ratio as discriminating axis) improved from 87.1% to 93.55% in colon dataset. In prostate dataset, nine out of the top 10 discriminating axes showed significant improvement. The mean accuracy for 1-gene classifier improved from 76.8% to 91.2% and 75.8% to 81.9% in both datasets. 4. The comparable classification accuracy achieved after data transformation suggested that there exist some cancer-related signals in the form of pair-wise gene expression ratio, especially prominent in the colon dataset. 5. Through the single gene analysis, we identified key biomarkers that agree with the findings by other researchers. In addition, study on gene-to-gene correlation and the classification outcome based on the pair-wise gene expression ratio suggested that genetic network within a cluster of cancer-related genes should be explored further. Competing interests The authors declare that they have no competing interests. Authors' contributions YLY proposed the idea, participated in the design, performed the statistical analysis and wrote the first draft of the manuscript. AD participated in the design and overall coordination of this study as well as in the writing of the manuscript. XWZ participated in the design of the study. YCW, XHW and MTL participated during the revision phase of this study. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC524507.xml
549072	Remembering George L. Wied, M.D., February 7, 1921-July 25, 2004	A personal tribute to George L. Wied, M.D., a founder of the medical subspecialty, cytopathology, who died July 25, 2004.	Tribute I arrived in Chicago last November for the ASC meeting, and reflexively reached for the phone to let Dr. Wied know that I had arrived, just as I had for each of countless trips to Chicago over the past 25 years. Then I remembered that he was no longer here, having passed away in Salzburg, Austria, on July 25th. But as the meeting unfolded, I realized that George's mark was deeply imprinted on the entire scientific program, and that his legacy was carried by most of the people in attendance. George Papanicolaou is attributed with the discovery of the cytologic method for detecting epithelial tumors. His laboratory at Cornell Medical Center in New York was the culture medium for the discipline, instructing such notable cytologists as Leopold Koss and George Wied. The medical specialty itself, however, owes its success to George Wied, for without him, it is doubtful whether it would have survived the skepticism of most other medical specialists. Dr. Wied had faith in the importance of cytology that was unwavering and his conviction was transmitted to all who followed him around the globe. A survivor of Nazi Germany, he was a fierce defender of individual freedoms, and he translated that zeal into inclusion of all peoples in his vision. He was an instinctual teacher and taught those around him to convey the criteria of those strange cellular samples via the Tutorials of Cytology. His attention to detail was impeccable, a trait that he insisted his faculty emulate. The TOC faculty quickly became internationally recognized experts, and the Tutorials were a successful enterprise for over 40 years. Until cytopathology became a mandatory part of the curriculum of U.S. pathology training programs, the Tutorials were among the few opportunities for physicians to become facile with the discipline. Repeat enrollment was common, as the specialty became more complex and its importance became apparent to the medical profession. For many nations TOC remained the sole source of first-hand and first-rate cytology education. Judging by the multinational backgrounds of presenters at the ASC meeting, and the excellence of the papers, Dr. Wied's goal has most assuredly been achieved. Setting and maintaining a level of excellence was a responsibility that Dr. George Wied eagerly assumed, and expected the rest of us to uphold. It was easy to follow his example, for his charisma infused us with commitment to those eager to learn. In order to validate the professionals involved in the practice, he established the examinations of the International Academy of Cytology. He personally traveled to every venue where the exam was held, usually following either a Tutorial or International Congress of the IAC. He savored each opportunity to bring another disciple into the fold of cytology. But where Dr. Wied had the most fun was in the realm of cytology automation. As I listened Sunday to the presentations of experiences with the "new" imaging systems and their integration into the clinical cytology laboratory, I recalled the numerous conferences devoted to development of computerized scanners. As early as 1951, Dr. Wied recognized that computers would become an essential part of our daily lives, for data management and communication. He also realized that the task of screening slides was work intensive, subjective, and fraught with opportunities for error. If Dr. Wied didn't have the knowledge himself, he immediately reached out to others who did, and drafted them to the cause of automating his specialty. One of those recruits was Peter Bartels, a gifted optical scientist and incredibly creative problem solver. Together they built a team of researchers at the University of Chicago that attacked each obstacle to success like an army of dragon slayers. Rather than seek the glory of discovery alone, Dr. Wied's generous spirit inspired him to organize meetings to discuss problems common to automation, bringing scientists from a variety of disciplines and numerous countries. If a potentially important contributor to the meeting did not have funding to attend, Dr. Wied would offer to support them, often personally. He was most supportive of young scientists and mentored them through their careers. These were usually pleasant meetings, as Dr. Wied's gentle and considerate nature didn't allow personal bickering. But he loved scientific controversy and he encouraged us to challenge each other to the next level of achievement. He fully believed that what was good for one research group was good for the entire profession. He was most distressed when the commercialization of automated scanners led to open fighting among the companies trusted to develop the fruits of so many years of collegial research. He was a frequent member of NIH study sections that reviewed research proposals in automation. He was also known to drop in unexpectedly on a research group thousands of miles from his home, just to see what was happening. He was free with his advice without being condescending. He would never knowingly offend anyone. His constant encouragement and validation of the importance of scientific discovery for this young specialty was manifested through the two journals he founded and edited, ACTA Cytologica in 1957 and Analytical and Quantitative Cytology and Histology in 1979. Recognition for his accomplishments came frequently and from various sources, including an Outstanding Investigator Award from the NIH. But if you were to reach into the heart of George Wied, I wager that he felt that his greatest accomplishment was being father to Kazutaka (George) Wied, son of his wife, Kay. An unlikely parent, Dr. Wied soon became a master at constructing dioramas out of shoe boxes, holding flash cards with German vocabulary words, and generally encouraging young George to treasure his education. Being a musician himself, Dr. Wied patiently tolerated the squawks and squeaks of his young son's efforts to be a violinist. As young George became more accomplished, after dinner performances soon became an expected treat whenever the occasion allowed. Dr. Wied and Kay continued to travel the world even though Dr. Wied's health was becoming increasingly fragile. His final trip could have been written by a romantic novelist. He accepted a speaking engagement in Japan, but instead of a simple round trip to Tokyo, he insisted on an around-the-world ticket, "because it's cheaper!" For one of the stops, he chose to go to Vienna, a city that he loved, to once more savor the delicious schnitzel at the Intercontinental Hotel, site of the Vienna Tutorials. Kay realized that the Salzburg Music Festival was nearby and would be a good place to relax and hear the music that Dr. Wied loved so dearly. Young George, having just graduated from Stanford University, was with them, not always the case. They spent the day at numerous concerts in Salzburg, all dedicated to Czech composers, an uncanny coincidence since Dr. Wied was born in what was then Czechoslovakia. After the concerts, Kay, young George and Dr. Wied spent the evening together, remarking on the beauty of the music. Dr. Wied died in the night, with his beloved family nearby, and across the street from the home of his most admired composer, Wolfgang Amadeus Mozart. There is an old adage, that when a loved one dies, you mourn each time for all the roles they played in your life. If it is a parent or sibling, you mourn once. However, if the person has been multiple influences in your life, you will mourn for each role. For Dr. Wied, many of us will mourn multiple times. For me, he was a teacher, mentor, advisor, friend, and role model. I will mourn many times, but will be comforted by having known him. I have already and will continue to repay his influence by transmitting his ideals to those who follow. His is truly a legacy that deserves to be perpetuated. George Wied was a man of rare breadth and depth, the kind of professional and human for which mankind will eternally thirst. Figure 1 Dr. Wied at the University of Chicago Cytopathology Laboratory, circa 1990.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC549072.xml
524249	Representation of Attended Versus Remembered Locations in Prefrontal Cortex	A great deal of research on the prefrontal cortex (PF), especially in nonhuman primates, has focused on the theory that it functions predominantly in the maintenance of short-term memories, and neurophysiologists have often interpreted PF's delay-period activity in the context of this theory. Neuroimaging results, however, suggest that PF's function extends beyond the maintenance of memories to include aspects of attention, such as the monitoring and selection of information. To explore alternative interpretations of PF's delay-period activity, we investigated the discharge rates of single PF neurons as monkeys attended to a stimulus marking one location while remembering a different, unmarked location. Both locations served as potential targets of a saccadic eye movement. Although the task made intensive demands on short-term memory, the largest proportion of PF neurons represented attended locations, not remembered ones. The present findings show that short-term memory functions cannot account for all, or even most, delay-period activity in the part of PF explored. Instead, PF's delay-period activity probably contributes more to the process of attentional selection.	Introduction Jacobsen (1935 , 1936 ) first discovered that damage to the primate prefrontal cortex (PF) appeared to cause a short-term memory deficit. In his experiments, monkeys and chimpanzees with bilateral damage to PF failed to retrieve food from one of two opaque cups when the food had been out of sight for even a few seconds. Intact animals could find the food 5 min or more after they had last seen it. Pribram et al. (1952) later identified the part of PF responsible for this deficit as area 46, also known as the dorsolateral prefrontal cortex (PFdl). More recently, temporary inactivations of portions of PFdl caused what appeared to be a short-term memory loss in localized regions of space ( Funahashi et al. 1993a ). Once the concept of working memory ( Baddeley 1986 ) became established in contemporary neuroscience (see Postle et al. 2003 ), these neuropsychological findings contributed to the theory that PF functions in working memory ( Goldman-Rakic 1987 ) and, in some extreme formulations, only in working memory. In the 1990s this theory developed a wide following, and the idea that PFdl functions in spatial working memory, with other parts of PF functioning in different kinds of working memory, became the predominant theory of PF function, especially for nonhuman primates. As important, the concept of working memory used by proponents of this theory focused mostly on the short-term maintenance of information, and rather less on the manipulation or monitoring of such information or on the use of that information for decisions. Accordingly, we refer to the former aspect of working memory as maintenance memory to distinguish it from the broader concept and do not use the phrase working memory elsewhere in this report. Note, however, that when we use the phrase maintenance memory, many authorities would use “working memory” instead. Consistent with the idea that PF functions predominantly in maintenance memory, delay-period activity in PF has often been interpreted as a memory trace (e.g., Funahashi et al. 1989 ; Romo et al. 1999 ; Constantinidis et al. 2001 ). The phrase delay-period activity applies to neuronal activity that follows the transient presentation of an instruction cue and persists until a subsequent “go” or “trigger” signal. The description of delay-period activity in PFdl appeared very early in the history of behavioral neurophysiology ( Fuster and Alexander 1971 ; Kubota and Niki 1971 ; Fuster 1973 ), and, in accord with the maintenance-memory theory, some PF cells appear to buffer activity representing remembered information, even when distracting stimuli appear during the delay period ( di Pellegrino and Wise 1993b ; Miller et al. 1996 ; Moody et al. 1998 ). Although the interpretation of delay-period activity in terms of the short-term memory of a stimulus has a long history, many studies have explored alternatives. Neurophysiological experiments designed to explore alternatives to the maintenance-memory interpretation of delay-period activity first attempted to dissociate sensory from motor signals. These studies showed that PFdl neurons preferentially reflected sensory signals, which supported the idea that these neurons encode stimulus memory over the short term. For example, one influential study used the “antisaccade” task ( Funahashi et al. 1993b ), in which a stimulus in one direction (from a central fixation point) instructed an eye movement in the opposite direction. More than twice as many PFdl neurons represented the location of the sensory stimulus as represented the target (or direction) of movement. In another experiment, when a given spatial cue guided two different reaching movements, motor factors affected PFdl neurons only rarely and weakly compared to neurons in the premotor cortex ( di Pellegrino and Wise 1993b ), especially when viewed at a population level ( Wise et al. 1996a ). These results supported the idea that more delay-period activity in PFdl reflected the memory of sensory cues than represented motor preparation or movement targets, but did not explore other alternative interpretations of delay-period activity. Neuroimaging studies have provided support for some of these alternatives. At first, neuroimaging studies appeared to back the maintenance-memory theory of PF function, which bolstered the interpretation of PF's delay-period activity in the context of that theory. After an initial period of nearly uniform support, however, subsequent neuroimaging studies have suggested that PFdl plays a role in aspects of attention and other functions instead of, or in addition to, maintenance memory. Indeed, one recent report disputed whether PF plays any role in short-term memory at all. To quote the investigators, “no part of frontal cortex, including PF, stores mnemonic representation[s] . . . reliably across distracted delay periods. Rather, working memory storage . . . is mediated by a domain-specific network in posterior cortex” ( Postle et al. 2003 ). Passingham and his colleagues have used the phrases attention to action, attention to intention, and attentional selection to describe certain PFdl functions ( Rowe et al. 2000 ; Rowe and Passingham 2001 ). Petrides and his colleagues have, likewise, emphasized a role for PFdl in monitoring items in memory ( Owen et al. 1996 ; Petrides et al. 2002 ). These alternative views of PF function point to a role in top-down control of attention and are supported by other neuroimaging and neuropsychological findings implicating PF in attentional functions (see Discussion). In sum, then, neuroimaging and neuropsychological findings bring into question the interpretation of PFdl's delay-period activity mainly in terms of maintenance memory. Previous neurophysiological experiments have ruled out motor factors, such as motor planning and the representation of the targets of movement, for most of PFdl's delay-period activity, but have typically lacked control over spatial attention. The present experiment tested an alternative to the maintenance-memory interpretation of PFdl's delay-period activity by pitting the representation of a remembered location against the representation of an attended location, when either location could serve as the target of an upcoming saccadic eye movement. Results Two monkeys performed the task depicted in Figure 1 A. Briefly, the monkeys maintained fixation on a spot presented at the center of a video screen, called the fixation point. A solid gray circle then appeared at a fixed distance from the fixation point in any one of the four cardinal directions ( Figure 1 A, part a): left, right, up, or down from center. Next, as central fixation continued, the gray circle revolved clockwise or counterclockwise around the fixation point, moving along a circular trajectory (arrow in Figure 1 A, part b). It then stopped at one of the four cardinal directions from center, after having revolved 90°, 180°, 270°, or 360° ( Figure 1 A, part b). After a variable delay period of 1.0–2.5 s, the circle brightened or dimmed for 150 ms ( Figure 1 A, part c) and then disappeared ( Figure 1 A, part d). The change in the circle's brightness served as the trigger signal for a saccadic eye movement (arrows in Figure 1 A, part d). On control trials, the circle either did not move or revolved 360° and stopped at its initial location for that trial. During those trials, both dimming and brightening of the circle instructed a saccade toward its location. During other trials, dimming and brightening of the circle guided both the timing of the response and the choice between two alternative saccade targets. Figure 1 Task and Behavior Behavioral task (A) and representative horizontal and vertical eye position records (B). (A) Each trial began when the monkeys pressed a button to make a fixation point (FP) appear at the center of the video monitor. Some time after the monkeys fixated the FP (dashed lines), a gray circle (depicted here as white) appeared at one of four peripheral locations. The figure illustrates its appearance at the 0° location (part a). The monkeys had to remember this location later in the task; hence we termed it the remembered location. On most trials, the circle subsequently revolved around the FP to a different location, as the monkeys maintained central fixation. The figure illustrates its termination at the 90° location (part b). A small change in the circle's luminance (part c) signaled the monkeys where to look next. This cue persisted for 150 ms, then disappeared. Because the monkeys depended on this subtle and brief cue for both timing and targeting information, we termed this the attended location. If the circle dimmed (dark gray, part c, top), the monkeys had to make a saccade to the attended location (Att-trials, part d, top). If the circle brightened (starburst, part c, bottom), the monkeys had to make a saccade to the remembered location (Rem-trials, part d, bottom). After saccade initiation, the central FP disappeared and, if the monkeys made a saccade to the correct location, a new FP appeared there (not shown). The monkeys had to fixate the new FP and, after it dimmed, release the button to produce a fruit juice reward. (Monkey drawing courtesy of Dr. Michael Shadlen.) Brightening of the circle indicated that the monkeys should make a saccade to the circle's initial location on that trial, which the monkeys had to remember in order to perform the task correctly ( Figure 1 A, parts c and d, bottom). Accordingly, we called these trials remembered-location trials (Rem-trials). Dimming of the circle signaled that the monkeys should make an eye movement to its current location ( Figure 1 A, parts c and d, top). We called these trials attended-location trials (Att-trials), for the following reasons. As a key feature of the experimental design, the circle's brightness changed only subtly and remained visible in its new form only briefly. Because the monkeys could not predict whether the circle would brighten or dim and because that subtle, short-lived event provided essential information about the time and target of the response, the monkeys had to attend to the circle intently during the period preceding the trigger signal. As a result of the central fixation requirement, this attention was necessarily covert, although it seems likely that the monkeys would have attended overtly to the circle (i.e., fixated it), had they been allowed to do so. Indeed, the monkeys did so during training. The Discussion takes up the issues of divided attention, multiple motor plans, default motor plans, and other interpretational issues. By varying the final location of the circle from trial to trial, we could test for significant spatial tuning for attended locations, and by varying the initial location of the circle, we could test for significant spatial tuning for remembered locations. In addition, we tested the monkeys' performance in a “no-memory” condition, which had the same the sequence of events as in the standard version of the task. In the “no-memory” condition, however, the initial location of the circle remained marked by a stationary stimulus identical to the circle that revolved around the fixation point. Behavior Figure 1 B shows selected eye-position records, matched to the trials illustrated in Figure 1 A. Table 1 shows that both monkeys achieved a high level of performance on this challenging task. For Rem-trials, these data show that the monkeys remembered the circle's initial location, and—because they could not know the trial type in advance of the trigger signal—they must have also done so for Att-trials. Table 1 Task Performance and Reaction Times for Each Monkey The percentage of correctly executed trials comes from the trials on which the monkey maintained fixation until the trigger signal occurred and then performed a saccade to the instructed (correct) or some other (incorrect) location. The reaction times come from correct trials only. Means (± SEM) are presented for different angular differences between the remembered and attended locations (0°, 90°, or 180°). For control trials, which correspond to a 0° difference (360° revolutions excluded), Att-trials are trials on which the circle dimmed, and Rem-trials are those on which the circle brightened Table 1 also shows the reaction times for each monkey. Taking the two monkeys together, saccades to the remembered location began approximately 36 ms later than those to the attended location, a difference that was highly significant (Wilcoxon rank sum test, p < 0.001). We can only speculate about the cause of this difference, but reaction times on Rem-trials may have been longer because attention had to be disengaged from the circle's location and reoriented to the remembered one prior to the response. For the “no-memory” condition (not given in Table 1 ), reaction times for Att-trials increased approximately 16 ms compared to the standard version of the task, whereas reaction times for Rem-trials decreased approximately 22 ms (both highly significant differences, Wilcoxon rank sum test, p < 0.001). These data are consistent with the idea that each of the two marked locations attracted attention in the no-memory condition, whereas the monkeys directed most of their covert attentional resources to the attended location in the standard version of the task. We acknowledge, however, that there are other interpretations of these data. On control trials, for example, when the saccade was always toward the circle, saccade initiation was approximately 18 ms slower when the circle brightened (as it did on Rem-trials) than on trials when it dimmed (as it did on Att-trials). Thus, factors other than the orientation of attention probably contributed to reaction-time differences. Single-Neuron Analysis Figure 2 illustrates the activity of a neuron tuned to the attended location during the delay period. Only activity collected during correctly executed trials appears in any of the analyses presented in this report. The figure shows histogram and raster displays of neuronal activity aligned on the trigger signal for Att-trials ( Figure 2 A) and Rem-trials ( Figure 2 B), arranged in the form of a matrix, as illustrated and labeled in Figure 2 C. Delay-period activity, enclosed by the red rectangles in Figures 2 A and 2 B, varied with the attended location (columns), but not with the remembered location (rows). The firing rate during the delay period was highest when the monkey attended to the 90° location (up from screen center, see Figure 1 A, part b). We called this the cell's preferred location. The lowest firing rate occurred when the monkey attended to the 270° location, termed the least preferred location. Figure 2 Example Neuron Representing the Attended Location In (A–C), the four rows correspond to different remembered locations and the four columns to different attended locations (see key in [C]). (A and B) PETHs and raster displays aligned on the trigger signal (vertical line). In the rasters, each dot represents a neuronal spike, and each line of dots shows a sequence of spikes during a single behavioral trial. (A) Trials in which the stimulus dimmed and the monkey made a saccade to the attended location (Att-trials). (B) Trials in which the stimulus brightened and the monkey made a saccade to the remembered location (Rem-trials). The activity of this neuron depended on where the monkey attended, with a preferred location of 90°. Note the large variation in firing rate from column to column (across the attended locations) and relative constancy of rate within columns (across remembered locations). (C) Compact representation of spatial tuning pattern shown in (A) and (B), combined. Each circle's area is proportional to the average firing rate during the 800-ms period immediately preceding the trigger signal (red rectangle in [A] and [B]). Note that the major diagonal of this firing-rate matrix, running from the upper left to the lower right corner, corresponds to the control trials, which lacked a memory requirement. F, maximal firing rate; sp/s, spikes per second. For each neuron, we assessed the extent of spatial tuning for the attended location with an index called attended-location index (I Att ), which measured the variability in discharge rate among attended locations. We assessed the extent of spatial tuning for the remembered locations with a related index called remembered-location index (I Rem ) (see Materials and Methods ). A neuron was considered spatially tuned if I Att , I Rem , or both significantly exceeded 1.0 (randomization test, p < 0.01; see Materials and Methods ). We classified neurons as attention cells if I Att attained statistical significance but I Rem did not, as memory cells for the opposite result, and as hybrid cells if both indexes showed statistical significance. Figure 3 A– 3 C shows examples of an attention cell, a memory cell, and two hybrid cells. ( Figures S1–S3 show the trial-by-trial activity for each of these four cells, both before and after the trigger signal.) Neurons tuned to the attended location (attention cells) dominated the neuronal sample in both monkeys, comprising 61% of cells spatially tuned during the pretrigger delay period ( Table 2 ). Neurons tuned to the remembered location (memory cells) made up 16% of the spatially tuned neurons, and those tuned to both locations (hybrid cells) amounted to 23%. For 27% of the hybrid cells, the attended and remembered locations associated with the highest firing rate were the same ( Figure 3 C, part a); in the remaining 73% of the hybrid cells, these preferred locations differed ( Figure 3 C, part b). Figure 3 Example Firing Rate Matrices and a Scatter Plot of Tuning Indexes PFdl neurons with different classes of spatial tuning. Firing rate matrices (A–C) in the format of Figure 2 C; (E) gives the key. Tuning selectivity indexes (I Att and I Rem ) and firing rate scale (F) appear adjacent to each firing rate matrix. (A) A neuron tuned to the attended location (different from the cell shown in Figure 2 ). (B) A neuron tuned to the remembered location. Its firing rate primarily varied across rows. (C) Two cells tuned to both the attended and remembered locations (hybrid neurons). One neuron (part a) exhibited a high firing rate when either the attended or remembered location was at 270°. The other neuron (part b) showed its highest activity when the remembered location was at 180°, but was inhibited when that was the attended location. (D) Scatter plot of spatial tuning indexes for attended (I Att ) and remembered (I Rem ) locations for each spatially tuned neuron in both monkeys. The different neuronal classes are color coded as in (A–C): blue corresponds to attention cells, red to memory cells, and green to hybrid cells. Table 2 Cell Classification Number of neurons that significantly ( p < 0.01) encoded the attended location (Attention), the remembered location (Memory), or both locations (Hybrid) during the 800 ms immediately prior to the trigger signal Figure 3 D illustrates the degree of tuning for both the attended (I Att ) and remembered ( I Rem ) locations. Each data point on the scatter plot represents a single spatially tuned neuron (both monkeys combined). Tuning for the remembered location (red symbols) was both weaker and less frequent than tuning for the attended location (blue symbols). Note that hybrid cells (green symbols) fill most of the space between the other two classes and that relatively few cells represent a single location exclusively. For example, many of the neurons classed as memory cells show some sensitivity to the attended location, albeit not a statistically significant one by the test that we employed. For the entire group of spatially tuned neurons ( n = 303, both monkeys and all three cell classes combined), the mean selectivity indexes (± SEM) for the attended and remembered locations were I Att = 1.84 ± 0.08 (median = 1.39, interquartile range [IQR] = 0.73) and I Rem = 1.21 ± 0.02 (median = 1.08, IQR = 0.23), which differed significantly at the p < 0.001 level (Wilcoxon matched-pairs test). Table 3 shows comparable data for each cell class and Figure S4 gives similar data for various combinations of these classes. The selectivity for the attended location also exceeded that for the remembered one when expressed in terms of firing rates. For the attended location, the difference in firing rate between the preferred and least preferred locations averaged 8.8 ± 0.5 spikes/s, which was significantly greater than the 5.3 ± 0.3 spikes/s for the remembered location (Wilcoxon matched-pairs test, p < 0.001). Table 3 Spatial Tuning Indexes Early Versus Late in the Trial Tuning indexes (mean ± SEM) were calculated from both the 800 ms immediately preceding circle movement (Early, I ) and the 800 ms immediately preceding the trigger signal (Late, I Att , I Rem ). For both attention and hybrid cells, spatial tuning to the attended location was significantly stronger (Wilcoxon matched-pairs test) late in the trial, when the monkeys awaited the trigger signal. Values for memory tuning (I Rem ) appear for completeness, not for statistical testing. See also Figure S4 We examined whether these results merely reflected the presence of a stimulus in the monkey's visual field and found strong evidence to the contrary. We compared tuning for the circle's location during the 800 ms before the circle started moving (called the early period) and during the last 800 ms of the delay period, immediately prior to the trigger signal (the late period). (Figures S5 and S6 show activity during a slightly different early period than measured here, but they illustrate the same basic result.) Despite the fact that the sensory inputs were identical in screen-centered, allocentric, retinocentric, fixation-centered, head-centered, and body-centered coordinates, the activity of PFdl neurons and their degree of spatial tuning differed in these two task periods. This result rules out a purely sensory response. For the entire PFdl sample, the late tuning index (1.29 ± 0.03) significantly exceeded the early one (1.16 ± 0.02; p < 0.001; Wilcoxon matched-pairs test). This measure is devoid of any bias caused by a cell's tuning properties in one task period or the other, but it includes the contribution of the spatially untuned cells. When we restricted the comparison to neurons that had any type of significant spatial tuning, in either the early or late periods, the late tuning index (1.76 ± 0.07) continued to exceed the early one (1.42 ± 0.05) significantly ( p < 0.001). Most important, we obtained similar results for neurons with significant tuning to the circle's location, which characterizes attention and hybrid cells (1.83 ± 0.08 late versus 1.46 ± 0.05 early; p < 0.001). Table 3 and Figure S4 present this analysis for all cell classes, alone, and in various combinations. Note that these indexes do not reflect a generalized increase in firing rate: They were normalized to remove the effects of firing rate per se. The section entitled Population Analysis presents a confirmatory result in terms of activity levels. Further confirming this result on a cell-by-cell basis, significant spatial tuning to the circle's location occurred more frequently during the late delay period (256 attention and hybrid cells) than during the early one (194 cells, of which 41 lost their spatial tuning in the late period). Thus, the representation of the circle's location in PFdl grew stronger around the time of the trigger signal, when it was important for the monkeys to attend to the circle. These findings rule out the mere presence of the circle in something akin to a visual receptive field as a complete account of the tuning of attention and hybrid cells. Histological Analysis Figures 4 and 5 show the locations of the cells in each class: Figure 4 as a function of electrode-penetration sites for both monkeys and Figure 5 as section reconstructions for monkey 2. The attention cells were concentrated more ventrolaterally than either the memory or the hybrid cells. Neurons located ventrolateral to the fundus of the principal sulcus ( n = 551) were predominantly attention cells (28% to 2% memory and 5% hybrid cells, with 65% lacking spatial tuning, both monkeys combined). Neurons dorsomedial to the fundus ( n = 412) fell into the three cell classes approximately equally (8% attention, 9% memory, and 10% hybrid cells, with 73% lacking spatial tuning). These regional differences within PFdl were highly significant for each monkey (p < 0.0001, χ 2 test). Cells with significant memory signals (memory and hybrid cells, combined) composed 70% of the spatially tuned population in dorsomedial PFdl, but only 20% in ventrolateral PFdl. Figure 4 Surface Projections Showing the Location of Neurons in Each Class All hemispheres are displayed so that rostral is to the left and dorsomedial is up. Reconstructed surface projections of the left hemispheres of monkey 1 (A) and monkey 2 (B). (C) Surface projection of the (inverted) right hemisphere of monkey 1. (D) A lateral view of the hemisphere shown in (C), with the region included in (C) approximated by the dashed box. The dotted ellipse encloses the cells deemed to lie inside the PFdl by histological analysis, but does not correspond to the cytoarchitectonic boundaries of area 46. Figure 5 Section Reconstructions for Monkey 2 (A–G) Coronal sections taken at the planes indicated in the surface drawing (H). Dashed lines mark the borders between PFdl (area 46) and area 12. Solid lines show the tracks of the marking pins (irregular outlines in sections [B] and [C]) and the estimated location of electrode penetrations. Colored hash marks show the estimated depth of neurons in each class, using the same color code as in Figures 3 and 4 . Longer hash marks indicate simultaneous recordings of more than one neuron of the same class. (H) Lateral view of left PF depicting surface projections of spatially tuned neurons. Black circles show the locations of pin holes used for localization, and gray squares show their predicted locations. Based on a cytoarchitectonic analysis conducted on two of the three hemispheres, all of the cells situated ventrolateral to the fundus of the principal sulcus were located within area 46 and none were located in area 12. The area 46/12 architectonic boundary was first described by Walker (1940) and was subsequently confirmed with different methods ( Preuss and Goldman-Rakic 1991 ). This boundary could be discerned in both monkeys as a distinct thinning of the internal granular layer in area 12 compared to area 46 and a more substantial departure in that area from the classic, homotypical appearance typical of area 46. The reconstructed location of recording sites showed that the small group of cells located caudomedially in both monkeys (see Figure 4 B and 4 C) was located in the postarcuate cortex (area 6) and in area 8, as indicated by the agranular and dysgranular cytoarchitecture of these two regions, respectively. This small group of cells was eliminated from the present analysis. Population Analysis Figure 6 displays the degree of spatial tuning for the different cell classes in the form of population histograms. The analysis of attention tuning ( Figure 6 A and 6 B) used the 800 ms immediately preceding the trigger signal to measure mean firing rates for different attended locations. We excluded control trials from this analysis. These rates were then ranked from the largest (i.e., the preferred attended location) to the smallest (the least preferred location). For each neuron, the preferred location chosen by this analysis was designated as preferred for all task periods displayed in the population histograms. (Similar results were obtained when the ranking was done for each individual task period.) The left side of the figure shows the mean attention signal for both attention ( Figure 6 A) and hybrid ( Figure 6 B) cells. After a transient response to the appearance of the circle (at a latency of approximately 100 ms), neuronal activity in both of these cell classes remained elevated when the circle stopped at the preferred location (blue curve) and became slightly suppressed when it was at the least preferred location (black curve). Figure 6 Attention and Memory Signals in Population Histograms (A) and (B) Representation of an attention signal by attention cells (A) and hybrid cells (B). (C) and (D) Representation of a memory signal by memory cells (C) and hybrid cells (D). In each panel, activity is shown centered on the appearance of the circle (left vertical line), on the time that the circle stopped moving (middle vertical line), and on the trigger signal (right vertical line). Attention and memory signals are reflected in the degree of separation in the average population histograms for different ranks. In (A) and (B), the data for the period immediately prior to the end of the circle's movement have been eliminated because the circle came from different initial locations. The right side of Figure 6 shows the mean memory signal for memory ( Figure 6 C) and hybrid ( Figure 6 D) cells. These population histograms were calculated on the basis of preferred remembered locations, ranked according to the pretrigger modulation. This location was then designated “preferred” for all task periods displayed in the plots. For memory cells ( Figure 6 C), the population averages were almost identical when the circle remained stationary at its initial location and that location did not yet need to be remembered. That is, on average it did not matter noticeably whether the circle initially appeared at a cell's preferred location or at its least preferred location ( Figure 6 C, red versus black curves). This finding is somewhat surprising because prior studies suggested that PFdl's memory cells had activity that began shortly after stimulus onset and continued throughout the delay period. In our memory cells, spatial tuning did not develop to any appreciable extent until after the circle began revolving around the central fixation point. This result shows that tuning to the remembered location developed during the trial and was not a simple replica of the tuning pattern during the initial presentation of the circle. Hybrid cells ( Figure 6 D) exhibited a weak spatial signal following the appearance of the circle consistent with their memory tuning prior to the trigger. Note that after the circle stopped moving, memory cells showed less of a difference between preferred and least preferred locations than did attention cells ( Figure 6 C versus 6A). This finding supports the results presented in Tables 2 and 3 and Figure 3 D, which show a predominance of nonmemory signals (see also Figure S4 ). Population representations of the attended and remembered locations were further analyzed using a neuron-dropping analysis. Neuron-dropping curves express the strength of spatial tuning as the ability to estimate a spatial variable from the activity of a neuronal ensemble, as a function of ensemble size. We randomly selected an ensemble from the population of recorded PFdl neurons and used a single trial of activity from each cell to estimate both the attended and remembered locations. The findings of the neuron-dropping analysis agree with those from the analysis of single-cell activity and the population histograms and thus provide independent support. However, neuron-dropping analysis offers several advantages over the population histograms, in addition to providing confirmation of those results. In neuron-dropping, the estimation of either an attended or remembered location does not depend on any assumptions about the nature of the spatial tuning curve or the relative importance of very active cells versus those showing less activity. It does not ascribe any special significance to increases in activity relative to baseline (excitation) versus decreases (inhibition) or to the most preferred and least preferred locations. Each cell's activity contributes to the population estimation for all locations regardless of the direction of its modulation relative to baseline and whether that modulation significantly differs from baseline levels. Furthermore, the computation makes no assumption about any relationship between tuning for attended locations and remembered ones. This analysis also has the advantage that its results are expressed as a percentage of correct estimations by the neuronal ensemble, thereby facilitating comparison with the monkeys' performance, which in this experiment always exceeded 75% correct and sometimes approached 100% ( Table 1 ). Figure 7 shows the neuron-dropping curves for each cell class (A–C) and all spatially tuned neurons combined (D) in monkey 1. Neuron-dropping curves for monkey 2 showed similar results, and Figure S7 presents the data for both monkeys combined. As expected, the neuron-dropping curves computed for attention cells yielded much better estimations of the attended location than the remembered one (see Figure 7 A, blue versus red curves). Note, however, that the attention cells also provided a better-than-chance estimation of the remembered location. This result reflects the fact that many cells with significant tuning for the attended location also showed some tuning for the remembered location (see blue data points in Figure 3 D with I Rem > 1.0). Figure 7 A also confirms the comparison of activity early versus late in the trial (blue versus gray curves), providing further evidence against a purely sensory account of this subpopulation's activity. Also as expected, memory cells yielded a better estimation of the remembered location than the attended one ( Figure 7 B, red versus blue curves), but these cells, too, yielded a fairly reliable estimation of the other spatial variable. Neuron-dropping curves for hybrid neurons showed comparable estimations for both locations ( Figure 7 C). When all spatially tuned neurons were combined ( Figure 7 D; see also Figure S7 D), the resultant neuron-dropping curves showed that PFdl activity was a much more reliable estimator of the attended location than the remembered one. Figure 7 Neuron-Dropping Curves for Different Subpopulations of PFdl Neurons in Monkey 1 Each curve represents the percentage of correct single-trial estimations of location as a function of the number of neurons in the assembled populations. The curves show predictions of the attended locations (blue lines) or remembered locations (red lines) during the 800 ms immediately preceding the trigger signal, after the circle had stopped revolving around the central fixation point. Also shown is the estimation for the 800-ms period immediately preceding the onset of the circle's movement (gray lines). The dotted line indicates the chance level of estimation, 25% correct. Neuron-dropping curves are shown for neurons tuned to the attended location (A), the remembered location (B), both locations (C), and all spatially tuned neurons (D). (E) and (F) Dynamic changes in estimations of the attended (blue) and remembered (red) locations for 20 spatially tuned neurons (marked by the dashed gray line and arrows), using a 200-ms sliding window. Dashed and solid lines in (E) and (F) are shown for consistency with Figure 8 . Note that the estimations in (D) are higher than in (E) and (F) because the former is based on an 800-ms interval, and the latter are based on only a 200-ms interval. The same analysis was applied to the ventromedial and dorsolateral regions within the PFdl, described in the section entitled Histological Analysis, above (not shown). The ventrolateral subpopulation of PFdl neurons (see Figure 4 A– 4 C) overwhelmingly represented the attended location. The dorsomedial subpopulation represented both locations comparably, with estimation of the attended location being slightly better in one monkey and estimation of the remembered location being slightly better in the other. Of the two subpopulations, the dorsomedial neurons showed a more reliable estimation of the remembered location. We also used a neuron-dropping analysis to examine the ensemble's properties during response selection and execution. Figure 7 E and 7 F show these time-dependent neural-estimation curves for monkey 1; Figure 8 does so for both monkeys combined. Note from Figure 7 D– 7 F that the time-estimation curves come from a random sample of neurons, much smaller than the sampled population, to avoid the effects of signal saturation. The estimations at each time point reflect activity averaged over the previous 200 ms. Prior to the trigger signal, the estimation of the attended location (blue curves in Figures 7 E, 7 F, 8 D and 8 E) was superior to that of the remembered location (red curves) for all spatially tuned neurons, as well as for attention cells ( Figure 8 A). This finding is consistent with the greater number and stronger spatial tuning of attention than memory cells. Figure 8 Time-Dependent Changes in Estimating the Attended Location and Remembered Location, for Both Monkeys Combined Solid lines, trials in which the monkeys made a saccade to the attended location; dashed lines, trials in which the monkey made a saccade to the remembered location. Blue lines, estimation of the attended location; red lines, estimation of the remembered location. All records are centered on the onset of the trigger signal (using data for the 200 ms prior to that time). Vertical lines at t > 0 show the average saccade latency on Att-trials (solid) and Rem-trials (dashed). The thick bar at the bottom of the plots shows the approximate onset of the peripheral fixation spot, which the monkeys continued fixating beyond the limit of the plot. Estimations for each monkey were calculated using the same methods as for Figures 7 E and 7 F, except that the ensemble size for monkey 2 was 60 neurons. This number of neurons was chosen to avoid ceiling effects (i.e., 100% correct). The plotted curves show the average for the two monkeys. Location estimations for attention (A), memory (B), hybrid cells (C), and all spatially tuned neurons on Att-trials (D) and Rem-trials (E). Above the plots are schematic depictions of an example trial, of the type illustrated in Figure 1 A. The red “R” marks the remembered location. In both D and E, prior to the trigger signal (left schematic), the monkeys fixated (dashed lines) centrally and covertly attended to the circle (yellow spot at the attended location). During this period, estimation of the attended location exceeded that of the remembered location. Following the saccade, the monkeys fixated a peripheral light spot (right schematic) and attended to this target to detect when it dimmed. On Att-trials (D), the monkeys' gaze shifted to the attended location, and the ensemble's estimation of this now overtly attended location improved (solid blue curve), while the representation of the now irrelevant, remembered location gradually decayed (solid red curve). On Rem-trials (E), the monkeys' gaze (dashed lines) and focus of attention (yellow spot) shifted to the (previously) remembered location. The estimation of this location consequently improved (red dashed curve), while the estimation of the previously attended (and now irrelevant) location gradually decayed. Abbreviations: Att, attended; Rem, remembered. On Att-trials, the estimation of the attended location (solid blue curves in Figures 7 E and 8 A–D) improved following the dimming of the circle and remained elevated during the saccade to that location. This improvement continued for the initial 200 ms of fixation there. Then the signal decreased. Note that the monkey maintained fixation at the target location for at least 1.0 s after the saccade. In contrast, the estimation of the remembered location on Att-trials (solid red curves) gradually decreased following the trigger signal. The fading of this representation most likely reflected the fact that the remembered location was no longer behaviorally relevant. On Rem-trials (dashed curves in Figures 7 F, 8 A– 8 C, and 8 E), the circle's brightening instructed a saccade to the remembered location (marked by the red “R” in Figure 8 E). We expected that redirecting attention toward the saccade target (yellow spot in Figure 8 E, right) would degrade the neuronal representation of the formerly attended location and improve the representation of the formerly remembered—but eventually fixated—one. The estimation of the attended location initially improved on Rem-trials following the trigger signal there (blue dashed curves in Figures 7 F, 8 A– 8 C, and 8 E). However, in accord with our expectation, that estimate decreased dramatically in accuracy after saccade onset, as the attended location became behaviorally irrelevant. In contrast, the estimation of the formerly remembered (and soon to be fixated) location (red dashed curves) improved sharply ( Figure 8 E), especially in attention cells ( Figure 8 A). Thus, PFdl neurons became more reliable encoders of that location. Given that these averages “look back” 200 ms, this development must have preceded the saccade. On both Att-trials and Rem-trials, the neuronal ensemble remained a reliable indicator of the saccade target relatively long after the target had been acquired (see solid blue and dashed red curves in Figures 7 E, 7 F, and 8). This signal might encode the fixated location, which could be important for monitoring performance, as suggested for nearby areas of frontal cortex ( Stuphorn et al. 2000 ; Ito et al. 2003 ). Alternatively, the saccade target may have been represented because the monkeys attended to the fixation spot at this location, so that when it dimmed they could quickly release the button to produce their reward (see Materials and Methods , below, for a description of that aspect of the task). Discussion In tasks involving short-term memory requirements, delay-period activity in PFdl has consistently been interpreted in terms of the maintenance-memory theory of PF function (e.g., Funahashi et al. 1989 ; Romo et al. 1999 ; Constantinidis et al. 2001 ), despite the existence of viable alternatives. However, our results show that much of PFdl's delay-period activity in such tasks reflects nonmemory functions. Accordingly, the maintenance-memory theory of PF function ( Goldman-Rakic 1987 , 1990 ), taken to its extreme, fails to account for PFdl's delay-period activity. Indeed, we found that, compared to the remembered location, the attended location was more frequently and more robustly encoded at both the neuronal and population levels. The present results thus support extensive neuropsychological ( Rueckert and Grafman 1996 ; Stuss et al. 1999 ; Koski and Petrides 2001 , 2002 ) and neuroimaging ( Corbetta et al. 1993 ; Gitelman et al. 1999 ; Kastner et al. 1999 ; Rosen et al. 1999 ; Cabeza and Nyberg 2000 ; Hopfinger et al. 2000 , 2001 ; Vandenberghe et al. 2000 ; Astafiev et al. 2003 ; Small et al. 2003 ; Thiel et al. 2004 ; Woldorff et al. 2004 ) research that points to a much more general role for PF than encompassed by the maintenance-memory theory, including the top-down control of selective attention. Interpretational Issues and Limitations The present experiment is the first neurophysiological study to achieve a degree of independent control over both spatial attention and spatial memory, so a detailed consideration of both its advantages and limitations is in order. A complete dissociation of these two spatial variables is probably impossible, but we achieved this goal to a considerable degree. Our experimental design, however, has several limitations and raises a number of questions. For example, is what we call attention really attention? We have elaborated on our usage of the term attention in the Results section. Although we did not quantify the degree of attention, it seems to us a reasonable assumption that the monkeys attended to the circle, given that its brightening or dimming was subtle, brief, and crucial to their correct performance. Moreover, the reaction-time data are consistent with the idea that the monkeys attended to the circle in the period immediately prior to the trigger signal. The remaining interpretational questions to be addressed, then, are: Do monkeys devote any attentional resources to what we call the remembered location? Do they “remember,” in some sense, what we call the attended location? Does the activity we interpret in terms of attention or memory reflect motor factors? And, given that the monkeys could anticipate and predict rewards, do the signals reflect these processes? We address each of these four questions, in turn, in the remainder of this section. First, although we contend that the monkeys must have devoted substantial attentional resources to the location of trigger signal, this does not necessarily rule out additional covert allocations of attention to the remembered location. However, there was no stimulus or expected signal at the remembered location to warrant the allocation of attentional resources there. In addition, the demands of fixating the central location (overt attention), while attending covertly to a stimulus located in peripheral visual space, make it unlikely that attention was further divided ( Hunt and Kingstone 2003 ; Muller et al. 2003 ). Accordingly, although we cannot completely rule out the possibility that the monkeys attended to the remembered location during the delay period, it seems implausible that they did so. If one adopts the view that they did, then some or all of the neurons we class as memory cells might instead have activity better interpreted as reflecting some aspect of highly divided attention. Second, the monkeys were required to remember the place where the circle first appeared on each trial, and their performance shows that they did so. Did they also “remember” the attended location? There is ample precedent for skepticism about the proposition that monkeys are not remembering some location. However, there is no basis for assuming a “memory” of a currently visible stimulus. It seems especially unlikely that the monkeys “remembered” the attended location in the context of the requirement that they centrally fixate while attending somewhere and remembering somewhere else. Third, we cannot rule out the participation of neurons we class as attention or memory cells in a variety of processes involved in preparing or planning the movement or selecting the response target. Prior to the trigger signal, the monkeys may have prepared to make a movement to the remembered location, to the attended location, to both, or to neither. Cisek and Kalaska (2002) have shown that some neurons in the premotor cortex encode a possible movement target before a particular one has been specified, but their experiment has yet to be done for PFdl neurons. In view of prior evidence arguing against interpreting much of PFdl's delay-period activity in terms of motor signals ( Funahashi et al. 1989 , 1993b ; di Pellegrino and Wise 1993b ; Asaad et al. 1998 ; Romo et al. 1999 ; Constantinidis et al. 2001 ) and the absence of a contemporary “motor theory” of PF function, the present experiment was not designed to address this issue. Future work along these lines, perhaps combining the design of di Pellegrino and Wise (1993b) with the present one, might be indicated by the present results. We believe, however, that a simple “motor” explanation for most of PFdl's delay-period activity is an unlikely outcome of such studies. A “motor” interpretation probably does, however, account for a small proportion of PFdl's delay-period activity, consistent with the results of Funahashi et al. (1993b) . On certain assumptions about a default motor plan, such neurons could have the tuning properties of the hybrid cell illustrated in Figure 3 C, part a. It is important to emphasize, however, that the present experiment tested whether the maintenance-memory theory could account for all delay-period activity in PFdl. It cannot. We view this result as supporting an important role for PF in the top-down control of attention. If one takes a motor theory of PF function more seriously than most expert opinion currently does, then it is possible to interpret the present result as indicating a role in context-dependent response or goal selection or in terms of the preparation of movements to remembered targets versus current stimuli. Neither interpretation is consistent with an interpretation of PFdl's delay-period activity entirely in terms of a maintenance-memory function. Fourth, we need to consider the possibility that the neural signals we observed reflect the prediction or anticipation of reward. Maunsell (2004) has recently pointed out that neural signals interpreted as arising from attention could instead reflect reward anticipation or prediction (and vice versa). In the present study, however, reward-related information processing could not have accounted for the properties of attention cells because, until the trigger signal, one alternative place (the remembered location) was associated with reward to the same degree as the attended location. Enhancement Effects The general term attention has been used to cover many disparate concepts, including the effects of attention on sensory processing and the mechanisms that mediate those influences. We emphasize that the present finding differs from previous ones describing effects of attention on phasic, sensory-like responses. Often called the enhancement effect, the finding that sensory responses are larger when a stimulus or location is more attended was first described for the superior colliculus ( Wurtz and Goldberg 1972 ) and has been repeatedly demonstrated for many cortical areas, including PFdl ( Mikami et al. 1982 ; Boch and Goldberg 1989 ; di Pellegrino and Wise 1993a ; Rainer et al. 1998 ; DeSouza and Everling 2004 ). In some instances, and especially in frontal cortex, the enhancement effect depends on the attended location being the target of a movement ( Goldberg and Bushnell 1981 ), but in other cases it does not ( Bushnell et al. 1981 ). It has often been suggested that the source of attention effects, including the enhancement effect, match enhancement, and related phenomena, depends on signals emanating from PF ( Miller et al. 1996 ; Kastner et al. 1999 ; Reynolds et al. 1999 ) or from the frontal eye field ( Thompson et al. 1997 ; Moore and Fallah 2004 ). The present results are consistent with this idea. They cannot, however, be considered as yet another example of the enhancement effect, which involves attention-dependent augmentation of a phasic sensory response. Neurons Encoding Both Attended and Remembered Locations Most neurons did not encode an attended or remembered location exclusively; rather, they exhibited varying degrees of tuning for both variables. The neuron-dropping curves (see Figure 7 ) show that attention cells were able to make limited, but above-chance, estimations of the remembered location and vice versa. As can be seen from the spatial tuning indexes in Figure 3 D, few individual neurons were pure attention or memory encoders (data points along the axes). Thus, the population of spatially tuned cells can be viewed as a continuum with attention and memory cells at the extremes, and hybrid cells in between. Interestingly, the neuron-dropping curves for the hybrid cells (see Figures 7 C and 8 C) showed effective estimation of both the attended and remembered locations. Hybrid neurons with dissimilar preferences for the two locations facilitated such estimations. For instance, the neuron shown in Figure 3 C, part b had a low firing rate when the monkey attended to the 180° location and a high firing rate when it remembered that place. Hybrid cells with dissimilar preferences can resolve the ambiguity inherent in cell activity like that illustrated in Figure 3 C, part a, which cannot distinguish between attended and remembered locations. Previous Neurophysiological Studies Previous neurophysiological studies of PFdl's delay-period activity have been interpreted in terms of the maintenance-memory theory. However, the lack of control over spatial attention in these studies raises questions about these interpretations. Constantinidis et al. (2001) , for example, trained monkeys to make delayed saccades toward the location of the brighter of two visual stimuli that briefly flashed on the video screen. They reported that the activity of PFdl neurons reflected the brightness of the stimuli. Although these authors interpreted their findings as demonstrating a purely sensory-mnemonic function for PFdl neurons, brighter stimuli, being more salient, are well known to attract attention to their location. Similar problems affect the interpretation of data from the “antisaccade” task ( Funahashi et al. 1993b ). In their antisaccade task, Funahashi et al. trained a monkey to respond to a stimulus to the left of a fixation point by making a saccade to the right and vice versa. They interpreted their data as demonstrating a function for PFdl in spatial memory because the largest number of neurons reflected the stimulus location rather than the movement target. They showed that during the delay period, when nothing was present on the screen, some neurons reflected where the stimulus had occurred, and these were interpreted as memory cells. Note, however, that where ever the stimulus appeared, whether in antisaccade or prosaccade trials, it served as an attention attractor. If the response to that signal persisted, then interpreting it exclusively as a sensory memory trace would be problematic. Many studies suggest that, for neurons in PF, the history of what has happened or the context in which it happens often affects neuronal activity in an important and persistent way ( Rainer et al. 1998 ; Asaad et al. 2000 ; Wallis and Miller 2003 ), sometimes regardless of relevancy ( Chen et al. 2001 ). Such persistent signals can be viewed as components of working memory in a general sense, but not in the narrow sense implied by the concept of maintenance memory. Neuroimaging and Neuropsychological Results from Humans Based on the idea that the principal or exclusive function of PFdl is to support maintenance memory ( Goldman-Rakic 1987 ), many neuroimaging papers on PF, including PFdl, have been interpreted as supporting this theory of PF function (see, for example, Courtney et al. 1996 , 1997 , 1998 ; Druzgal and D'Esposito 2003 ; Inoue et al. 2004 ). This idea has been defended ( Goldman-Rakic 2000 ), but a number of alternatives have been suggested. For example, several neuroimaging findings support a role for PF in the control of attention, and brain lesion studies also show attentional deficits after damage to various parts of PF ( Corbetta et al. 1993 ; Rueckert and Grafman 1996 ; Gitelman et al. 1999 ; Kastner et al. 1999 ; Rosen et al. 1999 ; Stuss et al. 1999 ; Cabeza and Nyberg 2000 ; Hopfinger et al. 2000 , 2001 ; Vandenberghe et al. 2000 ; Koski and Petrides 2001 , 2002 ; Astafiev et al. 2003 ; Small et al. 2003 ; Thiel et al. 2004 ; Woldorff et al. 2004 ; see also a recent review by Wood et al. 2003 ). In general, top-down attention has been assumed to result from signals emanating from the frontal cortex and biasing more posterior areas to favor some channels of information over others, and some neuroimaging papers have supported this idea ( Chawla et al. 1999 ; Kastner et al. 1999 ; Corbetta and Shulman 2002 ; Nakahara et al. 2002 ; Pessoa et al. 2003 ). In addition, a role in attentional selection and the related concepts of attention to action and attention to intention have been stressed as an alternative to the maintenance-memory theory of PF function ( Rowe et al. 2000 ; Rowe and Passingham 2001 ; Lau et al. 2004 ). Similarly, monitoring the items in short-term memory has been put forward as a principal function of PFdl, and this also is primarily an attentional function ( Owen et al. 1996 ; Petrides et al. 2002 ). Along these lines, a recent study by Nobre et al. (2004) indicated that PF plays a role in directing attention to locations within mental representations. Neuropsychological Results from Monkeys Previous research on monkeys has also suggested a role for PF (or nearby parts of the frontal lobe) in the orientation of spatial attention. Welch and Stuteville (1958) produced trimodal (auditory, visual, and tactile) neglect-like effects following ablations in the depths of the arcuate sulcus, including what was likely part of PF (although not PFdl). Rizzolatti et al. (1983) reported neglect for space beyond a monkey's reach after lesions targeting area 8. However, for at least one of the two monkeys they studied, the lesion may have included the area studied here. Deuel and Farrar (1993) also produced neglect-like symptoms by making cortical lesions that included much of the same region, and roughly similar observations have been interpreted as motor neglect ( Heilman et al. 1995 ). PF lesions also caused attention-like deficits in a conditional motor learning task (M.F.S. Rushworth et al., personal communication). In the context of the present results, the finding that inactivation of parts of PFdl ( Funahashi et al. 1993a ) produced what were termed “mnemonic scotomas” deserves reconsideration. In that experiment, a transient cue served as the target of a saccade after a delay period. Following local inactivations within PFdl, the monkeys in that study continued to make most of their responses to sites near the cue's remembered location, even with 3-s and 6-s delays after the disappearance of the cue (see their Figures 5, 9, and 13). The monkeys made the vast majority of their responses in the correct direction, but a few saccades fell outside the target zone. This inaccuracy contributed to significantly increased variance in the endpoints of the saccades, and Funahashi et al. (1993a) concluded on this basis that the monkeys were unable to remember the cue's location. We suggest, as an alternative explanation of their results, that their monkeys had a deficit in detecting the stimulus at the cued location, directing attention there, or maintaining their attention at the cued location. Thus, the results interpreted as “mnemonic scotomas” might be better understood as a localized neglect-like phenomenon or some combination of attention and memory deficits. This suggestion finds support in the results of a recent study in humans with PF lesions. Hornak et al. (2004) reported a failure of such patients to pay attention to information on a screen, and this problem accounted for their behavioral deficits. Therefore, the results of Funahashi et al. (1993a) provide little support for either the maintenance-memory theory of PF function or the interpretation of its delay-period activity in terms of that theory. The present results agree better with those of Rushworth et al. (1997) , who found that monkeys could remember nonspatial stimuli across relatively long delay periods after bilateral removal of the part of PF theorized to maintain such memories. The present results also agree with Petrides (2000) , who found that PFdl lesions do not affect the short-term memory for objects (as measured by a susceptibility to increasing delay periods), but do cause impairments in the ability to monitor which items have been selected from a group (as measured by a susceptibility to increasing group size). Conclusions The present study reexamined the interpretation of PFdl's delay-period activity in terms of the maintenance-memory theory. We found that other factors are more important than mnemonic ones. The present results do not argue against a short-term memory function for PF, as one among many contributions to behavior. Nor should they lead to the dismissal of interpretations of some delay-period activity in PF, or some neuroimaging signals from that region, in terms of short-term memory. However, spatial memory signals occur less frequently in PFdl than the maintenance-memory theory predicts. Our data thus accord better with neuroimaging and neuropsychological studies indicating that PF plays a major role in attentional selection, including the monitoring of information and actions ( Owen et al. 1996 ; Rowe et al. 2000 ; Rowe and Passingham 2001 ; Petrides et al. 2002 ; Manly et al. 2003 ; Lau et al. 2004 ). How do our findings mesh with the fact that damage to PF appears to produce deficits in short-term memory, as Jacobsen (1935 , 1936 ) first showed nearly 70 years ago? One possibility is that lesion studies speak more to the inability of other areas to compensate for the loss of PF than to the priority of functions within that region. Another is that an attentional deficit would likely have an important effect on the performance of tasks typically used to assess short-term memory in monkeys, such as matching-to-sample or delayed-response tasks, especially if monkeys use selective attention as a strategy for solving the problems posed by such tasks (see di Pellegrino and Wise 1993b ; Awh and Jonides 2001 ). Although attention could account for many findings about PF, we do not aim to replace one monolithic theory of PF function—the maintenance-memory theory—with an equally monolithic “attention theory.” Delay-period activity appears to reflect the learning and implementation of behavior-guiding rules ( Wise et al. 1996b ; White and Wise 1999 ; Wallis et al. 2001 , Wallis and Miller 2003 ), categorization of events and stimuli ( Freedman et al. 2001 , 2003 ), prediction of forthcoming events ( Rainer et al. 1999 ), task selection ( Hoshi et al. 1998 ; Asaad et al. 2000 ), and adaptive actions within structured-event sequences ( Barone and Joseph 1989 ; Quintana and Fuster 1999 ; Ninokura et al. 2003 , 2004 ; Hoshi and Tanji 2004 ), among other cognitive functions. According to one view, PF functions in general intelligence for the solution of any and all difficult cognitive problems ( Duncan and Owen 2000 ). Gaffan (2002) has likewise argued that PF resembles a global workspace, in the sense used by Baars et al. (2003) , implying a lack of domain selectivity. The present result, by showing that PFdl's delay-period activity lacks an account solely in terms of maintenance memory, supports these ideas to some extent. However, the finding of regional specializations among different parts of the PFdl (see Figure 4 ), in accord with similar findings ( Ninokura et al. 2003 , 2004 ; Hoshi and Tanji 2004 ), suggests that various parts of PF contribute to this global workspace differently, each by making some selective contribution to PF's overall function. Taken together, these observations suggest that delay-period activity in PF reflects functions extending far beyond maintenance memory to include all of the behaviors important to the life of primates. Materials and Methods Behavioral task, apparatus, and single-unit recordings We trained two rhesus monkeys (Macaca mulatta) to perform the task. Each monkey sat in a primate chair in front of a computer monitor placed 57 cm from the monkey's eyes. We recorded eye position with an infrared oculometer and sampled at 250 Hz. The monkeys pressed a waist-high button with their right hand to start each trial and did not release the button until the end of the trial. Once the monkeys pressed the button, a 0.2° fixation point appeared at the center of the screen. After they had fixated this stimulus for 1.0–1.5 s, a 2° solid, gray circle appeared 8° from the center of the screen in one of four places. Figure 1 A, part a illustrates the right (0°) location. After another 1.0–1.5 s, the circle revolved from this initial location to one of four final places ( Figure 1 A, part b) at 90°/s along a circular trajectory centered on the fixation point. For monkey 1, the circle revolved 90° or 180° either clockwise or counterclockwise. For monkey 2, the circle revolved 90°, 180°, or 270° either clockwise or counterclockwise. After the circle stopped, a 1.0 to 2.5-s delay period ensued. Then a trigger signal occurred, which provided an instruction as to the saccade target, as well as a “go” cue for the saccade. The trigger signal consisted of a 150-ms-long change in the circle's brightness ( Figure 1 A, part c), followed by its disappearance ( Figure 1 A, part d). If the circle dimmed, the saccade had to be directed to the circle's final (and current) location on that trial; if the circle brightened, the saccade had to be directed to the circle's initial location on that trial. After the monkeys started a saccade, the central fixation spot disappeared. If the monkeys made a saccade to the correct location, a new 0.2° fixation spot appeared there, and the monkeys had to fixate this spot for 1.0–1.5 s, after which it dimmed. The monkeys could then release the button to produce a fruit juice reward. If the monkeys broke fixation prior to the trigger signal, made an incorrect saccade, or released the button prematurely, the trial was cancelled, and the monkeys could begin a new trial. In control trials, the circle either did not move (both monkeys) or returned to its initial location (360° movement, either clockwise or counterclockwise, monkey 2 only), and the monkeys had to make a saccade to the location of the circle whether it dimmed or brightened. The initial and final locations of the circle and whether it brightened or dimmed were selected pseudorandomly, as was the duration of the delay period and the direction in which the circle revolved around the central fixation point. The monkeys had to complete one correct trial of each type (32 in all, including control trials) before repeating a trial type. After the monkeys learned the task, we implanted recording chambers over the left (monkeys 1 and 2) and right (monkey 1) PFdl. For monkey 1, we used a single-electrode microdrive to obtain single-neuron activity records; for monkey 2, we used a microdrive that independently moved up to seven electrodes. During recordings in monkey 1, we intentionally biased the selection of task-related neurons toward those with delay-period activity. In monkey 2, we recorded the activity of all isolated neurons, regardless of whether they were task related. For histological reconstruction of recording sites, we examined Nissl-stained sections of 40 μm thickness from the right hemisphere in monkey 1 and the left hemisphere in monkey 2. Quantification of tuning We represented firing rate data in a 4 × 4 matrix, F ij , with rows (i) corresponding to the remembered location and columns (j) to the attended location ( Figures 2 , 3 A– 3 C, S1–S3, S5, and S6). We assessed tuning for the remembered locations by comparing the variability of firing rate between trials in different rows with the variability of firing rate between trials from the same row. To avoid the influence of across-column modulations (i.e., an attention effect), both between-row and within-row variabilities were calculated only for matrix elements from the same column, one column at a time, and then we averaged these results. This procedure amounts to comparing different remembered locations, while holding the attended location fixed. To quantify the strength of tuning for the remembered location, we computed a ratio of between-row variability and within-trial type variability: (1) where l 1 and l 2 index individual trials, i, j, and k are matrix indexes that take on the values of 0°, 90°, 180°, and 270°, F ij (l) is the firing rate on the l th trial for which position i was the remembered location and position j was the attended location, and N 1 and N 2 are total number of elements in the respective sums. Control trials were excluded from the calculation by not considering the diagonal elements of F ij (i = j, j = k). We evaluated tuning to the attended location similarly by comparing across-column variability with within-column variability, one row at a time. The strength of representation of the attended location, was quantified as: (2) We used two task periods to compute the single trial firing rates F ij (l). To classify neurons into those representing remembered versus attended location, we used an 800-ms period preceding the trigger signal. We also evaluated spatial tuning (I) during the final 800-ms period before the circle started to move. (Figures S5 A and S6 A illustrate this “early” period slightly differently, averaging activity in an interval from 200 ms to 1,000 ms after the appearance of the circle.) The I Rem or I Att ratios approximated unity for untuned neurons and increased with tuning strength. To measure statistical significance, we used a randomization test. Trials were randomly shuffled among different remembered or attended locations, and the indexes were recomputed. This procedure was repeated 1,000 times to yield a distribution of index values from which we computed a probability p. We chose a statistical significance level of p < 0.01 to classify neurons as tuned for either I Rem , I Att , both, or neither (untuned). Population histograms We computed the population histograms of Figure 6 by first determining each neuron's preferred location, using firing rates during the 800 ms preceding the trigger signal. Then we ranked the trials as belonging to the preferred location, the next most preferred, the third most preferred, and the least preferred location. This ranking was then applied to the other task periods. Next we calculated peri-event time histograms (PETHs) for each rank, separately for each neuron. Averages of these single-neuron PETHs yielded the population histograms. To avoid biasing average histograms by statistical noise in the ranks, we used one half of the trials to compute the ranks and the other half to compute the histograms. If the spatial preference of a neuron merely reflected noise, this procedure tended to nullify the influence of the neuron on the population average. We ranked attended and remembered locations in separate computations. Neuron-dropping curves Neuron-dropping curves ( Figures 7 A– 7 D and S7) estimated how well ensembles of PF neurons represented the remembered and attended locations of the circle ( Wessberg et al. 2000 ). We excluded control trials, in which the circle either did not move or moved 360°, from this analysis. The method measured the probability that the attended and remembered locations could be correctly estimated using a single trial of activity from a neuronal ensemble as a function of its size. The calculation started with a random selection of n neurons from a population. Then, for a given condition (e.g., a remembered location i of 0° and an attended location j of 90°), we selected one trial of that condition randomly from each neuron (test trials). All the other trials for that neuron contributed to a look-up table of firing rates. This look-up table consisted of a matrix of average firing rates <F ij > for remembered locations, i, and attended locations, j. The differences between firing rates in the look-up table and the rate on the selected trial were rank ordered, with a smaller rank signifying a closer match. We then summed the ranks r ij across individual neurons and took the remembered and attended locations associated with the lowest combined rank as the population estimation. The estimated remembered location either agreed or disagreed with the actual remembered location of the selected trial, as did the estimated attended location in a separate computation. Repeating this procedure for a given number of neurons, n, more than 2,400 times—each time starting with a randomly selected set of test trials (more than 200 trials from each of the 12 conditions; four controls excluded)—yielded a percentage of correct estimations of the attended and remembered locations. We then calculated neuron-dropping curves for ensembles of size one to the total number of neurons, but typically the range 1–100 sufficed to capture the main features of the population estimation. To assess the representation of attended and remembered locations during the delays (see Figure 7 A– 7 D), we calculated neuron-dropping curves for the 800-ms period immediately preceding the onset of circle movement (gray curves in Figure 7 A– 7 D) and the 800-ms period immediately preceding the trigger signal (colored curves). Finally, we evaluated the time course of changes in these estimations, using neuron-dropping curves for a 200-ms window, which moved in 50-ms steps along the trigger-aligned records ( Figures 7 E, 7 F, and 8). The 200-ms window measured activity immediately before the time point plotted, to prevent the artifactual early appearance of a signal detection, and thus represents a “backward-looking” average. Supporting Information Figure S1 Rasters and Histograms from a Representative Attention Cell The activity matrix is the same as in Figure 3 A, measured in the 800 ms immediately prior to the trigger signal. This neuron is not the same as that illustrated in Figure 2 . Beneath the activity matrix, the rasters and histograms for each attended and remembered location are displayed in the format of Figure 2 A. (103 KB PPT). Click here for additional data file. Figure S2 Rasters and Histograms from a Representative Memory Cell The activity matrix is the same as in Figure 3 B, measured in the 800 ms prior to the trigger stimulus. Format as in Figure S1 . (95 KB PPT). Click here for additional data file. Figure S3 Rasters and Histograms from Two Representative Hybrid Cells The activity matrix in (A) is the same as in Figure 3 C, part a; the one in (B) is the same as in Figure 3 C, part b. Format as in Figure S1 . (151 KB PPT). Click here for additional data file. Figure S4 Activity Early Versus Late in the Delay Period A table of tuning indexes is given at the top for each of the cell classes (plotted in the bottom part of the figure), combinations of those classes, and other groups of cells as described in the left column. These population averages are divided into two groups of columns, those on the left showing data for the period before the circle began rotating (early) and those on the right showing data for the period after it had stopped and the monkey awaited the trigger signal (late). In the plot, the dashed line shows the median values, the dotted line shows the upper IQR. (56 KB PPT). Click here for additional data file. Figure S5 Activity Early Versus Late in the Delay Period Same PFdl neuron as in Figure 2 . The activity matrix in (C) comes from the data in (A), and the matrix in (D) comes from the data in (B), in the format of Figure 2 C. In (A), the red boxes enclose the measured period for the preferred location, 800 ms prior to the beginning of the circle's movement (200–1,000 ms after circle onset). In (D), the box shows the 800 ms immediately prior to the trigger stimulus. Note that the column-to-column variation in C necessarily results from chance variation because at that time the circle's final location is unknown. The figure shows, by example, that the spatial tuning in the period just before the triggering event strongly exceeds that before the circle begins moving, thus ruling out a strictly sensory account for spatial tuning (see also Figure S4 ). Note that after circle movement, responses to the circle were greater at the cell's preferred location (90°) but smaller at the least preferred location (270°). (188 KB PPT). Click here for additional data file. Figure S6 Activity Early Versus Late in the Delay Period Same PFdl neuron as in Figure S1 , in the format of Figure S5 . The red boxes show the measured period for the cell's preferred location in both (A) and (B). (156 KB PPT). Click here for additional data file. Figure S7 Neuron-Dropping Curves for the Two Monkeys Combined Format as in Figure 7 A– 7 D. (46 KB PPT). Click here for additional data file.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC524249.xml
554989	Automated migration analysis based on cell texture: method & reliability	Background In this paper, we present and validate a way to measure automatically the extent of cell migration based on automated examination of a series of digital photographs. It was designed specifically to identify the impact of Second Hand Smoke (SHS) on endothelial cell migration but has broader applications. The analysis has two stages: (1) preprocessing of image texture, and (2) migration analysis. Results The output is a graphic overlay that indicates the front lines of cell migration superimposed on each original image, with automated reporting of the distance traversed vs. time. Expert preference compares to manual placement of leading edge shows complete equivalence of automated vs. manual leading edge definition for cell migration measurement. Conclusion Our method is indistinguishable from careful manual determinations of cell front lines, with the advantages of full automation, objectivity, and speed.	Background The images we deal with are images of endothelial cells. Endothelial cells migrate to sites of injury in the body. They are involved in forming new blood vessels to help repair damaged areas [ 1 , 2 ]. In particular, we are interested in the effects of SHS on endothelial migration. By comparing automated migration analysis with varied exposure to SHS, for cells with and without specific genes, we can examine why exposure to SHS impairs endothelial cell migration and explore possible cures [ 3 , 4 ]. Cell migration is a basic biologic function that can be modified by changes in genetic code and in response to chemical and other stimuli. Upon 24 hours serum starvation, the cells were artificially wounded using P20 pipette tip across the plate, then cultured respectively in regular DMEM or DMEM containing Second Hand Smoke (SHS) (unpublished). The subsequent gaps were imaged at 0 and 6 hours post SHS exposure as previously described to determine the rate of migration of the front lines to close in the gap [ 5 - 8 , 23 ]. Figure 1 gives an example about the cell migration. Figure 1 Images of a plate of endothelial cells growing in agar. Cells appear as dark spots and contrast is limited because introduction of stains to increase contrast could affect cell function. An early time image (top row) shows a wide trough where a lane of cells was removed by the experimenter. A later time points (bottom row) show the effect of progressive narrowing of this lane as the cells migrate to fill in the gap. Our goal is to measure the width of the "clear lane" which corresponds to the amount of cell migration in the time interval. The automated borders are compared blindly by a team of domain experts to manual borders created by a technician to assess accuracy. Results are also evaluated blind to biologic significance to determine concordance and power to demonstrated biological effects. Biologists deal with this by making multiple manual measurements, to report an average. Observers have difficulty deciding where and how many times to measure the width. Besides, there are many pairs of images to be processed. Therefore, automatic measurement is desired. Implementation Generating texture The primary difference between cell-populated areas and the clear lane is texture. The cell-populated areas are speckled with cells, the clear lane is not. In order to capture the cellularity characteristic of the source images, we sought to compute a texture index that would emphasize the cellular attribute of the region of interest and also minimize the influence of non-cellular signal variations [ 11 - 14 ]. Because the image may have non-uniform background where the "clear lane" can be 'darker' than the 'cells' at other locations, the texture index should be generated from the relative gray value difference. Furthermore, we know the orientation of the experimentally produced clear lane, which we take to be vertical. Then our algorithm generates the texture in this way: For each point in the original image Search for darker point in this line vertically If find Set the distance between start and darker point as the gray value of the corresponding point in the texture map Search for continual darker points and set the distance as the value of them Scale the value to 0–255: where pv is the new pixel intensity value, cv is the distance value of the corresponding point and max and min are the minimum and maximum distance value. Panel b of figure 2 shows the texture of Panel a of figure 2 using this algorithm. Figure 2 An example image is shown in panel (a), followed by the derived texture index (b) and the resultant graphic overlay (c & d). Note that the region between the vertical lines of 2D is relatively devoid of cells, and each line represents the front of cell migration, as desired. Migration analysis Based on the texture map, the region we are interested in appears as a white vertical band. Thus the second stage of analysis must determine the position and width of this lane. As the information in each vertical column is equivalent to repeated measures, we can combine the data to marginal projection. From the histogram of this we can compute a classifier for lane vs. cells and determine the half-height width. The locations are then mapped to a graphic overlay on the original image to demarcate the front lines of cell migration. The change in distance between the front lines reports the amount of (bi-front) cell migration. 1. Project the texture values to a marginal profile "cellularity index profile" (Figure 3 ) which is an array of P --- P [1.. n ] where n is the width of the image. The value of each P [ i ] is the intensity sum of all the pixels in i column. Figure 3 The marginal cellularity index profile. 2. Compute the discriminant classifier (DC) which is average value of P [ i ]. 3. Locate the leading and trailing edges based on classifier crossing. If P [ i ] < DC and P [ i +1] > P [ i ], i is the leading edge. Conversely, if P [ i ] > DC and P [ i +1] < DC, i is the trailing edge. Then a few pairs of leading and trailing edges could be obtained. The target pair is identified based on the width, P [ i ] values between leading and trailing edges and "clear lane" location in the time neighboring image. 4. Record locations and generate graphic overlay for original image. Validation Since the manual assessment is the research gold standard for image processing [ 15 - 18 ], a technologist specially trained to identify the leading and trailing edges of cell migration was provided a computer tool to mark those edges manually in a manner compatible with the graphics overlay engine. These are called "manual edges." The manual edges and the automated edges were then presented to a team of domain experts in random order pairs (one of each on corresponding image) for preference scoring. The scores ranged from 1–5, where 1 is strong preference for first overlay, 2 mild preference for first overlay, 3 equivalency, 4 mild preference for second overlay, and 5 strong preference for second overlay. Results were analyzed by Kappa statistic as a measure of agreement. Results After analysis, results like the ones shown in the Figure 2 are obtained. Panel (a) shows a photograph of the cell cultures, while the remaining panels show various aspects of the analysis. Figure 4 shows the two worst cases of disagreement between automated and manual methods. Figure 4 Examination of width differences between automated and manual identified two outliers with relatively large differences. These are shown as two pairs. Note the uneven heterogeneous cell distribution in these cases, a result that is technical suboptimal and not desired, likely from an error in the excoriation (creation of clear channel). These are a poor sample pairs for technical reasons, and in retrospect domain expert still had no significant difference in preference of manual result over the automatic result. The results of domain expert preference by quality for automated vs. manual assignation of migration front lines, evaluated blinded to method, randomized, and subsequently decoded. Overall, there is complete equivalence of automated vs. manual with respect to expert preference for quality. The values ranged 2–4. In no cases was manual strongly preferred over automatic. Preference testing of analysis methods showed near equivalence, favoring preference for the automated borders (3.02 ± 0.11). Agreement between observers in preference was examined for two domain experts, revealing good agreement (Kappa = 0.59, p < 0.003). Agreement in preferences by a technologist without domain expertise was lower (Kappa = 0.23, 0.25, p > 0.10) but supported the same conclusion: the automated analysis is at least as good as manual selection by domain experts. Application of this method to determine the effect of SHS on endothelial cell migration demonstrates that SHS can reduce the cell migration rate, which is statistically significant (Figure 5 ). Figure 5 The migration distance of control group (NL) is 101.30 ± 10.32 and distance of SHS group 36.25 ± 2.71. Pair t test shows two groups are obviously different with p value = 2.24E-06. Discussion Our migration analysis is based on the texture index of the images. This index should reflect the attribute of the images. Since no global thresholding technique could be used in our images, the segmentation of regions and boundaries (edges) have to consider the local property [ 9 ]. Because the target boundaries always show as a vertical band, the line-based segmentation appears to be the most suitable approach for our task. Further analysis of regions and edges is based on a uniform data structure reflecting the texture character in each column. Our results show a robust automatic method with no detected errors. This study is a pilot study demonstrating feasibility and biologic significance in real application. Further collective experience in multi-center applications are needed to establish the full utility of the method. In addition, the program runs on the software platform, ImageJ [ 10 ] and the speed is fast. A normal process time for one study of images is less than 3 minutes. Results such as width and percentage can be shown as a table. It offers a convenient way for researcher to process their image data using excel. Conclusion We describe a novel method of cell migration analysis based on texture pre-processing and discriminant analysis. Domain expert preference testing demonstrates that this automated method compares favorably to the much more painstaking manual method. The further study is to apply this to evaluation of the impact of SHS on endothelial cell migration. For that purpose, we have constructed a SHS capture system in which we bubble the SHS through tissue culture medium to assess its impact on cell migration. Our results indicate that this analysis system is very sensitive to biological effects, documenting that SHS impairs cell migration [ 19 - 22 ]. Availability and requirements Project name: Cell migration measurement project Project home page: Operating system(s): Platform independent Programming language: Java Other requirements: Java 1.3.1 or higher, ImageJ License: Null Any restrictions to use by non-academics: Licence needed Authors' contributions JDP proposed and designed the method to evaluate cell migration objectively and automatically. JQ implemented the method as a plugin of ImageJ and validated the method. TWC performed the cell experiments and captured the images. LG performed manual edge definition and subsequently performed statistic analysis of expert preferences. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC554989.xml
552334	Anal screening cytology	This issue of CytoJournal contains an article on screening for anal intraepithelial neoplasia in high-risk male patients. This accompanying Editorial focuses on current understanding of this relatively new disease entity, with insights as to the potential role of screening cytopathology in the epidemiology, pathophysiology and clinical management of this HIV and HPV related anal lesion, which predominates in male patients living long-term with AIDS. Mention is made of techniques of obtaining samples, methods of preparation, and morphologic classification. Issues of anoscopic confirmation, as well as topical and surgical management are emphasized. The similarity of initial experiences in anal screening to problems encountered early in cervical cancer screening programs several decades ago, are highlighted.	Note For corresponding research article please see Arian et al, 2005 [ 15 ] The subject of anal screening cytology has entered the epidemiologic and cytopathologic literature as a topic of interest over the last decade, and is highlighted in this issue of CytoJournal in an article by Arain and colleagues. Until recently, anal cancer was not considered to be a neoplasm of major public health concern [ 1 ]. It occurred infrequently, usually in older people, affecting women more often than men; further, being a neoplasm of low incidence, it remained under the radar in terms of screening potential. In some respects, anal cancer mirrored cervical cancer in unscreened women, presenting late in the course of the disease with a variety of pelvic symptoms, and having a protracted, ultimately fatal, course. Patients invariably became social outcasts, suffering from intolerable fecal complications, which presented major nursing challenges. Two aspects of this scenario have changed. First was the fairly recent introduction of effective modern treatment regimes for invasive anal squamous cancers utilizing chemo-radiation, leading to improvement in morbidity and long term survival [ 2 , 3 ]. The second significant alteration was seen in epidemiology, and this is the area which has come to involve screening cytopathology. During the 1990s, in several European and North American cancer centers, an initially unaccountable increase in anal cancers was seen in younger people. It soon became apparent that HIV-positive homosexual males were affected in numbers greatly in excess of those expected. This was first noted in those urban areas in which large concentrations of homosexual men had been treated since the outbreak of the HIV epidemic. The term "males having sex with males" (MSM) was coined to cover these high-risk individuals. Preliminary information gleaned from screening programs in high HIV-positive incidence areas amongst MSM, showed detection rates of intraepithelial and early invasive neoplasia higher than any incidence ever recorded for cervical cancer screening. Further, HIV-negative homosexual MSMs, and HIV positive non-homosexual men (eg drug users) also exhibited an increased incidence of pre-malignant and invasive anal carcinomas. More recently, the syndrome of early onset anal cancer has been extended to include HIV-positive female patients, as well as females who are not HIV-positive, but who have genital HPV. All these groups show a higher incidence of abnormal anal cytopathology, though none quite as high as that found in HIV positive MSM. The unifying factor in most instances is ano-receptive intercourse, or extension of HPV infection from the genital tract to the anal mucosa, most obvious in the face of deficient immunity, or high viral load [ 4 - 6 ]. Epidemiologic studies have indicated that a prolonged preclinical phase precedes the onset of anal cancer in these high-risk groups. Just as in the cervix, there is a transitional zone (although not an abrupt squamocolumnar junction) in the anus. Rectal mucosa, with goblet cells, ends about one inch proximal to the external sphincter, giving way to a transitional epithelium, which in turn blends into a stratified squamous epithelium at the level of the anus. (External to the anus, any lesions which arise are considered to be primary skin lesions rather than anal lesions.) Almost all anal cancers develop in the transitional zone, where atypical, dysplastic and in-situ lesions are identifiable histologically. The cytologic counterparts are those of atypical cells of uncertain significance (ASCUS), low grade squamous intraepithelial lesions (LSIL) and high grade squamous intraepithelial lesions (HSIL). Once this natural history had been demonstrated and confirmed, it seemed natural that exfoliative cytology could be investigated as an "anal Pap smear" [ 7 , 8 ]. As with the cervix, the technique of obtaining the anal sample is critical to the success of screening. Standard colonic preparation is not required, but the rectum should be emptied prior to obtaining the anal sample. Brushes, brooms and Dacron swabs have all been used, the type of spatula probably being less important than the skill of the operator. The instrument is inserted to a depth of one and a half inches beyond the external sphincter, and subsequently withdrawn in a firm downward spiral movement incorporating 10–12 rotations, to ensure the device has made contact with the full surface area of the transformation zone. Some have used direct smears onto glass slides with immediate wet fixation; most centers, however, employ immediate insertion of the scraping device into liquid fixative for thin layer preparation. This appears both to improve adequacy and preservation, and also to eliminate any fecal contamination; residual material in the vial can be used for HPV studies if required, or for the creation of a bank of teaching slides. There has been a relative dearth of literature on the cytomorphology of anal samples [ 9 - 11 ]. Classification according to Bethesda guidelines has been advised, implying similarity of the exfoliative cytology of atypical squamous cells of undetermined significance (ASC-US), anal intraepithelial lesions (A-SIL) and invasive squamous-cell neoplasms to those encountered in the cervix. This early in the development of the science of anal screening, and evident also in the current article in CytoJournal, there are indications that cytologic evaluation may not fully anticipate the severity of some lesions, when compared with biopsies taken simultaneously. Several groups have advised that all abnormal anal Paps should be followed by anoscopic evaluation, with biopsy confirmation when necessary. Anoscopy essentially mirrors colposcopy, enabling the viewer to see vascular abnormalities at magnification, and direct biopsies to the most abnormal-appearing areas. Despite these basic similarities, it is strongly stated in the literature that expertise in colposcopy does not equate to immediate competence in anoscopy; a significant learning curve exists for those wishing to acquire excellence in anoscopic technique, with associated accurate biopsy sampling. It is important that the anoscopist not be sidelined by visible condylomata, which may merely be sentinels of deeper flat lesions of higher grade. At the present time, lack of available expertise in this interventional follow-up of abnormal anal Paps may be the single limiting factor in any new screening program. It thus behooves those interpreting anal samples to be as proficient as they can be in pre-interventional assessment of the anal transformation zone. Experience over the last decade suggests that anal cancer may be as highly appropriate a target for screening as is the cervix, in selected populations. The neoplasm is frequently encountered in well-defined high-risk groups. It has a detectable pre-malignant phase, and is amenable to easy cytologic sampling. Cytodiagnosis is reasonably sensitive and highly specific, and histologic confirmation is relatively easily obtained by well-trained personnel. If there is a current area of deficiency in such programs, it may well be in the treatment of intraepithelial lesions, which, as yet, has not been adequately assessed in large numbers of patients. It is known that highly active anti-retroviral therapy (HAART) does not appear to alter the pathophysiology of anal lesions once initiated. A variety of topical agents such as podophyllotoxin and imiquimod have been tried, as has intralesional interferon; superficial ablative therapies including liquid nitrogen, electrocautery, laser and LEEP have been attempted with varying success rates. Circumferential surgical resection almost inevitably results in unacceptable loss of sphincter control and soiling, but anoscopy-directed limited excision may prove less morbid. The fact that so many options are available implies perhaps that no single modality is yet considered sufficiently effective, with minimal complication [ 12 , 13 ]. This, too, is reminiscent of the early years of management of cervical pre-neoplasia, when a host of methods was pursued in attempted elimination of focal lesions of the squamocolumnar junction. As in the cervix, human ingenuity will undoubtedly prevail, and one or two forms of extirpation will emerge as both efficacious and uncomplicated. An interesting consideration is whether or not anal cancer, and thereby anal cytopathology screening programs, would be of value in the developing world, particularly in Africa, India and China, where the bulk of the global incidence of HIV resides. This will depend on two very different factors. First is the nature of transmission. Unlike the situation in the developed world, AIDS in these regions is not essentially a disease of homosexual males; thus, without anal intercourse predominating, an upward trend in the incidence of anal cancer would seem unlikely. Anal screening programs would be unnecessary or cost- in effective in these communities. The second feature dictating the institution of screening programs in developing countries relates to antiretroviral therapy. Anal intraepithelial neoplasia and cancer are not encountered early in the progression of HIV/AIDS. Rather, they are late complications of patients living long-term with AIDS, usually implying patients living long-term on HAART [ 14 ]. Unless affordable very low cost antiretroviral drugs could be manufactured and distributed widely, it seems unlikely that patients in developing countries would survive into the time zone in which delayed neoplasms such as anal cancer become a public health priority.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC552334.xml
552308	Quality of life in patients with personality disorders seen at an ordinary psychiatric outpatient clinic	Background Epidemiological studies have found reduced health-related quality of life (QoL) in patients with personality disorders (PDs), but few clinical studies have examined QoL in PDs, and none of them are from an ordinary psychiatric outpatient clinic (POC). We wanted to examine QoL in patients with PDs seen at a POC, to explore the associations of QoL with established psychiatric measures, and to evaluate QoL as an outcome measure in PD patients. Methods 72 patients with PDs at a POC filled in the MOS Short Form 36 (SF-36), and two established psychiatric self-rating measures. A national norm sample was compared on the SF-36. An independent psychiatrist diagnosed PDs and Axis-I disorders by structured interviews and rated the Global Assessment of Functioning (GAF). All measurements were repeated in the 39 PD patients that attended the 2 years follow-up examination. Results PD patients showed high co-morbidity with other PDs and Axis I mental disorders, and they scored significantly lower on all the SF-36 dimensions than age- and gender-adjusted norms. Adjustment for co-morbid Axis I disorders had some influence, however. The SF-36 mental health, vitality, and social functioning were significantly associated with the GAF and the self-rated psychiatric measures. Significant changes at follow-up were found in the psychiatric measures, but only on the mental health and role-physical of the SF-36. Conclusion Patients with PDs seen for treatment at a POC have globally poor QoL. Both physical and mental dimensions of the SF-36 are correlated with established psychiatric measures in such patients, but significant changes in these measures are only partly associated with changes in the SF-36 dimensions.	Background According to the DSM-IV [ 1 ] personality disorders (PDs) are characterized by enduringly deviating patterns of perceiving, relating to, and thinking about the environment and oneself that are exhibited in a wide range of social and personal contexts. Such patterns lead to "clinically significant distress or impairment in social, occupational, or other important areas of functioning". The DSM-IV does not indicate how "clinically significant distress or impairment" (page 633) should be evaluated, however, and a recent study showed that various formulations of this criterion hardly increased diagnostic validity [ 2 ]. Since the DSM-IV included the Global Assessment of Functioning Scale (GAF) as Axis V, it is reasonable to consider if the GAF should be used for evaluations of "significant distress or impairment". Two problems are implicit in such an approach: at what GAF cut-off score should "significant distress or impairment" be set, and the evaluation is only done by a professional. As to the first problem, Kessler et al [ 3 ] suggested a GAF score of 60 as a cut off for "serious mental illness". As to the second, in patients with somatic diseases "clinically significant distress or impairment" has for a long time been quantified by self-rating of the health-related quality of life (QoL) including mental health [ 4 ]. Several general instruments for the rating of QoL have been developed among which the Medical Outcome Study Short Form-36 (the SF-36) and its brief version the SF-12 have become the most popular [ 5 , 6 ]. Major treatment outcome studies of PDs like the Collaborative Longitudinal Personality Disorders Study [ 7 ], the Norwegian Network of Psychotherapeutic Day Hospitals [ 8 ], and the Cassel Hospital study [ 9 ] used the GAF, however, and did not included any QoL measurements, and the QoL was not included in a recommended core battery of instruments for measurement of changes in PD patients [ 10 ]. In contrast, two epidemiological studies of PDs have used the SF-12 as a measure of disability. In a national study from Australia, Jackson and Burgess [ 11 ] reported that the SF-12 Physical and Mental Component Summary Scales (PCS and MCS) were both significantly reduced in persons with one or more PDs diagnosed by a screening instrument, compared to persons without. When further examining the relationship between the SF-12 and PDs, they found that co-morbid Axis I and chronic physical conditions explained a considerable part of the MCS and PCS scores in PDs [ 12 ]. Mean MCS and PCS scores also became significantly lower with an increasing number of comorbid PDs present. A national study from the United States [ 13 ] confirmed the reduced MCS after controlling for co-morbid Axis I disorder in the avoidant, dependent, paranoid, schizoid, and antisocial PDs, but not in the histrionic PD, diagnosed by a more extensive diagnostic interview schedule than the Australian study. A review of the literature showed that the QoL had only been used as a disability measure in a few clinical research studies of PD patients. In depressed elderly patients, Abrams et al [ 14 ] found that the presence of criteria for cluster B PDs predicted lower QoL. Since the cluster B criteria overlapped considerably with symptoms of depression, it was unclear if they made any independent contribution to reduced QoL. Swinton et al [ 15 ] reported that male PD patients in a high security forensic setting were less satisfied with their overall QoL than patients with schizophrenia. The authors emphasized that the high security setting was quite unusual. Hueston et al. [ 16 ] showed that primary care patients with high risk for PDs, scored significantly lower on overall QoL and on several subscales of the SF-36, compared to patients with a low risk for PDs. Since prevalence of depression and alcohol dependence was higher in the high-risk group, the influence of PDs alone on QoL was difficult to tease out in that study. Nakao et al [ 17 ] examined the relationship between PDs and the GAF in 136 Axis I patients mainly with mood and anxiety disorders and found that patients with any comorbid PDs were more disabled than those without. They did not adjust for the presence of Axis I disorders, however. None of these clinical studies takes QoL as observed in PD patients seen at an ordinary psychiatric outpatient clinic (POC) as their point of departure. However PD patients are frequent at POC, and the QoL is an important self-rated measure of "clinically significant distress or impairment". Since QoL data on PD patients seen at at POC seems to be lacking from the literature, we found it relevant to study a consecutive sample of PD patients from a POC and collect QoL data with SF-36. We posed the following research questions: 1) How are the SF-36 dimensions mean scores in PD patients compared to age- and gender-adjusted norm data? 2) To what extent are the SF-36 scores in PD patients associated with co-morbid Axis I disorders? 3) How is the association between the SF-36 dimension and established patient- and professional-rated psychiatric measures in PD patients? and 4) What changes in the SF-36 dimensions of treated PD patients are observed from baseline to follow-up, and how are they related to changes in the psychiatric measures? Methods Setting Furuset POC serves a communality of Oslo City, Norway with a population of 28.000 people. At the time of the study, the staff consisted of three psychiatrists, three clinical psychologists, two psychiatric nurses, and two social workers. The intake rate was approximately 400 new patients a year. The first author (KN) invited the staff to take part in the study by referring to her new patients with probable PDs. Six professionals were willing to participate, while four declined due to heavy clinical burden, or lack of interest. At the start of the study in 1996, Furuset was a new suburb of Oslo, and the inhabitants were characterized by lower socio-economic conditions, high mobility, and a considerable prevalence of immigrants from Asian countries. The suburb had a high proportion of municipal housings, and the criterion for allotment to them was severe mental disorder and/or severe socio-economic problems. Many patients seen at Furuset POC were out of work due to mental disorders, and/or due to socio-economic circumstances. Patients Patients aged from 18 to 75 years were consecutively recruited from January 1, 1996 to June 30, 1998. The patients were referred from the local GPs, and physical examination and adequate treatment and follow-up of physical diseases were the responsibility of the GPs. The six therapists screened for probable PDs among new patients scheduled for treatment. Exclusion criteria were mental retardation, lifetime psychosis and bipolar disorder, organic mental disorders, current strong suicidal ideation, and insufficient knowledge of the Norwegian language. Eligible patients received oral and written information about the study from their therapists. Then the patients were invited to take part in the study, and they all gave written informed consent. The Ethical Review Board of Department of Psychiatry, Aker University Hospital approved the project. The six therapists did not miss out any patients at screening, but 5 (4%) eligible patients declined to take part in the study. Among 110 eligible patients referred to the study, only 91 filled in the SF-36 at baseline due to administrative misunderstandings. However, when they were compared to the 19 who did not fill in, the non-attenders only had significantly fewer co-morbid Axis I-disorders (data not shown). In order to answer the research questions, the sample was divided into three groups: cluster A+B PDs (n = 39), cluster C (n = 33), and Axis I-disorders (n = 19). The cluster A+B group could also contain co-morbid cluster C PDs and Axis I-disorders, and the cluster C co-morbid Axis I-disorders. Follow-up procedure Two years after baseline, the patients received a mailed written appointment for a follow-up interview. Those who did not show up were sent a written reminder. If they still did not meet, they were called by phone, and if there was no answer, their addresses and phone numbers were checked at the Census register. Appointments were mailed to new addresses, and phone-calls were made in case of non-response. Only a few patients responded to these extended search procedures. Norm sample Norm data on the SF-36 was obtained from the Survey of Level of Living in Norway 1998 [ 18 ] comprising 6.638 participants aged 23 to 75 years. The norm data were adjusted by gender and distribution into 5-year age groups in relation to the PD sample. Assessments At baseline, diagnoses of PDs were made with the use of the Personality Disorder Examination, and Axis I-disorders were diagnosed by the MINI-International Neuropsychiatric Interview. Anamnestic data were collected, and global assessment of function was rated. The professional-based interviews and examinations of all patients at baseline and follow-up were carried out by a single experienced psychiatrist (KN), who did not take part in any treatment given. All patients also filled in the following self-rating instruments at baseline: the SF-36, the Social Adjustment Scale, and the Symptom Checklist 90-Revised Personality Severity Index. At follow-up all these assessments were repeated, and additional information about treatment as well as job/education, social- and family changes was collected. Measures Professional-rated The Personality Disorder Examination (PDE) [ 19 ] is a structured clinical interview for PDs according to the DSM-III-R with good inter-rater reliability, and wide international application. Findings are reported as PD diagnoses, and as dimensional PD scores based on the sum of the scoring on each PD criterion (0: not present, 1: probably present, and 2: definitely present). Dimensional scores for the PD clusters are used as a main psychopathology variable, and the numbers of PDs are also reported. The MINI International Neuropsychiatric Interview [ 20 ] was used to diagnose Axis-I disorders according to DSM-IV. The MINI covers 18 Axis-I disorders, has been translated into many languages and has demonstrated good inter-rater reliability. Findings are reported as numbers and percentages of patients with positive Axis-I diagnoses, and as mean number of such diagnoses. The Global Assessment of Functioning (GAF) is a rating scale for the current evaluation of the overall functioning of a subject on a continuum from severe mental disorder to complete mental health that was defined as Axis V of the DSM-IV. Scale values range from 1 (sickest individual) to 100 (the healthiest person). The scale is divided in ten equal intervals from 1 – 10 to 91 – 100. Most outpatients will be rated between 40 and 70, although some individuals rated above 70 may seek therapy. The GAF is a reliable instrument [ 21 ], and the cut-off score for 'minimal impairment' has been set at 70 points or higher [ 22 ] and for 'serious mental disorder' at lower than 60 [ 3 ]. Patient-rated The SF-36 [ 5 ] was chosen for measurement of health-related QoL, since it is in widespread use, and has shown good psychometric properties in Norway [ 23 ]. The SF-36 has demonstrated sensitivity to change, and score changes can be interpreted as changes in the health-related quality of life of the patient. The SF-36 assesses eight dimensions of physical and mental health, and the range is from 100 (optimal) to zero (poorest): physical functioning (PF), physical role functioning (RP), bodily pain (BP), general health (GH), vitality (VT), social functioning (SF), emotional role functioning (RE), and mental health (MH). The Social Adjustment Scale (SAS-SR) [ 24 ] contains 42 questions which investigate expressive or instrumental roles in six major areas of functioning: work, social and leisure activities, relationship with extended family, role as a spouse/ partner, role as a parent, and role as member of the family unit. Each area is measured as a continuous variable, and the scales for individual items range from 1 (best) to 5 (worst). The scores within each role area are summed, and a mean for each area is obtained. By adding up the scores of all items and dividing by the number of items actually scored, an overall adjustment score is obtained. The Symptom Checklist 90-Revised (SCL-90-R), Personality Severity Index (PSI). The SCL-90-R is a 90-item self-report inventory assessing current levels of mental symptoms patterns. Each item is a description of a mental symptom rated on a five-point scale, and rates the degree of 'distress/discomfort' during the last week prior to its administration. Several indices based on the SCL-90-R scores have been defined, and the PSI is the mean value of 22 items covering the interpersonal sensitivity, anger/ hostility, and paranoid ideation subscales. The PSI reflects the presence and severity of relatively enduring characteristics of the patient, and is, therefore, relevant for the evaluation of severe PDs [ 25 ]. For the PSI, pathology is defined by a cut-off score of ≥ 1.0. Statistics Data were analyzed by SPSS version 12.0. Descriptive statistics were conducted with independent and paired-sample t-test as well as one-way ANOVA (with Bonferroni's correction for multiple comparisons) for metric variables, and with χ 2 and Fisher's Exact Test for categorical variables. The Mann-Whitney test and the Wilcoxon signed-rank test were applied for metric variables when the data distribution violated parametric assumptions. Spearman's rank correlation was used for associations. One-sample t-tests were applied when the SF-36 dimension scores of the age- and gender-adjusted norm groups were compared to the means of the three diagnostic groups. The comparison of mean scores on the SF-36 dimensions between the three groups was conducted with oneway ANOVA with Bonferroni's correction. The influence on the QoL scores of patients with PDs of Axis I disorders and of cluster C PDs in the cluster A+B PDs group, was examined with linear regression analyses. All tests were two-sided, and the level of significance was set at p < .05. Results Sample characteristics All the 91 patients included were Caucasian, and 48 (53%) were females. The mean age of the sample at baseline was 36.3 years (SD 10.5) and ranging from 19 to 74 years, with a median of 35 years. None of the patients had any significant somatic diseases as reported by their GPs. Patients belonging to the cluster A+B, cluster C, and Axis I- disorders groups did not differ at baseline on demographic variables (Table 1 ). Table 1 Demographic and psychopathological features at baseline in patients with cluster A+B, and cluster C personality disorders, and non-psychotic Axis I disorders. Variables Cluster A+B (n = 39) Cluster C (n = 33) Axis I (n = 19) p Age (mean, SD) 35.1 (11.2) 36.6 (10.9) 41.1 (10.4) .15 Gender (n, %) .74 Male 17 (44) 17 (52) 8 (42) Female 22 (55) 16 (48) 11 (58) Relationship (n, %) .53 Paired 23 (59) 18 (55) 7 (37) Non-paired 16 (41) 15 (45) 12 (63) Basic education level (n, %) .28 ≤ 9 years 15 (33) 13 (32) 8 (33) 10 – 12 years 14 (31) 16 (39) 13 (54) ≥ 13 years 16 (36) 12 (29) 3 (13) Early childhood loss (n, %) 12 (31) 7 (21) 1 (5) .22 Childhood sexual abuse (n, %) 7 (18) 7 (21) 1 (5) .22 Age of onset mental problems (mean, SD) 16.7 (8.9) 20.0 (8.4) 27.0 (11.8) <. 001 A+B, C vs Axis I Work income (n, %) 23 (59) 21 (64) 13 (68) .78 Economic support 16 (41) 12 (36) 6 (32) Income last year (1.000 NOK) (mean, SD) 154 (74) 178 (70) 179 (62) .27 On sickleave last year (n, %) .37 No 18 (46) 10 (30) 5 (26) ≤ 12 weeks 11 (28) 12 (37) 5 (26) ≥ 13 weeks 10 (26) 11 (33) 9 (52) No of PD cluster criteria (mean, SD) Total 41.8 (16.2) 26.6 (11.9) 8.1 (9.2) < .001 A+B vs C vs Axis I Cluster A 11.3 (8.2) 5.6 (4.6) 1.6 (2.0) < .001 A+B vs C, Axis I Cluster B 14.7 (10.9) 4.8 (5.4) 2.6 (3.7) < .001 A+B vs C, Axis I Cluster C 15.8 (10.0) 16.2 (5.7) 3.9 (5.4) < .001 A+B, C vs Axis I Comorbid Axis I disorder (n, % .60 No 10 (26) 8 (24) - Yes 29 (74) 25 (76) GAF (mean, SD) 41.5 (9.1) 51.0 (7.4) 55.4 (9.3) < .001 A+B vs C, Axis I PSI (mean, SD) 1.80 (.78) 1.24 (.71) 1.24 (.81) .005 A+B vs C, axis I SAS (mean, SD) Overall adjustment 2.70 (.66) 2.59 (.59) 2.16 (.52) .007 A+B, C vs Axis I Work 2.86 (1.62) 3.01 (1.80) 2.07 (.50) .94 Social and leisure 3.18 (1.18) 2.96 (1.06) 2.44 (.91) .06 Extended family 2.29 (.60) 2.00 (.51) 1.95 (.68) .05 Marital, partnership 2.21 (.56) 2.59 (.78) 2.14 (.63) .10 Parental 2.00 (.66) 1.91 (.58) 1.52 (.67) .14 Family unit 2.17 (.96) 2.21 (.95) 2.10 (.81) .94 As to psychopathology, the Cluster A and B PDs criteria sum scores were significantly higher in the Cluster A+B group compared to the two other groups, and both cluster A+B and cluster C group had significantly higher scores on cluster C criteria sum score than the Axis I group (Table 1 ). The Cluster A+B group had significantly lower GAF-score and higher PSI score than the two other groups which did not differ from each other. Mental problems had started significantly earlier in the PD groups compared to the Axis I-group. The SAS Overall adjustment was significantly poorer in the cluster A+B and cluster C groups, compared to the Axis I group. The diagnostic distribution of PDs and Axis I disorders in the three groups at baseline and follow-up are given in Table 2 . The mean number of PDs diagnosed in cluster A+B was 2.2, and in cluster C 1.3 per patient, and the most common PDs were avoidant, borderline, and dependent. One PD was observed in 37 patients (51%), two PDs in 20 (28%), and three PDs in 15 patients (21%). Fifty-four (75%) of the PD patient had at least one co-morbid Axis I-disorder. Table 2 Diagnostic distribution of the baseline and follow-up samples. Baseline Follow-up Cluster A+B (n = 39) Cluster C (n = 33) Axis I (n = 19) Cluster A+B (n = 18) Cluster C (n = 21) Axis I (n = 11) Personality disorders (DSM-III-R) N (%) N (%) N (%) N (%) N (%) N (%) Paranoid 14 (36) - - 7 (39) 2 (10) 1 (9) Schizotyp 6 (15) - - 2 (11) 1 (5) - Schizoid 4 (10) - - 4 (22) - - Antisocial 4 (10) - - 1 (6) - - Narcissistic 0 (0) - - 0 (0) - - Histrionic 4 (10) - - 0 (0) - - Borderline 19 (49) - - 2 (11) - - Dependent 5 (13) 7 (21) - 1 (6) 3 (14) - Avoidant 15 (39) 25 (76) - 11 (61) 15 (71) 3 (27) Obsessive-compulsive 5 (13) 8 (24) - 1 (6) 2 (10) - Passive-aggressive 8 (21) 2 (6) - 1 (6) 2 (10) - Mean no of PDs 2.2 1.3 - 1.7 1.2 .4 Axis I disorders (DSM-IV) Major depression 6 (15) 9 (27) 3 (16) 0 (0) 1 (5) 1 (9) Dysthymia 5 (13) 11 (33) 5 (26) 4 (22) 4 (19) 1 (9) Panic disorder 7 (18) 7 (21) 2 (11) 0 (0) 2 (10) 1 (9) Agoraphobia 5 (13) 7 (21) 2 (11) 2 (11) 2 (10) 2 (18) Social phobia 9 (23) 8 (24) 2 (11) 2 (11) 4 (19) 1 (9) GAD* 1 (3) 0 (0) 2 (11) 0 (0) 0 (0) 0 (0) OCD* 4 (10) 2 (6) 2 (11) 0 (0) 0 (0) 1 (9) Alcohol dependence 10 (26) 2 (6) 3 (16) 1 (6) 0 (0) 2 (18) Substance dependence 7 (18) 0 (0) 2 (6) 0 (0) 1 (5) 0 (0) Bulimia nervosa 4 (10) 1 (3) 0 (0) 0 (0) 1 (5) 0 (0) PTSD* 0 (0) 1 (3) 0 (0) 0 (0) 0 (0) 0 (0) Mean no of Axis I disorders 1.5 1.5 1.2 .5 0.7 0.8 GAD: Generalized anxiety disorders, OCD: Obsessive-compulsive disorder, PTSD: Post-traumatic stress disorder Axis-I disorders were equally common in the two PD groups (mean 1.5 disorder) and with slightly lower mean (1.2) in the Axis I-disorder group. Depressions, anxiety disorders, and alcohol dependence were the most frequent Axis I diagnoses in all groups. QoL in PD patients Figure 1 . shows that the mean scores on the eight dimensions of the SF-36 of the PD patients at baseline are significantly lower (p < .001 for all) than those of the age- and gender-adjusted norms. The mean difference was least (13 points) for PF, and highest for RF and RE (54 points and 49 points, respectively). Figure 1 SF-36 mean dimensional scores in personality disorder sample (N = 72) and the age- and gender-adjusted norm sample. Linear regression analyses showed that control for co-morbid Axis I disorders reduced the PF, GH, VT, SF, and MH scores of the total PD group significantly. Controlling for comorbid cluster C PDs did not influence the SF-36 scores of the cluster A+B PDs to any significant extent, while control for Axis disorders significantly reduced the MH scores in the cluster A+B and cluster C groups. No significant differences were found between genders on any of the SF-36 dimensions among the PD patients (data not shown). Both the cluster A+B, the cluster C, and the Axis I group differed significantly from their norms on all eight SF-36 dimensions (data not shown). No significant differences were observed on the eight SF-36 dimensions between the three diagnostic groups (Figure 2 ). Figure 2 SF-36 mean dimensional scores at baseline for Cluster A+B, Cluster C, and Axis I-disorders groups. When we compared the patients with one (n = 37), two (n = 20), and three or more (n = 15) PDs, we did not observe any significant differences in mean MCS and PCS scores. Correlation between SF-36 dimensions and other measures The eight dimensions of SF-36 are regularly divided into the four physical: PF, RP, BP, and GH, and the four mental ones: VT, SF, RE, and MH. In our PD sample the SF-36 mental dimensions had most significant correlations with the psychiatric measures of the GAF, the SCL-90-R PSI, the sum of positive PDs diagnostic criteria, and the dimensions of the SAS (Table 3 ). The SF-36 MH had a significant correlation to most of these measures, followed by VT and SF. The physical dimensions of the SF-36 had less frequently a significant correlation to the psychiatric measures. Table 3 Correlation of SF-36 dimensions with Global Assessment of Functioning (GAF), Personality Severity Index (PSI), and dimensions of Social Adjustment Scale (SAS). PF RP BP GH VT SF RE MH GAF .12 .32* .02 .32** .26* .36 .30 .40 Total no of positive PD criteria .03 -.11 .02 -.27 -.14 -.18 -.05 -.26** SCL-90-R PSI -.22* -.17 -.12 -.26* -.25* -.34 -.19 -.38 SAS Overall -.38 -.31 -.23* -.34 -.42 -.45 -.30 -.45** SAS Work -.17 -.22* -.10 -.16 -.44** -.31* -.26* -.41 SAS Social and leisure -.30 -.35** -.24* -.32 -.43 -.39 -.29 -.37** SAS Extended family -.20 -.18 -.23* -.21 -.26* -.32* -.28* -.43 SAS Marital/partner -.18 .01 -.04 -.18 -.24 -.08 -.05 -.10 SAS Parental -.04 -.06 .06 .02 -.07 -.09 -.07 -.04 SAS Family unit -.38 -.22 -.26* -.20 -.37** -.31* -.27* -.35** Sum significant correlation 4 4 4 4 6 7 6 8 * Correlation is significant at the .05 level (two-tailed), ** Correlation is significant at the .01 level (two-tailed) The SAS overall adjustment and the SAS social and leisure functioning had significant correlations to all the SF-36 dimensions, while the SAS marital/ partnership and the SAS parental functioning had none. The SAS work, extended family, and family unit fell in between. What changes in the QoL of PD patients can be observed from baseline to follow-up two years later? Although quite intensive search for patients was done for the follow-up examination, only 50 patients (53%) of the 91 patients who rated themselves on the SF-36 complied. The distribution of patients were cluster A+B group (n = 18), cluster C (n = 21), and Axis I-disorder group (n = 11). Due to small sample sizes, the Axis I disorder was dropped from further analysis and the two PD groups were pooled as to the study of changes after treatment. The 39 PD patient with SF-36 ratings both at baseline and follow-up were compared to the 33 PD patients only seen at baseline. The diagnoses at follow-up are shown in Table 2 , and among the non-compliant patients those with borderline PD, and alcohol and substance dependence were over-represented. Few significant differences were observed between compliant and non-compliant PD patients at follow-up (Table 4 ). In particular, no significant differences of the eight SF-36 dimensions were observed between the compliers and non-compliers at baseline. The compliers had significantly more depressive disorders and cluster C PDs at baseline. All of those who terminated treatment without the consent of their therapist (N = 22) were in the non-compliant group. The non-compliant patients also had a significantly longer mean duration of treatment. The mean treatment time for the PD patients attending follow-up was 16.6 months (SD 5.9), median 18 months, and range 4 to 24 months, and the mean follow-up time since treatment termination was 9.8 months (SD 6.4), median 10.4 months, and range 0 to 26 months. The majority of the patients had weekly individual psychotherapy, although a small proportion also had group psychotherapy in addition. Drug treatment was given to 20 patients of the 39 patients, and to 20 of the 33 non-compliers (ns). Among those seen at follow-up, had 15 got antidepressive and 5 antipsychotic medication, in addition to psychotherapy. Table 4 Demographic, psychopathological, and treatment features at baseline for patients with personality disorders with (N = 39) and without (n = 33) follow-up examination. Variable Follow-up + (n = 39) Follow-up - (n = 33) p Age (mean, SD) 37.9 (11.6) 33.3 (9.9) .08 Gender (n, %) .50 Male 17 (44) 17 (52) Female 22 (56) 16 (48) Relationship (n, %) .56 Paired 18 (46) 13 (39) Non-paired 21 (54) 20 (61) Basic education level (n, %) .64 ≤ 9 years 11 (29) 13 (40) 10 – 12 years 13 (34) 10 (30) ≥ 13 years 14 (37) 10 (30) ≥ 1 cluster A PDs 13 (33) 6 (18) .15 ≥ 1 cluster B PDs 10 (26) 15 (45) .08 ≥ 1 cluster C PDs 35 (90) 22 (67) .02 Mean (SD) of PD cluster criteria Total 34.8 (17.9) 34.8 (14.3) .99 Cluster A 9.5 (8.7) 7.7 (5.1) .28 Cluster B 7.6 (9.3) 13.1 (10.3) .02 Cluster C 17.6 (8.9) 14.0 (7.2) .07 ≥ 1 depressive disorder 19 (49) 7 (21) .02 ≥ 1 anxiety disorder 17 (44) 16 (49) .68 ≥ 1 substance use disorder 9 (23) 11 (33) .33 Comorbid Axis I disorder (n, %) .79 No 9 (23) 9 (27) Yes 30 (77) 24 (33) GAF (mean, SD) 46.0 (9.4) 45.7 (9.9) .91 PSI (mean, SD) 1.5 (.9) 1.6 (.7) .85 SAS Overall (mean, SD) 2.7 (.6) 2.6 (.6) .86 SF-36 (mean, SD)* Physical Functioning 79.4 (19.1) 76.7 (22.5) .72 Role Functioning 31.4 (34.3) 25.8 (36.2) .27 Bodily Pain 47.6 (28.9) 48.1 (26.7) .71 General Health 51.4 (23.5) 50.5 (20.39 .99 Vitality 35.0 (19.6) 29.1 (18.6) .16 Social Functioning 45.2 (28.5) 48.9 (21.5) .60 Role Emotional 42.7 (39.7) 32.3 (31.7) .35 Mental Health 42.5 (23.2) 38.3 (19.5) .52 No of sessions (mean, SD)* 16.6 (5.9) 18.8 (26.9) .01 Termination without consensus (n, %) 0 (0.0) 22 (67) < . 001 Treated by specialist (n, %) 14 (36) 16 (49) .28 Additional drug treatment (n, %) 20 (51) 20 (61) .43 *Mann-Whitney tests In the 39 PD patients who complied at both baseline and follow-up, significant improvement was seen in the RF and MH dimensions of the SF-36, while considerable, but non-significant changes were observed for BP and SF (Table 5 ). Table 5 Changes from baseline to follow-up in patients with personality disorders (n = 39). Measure Baseline Mean (SD) Follow-up Mean (SD) P SF-36 Physical Functioning 79.4 (19.2) 76.8 (24.6) .95 Role Physical 31.4 (34.3) 51.3 (38.5) .01 Bodily Pain 47.6 (28.9) 57.5 (25.6) .06 General Health 51.4 (23.5) 56.0 (26.6) .22 Vitality 35.0 (19.6) 36.3 (21.4) .70 Social functioning 45.2 (28.5) 53.5 (28.7) .09 Role-emotional 42.7 (39.7) 41.9 (38.0) .89 Mental Health 42.5 (23.2) 50.1 (22.3) .03 Global Assessment of functioning 46.0 (9.4) 54.6 (9.6) < .001 Total no of PD criteria 34.8 (17.9) 25.7 (11.5) < .001 SCL-90-R PSI 1.52 (.86) 1.30 (.80) .035 Social Adjustment Scale (SAS) Overall adjustment 2.66 (.63) 2.42 (.62) .007 Work 2.76 (1.49) 2.36 (1.39) .20 Social and leisure 3.17 (1.22) 2.87 (1.11) .045 Extended family 2.05 (.52) 1.97 (.50) .34 Both professional-rated measures the GAF, and the mean total number of PD criteria, showed significant improvement. Among the patient-rated measures significant better results at follow-up were found for the SCL-90-R PSI, the SAS overall adjustment, and the SAS social and leisure scales. Discussion The main findings of this study of mainly co-morbid PD patients treated at an ordinary POC, was that the QoL on both the physical and mental SF-36 dimensions was significantly lower than that of an age- and gender-adjusted general population sample. According to our knowledge, ours is the first report on QoL-data in such PD patients at a POC. This finding is in accordance with QoL studies of PD patients in the general population [ 11 , 13 ], and correspond to findings of clinical studies of patients with anxiety disorders, depression, schizophrenia, and substance dependence [ 27 - 30 ]. However, the SF-36 dimension mean scores of our PD sample are lower than those reported for these diagnoses, and for co-morbid disorders [ 31 ]. In our sample we did not find any significant differences between the SF-36 dimension scores of the cluster A+B, cluster C, or Axis I groups, and all groups had significantly lower scores on all dimensions than their age- and gender-adjusted norm groups. In contrast to the epidemiological study from Australia [ 12 ] we did not find worsening of MCS and PCS with increasing number of PDs present in our sample. This could be due to our small samples, but also due to the fact that our patients with 1 PD had considerably lower QoL than in the Australian survey [MCS: 33.7 (SD 10.6) versus 44.4 (SD 12.0), p < .001, and PCS: 43.8 (SD 8.6) versus 46.9 (SD 11.0), p = .03]. Comorbid Axis I disorders explained a significant part of scores of PF, GH, VT, SF, and MH scores of the total PD group. This is in accordance with the findings of the Australian study [ 12 ]. We found that the SF-36 dimensions had variable associations with established psychiatric measures. As expected the SF-36 MH was most strongly associated with the psychiatric measures, but so were also SF and VT. For the SAS we found that overall adjustment and social and leisure activities were significantly correlated to all the SF-36 dimensions. In our PD sample we observed a somewhat different pattern of significant correlations between the GAF and the SF-36 dimensions than reported by Meijer et al. [ 32 ] in patients with schizophrenia. Small sample sizes and different diagnostic classes could be the explanation. However, in sum the SF-36 had a considerable association with established psychiatric measures in our PD sample. For both the patient- and professional-rated psychiatric measures significant changes at follow-up after treatment was observed in the 39 patients who also scored themselves on the SF-36. We cannot say if these changes were related to treatment, and ours is not an outcome study. We wanted to examine if changes in established psychiatric measures were associated with changes in the QoL measured by the SF-36 in the PD patients seen at a POC. Significant changes at follow-up were found for only two of the SF-36 dimensions, however, one physical (RP) and one mental (MH). While the finding for MH was expected, the change in RP which covered problems with work or other daily activities as a result of physical health was more difficult to explain. The score on that dimension was extraordinarily low at baseline (mean 31.4), and regression towards mean could be a likely explanation. It seemed that only MH of the SF-36 changed in the same way as established psychiatric measures in our study. The SF-36 MH correlated significantly with most of such psychiatric measures, and MH is currently used as a valid measure for mental health in several studies [33]. This result could indicate that the other dimensions of the SF-36 are less valid as measures of changes in mental health of PD patients, or alternatively that most aspects of QoL measured by the SF-36 do not change in PD patients even if established psychiatric measures do. The main strength of our study was that we were able examine systematically various aspects of the QoL measured by the SF-36 in a clinically relevant sample of PD patients at a POC which is a common setting for such patients in psychiatry. Our study had a number of weaknesses. The study groups were small with limited statistical power, and there was a considerable risk of type II errors. More significant differences as to the SF-36 dimensions could turn up in larger samples. Although we put considerable efforts into location of patients, we had a lower follow-up rate than we had expected. However, the PD patients who did not show up at follow-up did not differ much from those who did. We cannot, therefore, generalize the discrepancy observed between significant changes in established psychiatric measures and lack of such changes in most of the SF-36 dimensions of PD patients treated at a POC to widely. The same experienced psychiatrists did all the interviews at baseline and follow-up. Although she was not involved in any treatments, we cannot exclude an expectation bias from her side. We think that our study has to be considered an exploratory one. Our finding of a generally strongly reduced QoL should be replicated in a PD sample with less comorbid Axis I disorders, although their influence was limited. The same is true for QoL as a valid measure for change in PD patient, since it was not recommended as part of a standard outcome battery and was not used by major treatment studies of PD patients. However, our study confirmed that the SF-36 MH dimension seemed to be a valid psychiatric measure in our PD patient sample. Conclusion In this study of the QoL in PD patients seen at an ordinary POC, we found that the PD patients had significantly lower mean scores on all the SF-36 dimensions compared to age- and gender-adjusted norm data. This is in accordance with the SF-36 measurements of other major diagnostic groups of mental disorders. Although the SF-36 dimensions correlated considerably with established psychiatric measures in our PD patients, they did not show the same significant changes over time as the established measures. The use of QoL measures like the SF-36 as an outcome measure in PD patients is in need of further investigation. List of abbreviations BP: SF-36 Bodily pain GAF: The Global Assessment of Functioning GH: SF-36 General health MH: SF-36 Mental health PD: Personality disorder PDs: Personality disorders PF: SF-36 Physical functioning POC: Psychiatric outpatient clinic PSI: Personality severity index of SCL-90-R QOL: Health-related quality of life RE: SF-36 Emotional role functioning RP: SF-36 Physical role SAS: The Social Adjustment Scale SCL-90-R: The Symptom Checklist 90-Revised SF-36: MOS Short Form 36 SF: SF-36 Social functioning VT: SF-36 Vitality Competing interests The author(s) declare that the have no competing interests. Authors' contributions KN conceived and planned the study, prepared the therapists at Furuset Outpatient Department, did all the psychiatric interviews at baseline and follow-up, and drafted the manuscript. AM helped designing of the study, supervised the statistic calculations, and drafted the manuscript. AAD participated in the design and coordination of the study, and drafted the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC552308.xml
553994	Mutational study of sapovirus expression in insect cells	Human sapovirus (SaV), an agent of human gastroenteritis, cannot be grown in cell culture, but expression of the recombinant capsid protein (rVP1) in a baculovirus expression system results in the formation of virus-like particles (VLPs). In this study we compared the time-course expression of two different SaV rVP1 constructs. One construct had the native sequence (Wt construct), whereas the other had two nucleotide point mutations in which one mutation caused an amino acid substitution and one was silent (MEG-1076 construct). While both constructs formed VLPs morphologically similar to native SaV, Northern blot analysis indicated that the MEG-1076 rVP1 mRNA had increased steady-state levels. Furthermore, Western blot analysis and an antigen enzyme-linked immunosorbent assay showed that the MEG-1076 construct had increased expression levels of rVP1 and yields of VLPs. Interestingly, the position of the mutated residue was strictly conserved residue among other human SaV strains, suggesting an important role for rVP1 expression.	Introduction The family Caliciviridae is made up of four genera, Sapovirus , Norovirus , Lagovirus , and Vesivirus , which contain sapovirus (SaV), norovirus (NoV), rabbit hemorrhagic disease virus, and feline calicivirus strains, respectively. Human SaV and NoV strains are agents of gastroenteritis. The prototype strain of human SaV, the Sapporo virus, was originally discovered from an outbreak of gastroenteritis in an orphanage in Sapporo, Japan, in 1977 [ 1 ]. Chiba et al. identified viruses with the typical animal calicivirus morphology, called the "Star of David" structure, by electron microscopy (EM). SaV strains were recently divided into five genogroups (GI to GV), of which GI, GII, GIV, and GV strains infect humans, while GIII strains infect porcine species [ 2 ]. The SaV GI, GIV, and GV genomes are each predicted to contain three main open reading frames (ORFs), whereas SaV GII and GIII have two ORFs. SaV ORF1 encodes for non-structural proteins and the major capsid protein (VP1). SaV ORF2 (VP2) and ORF3 (VP3) encoded proteins of yet unknown functions. The NoV genome is organized in a slightly different way than the SaV, since ORF1 encodes all the nonstructural proteins, ORF2 encodes the capsid protein (VP1), and ORF3 encodes a small protein (VP2). Human SaV and NoV strains are noncultivable, but expression of the recombinant VP1 (rVP1) in a baculovirus expression system results in the self-assembly of virus-like particles (VLPs) that are morphologically similar to native SaV [ 3 , 4 ] In a recent NoV expression study, a single amino acid substitution in the rVP1 gene affected VLP formation but not rVP1 expression [ 5 ]. In a different study, inclusions of NoV ORF3 and poly(A) sequences in a construct increased the expression levels of NoV rVP1 and the stability of VLPs when compared to constructs without these sequences [ 6 ]. Recently, cryo-EM analysis of SaV VLPs and X-ray crystallography analysis of NoV VLPs predicted the SaV shell (S) and protruding domains (subdomains P1 and P2) that were based the NoV domains [ 7 , 8 ]. Chen et al. also described strictly and moderately conserved amino acid residues in the capsid protein among the four genera in family Caliciviridae . The purpose of this study was to compare the time-course expression of two different SaV rVP1 constructs in a baculovirus expression system by Northern blotting, Western blotting, enzyme-linked immunosorbent assay (ELISA), and EM. Our novel results have indicated that nucleotide point mutations increased the yields of SaV VLPs in insect cells, offering an alternative explanation for the increased expression levels of rVP1 and yield of VLPs. Results Wt, MQG-1076, and MEG-1076 constructs Expression of SaV rVP1 in a baculovirus expression system results in the self-assembly of VLPs [ 4 ]. However, during PCR amplification nucleotide point mutations occurred in our initial MQG-1076 construct, at nucleotide positions 4 and 1076 in VP1, which resulted in two amino acid substitutions at residues 2 and 358, respectively, and a silent nucleotide mutation at position 1895 in VP2 (Fig. 1 ). Despite these two substitutions the MQG-1076 construct formed VLPs morphological similar to native SaV (data not shown). In order to further investigate these substitutions we expressed another construct (MEG-1076 construct) having only one substitution, at residue 358 in VP1 (Fig. 1 ). This construct also formed VLPs. Finally we expressed a construct (Wt construct) without these nucleotide point mutations, i.e., having the native sequence. The Wt construct also formed VLPs, however the expression level of rVP1 was noticeably lower than those of the MQG-1076 and MEG-1076 constructs in which had similar levels (data not shown). In order to compare expression levels, we infected Wt and MEG-1076 recombinant baculoviruses each at a multiplicity of infection (MOI) of 14.5 in 2.7 × 10 6 confluent Tn5 cells in 1.5 ml of Ex-Cell 405 medium followed by incubation at 26°C. RNA transcription and rVP1 expression experiments were run in parallel for the Wt and MEG-1076 constructs. Figure 1 Schematics of the SaV constructs, Wt, MEG-1076, and MQG-1076, containing the rVP1, rVP2, and poly(A) sequences. Each construct began at the predicted AUG start. The triangles show the positions of the nucleotide point mutations. The black triangle had an amino acid substitution in the VP1, whereas the open triangle in the VP2 gene did not change amino acid sequence. An RNA probe (anti-VP1) was used to monitor the transcription of rVP1 mRNA in which contained the native sequence, i.e., lacking the mutation at 1076. Northern blot analysis Total RNA was extracted from the cells at 1, 2, 3, 4, 5, 6, 7, and 8 days postinfection (dpi) for Wt and MEG-1076 constructs. Equal amounts (500 ng) of total RNA were added to a 2% agarose gel containing formaldehyde and stained with SYBR Gold (Fig. 2A ). The rVP1 mRNA was then analysed by Northern blot with a probe specific for the VP1 gene (native sequence) corresponding to the VP1 position 157 to 1283 (Fig. 1 ). The rVP1 mRNA transcript was predicted to be approximately 2300 nucleotides long. As shown in Figure 2B , rVP1 mRNA was detected for each construct. This result showed that the insert sequence and some part of the baculovirus vector, approximately 300 nt, was transcribed, although the exact location(s) on the vector has yet to be determined. Nevertheless, the MEG-1076 construct had increased band intensities, indicating an increased steady-state level, when compared to those of the Wt construct (Fig. 2B ). For the Wt construct, rVP1 mRNA was detected at 1 dpi, peaked at 2 dpi, decreased at 3 and 4 dpi, and then decreased to undetectable levels at 5, 6, 7, and 8 dpi. For the MEG-1076 construct, rVP1 mRNA was detected at 1 dpi, peaked at 2 dpi, had steady-state levels at 3 and 4 dpi, and then decreased at 5 dpi but could still be detected at 6, 7, and 8 dpi. These results indicated that the MEG-1076 rVP1 mRNA also had greater stability when compared to those of the Wt rVP1 mRNA. Figure 2 Northern Blot analysis of Wt and MEG-1076 rVP1 mRNA. The total RNA was purified from the cells at 1, 2, 3, 4, 5, 6, 7, and 8 dpi. (A) The relative amounts of total RNA for each construct. (B) The steady-state levels of rVP1 mRNA with an anti-VP1 probe specific for the VP1 gene, corresponding to the VP1 nucleotide position 157 to 1283. Western Blot analysis Western blot analysis was used to compare the expression levels of Wt and MEG-1076 rVP1. The culture medium was separated from the cell lysate 1, 2, 3, 4, 5, 6, 7, and 8 dpi as described in the Materials and Methods. Equal volumes of culture medium and cell lysate at each dpi were used for both constructs. Proteins were separated by SDS-PAGE, electrotransferred to PVDF, and detected with a 1:3000 dilution of hyperimmune rabbit Mc114 VLP antiserum. A band at the predicted rVP1 size (60 K) was first detected in the culture medium at 2 and 4 dpi for MEG-1076 and Wt constructs, respectively, which increased each day thereafter as evidenced by an increase in band intensity (Fig. 3A ). As indicated by increased band intensities, the MEG-1076 construct expressed increased levels of rVP1 (60 K) than those of the Wt construct. Similarly, these results were reproduced using different MOIs in order to address the variability in virus stock quality (data not shown). Figure 3 Western blot analysis of Wt and MEG-1076 rVP1. Confluent Tn5 cells were infected with Mc114 recombinant baculoviruses at MOI of 14.5 and incubated at 26°C. The culture medium, including the cells, were harvested 1, 2, 3, 4, 5, 6, 7, and 8 dpi as described in the materials and methods. (A) The cell culture medium was concentrated by ultracentrifugation, resuspended in 20 μl of Grace's medium, and 5 μl was mixed with loading dye and loaded into each well. (B) The cell lysate was separated from the culture medium, resuspended in 200 μl of Grace's medium, and 5 μl was mixed with loading dye and loaded into each well. A thin band of approximately 55 K was also detected in the culture medium that appeared at 4 and 5 dpi for Wt and MEG-1076 constructs, respectively, and increased each day thereafter. In a different experiment, we determined the amino acid sequence of the MQG-1076 upper and lower bands by an Edman's degradation method. We discovered that the first three amino acid residues were MQG for both the upper and lower bands. This result indicated that the 55 K bands for these constructs were likely truncated or C-terminal deleted forms of rVP1. A thin band of 60 K was detected at every dpi in the cell lysate for the MEG-1076 construct (Fig. 3B ), however the intensity of this band did not increase to the same extent as the MEG-1076 60 K band in the culture medium (Fig. 3A ). This suggested that immediately after translation the majority of rVP1 was rapidly exported from the cells to the culture medium, though a fraction accumulated within the cells. This may also explain why no 60 K bands were detected in the cell lysate for Wt construct. The VP2 amino acid sequence was the same in all constructs. We did not detect rVP2 during the time-course expression of the MQG-1076 construct using the antiserum raised against E. coli expressed VP2 (data not shown). Antigen ELISA and EM analysis of Wt and MEG-1076 VLPs An antigen ELISA system was used to compare the yields of Wt and MEG-1076 VLPs at 1, 2, 3, 4, 5, 6, 7, and 8 dpi. The ELISA incorporated hyperimmune rabbit (capture) and guinea pig (detector) antisera raised against purified Mc114 VLPs [ 4 ]. The ELISA first detected VLPs at 2 and 3 dpi for MEG-1076 and Wt constructs, respectively (Fig. 4 ). For both constructs, the yields of VLPs increased each day thereafter, however the MEG-1076 construct had increased yields of VLPs than those of the Wt construct at 4, 5, 6, 7, and 8 dpi, approximately 6-fold increase. EM was used to verify the VLP formation of each of these constructs. We first detected VLPs at 4 dpi in the culture medium for both constructs and the numbers of VLPs increased each day thereafter (data not shown). Figure 4 Antigen ELISA analysis of Wt and MEG-1076 VLPs. The ELISA used hyperimmune rabbit (capture) and guinea pig (detector) antiserum raised against Mc114 VLPs. For the antigen ELISA, purified Mc114 VLPs were used as the positive control at concentrations ranging from 500 ng to 0.24 ng. Amino acid analysis The MEG-1076 construct contained a nucleotide point mutation in which resulted in an amino acid substitution at position 358 in VP1. We aligned 21 different VP1 amino acid sequences of SaV GI, GII, and GV strains and found this residue was strictly conserved, but more importantly, there was a strictly conserved amino acid motif at this site, NGDV (data not shown). However, when we included a porcine SaV GIII strain and a recently identified SaV GIV strain (PEC and Hou-7, respectively), only the GD site was strictly conserved, though several other amino acids nearby were also strictly conserved (Fig. 5 ). Further analysis of other SaV GIV strains are clearly needed in order to examine the possibility that the NGDV motif was moderately conserved in other human SaV strains. Figure 5 also showed that the predicted SaV P2 domain had very few conserved amino acid residues. Apart from the strictly conserved GD motif, the only other strictly conserved motif in the P2 domain was at the 5' end. Figure 5 VP1 amino acid alignment of SaV GI, GII, GIII, GV, and GV strains. We originally aligned 21 SaV GI, GII, and GV sequences but to simplify the figure we used one representative strain from each genogroup. The green bar shows the SaV P2 domain predicted by Chen et al. [7]. The asterisks indicate conserved amino acids. We originally aligned 21 different VP1 amino acid sequences of SaV GI, GII, and GV strains and found the residue (N) at position 358 (yellow) was strictly conserved (data not shown), but SaV GIII and GIV strains (PEC and Hou-7, respectively) had other residues at this position. The alignment of the five SaV genogroups showed the amino acid motif, GD, was strictly conserved (red) and several other amino acids surrounding the residue at position 358 were also strictly conserved (red). Discussion Expression of the human SaV rVP1 in a baculovirus expression system was first reported in 1997 [ 9 ]. In that study, the full-length VP1 gene, ORF2, and poly(A) sequences were included in a construct (Sapporo strain, GI). The second human SaV reported to form VLPs was with a construct (Houston/90 strain, GI) using only the VP1 sequence, i.e., lacking ORF2 and poly(A) sequences [ 10 ], while the third human SaV reported to form VLPs used a construct (Parkville strain, GI) with only VP1 and ORF2 sequences, i.e., lacking poly(A) sequence [ 7 ]. We recently expressed human SaV GI, GII, and GV rVP1 with constructs (Mc14, C12, and NK24 strains, respectively) that included ORF2 and poly(A) sequences [ 4 ]. Additional information on human SaV rVP1 expression is lacking, although it appeared that the yields of human SaV VLPs were typically low for these three genogroups. In this study, we compared the time-course expression of two different Mc114 SaV rVP1 constructs in a baculovirus expression system (Fig. 1 ). The MEG-1076 construct had two nucleotide point mutations, one in the VP1 gene in which resulted in an amino acid substitution, and one in the VP2 gene in which was silent. Although both constructs formed VLPs morphological similar to native SaV, the levels of transcription, translation, and VLP formation were clearly different. As shown in Figure 2B , the MEG-1076 rVP1 mRNA had increased steady-state levels and greater stability when compared to those of the Wt rVP1 mRNA. This difference was understood to be due to the nucleotide mutations in the MEG-1076 construct, since a similar result was observed in a NoV expression study [ 6 ]. Bertolotti-Ciarlet et al. found that a nucleotide point mutation in a NoV rVP1 construct (ORF2- A U G → A C G -ORF3+3' UTR construct, represented in bold) had decreased levels of rVP1 mRNA at 36 hours post-infection, by approximately 50%, when compared to a construct without the mutation (ORF2+ORF3+3' UTR construct). Bertolotti-Ciarlet suggested that the RNA secondary structure or changes in the mRNA stability could be responsible for the different steady-state levels, but this was not proven. Also, the MEG-1076 construct had increased levels of rVP1 expression and yields of VLPs in the culture medium when compared to those of the Wt construct (Fig. 3A ). On the other hand, the concentration of rVP1 in the cell lysate remained more or less the same during the time-course expression for the MEG-1076 construct. And for the Wt construct, rVP1 was not detected in the cell lysate, although this may have been related to the low expression levels (Fig. 3B ). Our results showed that the MEG-1076 construct had a 6-fold increase in yields of VLPs in the culture medium (Fig. 4 ), which corresponded to approximately 80 μg of CsCl purified VLPs from 200 ml of culture medium (at 6 dpi), but less than 5 μg of CsCl purified VLPs in the cell lysate (data not shown). These results suggested that either (i) immediately after translation the majority of rVP1 was exported from the cells to the culture medium where the majority of VLPs were folded but a fraction were simultaneously folded within the cells or (ii) VLPs were folded within the cells and then the majority of VLPs were immediately exported from the cells to the culture medium, though a fraction remained within the cells. In a recent NoV expression study, a single amino acid substitution in the rVP1 gene affected VLP formation but not rVP1 expression [ 5 ]. In that study, a (native) histidine residue at position 91 (relative to NoV Snow Mountain Virus strain amino acid VP1 sequence) was found to be essential for VLP formation and a construct with a substituted (mutant) arginine residue at this position failed to form VLPs despite expressing rVP1. Interestingly, that study found a single amino substitution was critical for the formation of VLPs, whereas our results showed that a single amino acid substitution was beneficial, i.e., increased the yields of VLPs. Bertolotti-Ciarlet found that inclusions of NoV ORF3 and poly(A) sequences in a construct increased the expression levels of NoV rVP1 and the stability of VLPs when compared to constructs without these sequences; and suggested that expression of other caliciviruses (NoV and SaV) rVP1 that resulted in low yields or unstable VLPs may be due to constructs that lacked the VP2 gene [ 6 ]. An alternative explanation was that point mutations influenced steady-state levels of mRNA and stability, which in turn influenced VLP formation. In our case, one or two nucleotide point mutations caused an enhancement of transcription, leading to increased yields of SaV VLPs in insect cells. Furthermore, many of these studies that expressed calicivirus rVP1 in insect cells only examined rVP1 expression and yields of VLPs but not rVP1 mRNA transcription [ 11 - 14 ]. However, another reason for the increased yields of VLPs may be associated with adaptation of SaV rVP1 to the baculovirus expression system and insect cells, since a similar result was observed with porcine enteric calicivirus in primary kidney cells [ 15 ]. Although the growth rate and replication efficiency of the recombinant baculoviruses themselves and differences in the levels of virus replication might account for such variation, we observed similar results using other MOIs, that is, the MEG-1076 construct continued to express greater yields of VLPs than the Wt construct (data not shown). Another explanation may have been differences in the extents to which these baculoviruses induce apoptosis and all these may result from features in the baculovirus skeleton rather than from the inserted SaV sequence. Such effects might for instance affect the number of adherent cells harvested or the degradation rates of both proteins and RNAs. However, we found that the MQG-1076 construct, developed from a separate experiment, had similar expression levels to that of the MEG-1076 construct (data not shown), which may eliminate the possibility that the baculovirus skeleton played a role in the increased yields of VLPs. On the other hand, we could not demonstrate whether the nucleotide mutations in VP1 and/or in ORF2 affected the transcription, a construct with only one of these mutations would be needed. Nevertheless, our results indicate that translation was exclusively affected by the single amino acid substitution in VP1. Therefore, the final increase in yields of VLPs may have been coupled at multiple levels, involving one or both of the nucleotide mutations in VP1 and VP2. We did not detect rVP2 during the time-course expression of the MQG-1076 construct (data not shown). The Wt and MEG-1076 constructs had an identical amino acid sequence, which would suggest a similar negative-result. NoV studies have found that inclusion of VP2 increases the stability of VLPs, though the expression level of NoV rVP2 was low [ 6 ]. These results may suggest that (i) SaV rVP2 was expressed at undetectable levels, (ii) SaV rVP2 was not expressed in the insect cells, or (iii) SaV rVP2 was degraded in the insect cells. The SaV GI, GIV, and GV genomes are each predicted to encode a third ORF (ORF3) overlapping the VP1 gene, whereas SaV GII and GIII have only two ORFs. The functions of SaV ORF2 and ORF3 still remain unknown. The amino acid substitution (N → S) for the MEG-1076 construct occurred in the VP1 gene at residue 358. This asparagine residue was recently identified as a moderately conserved residue among the caliciviruses capsid proteins [ 7 ], but more importantly, the residue was strictly conserved among 21 different SaV GI, GII, and GV strains and belonged to a strictly conserved amino acid motif, NGDV (Fig. 5 ). However, when we included SaV GIII and GIV strains (PEC and Hou-7, respectively) we found that only the GD amino acids were strictly conserved though several other amino acids nearby were also strictly conserved (Fig. 5 ). These data further suggested that this site played an important role in the regulation of SaV VLP formation. Recently, the cryo-EM analysis of SaV was determined and compared to NoV X-ray crystallography structure [ 7 ]. Chen et al. analysed 30 different VP1 amino acid sequences of calicivirus strains belonging to the four genera in the family Caliciviridae and identified strictly and moderately conserved residues, and predicted the P1 and P2 domains of SaV VP1 based on NoV X-ray crystallography structure. Based on these predictions, the residue at position 358 (amino acid sequence) was found as a moderately conserved residue among the caliciviruses. This arginine residue was predicated to be in the P2 domain, which is defined as the outer most protruding domain for NoV and thought to provide strain diversity [ 16 ]. Further high-resolution structural analysis of SaV VLPs is clearly needed in order to determine the precise domains and regions of SaV. However, our expression results have indicated that only approximately 80 μg of purified VLPs from 200 ml of culture medium was possible (data not shown), thus in order to determine the X-ray crystallography structure of SaV, a minimum increase in expression level of about 20-fold would be required: a challenging feat. Materials and methods Virus strain, RNA extraction, cDNA synthesis SaV GI Mc114 strain (GenBank accession number, AY237422) was isolated from a male infant seven months of age from the McCormic Hospital, Chiang Mai, Thailand on the 7th May 2001 [ 17 ]. RNA extraction and cDNA synthesis were performed as previously described [ 18 ]. PCR and sequencing Our initial SaV rVP1 construct (MQG-1076 construct) was amplified with ExTaq DNA polymerase. However, this construct was later found to have two nucleotide point mutations in ORF1 at positions 4 ( G AG → C AG) and 1076 (A A T → A G T) and one nucleotide point mutation in ORF2 at position 1895 (GT G → GT A ) (relative to the VP1 start and represented in bold). Primer and PCR errors likely introduced these mutations. These three nucleotide point mutations resulted in two amino acid substitutions in the VP1 gene, one at the second residue, where glutamic acid (E) → glutamine (Q), and one at residue 358, where asparagine (N) → serine (S). The nucleotide point mutation in ORF2 did not result in an amino substitution. Despite the two amino acid substitutions, the MQG-1076 construct formed VLPs. We designed another construct (MEG-1076) using the pDEST8-MQG-1076 as template but with a new sense primer and used KOD-plus DNA polymerase according to the manufacture's instructions (Toyobo, Japan). The MEG-1076 construct had the same nucleotide point mutations at positions 1076 in VP1 and 1895 in VP2 as the MQG-1076 construct but not at nucleotide 4 in VP1 (Fig. 1 ). Lastly, we designed a third construct with the native sequence (Wt construct) using KOD-plus DNA polymerase and the original cDNA [ 4 ]. PCR-amplified fragments were cloned into the Gateway Expression System (Invitrogen, Carlsbad, Calif.) as previously described [ 4 ]. The insert sequences of the pDONR8 plasmids were confirmed, including the partial upstream and downstream sequences on the plasmids in which were found to be identical for the Wt and MEG-1076 constructs. Sequencing was performed as previously described [ 18 ]. Expression of rVP1 in insect cells Recombinant bacmids were transfected into Sf9 cells (Riken Cell Bank, Japan) and the recombinant baculoviruses was collected as previously described [ 4 ]. The expression of the rVP1 constructs were analyzed by infecting recombinant baculoviruses at a MOI of 14.5 in 2.7 × 10 6 confluent Tn5 cells in 1.5 ml of Ex-Cell 405 medium followed by incubation at 26°C. The total culture medium was harvested 1, 2, 3, 4, 5, 6, 7, and 8 dpi. The culture medium was centrifuged for 10 min at 3,000 × g , and further centrifuged for 30 min at 10,000 × g . The VLPs in the culture medium were further concentrated by ultracentrifugation for 2 h at 45,000 rpm at 4°C (Beckman TLA-55 rotor), and then resuspended in 30 μl of Grace's medium. The cell lysate from the first centrifuge was resuspended in 200 μl of Grace's medium and stored at 4°C. Northern blotting Total RNA was prepared from the attached cells at 1, 2, 3, 4, 5, and 6 dpi with 1 ml of Isogen (Nippon Gene, Japan). For 7 and 8 dpi, the cell culture medium (containing unattached cells) was collected and centrifuged for 5 min at 3,000 × g , the supernatant removed, and then the cells were dissolved with 1 ml of Isogen. The cells were stored at -80°C. RNA was purified by a chloroform/ ethanol method (Nippon Gene, Japan). Briefly, RNA was mixed with chloroform, centrifuged at 12,000 × g for 15 min at 4°C, and the aqueous layer collected. This was repeated once, and then the aqueous layer collected and mixed with isopropanol and stored overnight at -20°C. The solution was mixed, centrifuged at 12,000 × g for 15 min at 4°C, and the supernatant discarded. The pellet was resuspended in 80% ethanol, centrifuged at 12,000 × g for 15 min at 4°C. This was repeated once, and then the pellet air-dried and resuspended in 25 μl of TE, and stored at -80°C. The amounts of purified RNA were determined spectrophotometrically (Bio-Rad, USA). The same amounts (500 ng) of total RNA were loaded for each construct and each dpi onto a 2% denaturing agarose gel containing formaldehyde. The amounts of total RNA were compared using SYBR Gold staining (Invitrogen, USA). RNA was transferred to a positively charged nylon transfer membrane (Hybond-N+; Amersham Biosciences, Ireland) under vacuum (VacuGene XL; Pharamacia LKB, Sweden) and analyzed by Northern blotting according to the DIG Northern Starter Kit (Roche, USA), except for a minor modification. Briefly, a RNA probe corresponding to Mc114 VP1 position 157 to 1283 (anti-VP1) was generated from a PCR fragment (native sequence) according to the manufacture's instructions (Roche, USA). Hybridization was performed overnight at 68°C with anti-VP1 in 10 ml of ultrasensitive hybridization buffer (Ambion, Canada). After hybridization, immunological detection was performed according to the manufacture's instructions (Roche, USA). Western blotting, ELISA, EM, and protein sequencing Western blotting, ELISA, and EM were used to examine rVP1 expression as previously described [ 4 ]. However, it should be acknowledged that the hyperimmune rabbit and guinea pig antisera were raised against the MQG-1076 VLPs. Protein sequences were determined by an Edman's degradation method. Amino acid alignment VP1 nucleotide sequences were translated using Genetyx software (software development Co. Version 11.2.2) and submitted to online ClustalW at DDBJ . In total, we aligned different 21 SaV GI, GII, GIII, GIV, and GV sequences, and included: Arg39, AY289803; Bristol, AJ249939; C12, AY603425; Cruise ship/00, AY289804; PEC, AF182760; Dresden, AY694184; Hou-7, AF435814; Houston/86/US, U95643; Houston/27/90/US, U95644; London/29845/92/UK, U95645; Lyon/598/97/F, AJ271056; Manchester, X86560; Mc2, AY237419; Mc10, AY237420; Mex340/1990, AF435812; Mex14917/00, AF435813; NK24, AY646856; Parkville, U73124; Potsdam, AAG01042; Plymouth, X86559; Sapporo/82/Japan, U65427; and Sakaeo-15, AY646855. Competing interests The author(s) declare that they have no competing interests. Authors' contributions GH carried out the study and wrote the manuscript. KK, TO, KN, and NT participated in the design of the study and helped to draft the manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC553994.xml
551601	Two-part permutation tests for DNA methylation and microarray data	Background One important application of microarray experiments is to identify differentially expressed genes. Often, small and negative expression levels were clipped-off to be equal to an arbitrarily chosen cutoff value before a statistical test is carried out. Then, there are two types of data: truncated values and original observations. The truncated values are not just another point on the continuum of possible values and, therefore, it is appropriate to combine two statistical tests in a two-part model rather than using standard statistical methods. A similar situation occurs when DNA methylation data are investigated. In that case, there are null values (undetectable methylation) and observed positive values. For these data, we propose a two-part permutation test. Results The proposed permutation test leads to smaller p -values in comparison to the original two-part test. We found this for both DNA methylation data and microarray data. With a simulation study we confirmed this result and could show that the two-part permutation test is, on average, more powerful. The new test also reduces, without any loss of power, to a standard test when there are no null or truncated values. Conclusion The two-part permutation test can be used in routine analyses since it reduces to a standard test when there are positive values only. Further advantages of the new test are that it opens the possibility to use other test statistics to construct the two-part test and that it avoids the use of any asymptotic distribution. The latter advantage is particularly important for the analysis of microarrays since sample sizes are usually small.	Background The addition of a methyl group at the carbon-5 position of cytosine is a modification of DNA called DNA methylation. In mammalian cells, DNA methylation is essential for proper development [ 1 ]. The methylation patterns of tumor cells are altered compared to those of normal cells, moreover, there are also differences between different types of cancer as shown for subtypes of leukemia [ 2 ] and lung cancer [ 3 ]. Thus, DNA methylation analysis promises to become a powerful tool in cancer diagnosis [ 4 ]. DNA methylation data can be obtained using the MethyLight technology [ 5 ]. When the tested region is not or only partially methylated the result is negative (undetectable methylation, null values). In contrast, samples that show methylation will have a value greater than 0 [ 4 ]. Thus, DNA methylation data obtained with MethyLight have a clump of zero observations and a continuous nonzero part. For such a data structure, two-part models as proposed by Lachenbruch [ 6 - 8 ] are applicable. In that approach, the test statistic is the sum of two squared statistics, one comparing the proportions of zeros and one comparing the positive values. For example, one can use the binomial test and the Wilcoxon rank sum test. The asymptotic null distribution of the sum of the squares of the two test statistics is χ 2 with two degrees of freedom (df = 2). In microarray data it is relatively common that small and negative expression levels were clipped-off to be equal to an arbitrarily chosen cutoff value. Recent examples used different cutoff values: 1, 20, and 50, respectively [ 9 - 11 ]. Aside from the fact that negative values make no biological sense, there are, with regard to oligonucleotide arrays from Affymetrix, two primary reasons for truncating the values [ 12 ]. First, spots at the low intensity range are generally more vulnerable to noise, thus, it is thought that the technology produces a poor discrimination at low levels of expression [ 13 ]. Second, the focus is on expression of identified genes and expressed sequence tags. Differences at negative or low values may result from differences in binding to the mismatch probes. Since it is generally not known what binds to the mismatch probes, the differences at negative or low values cannot be attributed to target genes. Due to the truncation there are two different types of data: truncated values and original observations. Since the truncated values are not just another point on the continuum of possible values, it would be inappropriate to use a standard statistical method that would treat all values equally [ 14 ]. The two different types of data should be analyzed separately. Therefore, the two-part model, comparing the proportions of truncated values and the distribution of positive values, is applicable. Note that negative expression levels are not possible when the Affymetrix Microarray Suite (MAS) 5.0 software is used. However, small values are possible and may be truncated. Since, in microarray experiments, the sample sizes, i.e. the numbers of replications, are usually very small [ 15 , 16 ], the use of the asymptotic distribution of Lachenbruch's two-part test statistic may be questionable. Thus, we carried out permutation tests with the two-part statistic. For a permutation test all possible permutations under the null hypothesis are generated. In our situation we permute the group labels for the whole sample, i.e. for truncated values (or the null values in case of methylation data) and original observations. Then, the test statistic is calculated for each permutation. The null hypothesis can then be accepted or rejected using the permutation distribution of the test statistic, the p -value being the probability of the permutations giving a value of the test statistic as supportive or more supportive of the alternative than the observed value [ 17 , 18 ]. Thus, inference is based upon how extreme the observed test statistic is relative to other values that could have been obtained under the null hypothesis. We found that, in the case of a two-part model, the permutation test is not only a way to avoid the use of an asymptotic distribution, but also is a more powerful test, i.e. a test that produces, on average, smaller p -values. In addition, the permutation test reduces, without any loss of power, to a single test if no truncated (or null) values were present. Thus, the proposed test is applicable in routine use whether or not truncated (or null) values occur. After the definition of the tests in the following section, we present our findings for DNA methylation data and microarray data. We then confirm the results using simulations. Two-part tests As briefly mentioned above a two-part test statistic is the sum of two squared statistics, one comparing the proportions of truncated values and one comparing the positive values. Let n 1 and n 2 be the numbers of independent observations regarding one gene (or one region in case of methylation data, respectively), for two groups to be compared. The observed numbers of truncated values (or null values in case of methylation data) in the two groups are denoted by m 1 and m 2 . To compare these numbers m 1 and m 2 Lachenbruch [ 6 ] used the statistic where , , and . Under the null hypothesis the proportions of truncated values are not different between the two groups, and B 2 is asymptotically χ 2 -distributed with df = 1. B 2 is always well defined, unless there are only truncated values in both groups or no truncated values at all. For these two extreme cases we set B 2 = 0. For the second part in Lachenbruch's two-part model one can use different tests, Lachenbruch [ 6 - 8 ] considered the Wilcoxon rank sum test, Student's t test, and the Kolmogorov-Smirnov test. The use of the latter test in a two-part model, however, was too liberal, the type I error rate was close to 0.065 (for a significance level of α = 0.05 [ 7 , 8 ]). Both other tests work well. Here, we apply the Wilcoxon test for two reasons. This test was used in the original analyses of the data we use below [ 3 , 11 ]. Nonparametric tests based on ranks are more appropriate for non-normally distributed data such as microarray data [ 19 ]. The standardized rank sum statistic based on the non-truncated (or positive values in case of methylation data) values is defined as where RS is the rank sum, i.e. the sum of the ranks in group 1. When there are ties within the non-truncated values the denominator slightly changes [[ 20 ], p. 109]. For the extreme case that there are only truncated values in at least one group we set W = 0. The test statistic for the two-part test is X 2 = B 2 + W 2 . Under the null hypothesis of no difference between the groups, X 2 is asymptotically χ 2 -distributed with df = 2 [ 6 , 7 ]. Alternatively, a permutation test can be performed with the statistic X 2 . This test, called two-part permutation test here, is a permutation test based on the sum statistic X 2 . It is carried out by permuting the group labels for the whole sample. Thus, all observations, truncated and non-truncated values (or null and positive values in case of methylation data) are reallocated to the groups. When performing this two-part permutation test the exact permutation distribution of X 2 is determined. This distribution, computed by generating all possible permutations or, for the p -values given below in Table 1 , using a simple random sample of 20,000 permutations, is used to compute the p -value. Since it is a permutation test based on the sum X 2 , it is neither necessary to determine the permutation distributions of the summands B 2 and W 2 nor to calculate the p -values of the univariate tests related to B 2 and W 2 . Application to actual methylation data We use DNA methylation data from 7 regions and 87 lung cancer cell lines, 41 lines are from small cell lung cancer and 46 lines from non-small cell lung cancer [ 3 , 4 ]. The proportion of positive values for the different regions ranges from 39 to 100% for the small cell lung cancer and from 65 to 98% for the non-small cell lung cancer. The data are available at . Siegmund et al. [ 4 ] transformed the data by standardizing the positive values on the natural-log scale. However, for the tests applied here, this transformation has no influence. Table 1 presents the p -values of the two tests. For most regions the two-part permutation test gives a smaller p -value than the original two-part test. The only exception is the region APC . However, the original two-part test's p -value for this region is 0.1684 and, for a p -value of this size, a small change in the value is usually of no importance. Application to actual microarray data Tschentscher et al. [ 11 ] performed an experiment with HG-U95Av2 oligonucleotide arrays in order to compare patients with uveal melanomas with and without monosomy 3. Expression values were calculated by use of the MAS 4.0 software. The data are available at . The sample size in this microarray experiment is 10 per group. As mentioned above, expression levels below 50 were set to 50. This data truncation occurred in 2,215 (28%) out of 7,902 genes. First, we consider these 2,215 genes. Figure 1 displays the number of genes for the different number of truncated values per gene. Table 2 shows the frequencies of different size groups of the p -values. Often, the two-part permutation test gives a smaller p -value than the original two-part test. For instance, for 19 genes the p -value is ≤ 0.001 when the latter test is applied. The permutation test gives a p -value ≤ 0.001 for these 19 and for 39 additional genes. As usual, the majority of genes do not show any indication of being differentially expressed. For these genes a slight change in the p -value is of no importance. Thus, in Table 3 , we consider the genes for which the p -value of the original two-part test is ≤ 0.1. Out of the 2,215 genes 514 remain. As shown in Table 3 the p -values of the two-part permutation test are, on average, distinctly smaller than those of the original two-part test. For a large proportion of genes (72% in this data set) there are no truncated values. In that case, the two-part statistic reduces to the sum 0 plus the squared standardized rank sum W 2 . Of course, one has to define a priori whether the two-part test or the Wilcoxon test will be used to analyze the data. If the original two-sided (asymptotic) Wilcoxon test were chosen and applied, one could compare W 2 with critical values from a χ 2 distribution with one degree of freedom (df). If the two-part were chosen there is df = 2 and, in case of no truncated values, power is lost compared to the original Wilcoxon test. For instance, the 95% percentile of the χ 2 distribution with df = 1 is 3.84, but it is 5.99 for df = 2. The permutation test using the sum statistic X 2 does not suffer from this power loss: When there are no truncated values there is, of course, no difference in the proportions of truncated values, and the test statistic X 2 is, for every permutation, the sum 0 + W 2 . Thus, the two-part permutation test reduces to the exact two-sided Wilcoxon rank sum test when there are no truncated values. Consequently, this permutation test does not only give smaller p -values, but it is also applicable in routine use whether or not truncated values are present. Simulation study The two different tests, the original two-part test and the two-part permutation test, were compared in a Monte Carlo simulation study performed using SAS version 8.2, 5,000 simulation runs were generated for each configuration. The sample size of 10 per group was chosen as in the microarray experiment presented above. In some configurations some randomly chosen values were set to 0 according to binomial distributions with the probabilities p 1 and p 2 , and the remaining observations were generated according to a lognormal distribution (with median 1 and σ = 1). Then, the values of one group were shifted if applicable. In some other configurations all observations were generated according to the lognormal distribution and, in one group, shifted. Then, values smaller than a cutoff value were truncated. The type I error rates of the two tests are very similar. With e.g. p 1 = p 2 = 0.3 and a significance level of α = 0.05 the simulated type I error rates were 0.049 for both the original two-part test and the two-part permutation test. Table 4 displays results for situations with a difference between the two groups. As above, only those comparisons were regarded for which there is some indication of a difference, i.e. a p -value ≤ 0.1 of the original two-part test. In all considered configurations the median of the difference between the p -values of the original two-part test and the two-part permutation test is positive. The finding that the p -values of the permutation test are smaller corresponds to a higher power of this test. The power is given in Table 5 , as shown the power of the two-part permutation test is at least as high as that of the original two-part test. There is only one exception, the latter test is slightly more powerful in one situation, i.e. when the proportion of zeros is higher in group 1 and the positive values are larger in group 1. Discussion Previous research demonstrated that a two-part test is appropriate and powerful in the presence of a clump of zero observations (i.e. truncated or null values). In this paper we propose a permutation test for such situations with two types of data. Usually, nonparametric tests can be performed based on an asymptotic distribution or based on a permutation null distribution. The two approaches often give similar results, especially when the sample sizes are large. However, in the case of a two-part test one cannot simply replace the asymptotic distributions of B 2 and W 2 by the exact permutation distributions. If so, one would compute two exact p -values although the aim of a two-part test is to receive one p -value that combines information from both parts. Therefore, the two-part permutation test uses the exact permutation distribution of the sum statistic X 2 . That this permutation distribution of the sum is generated rather than to simply replace the asymptotic distributions of the summands by their exact permutation distributions may be the reason why the permutation test is more powerful. A disadvantage of a permutation test is that it can be computer-intensive. However, this issue is less relevant now due to faster algorithms [[ 18 ], chap. 13] and the advent of high-speed PCs. Furthermore, one can carry out a permutation test based on a random sample out of the possible permutations, as we did for the DNA methylation data (see Table 1 ). In microarray experiments it is common to investigate thousands of genes simultaneously. The approach presented here for the identification of differentially expressed genes is to consider a univariate testing problem for each gene. A correction for the multiplicity of genes is a subsequent step, that is, like the previous step of normalizing the data, outside the scope of this paper. A common approach to the multiplicity problem is to consider a procedure for testing the genes simultaneously for differential expression with the test on an individual gene being implied in the simultaneous test. For such a procedure different proposals have been made recently. For instance, there are methods based on the p -values of the tests from individual genes [ 21 - 23 ]. In a similar manner, the multiplicity of regions can be managed in DNA methylation data. Conclusion Aside from the shown improvement in power, the proposed two-part permutation test has three important advantages. First, it avoids the use of any asymptotic distribution and, therefore, can safely be applied in case of small sample sizes that are common in microarray experiments. Second, it reduces without any loss of power to the exact Wilcoxon test if there were no truncated (or zero) values. Thus, it can be used in routine analyses. Third, the permutation test opens the possibility to use other tests to construct the two-part test. Thus, tests with unknown or non-standard null distributions can be used. For instance, one could replace the Wilcoxon test by the Baumgartner-Weiß-Schindler test [ 24 ] that was recently recommended for the analysis of gene expression data [ 19 ]. Authors' contributions MN performed the statistical analyses and drafted the manuscript. TB prepared the microarray data. TB and KHJ participated in the design of the simulation study and helped to draft the manuscript. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC551601.xml
545648	Observed intra-cluster correlation coefficients in a cluster survey sample of patient encounters in general practice in Australia	Background Cluster sample study designs are cost effective, however cluster samples violate the simple random sample assumption of independence of observations. Failure to account for the intra-cluster correlation of observations when sampling through clusters may lead to an under-powered study. Researchers therefore need estimates of intra-cluster correlation for a range of outcomes to calculate sample size. We report intra-cluster correlation coefficients observed within a large-scale cross-sectional study of general practice in Australia, where the general practitioner (GP) was the primary sampling unit and the patient encounter was the unit of inference. Methods Each year the Bettering the Evaluation and Care of Health (BEACH) study recruits a random sample of approximately 1,000 GPs across Australia. Each GP completes details of 100 consecutive patient encounters. Intra-cluster correlation coefficients were estimated for patient demographics, morbidity managed and treatments received. Intra-cluster correlation coefficients were estimated for descriptive outcomes and for associations between outcomes and predictors and were compared across two independent samples of GPs drawn three years apart. Results Between April 1999 and March 2000, a random sample of 1,047 Australian general practitioners recorded details of 104,700 patient encounters. Intra-cluster correlation coefficients for patient demographics ranged from 0.055 for patient sex to 0.451 for language spoken at home. Intra-cluster correlations for morbidity variables ranged from 0.005 for the management of eye problems to 0.059 for management of psychological problems. Intra-cluster correlation for the association between two variables was smaller than the descriptive intra-cluster correlation of each variable. When compared with the April 2002 to March 2003 sample (1,008 GPs) the estimated intra-cluster correlation coefficients were found to be consistent across samples. Conclusions The demonstrated precision and reliability of the estimated intra-cluster correlations indicate that these coefficients will be useful for calculating sample sizes in future general practice surveys that use the GP as the primary sampling unit.	Background Cluster sample study designs are a cost-effective way of sampling difficult to reach populations. Examples include sampling schools to obtain cluster samples of students or medical practitioners to sample patients[ 1 ]. Cluster samples violate the simple random sample assumption of independence of observations, since observations are sampled from within the selected cluster – defined as the primary sampling unit. Observations within a cluster may be more alike than observations across clusters. This intra-cluster correlation leads to increased variation between clusters compared to the variation within clusters. Failure to account for intra-cluster correlation when designing a study where participants are recruited within clusters will lead to an under-powered study. To allow for any loss in power and precision, a cluster sample requires a larger sample size to answer the same research question as a study using simple random sampling [ 2 - 4 ]. Both the size of the intra-cluster correlation and the number of observations sampled within each cluster influence the power of the study. Even for a small intra-cluster correlation, as is often found in general practice and community samples, the loss of power can be appreciable, particularly if the size of the cluster is large[ 1 , 4 ]. Estimates of the size of intra-cluster correlations come from post hoc examination of studies that have used either allocation or sampling by cluster and a number of intervention studies have published observed intra-cluster correlation coefficients [ 4 - 6 ]. Many intervention studies however, still fail to report intra-cluster correlation coefficients[ 7 ] and there is even less information reported on survey studies that employ a cluster sample[ 1 , 8 ]. The lack of published estimated intra-cluster correlations continues to hamper the design of studies that employ a cluster sample[ 9 ]. Intra-cluster correlation varies within a study and depends on the outcome under analysis[ 1 , 4 , 6 ]. The intra-cluster correlation of the same outcome may also vary across studies depending on the primary sampling unit, and whether outcomes are reported as prevalence rates or modeled in association with other variables[ 1 , 4 ]. Researchers need reliable estimates of the intra-cluster correlations, specific to the primary sampling unit and selected outcomes of interest when making sample size calculations. These estimates will assist in deciding the trade off between cluster number and cluster sub-sample size in a study design[ 10 ]. There is however, little published on the estimated intra-cluster correlation coefficients in the Australian context, especially for primary health surveys where the health practitioner is the primary sampling unit. Research questions In one Australian study Carlin and Hocking[ 1 ] examined the intra-cluster correlation in two cross-sectional cluster surveys of school children that used the school as the primary sampling unit. The researchers observed that design effects for sociodemographic variables were larger than for morbidity related variables. Furthermore intra-cluster correlation was greater for descriptive outcomes such as prevalence estimates, means and proportions than for measures of association between variables such as regression coefficients and odds ratios. We wanted to examine whether these patterns could be generalised to other large cluster survey studies in the primary care setting. This paper reports some of the intra-cluster correlations observed in the Bettering the Evaluation and Care of Health (BEACH) program, a large cross-sectional survey of general practice patient encounters in Australia, where a random sample of general practitioners was used as the primary sampling unit. The BEACH study draws a new random sample of Australian general practitioners (GPs) each year, and this provided an opportunity to assess the stability of intra-cluster correlation coefficients across successive samples. If a population is re-sampled using the same cluster survey design, will the intra-cluster correlation coefficient for a particular outcome be the same across samples? This analysis takes an applied approach, examining the observed intra-cluster correlations for a range of demographic, morbidity and treatment outcomes. Methods The BEACH program is a continuous study of general practice activity commenced in 1998. The BEACH method is described in detail elsewhere and a brief summary is reported below[ 11 ]. Cluster sample design A random sample of approximately 1,000 general practitioners (GPs) is drawn each year from the Health Insurance Commission's sampling frame of the population of GPs in Australia. The GP population is randomly ordered into a list and GPs are recruited sequentially from the list, with re-randomisation of the sampling frame every three years[ 11 , 12 ]. GPs are sampled without replacement and have one chance of selection over three years. Sampling is continuous across the year, with around 20 GPs participating in the study in any one week. Each GP completes details of 100 consecutive patient encounters. The GP is the primary sampling unit (PSU), while the primary unit of inference is the patient encounter. Data elements A single page encounter form contains elements including: • Patient age and sex. • Whether English was the main language spoken at home. • Whether the patient holds an Australian health care concession card. • The problems managed by the GP at the encounter (up to four problems per encounter). • Treatments received at the encounter, including medications, other procedures, referrals and orders for pathology and imaging tests. Although sample weights are calculated each year for population estimates[ 13 ], the outcomes reported in this paper are unweighted to allow us to calculate estimates of the intra-cluster correlation based on the observed variance in the sample data. Descriptive outcomes Descriptive outcomes were defined as rates, means and percentages of single variables, e.g. mean age, per cent of encounters with female patients, per cent of encounters where at least one respiratory problem was managed. BEACH samples the GP-patient encounter, not independent patients. If a patient returns to the GP in the sampling period then that patient contributes two (or more) encounters to the sample. Therefore BEACH estimates are not true "prevalence" rates because the denominator, the population of GP-patient encounters, is many times larger than the population of all general practice patients. To avoid misunderstanding in this paper we have used the term "descriptive" rather than "prevalence" to report single variable estimates and their accompanying intra-cluster correlation coefficients. Descriptive rates are interpreted for example as "Proportion of patients at encounter who are female". Demographic variables Demographic variables include patient sex, patient age, whether the patient held a health care concession card and whether the main language spoken at home was not English. Morbidity variables Problems were classified using the International Classification of Primary Care (ICPC-2)[ 14 ]. The upper level of ICPC-2 classifies problems according to the body system involved, for example skin problems, respiratory problems, cardiovascular problems and problems of the digestive system etc. There are an additional three chapters for psychological problems, social problems and problems of a general or unspecified nature. Morbidity estimates are expressed as the percent of patient encounters where at least one problem from the chapter was managed. The total number of problems managed by the GP at the encounter was also included as an outcome. Treatment outcomes Treatment outcomes included the proportion of encounters that resulted in at least one medication, the proportion that received at least one referral, the proportion that received at least one order for an imaging test and the proportion receiving at least one order for a pathology test. Association outcomes Intra-cluster correlation coefficients were calculated for associations between variables using logistic regression e.g.: the effect of patient age (predictor) on the rate of cardiovascular problems (outcome). Design effect Obtaining the sample size for cluster designs involves calculating the sample size under the assumption of simple random sampling and then inflating the number of observations to allow for the design effect of the cluster sample. The design effect (Deff) of an outcome has been defined as the ratio of the variance taking into account the cluster sample design and the variance of a simple random sample (srs) design with the same number of observations[ 1 ]. Deff = Variance (clustersample) /Variance (srs) Intra-cluster correlations and their standard errors for the outcome variables were calculated using the method described by Carlin & Hocking[ 1 ]. Specifically STATA 7 was used to calculate the design effects using the "survey estimator" procedures, which were purposefully designed to analyse complex survey data. STATA 7 calculates the design effect directly from the ratio of the estimated variances[ 15 ]. The intra-cluster correlation coefficient (ICC) was then calculated from the design effect using the formula: ICC = ( Deff - 1)/( k - 1) and the approximate standard error (SE) of the intra-cluster correlation was calculated using the formula[ 1 , 6 ]: where m = number of clusters, k = mean number of observations per cluster. The intra-cluster correlations and respective 95% confidence intervals for the second BEACH year sample from the period April 1999 to March 2000 were compared against those in the year 5 sample (April 2002 to March 2003) to assess whether the intra-cluster correlations were consistent across samples over time. All calculations specified the GP as the primary sampling unit. Results From April 1999 to March 2000, 1,047 GPs were recruited, recording a sample of 104,700 patient encounters. From April 2002 to March 2003, 1,008 GPs were recruited and 100,800 encounters recorded. Table 1 shows the age and sex distribution of the two samples of GPs compared with the sampling frame of the population of Australian GPs in the year April 2002 to March 2003[ 13 ]. The two GP samples were comparable to the GP population in terms of distribution by age, sex and state. Table 1 Comparison of GP participants and all active recognised Australian GPs. BEACH April 1999–March 2000 % (95%CI) (N = 1,047) BEACH April 02–March 03 % (95%CI (N = 1,008) Australian GPs April 02 to March 03 % (N = 17,884)[13] Males 69.6 (66.8,72.4) 64.8 (61.8,67.7) 66.8 Age group <35 8.4 (6.7,10.1) 7.3 (5.7,9.0) 9.7 35–44 32.4 (29.6,35.3) 26.6 (23.9,29.3) 25.1 45–54 32.4 (29.6,35.3) 35.2 (32.3,38.2) 33.1 55+ 26.7 (24.1,29.4) 30.9 (28.0,33.7) 32.0 State NSW 37.4 (34.5,40.4) 39.6 (36.7,42.7) 33.6 Victoria 20.1 (17.7,22.5) 18.8 (16.4,21.3) 24.5 Queensland 20.2 (17.8,22.6) 21.2 (18.7,23.8) 18.5 South Australia 9.1 (7.3,10.8) 6.2 (4.7,7.6) 8.7 Western Australia 8.8 (7.1,10.5) 8.9 (7.2,10.7) 9.5 Tasmania 2.4 (1.5,3.3) 2.8 (1.8,3.8) 2.9 ACT 1.1 (0.5,1.8) 1.4 (0.6,2.0) 1.5 NT 0.9 (0.3,1.4) 1.1(0.4,1.7) 0.8 The two samples of patient encounters were similar in terms of demographics (Table 2 ) In the year 1999–00, 59.0% of encounters were with female patients compared with 59.3% in 2002–03. The samples were comparable in terms of the mean age of patients, the proportion of health care card holders, and encounters with patients from a non-English speaking background. Table 2 Descriptive parameters of demographic, morbidity and treatment variables with design effects (Deff), intra-cluster correlation coefficients (ICC) and standard errors of ICC (SE) for sample year April 1999 to March 2000 (N = 1,047 general practitioners): compared with ICC and SE for sample April 2002 to March 2003 (N = 1,008 GPs). 1999–2000 (N = 1,047 GPs) 2002–2003 (1,008 GPs) Parameter Estimate(SE) Deff ( a ) ICC(SE) Estimate ICC(SE) Demographics Sex (% female) 59.0 (.39) 6.4 .055 (.003) 59.3 .066 (.003) Age (years) – mean 44.5 (.31) 16.6 .159 (.006) 45.4 .153 (.006) Holds health care card (%) 40.1 (.70) 21.4 .206 (.007) 42.7 .209 (.008) Patient language ( c ) (%) 7.0 (.53) 45.6 .451 (.011) 8.8 .423 (.011) Morbidity Number of problems (per 100 encounters) 149.5 (.86) 13.6 .127 (.005) 148.7 .141 (.006) Problem by ICPC-2 chapter (b) Cardiovascular (%) 15.2 (.29) 6.7 .057 (.003) 15.3 .056 (.003) Respiratory (%) 21.0 (.26) 4.2 .032 (.002) 19.0 .040 (.002) Psychological (%) 10.6 (.25) 6.8 .059 (.003) 10.6 .061 (.003) Endocrine/Metabolic (%) 8.8 (.18) 4.2 .032 (.002) 10.1 .031 (.002) Blood (%) 1.7 (.08) 3.7 .027 (.002) 1.4 .007 (.001) Digestive (%) 9.6 (.12) 1.8 .008 (.001) 9.7 .010 (.001) Eye (%) 2.8 (.06) 1.5 .005 (.001) 2.6 .003 (.001) Musculoskeletal (%) 16.3 (.23) 4.1 .032 (.002) 16.5 .045 (.002) Skin (%) 16.1 (.19) 2.7 .017 (.001) 15.9 .042 (.002) General unspecified (%) 14.0 (.22) 4.3 .034 (.002) 15.8 .043 (.002) Treatment (% of encounters) Any medications 67.0 (.37) 6.6 .056 (.003) 64.4 .068 (.003) Any referrals 11.2 (.20) 4.1 .031 (.002) 12.0 .033 (.002) Any pathology tests ordered 14.7 (.26) 5.8 .048 (.002) 16.0 .046 (.002) Any imaging tests ordered 6.9 (.15) 3.8 .028 (.002) 7.8 .029 (.002) ( a ) Average number of observations per cluster k = 100, except age ( k = 99.2) and sex ( k = 98.8). ( b ) Per cent of encounters where at least one problem from the chapter was managed. ( c ) Patient speaks a language other than English at home. Descriptive ICCs (March 1999–April 2000) Demographics For descriptive estimates of demographic variables the intra-cluster correlation ranged from 0.055 for sex of patient at encounter to 0.451 for language spoken by the patient at home. (Table 2 ). With a standard cluster size of 100 encounters this produced design effects ranging from 6.4 for patient sex to 45.6 for non-English speaking background. Morbidity (ICPC body chapter) For descriptive estimates of the management rates of morbidity problems, the intra-cluster correlations ranged from 0.005 for estimates of eye problems to 0.059 for estimates of psychological problems, with design effects of between 1.5 and 6.8 respectively. Treatments The intra-cluster correlation coefficients for treatments received ranged from 0.028 for any imaging tests ordered to 0.056 for any medications. Association ICCs For bivariate relationships between an outcome and predictor, the association ICCs were considerably smaller than the descriptive ICCs (Table 3 ). This pattern was observed for both demographic and morbidity outcomes. When analysing the association between holding a health care card and other demographic variables, the ICCs ranged from 0.012 for patient sex to 0.128 for language background (Table 3 ), which were smaller than for the descriptive estimate of the percentage holding a health care card (Table 2 ). Table 3 Associations between demographic and morbidity variables, measured as odds ratios, with design effect (Deff) and intra-cluster correlation coefficients (ICC) with standard errors (SE) for sample year April 1999 to March 2000 (N = 1,047 general practitioners): and ICC and SE for sample April 2002 to March 2003 (N = 1,008 GPs). 1999–2000 (N = 1,047 GPs) 2002–2003 (1,008 GPs) Outcome* Predictor Odds Ratio Deff ( a ) ICC (SE) ICC (SE) a) Demographic Patient holds health care card Female patient 1.07 2.2 .012 (.001) .018 (.001) Age (years) 1.03 6.5 .056 (.003) .073 (.003) Patient language ( b ) 1.26 13.7 .128 (.005) .114 (.005) Patient language ( b ) Female patient 0.94 3.7 .028 (.002) .024(.001) Age (years) 1.00 11.2 .104 (.004) .098 (.004) b) Morbidity Chapters Cardiovascular Female patient 0.90 1.3 .003 (.001) .004 (.001) Age (years) 1.05 2.1 .011 (.001) .017 (.001) Holds health care card 2.55 2.4 .014 (.001) .018 (.001) Patient language ( b ) 1.17 5.2 .042 (.002) .034 (.002) Respiratory Female patient .86 1.3 .003 (.001) .003 (.001) Age (years) .99 2.5 .015 (.001) .022 (.001) Holds health care card .89 2.1 .011 (.001) .017 (.001) Patient language ( b ) 1.17 2.9 .020 (.001) .025 (.002) Psychological Female patient 1.13 2.2 .013 (.001) .008 (.001) Age 1.01 3.3 .024 (.001) .022 (.001) Holds health care card 1.89 2.4 .014 (.001) .020 (.001) Patient language ( b ) .74 5.0 .040 (.002) .026 (.002) Endocrine/metabolic Female patient .95 1.6 .006 (.001) .004 (.001) Age 1.03 2.1 .011 (.001) .012 (.001) Holds health care card 1.63 2.4 .014 (.001) .009 (.001) Patient language ( b ) 1.58 3.3 .023 (.001) .019 (.001) * Each predictor is fitted alone, each line represents a separate model. ( a ) Average number of observations per cluster k = 100, except age ( k = 99.2) and sex ( k = 98.8). ( b ) Patient speaks a language other than English at home. When analysing the association between cardiovascular problems as the outcome and selected demographic variables, the ICCs ranged from 0.042 (patient language as the predictor) to 0.003 (patient sex as the predictor)(Table 3 ) compared with the larger ICC of 0.057 when describing the rate of cardiovascular problems (Table 2 ). Comparison of year 2 (April 1999 to March 2000) and year 5 (April 2000 to March 2003) For descriptive outcomes the intra-cluster correlations for year 2 and year 5 samples there was consistency in the patterns of ICCs across samples. (Table 2 and Figure 1 ). One exception was for the management of problems related to the blood system, where the descriptive ICC in 1999–00 was 0.027 (95% CI: 0.024–0.030), three times that observed in 2002–03 (0.007, 95% CI: 0.005–0.008). This was influenced by one GP in the 1999–00 sample who managed blood-related problems at more than 50% of encounters. When this GP was removed, the descriptive ICC for blood related problems in 1999–00 was 0.011 (95%CI: 0.009–0.013), much closer to the ICC observed in 2002–03. Figure 1 Intra-cluster correlation(ICC) and 95% confidence intervals for descriptive and morbidity outcomes in two BEACH samples, April 1999–March 2000 (N = 1047 GPs) and April 2002–March 2003(N = 1008 GPs) * Total problems = the number of problems managed at the current encounter. The intra-cluster correlation for associations between morbidity outcomes and demographic predictors are shown in Table 3 and Figure 2 . Although the intra-cluster correlations for associations between variables across each year were statistically significantly different for some outcomes, in these instances the ICCs were very small and the difference between samples was less than 0.01. Figure 2 Intra-cluster correlation (ICC) and 95% confidence interval for association between morbidity outcomes with health care card status as predictor in two BEACH samples, April 1999–March 2000 (N = 1,047 GPs) and April 2002–March 2003 (N = 1,008 GPs) Discussion The pattern of intra-cluster correlation and design effects observed in the BEACH study agree with Carlin and Hocking's observations in other cluster sample surveys[ 1 ]. Generally we found that sociodemographic variables had larger intra-cluster correlation coefficients than morbidity or treatment variables and outcomes fitted with explanatory variables had smaller intra-cluster correlation coefficients than outcomes reported as descriptive rates. Therefore when designing cluster sample surveys, the effect of the intra-cluster correlation on power calculations, depends on whether the main outcomes of interest are demographic or morbidity variables, and whether the main aims of the study are descriptive or predictive[ 1 ]. We further demonstrated that for a large range of variables the size and patterns of intra-cluster correlation coefficients for particular outcomes were mostly consistent over different sample periods. This indicates that intra-cluster correlation is quite stable when re-sampling a population using the same primary sampling unit, where the number of clusters is sufficiently large. This repeatability demonstrates the validity of using published intra-cluster correlation coefficients to predict intra-cluster correlation in future studies of similar design. Precision can be an issue for estimating intra-cluster correlation, especially for studies with a small number of clusters[ 10 ]. The large number of clusters in this study gave good precision in the estimated intra-cluster correlation coefficients[ 10 ]. There are no other published studies in general practice in Australia with such a large sample of clusters and a large balanced sample of observations per cluster, thus estimating intra-cluster correlation with a high degree of precision. The BEACH study also has the advantage of being a nationwide survey of general practice where the generalisability to Australian general practice has been well-described[ 11 ]. Most research in primary care in Australia is done through general practice, so estimating the intra-cluster correlation for a range of outcomes is important for future researchers who intend to use the GP as the primary sampling unit. The good representation of general practice in the BEACH study, the large sample of clusters and the large cluster size, allow the intra-cluster correlation coefficients reported here to be generalisable to other general practice surveys. These reported intra-cluster correlation coefficients are also likely to be useful for intervention studies that use the GP as the unit of randomisation[ 1 ]. Treatments received at the encounter are outcomes that arise as a result of the GP-patient interaction. Treatments are directly related to GPs' behaviour and so might be expected to be highly correlated within clusters. However we found that the intra-cluster correlation coefficients for medications, referrals, imaging and pathology orders were of a similar order to those for health problems managed. The difference across samples in the intra-cluster correlation coefficients for the management of blood system problems indicates that, even in large samples, intra-cluster correlation may be influenced by GPs in the sample who specialise in particular areas of health. Demographic variables are collected in the BEACH study for the purpose of understanding health status and health service use and these variables are likely to be correlated to a patient's choice of GP. Furthermore a patient can be sampled more than once if they return to the GP during the survey period. Therefore the intra-cluster correlation estimated for demographic variables may be larger than those that have been reported in community based surveys[ 1 , 8 ]. Conclusions As with cluster randomised trials, researchers in primary health care need access to a range of estimates of intra-cluster correlation for the successful planning of cluster survey study designs. We have reported relatively stable intra-cluster correlation coefficients for a range of outcomes across two independent random samples in a large-scale representative survey of general practice in Australia. The demonstrated precision and reliability of the estimated intra-cluster correlations indicate that these coefficients will be useful for calculating sample sizes in future general practice surveys that use the GP as the primary sampling unit. Abbreviations GP: General Practitioner ICC: Intra-cluster correlation coefficient Competing interests The author(s) declare that they have no competing interests. Authors contributions SK conceived the research questions, undertook the analysis and wrote the main draft of the manuscript. PC participated in formulating the research questions and the design of the analysis, undertook a literature search and assisted in the writing of the main draft and subsequent revisions of the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC545648.xml
553995	Does angiotensin-1 converting enzyme genotype influence motor or cognitive development after pre-term birth?	Background Raised activity of the renin-angiotensin system (RAS) may both amplify inflammatory and free radical responses and decrease tissue metabolic efficiency and thus enhance cerebral injury in the preterm infant. The angiotensin-converting enzyme (ACE) DD genotype is associated with raised ACE and RAS activity as well as potentially adverse stimuli such as inflammation. The DD genotype has been associated with neurological impairments in the elderly, and thus may be also associated with poorer motor or cognitive development amongst children born preterm prematurely. Methods The association of DD genotype with developmental progress amongst 176 Caucasian children born at less than 33 weeks gestation (median birthweight 1475 g, range 645–2480 g; gestation 30 weeks, range 22–32; 108 male) was examined at 2 and 5 1/2 years of age. Measured neuro-cognitive outcomes were cranial ultrasound abnormalities, cerebral palsy, disability, Griffiths Developmental Quotient [DQ] at 2 yrs, and General Cognitive Ability [British Ability Scales-11] and motor performance [ABC Movement], both performed at 5 1/2 yrs. All outcomes were correlated with ACE genotype. Results The DD genotype was not associated with lower developmental quotients even after accounting for important social variables. Conclusion These data do not support either a role for ACE in the development of cognitive or motor function in surviving infants born preterm or inhibition of ACE as a neuroprotective therapy.	Background Delight over recent survival gains for the very premature infant has been tempered by the frequent presence of cerebral injury and developmental impairment. One quarter of those born before 26 weeks postmenstrual age (at least 11 weeks premature) show evidence of severe cerebral injury including cognitive dysfunction by 30 months of age [ 1 ]. Preterm children without any disability remain at risk of a range of motor, cognitive, behavioural and psychological deficits during childhood even if not born so close to the margin of viability [ 2 ]. To date, the pathophysiological processes leading to such impairment remain largely occult. In particular, cerebral imaging has failed to identify structural correlates of impaired higher function [ 3 ] although imaging can predict many cases of motor abnormality (such as cerebral palsy) due to the presence of periventricular white matter injury [ 4 ]. Three factors seem to play important roles in the aetiology of preterm cerebral injury. Firstly, exposure to inflammatory stimuli is associated with white matter injury and cerebral palsy in the preterm [ 5 ]. Secondly, reduced glucose and oxygen delivery to the developing brain (hypoxia-ischaemia: local cerebral or systemic) may cause excito-toxic neurotransmitter release followed by neuronal death [ 6 ]. Thirdly, free-radicals may damage the oligodendrocytes of white matter of the preterm brain [ 6 ]. Damage to the primitive white matter prevents the normal formation of grey matter connections which may influence cognitive development in childhood [ 7 ]. Candidate systems that might influence motor or cognitive outcome after premature birth are likely to be those which affect these responses. The human renin-angiotensin systems may be such a system. Angiotensin converting enzyme (ACE), a key component of the circulating (or endocrine) renin-angiotensin system (RAS), cleaves angiotensin I to yield the potent vasoconstrictor angiotensin II. In addition, ACE degrades vasodilator kinins. In these ways, endocrine RAS plays an important role in circulatory homeostasis. However, local RAS also exist in diverse human tissues including lung, myocardium, vasculature, lymphocyte and brain tissue. These are powerful regulators of mitochondrial respiration and whole-cell metabolism [ 8 ] and exert profound effects on whole-human metabolism and metabolic efficiency: elevated ACE may impair cellular aerobic metabolism [ 9 ]. RAS also plays a key role in the regulation of tissue inflammatory responses; ACE, through generation of angiotensin II, stimulates the synthesis of pro-inflammatory cytokines, including IL-6 which itself is thought to exert major neurocytotoxic effects with the genesis of functionally significant lesions in the developing preterm brain [ 5 ]. It has also been noted that the inhibition of RAS may reduce the effects of excitotoxic neurotransmitters and free radicals [ 10 ]. It is possible therefore that enhanced ACE activity may adversely influence the development of the child born prematurely. A common variant of the human ACE gene provides a tool to determine if ACE activity does influence developmental progress after preterm birth. The presence (insertion, or 'I' allele) rather than the absence (deletion, or 'D' allele) of a 284-base-pair fragment in the human ACE gene is associated with lower ACE activity in organs including both circulating inflammatory cells [ 11 ] and the circulation itself [ 12 ]. Given the likely causal association of pro-inflammatory responses, ischaemic-hypoxia, excitotoxic neurotransmitters, and free radical attack with impaired neuro-outcome; and given the potential role of increased RAS activity in amplifying these effects, we might expect the DD genotype (encoding raised ACE activity) to be associated with poorer neuro-developmental progress after pretem birth. Comparable findings have been described with respect to the deterioration of cognitive function in the elderly by some authors [ 13 - 15 ]. We have tested this hypothesis by studying the association of the ACE I/D polymorphism with measures of neuro-developmental progress at 2 and 5 1/2 years of age in children who had participated in a neuro-developmental outcome study (The Avon Premature Infant Project, APIP [ 16 ]). All the patients were born at less than 33 weeks postmenstrual age (normal gestation is 37–40 weeks). Methods Patients The study was approved by the ethical committees of Southmead Hospital and United Bristol Health Care Trust. Parental consent was obtained for participation in neurodevelopmental follow-up [ 16 ] (see below). Consent was not required for the genetic component of this study as all personal information was held separately from the genetic information and patients were identified only by study codes. All children were born at 32 weeks gestation or less, between December 1990 and July 1993 at Southmead Hospital or St. Michael's Hospital, Bristol. All had participated in the Avon Premature Infant Project (APIP) [ 16 ]. Briefly, this was a randomised controlled trial in which developmental support (Portage) or supportive counselling (parental adviser), each started at discharge and continued for up to 2 years, were found to confer some measurable (3–4 DQ points (below)) but clinically insignificant benefit to development at 2 years of age, when given in addition to appropriate primary care and community support, after adjusting for social variables. Neuro-developmental outcome The Griffiths Mental Development Scales, used to assess motor and cognitive performance, was performed at 2 years corrected age [ 17 ]. The Griffiths scales comprise five subscales, including personal and social, hearing and speech, locomotor, eye hand co-ordination and performance domains, from which is derived an overall developmental quotient (DQ). A lower Griffiths DQ reflects a poorer neuro-developmental performance, with a difference DQ of five points being clinically apparent. DQ was standardised originally to a mean of 100, with a standard deviation of 15, but secular drifts in population scores have resulted in a higher population mean. Thus for severe disability a score of 70 (-2 standard deviations (sd)) would indicate severe disability. Cognitive developmental progress at 5.5 years of age was assessed using the British Ability Scales [ 18 ]. The BAS-II was standardised in the early 1990s and was used to compute general cognitive ability (GCA) together with visuospatial, verbal and non-verbal subscales. The GCA is a developmental quotient, equivalent to an IQ estimate, normalised at 100 (sd +/- 15) in which a lower score again indicates poorer conceptual ability. The Movement ABC scales were used to assess manual dexterity, ball skills, and balance over ten tests at 5 1/2 years of age. Scores of each component are summed to produce a composite score ranging from 0–40, with high scores indicating a more impaired motor skills and 0 indicating normal skills. A psychologist performed the Griffiths Scales of Mental Development and a second psychologist performed the British Ability Scales (second edition) (BAS). The ABC Movement tests were performed by a trained research nurse. All assessments were blind to the child's neonatal course and subsequent progress. ACE genotyping DNA was extracted from the Guthrie card blood spots (newborn metabolic screening cards). ACE genotype was determined using 3-primer PCR amplification [ 9 ]. Primer ratios corresponded to 50 pmol of an I-specific oligonucleotide in a 20-υl reaction volume. The PCR was performed using Taq polymeraase yielding amplification products of 84 bp for the D allele, and 65 bp for the I allele. Amplification products were visualised using a 7.5% polyacrylamide gel stained with ethidium bromide. Genotyping was performed by staff blind to all clinical data. Study Size An estimate of sample size suggested that 144 patients would be needed for this study. The assumptions made for this calculation were that DD genotype infants had a mean DQ of 92.5 (1/2 SD below the norm) compared to a mean DQ of 100 in the ID+II group, assumed typical genotype distributions, and a significance of 0.05 with 80% power. Statistical analysis Data were stored in SPPS v9.0 for Windows. Lymphocyte [ 11 ] and tissue ACE [ 12 ] activity is primarily raised in DD genotype when compared to either ID or II genotype, and so data for those of DD genotype were compared to those from I-allele carriers. Categorical data were analysed by Chi square and continuous data by Student's T Test if normally distributed or Mann-Whitney U test as appropriate. Results Guthrie cards were located for 230 of 308 children. After exclusion of non-Caucasians and, at random, 1 child of any identical twin pairs (based on genotypes and gender) 176 babies with ACE genotype formed the study population (median birthweight 1475 g, range 645–2480 g; gestation 30 weeks, range 22–32) with follow-up data at 2 years. 122 of these also had follow-up at 5 1/2 years. The ACE genotype distribution was 49 [27.8%] DD, 73 [41.5%] ID, 54 [30.7%] II, demonstrated Hardy-Weinberg equilibrium, and was similar to that observed in the newborn term population from the same region of the UK (203 [24.1%] DD, 433 [51.5%] ID, 205 [24.4%) II). Baseline characteristics were independent of genotype, except that fewer individuals of DD genotype were from twin births ( p = 0.047) (table 1 ). There was no association between markers of neonatal cerebral injury: severe intraventricular haemorrrhage or white matter injury (table 1 ). There was no association with the presence of any disability at 2 years of age (DD 17% vs ID/II 15%, p = 0.65). Table 1 Perinatal and social factors DD Genotype (n = 49) ID/II Genotype (n = 127) No maternal antenatal corticosteroids 44 (80%) 112 (81%) No. of children from twin pregnancy* 4 (8%) 27 (21%) Male 32 (65%) 76 (60%) Gestation, weeks (± SEM) 29.7 (± 0.3) 30.0 (± 0.2) Birth weight, g (± SEM) 1453 (± 56) 1461 (± 34) Portage, parent adviser 17 (31%), 19 (35%) 46 (33%), 42 (30%) Severe intraventricular haemorrhage 5 (11%) 7 (6%) White matter injury 7 (14%) 14 (11%) Maternal age (± SEM) 27.2 (± 0.8) 27.4 (± 0.8) Manual occupation 28 (57%) 76 (60%) Maternal car use 30 (61%) 75 (60%) Mother educated beyond 16 yrs. 17 (35%) 48 (38%) *p = 0.047 (Fisher's Exact Probability Test) Continuous data is shown as mean (± standard error of mean). Measures of developmental cognitive and motor outcome were entirely independent of genotype (table 2 ). The findings were unchanged after post hoc subgroup analysis of singletons, infants with normal cranial scans, amongst children without disability and after adjusting for potential influential variables (including twin birth) using multiple regression (data not shown). Table 2 ACE genotype and developmental performance at 2 and 5 1/2 years of age. Data shown is mean (± SEM). Developmental tests DQ for DD DQ for ID/II p Griffith DQ at 2 years 96.2 (3.1) 96.3 (1.3) 0.95 Locomotor subscale 92.7 (2.7) 92.4 (1.3) 0.92 Personal & social subscale 101.9 (3.0) 101.0 (1.6) 0.80 Hearing and speech subscale 92.9 (4.1) 94.0 (2.1) 0.80 Eye hand co-ordination subscale 90.8 (3.1) 92.8 (1.2) 0.46 Performance subscale 102.2 (4.3) 101.3 (1.6) 0.79 Griffith DQ at 2 years (adjusted for social variables) 100.0 (0.9) 99.3 (0.6) 0.43 ABC Movement summative score 8.1 (1.8) 8.0 (0.9) 0.97 GCA at 5 1/2 years 99.2 (3.4) 100.2 (2.0) 0.80 Verbal ability subscale 98.0 (4.0) 103.2 (1.7) 0.22 Pictoral ability subscale 99.9 (3.3) 98.7 (1.7) 0.99 Spatial ability subscale 98.4 (3.3) 97.3 (1.9) 0.67 Discussion After a search of Embase and Medline we believe that this study is the first to attempt to dissect out the contribution of genetic variation in the ACE gene to developmental progress after pre-term delivery. Despite much physiological and biochemical evidence to support our hypothesis, we found that ACE DD genotype was not associated with adverse long term developmental outcome in infants of < 33 weeks gestation in this study. These data are perhaps at variance with previous studies of Alzheimer's disease, age-associated memory impairment and vascular dementia, all of which have implicated the ACE D allele in having a role in mental decline [ 13 - 15 ]. However this is not a universal finding. Furthermore although ACE inhibitors appear to reduce inflammatory responses, ischaemic effects, and excitotoxic and free radical induced injury [ 10 ], angiotensin II does not (indeed angiotensin II may actually enhance ischaemic and excitotoxic neural injury via the AT2 receptor). In addition, both captopril and losartan (RAS inhibitors) appear to improve cognitive performance in mice [ 19 ] and humans [ 20 ]. It should be noted however that little is known about the ontogeny of the RAS in the human foetus. Certainly RAS (and angiotensin II receptors in particular) play a role in blood-brain barrier and central nervous system development in mice, and alterations in RAS receptor expression over foetal and neonatal life are recognised. It is thus possible that developmentally regulated patterns of AT1 receptor expression might offer some level of protection against the potentially detrimental effects of ACE-mediated angiotensin II synthesis. Although there may be similar molecular pathways that effect cerebral injury in the preterm infant and the elderly, ontological differences in the expression of genes involved in predisposition to neural injury are well described. In particular reactive production of nitric oxide may be enhanced in the elderly and the ability to protect the brain from oxidants may be reduced in the elderly (22). Thus the effect of any one polymorphism, with a relatively minor effect, may be swamped in the newborn infant by other protective mechanisms. The lack of any association between ACE genotype and scores of developmental progress was also surprising because we have demonstrated an association between DD genotype and markers of poor cardio-respiratory instability in the perinatal period in this patient group [ 21 ]. This association (between genotype and worse early cardio-respiratory status) could predispose to death, which would in turn weaken any association (if it exists) between DD genotype and worse developmental quotients. It is of course possible that our sample size was insufficient to demonstrate any association with ACE genotype and developmental progress. However, similar-sized studies have been sufficient to demonstrate an association between ACE D allele and cognitive decline in the elderly [ 13 - 15 ], and power calculations suggested we had enough patients to demonstrate at least a trend. If an undetected genotype-association does exist such an effect is weak. Conclusion We cannot support an association of ACE genotype with cognitive or motor development in survivors born preterm or, thus, the use of RAS inhibition as a neuroprotective agent in the preterm. Given the current lack of understanding of the mechanisms leading to cerebral injury and subsequent impairment – particularly of higher function – in such patients, further genetic association studies of other candidate genes are warranted. List of abbreviations used ACE, angiotensin-1 converting enzyme; DQ developmental quotient, BAS, British ability scales (second edition); GCA, general cognitive ability; RAS, renin angiotensin system; PCR, polymerase chain rection. Competing interests The author(s) declare that they have no competing interests. Authors' contributions DH, HM, AW, NM conceived the study and its design and wrote the manuscript. DH, DD and SD performed data collection, DNA extraction and PCR and participated in analysis of the data with SH and HM. NM reviewed all cranial imaging. All authors participated in the writing of the manuscript and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC553995.xml
553980	The Arabidopsis Mei2 homologue AML1 binds AtRaptor1B, the plant homologue of a major regulator of eukaryotic cell growth	Background TOR, the t arget o f the antibiotic r apamycin in both yeast and mammalian cells, is a potent cell growth regulator in all eukaryotes. It acts through the phosphorylation of downstream effectors that are recruited to it by the binding partner Raptor. In Arabidopsis, Raptor activity is essential for postembryonic growth. Though comparative studies suggest potential downstream effectors, no Raptor binding partners have been described in plants. Results AtRaptor1B, a plant Raptor homologue, binds the AML1 ( A rabidopsis M ei2- l ike 1) protein in a yeast two-hybrid assay. This interaction is mediated by the N-terminal 219 residues of AML1, and marks AML1 as a candidate AtTOR kinase substrate in plants. The AML1 N-terminus additionally carries transcriptional activation domain activity. Plants homozygous for insertion alleles at the AML1 locus, as well as plants homozygous for insertion alleles at all five loci in the AML gene family, bolt earlier than wild-type plants. Conclusion AML1 interacts with AtRaptor1B, homologue of a protein that recruits substrates for phosphorylation by the major cell-growth regulator TOR. Identification of AML1 as a putative downstream effector of TOR gives valuable insights into the plant-specific mode of action of this critical growth regulator.	Background TOR, the t arget o f the antibiotic r apamycin in both yeast and mammalian cells, is a major regulator of cell growth and translation [ 1 ]. TOR is a large (over 2,400 residues) protein kinase [ 2 ] present in all eukaryotes analyzed. It is thought to act in a nutrient-sensitive complex TORC1 with Raptor ( r egulatory a ssociated p rotein of TOR) and another protein to regulate cell growth [ 3 - 7 ] – though there is some debate on the nutrient sensitivity of the complex [ 8 ]. Raptor, a protein with HEAT and WD-40 protein interaction domains, recruits substrates for phosphorylation by TOR in yeast and mammals [ 3 , 7 ]. TOR also acts in a second, nutrient-insensitive complex without Raptor to regulate the cytoskeleton [ 3 , 8 , 10 ]. Disruption of the Arabidopsis TOR homologue AtTOR is lethal early in plant embryonic development [ 11 ]. Disruption of AtRaptor (encoded by two paralogous loci in Arabidopsis) causes seedling developmental arrest but allows normal embryonic development [[ 12 ], manuscript in preparation], suggesting that AtTOR embryonic activity is independent of AtRaptor and that the TOR-Raptor complex has been adapted in the ancestor of the angiosperms to regulate post-embryonic growth. In support of this, AtTOR has been show to be expressed in dividing and expanding cells [ 11 ]. Thus, identifying downstream activators of TOR signaling may provide insights into the activity of AtTOR in post-embryonic growth. Mei2 is a putative TOR substrate and potent meiosis-signaling molecule identified in the fission yeast Schizosaccharomyces pombe [ 13 , 14 ]. Mei2 is bound by the Raptor homologue Mip1 [ 15 ], and is an inactive phosphoprotein under high nutrient conditions [ 16 , 17 ] – conditions which promote TOR kinase activity. The kinase governing two of the three Mei2 phosphorylation sites is known [ 16 ]; TOR is a strong candidate for the kinase governing the third. In diploids under low nutrient conditions, unphosphorylated Mei2 accumulates and localizes to the nucleus, where it binds to a noncoding, mRNA-like RNA molecule meiRNA in an interaction mediated by the third of its three RNA Recognition Motifs (RRMs)[ 14 , 16 , 18 ]. The Mei2-meiRNA interaction occurs as meiRNA is being transcribed, tethering Mei2 to the meiRNA locus [ 19 ]. Accumulation of Mei2 at this focused point immediately precedes meiosis. Mei2-like proteins are predicted in a wide range of organisms [ 20 , 21 ] including some fungi, alveolates, a diatom, and all land plants, but they are absent from metazoans and budding yeast. In land plants, predicted Mei2-like proteins form a small conserved gene family, many of whose members' transcripts accumulate in the shoot apical meristem specifically or in the shoot apical meristem in addition to a range of mature tissues [ 20 ]. A rabidopsis M ei2- l ike 1 (AML1), the first member of this family to be described, was isolated in a screen for plant cDNAs whose expression could complement defects in the fission yeast meiosis signaling pathway [ 22 ]. Like Mei2, AML1 has three RRMs. Expression of a protein fragment containing only the third AML1 RRM was sufficient for restoration of meiosis signaling in fission yeast lines with defects upstream of Mei2, but not in lesions of the mei2 locus itself. T erminal E ar 1 (te1), a more divergent member of the mei2 -like family of genes, regulates leaf initiation in maize; the tassel of mutant plants is encased a whorl of leaves superficially resembling a maize ear [ 23 ]. Given the potential role of downstream AtTOR effectors in post-embryonic growth, the intriguing signaling activity of Mei2 in fission yeast, and the known interaction between Mei2 and the fission yeast Raptor protein, we asked if the Mei2-Raptor interaction was conserved between its Arabidopsis orthologues AML1 and AtRaptor1B. Results AML1 and AtRaptor1B interact in a yeast two-hybrid assay To test for an interaction between the Arabidopsis proteins AML1 and AtRaptor1B, the open reading frames (ORFs) of each of the transcripts predicted to encode these proteins were amplified via PCR with primers carrying suitable restriction sites for cloning into the yeast two hybrid vectors pGADT7 and pGBKT7. pGADT7 encodes a transcriptional activation domain (AD) which can recruit the yeast transcription machinery. pGBKT7 encodes a DNA binding-domain (BD) which binds to the promoters of ADE2 and HIS3 . pGADT7 and pGBKT7 additionally carry the genes LEU2 and TRP1 . When transformed into the yeast line AH109, which is leu2 - trp1 - ade2 - his3 - , co-transformed cells will grow on yeast medium lacking leucine and tryptophan. Cells co-transformed with two-hybrid constructs encoding proteins that interact will grow on media additionally lacking histidine and adenine (selective media). AH109 yeast cells co-transformed with clones encoding AML1 and AtRaptor1B in complementary two-hybrid vectors grew on selective media. Co-transformations of control pGBKT7 and pGADT7 empty vectors, or either control vector co-transformed with its complement harboring the AML1 or AtRaptor1B ORF, yielded cells able to grow on media lacking leucine and tryptophan but not on selective media. Evidence from other systems indicates that Raptor protein fragments lose the ability to bind substrates [ 4 ]. Therefore, only full length AtRaptor1B was tested in this assay. Mei2, the fission yeast AML1 homologue, is highly modular. It is divided into distinct N-terminal and C-terminal domains. The N-terminal half of the protein appears to play a regulatory role [ 24 ]. The C-terminal half of Mei2 is sufficient to complement lesions in the mei2 locus [ 16 ]. Additionally, the AML1 C-terminal half, expressed in fission yeast meiosis signaling mutants, is able to complement meiosis signaling defects upstream of Mei2 [ 22 ]. Therefore we generated clones encoding fragments of AML1 and assayed them for interactions with full length AtRaptor1B. AML1 fragments N412 and N219, comprised of the first 412 or the first 219 residues of the 915 residue AML1 protein, restored growth on media lacking leucine, tryptophan, histidine and adenine when cloned into pGADT7 and co-transformed with pGBK-Raptor (Fig. 1 ). Neither N163, nor 155–219, (the fragments which together comprise N219), nor 155–412 (which with N163 comprises N412), could restore growth when cloned into pGADT7 and co-transformed with pGBK-Raptor. AML1 fragment 402C, comprised of residues 402 to the C-terminus of the protein, and all C-terminal fragments tested (695C, 402–704) failed to restore growth on selective medium. None of the AML1 fragments cloned into pGADT7, and cotransformed with empty pGBKT7 control vector, yielded transformants able to grow on selective medium. AML1 fragments N412 and N163 harbor activation domain activity in a yeast one-hybrid assay AML1 N412, cloned into pGBKT7, restored growth on selective medium to cells co-transformed with either pGAD:Raptor or pGADT7. To investigate this result further, we tested all AML1 fragments for native transcriptional activation domain activity in a yeast one-hybrid assay. AML1 fragments were cloned into pGBKT7, singly transformed into AH109 yeast cells, and assayed for growth on media lacking tryptophan (to confirm transformation) and media additionally lacking adenine. The DNA binding-domain of the pGBKT7 tethers any C-terminally fused fragments to the ADE2 promoter. In the absence of a binding partner, BD-fusion chimeric proteins trigger transcription of ADE2 only if the protein fused to the BD contains native transcriptional activation activity. AML1 N412 and AML1 N163, but not full length AML1, AML1 N219 or any C-terminal AML1 fragments, were able to restore growth on media lacking tryptophan and adenine (Fig. 2 ). AML1 N122 was similarly unable to restore growth. AML1 fragments 155–219 and 155–412, in pGBKT7, could not be stably transformed into yeast. These results were observed in multiple independent transformant lines for a given construct. Cotransformation of any of the pGBKT7-derived constructs with pGADT7 did not affect the growth of any of the transformed lines on media lacking tryptophan and adenine. Plants homozygous for insertion alleles of AML1 and of all five AML family members show early flowering An interaction with AtRaptor1B points to AML1 as a downstream effector of TOR signaling in plants. Additionally, the dramatic phenotype mei2 disruption and the intriguing mode of Mei2 action led us to ask what the consequences would be of disruption of the AML1 locus and of all five AML gene family members. To obtain insertion alleles in AML1 and other AML gene family members, we screened the insertion allele populations at the University of Wisconsin Arabidopsis Knockout facility [ 25 ], obtaining alleles harboring insertions in AML1 , AML3 , AML4 and AML5 (Fig. 3 ). An AML2 insertion allele was obtained from the SIGnAL collection at the Salk Institute [ 26 ]. By RT-PCR using primers which anneal to the cDNA at sites spanning the insertion site of each insertion allele, we established that no wild-type transcripts accumulate in homozygous mutants (Fig. 4 ). By a series of crosses, we then generated higher-order insertion homozygotes, culminating in the quintuple insertion homozygotes Q6 and Q17. All lines were viable and fertile. AML insertion homozygotes bolted earlier than wild-type lines (Fig. 5A and 5B ). This effect was independent of the number of insertion alleles carried by the mutants; AML 'quint' lines Q6 and Q17 were not qualitatively different than lower order insertion allele homozygotes. Additionally, AML insertion homozygote seedlings were assayed for a differential response to a range of signaling molecules. Seedlings were germinated on culture medium supplemented with the gibberellic acid GA 3 , paclobutrazol, the auxin 2,4-D, 1-amino-cyclopropane-1-carboxylic acid, 1% sucrose, 6% sucrose and kinetin, and in the dark. Quintuple insertion mutants responded slightly more than wild-type seedlings to GA 3 as measured by the change in seedling length in the presence vs. in the absence of the hormone. This effect was repeatable but weak, and no other differential hormonal response was observed (data not shown). Sequence downstream of the mutant allele insertion sites is transcribed Given the mild phenotype of the AML insertion homozygotes, we further investigated the extent of the effect of the insertions at each locus. RT-PCR, as previously stated, showed that no wild-type transcripts accumulate in lines Q6 and Q17. In fission yeast, however, the C-terminal half of the protein is sufficient to complement lesions of the mei2 locus [ 16 ]. The insertion alleles of all but AML4 are disrupted at or near the 5' ends of their predicted coding regions. We therefore designed primers that anneal to the region downstream of the insertion site in each allele and performed polymerase chain reactions to assay for accumulation of fragments capable of encoding the C-terminal half of any of the AML proteins (Fig. 4 ). To determine whether the amplified fragments corresponded to AML cDNAs, we performed restriction digests on the PCR products, which confirmed that the cDNAs originated from AML gene transcripts. Weak amplification of cDNA representing transcripts originating downstream of the insertion site was observed for AML1, AML2 and AML3; amplification of the AML5 3' region was indistinguishable from the amplification seen from of wild-type cDNA template. Transgenic lines overexpressing AML1:GFP or GFP:AML1 fusion proteins could not be recovered The AML1 ORF was separately cloned into the pCambia1302 35S::GFP plant transformation vector both 5' and 3' of the GFP ORF, and the construct was transformed into Arabidopsis via Agrobacterium -mediated floral dip [ 27 ]. Transformants, identified by resistance to the antibiotic hygromycin and confirmed through PCR, were recovered at a very low rate of less than .01%. No fluorescence was observed in any tissues of any transformants assayed, and AML1:GFP transcripts could not be detected via RT-PCR performed on cDNA transcribed from RNA extracted from bulk shoot tissue (data not shown). Discussion Raptor proteins in yeast and mammals function by recruiting substrates for TOR, a central regulator of cell growth in response to nutrients [ 4 , 7 , 28 ]. An interaction with Raptor therefore strongly suggests that a given protein is a TOR substrate and downstream effector of TOR signaling. Plants homozygous for lesions at both AtRaptor loci show normal embryonic development but are unable to maintain shoot meristem activity [ 12 ]. TOR substrates, then, may play a role in regulating meristem-driven post-embryonic growth. The interaction between AML1 and AtRaptor1B implicates the AML family of proteins in TOR signaling. It points specifically to a role for the AML proteins in regulation of shoot meristem activity. An interaction between Mei2 and the fission yeast Raptor homologue Mip1 has been reported previously; indeed, Mip1 ( M ei2 i nteracting p rotein 1) was the first Raptor homologue characterized in any eukaryote [ 15 ]. The conservation of this interaction from fission yeast to plants suggests that the well-characterized Mei2 signaling pathway may provide insight into the function of the AMLs. Mei2 is a potent meiosis-signaling molecule. It triggers pre-meiotic cell differentiation and meiosis in response to nutrient stress [ 13 , 14 ]. Meiosis signaling in fission yeast is a model for cell differentiation in response to external nutrient cues. Thus the AMLs may also play a role in cellular differentiation or in meiosis signaling. Aside from the effect of Mei2 in development, there is the intriguing issue of its mode of action. Mei2 sub-cellular localization is mediated by an interaction with a noncoding, mRNA-like molecule [ 14 , 19 ]. There is a fairly large population of mRNA-like transcripts conserved among land plants despite lacking large conserved open reading frames [ 29 ]. Of these, the conserved alfalfa transcript ENOD40 has been shown to mediate the sub-cellular localization of an RNA-binding protein [ 30 ] and to mediate phytohormone responses [ 31 ]. AML1 may be a binding partner of one or more of these mRNA-like noncoding molecules in plants. The transcriptional activation activity of the AML1 N-terminus observed in the yeast one hybrid assay has not been ascribed to Mei2 and may represent a novel activity of plant Mei2-like proteins. This activation activity localizes to the N-terminal 163 residues, but is strongly influenced by the adjacent residues. Activity is lost in N219, regained on N412 and lost again in full length AML1. This suggests that the AML1 N-terminal half has multiple configurations, and that the accessibility of the activation domain varies among configurations. We should emphasize, however, that the AML1 N-terminal fragment transcriptional activation activity has yet to be shown in planta , and that the activity may result from fusion to the DNA binding domain rather than being present in native AML1. AtRaptor1B binding to AML1 is also localized to the N-terminus, and appears to be mediated by multiple sites in this region. This suggests that the N-terminus may contain a TOR phosphorylation site, and that this site may influence the configuration of the N-terminus. The repeated failure to recover transgenic lines expressing AML1 suggests that its unregulated overexpression is lethal. Future efforts to characterize the AML proteins in transgenic plants may benefit from the use of inducible promoters driving transgene expression to circumvent the putative lethality of unregulated AML expression. Disruption of any of the AML loci causes early bolting in plants grown under long days. However, lines homozygous for insertions in all five AML loci did not differ dramatically from lower-order insertion homozygotes, despite the fact that RT-PCR performed with primers spanning the insertion sites show that the wild-type transcript does not accumulate. Transcripts originating downstream of the insertion sites but still capable of encoding the C-terminal half of the wild-type protein accumulate from all loci but AML4 . This raises the possibility that the AML quintuple insertion homozygote lines do not represent total disruption of AML activity. Four of the five AML open reading frames in the insertion mutant are apparently truncated and all are divorced from their native promoters, but some promoter activity (perhaps from the 35S viral promoters harbored within the inserted DNA) remains and may be sufficient to cause transcription of AML coding region DNA downstream of the insertion site. Viewed in this light, the early flowering phenotype of AML insertion homozygotes may arise not from the total disruption of AML activity but from the accumulation of AML proteins which, due to the truncations in their N-termini caused by the insertions, are no longer bound by AtRaptor1B, no longer phosphorylated by AtTOR, or no longer able to activate transcription of floral repressors. Finally, these results provide a cautionary tale. RT-PCR performed using primers which span an insertion site may not be sufficient to conclude that all activity of a protein of interest is abolished. Conclusion TOR is a major regulator of cell growth in eukaryotes, but little is known about its downstream effectors in plants. This work shows that AML1 binds AtRaptor1B, and suggests that the AML protein family may be phosphorylated by AtTOR in an AtRaptor1B-mediated interaction. The interaction with AtRaptor1B implicates AML1 as a downstream effector of AtTOR kinase signaling, and provides insight into the mode of action of this critical growth regulator. Methods Generating the two-hybrid constructs AML1 was cloned via RT-PCR. The cDNA template was reverse-transcribed using Omniscript (Qiagen) from RNA extracted from bulk shoot tissue using Trizol Reagent (Invitrogen). Restriction sites Nco I and Xma I/ Sma I were added to the 5' and 3' ends of the ORF and of all smaller AML1 fragments via PCR using ExTaq high-fidelity polymerase (Takara). An EST clone (RZL03b06) tagging AtRaptor1B was obtained from Kazusa DNA institute and sequenced. Restriction sites Nco I and Eco RI were added at the 5' and 3' ends of the ORF via PCR. pGBKT7 and pGADT7 are distributed by BDBiosciences. Yeast two-hybrid assay AH109 cells ( leu2 - trp1 - ade2 - his3 - ) were grown in YEPDA liquid plates or on YEPDA plates with 17 g/L Agar-Y (Bio101 Systems). Cells were transformed using the Yeastmaker Yeast Transformation System2 (BD Biosciences) and plated on medium lacking the appropriate macronutrients (Bio101 Systems). Colonies were observed 3–7 days after transformation. Genotyping of insertion alleles DNA from lines harboring insertion alleles was extracted using the alkaline boiling method [ 32 ]. Provisional homozygotes were confirmed via a second extraction using the C-TAB DNA extraction protocol. PCR to assay for wild-type and insertion alleles was performed in 20 uL volumes using ExTaq polymerase and buffers and the following cycling parameters: 94°C, 15 seconds; 61°C, 30 seconds; 72°C, 2 minutes; 35 cycles. Genotyping primers were as follows: AML1-5sm 5'atagaaggaaacaaaaaggaaaggaggaa3'; AML1-3sm 5'tagcatatcacttccctgtagccgcactg3'; AML2-5sm 5'attgctctgtctctgatgatgttttgtcg3'; AML2-3sm 5'gcagcaatatttctaaagcatcgggttca3'; AML3-5sm 5'ctttagttccctctttcctctgctgtgat3'; AML3-3sm 5'ctgccaagaacgggaaaacaaacataaa3'; AML4-5sm 5'ttgcaagcggtagtccatataaatcctc3'; AML4-3sm 5'atgctaccgggagaacctaagtgaaatc3'; AML5-5sm 5'tctttagccacatcaatcattctcatcct3'; AML5-3sm 5'atcagcgtcaagttccattcctcctccac3'; JL-202 5'cattttataataacgctgcggacatctac3'; JL-270 5'tttctccatattgaccatcatactcattg3'; pROC-737 5'gggaattcactggccgtcgttttacaa3'. The wild-type loci were assayed with the above pairs. The insert was assayed using the following pairs: AML1-5sm or AML1-3sm with JL-270 or JL-202, AML2-5sm with pROC-737, AML3-5sm with JL-270 or JL-202, AML4-3sm with JL-270 or JL-202, AML5-5sm with JL-202. Insertions in the AML1 and AML5 loci were obtained from the University of Wisconsin alpha collection using their described protocol and are in the Wassiljewskia (Ws) ecotype background. The AML1 insertion was found in pool CSJ8-46-H35. The AML5 insertion was found in pool CSJ1091-H45. Insertions in the AML3 and AML4 loci were obtained from the University of Wisconsin Basta collection and are in the Ws background. The AML3 insertion was found in pool 67-6-F. The AML4 insertion was found in pool 18-2-H. The insertion in AML2 was obtained from the line 029713 from the Salk collection and is in the Columbia (Col-0) ecotype background. Aside from regions genetically linked to any of the insertion loci, the AML quintuple insertion allele homozygote lines are in the genomic background of a Col-0 × WS F 3 individual once backcrossed to the WS background. RNA extraction, RT-PCR RNA extraction was performed using TRIzol™ Reagent (Invitrogen) essentially according to manufacturer's instructions. Total RNA was treated with DNA- free ™ DNase (Ambion) and reverse transcribed using Omniscript reverse transcriptase (Qiagen) with an oligo-dT primer. Primer pairs spanning the insertion site for RT-PCR on all five lines are as follows: AML1 117–138 5'gtgatggatgcgattggataga3', AML1 556-534rc 5'attgtggcttcagctggtaactt3'; AML2 86–109 5'tttgcttctccgattctcttcctt3', AML2 456-435rc 5'agcatcgggttcaacatcttcc3'; AML3 548–568 5'gtagcggaggaggtcttgaat3', AML3 1060-1039rc 5'tctccttgatctcgccataaac3'; AML4 1932–1955 5'aagcggtagtccatataaatcctc3', AML4 2944-2927rc 5'tcccctgaatccgaccat3'; AML5 104–124 5'cgtgatcatcgtcggtgttgg3', AML5 1047-1024rc 5'ctcgacgaatttgtgatgcctctta3'. Reactions were performed using Takara ExTaq and 35 cycles of 94°C for 30 sec, 58°C annealing ( AML1,2,4 ) or 60°C annealing ( AML3,5 ) for 30 sec, and 72°C for one minute. Primer pairs amplifying a region 3' of the insertion site for RT-PCR on AML1 , AML2 , AML3 and AML5 insertion homozygotes are as follows: AML1 +1635 5'aggctctcgccgccctatta3', AML1 -2466 5'cgttgccaccttctcgctatt3'; AML2 +1779 5'accggggaacagtagtgaac3', AML2 -2107 5'ctgtcggcaagcatagaaag3'; AML3 +1756 5'tctggcctgctgctacaatgg3', AML3 -2326 5'cgccgacaagaagatgagaaaac3'; AML5 +1268 5'gcaacggcttccaacagtca3', AML5 -1869 5'acgaggcctaccattttcatacaa3'. All reactions were performed with a 59°C annealing temperature. Authors' contributions GHA conceived of the study and performed all experimental manipulations. MRH provided guidance and arranged funding for the project. GHA and MRH drafted the manuscript, and both authors approve the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC553980.xml
544543	Unexpressed but Indispensable—The DNA Sequences That Control Development	null	Amidst the hoopla over the exact number of genes we have in our genome—more than a fruitfly, fewer than a rice plant—a more fundamental genetic truth has often been obscured. The expression of 20,000–30,000 genes is under the control of an uncounted host of non-coding sequences, which bind transcription factors and thereby regulate when and where genes are expressed. Unlike coding sequences, whose signatures are easy to spot, the characteristic features of non-coding regulatory elements are largely unknown, making their discovery by simple sequence analysis difficult. In this issue, Greg Elgar and colleagues attack this problem by comparing the non-coding sequences of the human and the pufferfish. Since the last common ancestor of these two species existed 450 million years ago, the authors reasoned that non-coding sequences conserved between them are likely to be fundamental to vertebrate development. Through sequence alignment with increasingly strict criteria, they identified 1,373 highly conserved non-coding elements (CNEs), with an average length of about 200 base pairs. The average sequence match is 84%: not perfect, but much higher than for coding regions shared by humans and pufferfish. A quick check showed that virtually all the sequences also occurred in rodents, chickens, and zebrafish, but not in the nematode, fruitfly, or even the sea squirt, a primitive non-vertebrate chordate. Highly conserved vertebrate non-coding elements direct tissue-specific reporter gene expression CNEs are not spread uniformly throughout the genome. Instead, they are bunched together in fewer than 200 clusters, most of them in close proximity to genes implicated in transcriptional regulation or development. This clustering of CNEs suggests they may not only attract transcription factors, but may also influence the local topology of the DNA, thereby controlling access to their associated gene. Several clusters also appear in regions without any known genes—the identification of these clusters might lead to the discovery of new developmentally significant genes. While “in silico” discoveries such as this can be the jumping-off point for whole new areas of investigation, their validity must be tested “in aqua,” in the wet biology of real organisms. For this Elgar and colleagues chose the zebrafish, because its transparent embryo is ideal for observing developmental events. They injected individual CNEs into embryos, along with a green fluorescent protein (GFP) reporter. By day two of development, 23 out of 25 CNEs injected had upregulated GFP expression, indicating interaction of these sequences with endogenous transcription factors. Different CNEs caused different regional patterns of expression, in keeping with their presumed roles in distinct developmental processes. The discovery of these developmentally important sequences opens several avenues of new research. For example, analyzing the sequence and location of these CNEs may help point the way to other non-coding elements that remain undiscovered. It is also likely that mutations in these critical sequences cause human diseases. Studying how such mutations drive development astray may lead to better understanding not only of these diseases, which are likely to be rare, but also of normal human development.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544543.xml
552335	Advances in wearable technology and applications in physical medicine and rehabilitation	The development of miniature sensors that can be unobtrusively attached to the body or can be part of clothing items, such as sensing elements embedded in the fabric of garments, have opened countless possibilities of monitoring patients in the field over extended periods of time. This is of particular relevance to the practice of physical medicine and rehabilitation. Wearable technology addresses a major question in the management of patients undergoing rehabilitation, i.e. have clinical interventions a significant impact on the real life of patients? Wearable technology allows clinicians to gather data where it matters the most to answer this question, i.e. the home and community settings. Direct observations concerning the impact of clinical interventions on mobility, level of independence, and quality of life can be performed by means of wearable systems. Researchers have focused on three main areas of work to develop tools of clinical interest: 1)the design and implementation of sensors that are minimally obtrusive and reliably record movement or physiological signals, 2)the development of systems that unobtrusively gather data from multiple wearable sensors and deliver this information to clinicians in the way that is most appropriate for each application, and 3)the design and implementation of algorithms to extract clinically relevant information from data recorded using wearable technology. Journal of NeuroEngineering and Rehabilitation has devoted a series of articles to this topic with the objective of offering a description of the state of the art in this research field and pointing to emerging applications that are relevant to the clinical practice in physical medicine and rehabilitation.	The potential impact of wearable technology on physical medicine and rehabilitation Understanding the impact of clinical interventions on the real life of individuals is an essential component of physical medicine and rehabilitation. While assessments performed in the clinical setting have value, it is difficult to perform thorough, costly evaluations of impairment and functional limitation within the time constraints and limited resources available in outpatient units of rehabilitation hospitals. Furthermore, it is often questioned whether assessments performed in the clinical setting are truly representative of how a given clinical intervention affects the real life of patients. While this observation has fostered a great deal of interest for the development and validation of outcome measures that largely rely on the use of questionnaires [ 1 ], researchers and clinicians have looked at recent advances in wearable technology intrigued by the possibility offered by this technology of gathering sensor data in the field [ 2 , 3 ]. Likely to be complementary to outcome measures, the use of wearable systems in the clinical management of individuals undergoing rehabilitation is very attractive because it provides the opportunity of recording quantitative data in the settings that matter the most, i.e. the home and the community. A number of clinical applications of wearable systems in physical medicine and rehabilitation emerged in the past few years. They range from simple monitoring of daily activities, for the purpose of assessing mobility and level of independence in individuals, to integrating miniature sensors to enhance the function of devices utilized by patients to perform motor tasks that they would be otherwise unable to accomplish. Monitoring functional motor activities was one of the first goals of research teams interested in clinical applications of wearable technology. The focus was initially on using accelerometers [ 4 - 8 ] or a combination of accelerometers and electromyographic sensors [ 9 ] to capture movement and muscle activity patterns associated with a given set of functional motor tasks. The set of tasks to be identified varied according to the clinical application. Their study was combined with monitoring systemic responses when the clinical assessment required combining motor activities and cardio-respiratory data such as in the clinical management of patients with chronic obstructive pulmonary disease [ 10 ]. A level of complexity was added when researchers started investigating motor disorders and the possibility of utilizing wearable technology to assess the effect of clinical interventions on the quality of movement observed while patients performed functional tasks. Two applications worth mentioning are the one to assess symptoms and motor complications in patients with Parkinson's disease [ 11 - 14 ] and the study of motor recovery in post-stroke individuals [ 15 - 17 ]. This shift from identifying functional motor activities to studying motor patterns associated with motor disorders generated significant interest for more complex ways to monitor movement, i.e. utilizing not only accelerometers but also gyroscopes and magnetometers or inclinometers. The combination of multiple sensors allows one to estimate the kinematics of movement [ 18 - 21 ] with a reliability that cannot be obtained by solely relying on accelerometers [ 22 ]. Finally, recent studies have been focused on integrating wearable, miniature sensor technology with orthoses, prostheses, and mobility assistive devices. Sensor technology is particularly appealing in these applications because it allows implementing closed-loop strategies that take advantage of the increased complexity and flexibility that robotics is contributing to the design of orthoses, prostheses, and mobility assistive devices. Namely, the characteristics of such devices can be constantly modified as a function of the task individuals are engaged into and environmental disturbances [ 23 , 24 ]. In all the emerging applications summarized above, either continuous recording of sensor data or at least monitoring over extended periods of time are necessary to design and implement an effective clinical intervention. Unobtrusive, wearable systems providing ease of data gathering and some processing capabilities are essential to achieve the objective of making the leap between the preliminary results obtained as part of the research carried on so far and the daily clinical practice of physical medicine and rehabilitation. Three areas of work are essential to achieve this objective: 1)the development of wearable sensors that unobtrusively and reliably record movement and other physiological data relevant to rehabilitation; 2)the design and implementation of systems that integrate multiple sensors, record data simultaneously from wearable sensors of different types, and relay sensor data to a remote location at the time and in the way that is most appropriate for the clinical application of interest; and 3)the development of methodologies to manipulate wearable sensor data to extract information in a clinically relevant manner to perform clinical assessments or control devices aimed at enhancing mobility in individuals with conditions that limit their level of independence. A series of papers have been assembled to provide the readership of Journal of NeuroEngineering and Rehabilitation with a description of the state of the art of the application of wearable technology in physical medicine and rehabilitation. Wearable sensors to measure movement and physiological signals A first set of the papers that have been assembled for publication on Journal of NeuroEngineering and Rehabilitation on the topic of wearable technology in physical medicine and rehabilitation has the objective of describing recent advances in wearable sensor technology. Two manuscripts describe attempts by different groups of measuring angular displacements for upper and lower extremity joints by embedding conductive fibers into the fabric of undergarments. The paper by Gibbs and Asada, entitled "Wearable conductive fiber sensors for multi-axis human joint angle measurements", reports encouraging preliminary results concerning monitoring lower limb joint displacements during ambulation by utilizing such technology. The manuscript by Tognetti et al, entitled "Wearable kinesthetic system for capturing and classifying upper limb gesture in post-stroke rehabilitation", describes the design and implementation of a system similar to the one proposed by Gibbs and Asada but geared toward monitoring movements of the upper extremities. The authors also explore the application of these wearable sensors to monitoring motor recovery in post-stroke individuals. Simone and Kamper focus their contribution on unobtrusively measuring finger movements in patients undergoing rehabilitation. Their manuscript "Design considerations for a wearable monitor to measure finger posture" summarizes the authors' recent work toward developing ways to record fine motor control tasks involving manipulation of objects requiring fine motor control of the hand and fingers. This technology has immediate application in patients such as post-stroke individuals undergoing rehabilitation that targets fine motor control skills. While initial research in the area of wearable technology was aimed at combining existing, miniature sensors with special fabrics or wireless technology, recent advances in this field have been focused on the development of sensing elements that can be even more easily embedded in clothing items. An example of such effort is reported in the paper by Dunne et al entitled "Initial development and testing of a novel foam-based pressure sensor for wearable sensing". This paper summarizes positive preliminary results by the research team aimed at measuring shoulder movements, neck movements, and scapular pressure. The sensing elements can also be used to monitor respiratory rate. Devoted to monitoring systemic responses is the last of the papers focused on wearable sensors. In this manuscript, Yan et al describe a new method to reliably measure heart rate and oxygen saturation. The paper is entitled "Reduction of motion artifacts in pulse oximetry by smoothed pseudo Wigner-Ville distribution" and demonstrates how advanced processing techniques may be necessary to derive reliable data when recordings are performed in the field. Wearable systems to gather data unobtrusively and reliably over extended periods of time A second area of research relevant to the application of wearable technology in physical medicine and rehabilitation concerns the integration of wearable sensors into systems. Following the seminal work by Park and Jayaraman [ 25 ], several researchers relied on conductive fabrics to deliver sensor data to a data-logger and then integrated it into a system that allowed remote access to the data. Other researchers explored the use of wireless technology as a means to relay wearable sensor data to a base station for data recording and remote access to clinically relevant information. Jovanov et al summarize recent advances by their research team toward developing body area networks in the manuscript entitled "A wireless body area network of intelligent motion sensors for computer assisted physical rehabilitation". Key points concerning the use of wireless technology in field monitoring of patients undergoing rehabilitation are the design of low-power transmission devices, the integration of multiple sensors, and the ability of providing processing capability that may reduce the amount of information to be transmitted. These issues are addressed in the above-referenced paper as well as in the manuscript by Sung et al entitled "Wearable feedback systems for rehabilitation". Sung et al describe a platform of wearable sensors recently developed by their team as well as potential applications currently under investigation. Clinical applications of wearable technology in physical medicine and rehabilitation A final set of papers is focused on applications that are relevant to physical medicine and rehabilitation. Sherrill et al describe in their paper entitled "A clustering technique to assess feasibility of motor activity identification in COPD patients via analysis of wearable-sensor data" a method to design classifiers of motor activities such as walking and stair climbing. The proposed technique relies on the examination of small datasets via clustering methods. Measures are derived from clusters associated with different motor activities to evaluate whether the set of wearable sensors and features derived from the recorded data are suitable to reliably identify the motor tasks of interest. Wang and Winters put the information gathered via wearable systems into a clinical context via processing that relies on neuro-fuzzy models. Their paper entitled "A dynamic neuro-fuzzy model providing bio-state estimation and prognosis prediction for wearable intelligent assistants" presents encouraging results indicating that the proposed method can put in the correct context dynamic changes observed in post-stroke individuals undergoing rehabilitation. Wang and Kiryu in their manuscript entitled "Personal customizing exercise with a wearable measurement and control unit" summarize their results on customizing machine-based exercise routines on the basis of physiological data that are continuously gathered from individuals performing such routines. Their results demonstrate the feasibility of a closed-loop system that optimally adapts workload. Dozza et al describe a wearable system designed to reduce body sway in individuals with severe vestibular problems. Their manuscript entitled "Influence of a portable audio-feedback device on structural properties of postural sway" summarizes positive results obtained with a prototype wearable system that utilizes audio-feedback to improve balance. Finally, Mavroidis et al describe how miniature sensor technology can be used to design a new generation of smart rehabilitation devices. Three devices are described in their paper entitled "Smart portable rehabilitation devices": a passive motion elbow device, a knee brace that provides variable resistance by controlling damping via the use of an electro-rheological fluid, and a portable knee device that combines electrical stimulation and biofeedback. These devices combine sensing technology and control strategies to enhance rehabilitation. Conclusion This collection of papers provides an up-to-date description of the state of the art in the field of wearable technology applied to physical medicine and rehabilitation. The field is rapidly advancing and numerous research groups have already demonstrated applications of great clinical relevance. The potential impact of this technology on the clinical practice of physical medicine and rehabilitation is remarkable. A significant shift in focus is possible thanks to wearable technology. While the main focus of clinical assessment techniques is currently on methods that are implemented in the clinical setting, wearable technology has the potential to redirect such focus on field recordings. This is expected to allow clinicians to eventually benefit from both data gathered in the home and the community settings during the performance of activities of daily living and data recorded in the clinical setting under controlled conditions. Complementarities are expected between field and clinical evaluations. Future research will surely address optimal ways to combine these two types of assessment to optimize the design of rehabilitation interventions.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC552335.xml
544542	Neuronal Encoding of Texture in the Whisker Sensory Pathway	A major challenge of sensory systems neuroscience is to quantify brain activity underlying perceptual experiences and to explain this activity as the outcome of elemental neuronal response properties. Rats make extremely fine discriminations of texture by “whisking” their vibrissae across an object's surface, yet the neuronal coding underlying texture sensations remains unknown. Measuring whisker vibrations during active whisking across surfaces, we found that each texture results in a unique “kinetic signature” defined by the temporal profile of whisker velocity. We presented these texture-induced vibrations as stimuli while recording responses of first-order sensory neurons and neurons in the whisker area of cerebral cortex. Each texture is encoded by a distinctive, temporally precise firing pattern. To look for the neuronal coding properties that give rise to texture-specific firing patterns, we delivered horizontal and vertical whisker movements that varied randomly in time (“white noise”) and found that the response probabilities of first-order neurons and cortical neurons vary systematically according to whisker speed and direction. We applied the velocity-tuned spike probabilities derived from white noise to the sequence of velocity features in the texture to construct a simulated texture response. The close match between the simulated and real responses indicates that texture coding originates in the selectivity of neurons to elemental kinetic events.	Introduction One goal of sensory systems neuroscience is to understand how the representations of complex, natural stimuli arise from the basic response properties of neurons. The present experiments explore the representation of textures in the rat somatosensory system. Rats have texture discrimination capacities rivaling those of humans [ 1 ]. In rats, as in humans [ 2 ], object exploration in the tactile modality derives from active palpation. Thus, rats create sensory signals by sweeping their whiskers across surfaces in a rhythmic forward-backward cycle with a frequency ranging from 5 to 15 Hz [ 1 , 3 , 4 , 5 ]. Several hundred primary afferent fibers—“first-order neurons”—innervate specialized receptors on each whisker shaft [ 6 ], and these are excited by whisker movement. Signals travel along the sensory nerve, past the cell body in the trigeminal ganglion, to the brain stem. Here the first synapse is located. The axons of second-order neurons cross the brain midline and travel to the thalamic somatosensory nuclei, where the second synapse is located. Thalamic neurons project to the primary somatosensory cortex, conveying information to layer IV cell populations called “barrels” [ 7 , 8 ]. There have been no reports concerning the cortical or subcortical neuronal activity generated by whisking along irregular surfaces, and the differences in activity associated with two surfaces remain unknown [ 9 ]. However, recent work suggests the framework for a texture coding model. For nonnatural whisker deflections such as ramps [ 10 ], sinusoids [ 11 , 12 ], and temporally unstructured movement [ 13 ], first-order sensory neurons and cortical neurons emit spikes with probabilities that increase in proportion to stimulus velocity. This raises the possibility that neurons represent texture by encoding the kinetics of whisker vibrations. However, key elements of the model are untested. Does whisker movement across different textures produce distinct vibrations? If so, do neurons in the central pathway reliably report these vibrations? Through what coding mechanisms? To answer these questions, the model must be challenged under conditions where the sensory input is precisely controlled and yet resembles what occurs during natural tactile behavior. Guided by this strategy, in anesthetized rats we produced whisker movements across textures while measuring vibrations of the whisker shaft. We then played back the identical vibrations to other rats, and measured the neuronal activity at two stages of the sensory pathway—the first-order neurons that innervate the sensory receptors, and the barrel cortex neurons, which are the first site of cortical integration. Texture discrimination depends on the integrity of the cortical barrels [ 14 ]. By measuring the activity of trigeminal ganglion neurons (the cell bodies of the first-order neurons), we investigated how the sweeping motion of whiskers along a surface is converted to a neuronal impulse code. By measuring activity in the cortex, we explored the neuronal representation that rats rely on to judge the identity of external objects [ 15 , 16 ]. Comparing two levels of the pathway, we reveal the transformation of neuronal signals at successive levels of integration, and show how the neuronal signals emerge from elemental feature extraction. Results Kinetic Signatures of Textures The experimental strategy ( Figure 1 ) was to collect records of the natural movement of whiskers across surfaces ( Figure 1 A) and use them as a stimulus set to probe the neuronal representation of texture ( Figure 1 C). In one group of rats ( n = 3), we electrically stimulated cranial nerve VII, generating 8-Hz whisking movements [ 17 , 18 ] that resemble whisker trajectories in awake rats [ 4 ]. Meanwhile, whisker displacements transmitted to the receptors in the follicle were measured by an optical sensor placed 1 mm from the skin. The vertical and horizontal channels of the sensor ( Video S1 ) reported whisker position with less than 3-μm spatial and 0.13-ms temporal resolution. Movements were measured under different conditions ( Figure 1 B, “texture” column): whisking with no object contact (“free whisk”), whisking on compact disk surface (smooth), and whisking on sandpapers of four different grades: P1200, P400, P280, P100 (from fine-grained to coarse-grained; Table 1 ). The surface was oriented so that the whisker rested on it and remained in contact during the entire whisk trajectory. The proximal edge of the surface was 7 mm from the base of the whisker. The illustrated data ( Figure 1 ) come from whisker C3 in rat EW3. Figure 1 Collection and Playback of Texture Library (A) Whisker vibration data were collected during “electrical whisking,” induced by stimulation of the facial nerve (1) with pulse trains (2) in rat EW3. An optical sensor, shown schematically by two orthogonal light paths (3), monitored vertical and horizontal whisker motion of whisker C3. (B) “Texture” column: Photographs of the 5 surfaces used. “Trajectory” column: Sample whisker trajectories (first whisk of trial 50) associated with free whisking and the five surfaces. Each point, separated by 1 ms, gives the horizontal and vertical position; the trajectory begins with protraction (P) at t = 0 and terminates 125 ms later at the end of retraction (R). Speed is given by the color of each point. Note the irregularities—jumps, stops, and starts—induced by whisking on sandpaper. “Velocity profile” column: Whisker trajectories displayed according to the horizontal and vertical velocities (V H and V V, respectively). P refers to protraction phase (positive V H ), and R to retraction phase (negative V H ). In this and all figures, V H and V V were calculated 7,634 times per second. “Velocity spectrogram” column: Velocity spectrograms for each texture (see Materials and Methods ). (C) Playback of the whisker trajectories to a second group of rats through a piezoelectric motor (4), shown schematically by the horizontal and vertical arrows at the base of the whisker. Table 1 Parameters of the Sandpapers See http://www.fepa-abrasives.org ND, no data Under free whisking conditions the trajectory was a smooth ellipsoid [ 4 ], the principal axis aligned with protraction and retraction movements (P and R, respectively, in Figure 1 B, “trajectory” column). As the rat whisked on the compact disk surface, the trajectory was similar but covered a more restricted vertical range. In contrast, whisking across grainy surfaces produced irregularities in the trajectory, and each texture was associated with a characteristic whisker shaft vibration. These distinct “kinetic signatures” are evident in the velocity profile—that is, the temporal sequence of velocity features across the course of a whisk ( Figure 1 B, “velocity profile” column). Each velocity profile covers 125 ms (one complete forward and backward whisk) and consists of two histograms—horizontal (V H ) and vertical (V V ) velocity. For V H , whisker protraction (forward movement) is positive and whisker retraction (backward movement) negative. For V V , upward movement is positive and downward movement negative. To better visualize the time-varying frequency content of the velocity profiles, the velocity spectrograms are also plotted ( Figure 1 B, “velocity spectrogram” column). The spectrograms were formed by computing the magnitude of all sinusoidal components (0–500 Hz) of the velocity profile in a 6-ms wide window, and then sliding the window with 0.13-ms time steps (see Materials and Methods ). The velocity profile and the velocity spectrogram, taken together, illustrate the kinetic features that make each texture unique—the duration and frequency content of each velocity peak, as well as the number of peaks and the temporal spacing between them. In a different group of rats, the texture-induced vibrations were played back to the base of a whisker ( Figure 1 C and Video S2 ) and neuronal activity was recorded. Construction of the stimulus sequence off-line allowed us to smoothly “stitch together” the vibrations: The transitions between textures and free whisks were always inserted at time zero, corresponding to the point of maximal retraction, when whisker velocity was zero. The replay was an accurate replica of the motion recorded during electrical whisking ( Figure S1 ). Receptor and Cortical Coding Properties The physiological dataset (seven rats) consists of six first-order neuron recordings, five cortical cluster recordings, and seven “paired” recordings—simultaneous first-order neuron and cortical cluster. The principal result is that time-varying neuronal activity in the trigeminal ganglion and cortex captured the kinetic features of the texture-induced vibrations. From the same texture library given in Figure 1 (rat EW3, whisker C3), Figure 2 A gives the velocity profile, averaged across 100 trials, for two free whisks (−250 to 0 ms) followed by two whisks on P280 sandpaper (0 to 250 ms). Note the distinct kinetic signatures of whisker movement: Unlike free whisking, the coarse (P280) sandpaper caused irregular bursts of high and low velocity, particularly during whisker retraction. The response of one first-order neuron (named Zurvan) is shown in Figure 2 B as a raster plot of 100 trials and in Figure 2 C as a peristimulus time histogram (PSTH). Several coding properties are evident: (i) The first-order neuron fired a greater number of spikes for the coarse texture than for free whisks; (ii) spikes were closely aligned to instants in which the whisker moved at high velocity (blue arrowheads); (iii) it fired in a reproducible manner across trials—the spikes were aligned; and (iv) it was selective to whisker retraction (did not fire for high-velocity protractions; red arrowhead). Figure 2 Sensory Receptor and Cortical Coding Properties (A) V H and V V for two free whisks followed by two P280 whisks. The labeling conventions are as in Figure 1 B. Each presented trial was unique due to small variations in whisker trajectory even on the same surface ( Figure 7 ); the illustrated velocity profiles are the averages of 100 trials. The red arrowhead indicates the time of the first V H peak during whisker protraction on P280; blue arrowheads indicate the times of the three V H peaks during whisker retraction on P280. (B) Raster plot of first-order neuron aligned with the whisker trajectories, in response to 100 unique trials. Stimuli were applied to whisker E4. (C) PSTH of first-order neuron with 0.2-ms bins. Blue arrowheads indicate the times of maximum response to the three peaks in retraction velocity. The red arrowhead indicates the expected time of response to the peak in protraction velocity; however, the neuron did not respond to whisker protraction. (D) Raster plot for the cortical neuron cluster recorded simultaneously with the first-order neuron. (E) Two cortical PSTHs, both with 2-ms bins. The upper PSTH corresponds to the raster plot in (D); the lower PSTH is from a second cortical neuron cluster recorded simultaneously at a neighboring electrode (distance 560 μm). Blue and red arrowheads indicate the times of maximum response to the peaks in whisker protraction and retraction velocity, carried down from (A). The cortical neuron clusters responded to high velocities for both protraction and retraction. Because the first two peaks in retraction velocity were separated by just 7 ms, the resulting peaks in cortical response were fused. All PSTHs are extended to 260 ms to show responses to the final velocity feature. Two barrel cortex neuron clusters were recorded simultaneously with the first-order neuron, allowing direct comparison of different stations along the sensory pathway. Figure 2 D shows the raster plot for one of the cortical clusters, while Figure 2 E shows PSTHs for both cortical clusters. Like the first-order neuron, the cortical clusters responded to high velocities (arrowheads, Figure 2 A, 2 C, and 2E) and, as a result, fired a greater number of spikes for P280 sandpaper than for free whisks. Key differences from the first-order neuron are clear: (i) The cortical neuron clusters fired in a less reproducible manner across trials—there was more variability in the number of spikes per whisk and in the temporal alignment of spikes; and (ii) they fired for both whisker protraction and retraction (red and blue arrowheads, respectively). The selectivity of first-order neurons and cortical clusters for the direction of whisker movement is described in more detail in Figure 3 . Figure 3 Directional Selectivity in First-Order and Cortical Neurons (A) Mean spike count per whisk for ten first-order neurons separated into protraction and retraction phases. Responses to free-whisk and all textures were combined, giving a total of 8,000 whisks. First-order neurons are arranged from left to right according to their retraction:protraction spike count ratio. Five first-order neurons preferred retraction, three preferred retraction, and two responded to both phases. Principal whisker of each neuron is indicated. The neuron Zurvan is indicated by an asterisk. (B) Same analysis for 12 cortical clusters. Individual cortical neuron clusters did not present a clear preference for either retraction or protraction. Conclusions about single unit directional selectivity cannot be drawn, however, because the directional selectivity of any cluster must always be less than that of the most selective single unit in the cluster. The neuron cluster ( Figure 2D, 2E ) recorded simultaneously with Zurvan is indicated by an asterisk. Texture Coding by Firing Rate To permit sensory discriminations, some properties of neuronal firing must vary systematically from texture to texture. Earlier work [ 11 , 12 ] showed that neuronal firing rate, in response to sinusoidal whisker movement, is dictated by mean vibration speed, proportional to the product of amplitude and frequency, Xω (referred to in previous publications as Af ). The generalization of Xω to the natural, texture-induced vibration is 〈\| X(ω,τ) \| ω 〉 Ω,Τ , a quantity known as “equivalent noise level” (see Materials and Methods ). In Figure 4 , we compare response magnitude for the full population of first-order and cortical neurons to each texture's equivalent noise level. The Pearson correlation coefficient between equivalent noise level and spike count was 0.93 for the first-order neurons and 0.99 for the cortex. This finding indicates that neuronal spike count is a function of the magnitude of the composite frequency components of the whisker vibration, whether the stimulus is a simple sinusoid (where all the power is at a single frequency) or a complex, texture-induced vibration. Figure 4 Texture Coding by Firing Rate Equivalent noise level (plus SD) of texture-induced vibrations averaged across 100 trials of 500 ms each (see Materials and Methods ). Average spike count per trial (plus SD) pooled from ten first-order neurons and 12 cortical neuron clusters. Note separate scales for spike counts of neurons recorded in Ganglion and Cortex. Texture Coding by Firing Patterns Since different sandpapers can induce vibrations with similar equivalent noise levels and thereby evoke similar spike counts (e.g., P400, P280, and P100 in Figure 4 ), we must expect additional texture coding mechanisms to be at work. We therefore examined more closely the neurons shown in Figure 2 , on the hypothesis that spike patterns might carry texture-specific information. V H and V V profiles associated with two whisks on each texture are plotted ( Figure 5 A), together with PSTHs for the first-order neuron ( Figure 5 B) and the cortical neuron cluster ( Figure 5 C). Textures evoking similar spike counts due to similar equivalent noise levels were readily distinguished by spike patterns. The patterns arose from the alignment of spikes to the velocity profile of the input vibration. An assessment of spike alignment to other stimulus features (whisker position and whisker acceleration) is given in Figure 6 and indicates that these features were reported less reliably than whisker velocity. The first-order neuron reported the velocity profile for whisker retraction, while the cortical neuron cluster reported both protraction and retraction profiles, albeit with lower fidelity to individual velocity features. Figure 5 Texture Coding by Firing Patterns (A) V H and V V for two whisks on texture P400 (left), P280 (middle), and P100 (right). Each illustrated velocity profile is the average of 100 unique profiles. (B) First-order neuron PSTHs (0.2-ms bins) aligned with the whisker trajectories. (C) Cortical PSTHs (2-ms bins). PSTHs are extended to 260 ms. The arrowheads on the left side of PSTHs indicate mean firing rates. Figure 6 Test for First-Order Neuron Encoding of Position and Acceleration To investigate whether first-order neurons represented stimulus features other than velocity, we repeated the same analysis as in Figure 5 , in relation to whisker position (A) and acceleration (B), because it has been suggested that neuronal activity is determined by these stimulus parameters [ 10 , 13 ]. Alignment between the PSTH (C) and stimulus position or acceleration revealed no consistent correlation. For texture P100, the boxes extending across A, B, and C highlight the absence of correlation. For example, two periods with similar positions produced first no spikes (red-outlined box on left) and then a large response (red-outlined box on right). Moreover, high acceleration (left box) produced no spikes, while lower levels of acceleration (right box) produced a large response. For this neuron, only velocity was encoded. Sources of Neuronal Variability In a number of sensory modalities, first-order neuron responses can be remarkably reliable when a stimulus is presented repeatedly [ 19 ], whereas cortical responses vary across trials [ 20 ]. It is of interest to elucidate the mechanisms that permit reliable first-order neuron responses and, by the same token, to identify the sources of trial-to-trial variability among cortical neurons. In the data shown so far, the 100 trials for a given texture were composed of 400 unique whisks (four whisks per trial). Each whisk differed in the minute details of its trajectory ( Figure 7 ). To discover the origin of neuronal variability, we selected trial 50 for each texture and repeated the four-whisk sequence 100 times. If neuronal variability originates purely in stimulus variability, it will disappear across repeated trials; variability due to internal brain fluctuations, however, will remain. Figure 7 Velocity Profile Variability across Trials Ten successive trials are shown (numbers 46–55), each trial composed of the final two free-whisks (–250 to 0 ms) and the first two whisks on P280 (0 to 250 ms). Free whisk velocity profiles varied little across trials. When the whisker swept across P280 repeatedly, the fundamental kinetic signature was conserved (e.g., the three peaks in retraction velocity for P280) but minute details of the profile varied—note, for example, the velocity event (red asterisk) that occurred uniquely on trial 50. The velocity profile for the final two free whisks (−250 to 0 ms) and the first two P280 whisks (0 to 250 ms) of trial 50 is given in Figure 8 A. The response of the first-order neuron is shown as a raster plot ( Figure 8 B) and a PSTH ( Figure 8 C). These can be compared to responses of the same cell in Figure 2 B and 2 C. Response was nearly identical on each trial, because of the precise temporal alignment of spikes on the high-velocity events. Indeed, some stimulus features evoked 0.7–0.8 spikes per bin per trial, meaning that neuronal jitter fell within the 0.2-ms PSTH bin size. Figure 8 Sources of Neuronal Variability (A) V H and V V across the final two free whisks and the first two P280 whisks of trial number 50. Here, as in Figure 2 , the red arrowhead indicates peak whisker velocity during protraction, and blue arrowheads indicate the peak whisker velocities during retraction. (B and C) First-order neuron raster plot (B) and PSTH (C), aligned with the whisker trajectories, for 100 stimulus repetitions. Due to the temporal precision of neuronal responses, the vertical scale of the PSTH has been altered (compare to Figures 2 and 5 ) to reflect the large numbers of spikes within single bins. (D) Cortical neuron cluster raster plot. (E) Two cortical PSTHs from activity recorded simultaneously with the first-order neuron. The upper PSTH corresponds to the raster plot in (D); the lower PSTH is derived from a second cortical neuron cluster recorded simultaneously at a neighboring electrode (distance of 560 μm). PSTHs have 0.2-ms bins for the first-order neuron and 2-ms bins for the cortical neuron clusters. All PSTHs are extended to 260 ms to show responses to the final velocity feature. Response peaks are signaled by red and blue arrowheads according to the velocity events that evoked them. In Figure 8 D and 8 E, the cortical response to repeated trials is presented. Direct, quantitative comparisons between the variability of first-order responses and that of cortical responses cannot be made, because the cortical recordings were made from multi-neuron clusters. However, the cortical response to repeated trials can be compared to the same cluster's response to 100 unique trials in Figure 2 D and 2 E. Eliminating trial-to-trial variability in the timing of stimulus features reduced but did not eliminate neuronal jitter. The remarkable response locking of first-order neurons to stimulus features is further highlighted in Figure 9 . Unlike Zurvan, the illustrated neuron was selective to whisker protraction. For texture P280, the velocity histograms ( Figure 9 A) contained well-separated peaks during protraction. With 100 repetitions of trial number 50, the raster plot ( Figure 9 B) and PSTH ( Figure 9 C) yielded discrete response peaks aligned with high-velocity protraction events. Applying the green horizontal line to the PSTH as a threshold, we extracted six separable response clusters. Every cluster contained exactly 100 spikes resulting from one spike per trial for each velocity event. The final response was evoked by a clear protraction event (red asterisk in Figure 9 A) and was selected for closer inspection (red inset). Here, the 100 spikes spanned a range of 0.38 ms; standard deviation (SD) in spike time was 0.08 ms. For the five preceding response clusters, spike time SDs were 0.13, 0.09, 0.09, 0.13, and 0.11 ms. The minimum measured spike time SD in the dataset was 0.07 ms. These must be taken as underestimates of spike time precision, given that they include measurement noise inherent to the recording system (e.g., the SD in spike time caused by digitizing the action potential threshold crossing time at 30 samples per ms is nearly 0.02 ms). Figure 9 Precision of a First-Order Neuron (A) V H and V V across the first two P280 whisks of trial number 50 (see Figure 7 ). (B and C) Raster plot (B) and PSTH (C) of the first-order neuron for 100 repetitions of the stimulus given in (A). Inset in red frame shows a magnified view of spikes emitted in response to a single velocity event (red asterisk in [A]) and their SD in time. The same measurement of jitter was carried out for each of the response peaks that surpassed the green horizontal line (see text). From these observations we conclude that, under our experimental conditions, the trial-to-trial response variability of first-order neurons is caused exclusively by stimulus jitter, whereas that of cortical neurons results mainly from variations across time in sensory integration, and must emerge at some integration site between the trigeminal ganglion and cortex. A question of current interest is whether the variability in cortical responses results from noise and imprecision in neuronal integration [ 20 ], or else reflects functionally significant modulations in responsiveness [ 21 , 22 , 23 ]. From Response Properties to Natural Responses We hypothesize that the firing patterns of first-order and cortical neurons during presentation of textures can be explained by their extraction of elemental features from the complex input signal, and that these elemental features are bursts of high velocity. To test this directly, we presented a “white noise” stimulus in which the two stimulus features V H and V V varied randomly across time. Responses to noise stimuli allowed us to quantify velocity sensitivity and then to generate simulated spike trains based on the sequence of velocity events in the actual texture-induced vibrations. Finally, comparison between simulated and observed responses reveals the extent to which the responses to natural stimuli are explained by neuronal selectivity to velocity features: If simulated responses closely resemble real responses, we can conclude that neurons are in fact operating on natural texture stimuli according to their tuning to elemental kinetic events. Typically, neuronal tuning curves are mapped out using an “unbiased” stimulus—a stimulus that avoids the temporal correlations present in natural stimuli. The first step, therefore, was to map out how first-order and cortical neurons encode whisker kinetic features when these features are extracted from the context of the natural stimulus. We applied a stimulus that varied randomly in velocity—Gaussian velocity noise—and therefore was not constrained by the velocity patterns present in texture trajectories (see Materials and Methods ). Figure 10 A tracks V H and V V (white circles) across 5 ms of filtered white noise. Figure 10 Velocity Tuning Curves and Simulated Texture Responses (A) A 5-ms trajectory of velocity white noise. Radial coordinates give V H , V V . Velocity space was subdivided such that each segment included the same number of events (3,435,300). One segment (red outline) is selected for further explanation (see text). (B) 100-ms ganglion and cortical spike train aligned below occurrences of the velocity event of interest (red bar). After each such event, spike times were accumulated to build up a spike probability profile. (C) First-order neuron spike probabilities, given by color scale, in relation to joint A,R events. To estimate the tuning curve in finer detail, the number of angles was increased to 20. Each segment now contains about 1,374,120 velocity events. One P280 whisk trajectory is superimposed. (D) Spike probabilities for cortical neuron cluster, given by color scale, in relation to joint A,R events. One P280 whisk trajectory is superimposed. (E) Simulated raster plot for first-order neuron and simulated (black) and real (red) PSTHs. (F) Simulated raster plot for cortical neuron cluster and simulated (black) and real (red) PSTHs. We then constructed firing probability profiles in relation to millions of occurrences of each velocity event, such as the velocity event in the fifth angular sector and ninth radial sector, or A 5 ,R 9 (red outline in Figure 10 A). Figure 10 B shows how response probabilities were constructed in relation to this particular velocity event. The first trace shows the occurrences of A 5 ,R 9 across a 100-ms window—the first occurrence of the event (asterisk in Figure 10 A) corresponds to the crossing of A 5 ,R 9 in Figure 10 A (also marked by an asterisk). Below, first-order sensory neuron and cortical spike times are shown. After 10 min of stimulus noise, the postevent spike probability profiles associated with A 5 ,R 9 and all other events could be constructed. For the first-order neuron Zurvan, spike probabilities in the 1–2-ms postevent interval are given in Figure 10 C for all joint events ( A,R ). To construct the neuron's “tuning curve” in finer detail, velocity space was subdivided into 20 angular and 10 radial segments. The neuron emitted spikes with increasing probability as velocity increased, but only for restricted directions, preferring high speeds that combined retraction (negative horizontal velocity) and upward movement (positive vertical velocity). For the simultaneously recorded cortical neuron cluster, spike probabilities in the 5–20-ms poststimulus interval are given in Figure 10 D; like the first-order neuron, the cortical cluster emitted spikes with increasing probability as speed increased, but its directional selectivity was less pronounced and was radially symmetric. Can the neurons' responses to complex, natural stimuli be explained as the outcome of these elemental tuning properties? To find out, we projected whisk velocity trajectories upon the white noise-derived tuning curves. One whisk (first whisk of trial 50) on P280 sandpaper is depicted on both tuning curves. The first observation is that the intersection of the velocity trajectory with the tuning curves explains why the first-order neuron was selective for whisker retraction while the cortical cluster was directionally nonselective (see Figures 2 and 5 ). Figures 10 E and 10 F show the simulated responses of the first-order neuron and cortical cluster to two P280 whisks, delivered 100 times. The simulation was of 100 unique trials (see Figure 2 ) rather than repeated trials (see Figure 8 ). Thus, on each trial the minute details of the whisks gave rise to a unique sequence of P ( t ) and a corresponding raster plot for that trial. The 100-trial raster for one run of the simulation was summated to form a PSTH. The close match between the simulated PSTHs (black) and the real PSTHs (red lines, reproduced from Figure 2 C and 2 E) indicates that the real responses to natural stimuli could be explained by neuronal selectivity to velocity features. The Pearson correlation coefficients between the predicted and observed PSTHs were 0.94 for the first-order neuron and 0.84 for the cortical cluster. Because these correlation values fall within the range obtained by comparing two real PSTHs generated from separate sets of 50 trials, we conclude that simulated spike trains are as similar to real spike trains as real spike trains are to each other. Thus, the velocity feature extraction properties of the neurons are sufficient to explain texture responses. Discussion Texture coding appears to derive from two fundamental processes: First, the whisker transmits a “kinetic signature” of the palpated surface to the receptors in the follicle. Second, the first-order neurons relay to the whisker region of cortex (through intervening stations) precise information about the kinetic features transmitted to the follicle. One kinetic feature is the “equivalent noise level”: Spike counts per whisk both for first-order neurons and for cortical neurons are proportional to the equivalent noise level of the texture-induced vibration. Thus, when texture vibrations differ in this quantity, neuronal spike counts also differ and thereby carry information that could, by itself, separate the textures. By the same token, when texture vibrations have similar equivalent noise levels, spike counts per whisk appear not to carry sufficient information. The second kinetic feature, then, is the temporal sequence of velocity events—distinctive velocity profiles induce distinctive temporal patterns in the spike trains with spike alignment of better than 0.2 ms in the first-order neurons and a few ms in the cortex. The stimulus playback method used here might not produce the identical input to the sensory receptor as occurs during active whisking [ 18 ]. The optical sensor at the base of the whisker did not register the bending nor tension (pulling) of the whisker. Moreover, active muscle contractions might affect sensory receptors at the interface between the whisker shaft and the inner membrane of the follicle. Thus, additional information related to surface texture might be available to the sensory system. In the present dataset, even after the possible loss of some texture-dependent information due to passive stimulation, the neuronal responses afforded a high degree of discriminability. We interpret the dataset as showing that vibration patterns, by themselves, must be a fundamental feature supporting the neuronal coding for texture. This awaits confirmation in experiments in actively whisking rats. A classical approach to investigating sensory coding is to map the relationship between well-controlled artificial sensory stimuli and evoked neuronal activity. This can provide a complete description of neuronal feature extraction properties [ 24 , 25 ], but it sheds little light on the brain activity underlying normal perceptual experiences. Moreover, the processing mechanisms that have evolved to extract behaviorally relevant information may operate inefficiently during artificial stimulation [ 26 ]. Another approach [ 27 , 28 ] is to measure neuronal activity during natural stimuli (i.e., visual scenes or animal calls). Here, the drawback is that the features evoking spikes during natural stimulation can be multidimensional, complex, and difficult to quantify, offering only limited insight into sensory processing mechanisms [ 27 ]. In principle, one can bridge the gap between artificial and natural stimuli by (i) measuring neuronal activity during ecologically relevant stimuli, stimuli that are collected from an animal's normal interaction with the environment, (ii) constructing tuning curves under artificial stimulation (usually white noise), and (iii) applying the tuning curves to the natural stimuli to test whether they account for the observed response. Because artificial stimuli only partially cover dimensions of stimulus space present in natural stimuli, and because of neuronal nonlinearity, this procedure typically provides neuronal simulations that match real neuronal output with a correlation of less than 0.5 [ 27 , 29 , 30 ]. Yet, the present experiments followed this same procedure and generated simulated PSTHs that were highly correlated with real responses (first-order neuron, 0.94; cortex, 0.84), approaching the upper bound set by the trial-to-trial variability that limits the correlation even between two real PSTHs. The simulations were successful for two reasons. First, recent work [ 11 , 12 ] uncovered the critical physical dimension—velocity—encoded by neurons. Second, receptor and cortical stimulus integration is linear to a first approximation—ongoing responses depend upon an integration process where preceding events affect the neurons independently of one another. As an alternative to the temporal model outlined here, a spatial model for texture coding has recently been proposed. It begins from the observation that whisker length varies systematically across the anterior-posterior dimension of the rat's snout [ 31 ]. When stimulated near the distal end, the posterior whiskers resonate at lower frequencies than do the shorter, anterior whiskers [ 32 ]. If different textures cause systematically different vibration frequencies, there might be texture-specific differences in the magnitude of vibration of posterior versus anterior whiskers [ 33 ]. Because whisker position is relayed in a somatotopic manner to the cerebral cortex [ 7 ], texture could be encoded by the location of the focus of activity in barrel cortex, much like frequency is encoded by the tonotopic organization of the cochlea and the auditory cortex. The resonance frequency hypothesis predicts that rats would fail to discriminate between surfaces using just a single whisker, yet they have been shown to perform texture discriminations after progressive clippings down to one or two whiskers [ 34 ]. Our results help explain the behavioral findings by emphasizing that, although additional information might be available due to differences in the mechanical properties among whiskers, even a single whisker can transmit large amounts of texture-specific information to its central neural circuits. This occurs because of the match between the feature of the whisker output signal that best distinguishes one texture from another (the kinetic signature of the vibration) and the tuning properties of first-order neurons. Cortical neurons conserve this kinetic signature in their firing patterns. Materials and Methods Recording the texture library and construction of the stimulus set Experiments were conducted in accordance with NIH and institutional standards for the care and use of animals in research. Subjects were ten adult male 250–350-g Wistar rats. In one set of anesthetized rats (urethane, 1.5 g/kg), “electrical whisking” [ 17 , 18 ] was generated by stimulating the right facial nerve (see Figure 1 A) with 1–2-V pulses of 100 μsec at 200 Hz for 60 ms to produce whisker protraction, followed by a passive 65-ms whisker retraction. For a selected whisker, horizontal and vertical movements at the base were registered by a two-channel optical sensor, each channel consisting of an LED light source and phototransistor ( Video S1 ). The two voltage signals were digitized (7,634 samples per second). Whisker movement was studied for 10 min under each of six conditions (see Figure 1 B). The angle traversed at the whisker base, averaged across all trials and all textures, was 25 degrees (SD ± 1 degree). Average translation across the edge of the textured surface was 3.08 mm (SD ± 0.14 mm). For each texture, a 50-s continuous record was extracted and sliced into 100 trials of 500 ms, each trial composed of two-dimensional position signals across four 125-ms whisks. For free whisks, a 250-s record was sliced into 500 unique trials. The stimulus set was constructed by splicing trials together at the point of maximum retraction (V H = 0), avoiding the introduction of any position or velocity discontinuity. A free whisk trial always separated two successive texture trials. A block was composed of five different texture trials (t 1–5 ) with free whisk trials (fw) interspersed, e.g., fw-t 3 -fw-t 5 -fw-t 1 -fw-t 2 -fw-t 4 . Before stimulus delivery, signals were low-pass filtered at 500 Hz. All data were manipulated in MATLAB software ( http://www.mathworks.com ). Analysis of vibration kinetics We sought to quantify the kinetic features that characterized each texture-induced vibration. We refer to the whisker trajectory in one dimension as x(t) and the whisker velocity profile as For each texture, we computed the spectrogram \| V(ω,τ n ) \| of the velocity profile where Δt is the 6-ms interval within which each spectrum was computed and τ n represents the series of N sequential time windows. Thus, each spectrogram (see Figure 1 , “velocity spectrogram” column) was composed of N = 906 spectra. Considering the duality between the direct and inverse Fourier space we can substitute Equation 1 into Equation 2 , to rewrite Equation 2 as Note that \|X ( ω,τ n )\| also corresponds to the spectrogram of the trajectory in position. Previous studies have shown that, when the stimulus set consists of sinusoidal whisker movements, the spike count per stimulus for cortical neurons is proportional to the product of the sinusoid's amplitude and frequency, Xω [ 11 , 12 ]. To test whether this coding principle extends to natural stimuli, we generalized the measure of Xω to the texture-induced vibration. X becomes a time-varying spectrum X(ω,t) where T is the entire time domain and Ω is the entire domain of frequency ω. This quantity—known as the “equivalent noise level”—represents the average amplitude of white noise velocity that dissipates the same average power as the signal of interest. It serves to characterize the entire kinetic signature by a single quantity, equivalent to mean value of the product Xω across all time intervals and all values of ω . Our experiments measured velocity in two dimensions, V H and V V . Equations 1–5 were generalized to a second dimension by adding, for each time window τ n , the separately measured spectrograms for the two dimensions. The “velocity spectrogram” column in Figure 1 B gives two-dimensional spectrograms as above. Similarly, in Figure 4 the equivalent noise level was calculated in two dimensions, averaged across 100 trials of each texture vibration, and then plotted in relation to neuronal spike count. Measurement of neuronal responses In a second set of urethane-anesthetized rats, neuronal recordings were made simultaneously from two sites. First-order neurons were recorded by advancing a single electrode (FHC, Bowdoinham, Maine, United States; http://www.fh-co.com ) into the right trigeminal ganglion according to stereotaxic coordinates. Cortical recordings were obtained by inserting a 100 microelectrode array (Cyberkinetics, Foxborough, Massachusetts, United States; http://www.cyberkineticsinc.com ) to a depth of 700–1,000 μm in the left barrel cortex [ 35 , 36 ]. Ganglion recordings were always single units, whereas cortical recordings consisted of a multiunit cluster at each channel. The principal whiskers (receptive field centers) in the first-order-only recordings were C3, E1, D6, E6, and γ (twice). The principal whiskers in the cortex-only recordings were A1, B4, E5, and E3 (twice). The principal whiskers in the paired first-order neuron-cortex recordings were δ (twice), E3 (twice), C2 (twice), and E4. Texture stimuli were delivered to a single whisker using a motor constructed from two orthogonal pairs of parallel piezoelectric wafers driven independently by horizontal and vertical signals ( Video S2 ). The whisker was inserted into a metal tube (0.33 mm inner diameter) with opening 1 mm from the skin. By optically monitoring the whisker shaft, we verified that movements precisely reproduced the previously recorded signals ( Figure S1 ). The second set of rats thus received whisker vibrations identical to those previously recorded during active whisking in the first set of rats. Construction of neuronal tuning curves and response simulations We selected V H and V V independently from a Gaussian distribution 7,634 times per second. The stimulus was then low-pass filtered (Chebyshev type II) at 500 Hz so as to not exceed the physical capacities of the piezoelectric wafer stimulator (instantaneous reversals of velocity cannot be achieved by any device). The noise stimulus was presented for 10 min after conclusion of the texture stimuli. We adopted a method of “forward correlation” between stimulus and response where, for all occurrences of a particular stimulus event, the ensuing neuronal spike trains were averaged to construct a response probability profile for that event. This required subdividing velocity space into discrete segments. In Figure 10 A, velocity space was partitioned into eight 45-degree angular sectors ( A 1–8 ) and ten radial sectors ( R 1–10 ). At each time point, angle corresponds to the direction of whisker movement, while radial distance corresponds to instantaneous speed. The positions of the radial boundaries were chosen to make each segment contain an equal number of instantaneous velocity events: Because velocity had a Gaussian distribution with a mean of zero, high-velocity events were less common, and consequently the radial boundaries were increasingly widely spaced as distance from zero increased. To test whether the complex, texture-induced spike patterns followed directly from the tuning curves, a more elaborate analysis was necessary. Each instantaneous velocity ( A,R ) during the whisk gave an ensuing spike probability profile. To simulate a spike train, a spike was generated in each time bin t (size 0.13 ms) with probability P ( t ), given by the average of the overlying spike probabilities associated with velocity events in the time window before t (time window was 1–2 ms before t for first-order neurons and 5–20 ms before t for cortical neurons). After completion of the simulation, P ( t ) was normalized so that the overall simulated spike count matched that in the real data; this normalization did not affect the temporal profile of the simulated PSTH. Supporting Information Figure S1 Comparison of Recorded and Played Back Whisking Trajectories The whisker movements presented as sensory stimuli were accurate reproductions of the movements recorded during electrical whisking in other rats. (A) Trajectories of two whisks (only horizontal channel shown) recorded during contact with sandpaper P280. (B) To test playback, a whisker was inserted in the piezoelectric motor guide tube and whisker displacements were measured by the optical sensor placed adjacent to the insertion point of the whisker. The trace shows recordings of the playback of the same two whisks of part A. (C) Magnified view of the traces indicated in the rectangle in (A) and (B). (161 KB PDF). Click here for additional data file. Video S1 Two-Dimensional Optic Sensor and Electrical Whisking Each optic sensor channel consisted of a pair of light tubes (one is a light source, the other leads to a photodiode). The two channels were mounted normal to each other in a metallic ring. Electrical stimulation of cranial nerve VII with 1-V pulses of 100 μs at 200 Hz for 60 ms produced whisker protraction that was followed by a passive 65-ms whisker retraction. The film shows four 125-ms free whisks in the air (8-Hz whisking), consisting of protraction (right to left) and retraction (left to right). Recorded at 500 frames per second with a Motion Scope 500 digital camera (Redlake, San Diego, California, United States; http://www.redlake.com ). Rat EW3, whisker C3. (9.1 MB MOV). Click here for additional data file. Video S2 Texture Playback with the Two-Dimensional Piezoelectric Motor The motor consists of two pairs of piezoelectric wafers with axes meeting at a mobile joint. The whisker to be stimulated was placed inside the metal tube, which is orthogonal to the joint. The film shows playback of four 125-ms free whisks in the air, consisting of protraction (left to right) and retraction (right to left). Recorded at 500 frames per second. (9.0 MB MOV). Click here for additional data file.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544542.xml
552321	Malaria and urbanization in sub-Saharan Africa	There are already 40 cities in Africa with over 1 million inhabitants and the United Nations Environmental Programme estimates that by 2025 over 800 million people will live in urban areas. Recognizing that malaria control can improve the health of the vulnerable and remove a major obstacle to their economic development, the Malaria Knowledge Programme of the Liverpool School of Tropical Medicine and the Systemwide Initiative on Malaria and Agriculture convened a multi-sectoral technical consultation on urban malaria in Pretoria, South Africa from 2nd to 4th December, 2004. The aim of the meeting was to identify strategies for the assessment and control of urban malaria. This commentary reflects the discussions held during the meeting and aims to inform researchers and policy makers of the potential for containing and reversing the emerging problem of urban malaria.	Introduction Africa's population will almost triple by the year 2050. This expansion will occur primarily in urban areas and by 2025, 800 million people will live in urban communities. Especially affected will be West Africa, where the urban population annual growth rate of 6.3% is more than twice the rate of the total population growth. Today in the humid forest zone, more people live in cities than in rural areas and in twenty years time, two out of three West Africans will live in urban centres. While many of Africa's health problems are common to both urban and rural environments, recognizing and meeting the public health challenges in these growing cities is becoming increasingly urgent. Malaria has been considered a predominantly rural disease in Africa, primarily because suitable vector breeding sites are scarce in highly populated areas. Yet, although studies have shown that Anopheles mosquito breeding decreases with increasing proximity to the centre of urban areas [ 1 - 3 ], transmission of malaria still occurs. Clearly, the complex factors contributing to malaria risk in urban areas are not fully understood [ 3 ] but evidence is rapidly accumulating that the urban poor are at far higher risk from malaria than previously acknowledged [ 4 , 5 ]. The Malaria Knowledge Programme of the Liverpool School of Tropical Medicine and the International Water Management Institute/ Systemwide Initiative on Malaria and Agriculture convened a meeting in Pretoria 2 nd -4 th December 2004 to develop an evidence-based approach for evaluating and controlling urban malaria. Participants were drawn from seven sub-Saharan countries, Europe, North America and South Asia (see additional file). Recognizing the need for extensive cross-sectoral involvement and collaboration in dealing with the challenge of urban malaria, representatives from the research/ academic, NGO, development, policy-making and donor communities co-operated in the process to identify key knowledge gaps and opportunities for control. Included in the group were sociologists, clinical epidemiologists, entomologists and control specialists. Discussion Identifying the populations at risk in urban areas Urbanization is a recent phenomenon in Africa: in 1960 there were no African cities with one million inhabitants, today there are forty. Has malaria become a serious problem within these huge cities and their peri-urban environs? Data presented from studies in a number of sub-Saharan African cities (Brazzavile, Congo; Dakar, Senegal; Abidjan, Cote d'Ivoire; Cotonou, Benin; Ouagadougou, Burkina Faso; Dar es Salaam, Tanzania, and Accra and Kumasi, Ghana) showed clearly that malaria is a considerable urban health problem in Africa. The studies demonstrated great heterogeneities in malariometric indices both between and within cities. It was recognized that not only the major cities of Africa, but also many medium sized regional towns, home to a large proportion of the Africa population, have considerable levels of malaria [ 5 ]. With malaria risk unevenly distributed across urban environments, interventions must be preceded by the identification and prioritization of the most vulnerable. Vulnerability is not simply the result of low socio-economic status [ 6 ], although this is often a major contributory factor, but reflects factors beyond the individual level such as the proximity of the household to sites of urban agriculture or environmental/cultural factors working at the community level. Discussion focussed on research to define this risk, to improve access to correct diagnosis and appropriate treatment and effective preventative measures, and to identify accurate monitoring and evaluation tools tailored to the urban context. Prioritizing improved diagnosis and treatment for the vulnerable Misdiagnosis of malaria is a serious problem everywhere, but in areas of low malaria endemicity presumptive treatment of all fevers as malaria can result in over 75% of cases being misdiagnosed as malaria [ 7 ]. The effect of malaria misdiagnosis on the vulnerable will result in more ill health due to delayed diagnosis and repeat visits, overburdened health services, more severe malaria, loss of faith in health services, increase in real and perceived malaria resistance, chronic disease secondary to untreated infection, increased cost to patient and to health facilities and consistent misdiagnosis that will encourage detrimental health-seeking behaviour [ 7 ]. Effective provision of appropriate treatment also remains a serious challenge in urban settings. The Abuja Declaration stated that by 2005 "At least 60% of those suffering from malaria have prompt access to and are able to use correct, affordable and appropriate treatment within 24 hours of onset of symptoms." Despite the fact that access to quality health care is better on average in urban compared to rural zones, the formal public health facilities are often the last source of treatment used along the pathway to cure. Often malaria care initially involves leftover medicines from the home (from previously incomplete malaria or other treatment regimes), the purchase of cheaper herbal medicines or unprescribed conventional medicines. The problems of obtaining treatment from a health facility may be exacerbated by the need to obtain permission from an authority figure, absence from work and loss of income, the need to raise money to fund both the treatment and associated costs such as travel [ 6 ]. As a result, in Africa over 70% of malaria episodes in rural and over 50% in urban areas are self-diagnosed and self-treated [ 8 ]. With Home Management of Malaria proposed as an integral part of the Roll Back Malaria strategy, the consequences of presumptive treatment policies for malaria in the context of the introduction of newer and more expensive anti-malarial drug combinations urgently require further investigation [ 9 ]. Ensuring malaria prevention measures reach the vulnerable The highly focal nature of urban malaria requires targeting of interventions to specific urban districts, and therefore, requires detailed information on each area in advance. However, relationships between administrative boundaries, environment and population distribution are complex in urban areas, which makes them difficult to sample and characterize in a representative way. Strategies for population-representative sampling must incorporate a range of environments and populations to identify accurately environmental and other risk factors. This may be further complicated as urban populations can be highly mobile and in peri-urban areas there may be a high rate of turnover in groups of lower socio-economic status. Presentations from South, East and West Africa clearly demonstrated that Geographic Information Systems (GIS)-based approaches are valuable tools for assessing heterogeneities in risk factors for urban malaria, and for subsequent implementation and monitoring of interventions. Experienced researchers believe that the urban environment has advantages for the effective delivery of appropriate interventions. A number of studies have demonstrated that higher rates of coverage with insecticide-treated bednets can be achieved in urban areas [ 10 , 11 ], although whether or not the most vulnerable groups benefit, remains to be confirmed. Moreover, there is a growing realization within the commercial sector of the need to engage in health and broader social issues. The management of malaria can bring economic benefits to both businesses and the communities in which they operate. This has been powerfully demonstrated in two public-private partnership programmes in southern Africa that utilised indoor residual spraying to control malaria [ 12 , 13 ]. Larval control, achieved either by source reduction or larviciding, can be community directed and may be feasible in certain settings as part of a comprehensive, integrated vector management strategy. There is optimism in some communities about its efficacy and the results of further research into the costs and benefits of such interventions are awaited with interest. Environmental modifications may also be feasible if partners from the community and outside the health sector are engaged. Work from Sri Lanka has demonstrated how a very effective scheme to control malaria by modification of irrigation structures was accepted by the agricultural community because of the financial and water savings that the scheme introduced [ 14 ]. However, it was clear that obvious benefits from the intervention must exist to attract the involvement of non-health sectors. Conclusions The conclusions of the meeting have been summarised in the Pretoria Statement on Urban Malaria (Figure 1 ). While it is clear that urban malaria represents a major challenge for public health in Africa, the statement highlights that the unique nature of the urban environment provides an opportunity for malaria control. There are a number of reasons for this: the high population density in urban areas may facilitate increased coverage and impact of both interventions and health education programmes; the activities of departments in urban municipal authorities are typically better resourced and more easily mobilized than in rural areas; the extensive private health sector found in urban settings can be engaged to improve diagnosis, treatment and prevention of malaria. Solutions to the urban malaria problem must include groups from outside the health sector. The disease burden in the most vulnerable communities is a major obstacle to the economic growth of sub-Saharan countries and the challenge is to engage stakeholders at all levels in effective and sustainable intersectoral collaboration [ 15 ]. Urban malaria is uniquely amenable to prevention and control as the existing health, urban planning, agricultural and governance structures present opportunities for collaborative approaches that can include both the community and the substantial private sector. Figure 1 The Pretoria Statement on Urban Malaria. Authors' contributions All authors participated as session chairs in the technical workshop and were instrumental in producing the summary conclusions. All authors read and approved the final manuscript. Supplementary Material Additional File 1 List of attendees and affiliations Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC552321.xml
544557	Identification of α-type subunits of the Xenopus 20S proteasome and analysis of their changes during the meiotic cell cycle	Background The 26S proteasome is the proteolytic machinery of the ubiquitin-dependent proteolytic system responsible for most of the regulated intracellular protein degradation in eukaryotic cells. Previously, we demonstrated meiotic cell cycle dependent phosphorylation of α4 subunit of the 26S proteasome. In this study, we analyzed the changes in the spotting pattern separated by 2-D gel electrophoresis of α subunits during Xenopus oocyte maturation. Results We identified cDNA for three α-type subunits (α1, α5 and α6) of Xenopus , then prepared antibodies specific for five subunits (α1, α3, α5, α6, and α7). With these antibodies and previously described monoclonal antibodies for subunits α2 and α4, modifications to all α-type subunits of the 26S proteasome during Xenopus meiotic maturation were examined by 2D-PAGE. More than one spot for all subunits except α7 was identified. Immunoblot analysis of 26S proteasomes purified from immature and mature oocytes showed a difference in the blots of α2 and α4, with an additional spot detected in the 26S proteasome from immature oocytes (in G2-phase). Conclusions Six of α-type subunits of the Xenopus 26S proteasome are modified in Xenopus immature oocytes and two subunits (α2 and α4) are modified meiotic cell cycle-dependently.	Background Eukaryotic cells, from yeast to human, contain large nonlysosomal proteases called proteasomes [ 1 ]. The 26S proteasome is part of the ubiquitin-dependent proteolytic system, which regulates proteins through a mechanism of selective degradation [ 2 - 4 ]. The 26S proteasome is composed of a 20S proteasome as a catalytic core and regulatory particles at either end. The subunits of the 20S proteasome subunits can be classified into two families, α and β. In eukaryotes, the 20S proteasome contains seven α-type subunits and seven β-type subunits. The fourteen kinds of subunits are arranged in four rings of seven subunits and form an α7β7β7α7 structure [ 5 ]. Fully grown frog oocytes arrest in the late G2 phase of meiosis. Maturation-inducing hormone (MIH) acts on the oocytes, inducing final maturation and triggering germinal vesicle breakdown (GVBD), and the oocytes arrest again at the second meiotic metaphase until fertilization. The proteasomes are thought to be involved in regulating the maturation and fertilization of oocytes [ 6 , 7 ]. Previously we identified the proteasomal subunit modified during oocyte maturation in Xenopus and goldfish as α 4 [ 8 , 9 ]. In the present study, we cloned three unidentified α-type subunits of Xenopus and prepared antibodies for a total of five subunits. Using a set of specific antibodies, we analyzed changes in all α subunits composing the 26S proteasome during the meiotic cell cycle. We demonstrated that 6 of the subunits exist as a heterogeneous population in frog oocytes and identified another subunit in addition to α4 which was modified meiotic cell cycle dependently. Results and discussion Isolation and characterization of cDNA clones A BLAST search of the Xenopus EST database was conducted using known proteasomal subunit α cDNAs. From the data for each subunit, full-length ORFs were obtained by PCR. The amplified cDNAs were 741, 726 and 786 bp long. The clones encode proteins of 246, 241 and 261 amino acid residues with a predicted molecular mass of 27463, 26402 and 29327 daltons, respectively (Fig. 1 ). Comparison of the amino acid sequence revealed that these molecules are highly homologous to the α1, α5 and α6 subunits in humans (overall identity 91.5–95.4%) [ 10 , 11 ], Drosophila (53.2–69.1%) [ 12 , 13 ] and yeast (53.2–61.7%) [ 14 - 16 ] (Fig. 2 ). Thus, we concluded that the cDNAs isolated in this study encode the α1, α5 and α6 subunits of the Xenopu s 20S proteasome. We named these clones α1_xl, α5_xl and α6_xl (α1, α5 and α6 subunits of Xenopus laevis ) according to a systematic nomenclature [ 5 ]. Figure 2 represents a comparison of amino acid sequences predicted from cDNA sequences of α-type subunits of the Xenopus 20S proteasome. Overall identity between the subunits was 25.1–38.4 %. A consensus sequence for α-type proteasomal subunits was conserved. Interestingly, a conserved sequence for β-type proteasomal subunits was found in the α3 subunit [ 17 ]. Figure 1 Amino acid sequence comparison of the Xenopus , human, Drosophila , and Yeast α 1 , α 5 and α 6 proteasome subunits. Amino acid sequence comparisons of α1 (A), α5 (B) and α6 (C) proteasome subunits are indicated. Matched sequences are boxed. Consensus sequences for calcium/calmodulin-dependent kinase II (CaMKII), cAMP/cGMP-dependent kinase (cAMP/cGMP), casein kinase II (CKII) and Ca 2+ -dependent kinase (PKC) are indicated. The numbers refer to the amino acid position at the end of each line. Figure 2 Amino acid sequence comparison of the Xenopus proteasomal α subunits. Matched sequences are boxed. The proteasomal α-type and β-type signatures were detemined by using the 'PROSITE' database [ 17 ] and are boxed. The numbers refer to the amino acid position at the end of each line. Comparison of proteasomes purified from immature and mature oocytes Polyclonal antibodies specific for five subunits (α1, α3, α5, α6, and α7) were raised against purified recombinant proteins. The specificity of the antibodies was examined by immunoblotting with the cytosol fraction and the purified 26S proteasome (Fig. 3 ). Each antibody preparation displayed a specific reaction for different polypeptides in both samples. Recombinant proteins from the cDNAs clearly cross-reacted with each antibody (data not shown). Thus, specific antibodies for each subunit were prepared. With these antibodies and previously described monoclonal antibodies for subunits α2 and α4 [ 18 ], changes to all α-type subunits during Xenopus meiotic maturation were analyzed. The modifications were demonstrated by 2D-PAGE (Fig. 4 ). The α7 subunit antibodies gave a single spot but all of the other antisera produced more than one spot, suggesting that the α1–α6 subunits undergo some type of modification in oocytes of Xenopus as demonstrated in other species [ 19 , 20 ]. A difference in the spots between the 26S proteasome from immature and mature oocytes was detected in the blots of subunits α2 and α4. In blots of α2 and α4, only a major spot was detected in the 26S proteasome from mature oocytes (in M-phase). It is suggested that the α4 subunit is phosphorylated in immature oocytes and dephosphorylated in mature oocytes [ 8 ]. Likewise, it is speculated that part of the α2 subunit is phosphorylated in interphase and dephosphorylated in metaphase. These results suggest that the subunits of 26S proteasomes are changed by meiotic cell cycle-dependent modifications. It can be speculated that these modifications are involved in the regulation of the meiotic cell cycle. Figure 3 Immunoblotting of the cytosol fraction and purified 26S proteasome. The cytosol fraction and purified 26S proteasome were electrophoresed under denaturing conditions (10.0% gel) and stained with Coomassie Brilliant Blue (CBBR), or immunostained with antibodies for α subunits of the 20S proteasome. Lanes cyt and 26S indicate the cytosol fraction and the 26S proteasome from immature oocytes, respectively. Molecular masses of standard proteins are indicated at the left. Protein bands of each subunit are indicated by arrows. Figure 4 2D-PAGE analysis of 26S proteasomes from immature and mature oocytes. The 26S proteasomes from immature (I) and mature (M) oocytes were subjected to 2D-PAGE followed by immunostaining with polyclonal antibodies against each of the Xenopus 20S proteasome subunits as indicated. The spots detected by each antibody are represented at high magnification and indicated by arrows. The spots differing between immature and mature oocytes are indicated by asterisks. The modification of proteasomal subunits and factors interacting with proteasomes may be involved in the regulation of proteasome function [ 21 ]. By two-dimensional polyacrylamide gel electrophoresis, up to 20 different polypeptides were separated from the 20S proteasome which was shown to be composed of 14 gene products [ 22 ]. Furthermore, changes in proteasomal subunit composition under different physiological conditions and the likely existence of a different subpopulation of proteasomes have been reported [ 12 , 23 ]. All these results suggest that the subunit composition of proteasomes, and likely their activity, is under complex control in vivo . Some of these changes may be due to post-translational modifications of the proteasomal subunits. Regarding protein modification, there have been several reports about the phosphorylation of proteasomal subunits. Phosphorylated proteasomal subunits were detected in crude preparations from cultured Drosophila cells [ 22 ]. Several subunits of the 20S proteasome could be phosphorylated in vitro by a cyclic AMP-dependent protein kinase copurifying with the bovine pituitary 20S proteasome [ 24 ]. Castaño et al. [ 25 ] (1996) identified the CKII phosphorylating subunit and its phosphorylation sites as the C8 component (α7 subunit) and serine-243 and serine-250, respectively. CKII was also reported to phosphorylate the C2 component (α6 subunit) in rice [ 26 ]. The phosphorylation of subunits in the 26S proteasome in vivo was investigated using cultured human cells. Mason et al. [ 27 ] (1996) showed the phosphorylated subunits to be the C8 (α7 subunit) and C9 (α3 subunit) components in the 20S core, and the S4 (Rpt2p) subunit and several other components in regulatory particles [ 28 ]. Recent approaches have revealed post-translational modifications to many of the subunits. In the yeast 20S proteasome, the α2- and α4-subunits are phosphorylated at either a serine or threonine residue, and the α7-subunit is phosphorylated at tyrosine residue(s) [ 20 ]. In the human 20S proteasome, more than two spots were identified in all α-type subunits except α5 and phosphorylation of the α7-subunit at serine-250 was revealed [ 19 ]. However the sites and kinases responsible for the phosphorylation of the α2 and α4 subunits of the 20S proteasome have yet to be demonstrated. The modification of these proteins is one possible mechanism regulating the functions of the 26S proteasome during the meiotic cell cycle. Consensus sequences for phosphorylation sites are conserved in these subunits [ 8 , 29 ]. Cyclic-AMP dependent protein kinase is responsible for the G2/M and metaphase/anaphase transitions [ 30 ]. Calcium/calmodulin-dependent protein kinase II is shown to be involved in the exit from metaphase II arrest at fertilization in Xenopus [ 31 ]. It can be hypothesized that these kinases are involved in the regulation of 26S proteasome activity. The identification of kinases and the phosphorylation sites of the α2 and α4 subunits may reveal how the modification of proteasomal subunits is involved in controlling the cell cycle. Currently, we have identified one of the protein kinase for α4 subunit as Casein KinaseIα [ 32 ]. Possible regulation of 26S proteasome activity by this kinase is under investigation. Recently, alternative subunits of proteasomes have been identified. In Drosophila where alternative α-type, β-type and 19S cap subunits are expressed from separate genes during spermatogenesis [ 33 ] and in Arabidopsis and rice where alternative isoforms of most proteasome subunits are differentially expressed from separate genes during development [ 34 , 35 ]. There are also examples of alternative β-type subunits in mammals (e.g., γ-interferon inducible "immunoproteasome" subunits β1i, β2i and β5i) [ 36 ]. Alternative subunits have yet to be identified in Xenopus , there is a possibility that the changes in the spots identified in this study may derive from differential expression of alternative subunits from paralogous genes. Conclusions (1) cDNAs for three α-type proteasome subunits ( α1_xl, α5_xl and α6_xl ) of X. laevis were identified. (2) Six subunits but not α7_XL are modified in immature oocytes in X. laevis . (2) α2, α4_XLs are modified during the meiotic cell cycle in X. laevis . Methods Purification of proteasomes Frogs ( Xenopus laevis ) were purchased from Jo-hoku Seibutsu Kyozai (Shizuoka, Japan) and maintained till used. 26S proteasomes were purified from immature oocytes and ovulated oocytes as described [ 37 ]. Electrophoresis and immunoblotting SDS-PAGE was carried out according to the method of Laemmli [ 38 ] (1970). 2D-PAGE (first dimension, NEPHGE; second dimension, SDS-PAGE) was carried out as described by O'Farrell et al. [ 39 ] (1977) using a precast polyacrylamide gel for NEPHGE (Immobiline Dry Strip pH3-10NL and pH6-11L for α4 subunit, Amersham biosciences) as reported [ 8 ]. Electroblotting and detection using antibodies were conducted as described [ 18 ]. cDNA cloning and sequencing Identification and sequence analysis of cDNAs. A BLAST search of the Xenopus EST database was conducted using known proteasomal subunit α cDNAs. From the data obtained for each subunit, independent sequences were linked and the full-length ORF sequences were eliminated(α1: BG347128 and CB558360, α5: BQ398972 and BJ043946, α6: BJ072624 and BJ091555). The specific primers for amplification of the full-length ORF were α1: 5'-GGAATTCCATATGTCTCGGGGATCTAGCGCG-3' and 5'-CCGCTCGAGGTCACGCTCAGCTAGTGCAAC-3', α5: 5'-GGAATTCCATATGTTCCTAACCCGCTCCGAG-3' and 5'-CCGCTCGAGGATGTCCTTAATAACTTCCTC-3', and α6: 5'-GGAATTCCATATGTTTCGCAATCAGTATG-3' and 5'-CCGCTCGAGGTGCTCCATAGGCTCCTCCTGC-3'), in which EcoRI (5'end) and XhoI (3'end) recognition sequence was added for cloning to the vector pET21a (Novagen). PCR was carried out using KOD DNA polymerase (TOYOBO) or LA taq DNA polymerase (TaKaRa), with Xenopus ovarian cDNA as a template, and the product was cloned to pET21a. The DNA sequencing was performed using a 377A DNA sequencer (Applied Biosystems). The sequences that include the full-length ORF identified here were deposited into GenBank (accession nos. AB164677, AB164678 and AB164679 for α1_xl, α5_xl and α6_xl , respectively). Pairwise comparisons of sequence homology were conducted using the Genetyx-Mac ver.12 computer program (Software Development, Tokyo, Japan). Production of recombinant proteins and preparation of antibodies The recombinant proteins were produced in E. coli BL21 (LysE) and purified by SDS-PAGE as described [ 6 ]. Polyclonal antibodies specific for each subunit were raised against purified recombinant proteins according to a procedure described before using guinea pigs [ 40 ]. Anti serums, which recognize the bands of each subunit, were obtained. Abbreviations bp, base pair; BLAST, basic local alignment search tool; cDNA, DNA complementary to RNA; EST, expressed sequence tags; kDa, kilodalton; NEPHGE, non-equilibrium pH gradient gel electrophoresis; PCR, polymerase chain reaction; SDS-PAGE SDS-polyacrylamide gel electrophoresis; 2D-PAGE, two-dimensional-PAGE. Authors' contributions YW carried out cDNA cloning, expression of recombinant proteins, antibody production and 2D-PAGE analysis. MT and RH participated in cDNA cloning, expression of recombinant proteins. YN and KI participated in coordination of the study. TT carried out the protein purification and also participated in the design of the study and drafted the manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544557.xml
544556	The effect of short-duration sub-maximal cycling on balance in single-limb stance in patients with anterior cruciate ligament injury: a cross-sectional study	Background It has previously been shown that an anterior cruciate ligament (ACL) injury may lead to impaired postural control, and that the ability to maintain postural control is decreased by fatigue in healthy subjects. To our knowledge, no studies have reported the effect of fatigue on postural control in subjects with ACL injury. This study was aimed at examining the effect of fatigue on balance in single-limb stance in subjects with ACL injury, and to compare the effects, and the ability to maintain balance, with that of a control group of uninjured subjects. Methods Thirty-six patients with unilateral, non-operated, non-acute ACL injury, and 24 uninjured subjects were examined with stabilometry before (pre-exercise) and immediately after (post-exercise) short-duration, sub-maximal cycling. In addition, the post-exercise measurements were compared, to evaluate the instantaneous ability to maintain balance and any possible recovery. The amplitude and average speed of center of pressure movements were registered in the frontal and sagittal planes. The paired t-test was used for the intra-group comparisons, and the independent t-test for the inter-group comparisons, with Bonferroni correction for multiple comparisons. Results No differences were found in the effects of exercise between the patients and the controls. Analysis of the post-exercise measurements revealed greater effects or a tendency towards greater effects on the injured leg than in the control group. The average speed was lower among the patients than in the control group. Conclusions The results of the present study showed no differences in the effects of exercise between the patients and the controls. However, the patients seemed to react differently regarding ability to maintain balance in single-limb stance directly after exercise than the control group. The lower average speed among the patients may be an expression of different neuromuscular adaptive strategies than in uninjured subjects.	Background The anterior cruciate ligament (ACL) is the most commonly injured ligament in the knee. The risk of future joint problems, in the form of functional limitations, secondary lesions, and arthrosis, is increased following such an injury. Secondary effects commonly seen after an ACL injury include defective neuromuscular function with reduced strength and functional performance, a different movement and activation pattern, defective proprioception and impaired postural control [ 1 ]. Impaired postural control has been reported after acute [ 2 ], and chronic ACL injury [ 3 - 5 ], as well as after ACL reconstruction [ 6 - 8 ]. Higher amplitude values [ 2 - 5 ] and longer reaction time when subjected to perturbations [ 4 , 6 , 7 ] have been observed among patients compared to controls. Studies have also shown that patients with better subjective function have lower amplitude values [[ 8 ], Ageberg E, Roberts D, Holmström E, Fridén T: Balance in single-limb stance in individuals with anterior cruciate ligament injury – relation to knee laxity, proprioception, muscle strength, and subjective function. Manuscript submitted]. The present study was initiated by the clinical knowledge that although patients with ACL injury have had extensive neuromuscular training and function well during daily life and (modified) physical activities, they experience a decreased ability to maintain balance during weight-bearing on the injured leg in demanding situations while fatigued. This may be related to an increased risk of further injuries. Fatigue is caused by a combination of different physiological mechanisms occurring at both the central and peripheral levels [ 9 ], affecting afferent neuromuscular pathways, observed as proprioceptive deficiency [ 10 - 12 ], and efferent neuromuscular pathways, seen, for example, as a delay in muscle response [ 13 , 14 ]. Thus, muscular fatigue leads to a decline in work performance, which may also include effects on postural control. A decreased ability to maintain balance in bilateral stance [ 15 - 17 ], and single-limb stance [ 15 , 18 - 20 ] after fatiguing exercise (i.e., higher values after exercise) has been reported in uninjured subjects, and it has been suggested that individuals are therefore at increased risk of injury when fatigued [ 15 , 19 , 20 ]. Studies of balance in single-limb stance are of importance and of interest since these movement patterns resemble the stance phase, and since many knee injuries occur during weight-bearing on one leg [ 21 ]. To our knowledge, no studies evaluating the effect of fatigue on postural control in subjects with ACL injury have been reported. The main purposes of this study were: 1) to examine the effect of short-duration, sub-maximal exercise performed on a cycle ergometer, on postural control, measured by stabilometry in single-limb stance on a force platform, in individuals with ACL injury in comparison with that of a control group of uninjured subjects, and 2) to explore the patients' instantaneous ability to maintain balance in single-limb stance after exercise, in comparison with that of the control group. Furthermore, the patients were compared to the control group in order to verify previous findings that postural control is affected in both legs by a unilateral ACL injury [ 2 - 5 ]. No comparisons were, therefore, made between the injured and uninjured legs. Our hypothesis was that the patients with ACL injury would be more affected by exercise than the uninjured subjects, since fatigue has been shown to reduce postural control in healthy subjects, and since postural control may already be impaired due to the injury. Methods Patients Thirty-six patients (18 men and 18 women) were included in the study. Inclusion criteria were: 1) age between 15 and 35 years, 2) unilateral, non-operated, non-acute ACL deficiency with or without associated lesions of other structures of the knee, 3) an uninjured contralateral extremity, back and neck, and 4) no history of neurological disease, vestibular or visual disturbance. Their mean age was 26 years (SD 5 years), mean height 174 cm (SD 9 cm), and mean body mass 72 kg (SD 13 kg). Their median activity level before injury was 6.5 (range 3 to 9) and on the test occasion 4 (range 1 to 9) according to the Tegner activity level scale [ 22 ]. The mean time elapsed from injury to the test occasion was 3.8 years (SD 3, range 0.5 to 11 years). The patients had undergone an extensive neuromuscular training program [ 23 ] after the injury under the supervision of physical therapists, with a mean duration of 7 months (SD 5 months). A visual analog scale graded from 0 to 100 mm was used for subjective evaluation of extremity function, where 0 was "as if the knee had been recently injured" and 100 was "perfect" [ 24 ]. The patients' mean value and median value on this scale were 68 mm and 59 mm (range 12–95 mm), respectively. Control group The measurements of twenty-four uninjured volunteers (11 men and 13 women) from a previous study [ 18 ], with no history of neurological disease, major orthopedic lesion, vestibular or visual disturbance, constituted control values. Their mean age was 24 years (SD 3 years), mean height 176 cm (SD 8 cm), and mean body mass 71 kg (SD 13 kg). Their median activity level was 5 (range 2 to 9) according to the Tegner activity level scale [ 22 ]. The subjects in the control group were chosen in order to have the same distribution in age, sex, and physical activity as the patients [ 25 ]. No significant difference was found between the groups in age, height, body mass or activity level. The Research Ethics Committee at Lund University approved the study. All subjects gave their written informed consent to participate in the study. Assessment Stabilometry Balance in single-limb stance was tested by means of a strain gauge force plate (33 × 38 cm) with the subject barefoot in a standardized position [ 5 , 26 , 27 ] (Figure 1 ). This measurement was performed before (pre-exercise) and immediately after fatiguing exercise (post-exercise). The foot was placed pointing straight forward in relation to reference lines in the frontal and sagittal planes (origin of coordinates). The other leg was flexed 90° at the hip and knee joints with both arms hanging relaxed at the sides. The subjects were instructed to stand as motionless as possible, looking straight ahead at a point on the wall 65 cm away; they were allowed to practice maintaining this position for about 20 s before three measurements on each leg were made, with the subjects standing alternately on their right and left leg. The test order between legs was randomized regarding injured/uninjured leg in the patient group (injured leg n = 20, uninjured n = 16), and regarding right/left leg in the control group (right leg n = 13, left n = 11). No differences were observed in the stabilometric measurements between these randomization groups. Accordingly, the assessment included three measurements made on each leg, giving a total of six measurements pre- and post-exercise, respectively. These six measurements lasted for approximately 3 1/2 minutes, with about 10 seconds between each measure. The median value of the three measurements on each leg was used to compare pre- and post-exercise values. Decreasing values in the three measurements have been observed in a previous study, indicating a learning effect [ 26 ]. Some degree of recovery may, therefore, occur during the three post-exercise measurements. For this reason, the first and third of the three post-exercise measurements on each leg were used, to evaluate the instantaneous value of the ability to maintain postural control (first measurement) and the possible recovery (third measurement). Movements of the center of pressure (CP) in the frontal plane (FP) and sagittal plane (SP) were recorded for 25 s at a sampling frequency of 20 Hz. A computer program (Viewdac 2.1, Keithley Instruments, Inc., Cleveland, Ohio, USA), was used to analyze the following variables: 1) average speed of CP movements in mm·s -1 ; and 2) number of movements exceeding 10 mm from the mean value of CP (DEV 10) , giving a total of four variables (two variables in each plane). The mean value of CP is the average distance (mm) of the CP from the reference lines, and DEV 10 is the number of movements exceeding 10 mm from the mean value of CP. DEV 10 (n) reflects the deviation of CP (i.e., displacement of CP), and the average speed (mm·s -1 ) reflects the amplitude and frequency of CP movements. Figure 2 shows raw data from a stabilometry test. Average speed and DEV 10 were used in the present study, since our previous studies have shown that these variables are reliable [ 18 , 26 ], and sensitive in detecting differences between patients and uninjured subjects [ 2 ], and sensitive in detecting the effects of exercise [ 18 ]. We expected to find higher values after exercise [ 18 ]. Short-duration sub-maximal exercise Short-duration, sub-maximal exercise was performed on a cycle ergometer. The subjects' heart rate was continuously recorded during the entire test. Borg's scale for Rating of Perceived Exertion (RPE scale) was used to assess the subjective effort level during exercise [ 28 ]. On this scale, numbers ranging from 6 to 20 are matched with descriptors (e.g., 6 = No exertion at all , 13 = Somewhat hard , 15 = Hard , 17 = Very hard , 19 = Extremely hard , and 20 = Maximal exertion ). The RPE scale was designed to increase linearly with exercise intensity and heart rate for work on a bicycle ergometer, and correlates closely with several physiological variables, including heart rate and blood lactate concentration [ 28 ]. A linear relationship exists between heart rate and oxygen consumption with increasing rate of work. A given percentage of the maximum oxygen consumption (VO 2max ) results in a higher percentage of the maximum heart rate (HR max ); e.g., 75% of VO 2max represents an intensity of 86% of HR max [ 29 ]. The maximum heart rate can be estimated from the following equation: maximum heart rate (beats/min) = 220 – age (years) [ 29 ]. Effects of fatigue are likely to occur after a few minutes of sub-maximal exercise [ 9 ]. The rate of pedaling was kept constant at 60 revs/min. The level of exercise was calculated so as to be similar to that perceived during a general exercise session. The workload (W) was set individually, depending on the sex and physical condition of each subject, with the aim of reaching a heart rate above 60% of the predicted HR max [ 30 ] in all subjects. Cycling was stopped when the subjects had reached a heart rate exceeding 60% of the predicted HR max , perceived the exercise as hard or very hard (values 14–17 of the RPE scale), and had reached steady-state heart rate, i.e., after approximately 5 min. Statistical analysis The average of the right and left legs; i.e., (right+left)/2, was used for statistical analysis in the control group, since there were no clinically or statistically significant differences between the legs. The use of the mean value of both legs when performing parametric statistics can be questioned, since this may affect the data variability. It cannot, however, be excluded that a dominance of one or the other side exists, which is difficult to define [ 25 ], and therefore it is hard to determine which leg to use in comparison with the patients. For this reason we used the average of the right and left legs. However, the results were confirmed using the right and left legs separately as the control leg. The median value of the three measurements was used to compare pre- and post-exercise values. In addition, the first and third of the three post-exercise measurements were compared, to evaluate the instantaneous ability to maintain postural control in single-limb stance (first measurement) and the possible recovery (third measurement). We used the paired t-test for the intra-group comparisons, and the independent t-test for the inter-group comparisons, with Bonferroni correction for multiple comparisons. The present study is of exploratory character, and the level of correction for multiple comparisons was chosen with regard to this. For each stabilometric variable, five separate t-tests were performed in comparisons between pre- and post-exercise values for the injured leg and the control group: 1) injured leg pre-exercise vs. post-exercise, 2) control group pre-exercise vs. post-exercise, 3) injured leg vs. control group pre-exercise, 4) injured leg vs. control group post-exercise, and 5) effects of exercise (post-exercise minus pre-exercise) injured leg vs. control group. These five t-test were also performed in the analysis of possible differences between pre- and post-exercise values for the uninjured leg and the control group. Since five comparisons were made in the above-mentioned analyses, the alpha level was set at 0.05/5 = 0.01. For each stabilometric variable, three separate t-tests were performed in comparisons between post-exercise measurements 1 and 3 for the injured leg and the control: 1) injured leg measurement 1 vs. measurement 3, 2) control group measurement 1 vs. measurement 3, and 3) effects of exercise (measurement 3 minus measurement 1) injured leg vs. control group. These three t-tests were also performed in the analysis of possible differences between post-exercise measurements 1 and 3 for the uninjured leg and the control group. Since three comparisons were made in the above-mentioned analyses, the alpha level was set at 0.05/3 = 0.02. The statistical analyses were performed using the program package SPSS 11.0 (SPSS Inc., Chicago, Illinois, USA). Results Fatiguing exercise All subjects exceeded the 60% value of the predicted HR max ; the mean level being 82% (SD 6%, range 66 to 92%) among the patients and 81% (SD 7%, range 68 to 99%) among the controls. The median power output produced by the patients and the control group at the end of fatiguing exercise was 125 W (range 75 to 200 W) and 150 W (range 100 to 200 W), respectively, and the mean value of perceived exertion, rated according to the RPE scale, was 15.8 (SD 1.1) and 15.4 (SD 0.9), respectively. The final heart rate attained among the patients and the control group was 159 beats/min (SD 11 beats/min) and 159 beats/min (14 beats/min), respectively, and the heart rate after the stabilometric assessment, approximately 3 1/2 minutes after exercise, was 112 beats/min (SD 14 beats/min) and 117 beats/min (SD 16 beats/min), respectively. No significant differences were found between the patients and controls with regard to the above-mentioned variables. Average speed of CP movements Higher values were noted post- than pre-exercise in the FP and SP in the injured and uninjured legs, but only in the FP in the control group (Table 1 ). No differences were noted between the groups regarding the effects of exercise (mean difference of post-exercise minus pre-exercise values) (Table 2 ). Figures 3 and 4 show the pre-and post-exercise values for the injured leg and the control group. A lower value was observed in the third than in the first of the post-exercise measurements on the injured leg in the FP, but no differences were noted on the uninjured leg or in the control group (Table 3 ). The injured leg of the patients was more affected by exercise directly after cycling than the legs of the control group in the FP (Table 4 ). Figures 7 and 8 show the first and third of the post-exercise measurements on the injured leg and in the control group. Lower values were observed pre-exercise in the SP in the injured and uninjured legs of the patients than in the control group (Table 5 ). Number of movements exceeding 10 mm from the mean value of CP A higher value was found post- than pre-exercise in the uninjured leg in the FP, and the post-exercise value tended to be higher in the injured leg and in the control group (Table 1 ). No differences were found between pre- and post-exercise values in the SP (Table 1 ), or between the groups regarding the effects of exercise (mean difference of post-exercise minus pre-exercise values) (Table 2 ). Figures 5 and 6 show the pre-and post-exercise values for the injured leg and the control group. The third of the post-exercise measurements was lower than the first in the injured leg in both planes, but no differences were found for the uninjured leg or in the control group (Table 3 ). No differences were noted between the groups regarding the effects of exercise directly after cycling (Table 4 ). Figures 9 and 10 show the first and third of the post-exercise measurements on the injured leg and in the control group. No differences were found between the injured leg and the control group, or between the uninjured leg and the control group (Table 5 ). Discussion Short-duration, sub-maximal exercise on a cycle ergometer resulted in increased average speed in both planes, and in the amplitude of CP movements (DEV 10) in the FP during balance in single-limb stance among the patients with ACL injury. In the intra-group comparisons, three of four variables showed higher values post- than pre-exercise in the uninjured leg, and two of four variables were higher post-exercise in the injured leg. In the control group, one of four variables was higher post- than pre-exercise (Table 1 ). However, no differences in the effects of fatigue (mean difference of post-exercise minus pre-exercise values) were found in the inter-group comparisons (Table 2 , and Figures 3 , 4 , 5 , 6 ). The variables were more sensitive in detecting the effects of exercise in the FP than in the SP. The primary motions of the knee joint occur in the SP, and the joint has limited capacity to make postural adjustments in the FP due to anatomical constraints, whereas the hip joint and ankle are involved in postural corrections in both the FP and SP during weight-bearing [ 31 ]. Since many injuries to the knee occur during weight-bearing on one leg [ 21 ]; i.e., in a closed kinetic chain including the hip joint and ankle, it is of interest to examine postural control in both the FP and SP in individuals with ACL injury. The results of a previous study [ 18 ] and the present one indicate that measurements in the FP may be more sensitive and revealing in detecting effects of exercise than measurements in the SP. It has been demonstrated that afferent information has an effect on the neuromuscular function of both the ipsilateral and contralateral limb muscles [ 32 ], which may explain why more variables were higher post- than pre-exercise not only in the injured leg, but also in the uninjured one, than in the control group. Several studies have reported bilateral defects in postural control after an ACL injury or reconstruction [ 2 - 7 ], which may be due to central nervous system modifications following the loss of knee mechanoreceptors after the injury [ 33 , 34 ]. Another explanation may be that the patients had inherently poor balance, which might have contributed to the original injury. This has been reported by Tropp et al. [ 35 ], where soccer players with abnormal stabilometric values (defined as a value exceeding 2 SD of the mean value in a control group), ran a higher risk of sustaining an ankle injury than players with normal values. In a previous study [ 26 ], we observed decreasing values in the three measurements, indicating a learning effect. In another study [ 36 ], fatigue was shown to interfere with this learning process, which is in agreement with the results that we found on the uninjured leg and in the control group. However, the injured leg reacted differently from the uninjured one, and the control group when the first and third of the post-exercise measurements were compared. It was assumed that the first measurement could provide us with the instantaneous value of the ability to maintain postural control in single-limb stance. The results showed that the third measurement was lower, or tended to be lower, than the first in the injured leg, regarding average speed and DEV 10 in both planes. No such effect was, however, found in the uninjured leg or in the control group (Table 3 ). The inter-group comparisons for these post-exercise measurements showed greater effects in the injured leg than in the control group in average speed in the FP, and a tendency towards greater effects in the other three variables (Table 4 and Figures 7 , 8 , 9 , 10 ). This finding indicates that balance standing on one leg may be improved during the recovery period, and that a learning process may be needed in the injured leg after exercise. A different strategy in the injured leg than in the uninjured one has been reported in individuals with ACL injury [ 37 ]. In that study, Di Fabio et al. [ 37 ], found that postural responses, measured with external perturbations while standing on a force platform, could be unilaterally restructured and preprogrammed to compensate for the injury. Mechanoreceptors in the ACL contribute to the neuromuscular control of the muscle tonus around the knee joint via the reflex arc (i.e., reflex from joint afferents to the muscle spindles via the gamma motoneurons), and therefore to the stabilization of the knee joint [ 32 ]. Decreased proprioception [ 11 , 12 ], increased joint laxity in the knee joint [ 14 , 38 ], and a delay in muscle response in leg muscles [ 13 , 14 ] have been described after fatiguing exercise. In these studies, uninjured subjects were tested. The activity of joint receptors, muscle spindles and Golgi tendon organs may be reduced by fatigue, resulting in proprioceptive deficiency in muscle receptors and loss of muscular reflexes responsible for joint stability [ 10 ]. Since this afferent information is important for the maintenance of postural control [ 32 ], this may lead to decreased muscle response and poorer ability to maintain balance. The increase in joint laxity following fatigue has been suggested to be due to reduced muscle tone [ 38 ], viscoelastic changes in the collagenous tissues of the knee and fatigued muscle stabilizers [ 14 ], and results in inadequate ligament mechanoreceptor feedback, which is required to elicit the muscular reflexes responsible for joint stability [ 10 ]. It has been suggested that muscle receptors are the primary determinant of joint position sense, and capsular receptors may have a secondary role [ 12 , 32 ]. Therefore, the decreased proprioceptive ability following fatigue has been proposed to be due to the decrease in muscle receptor activity [ 11 , 12 ]. Since defects in proprioception [ 39 ], impaired postural control [ 2 - 5 ], increased joint laxity [ 32 ], and a delay in muscle reaction time [ 4 , 40 , 41 ] are present already in an unfatigued state in individuals with ACL injury, they may, at least theoretically, be more affected by fatigue than uninjured subjects. Although we found effects of exercise after a short period of cycling above 60% of the predicted HR max , it is possible that greater effects of exercise on balance in single-limb stance may be seen after longer durations of exercise than in the present study. It is also possible that larger effects of exercise may be reflected in more challenging measures of postural control, such as dynamic balance tests. Since, to our knowledge, this is the first study on the effects of fatigue on postural control in patients with ACL injury, the clinical relevance of our results remains unclear. More research is needed to further study whether this may be related to an increased risk of further injuries. The lower average speed and lack of difference in DEV 10 in the patients compared to the control group, indicate sway movements at a lower speed with retained amplitudes to be neuromuscular adaptive strategies, rather than more rapid, smaller adjustments (Table 5 ). These strategies may be the result of decreased proprioception [ 39 ], and a delay in muscle reaction time [ 4 , 40 , 41 ], which has been reported after an ACL injury, and thus, these strategies may be needed to generate sufficient afferent impulses to obtain dynamic stabilization of the knee joint. Another possible explanation may be that the patients had all undergone neuromuscular training, which may have affected the strategies of maintaining balance in single-limb stance compared with the control group who had not undergone such training. The clinical relevance of the fact that the patients' post-exercise values approached those of the control group, remains, however, unclear. More research is needed to elucidate this further. Conclusions The results of the present study showed no differences in the effects of exercise between the patients and the controls. However, the injured leg was more affected or tended to be more affected directly after exercise than the control group, which indicates that patients with ACL injury react differently regarding their ability to maintain balance in single-limb stance after short-duration, sub-maximal cycling, than a control group of uninjured subjects. The patients used sway movements at a lower speed with retained amplitudes, which may be an expression of neuromuscular adaptive strategies. Competing interests The author(s) declare that they have no competing interests. Authors' contributions EA participated in the design of the study, participated in collecting the data, performed the statistical analysis, and drafted the manuscript. DR participated in collecting the data. EH participated in the progress and revision of the manuscript. TF participated in the design of the study, and in the progress and revision of the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544556.xml
549066	Wound healing and inflammation genes revealed by array analysis of 'macrophageless' PU.1 null mice	To define the events in wound healing that are independent of inflammation, gene expression during wound healing was profiled in wild-type mice and PU.1 null mice, which cannot raise the standard inflammatory response but which can repair skin wounds rapidly.	Background Much is known about the sequence of cell and tissue behavior that leads to repair of a mammalian skin wound [ 1 , 2 ] but we still have a rather incomplete knowledge of the portfolio of genes that drives these events. From late fetal stages onwards, tissue repair is always accompanied by a robust inflammatory response and this intimate association between wound healing and inflammation has made it difficult to dissect out the key elements of the repair process from those that are simply a consequence of inflammation and not necessary for healing. For this reason, and because adult skin healing is a complex process drawn out over several days to weeks, no systematic microarray analysis has yet been undertaken to encompass all those episodes from initial injury to the final sealing of the wound. The compelling argument for performing such a study comes from microarray investigations of genes upregulated in fibroblasts in response to serum exposure. Cluster analysis of these results hints at the roles of hundreds of genes by the similarity of their temporal profile with genes whose function as part of the serum response cascade is well characterized [ 3 , 4 ]. To overcome the problems of extended wound repair time course and to distinguish repair genes from those involved in, or a consequence of, inflammation, we have developed an incisional wound model in neonatal mice where healing is rapid and largely complete by 24 hours and we have used this model to compare wound-expressed genes in wild-type mice versus PU.1 null sibs which are genetically incapable of raising an inflammatory response because they lack key leukocyte lineages. PU.1 is an ETS family transcription factor that is crucial for several lineage decisions in hematopoetic cells; consequently, PU.1 null mice lack a number of hematopoetic cell types [ 5 ]. They are born with no macrophages or osteoclasts, and there is a late onset of neutrophil and T-cell development [ 5 ]. However, although there are no neutrophils or macrophages for recruitment to sites of tissue damage, neonatal PU.1 null mice can efficiently heal skin wounds [ 6 ]. Indeed, repair in the PU.1 null mice results in less indication of fibrosis and an altered cytokine and growth-factor profile compared to wild-type. For example, interleukin 6 (IL6) mRNA, which is robustly expressed at wild-type wound sites, is almost undetectable in PU.1 null wounds, and TGFβ1 mRNA, previously implicated in several fibrosis scenarios, is significantly reduced in PU.1 null wounds, as revealed by RNase protection analyses [ 6 ]. In this study we use Affymetrix GeneChip analysis of mRNAs collected at various time points after wounding of wild-type versus PU.1 null skin, to distinguish those key transcriptional events that are part of the tissue repair process but independent of whether or not there is an accompanying inflammatory response, from those genes that are 'inflammation dependent'. The latter are expressed either by inflammatory cells recruited to the wound, or upregulated by fibroblasts, endothelial cells, muscle cells and others at the wound site, as a consequence of inflammation. Using cluster analysis we have grouped more than 1,000 wound-induced genes according to their temporal profiles, with each cluster having a unique temporal profile of expression that correlates with a clear physiological episode during the repair process. For a small sample of genes from each of these clusters, we show in situ hybridization data that also reveals spatial resolution. Results and discussion For our wound model we chose neonatal mouse back skin which raises a robust inflammatory response to wounding that is not dissimilar to that seen at sites of tissue damage in adult skin, but which heals rapidly, such that incisional lesions are generally fully re-epithelialized by 24 hours. This compression of the repair process reduces the temporal 'noise' and thus the potential loss of gene-expression synchrony between wild-type and PU.1 null animals, which naturally will increase with time after the initial wound insult. Wounding of neonatal back skin results in rapid healing with or without an associated inflammatory response Resin histology of healing incisional wounds in neonatal mouse skin reveals closure of the wound commencing within 3 hours of the lesion; by 24 hours, the epidermal wound edges have generally met and fused along much, if not all, of the length of the wound. This is true for both wild-type neonates and for PU.1 null sibs, with the only obvious differences apparent in the histology being an absence of inflammatory cells in the PU.1 null wounds (Figure 1c-j ). As previously described, in situ hybridization studies using a c-fms macrophage-specific probe reveal large numbers of these cells drawn to the wound connective tissue just beneath the epidermal fusion seam at 24 hours after wounding wild-type skin, but their complete absence in PU.1 null equivalent wound sections (Figure 1k,l ). The same difference, although with an earlier temporal profile, is observed if wounds of wild-type and PU.1 null skin are probed with histochemical stains that reveal neutrophil influx (data not shown). These wound dynamics lead us to believe that the neonatal mouse skin wound model provides a good opportunity to analyze the transcriptional events that regulate the various tissue-repair episodes from initial activation steps through to the 'stopping' signals that occur when the tissue defect has been filled in, both in the presence and absence of an inflammatory response. More than 1,000 genes are differentially expressed post-wounding For microarray comparison, a consistent series of horizontal and vertical incisional (criss-cross) wounds were made to the back skin of 2-day-old neonatal PU.1 null mice and their wild-type littermates. Each time-matched pair ( PU.1 null and wild-type) were chosen from the same litter to reduce the possibility of differential expression 'noise' due to environmental differences. Wound tissues were harvested at either 30 minutes to identify immediate early genes, 3 hours for early tissue repair effector genes, and 12 or 24 hours to reveal later tissue repair effectors as well as inflammatory genes. Total RNA was then extracted and hybridized to Affymetrix GeneChips, and differentially expressed genes were identified by comparison of expression levels for each time point with unwounded skin samples that served as baseline controls. Genes were selected if transcript levels exceeded a twofold increase over either the unwounded baseline, or between time points, or between the wild-type and PU.1 null wounds. On the basis of these criteria, 1,001 genes were identified as wound-induced (see Additional data file 1 for an annotated database of all these genes together with full details of expression levels at all time points). Cluster analysis to group these genes reveals temporal profiles that correlate with distinct physiological episodes in the repair process Cluster analysis with Spotfire Array Explorer 3.0 software was used to organize the 1,001 wound-induced genes into groups according to the cosine coefficient similarity measurement; this includes within a group all those genes that have a similarly shaped temporal profiles, regardless of the levels of gene expression. Nine clusters were identified in this way, and of these, seven correlated with clear episodes in the repair process. The other two had profiles that, as far as we can tell, do not correspond to any currently understood step in the repair process and so were discarded for further analysis, although they appear in our supplementary data (see Additional data file 2 for median graphs of these clusters). Of the seven clusters associated with known repair episodes, five contain one or more known genes with good functional associations to that repair episode, and this encourages us to name each cluster according to that physiological episode. This does not provide definitive proof of function for any gene in that cluster, but it gives the best opportunity to predict function, particularly for expressed sequence tags (ESTs) with no further sequence information. Four clusters have profiles that are independent of an inflammatory response Four clusters of genes have profiles that are largely independent of inflammation. Genes in these clusters are expressed with similar profile whether wounds are in wild-type skin, where there is an influx of inflammatory cells, or in PU.1 null skin, where there is none. In both these situations there is full and complete repair, and so we propose that these four clusters represent the basic repertoire of repair genes that are activated during the repair response. Figure 2 shows line graphs that display the temporal profile of the median expression levels at each time point to give a representation of that cluster and these have been termed the 'activation' (Figure 2a ), 'early effector' (Figure 2b ), 'late effector' (Figure 2c ) and 'stop' (Figure 2d ) clusters. The number of genes found in each cluster is displayed on each graph. Three gene clusters correlate with various phases of the inflammatory response Three further clusters of genes represent expression profiles that correlate with the onset of inflammation and thus we consider them inflammation-associated genes. In neonatal animals, the inflammatory response is generally induced by 12 hours and is well established by 24 hours post-wounding. Two of these inflammation-associated gene clusters contain genes that are not expressed in unwounded skin or at early stages of repair; rather, they are upregulated in wild-type skin directly coincident with the onset of the wound inflammatory response but are generally not upregulated in the PU.1 null wound site at any stage. We have called these two groups of genes, the 'early inflammatory' cluster (Figure 2e ) and the 'late inflammatory' cluster (Figure 2f ). A third cluster does not display the standard inflammatory response profile as typified by the early and late inflammatory clusters. Rather, this cluster contains genes that are expressed at early stages of repair in both PU.1 null and wild-type mice but, whereas expression appears to increase in the wild-type wound coincident with the inflammatory response, the same genes are downregulated in the PU.1 null wound, where there is no inflammatory response; we have called this group of genes the 'inflammation-maintained' cluster (Figure 2g ). Nearly 100 genes are expressed with an immediate early gene profile at the wound site One of the most clear-cut clusters of genes is of those whose temporal expression profiles are suggestive of a transient, immediate early response to wounding. These genes show almost identical profiles, whether in the wild-type or PU.1 null situation, and thus are independent of an inflammatory response. We have named this group the activation cluster, as many will be kick-start activators of the various cell behaviors that together comprise the wound-healing process. This cluster is dominated by transcription factors and contains several well known immediate early genes, such as Egr1 ( Krox 24 ), JunB , Myc , and I-Kappa-Bα ( Nfkbia ). We present a heatmap for the 90 genes in this cluster arranged with the most highly expressed at the top of the map (Figure 3a ). Heatmaps provide a visual representation of temporal profiles only, and so for a small sample of these genes we also include in situ hybridization data on wounded skin sections to illustrate which cells and tissues express that particular gene. This spatial expression profile reveals expression in the in vivo setting, giving clues to the function of that gene during repair. Krox24 (Figure 4a ) has previously been shown to be transiently induced in both embryonic and adult mouse wounds [ 7 ]. In situ hybridization reveals Krox24 to be expressed by those epidermal cells extending back 10-12 cell diameters from the cut edge of neonatal wounds and all the associated hair follicles within this zone also (Figure 4b ). MKP-1 (Figure 4c ) is a dual-specificity phosphatase with close homology to Drosophila puckered, which has been shown genetically to be key in braking the Jun N-terminal kinase (JNK) cascade activated during morphogenetic episodes such as dorsal closure in the fly embryo [ 8 ]. In situ hybridization shows that the front few rows of wound epidermis express MKP-1 , although expression extends less far back from the wound edge than for Krox24 (Figure 4d ). By analogy to Drosophila morphogenetic episodes, it may be that MKP-1 operates as suppressor of MAP kinase (MAPK) signaling by phosphorylation of extracellular-regulated kinases 1 and 2 (ERK1 and 2), and so may actually function as a brake on the earliest tissue movements activated at the wound site. Expression of Fos-like antigen 1 ( Fosl1 , Fra1 ), has previously been associated with epithelial migrations during tumorigenesis but has not been analyzed in a wound-repair model [ 9 , 10 ]. Fosl1 has a classic activator temporal profile (Figure 4e ) and a similar spatial profile to Krox24 , with high levels of expression in wound-margin epidermal cells but somewhat weaker expression in damaged hair follicles at the wound site (Figure 4f ). Its close relative c-fos has previously been shown to be upregulated during repair of embryonic skin wounds [ 11 ], and in vitro studies show that blocking wound-induced fos induction may hinder cell migration [ 12 ]. Because cluster analysis allows us to group genes together that are likely to have similar functions [ 13 ], the temporal profiles of, as yet, uncharacterized ESTs in the activation cluster implicates them as having an immediate-early activator function during repair. A good example of such a gene is EST GenBank accession number AI853531 (Figure 4g ), which is weakly homologous to human Mitogen-Inducible-Gene-6 ( Mig-6 , Gene 33 ). The exact function of Mig-6 remains elusive but it has been shown to interact with Cdc42, a member of the Rho family of GTPases, via the activation of stress-activated protein kinases (SAPKs) [ 14 ]. In situ hybridization reveals clear expression of this gene in wound fibroblasts (Figure 4h ); together with its potential Cdc42 effector status and its induction in quiescent fibroblasts upon mitogenic stimulation and expression in many human cancer cell lines [ 15 ], this suggests that Mig-6 may mediate a fibroblast migration signal. The remaining genes in the activation cluster all have very similar temporal profiles, suggesting that they too may have important roles in activating or modulating early cell behavior at the wound edge. A further 200 genes are also expressed independently of inflammation, but with later onset and a less transient time course Two further clusters of genes have increased expression levels post-wounding in a manner that is also inflammation-independent but where expression occurs at a later time than with the activation genes. The profiles of these two clusters are temporally distinct from one another and so we have called them the early effector and late effector clusters. Between them they contain 184 genes that fit the expected profile of genes that might direct re-epithelialization and granulation tissue assembly events. The temporal profiles of all these genes can be seen by heatmap in Figure 3b (early effector cluster) and Figure 3c (late effector cluster). These two clusters contain varied types of tissue repair effectors such as tissue remodelers, genes encoding extracellular matrix (ECM) proteins, those involved in the signaling machinery and structural genes required for cell migration. Again, we provide here several examples of genes within these clusters with accompanying in situ hybridization data to provide an insight into the spatial localization of some genes in these clusters. Map4k4, a member of the serine/threonine protein kinase family that activates the JNK and MAPK signaling pathways in response to stress signals, cytokines and growth factors [ 16 ], is a member of the early effector cluster. The temporal profile (Figure 4i ) and expression of Map4k4 , in both keratinocytes up to 10-12 cell diameters from the wound edge and a subset of dermal fibroblasts extending a similar distance back from the wound edge (Figure 4j ), confirms the activation of this intracellular signaling cascade at sites of tissue repair. The JNK pathway has recently been shown to have a role in Paxillin regulation during fibroblast migrations triggered by in vitro scratch wounds [ 17 ], and so expression of Map4k4 is also suggestive of a cell migratory regulatory role for this signaling pathway in keratinocytes and fibroblasts during in vivo repair. Also in the early effector cluster, retinol binding protein-1 (Rbp1), a Fabp/p2/Crbp/Crabp family retinol transporter is expressed in wound epidermal cells approximately 15 cell diameters back from the wound site (Figure 4k and 4l ). This suggests a role for retinoids in re-epithelialization of the wound, and indeed, there is some evidence that these molecules can trigger epidermal proliferation via heparin-binding epidermal growth factor (HB-EGF) expression in suprabasal epidermal cells [ 18 ]. Typifying the late effector profile is Keratin 6 ( K6 ), a classic wound-induced gene [ 19 ] (Figure 4m ). K6 encodes a nonconventional keratin which is thought to facilitate the packaging up of other intermediate filaments in activated keratinocytes, so that these cells can migrate forward to re-epithelialize the wound [ 19 ]. High levels of expression of K6 by the front 10-12 rows of wound-edge keratinocytes were confirmed by in situ hybridization (Figure 4n ). Interestingly, another member of the late effector cluster, the intracellular Ca 2+ -binding protein MRP8 (S100A8) is expressed in a similar temporal and spatial pattern to K6 (Figure 4o and 4p ). MRP8 binds to keratin filaments as an MRP8/14 heterodimer in a Ca 2+ dependent manner [ 20 , 21 ] and is postulated to interact with these keratin filaments and guide cytoskeletal rearrangements during tissue repair [ 22 ]. The temporal and spatial coexpression of K6 with MRP8 may highlight a relationship between them and as such reveals another advantage of cluster analysis - the ability to identify potential interactions between genes and genetic pathways within the same cluster. Not all functionally related genes cluster together, however. The heterophilic binding partner of MRP8 is MRP14, which does not appear in the same cluster but rather is expressed within the early inflammation cluster (see later), since, in addition to keratinocyte expression, it is expressed at high levels by wound leukocytes. As both the MRP8/MRP14 heterodimer and a homodimer, MRP8 is a potent chemoattractant [ 22 , 23 ] and, interestingly, the MRP8/14 heterodimer also has an entirely different role, operating as a wound antimicrobial factor, although the MRP14 subunit seems to be responsible for this activity [ 24 ]. The pleiotropic activities of MRP8/MRP14 may reflect different functions of monomeric versus complexed subunits. A final cluster of inflammation-independent genes may indicate players in the 'contact inhibition' stopping process At the end of the repair process many of the cell behaviors that drive repair - such as migration and proliferation - clearly need to cease as tissues re-establish approximately their pre-wound state. This will be a gradual process and yet we might expect to see such genes depressed during the repair period and becoming upregulated as wound edges meet and closure is finishing. We see a cluster of genes with exactly this profile, suggesting that some of these genes are re-expressed to control the later stages of repair. We have loosely termed this the stop cluster. Because of their known biology, several genes in this cluster make ideal candidates for players in the processes of contact inhibition and epithelial fusion that occurs as cells from the two epidermal wound fronts confront one another. The Eph receptors and their ligands, the ephrins, have features that might make them ideal for sensing and responding to stop cues. In vitro studies show that both ligand- and receptor-bearing cells become activated upon cell-cell contact [ 25 , 26 ], and this interaction leads to a repulsive response by receptor-expressing growth cones during the developmental wiring of the nervous system [ 27 ]. Further evidence for ephrin-mediated control of epithelial sheet movement and fusion comes from studies in Caenorhabditis elegans , where Eph receptor mutants display defects in the movement of epidermal cells over neuroblasts, and in Eph knockout mice, where various morphogenetic epithelial fusions fail, leading, for example, to cleft palate and hypospadius [ 28 , 29 ]. All these results suggest that the transcriptional regulation of EphB1 revealed in the heatmaps for our stop cluster (Figure 5a ) may reflect a functional role in the stopping or final fusion episodes of wound re-epithelialization. Similarly, the expression levels of the receptor Notch also dip and rise during the repair period, and in situ hybridization studies reveal that this transcriptional regulation is also occurring within leading wound-edge epidermal cells (Figure 5b-e ). Notch has exceptionally complex biology with several ligands, including Delta and Serrate, and is a widely used as a signaling cassette at various stages of embryogenesis, and has been shown to be downregulated in several invasive tumors [ 30 ]. In Drosophila , Notch signaling has been implicated in the contact inhibition and fusion events that occur during dorsal closure at the end of embryogenesis (A. Martinez-Arias, personal communication), and during gut cell migratory episodes, which are also dependent on transcriptional activation of the short stop gene [ 31 ], the mammalian orthologue of which, Actin crosslinking family 7 (ACF7), is another member of our wound stop cluster. Several other genes within the stop cluster have characteristics that indicate they may be involved in sensing contact-inhibition cues or be downstream of these signals and operate to adhere epidermal fronts together. They include genes for Plexin 3 (Plxn3), a member of the plexin family of semaphorin receptors [ 32 ], Desmocollin 3 (Dsc3), which is a cadherin component of intercellular desmosomal junctions [ 33 ] and ACF7, a cytoskeletal linker protein [ 34 ]. As with the other clusters, suggestive biology is no proof of function, and it is worth noting that several other genes with this temporal profile do not have biology suggestive of a role in these late stages of wound healing. We feel that this cluster, more than any other, can only hint at function, and definitive function testing using knockout or knockdown assays will be necessary to investigate any speculative roles in the repair process. Expression of 200 genes at the wound site is dependent on the inflammatory response A comparison of those genes expressed during the repair process in wild-type versus PU.1 null mice reveals most clearly genes that are dependent on the presence of an inflammatory response at the wound site. The heatmaps for early and late inflammatory gene clusters strikingly reveal robust expression in wild-type wounds, but little or no expression in the PU.1 null mice for these genes (Figure 6 ). Together, the early and late inflammatory clusters comprise 169 genes that are not expressed in unwounded wild-type skin or at early stages of repair but appear to be upregulated in the wild-type wound directly, coincident with the onset of the inflammatory response. The early inflammatory cluster typically contains genes whose expression is upregulated rapidly in the wild-type, often reaching a peak by 12 hours (Figure 6a ), coincident with the influx of neutrophils to the wound site. In the late inflammatory cluster, expression typically peaks a little later, at 24 hours post-wounding (Figure 6b ), more suggestive of a link to the later influx of macrophages. A further 17 genes are initially expressed at both the wild-type and PU.1 wound site, but are maintained at high level only in the wild-type wound, where there is an influx of leukocytes. In PU.1 null wounds, where there is no such influx, these genes are only transiently expressed. We assume that expression of these inflammation-maintained genes (Figure 6c ) is directly or indirectly dependent on signals released by inflammatory cells. Inflammation-dependent genes may be expressed by leukocytes or by host cells as a 'response signature' to inflammatory signals Genes that are expressed only in wild-type wounds and whose temporal expression patterns are coincident with the influx of neutrophils and/or macrophages will include those genes that are constitutively expressed by one or both of these lineages, or genes that are upregulated as part of the leukocyte activation state, or may be expressed by cells other than the invading leukocytes as a downstream consequence of host fibroblast, endothelial and muscle cells being exposed to signals from these leukocytes. We present a selection of in situ hybridization studies to illustrate each of these scenarios as revealed by very distinct classes of spatial expression pattern. Early inflammatory cluster L-plastin (Lcp1) is a pan-leukocyte, calcium-dependent, actin-bundling protein that has previously been implicated in macrophage activation and migration, although it is also overexpressed in many types of malignant human tumors [ 35 ]. It is first expressed in the wild-type wound coincident with the early stages of the wound inflammatory response, with a peak of expression at 12 hours post-wounding; our temporal data indicate no expression at any stage in PU.1 null wounds (Figure 7Aa ). In situ hybridization studies reveal intense expression by leukocytes clustered within the wild-type wound site but no expression in surrounding skin (Figure 7Ab ), and they confirm the absence of expression in PU.1 null wounds (Figure 7Ac ). The wound-restricted expression pattern of L-plastin suggests that expression of this gene is limited to activated leukocytes only. Also expressed by leukocytes in the early inflammatory cluster are C3, a key component of the classical and alternative complement pathways, and its receptor, C3R. C3 is expressed at similar levels in unwounded PU.1 null and wild-type skin, but whereas expression is rapidly upregulated by 30 minutes post-wounding and continues until 24 hours in wild-type wounds, upregulation of C3 is delayed and much weaker in the PU.1 null wound (Figure 7Ad-f ). This delay in C3 expression suggests that inflammation has a significant role in raising and maintaining a rapid complement response at the wound site. Onzin also appears as a member of the early inflammatory cluster; it encodes a leukaemia-inhibitory factor-regulated protein that has previously been identified in a screen for genes controlling inflammatory dermatitis [ 36 ]. Unwounded wild-type skin expresses Onzin at low levels but it is completely absent in PU.1 null, unwounded skin and remains so until 12 hours post-wounding, when it is upregulated, but to a much lesser extent than in wild-type (Figure 7Ag ). In situ hybridization studies reveal a rather similar expression pattern in both wild-type and PU.1 null wounds (Figure 7Ah,i ). This suggests that Onzin might be expressed in wild-type skin by resident inflammatory cells and in the PU.1 null wound, either by inflammatory cells whose development is delayed, such as T cells, or that there may be an alternative or compensatory mechanism of gene regulation in non-inflammatory cells at the wound site. As discussed previously, both the genes for MRP8 and its binding partner MRP14 are upregulated by wound-edge keratinocytes. Both are also expressed by leukocytes, and in the case of MRP14 this expression predominates and leads to cluster separation of the two genes, with MRP14 categorized as part of the early inflammatory cluster. In the wild-type wound, it is expressed from 3 hours, with expression peaking at 12 hours post-wounding, whereas in the PU.1 null, expression does not begin until 12 hours post-wounding and levels are much reduced compared with wild-type (Figure 7Aj ). In situ hybridization clearly shows MRP14 to be expressed, in addition to expression in keratinocytes, in the region of the wound populated by inflammatory cells in the wild-type only (Figure 7Ak,l ), and indeed, previous experiments suggest that both neutrophils and macrophages express MRP8 and MRP14 [ 22 ]. It may be that genes expressed by host connective-tissue cells at the wound site as a consequence of inflammatory signals are detrimental to healing and lead to some of the imperfect aspects of repair seen in adult healing such as fibrosis and scarring. One candidate for such a gene is Osteopontin ( Spp1 , minopontin ), encoding a glycoprotein that can mediate cell-matrix interactions via the engagement of a number of adhesive receptors (reviewed in [ 37 ]). Previous wound-healing studies on Spp1 null mice report differences from wild-type in that repair is characterized by abnormal macrophage debridement and abnormal maturation of collagen bundles [ 38 ]. Osteopontin has a clear inflammation-related profile (Figure 7Am ) and in situ hybridization reveals an unusual pattern of expression at the wild-type wound site, with some expression by a subset of leukocytes but with most positive cells located in what appears to be the deep dermal or muscle layers of the wound region (Figure 7An,o ). Both the early and late inflammatory clusters contain chemokine and growth factor receptors unique to leukocytes, and presumably used by these cells to detect various chemotactic cues that will guide them to the wound site. For example, the gene for chemokine receptor 1 ( CCr1 ), a receptor for several chemokines including MIP-1α, CCL5 and Scya7, is expressed as early as 3 hours post-wounding, with expression levels peaking by 12 hours. There is no expression at the PU.1 null wound site (Figure 7Ap ). In situ studies show CCr1 to be expressed in the wild-type wound by leukocytes recruited to the wound site (Figure 7Aq,r ). As well as chemokine receptors, chemokines themselves are found in these clusters. CXCL10 ( IP-10 ) encodes an α-chemokine that functions as a potent chemoattractant for macrophages and T cells, and is upregulated by 12 hours in wild-type wounds but is absent in PU.1 null wounds (Figure 7As ). In situ studies reveal intense staining by what could be either leukocytes or host fibroblasts at the wild-type wound site (Figure 7At,u ). Either this chemokine is an amplifying chemotactic signal expressed by leukocytes to draw in further leukocytes, or its expression is triggered in fibroblasts, but only if they receive signals from the first influx of neutrophils. Late inflammatory cluster Cathepsin S is a typical gene of the late inflammatory cluster, being highly upregulated at 24 hours post-wounding in the wild-type, but with no expression in the PU.1 null wound (Figure 7Ba ). Cathepsin S is one of a large family of leukocytic proteases - this one largely macrophage-specific - that catalyze the remodelling of ECM proteins. In situ hybridization studies in the wild-type wound show Cathepsin S to be expressed by macrophages clustered around the wound site, but also by cells in the dermis at skin sites well away from the wound (data not shown), suggesting that it is constitutively expressed by cells of the monocyte lineage, rather than being part of the macrophage activation profile. No expression of Cathepsin S is seen in wounded or unwounded skin of the macrophageless PU.1 null mouse (Figure 7Bb,c ). Repetin is an epidermal differentiation gene and a member of the fused gene subgroup of the S100 family that encodes multifunctional epidermal matrix proteins [ 39 ]. This temporal profile at the wound site implicates Repetin as being responsive to inflammatory signals (Figure 7Bd ), and yet in situ hybridization studies reveal it is not expressed by inflammatory cells, but rather by leading-edge keratinocytes in both wild-type and PU.1 null wounds (Figure 7Be,f ). While not absolutely dependent on inflammatory signals, it appears that Repetin expression by wound keratinocytes is significantly enhanced by inflammatory cues. As several studies have shown somewhat enhanced rates of re-epithelialization where one or more components of the inflammatory response are reduced during healing [ 6 , 40 , 41 ], it is tempting to speculate that genes like Repetin , which are upregulated in the wound epidermis in response to inflammatory signals, may in some way retard the re-epithelialization process. As with the early inflammatory cluster, there are several genes in the late inflammatory cluster that may directly or indirectly, via their effects on signaling pathways, be responsible for wound fibrosis. The angiotensin II receptor has previously been implicated in mediating the fibrotic response in several tissue injury situations, such as myocardial infarction [ 42 - 45 ]; its gene is also a member of the late inflammatory cluster but is expressed at both wild-type and PU.1 null wounds. Expression is clear in both wild-type and PU.1 null wounds but significantly higher in the wild-type (Figure 7Bg ). The spatial expression pattern of Angiotensin II receptor is reminiscent of Osteopontin in the early inflammatory cluster, with the brightest staining in the deep dermal or muscle layer of the wild-type wound and only very faint expression seen at the PU.1 null wound site (Figure 7Bh,i ). Presumably, a subset of genes found in these inflammatory clusters, which are upregulated by host granulation tissue lineages rather than by leukocytes, may turn out to be markers, or direct regulators, of the fibrotic response that is routinely activated in adult wound granulation tissue. Clearly, therapeutic reduction of the products of these genes at the wound site might result in the reduction of wound fibrosis. Inflammation-maintained cluster A final cluster of genes appears to be regulated by the inflammatory response in that they are generally expressed at early stages post-repair in both PU.1 null and wild-type mice, but, whereas their expression subsequently diminishes in the PU.1 null mouse, expression is maintained, or increases, coincident with the inflammatory response in wild-type wounds. This temporal expression profile is most clearly visualized from heatmap data (Figure 6c ). Some of the genes in this cluster implicate mast cells in the recruitment of other leukocyte lineages which then amplify the inflammatory signal. For example, Mast Cell Protease 5 (Mcpt5) is a serine chymase stored in the secretory granules of mast cells and acts as a potent chemoattractant [ 46 ]. Mcpt5 is rapidly and transiently upregulated immediately post-wounding and by 12 hours is back to near basal levels in the wild-type wound. However, it is secondarily upregulated at 24 hours. Expression is also clear at the PU.1 null wound site as an immediate response but levels remain low and there is no second peak of expression (Figure 7Ca ). In situ hybridization studies show expression by scattered cells within the wild-type wound, with low levels of expression detected at the PU.1 null wound site also (Figure 7Cb,c ). These data suggest that Mcpt5 is initially expressed independently of signals from macrophages and neutrophils, but that leukocytes are subsequently responsible for a secondary expression, either directly by expressing Mcp5 themselves, or indirectly by triggering expression in another cell type, possibly supplying cues that reinforce expression by mast cells or prevent their dispersal from the wound site. Chemokines are also represented in this inflammation-maintained cluster. CCL2 and CCL7 are C-C chemokines with roles in directing the cellular composition of the inflammatory response. They are upregulated at 3 hours with expression tailing off by 24 hours post-wounding in the wild-type. In the PU.1 null wound, CCL2 and CCL7 are also upregulated at 3 hours but to a lesser degree than in the wild-type, and unlike in the wild-type, expression is immediately downregulated, so that by 12 hours post-wounding there is a complete absence of expression (Figure 7Cd,g ). This suggests that expression is enhanced and maintained in the wild-type by the presence of macrophages and neutrophils, whereas in the PU.1 null wound, initial expression is independent of these leukocytes but without them expression cannot be amplified and maintained. Our in situ studies suggest that these chemokines are expressed by host wound connective-tissue cells rather than leukocytes at both the wild-type and PU.1 null wound sites (Figure 7Ce,f , and Ch,i ). Conclusions Here we report an Affymetrix GeneChip microarray study of in vivo wound healing using a neonatal mouse wound model where all phases of the repair process are compressed into a 24-hour period. Cluster analysis of wild-type wounds versus those of PU.1 null mice that are genetically incapable of raising an inflammatory response allow us to distinguish repair genes from those involved in, and a consequence of, wound inflammation. Several previous studies have modeled wound healing in vitro by exposure of fibroblasts to serum, as tissue damage to blood vessels in vivo leads to exposure of connective-tissue cells to blood serum [ 3 ]. Our results show that for the earliest phases of the repair process, this model does indeed mirror the in vivo repair response. When we consider only those genes present on both experimental microarrays, we see that more than 40% of the genes that are upregulated with an immediate early profile at the wound site have previously been shown to have similar temporal profiles after in vitro activation of fibroblasts. Other in vitro models of repair also turn out to be rather good predictors of the in vivo response. For example, exposure of keratinocytes to keratinocyte growth factor (KGF) in vitro reveals many of the early and late effector genes that are expressed by keratinocytes at the wound edge [ 22 ]. Other aspects of the repair process, in particular the stopping phase where migratory and proliferative behaviors cease as wound edges confront one another, have not yet been successfully modeled in vitro , but our study shows that even in the complexity of in vivo healing, many hints as to the genes responsible for these episodes can be gleaned by microarray surveys. Clearly, the most novel aspect of our study is its capacity to highlight those gene responses that are specifically associated with, or a consequence of, the wound-activated inflammatory response. Those genes expressed at the wound site by virtue of their being expressed by the invading leukocytes provide clues as to the migratory machinery of leukocytes in vivo , informing us, for example, which chemokines might be key attractive cues by revealing which of the chemokine receptors are expressed by these cells. Of most therapeutic significance are those genes expressed as a consequence of inflammation by host wound fibroblasts, endothelial and muscle cells. These genes are clearly not absolutely essential for repair, or PU.1 skin wound not heal. Rather, they will include genes that contribute to the negative side effects of inflammation at the wound site including retarded re-epithelialization and fibrosis. Dissecting out exactly which genes from the inflammation-associated clusters might sit in such a category will be a major goal of our future studies. How full a survey of the wound healing process is revealed by our microarray study? The Affymetrix GeneChip we used covered approximately half of the mouse genome and so we cannot claim this to be a saturation screen. Moreover, it has not escaped our attention that several well established wound players that we know to be represented on the chips are apparently absent from any of the wound clusters. This is true for several growth factors, most notably TGFβ1, which we have previously shown to be differentially expressed in wild-type versus PU.1 null wounds in RNase protection assays [ 6 ], and the same may be true for several other classes of genes expressed at low copy number. A similar observation was made in a recent serial analysis of gene expression (SAGE) study of Drosophila genes expressed downstream of JNK signaling, which highlighted many such genes but revealed barely any change in expression of the TGFβ family member dpp , for which there is excellent genetic evidence for downstream activation by JNK signaling [ 47 ]. For these reasons it is clear that our screen underestimates the numbers of genes associated with each of the repair episodes and is perhaps somewhat biased towards genes expressed at higher copy number. As we have highlighted throughout this paper, revealing a temporal expression profile that coincides with one of the physiological episodes of the repair process in no way proves function for a wound-expressed gene. Although limited to a small sample of genes, our in situ hybridization studies add spatial resolution to this dataset, revealing whether a gene is expressed by the wound epidermis or connective tissue cells, or by inflammatory cells, but given the vast array of genes expressed at the wound site, how can one dissect each of their roles during the repair process? Using recent developments in Cre driver lines, it will be possible to knockout genes specifically in appropriate cell lineages within the mouse, so that the requirement for a particular chemokine receptor in the recruitment of inflammatory cells can be assessed, or the link between expression of a candidate 'fibrosis' gene by wound fibroblasts and subsequent scarring, can be tested. Complementary studies in which mRNAs for candidate inflammation/fibrosis genes are simply knocked down by local delivery to the wound site of antisense oligodeoxynucleotides (AS ODNs) [ 48 ] will provide a testbed for whether such approaches may be of therapeutic benefit to improve the repair process. While mammalian models may remain the best guide for potential clinical application, it may be faster and more efficient to turn to simpler, more genetically tractable organisms to trawl through the vast numbers of candidate repair and inflammation genes revealed by microarray studies. Indeed, we and others [ 49 - 51 ] have begun to use Drosophila as a testbed to dissect the genetics of some aspects of the repair process and to determine by mutant analysis precisely which genes are required for which repair episodes. In summary, we present here a portfolio of genes expressed during the in vivo wound healing process and categorized according to the physiological episodes that best correlate with their temporal expression profile. Such a classified listing provides good clues to the genetic regulation of all the cell behaviors that contribute to healing, and supplies us with a pool of genes whose modulation may prove to be therapeutically beneficial to the repair process. Materials and methods Mice and wounding Generation and PCR genotyping of PU.1 mice has been described previously [ 6 ]. Tail-tip blood smears were stained with Giesma (Sigma) for rapid identification of null individuals by the absence of neutrophils. Two-day pups received local anesthetic and full-thickness incisional wounds were made to a 1 cm × 0.5 cm area of the back skin. For the microarray study, a criss-cross network of 10 × 8 incisional wounds were made with a scalpel (Figure 1a ) such that all cells within the rectangle of skin were adjacent to a cut. For in situ hybridization studies, three incisional wounds were made along the long axis of this patch of skin (Figure 1b ). Subsequently, PU.1 null mice and control sibs were maintained with daily antibiotic injections until sacrifice at appropriate times. For microarray experiments, full-thickness back skin was dissected and immediately transferred, without fixation, into liquid nitrogen for subsequent RNA extraction. Wound harvesting and cryostat tissue sectioning for immunohistochemistry and in situ hybridization were performed after perfusion fixation with 4% paraformaldehyde in PBS as described [ 6 ] with tissue sections cut at 14 μm. RNA extraction and preparation for hybridization Total RNA was isolated from all skin samples using RNAzol (Biogenesis) according to the manufacturer's instructions with a secondary clean-up stage using an RNeasy kit (Qiagen). RNA was amplified and labeled with biotin as described previously [ 52 ]. Array hybridization and scanning Double-stranded cDNA was generated from 10 μg total RNA using Superscript Choice kit (Life Technologies) with a T7-poly(T) primer. Approximately 1 μg cDNA was used to generate biotinylated cRNA by in vitro transcription using Bioarray High Yield RNA Transcript Labelling kit (Enzo Diagnostics Inc). Fragmented cRNA (10 μg) was hybridized in 100 mM β-mercaptoethanol, 1 M NaCl, 20 mM EDTA, 0.01% Tween20, 0.1 mg/ml herring sperm DNA, 0.5 mg/ml acetylated BSA, 50 pM control oligonucleotide and eukaryotic hybridization controls, to Affymetrix MGU74A arrays at 45°C for 16 h. Arrays were washed using Affymetrix protocols in nonstringent buffer (6x SSPE buffer, 0.01% Tween20, 0.005% antifoam) at 25°C and stringent wash buffer (100 mM MES buffer, 0.1 M NaCl, 0.01%Tween20) at 50°C and stained with streptavidin phycoerythrin (10 μg/ml) including an antibody amplification step. Arrays were scanned using an Affymetrix confocal scanner. Analysis of GeneChip data The data were analyzed using Microarray Analysis Suite version 4.0 (Affymetrix). The data were scaled to a target intensity of 300. Values representing genes not expressed are unreliable and are given an absent call. All genes were subjected to a filter to identify genes with at least one present call across the full eight time points and that had on one or more occasion a greater than twofold change in gene expression levels between time points or between the wild-type and PU.1 null. The remaining genes were sorted into nine clusters using Spotfire Array Explorer 3.0 software, of which seven correlated with clear physiological episodes of repair or inflammation and we have loosely named those clusters according to these criteria. Resin histology and c-fms in situ hybridization Wound tissues were processed for resin histology with sections cut at 5 μm and stained with Toluidine Blue as previously described [ 6 ]. To visualize macrophages we carried out in situ hybridization studies using the macrophage-specific c-fms probe, using the protocol outlined in [ 6 ] and see below. In situ hybridization Probe details are available in Additional data file 3. In situ hybridization on frozen sections was performed as previously described [ 53 ], with probe hybridization carried out in a humidity chamber at 55°C for 16 h. Expression was visualized by BCIP/NBT precipitation (Roche Biochemicals) and sections viewed on a Zeiss Axiphot microscope after mounting in Citifluor (UKC). This spatial expression information gave us a good indication of which were the expressing cell lineages in the wound site, particularly for epidermal keratinocytes (most superficial cell layer), and leukocytes (scattered individual cells in the wound granulation tissue), but without double staining with lineage-specific antibodies we cannot be definite, particularly in distinguishing specific leukocyte lineages from subpopulations of wound fibroblasts and so forth. Additional data files The following additional data are available with the online version of this paper. Additional data file 1 , wound gene bioinformatics, is an Excel file containing an annotated database for the 1,001 differentially expressed genes in the nine temporal gene clusters: activation, early effector, late effector, stop, early inflammatory, late inflammatory; inflammation-maintained, and excluded clusters 1 and 2. For each gene this database provides an Affymetrix ID and GenBank description, together with absolute fluorescence levels and absence/presence calls for each time point, and known functional information gained from GenBank and Swiss-Prot databases. Additional data file 2 , excluded clusters, contains line graphs displaying the temporal profile of the median expression levels at each time point to give a representation of excluded clusters 1 and 2. Additional data file 3 , in situ hybridization probe source, is a Word document listing the origins of the various RNA probes used in the in situ hybridization studies. Additional data file 4 , full array expression data, is an Excel file containing the raw data - absolute fluorescence levels and absence/presence calls - for all genes on the Affymetrix MGU74 arrays at all experimental time points. Supplementary Material Additional data file 1 An Excel file containing an annotated database for the 1,001 differentially expressed genes in the nine temporal gene clusters: activation, early effector, late effector, stop, early inflammatory, late inflammatory; inflammation-maintained, and excluded clusters 1 and 2. For each gene this database provides an Affymetrix ID and GenBank description, together with absolute fluorescence levels and absence/presence calls for each time point, and known functional information gained from GenBank and Swiss-Prot databases Click here for additional data file Additional data file 2 Line graphs displaying the temporal profile of the median expression levels at each time point to give a representation of excluded clusters 1 and 2 Click here for additional data file Additional data file 3 A Word document listing the origins of the various RNA probes used in the in situ hybridization studies Click here for additional data file Additional data file 4 An Excel file containing the raw data - absolute fluorescence levels and absence/presence calls - for all genes on the Affymetrix MGU74 arrays at all experimental time points Click here for additional data file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC549066.xml
552320	Cancer/testis antigens and gametogenesis: a review and "brain-storming" session	Genes expressed both in normal testis and in malignancies (Cancer/ Testis associated genes – CTA) have become the most extensively studied antigen group in the field of tumour immunology. Despite this, many fundamentally important questions remain unanswered: what is the connection between germ-cell specific genes and tumours? Is the expression of these genes yet another proof for the importance of genome destabilisation in the process of tumorigenesis?, or maybe activation of these genes is not quite random but instead related to some programme giving tumours a survival advantage? This review collates most of the recent information available about CTAs expression, function, and regulation. The data suggests a programme related to ontogenesis, mostly to gametogenesis. In the "brain-storming" part, facts in conflict with the hypothesis of random CTA gene activation are discussed. We propose a programme borrowed from organisms phylogenetically much older than humans, which existed before the differentiation of sexes. It is a programme that has served as a life cycle with prominent ploidy changes, and from which, as we know, the germ-cell ploidy cycle – meiosis – has evolved. Further work may show whether this hypothesis can lead to a novel anti-tumour strategy.	Introduction Cancer/Testis (CT) antigens are a group of tumour antigens with gene expression restricted to male germ cells in the testis and to various malignancies. Their function in tumours is enigmatic and a common between testis genes (gametogenesis) and cancer remains elusive. When the causal link is not evident, it is tempting to believe the association is random, and assign it to general aspects of "genome instability in cancer". However, we believe a more direct link may exist. As suggested in this review, possible clues may be found in the common evolutionary pathway between ploidy cycles in meiosis and polyploidy in tumour cells. The latter, along with CT antigen expression, is a characteristic feature of well-progressed tumours. However, before discussing such possibilities, it is necessary to review the established literature. The search for tumour antigens began in the 1960's with two groups identifying first alpha-fetoprotein (AFP), a serum marker for hepatoma and germ-cell tumours [ 1 ], and then carcinoembryonic antigen (CEA), a serum marker for colon and other epithelial cancers [ 2 ]. These antigens were discovered using heterogenous sera acquired by immunizing laboratory animals with human tumour material. However, only during the 90's did both cellular [ 3 , 4 ] and humoral [ 5 ] immune responses to human tumours get proper molecular definitions. The first CTA, MAGEA-1, was identified in 1991 by Boon and colleagues using T-cell epitope cloning, a very complicated and time-consuming method [ 3 ]. In 1995 the SEREX (serological expression cloning) technique to identify tumour antigens was developed by Pfreundschuh and colleagues [ 5 ], which remains the leading approach to identifying new antigens that elicit humoral immune responses. Besides MAGEA1, BAGE, and GAGE1 discovered by T-cell epitope cloning, SEREX very soon displayed more tumour antigens with a cancer/testis restricted expression profile (SSX2, NY-ESO-1, and SYCP-1). The term "cancer-testis (CT) antigen" was introduced by Chen et al. [ 6 ], who recognized this group of genes had little in common except their expression profile. By initial definition, expression of genes coding for CT antigens should be restricted in normal tissues to male germ cells in the testis and to malignancies of various histological types. However, the criteria proposed in the 90's are not true for all antigens of this group as seen today. Furthermore, for many of the recently discovered gene products with the described expression profile, no T-cell recognized epitopes have hitherto been identified. This is why CTA – "Cancer/Testis Associated" is a more appropriate name for this family of genes (and will be used in this context further in this review), because a lot of its members still need to be proven as possessing antigenic properties in cancer patients. Attributing genes to the CTA gene family is based on several characteristic features [ 7 , 8 ]: 1. Predominant expression in germ cells of the testis and generally not in other normal tissues. 2. Expression in a number of malignant tumours of various histological types. 3. Mapping of the gene to the X-chromosome 4. Membership of a multigene family. 5. Antigenic properties in tumour-bearing patients. Some exceptions to these criteria for certain CTAs will be described and discussed later. Expression of CTA genes To date, 89 individual CTA genes or isoforms have been described, which are organised in to 44 families (see additional file 1 ). From these, 19 families are testis-restricted, and 11 show additional expression in one or two somatic tissues. Nine are expressed in 3–6 tissue types besides testis, and 5 are ubiquitously somatically expressed. With the exception of the testis-restricted CTAs, the others also show expression in the pancreas but at levels as much as 10 × lower than in testis (based on mRNA expression levels from [ 9 ]). Expression of CTA was first shown in melanoma and all the classic CTA are expressed in this type of tumour, but since the 1980's, expression in various other tumours has been recognised ( additional file 2 ). The expression pattern of CTAs during spermatogenesis is of special interest. Functional analysis of these genes during gametogenesis might well give some clues about their possible role in tumours. Their expression is restricted exclusively to spermatogenic germ cells with other tubular cells (e.g. Leydig and Sertoli) being negative. This fits well with the findings of Yuasa et al. [ 10 ] who demonstrated that CTAs have much higher expression frequencies in the germ cell cancers (seminomas) than non-seminomas.. Different CTAs are expressed during different stages of spermatogenesis (Fig. 1 .), so one may imagine that their functions are versatile, starting from regulation of mitotic cycling in spermatogonia, association with the meiotic cycle in spermatocytes, and finalizing with acrosome maturation in sperm. Figure 1 Expression of CTAs during male germ cell development. References for Fig. 1: [25] [27] [28] [37] [40] [42] [53] [92] [93] [94] [95] [96] In normal tissues, expression of NY-ESO1, MAGE-A3, -A4, and -A8 through -A11 as well as of several members of the XAGE gene family is found in the placenta. NY-ESO1 and several XAGEs are also expressed in the fetal ovary [ 11 - 13 ]. Regulation of CTA expression The mechanisms involved in regulation of CTA expression have recently been comprehensively reviewed by Albert Zendman et al [ 14 ]. Thus, only a short recitation of some of the main points is provided here. Mostly, methylation processes are responsible for the ectopic derepression of CTA genes. Using the demethylating agent, 5-aza-2-deoxycytidine (5DC), expression of several CTAs in cultured tumour cell lines can be induced/upregulated [ 15 ]. 5DC entraps DNA methyltransferases in a complex with DNA, which leads to progressive loss of DNA methylation, thereby releasing transcriptional blockage. Such upregulated expression has been reported for several MAGE members – LAGE-1, SSX-2, CAGE, NY-ESO-1. For MAGE-A1, demethylation is necessary and sufficient for gene expression, suggesting demethylation is the primary mechanism of transcription control [ 16 ]. In this context, a recent discovery of a CTA, viz . 'Boris', is interesting. Boris is reported as being expressed in several types of malignancies, and normally plays a major role in regulating methylation processes during spermatogenesis – it removes imprinting from genes during the last mitotic division of spermatocytes (reviewed in [ 17 ]). Several lines of evidence indicate that expression of some CTAs is dependent not only on demethylation, but on other transcriptional mechanisms. Histone deacetylase (HDAC) inhibitors, on their own or in combination with 5DC, can also induce CTA expression, including MAGE, SSX, and NY-ESO-1 family members [ 15 ]. The CTA-rich region in Xp11.21-22 (e.g. SSX, MAGE-B) may escape X-chromosomal inactivation, but these genes are not normally expressed in females [ 18 ]. While global hypomethylation is common and prominent in colorectal cancer, few CTAs have ever been reported as expressed in this type of cancer [ 19 ]. Non-demethylation dependent induction of MAGE expression has been demonstrated by Park et al. [ 20 ], demonstrating that 40 mM NaCl induces the transcriptional and translational activation of MAGE-B1 and -B2 in specific tissues at hypertonic conditions. There exist definite expression patterns (sets) of different CTAs in certain tumours. Marked heterogeneity of CTA expression is found in cells of some tumours, which cannot easily be explained by a global demethylation process [ 21 - 24 ]. The mechanisms of ectopic transcriptional activation of CTA genes clearly needs more investigation. Function Information regarding the function and cellular localization of CTAs is far less comprehensive. Often the proposed function is based purely on sequence homology with another protein of a known function. The only CTA proteins functionally established in gametogenesis are SCP-1, involved in chromosome pairing during meiosis [ 7 ], OY-TES-1 which functions in acrosin packaging in the acrosome of sperm heads [ 25 ], SPO11 acting as a meiosis-specific endonuclease [ 26 ], and BORIS, which is involved in cancellation of imprinting by epigenetic reprogramming during the final round of mitosis in spermatogenesis [ 27 ]. However, contrary to the situation in meiosis where it is rapidly degraded after the meiotic prophase in spermatocytes, SCP-1 expression in tumours is not cell cycle restricted [ 7 ]. BORIS is a paralog of CTCF. CTCF is a highly versatile 11 zinc-finger factor involved in various aspects of gene regulation – X chromosome inactivation, reading of imprinting sites, etc. [ 27 ]. During spermatogenesis, Boris is expressed later than many other CTA genes ([ 27 ]; Fig. 1 .). Suggestions for the functions of other CTAs mostly arose from studies of their homology with some well-known proteins and their domains, TSP50 being protease-like, CT17 phospholipase-like, and CT15 metalloproteinase-like [ 28 ]. The CT15 gene encodes a disintegrin and metalloproteinase (ADAM) domain 2, which is a member of the ADAM protein family [ 29 ]. Members of this family are membrane-anchored proteins structurally related to snake venom disintegrins, which have been implicated in a variety of biologic processes involving cell-cell and cell-matrix interactions, including fertilization, muscle development, and neurogenesis. This member is a subunit of an integral sperm membrane glycoprotein called fertilin, which plays an important role in sperm-egg interactions [ 30 ]. It is a membrane metalloproteinase with a possible role in tumour evasion and metastasis. LDHC, the germ cell-specific member of the lactate dehydrogenase family, escapes from transcriptional repression, resulting in significant expression levels in virtually all tumour types tested. It might contribute to the constitutive activation of an anaerobic pathway in tumours, because its expression in tumours is not dependent on hypoxia [ 31 ]. Suggestions as to the role of MAGEs with a CTA expression profile mainly depend on studies of their ubiquitously expressed family members (e.g. Necdin, MAGE-D1, NRAGE, Dlxin-1). In general, the data point to a role for MAGEs via transcriptional regulation in cell cycle control and apoptosis. F.ex, Necdin-related MAGE proteins differentially interact with the E2F1 transcription factor and the p75 neurotrophin receptor [ 32 ]. The high level of homology among members of the MAGE family in both mouse and human suggests an important function both in testis and cancer. The products of the SSX genes belong to the family of highly homologous synovial sarcoma X (SSX) breakpoint proteins. These proteins may function as transcriptional repressors; SSX1, SSX2 and SSX4 genes have been involved in the t (X;18) translocation characteristically found in all synovial sarcomas [ 33 - 35 ]. This translocation results in the fusion of the synovial sarcoma translocation gene (SYT) on chromosome 18 to one of the SSX genes on chromosome X. The encoded hybrid proteins are probably responsible for transforming activity. In the nucleus of sarcoma cells, both diffuse and speckled localisations of SSX protein have been reported [ 36 , 37 ]. The HOM-TES-85 protein has structural peculiarities that are shared exclusively with the N-myc oncoprotein. However, functional studies are required for confirmation [ 38 ]. Besides, the C-myc proto-oncogene is a normal participant of spermatogenesis (Fig. 2 ). Figure 2 Oncogene expression during spermatogenesis (mainly in mice). From [70]. BRDT is similar to the RING3 protein family. It possesses 2 bromodomain motifs and a PEST sequence (a proline, glutamic acid, serine, and threonine cluster) characteristic of proteins that undergo rapid intracellular degradation). The bromodomain is found in proteins that regulate transcription [ 39 ]. PLU-1, a large multi-domain nuclear protein also has a strong transcriptional repression activity. It is a member of the ARID family of DNA-binding proteins. Plu-1 mRNA and PLU-1 protein are both highly expressed in the mitotic spermatogonia. The expression is reduced in the early prophase I stages (leptotene, zygotene), but reappears at pachytene, still being detectable in diplotene cells. It is located diffusely over the nucleus. PLU-1 might have a role in regulating meiotic transcription, restricted to certain meiotic stages [ 40 ]. The protein encoded by the SPANX gene targets the nucleus and associates with nuclear vacuoles and the redundant nuclear envelope in sperm cells [ 41 ]. In situ hybridization of human testis sections showed SPAN-X mRNA expression in round and elongated spermatids [ 42 ]. These redundant nuclear envelopes have a unique structure of limited chromatin sheets continued as annulate lamellae. Both these enigmatic structures have also been described in intact lymphomas [ 43 ] and irradiated lymphomas [ 44 ]. The protein encoded by IL13RA1 gene is a subunit of the interleukin-13 receptor. This subunit forms a receptor complex with IL-4 receptor alpha, a subunit shared by IL-13 and IL-4 receptors. This subunit serves as a primary IL-13-binding subunit of the IL-13 receptor, and may also be a component of IL-4 receptors. This protein binds tyrosine kinase TYK2, and thus may mediate the signalling processes that lead to the activation of JAK1, STAT3 and STAT6 induced by IL-13 and IL-4 [ 45 ]. SGY-1 (soggy-1), a secreted protein related to the Dickkopf protein family, is involved in suppressing the Wnt signal-transduction pathway controlling transcription activation of genes such as c-myc, c-jun, Fra, and cyclin D1 by preventing the accumulation of beta-catenin [ 46 , 47 ]. Wnt proteins are implicated in a wide variety of biologic processes including cell fate determination and patterning in early embryos, and in cell growth and/or differentiation in certain adult mammalian tissues [ 48 ]. Wnts can induce proliferation in different types of stem cells [ 49 , 50 ]. The importance of Wnt signalling during tumorigenesis has been recently emphasised [ 51 , 52 ]. NY-ESO-1 is one of the most immunogenic and therapeutically promising CTAs but functional studies on this gene are severely lagging behind its practical application. Unlike the majority of CTA genes, NY-ESO-1 stops its expression in well-progressed tumours, so it can be used as a marker to follow the early progression of testicular tumorigenesis [ 53 ]. CAGE [ 54 ] and HAGE [ 55 ] code for proteins with helicase-like features. Probably, it might be involved in recombination exchange in testis and recombination DNA repair in tumours. TPX-1 is now seen as an integral protein of the outer dense fibres and the acrosome of spermatids in rats [ 56 ]. In summary, we see that the functions of individual CTA, when known, are very diverse, including, for example, both activators and repressors of proliferation and transcription. Immunogenicity Immunogenicity in cancer patients is elicited only by short peptide sequences of CTA epitopes, which are presented on the tumour cell surface by HLA Class I molecules in the case of cytotoxic T lymphocyte (CTL) mediated immune responses and by HLA Class II molecules on the surface of APC (antigen presenting cells), in the case of T-helper cell (T H ) mediated immune responses. Identification of these epitopes is one of the main goals of CTA research. Knowledge of epitopes recognized by the immune system allows the creation of the tumour-specific vaccines. In the vaccination process, T-cell epitopes are often administered together with different adjuvants or cytokines, or delivered using peptide pulsed autologous dendritic cells, all of which are aimed at enhancing the immune reaction [ 57 ]. Currently efforts are being made to identify HLA class II restricted epitopes in order to promote T H responses, which are required to support the activity of CTLs, and provide a more "complete" immune response. However, vaccines designated to prime the immune system against tumours expressing various CTA have so far shown only partial clinical success [ 57 - 59 ]. Since the technique used to identify candidate tumour antigens has changed from T-cell epitope cloning [ 3 , 60 ], and SEREX (serological analysis of cDNA expression libraries) [ 5 , 61 , 62 ] to differential gene expression analysis by various techniques like RDA (representative difference analysis) [ 63 ], DD (differential display) [ 64 ] and SSH (suppression subtractive hybridization) [ 65 ] – and even further to bioinformatics – we cannot really be sure about the adequate use of the definition "antigen" in relation to CTA gene products. Whilst the former two techniques are dependent on the immunogenicity of specific epitopes in cancer patients, the latter ones are simply based on mRNA expression levels or even sequence homologies detected via search engines and provide no answer about immunoreactivity. In additional file 1 , one can see that an immune response is documented against only 19 of 44 CTAs. Immune recognition of the majority of these CTAs is cancer-related, occurring spontaneously in cancer patients, not in cancer-free individuals. The exceptions to this are humoral immune responses to MAGEB1/CT3.1 in systemic lupus erythematosus patients [ 66 ] and to SPA17/CT22 in vasectomised men [ 67 ]. CTA can be considered to be tumour specific. There exists the blood-testis barrier [ 68 ], which prevents the immune system from contacting with CTA gene products. Besides this, germinative cells do not express HLA class Ia molecules [ 69 ], so they cannot present their expressed proteins to the immune system. For these reasons, the immune system never comes into contact with these proteins and recognizes them as "non-self" structures. Brain-storming The function of most CTAs is unknown, although some role in regulation of gene expression (both activating and repressing) seems likely [ 8 ]. Are CTAs oncogenes? By definition, oncogenes are normal cellular genes participating in proliferation cascades, which are abnormally activated in tumours [ 70 ]. Therefore, with the exception of one (HOM-TES-85, which has structural homology with the myc-oncogene) CTAs can not formally be regarded as oncogenes. Are CTAs simply activated by the imbalanced genome, due to its instability in tumours? Some, such as SSX, whose ectopic expression is caused by the SYT-SSX fusion due to translocation t (X;18) in synovial sarcomas, certainly are. However, a large number of CTA genes are only located on the X-chromosome and this chromosome is not a specific site of chromosome breaks and translocations usually associated with tumours [ 71 ]. However, the chromosome region 20q13.2 containing the BORIS gene is commonly amplified or exhibits moderate gains of material in many human cancers. This has strengthened the idea that this region contains a major oncogene [ 72 , 73 ]. A preliminary report using RT-PCR has found that BORIS expression is detectable in over half of ~200 cancer cell lines studied, representing most of the major forms of human tumours, discussed in [ 17 ]. However, this observation awaits further confirmation. Theoretically, the aberrant expression of the amplified (by chance) Boris, which by analogy with CTCF may demethylate CTA genes located on X-chromosome, may in turn activate a large body of CTA genes in human malignancies. However, why are other gametogenesis-related CTA genes also activated from other chromosomes? It does not look like a chance event and therefore amplification of "Boris" may still not be random. Diversity of functions, including genes involved in ontogenesis suggest that CTAs are activated either as a result of the genome instability (however, why testicular genes?) or as part of a complex program. Also from this point, this looks like a program related to gametogenesis. The same idea was proposed by Old [ 8 ]. If CTAs generally do not enhance tumour growth except by stimulating proliferation, like oncogenes, another possibility is that they could do it by stimulating DNA repair or inhibiting apoptosis (combination of all three is possible). Indeed, some CTAs have relation to the DNA repair factors by homologous recombination. These are SPO11, SYCP1, helicase-like CAGE and HAGE acting in meiotic prophase of gametogenesis. In turn, homologous recombination in tumours was shown to act anti-apoptotically [ 74 ]. Are these CTAs restricted only to prophase of meiosis, where recombination takes place? It appears not. For example, some are associated with the spermatogonial stage (Plu-1 mRNA and PLU-1), and some with maturation of the acrosome (see Fig. 1 ). It is a program again, and why only male gametogenesis? But it also occurs in oogenesis [ 8 ]. Thus, prophase of meiosis (pairing and recombination) may be still the most important. So, ectopic activation of CTA genes is not entirely random, being induced from the sexual X-chromosome but also from loci on other chromosomes, relating to gametogenesis. However, it is curious then that the very common proto-oncogenes of proliferatives cascades also participate in gametogenesis, e.g. myc, ras, jun, etc. This can be seen in Fig 2 , (taken from the Janis Erenpreiss [ 70 ]). He and others postulated a link between gametogenesis and cancerogenesis before CTAs were revealed [ 75 - 77 ]. In turn, Old [ 8 ] looked at the problem from a new angle and suggested that CTAs provide a causal link between gametogenesis and cancer. This seems plausable, but does this illegitimate program in tumours embrace, even mosaically, only gametogenesis? May be only the DNA recombination repair component is common? But the CTA genes are repressing a wide Wnt family, which also functions in early embryogenesis. The program sounds more like an ontogenetic (life-cycle) one. Let us remember the experiments by Mintz and Illmeisee [ 19 ] who cloned normal genetic mosaic mice by introducting the nuclei of malignant teratocarcinoma into enucleated eggs. Both gametic, parthenogenetic and trophoblastic theories of cancer have also been proposed in the past [ 8 , 70 ] and can be viewed as embryonal or ontogenetic theories of cancer. Another question is whether CTA genes govern a life-cycle-like program with its key events similar to meiosis? In turn, to which process does meiosis provide a key – recombination and reduction division? Are tumours capable of these? We also have to consider why X-chromosomes are involved, and not Y-chromosomes, which are responsible for sex? Perhaps this is because the X-chromosome is better conserved evolutionary and appeared prior to sex discrimination. So, this looks like an ontogenetic program, which evolutionary preceded sexual (amphimictic) life-cycle, with the key in recombination and reduction division; but what is it? The answer is evident – ancient ploidy cycles [ 78 , 79 ]. In general, ploidy cycles are displayed as a cyclic increase and reduction of ploidy (chromosome number), and these involve the pairing of homologous chromosomes and their segregation, omitting one round of DNA replication. There may be reduction of ploidy 2n-n (sexual meiosis) or from nx to 2n DNA numbers, in the asexual ploidy cycles characteristic for a few protists, as in Amoebae and foraminiferans [ 80 , 81 ] as well as part of ontogenetic programs in the more developed taxons [ 82 , 83 ]. Contrary to gametic reduction in sexual meiosis, the reduction of ploidy in polyploid somatic cells is called "somatic reduction". In this context, expression of some CTAs by the placenta is of interest because trophoblast and decidua are the only mammalian tissue capable of endoreduplication creating enormously large ploidies [ 84 ]. an ability shared by many tumour cells. The latest studies on a silver fox revealed somatic reduction in the giant cells of a trophoblast [ 85 ]. Now let us return to DNA recombination repair in tumours as a means of survival, as already mentioned. Repair by homologous recombination can protect malignant tumour cells from apoptosis. In particular, as shown in our laboratory, endopolyploid cells employ this mechanism [ 86 ]. Likewise, expression of CTAs – endopolyploidy – is a hallmark of malignant tumour progression where there is deficient TP53 function [ 87 ]. In turn, some giant tumour cells show the capability to segregate their genomes and return to mitosis ([ 86 , 88 - 91 ], see Fig. 3 ). Figure 3 Reductional mitotic divisions generating low ploidy cells from one large polyploid cell. Non-treated Burkitt's lymphoma cell line, DNA staining with Toluidine-blue. × 2,500. Conclusion A hypothesis is put forward that activation of at least some of the CTA genes in p53-deficient human tumours could be due to the genetic program running "relic" ploidy cycles in tumour cells. This hypothesis offers new opportunities for the design of novel tumour treatment strategies. In particular, passive therapy using CTAs to prime the host immune system against the tumour could be replaced with gene therapy aimed to block the function of CTA gene products or even their expression. This approach is promising, because normally only the germ cells in the testis express CTA genes and they are well protected by the blood-testis barrier. Thus, there should not be any problem with tumour-specific priming, and respectively we would predict there to be no side effects. Supplementary Material Additional File 1 "Chromosomal localization, type of immune response, identification method and identification references for all known 44 CTA gene families". Click here for file Additional File 2 " CTA Frequency (%) of expression in various tumour types". Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC552320.xml
549067	Full-length cDNAs from chicken bursal lymphocytes to facilitate gene function analysis	This article reports a cDNA collection representing more than 2000 new, full-length transcripts from a high-quality cDNA library.	Background Large-scale genomic and cDNA sequencing projects have revealed thousands of new genes whose open reading frames (ORFs) are highly conserved during vertebrate evolution, but whose precise cellular functions remain unclear. Although functional analysis by gene disruption is possible after transfection of murine embryonic stem cells and the breeding of knockout mice [ 1 ], these whole-animal studies are laborious and expensive. If the mutant phenotype can be distinguished in cell culture, the chicken B-cell line DT40 is a valid alternative to murine knockouts because of its high ratio of targeted gene integration [ 2 - 4 ]. Additional advantages of DT40 are tightly regulated conditional gene-expression systems for the analysis of essential genes [ 5 - 7 ] and the ability to study genetic interactions by the stepwise modification of multiple loci [ 8 ] and marker recycling [ 7 ]. The recent release of the chicken genome sequence [ 9 ] greatly benefits the DT40 research community. For the first time, the entire genome can be searched for sequences that are conserved during vertebrate evolution and whose function might be clarified after genetic modification in DT40. However, in silico gene structure prediction methods have a high error rate and often do not correctly annotate the intron-exon structure of genes. Only full-length cDNAs unambiguously define the boundaries of the transcription units within whole-genome assemblies and cloned full-length cDNAs are also of immense practical value to complement mutant phenotypes and artificially express the encoded protein [ 10 ]. For these reasons, many genome sequencing projects in higher eukaryotes have been complemented by large-scale efforts to obtain a maximum number of full-length cDNAs [ 11 , 12 ]. Although relatively large expressed sequence tag (EST) databases from bursal lymphocytes [ 13 ] and other tissues have been described [ 14 ], relatively few chicken cDNA sequences had been deposited in the public databases. Here we describe a project to sequence and characterize a large number of full-length cDNAs from bursal lymphocytes. The corresponding genes are likely to be expressed in DT40 and this should facilitate their analysis by targeted gene modifications. In combination with the recently released cDNAs from other tissues [ 15 ], the bursal cDNAs will be a valuable resource for many laboratories working with the chicken as a model organism. Results and discussion Generation of bursal cDNA sequences The overall strategy for producing the greatest possible number of new full-length cDNAs expressed in bursal lymphocytes is outlined in Figure 1 . We previously described a cDNA library of bursal lymphocytes, but it contained only a low number of full-length cDNA clones [ 13 ]. It was therefore decided to synthesize a new cDNA library, called 'riken1', using the biotinylated cap trapper method which is optimized to generate a large percentage of full-length cDNA inserts [ 16 ]. To assess the quality of the library and guide the selection of clones for full insert sequencing, the 5' ends of over 14,000 clone inserts were sequenced. BLAST [ 17 ] searches against the public protein databases indicated that about 80% of the 11,116 high-quality ESTs obtained showed significant homology to existing entries and more than 80% of these extended further upstream than the methionine start codon of their homologs in the databases. This indicated that the riken1 library indeed contains an extraordinary high percentage of full-length cDNA inserts. Only clones whose ESTs showed significant BLAST matches against the public protein databases and covered the methionine start codon of their homolog were considered for full-length sequencing, as evolutionarily conserved genes are of highest interest for the DT40 research community. The remaining ESTs were clustered to remove duplicates corresponding to the same gene. In addition, ESTs corresponding to already known chicken genes in the public databases were removed. The plasmids corresponding to the remaining 2,796 ESTs were chosen for full insert sequencing by bidirectional primer walks. Once the end of the walks had been reached, the sequences of the full-length cDNA inserts were assembled. From the BLAST search results the most likely methionine start codon was assigned to each sequence. About 15% of the cDNA sequences showed evidence for premature frameshifts in the form of short ORFs and stretches of conserved sequence in a different frame further 3'. If overlapping ESTs were found in the public databases, the cDNA sequences were edited to correct the likely reverse transcription error, otherwise these sequences were discarded. Length distribution and GC content A total of 2,272 high-quality chicken full-length cDNA clones were sequenced and assembled, manually annotated with respect to their likely translation start codon and deposited both at The Bursal Transcript Database website [ 18 ] and in the public databases. The lengths of the proteins encoded by the annotated ORFs were compared with the lengths of UniProt [ 19 ] database entries and the lengths of the untranslated region (UTR) sequences were compared with the lengths of known vertebrate UTRs available from the UtrDB collection [ 20 ] (Figure 2 ). The distributions obtained for the bursal cDNAs closely resemble those calculated for known sequences. Most of the 5' UTRs have lengths in the range of 100 base-pairs (bp) [ 21 ], a value conserved in diverse taxonomic classes. The length distribution of 3' UTRs is much broader, with a significant number of long sequences exceeding 1 kilobase (kb). The similarity between the length distributions observed for the collection presented here and those sequences stored in public databases suggests that most of our sequences are full-length cDNAs with correctly annotated start codon positions. The most remarkable feature noted in the analysis of 5' UTRs of the bursal cDNAs is a very high GC content (67%). This supports the observation that the GC content of 5' UTRs is particularly high in warm-blooded species [ 22 ]. On the other hand, the percentage of GC base-pairs in 3' UTRs of the bursal cDNAs (41%) is close to the value observed for database sequences (42%). The ORFs of the bursal full-length cDNAs contain 49% GC base-pairs. Analysis of start codon context The accurate prediction of the translation start codon remains difficult and in some cases our annotations remain tentative. Sequences surrounding the translation start codons are not random and in mammals match the consensus GCCRCCaugG (where aug is the start codon and R is either A or G) [ 23 ]. The most conserved nucleotides in the consensus are a purine, usually A, at position-3 and G at position 4. It has also been observed that a large fraction of 5' UTRs contain AUG codons upstream of the translation start site, but these codons are unlikely to be flanked by the consensus sequence [ 21 ]. A detailed analysis shows that the riken1 collection of cDNA sequences contains 4,406 AUG codons upstream of the annotated translation start codons in 2,218 of the bursal cDNAs. Nine hundred one of these alternative start codons were in the same reading frame as the annotated ORF. An in-frame stop codon within the 5' UTR region was present downstream of 501 of these 901 alternative start codons. The total number of ORFs present in 5' UTR regions of riken1 cDNAs was 1,289. We have checked whether the context of the annotated AUG start codons differs from the context of the alternative upstream AUG sequences of the bursal cDNAs. We therefore extracted 10-bp long sequences surrounding the annotated start codons and the alternative upstream AUGs and visualized sequence variability using the sequence logo software [ 24 ] (Figure 3 ). The annotated start codons closely match the consensus, but the alternative upstream AUG codons do not exhibit flanking nucleotide preferences. This provides further evidence that the ORFs in our collection are correctly annotated. Similarity to predicted Ensembl transcripts and UniProt protein sequences All full-length cDNAs were compared to the collection of transcripts predicted from the chicken genome sequence by the Ensembl system [ 25 ]. The transcripts were downloaded before the Ensembl team used our collection of full-length bursal cDNA sequences to improve transcript predictions. Distribution of the percent identity and coverage of the best BLASTN alignments are shown in Figure 4a . Only 494 of the chicken full-length transcripts matched predicted mRNAs with a length coverage greater than 90%. This is not surprising taking into account that computational prediction of untranslated regions, based on the genome sequence alone, is very difficult, if not impossible. However, there were also significant differences between sequenced and predicted cDNAs within ORF regions. There are 1,463 sequences in which either the 5' or the 3' end of the ORF was not covered by predicted transcripts. In most cases (1,106), the discrepancy concerned the 5' end. The statistics presented above and summarized in Table 1 indicate that our collection of full-length cDNA sequences may be used to significantly improve the annotations of more than 1,400 chicken genes. This analysis is further supported by the mapping of bursal serial analysis of gene expression (SAGE) tags to Ensembl transcripts and the genome sequence [ 26 ]. Figure 4b shows the distribution of the percent identity and coverage statistics of the BLASTP comparison of the proteins encoded by the bursal cDNAs to the UniProt collection of protein sequences. In most cases (1,524), the proteins encoded by riken1 cDNAs were almost fully covered in the alignments (more than 90% coverage) and showed a high percentage identity (greater than 70%) to known protein sequences. When compared to available chicken ESTs or cDNAs in the public databases, some of the bursal cDNAs showed significant structural differences most likely due to differential transcript processing. In addition, the bursal cDNA collection has been used to define a large number of intragenic single-nucleotide polymorphisms (SNPs) [ 27 ]. Functional domain assignment All full-length cDNAs were compared to the Pfam database [ 28 ], which stores sequence profiles representing functional domains and the 10 most frequently occurring domains are shown in Table 2 . Subsequently, we have used the Gene Ontology (GO) [ 29 ] annotation of Pfam domains provided by the InterPro [ 30 ] database to assign functional descriptors to the domains detected in our sequences. It is important to note that the assignment of a GO term to a given cDNA sequence indicates only the presence of a functional domain rather than an orthologous relationship to other genes annotated by the term. Determination of orthologous relationships is best done at the level of whole-genome comparisons and is therefore beyond the scope of this study. This classification will be valuable for the selection of candidate genes for further analysis in DT40, but it is unlikely to be representative for the whole chicken genome because only a selected subset of cDNAs expressed in bursal cells were chosen for sequencing. Tables 3 and 4 list the assignments of GO molecular function and biological process descriptors to functional domains detected in riken1 full-length cDNAs. The most frequent molecular function associated with a domain is 'ATP binding' (166 cDNAs) assigned to the protein kinase domain and other domains such as the AAA ATPase family, ABC transporters and others. The 64 DNA-binding proteins represented in our collection include RNA polymerase II, C5-cytosine-specific DNA methylase and 19 proteins exhibiting transcription factor activity according to biological process annotation. GTP-binding proteins are involved in translation initiation (eIF-2-gamma), cell-cycle regulation (Septin 5) and regulation of transport from the endoplasmatic reticulum to the Golgi apparatus. There are 22 full-length cDNAs in our collection containing Pfam domains annotated by the GO term 'molecular function unknown'. Experimental information concerning the molecular mechanisms of action is very sparse or nonexistent for proteins sharing these evolutionarily conserved domains. Highly similar human proteins exist for the chicken proteins, an example being the human protein BM02. Taking into account the ease of targeted genome modification and availability of numerous functional assays, the DT40 cell line is an attractive model system to provide first insights into the functions of the evolutionarily conserved domains described above. Bursal Transcript database All the full-length cDNA sequences are stored within the Bursal Transcript database [ 18 ]. This database links the previously published EST data with the new cDNAs and can be searched by keyword or by using BLAST. Browsing of functional categories is also available as dynamically generated web pages link the bursal cDNAs to Ensembl, UniProt, Pfam and to GO data. To highlight gene expression differences between DT40 and bursal cells, the bursal cDNAs are also linked to SAGE data from both of these types of cells [ 26 ]. Conclusions The cDNAs from bursal lymphocytes represent one of the largest full-length cDNA collections in the chicken, comprising about one third of all currently available, experimentally verified transcripts and will be of general interest to researchers using the chicken as an experimental model as well as to the poultry industry. The resource has already been integrated with the chicken genome sequences to build a unigene catalog [ 9 ], to define the nature and frequency of intragenic chicken strain polymorphisms [ 27 ] and to develop a chicken gene microarray for gene-expression profiling (B. Wong, T. Makeev and C. Davies, unpublished data). However, the main beneficiary of the full-length cDNAs is the DT40 research community. Although the release of the genome sequence has greatly simplified the identification of candidate genes for disruption and the design of the knockout constructs, it is still not a trivial task to predict the ORFs as well as 3' and 5' UTRs without cDNA sequences. Other uses are the expression of the cDNAs in vitro or for complementation of mutant DT40 phenotypes with the added convenience that the cDNA sequences are not only known, but also available as cloned pieces of fully sequenced DNA. Materials and methods Construction of the riken1 cDNA library and 5' EST sequencing The riken1 library was synthesized from mRNA of 2-week-old CB strain bursal lymphocytes using the biotinylated cap trapper method [ 16 , 31 ]. The resulting phage library was converted into pKS-derived plasmids and individual clones were then selected on ampicillin-containing agarose plates. About 45,000 colonies were picked and transferred into 384-well microtiter plates to prepare a permanent clone stock. Plasmids from 14,976 of the arrayed clones were sequenced on an Applied Biosystems automated sequencer using a primer that anneals to the plasmid backbone upstream of the 5' end of the cDNA inserts (see [ 18 ] for details of the cloning vector sequence). The ABI sequencing files were processed as described previously [ 13 ]. About 5% of the riken1 clones contained an insert sequence which was 100% identical to the GenBank entry AJ277662, annotated as a human genomic fragment including the LMO1 locus. This sequence was present as a stuffer of the lambda vector used for the library construction and the clones containing it were removed from further analysis. In total, the 5' single-pass sequencing of 14,976 clones yielded 11,116 high-quality ESTs of the riken1 library. Selection of clones for full-length insert sequencing BLAST searches against the 'All non-redundant GenBank CDS' database showed that approximately 80% of the 5' EST sequences matched GenBank entries with a score of at least 50. This score threshold was chosen because it allowed us in most cases to align the putative start codon of the query sequence to the EST. These sequences were chosen and clustered [ 32 ] to remove duplicates. In addition, all sequences matching chicken entries in the public databases with a score of over 300 were not considered further. The BLAST results of all remaining sequences were manually inspected and only those sequences which covered the methionine start codon of their closest match in the public databases were retained. In the end, the cDNA inserts corresponding to 2,796 ESTs were chosen for full insert sequencing. Full-length insert sequencing Sufficient plasmid template for numerous sequencing reactions was prepared from the clones corresponding to the selected ESTs. All plasmids were then sequenced with a primer complementary to a plasmid sequence 3' of the cDNA insertion site. Subsequently, custom-made 20-mer primers based on available sequences were used for sequencing until the 3' and 5' ends of the cDNA inserts were reached. All sequences were processed as described previously [ 13 ], except that a routine of manual proofreading and editing of the chromatograms within the Staden pregap program was implemented to increase the quality of the base calling and to decrease the failure rate of the next primer walk. The FOUNTAIN software [ 32 ] was extended to automatically design primers in 96-well format suitable for these walks. The primers were positioned to give an average of a 70-bp overlap between sequences. Once both ends of the insert were reached by the primer walks, the Staden gap4 program [ 33 ] was used to produce a double-stranded consensus of the cDNA insert. A total of 2,565 high-quality cDNA contigs were assembled for further analysis. Quality check and correction of frameshifts in the cDNA sequences The integrity of the conserved ORF within each assembled cDNA sequence was manually examined by inspecting BLAST search results against the public protein and EST databases. To facilitate this task, a new EstSet module was added to FOUNTAIN [ 32 ]. The user interface displays the sequence of the cDNA insert together with its three possible translations and its BLAST search results against the public protein and EST databases. On the basis of this information a likely methionine start codon can be assigned to the cDNA. Around 15% of the cDNA sequences showed evidence of an artificial frameshift in the form of suspicious BLAST matches in two or more ORFs, presumably due to errors in the reverse transcription process. These sequences were compared to other Gallus gallus ESTs from the public databases. If the short ORF could be corrected by adopting the sequence of an overlapping EST, the cDNA sequence was edited. The type of editing was recorded and the corresponding riken1 clone was annotated as likely to be defective. In total, 293 cDNAs were removed because either a likely artificial frameshift could not be corrected by using sequences of overlapping ESTs or they contained multiple stop codons in all three reading frames or they showed evidence for unspliced introns. All cDNA clones are freely available upon request to the corresponding author and their sequences have been submitted to the EMBL public database (accession numbers AJ719267-AJ721138 and AJ851370-AJ851825). Analysis of the start codon context The sequences surrounding the annotated start codons (10 bp upstream and downstream) were exported and submitted for information content visualization by the WebLogo software [ 24 ]. Subsequently, we have exported the ± 10 bp context of every ATG codon located upstream of the annotated start of the coding sequence. These sequences were also submitted to analysis by WebLogo software. Sequence-similarity searches and functional class annotation The riken1 cDNAs were compared with the collection of predicted chicken transcripts downloaded from the Ensembl ftp site [ 34 ] using the BLASTN program. BLASTP software was used to compare translated ORFs with the protein sequences stored in the UniProt database. Functional domains were assigned by comparing riken1 cDNAs with sequence profiles representing Pfam domains. This comparison was performed with RPSBLAST software (e-value cut-off of 10 -6 ) run on the binary database files downloaded from the National Center for Biotechnology Information (NCBI). Functional classes were assigned according to Pfam to GO mapping provided by the InterPro database. The XML information exchange standard was used to interface the BLAST program outputs with the FOUNTAIN package.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC549067.xml
497041	Assessing harmful effects in systematic Reviews	Background Balanced decisions about health care interventions require reliable evidence on harms as well as benefits. Most systematic reviews focus on efficacy and randomised trials, for which the methodology is well established. Methods to systematically review harmful effects are less well developed and there are few sources of guidance for researchers. We present our own recent experience of conducting systematic reviews of harmful effects and make suggestions for future practice and further research. Methods We described and compared the methods used in three systematic reviews. Our evaluation focused on the review question, study designs and quality assessment. Results One review question focused on providing information on specific harmful effects to furnish an economic model, the other two addressed much broader questions. All three reviews included randomised and observational data, although each defined the inclusion criteria differently. Standard methods were used to assess study quality. Various practical problems were encountered in applying the study design inclusion criteria and assessing quality, mainly because of poor study design, inadequate reporting and the limitations of existing tools. All three reviews generated a large volume of work that did not yield much useful information for health care decision makers. The key areas for improvement we identified were focusing the review question and developing methods for quality assessment of studies of harmful effects. Conclusions Systematic reviews of harmful effects are more likely to yield information pertinent to clinical decision-making if they address a focused question. This will enable clear decisions to be made about the type of research to include in the review. The methodology for assessing the quality of harmful effects data in systematic reviews requires further development.	Background Systematic reviews are important tools for evidence-based health care. They are certainly one of the reasons for the progress that has been made in obtaining reliable evidence on the beneficial effects of interventions. A recent study of the medical literature, using Medline and the Cochrane Library, showed that the number of systematic reviews published has increased dramatically, from a single publication in the years 1966 to 1970, to 23 in 1981 to 1985, and 2467 in 1996 to 2000 [ 1 ]. Most of the systematic reviews focused on efficacy or effectiveness. However, to make a balanced decision about any intervention it is essential to have reliable evidence on the harms as well as the benefits. Although the coverage of harmful effects has increased over time, only 27% of the reviews published between 1996 and 2000 included any information about safety, and only 4% focused primarily on the safety of the intervention reviewed [ 1 ]. This is perhaps unsurprising as many authors of systematic reviews restrict inclusion to randomised controlled trials (RCTs) to minimise bias, and harmful effects are often inadequately assessed and/or reported in RCTs [ 2 , 3 ]. Another important reason for the relative lack of reliable evidence on harmful effects is that RCTs are not always suitable to evaluate them and other types of study design need to be considered [ 4 ]. The methodology for conducting systematic reviews of beneficial effects from RCTs is well established, whereas the methods for systematically reviewing randomised or observational data on harmful effects are less well developed and less often used. Only 1.25% of 3604 publications cited in the 2001 edition of Side Effects of Drugs (SEDA-24) were systematic reviews [ 5 ]. At present researchers, like us, who conduct systematic reviews have limited sources of guidance, such as the suggestions offered by the Cochrane Collaboration [ 6 ]. Fortunately, research into the methodology of incorporating harmful effects data in systematic reviews is on the increase, from which we expect more sources of guidance to emerge. It is not uncommon, even among experienced reviewers, to assume that the objective of a systematic review of harmful effects should encompass all known and previously unrecognised harmful effects and that data from all types of study design should be sought. We have re-visited three systematic reviews of drug interventions in which we had reviewed harmful effects, to evaluate our own recent experience, identify areas for improvement and to share our ideas with other researchers undertaking reviews. Methods We used three reviews for this study on the basis that they had been completed recently (between 2001 and 2003) and that one of us had been the lead reviewer of harmful effects in each review. The reviews were conducted as Health Technology Assessments for the National Coordinating Centre for Health Technology Assessment (NCCHTA) on behalf of the National Institute for Clinical Excellence (NICE). The reviews, in order of completion, were: nicotine replacement therapy (NRT) and bupropion sustained release (SR) for aiding smoking cessation [ 7 ], atypical antipsychotics for schizophrenia [ 8 ], and newer antiepileptic drugs for epilepsy in adults [ 9 ]. We described and compared the methods used in each review and the problems we encountered in applying those methods. We focused our evaluation on the review objectives, the inclusion criteria for study design and the quality assessment of the primary studies. We do not report on the matter of searching for studies about harmful effects which presents another challenge to those who conduct systematic reviews [ 10 , 11 ], because exploratory work following from the reviews described here is underway and preliminary results are reported elsewhere [ 12 , 13 ]. Results The main components of the three systematic reviews of harmful effects are described in Table 1 . Our evaluation highlighted the following aspects of the methodology that could have been improved on and others that require further development. Table 1 Description of the assessment of harmful effects in the three systematic reviews Review Schizophrenia Smoking cessation Epilepsy Intervention evaluated 8 atypical antipsychotics. NRT and bupropion SR. 7 newer antiepileptic drugs. Objective / Scope Review commissioned by the HTA programme and an update of the HTA report commissioned by NICE. The objective regarding harmful effects was to determine the incidence of specific rare adverse events to populate an economic model. Scope provided by NICE: To review all known or unknown harmful effects that might be associated with the interventions. Scope provided by NICE: To include adverse effects in a review of RCTs of clinical effectiveness in adults with epilepsy. The reviewers undertook a supplementary review of serious, rare and long-term harmful effects. Serious was defined by WHO criteria [25], long-term as longer than 6 months, and rare as defined by the authors of primary studies. Study designs included Randomised trials RCTs of atypical antipsychotics versus alternative drug treatment or placebo in schizophrenia. An existing Cochrane review was used as a source of summary data on adverse effects from RCTs of effectiveness [26]. Studies that assessed safety as the primary objective were included in the review of primary studies of harmful effects. This included RCTs that investigated aspects of clinical pharmacology that might impact on the drugs' tolerability and safety. The five most commonly reported adverse effects were extracted from RCTs as part of the review of clinical effectiveness in epilepsy. RCTs in indications other than epilepsy and dose comparisons were eligible for inclusion in the supplementary review of harmful effects. Non-randomised studies Cohort studies and case series with 2000 or more participants or at least 2 years follow-up, and case-control studies of any size or duration. Uncontrolled trials, prospective and retrospective observational studies, data from adverse events monitoring systems (e.g. UK yellow card scheme) and case reports. Non-randomised controlled trials, cohort and case-control studies, prospective case series and other uncontrolled trials, and open-label extension phases of trials. More than 300 participants had to be exposed or follow-up more than 6 months unless the study objective was to investigate a specific adverse effect. Prescription event monitoring [27], and post-marketing surveillance reports were also included. Studies identified 6477 items screened, 924 articles retrieved, and 223 studies included: 171 RCTs, 13 cohort studies, 1 case-control study, 38 case series. 1280 items screened, 353 articles retrieved, and 123 studies included: 25 RCTs, 4 non-randomised controlled trials, 30 uncontrolled trials, before/after studies or cohort studies, 1 case-control study, 9 surveillance studies, 1 survey, 53 case reports or case series. 108 RCTs were included in the review of effectiveness, selected from 4211 items screened and 887 articles retrieved. In the supplementary review of harmful effects 3884 items were screened, 227 articles retrieved, and 77 studies included: 2 RCTs, 2 non-randomised controlled trials, 26 uncontrolled trials, 14 open-label phases, 25 cohort studies, 1 case-control study, 4 prescription event monitoring studies, 3 post-marketing surveillance studies. Quality assessment Quality checklists for various study designs provided in CRD Report 4 were used [28]. The quality checklist for RCTs provided in CRD Report 4 [28], and checklists for the other study designs published elsewhere [29], were used. Published checklists were used as a starting point. Questions were amended and others added to capture information specifically on the reliability of harmful effects data. Findings of review Very few studies with useful data were found, so the economic model could not be populated with incidence rates of the adverse events of interest. Primarily the findings merely reflected the accepted side-effect profiles for NRT and bupropion SR. The review did not identify any previously unknown harmful effects. The supplementary review of harmful effects did identify reports of potential adverse effects not reported in the RCTs of clinical effectiveness. However, these were mostly effects already documented in tertiary sources. There was insufficient evidence to attribute causality of other reported effects to the test drugs. Review objectives The schizophrenia review objective appeared to be appropriate in seeking to determine the incidence of named outcomes that were considered by health economists to be most likely to lead to a change in prescribed treatment [ 14 ]. The objectives of the smoking cessation and epilepsy reviews were very broad in comparison. Given that the side-effect profiles of the drugs for smoking cessation were well established, with details available in various published standard reference texts [ 15 , 16 ], it would have been more efficient to focus the review effort on a clear question, such as the significance of seizures for bupropion SR and the cardiovascular effects of nicotine in NRT. The objective of the review of harmful effects of the antiepileptic drugs did not target clinical decision-making; the supplementary review of harmful effects might have been of real use to decision makers if we had focused on a crucial clinical question such as the safety of the drugs in pregnancy. Study designs All three reviews included study designs other than RCTs to assess harmful effects. The types of non-randomised studies included for each review reflected differences in the reviews' objectives, our judgment as reviewers as to where the most useful data were likely to be found, and was to some extent pragmatic in terms of the time available to complete the reviews. The reviews with the broad objectives included more non-randomised studies and more diverse study designs. The schizophrenia and epilepsy reviews specified a minimum size and duration of study to be included (see table) in an attempt to add data over and above what was available from the largest and longest RCTs. Doing this did involve some indeterminable risk of missing important information. The review of observational studies carried out in the schizophrenia review was necessary because the pre-determined harmful effects of interest were known to be under-reported in RCTs [ 8 ]. The inclusion of non-randomised studies in the smoking cessation review might have targeted observational data on specific questions about harmful effects had we reviewed beforehand the RCTs that were summarised briefly in the Cochrane review. Similarly in the epilepsy review all the adverse events (not just the most common) reported in the RCTs of clinical effectiveness should have been reviewed before moving on to observational studies. Applying the inclusion criteria Once the inclusion criteria for study design had been defined, applying them was problematic. Reports of primary studies rarely described the study design in sufficient detail. Many of the studies included in the schizophrenia review purported to be cohort studies but on closer examination were in fact large case series involving more than one intervention. Some of the 'cohort study' data on bupropion SR included in the smoking cessation review had actually been derived retrospectively from RCTs. How exactly the 'cohorts' had been established in studies of epilepsy was often unclear in terms of the source population, eligibility criteria, and selection, or was simply not reported. Had we, in all three reviews, only included reports of studies fitting textbook definitions of particular study designs, virtually all of the primary study reports we identified would have been excluded. The inclusive approach we took turned out to be unrewarding. In the smoking cessation review, in addition to difficulties with the study design inclusion criteria, application of the criterion to only include studies in which assessment of adverse events was the primary objective was problematic because it involved a high degree of subjective judgment. Quality assessment We encountered problems when applying published checklists in our reviews of harmful effects. The response to some questions depended on the outcome of interest, for example, follow-up may have been adequate for the assessment of the primary (usually a beneficial) outcome of the study but not for the collection of data on harmful effects. We also found that published checklists omit key features such as how harmful effects data were recorded. In the epilepsy review we were in a position to learn from the earlier reviews and spent time clarifying the questions in the checklists so that they would provide information relevant to the reliability of the harmful effects data. We also added items pertinent to reports of harmful effects such as how and when events were recorded and whether the time at which they occurred during the study was reported. Although this informed approach was a step in the right direction, the major hindrance to applying checklists in all three reviews was inadequate reporting of the basic design features of the primary studies. Once the quality criteria had been applied there remained the challenge of interpreting the results. In our reviews we described the evidence identified and tabulated the response to each checklist question for each primary study. This generated lengthy summaries that had limited utility. Even comparing validity within study designs (not across them) we found it impossible to synthesise the information as all the included studies had methodological flaws and features that could not be assessed due to inadequate reporting. Reaching a decision about which studies were likely to give the most reliable results was not straightforward. Discussion Our experience of reviewing harmful effects mirrors that of other researchers in that a significant investment of effort failed to yield significant new information [ 6 , 17 ]. A focused review question is standard practice for assessing beneficial outcomes in systematic reviews and should also be so when reviewing harms. Researchers conducting reviews need to make sure that they address a well-formulated question about harms that are likely to impact on clinical decisions. Focusing a review question about harmful effects will not necessarily mean restricting it to specific adverse events but may mean, for example, addressing a particular issue such as long-term effects, drug interactions, or the incidence of mild effects of importance to patients. If the aim of the research is to look for previously unrecognised harmful effects, analysis of primary surveillance data may be more appropriate than a systematic review [ 18 ]. Researchers also need to be aware that scopes set by external commissioning bodies, despite having consulted with national professional and patient organisations, may not be a suitable question to address in a systematic review. The wisdom of broad and non-specific questions about harmful effects should be questioned because the resources, especially time, needed to do this comprehensively are usually insufficient. It is important to realise that an unquestioning belief that observational studies are the best source of harmful effects data simply because they are not RCTs can be a pitfall. It is essential to think carefully about the review question before widening the inclusion criteria to include non-randomised study designs. Some harmful effects, such as very rare events or those emerging in the long-term, are unlikely to be addressed adequately in RCTs. But, even if observational studies are appropriate to the review question researchers should be prepared for the difficulty of interpreting observational study data outweighing the anticipated benefits. The importance of quality assessment of RCTs in systematic reviews of effectiveness is well established [ 19 ], but debate continues over the usefulness of checklists and scales. Quality assessment of other study designs in systematic reviews is far less well developed [ 20 ]. Although the feasibility of creating one quality checklist to apply to various study designs has been explored [ 21 ], and research has gone into developing an instrument to measure the methodological quality of observational studies [ 22 ], and a scale to assess the quality of observational studies in meta-analyses [ 23 ], there is as yet no consensus on how to synthesise information about quality from a range of study designs within a systematic review. Our appraisal of our reviews has shown that these difficulties are compounded when reviewing data on harms. It is essential that quality assessment is able to discriminate poor from better quality studies of harmful effects. Levels of evidence hierarchies have several shortcomings. The hierarchy of evidence is not always the same for all harmful or beneficial outcomes. For example, an RCT with adequate internal validity but limited sample size or follow-up may be a less reliable source of information about relatively uncommon harmful effects emerging in the long-term than a large well-conducted cohort study with many years of follow-up. Another problem with ranking evidence in a hierarchy is that different dimensions of quality get condensed into a single grade, resulting in a loss of information. Furthermore, the dimensions included in current hierarchies may not be the most important in terms of reflecting the reliability of a particular study's findings [ 24 ]. Researchers need to clarify a priori what exactly they need to glean from their quality assessment of the primary studies in their own review of harmful effects and it may be necessary to differentiate clearly between internal and external validity. We suggest that further research is needed to collate, assimilate and build on the existing information relevant to systematically reviewing primary studies for harmful effects of health care interventions. This should include a review of the literature pertinent to the methodology of incorporating evidence of harmful effects in systematic reviews; a description and categorisation of the methods used in systematic reviews published to date, and any evidence from methodological research on which they are based; and the development of quality assessment methods. Conclusions Appraisal of our recent experience highlighted some of the problems inherent in conducting systematic reviews of harmful effects of health care interventions. Such reviews need to address a well-formulated question to facilitate clear decisions about the type of research to include and how best to summarise it, and to avoid repeating what is already known. The review question about harmful effects needs to be relevant to clinical decision-making. A systematic review of the methodology pertinent to systematic reviews of harmful effects is warranted. Competing interests None declared. Authors' contributions AMB, HMM and NFW conducted the review work described. NFW conceived of the study. NFW and HMM drafted the manuscript. All authors contributed to, read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC497041.xml
551600	A note on generalized Genome Scan Meta-Analysis statistics	Background Wise et al. introduced a rank-based statistical technique for meta-analysis of genome scans, the Genome Scan Meta-Analysis (GSMA) method. Levinson et al. recently described two generalizations of the GSMA statistic: (i) a weighted version of the GSMA statistic, so that different studies could be ascribed different weights for analysis; and (ii) an order statistic approach, reflecting the fact that a GSMA statistic can be computed for each chromosomal region or bin width across the various genome scan studies. Results We provide an Edgeworth approximation to the null distribution of the weighted GSMA statistic, and, we examine the limiting distribution of the GSMA statistics under the order statistic formulation, and quantify the relevance of the pairwise correlations of the GSMA statistics across different bins on this limiting distribution. We also remark on aggregate criteria and multiple testing for determining significance of GSMA results. Conclusion Theoretical considerations detailed herein can lead to clarification and simplification of testing criteria for generalizations of the GSMA statistic.	Background Wise, Lanchbury and Lewis [ 1 ] introduced a rank-based statistical technique for meta-analysis of genome scans, the Genome Scan Meta-Analysis (GSMA) method, and derived its exact null distribution using a clever inclusion/exclusion argument. Koziol and Feng [ 2 ] provided an alternative derivation of the null distribution of the GSMA statistic via a combinatoric approach involving probability generating functions, and suggested an Edgeworth series approximation to its exact null distribution that improves upon the Wise [ 1 ] normal approximation. Levinson [ 3 ] described two generalizations to the GSMA statistic: (i) a weighted version of the GSMA statistic, so that different studies could be ascribed different weights for analysis; and (ii) an order statistic approach, reflecting the fact that a GSMA statistic can be computed for each chromosomal region or bin across the various genome scan studies. Wise [ 1 ] had suggested that each chromosomal region (bin) be about 30 cM, leading to a total of about n = 120 bins spanning the entire genome, and correspondingly 120 GSMA statistics. Wise [ 1 ] and Koziol and Feng [ 2 ] had investigated the marginal distribution of any of these (exchangeable) GSMA statistics, whereas under the order statistic formulation of Levinson [ 3 ], the joint distribution of the entire set of GSMA statistics is taken into account. In this note, we consider both generalizations in turn. In particular, (i) we provide an Edgeworth approximation to the null distribution of the weighted GSMA statistic, analogous to that in Koziol and Feng [ 2 ]; and (ii) we examine the limiting distribution of the GSMA statistics under the order statistic formulation, and quantify the relevance of the pairwise correlations of the GSMA statistics across different bins on this limiting distribution. We conclude with remarks concerning the Levinson [ 3 ] aggregate criteria and multiple testing for determining significance of GSMA results. Results The GSMA statistics We first introduce some notation. Let X ij , i = 1, ..., m , j = 1, ..., n , denote the rank of any particular linkage test statistic (e.g., LOD score) in the j th chromosomal region (bin) from the i th study, with each study being ranked separately. Levinson [ 3 ] rank the bins from 1 = "best" to n = "worst" on the basis of, say, maximum LOD score or lowest p value observed within each bin, but the reverse ranking from 1 = "worst" to n = "best" is also feasible. In practice, m can be as few as 4 (e.g., [ 4 , 5 ]); and, following Wise [ 1 ], n is generally about 120. The GSMA statistics are then S 1 , ..., S n , where . The exact (marginal) null distribution of each S j was derived in Wise [ 1 ]; in the notation of Levinson [ 3 ], P AvgRnk , the "pointwise probability" of any S j , is obtained from its marginal null distribution. The normal approximation to the exact distribution of the S j is straightforward: the S j are identically distributed, and each S j has an approximate normal distribution with mean and variance under the null hypothesis that ranks are randomly assigned within each study. Koziol and Feng [ 2 ] provided an Edgeworth correction to this approximation, and recommended that the correction be used, at least for m ≤ 12. The weighted GSMA statistic Levinson [ 3 ] proposed a weighted version of the GSMA statistic, namely, , with the weight w i ascribed to the i th study reflecting the relative linkage information from that study. (We are temporarily omitting the j subscript for clarity.) The normal approximation to the marginal null distribution of S w is straightforward, and depends on the two parameters and . The combinatorial argument utilized by Koziol and Feng [ 2 ] to derive the exact distribution of the unweighted GSMA statistic (which relies on probability generating functions) is generally no longer applicable in the weighted setting. Nevertheless, as in Koziol and Feng [ 2 ], we here provide an Edgeworth correction that may be applied to the weighted GSMA statistic. To this end, we equivalently consider the linear transform , where . We then have E [ R w ] = 0, , and (We have used Koziol and Feng [ 2 ], eqn. 11, for .) The Edgeworth Type A series approximation to the density of up to 4 th order terms, is f ( z ) = φ ( z )[1 + c 4 H 4 ( z )], where φ (·) is the standard normal density function, , H 4 (·) is the 4 th degree Hermite-Chebyshev polynomial, H 4 ( z ) = z 4 - 6 z 2 + 3, and the constant c 4 given by (Stuart and Ord [ 6 ], eqn. 6.42). Furthermore, the cumulative distribution function of the Edgeworth series is given simply by where Φ(·) denotes the cumulative distribution function of the standard normal distribution, c 4 and φ (·) are as above, and H 3 (·) is the 3 rd degree Hermite-Chebyshev polynomial, H 3 ( z ) = z 3 - 3 z (Stuart and Ord [ 6 ], eqn. 6.43). In practice, we would expect the Edgeworth series approximation to provide an adequate representation of the exact distribution of the weighted GSMA statistic, in a manner analogous to the unweighted case [ 2 ]. Here, we briefly investigate the adequacy of the Edgeworth approximation, using an example from Lewis et al. [ 7 ]. They had applied the GSMA methodology to data from m = 20 schizophrenia genome scans, and found strong evidence for linkage on chromosome 2q, as well as suggestive evidence for linkage at several other chromosomal locations. The rank data for each scan are available online at D.F. Levinson's website (accessed July 14, 2004) [ 8 ], and we use these data to reconstruct first the unweighted GSMA statistics S j , j = 1, 2, ..., 120, corresponding to the 120 bins spanning the entire genome, then their preferred weighted versions. Lewis [ 7 ] had recommended weights for each individual study proportional to the square root of the number of affected cases for that study. From Levinson's website [ 8 ], the individual weights w i , i = 1, 2, ..., 20, are 2.32, 1.77, 1.20, 1.17, 1.17, 1.16, 1.15, 1.08, 1.03, 1.01, 0.95, 0.88, 0.80, 0.80, 0.68, 0.67, 0.59, 0.54, 0.53, 0.51, greater than a four-fold range. We simulated the null distribution of the weighted GSMA statistic , where with m = 20, n = 120, by drawing each X i as an independent random integer from 1 to 120 (that is, a uniform distribution of the integers from 1 to 120), then forming R w with the Levinson [ 8 ] weights. We used the random number generator in R [ 9 ], to produce 10,000 values for R w . We then formed , and compared the empirical distribution of the 10,000 Z values with the Edgeworth approximation as described above; for comparative purposes, we also computed the normal approximation, which is based on matching the first two moments rather than the first four moments with Edgeworth. Figure 1 shows the resulting quantile-quantile plot of the empirical distribution of the weighted GSMA Z values with both a normal approximation, panel A, and the Edgeworth approximation, panel B. Note that, even in this setting of the weighted combination of m = 20 individual GSMA statistics, the normal approximation is particularly ill-fitting in the tails. Agreement in the tails would be of particular relevance in practical applications, as these represent the areas of potentially significant findings (p-values). The noticeable disagreement in the tails between the weighted GSMA statistic and its normal approximation is largely ameliorated with the Edgeworth approximation. With attendant computational savings, the Edgeworth approximation provides a practical means of determining significance of weighted GSMA results compared to simulation; tail probabilities derived from the normal approximation should only be used with extreme caution. The GSMA order statistics We turn next to order statistic considerations (and reintroduce the subscript j ). The Levinson [ 3 ] order statistic approach to inference relating to the GSMA statistics takes into account the inherent ordering of the S j : their P ord refers to the probability of any observed S k given the k th bin's place in the ordering of all of the S j . We here derive approximations to this distribution. Let , j = 1, ..., n , and T (1) < T (2) < ... T ( n ) denote the order statistics. We note first that the T j have (approximately) a singular symmetric multivariate normal distribution, with means 0, variances 1, and correlations . That the joint distribution is singular follows from the observation that for all i , hence, is identically 0. If we dismiss the correlations as negligible (of absolute magnitude < 0.01 for n > 100), then the T j are (approximately) independent, identically distributed N (0,1) (standard normal) random variates, and the cumulative distribution function (cdf) F k of the k th order statistic T ( k ) is given by with Φ(·) as above (David [ 10 ], eqn. 2.1.3). We briefly examine whether correlations can be ignored when determining the distributions of the T ( j ) . Numerical computation of the distributions of the order statistics from a symmetric multivariate normal distribution is feasible in a number of cases; we here examine perhaps the most relevant case, concerning the extreme T ( n ). Note that Prob ( T ( n ) ≤ x ) = Prob ( T 1 ≤ x , T 2 ≤ x , ..., T n ≤ x ); (2) this latter probability may be calculated in R using the mvtnorm package [ 9 ], based on methodology by Genz [ 11 , 12 ]. With n = 120, we depict in Figure 2 a Q-Q plot of the (approximate) distribution of T ( n ) under independence, eqn. (1), compared to the distribution from eqn. (2) with pairwise correlations . The independence model tends to agree quite closely to the correlation model in this particular case, especially in the critical right tail, and has the virtue of numerical simplicity. We remark that one might improve slightly on the normal independence model by incorporating the Edgeworth correction into the individual cumulative distribution functions in equation (1). Aggregate criteria and multiple testing Levinson [ 3 ] had proposed an aggregate criterion for detecting linkage based on both the marginal distributions and the order statistic distributions of the GSMA statistics. In particular, they argued that bins that have achieved both P AvgRnk < 0.05 and P ord < 0.05 "are the most likely to contain linked loci or to be adjacent to linked bins". Note that their criterion entails both the marginal distribution of the T j , through P AvgRnk , and the (joint) order statistic distribution of the T j , through P ord . We remark that there is some redundancy to the aggregate criterion { P AvgRnk < 0.05 and P ord < 0.05}, as can be seen through consideration of critical values relating to their aggregate criterion. With the normal approximation to the distribution of each normalized GSMA statistic T j , the criterion { P AvgRnk < 0.05} is equivalent to the criterion { T j > 1.645}. The criterion { P ord < 0.05} may be computed from eqn. (1), and depends on the ordering of the individual T j . With n = 120, then for the ten largest order statistics T (120) , T (119) , ..., T (111) , the criterion { P AvgRnk < 0.05 and P ord < 0.05} reduces to { P ord < 0.05}, since their 95 th percentiles under their joint order statistic distribution exceed 1.645 [implying that, if { P ord < 0.05} obtains, then { P AvgRnk < 0.05} will automatically be satisfied]; and, for the remaining order statistics T (110) , T (109) , ..., T (1) , the criterion { P AvgRnk < 0.05 and P ord < 0.05} reduces to { P AvgRnk < 0.05}, equivalently, { T ( j ) > 1.645}, as their 95 th percentiles under the order distribution, eqn. (1), are less than 1.645 [implying that, if { P AvgRnk < 0.05} obtains, then { P ord < 0.05} will automatically be satisfied]. We conclude with a remark concerning multiple testing. Levinson [ 3 ] suggested a simple Bonferroni correction for multiple testing when determining the significance of GSMA results. In particular, they used the criterion { P AvgRnk < 0.000417} (0.05 corrected for 120 tests) for evidence that a bin is likely to contain a linked locus or loci. One can improve on this procedure by using Holm's [ 13 ] paradigm for multiple testing rather than Bonferroni. We illustrate Holm's [ 13 ] procedure by returning to the Lewis [ 7 ] study with m = 20 schizophrenia genome scans. As noted above, we used the online data to reconstruct the normalized unweighted GSMA statistics T j , j = 1, 2, ..., 120, corresponding to the 120 bins spanning the entire genome. With m = 20 studies, we shall invoke the normal approximation to the distributions of the individual T j . Lewis [ 7 ] had extensively investigated various criteria for linkage from the 20 schizophrenia genome scans, and we shall not reproduce their analyses. Rather, we here illustrate a graphical procedure for the simultaneous evaluation of p-values from tests on the same data; this procedure is immediately applicable to the simultaneous consideration of the 120 GSMA statistics. The procedure, originally suggested by Schweder and Spjøtvoll [ 14 ], consists of a probability plot of the p-values versus the uniform distribution. Koziol [ 15 ] subsequently suggested that Holm's [ 13 ] paradigm for multiple testing be applied to Schweder and Spjøtvoll's [ 14 ] scenario, for a graphical determination of the number of true hypotheses. Let us briefly review the Holm [ 13 ] method, which is an extension of the Bonferroni method for multiple comparisons. Suppose we compare the smallest p-value P (1) among n p-values with α / n and we find that the p-value is less than α / n . Then our multiple testing problem has been reduced by one test, and we should compare the next smallest p-value P (2) to . In general, we would compare P ( i ) with . Holm's [ 13 ] step-down test begins with i = 1, comparing P ( i ) with , and stops as soon as P ( i ) exceeds , rejecting at overall level α all prior tests with smaller p-values. The Holm [ 13 ] method, like Bonferroni, makes no assumption on the dependence of tests, but beyond P (1) is less conservative than Bonferroni. In Figure 3A we present a probability plot of the 120 p-values corresponding to the 120 individual T j statistics, which we have recomputed from the online Levinson dataset [ 8 ]. On this plot, the points corresponding to the "true" hypotheses of no linkage in individual bins should roughly fall along a straight line passing through the origin. We have also superimposed the Bonferroni and Holm boundaries for overall alpha level 0.05 and n = 120 p-values; but, the two boundaries are virtually indistinguishable. There is little indication of large departures from the global null hypothesis of no linkage. In Figure 3B we rescale the y-axis, and focus solely on the Bonferroni and Holm boundaries. Differences are most readily apparent for the largest ordered p-values. On the other hand, with a large number of hypotheses (here 120), the improvement of Holm over Bonferroni at the smallest ordered p-values is marginal at best. As a reviewer has presciently remarked, the Holm procedure generally is most helpful (advantageous) relative to Bonferroni with only a small number of hypotheses. In Figure 3C we zoom in on the part of the probability plot nearest the origin; we here have superimposed the Holm [ 13 ] boundary. In accord with Lewis [ 7 ], we find that only one GSMA statistic achieves statistical significance at overall alpha level 0.05, namely, the statistic corresponding to bin 2.5. [Recall that the Holm and Bonferroni boundaries coincide at the smallest p-value, P (1) .] That is, in this particular instance, the unweighted GSMA statistics with either Bonferroni or Holm [ 13 ] correction for multiple testing identify statistically evidence for linkage on chromosme 2q. Conclusion For practitioners utilizing GSMA statistics, the question arises as to whether the methods proposed here as well as in Koziol and Feng [ 2 ] are merely of theoretical interest, or have practical import. If one utilizes solely the unweighted GSMA statistic, and chooses to consider its marginal distribution (corresponding to the P AvgRnk formulation of Levinson [ 2 ]), then the exact null distribution of the GSMA statistic is available from Wise [ 1 ] or Koziol and Feng [ 2 ], and should be preferred over any approximate methods. If the exact null distribution is computationally intractable for practitioners, then the Edgeworth approximation of Koziol and Feng [ 2 ] provides a simple and accurate means of assessing significance; we would argue that the Edgeworth approximation is preferable to a normal approximation in this instance. When weights are introduced into the GSMA statistic, then the combinatoric arguments of Wise [ 1 ] and Koziol and Feng [ 2 ] will typically be insufficient to derive the exact null distribution [though we remark that a moment generating function approach patterned after the probability generating function formulation of Koziol and Feng [ 2 ] can be brought to bear on this problem.] One can either simulate the null distribution or derive an Edgeworth approximation: we do not believe either method enjoys global advantages over the other. We caution against simple reliance on a normal approximation: in the situation investigated here, Figure 1 , the weighted combination of m = 20 individual GSMS statistics, the normal approximation is particularly ill-fitting in the tails. [Agreement in the tails is of particular relevance to practitioners, as these represent the areas of potentially significant findings (p-values).] As for the order statistic formulation and the aggregate criteria of Levinson [ 3 ], we believe that the theoretical considerations given in this paper can lead to clarification and simplification of testing criteria. Authors' contributions JAK conceived, designed and drafted the manuscript. ACF performed the statistical simulation. Both authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC551600.xml
554988	Admission to hospital following head injury in England: Incidence and socio-economic associations	Background Head injury in England is common. Evidence suggests that socio-economic factors may cause variation in incidence, and this variation may affect planning for services to meet the needs of those who have sustained a head injury. Methods Socio-economic data were obtained from the UK Office for National Statistics and merged with Hospital Episodes Statistics obtained from the Department of Health. All patients admitted for head injury with ICD-10 codes S00.0–S09.9 during 2001–2 and 2002–3 were included and collated at the level of the extant Health Authorities (HA) for 2002, and Primary Care Trust (PCT) for 2003. Incidence was determined, and cluster analysis and multiple regression analysis were used to look at patterns and associations. Results 112,718 patients were admitted during 2001–2 giving a hospitalised incidence rate for England of 229 per 100,000. This rate varied across the English HA's ranging from 91–419 per 100,000. The rate remained unchanged for 2002–3 with a similar magnitude of variation across PCT's. Three clusters of HA's were identified from the 2001–2 data; those typical of London , those of the Shire counties , and those of Other Urban authorities. Socio-economic factors were found to account for a high proportion of the variance in incidence for 2001–2. The same pattern emerged for 2002–3 at the PCT level. The use of public transport for travel to work is associated with a decreased incidence and lifestyle indicators, such as the numbers of young unemployed, increase the incidence. Conclusion Head injury incidence in England varies by a factor of 4.6 across HA's and PCT's. Planning head injury related services at the local level thus needs to be based on local incidence figures rather than regional or national estimates. Socio-economic factors are shown to be associated with admission, including travel to work patterns and lifestyle indicators, which suggests that incidence is amenable to policy initiatives at the macro level as well as preventive programmes targeted at key groups.	Background It has been estimated that 6.6% of those attending A&E in any given year have a head injury [ 1 ] and over 100,000 people are admitted as a consequence [ 2 ]. This incidence of admission to hospital following a head injury is known to vary considerably from locality to locality [ 3 ]. Although some technical issues, such as case identification and inaccurate coding may contribute [ 3 , 4 ], there is also evidence to support that demographic- and social factors such as age, gender, environment and ethnicity cause variation in incidence and outcome [ 5 - 12 ]. For example the adjusted odds ratio for age 15–24 years, and the male sex have been found to be significant independent predictors for medically attended brain injury in the USA [ 13 ]. Thus the evidence suggests that there is considerable potential for complex interactions which could elevate or depress incidence rates at the local level to a significant degree. Yet, within the UK, evidence upon which to base local service planning is scarce, as recognised in the recent report of the Health Committee of the UK House of Commons [ 14 ]. Consequently the committee recommended that a way is found 'of improving the methods of data collection on incidence, prevalence and severity of head injury and subsequent disability...' (vii). This paper makes an initial response to that recommendation and reports on the incidence of head injury leading to a hospital admission at the local level in England, and examines the socio-economic associations of any variation shown to be present. Methods Hospital Episodes Statistics (HES) record all episodes of continuous in-patient care in hospitals in England and can be obtained from the UK Department of Health. For the year 2001–2, statistics are available for the 95 Health Authorities (HA) extant at the time, and for 2002–3 for Primary Care Trusts (PCT's). Concerned with incidence of new cases, data in the current study is based upon the postcode of residence of the patient. 'First episode' was chosen as a filter variable (as approximately 10% of patients have more than one episode, that is, fall under the care of more than one consultant during their stay). A second filter identified the relevant primary cause ICD10 codes for head injury (S00.0 to S09.9). There is a long history of debate about the appropriate codes for inclusion in such studies, often surrounding the debate between 'head injury' and 'brain injury' [ 15 ]. Much was made of the fact that 'fracture of the facial bones' (ICD-9 code 802) was not indicative of brain injury, but new research has shown that excluding this group will omit many with brain injury [ 16 ]. The current study is inclusive of all codes, and so includes 'superficial injuries to the head', which nevertheless required admission to hospital. Using census population data for the same areas as the denominator, admission rates per 100,000 are calculated for all ages, and age-specific rates for 0–15 year olds, 16–74 year olds, and those aged 75 years and over. Rates are thus for admission of residents of England and their respective HA or PCT, in an English hospital. Admissions are defined as the first period of in-patient care under one consultant within one healthcare provider. The 16–74 year old band was chosen explicitly to match the census key statistics which report on various indicators for the economically active population, deemed to be 16–74 years of age. Census Key Statistics are available from the UK Office of National Statistics and include a range of demographic, social and economic variables that can be presented as percentage indicators. Those chosen for inclusion in the current study are shown in Table 1 . The indicator for qualification at level 4/5+ represents those with a first degree, a higher degree, NVQ levels 4 and 5, HNC, HND, qualified teacher, medical doctor, dentist, nurse, midwife or health visitor. Table 1 Key statistics included in the study and presented as percentage indicators. Indicator % born outside the UK % with limiting long-standing illness % permanently sick of working age % 16–24 who are unemployed % age 50+ who are unemployed % Unemployed % without any qualifications % with qualifications at grade 4/5+ % using private transport to work % using public transport to work % of households without a car % who own (or buying) their home % who rent home privately % living in overcrowded homes % lone parent families In addition, the Townsend Deprivation Index is calculated using the percentage of households with no car, not owner occupied, overcrowded and those economically active who are unemployed [ 17 ]. The latter two are transformed logarithmically, and then each variable is transformed to a normal distribution using the means and standard deviations for England as a whole. The variables are summed giving an average for England of zero, with negative values indicating less deprived areas, positive values more deprived areas. The UK Census key statistics are currently made available at the Primary Care Trust (PCT) level, and thus it was necessary to aggregate these data up to the HA level to match the 2001–2 admission data which was provided at the Health Authority level. The majority of PCT's fit neatly into the 95 Health Authority areas extant in 2002. However, there are some slight variations such that aggregated populations of the HA derived from their respective PCT's match exactly the independent figures (provided separately from the ONS) of 2002 HA populations in 90% of cases, and differ by up to 5% for the rest. For example, a small number of Enumeration Districts (the lowest level census tract) belonging to the Preston PCT, which belongs in North West Lancashire HA, are allocated to East Lancashire. No attempt was made to adjust for these slight variations in boundaries. Key statistic indicators for 2001–2 were thus derived from the aggregated PCT data, based on their aggregated population which, for a few HA's, will differ slightly from the population used to estimate the admission rates for head injury. There was a one-to-one relationship between admission data and census data at the PCT level for the 2002–3 data. Patterns of head injury and socio-economic indicators are determined by Two-Step Cluster Analysis. The procedure is an exploratory tool designed to reveal natural groupings (or clusters) within a data set that would otherwise not be apparent. The association between the various socio-economic factors, and the overall rate for head injury is explored through a multiple regression model using the 2001–2 data at the HA level. This model is validated on the 2002–3 data at the PCT level. Funding and ethics The project was funded by the UK Department of Health. The author is independent of the funding body. Ethical approval was not required. Results A total of 112,718 admissions were recorded for 2001–2. The gives an incidence rate for admission to hospital following a head injury in England in 2001–2, for all ages, of 229.4 per 100,000 (Table 2 ). Of these, 31.2% were aged 0–15; 56.2% were aged 16–74 and 12.6% aged 75 years and over. The total incidence varies by a factor of 4.6 from 90.7 per 100,000 in Brent and Harrow, to 419.4 in Liverpool. Rates for children and the elderly are much higher than for those aged 16–74 years. The highest incidence rate for children was East Lancashire at 637.7 per 100, 000 aged 0–15; the highest for the elderly was North Cheshire at 799.8 per 100,000 aged 75 years and over (See Additional File 1 ). The incidence for 2002–3 was identical at 229.1 per 100,000 all ages (Table 3 ). While the magnitude of difference for the overall all-age incidence remained the same at 4.6, at the PCT level the age-specific variability appears greater, with the highest rate for children being 881.7 per 100,000 (North Manchester, see Additional File 2 ) and that for those aged 75 years and over, 1116.0 per 100,000 in Central Liverpool. Table 2 Hospitalised Incidence rate for head injury in England in 2001–2. Estimated rate per 100,000; for those aged 0–15; 16–74; and 75 years and over, and in total. Estimates for England, and the highest and lowest incidence for health authorities. Health Authority RATE 0–15 RATE 16–74 RATE 75+ Total RATE England 355.8 178.1 383.8 229.4 Liverpool 465.9 376.9 741.5 419.4 Tees 621.7 346.9 514.5 416.6 North Cheshire 488.0 356.0 799.8 411.5 East Lancashire 637.7 298.5 638.3 399.9 Sunderland 461.4 344.2 609.9 385.0 South Essex 235.0 116.4 223.2 148.9 Barking and Havering 223.9 117.4 210.0 147.4 North Essex 256.0 103.0 268.1 146.8 Bexley, Bromley & Greenwich 218.1 105.4 181.7 134.4 Brent and Harrow 119.5 81.7 106.7 90.7 Table 3 Hospitalised Incidence rate for head injury in England in 2002–3. Estimated rate per 100,000; for those aged 0–15; 16–74; and 75 years and over, and in total. Estimates for England, and the highest and lowest incidence for PCT's. PCT RATE 0–15 RATE 16–74 RATE 75+ Total RATE ENGLAND 339.0 179.5 410.8 229.1 Preston 577.4 478.3 611.9 508.0 Central Liverpool 516.5 429.0 1116.0 488.6 Central Derby 698.0 362.9 895.5 479.0 Middlesbrough 582.4 431.1 550.6 472.5 Birkenhead and Wallasey 473.0 436.3 777.3 471.0 Colchester 175.5 95.4 195.6 118.5 Cherwell Vale 193.7 88.9 174.9 117.1 Brent 134.2 108.7 95.4 113.1 South West Oxfordshire 153.2 98.5 136.2 112.5 Harrow 124.7 97.0 198.5 109.8 Taking the overall incidence rate for 2001–2, together with the key statistics in Table 1 , a Cluster Analysis indicated three clear groupings of Health Authorities in England (Table 4 ). The first can be described as 'London', and includes those authorities located within London. Here, 26.7 % of the population was born outside of the UK and the workforce is better qualified than elsewhere. A lower than average number of people own their own homes, and more people travel to work by public transport than by their own car. It is more overcrowded which contributes to a high deprivation index at 5.5. The admission rate is lower than average at 176 per 100,000 all ages. Table 4 Cluster analysis of socio-economic indicators associated with head injury in England. Cluster Characteristic London Shire Other Urban England Admission Rate – all ages 176 206 288 229 % Born out of UK 26.7 5.7 4.8 9.2 % of working age permanently sick 4.5 4.2 7.6 5.7 % age 16–24 unemployed 5.5 4.6 7.4 5.9 % without qualifications 22.4 26.7 35.1 29.7 % qualified at level 5+ 33.2 19.6 14.8 19.3 % using private transport to work 35.2 67.0 65.3 62.2 % using public transport to work 43.8 8.9 14.2 15.6 % households without a car 38.3 20.1 32.3 27.6 % owning (or buying) own home 55.8 74.0 66.5 68.6 % Lone parent household 10.9 8.2 10.9 9.4 % living in overcrowded homes 18.0 5.1 5.7 7.0 Average Townsend Index 5.5 -2.7 1.2 0.0 The second cluster can be described as the 'Shire' counties, typical of the more rural areas found within England, including Cambridgeshire and Worcestershire. They are characterised by a large proportion of people owning their own home, travelling to work in their own car, and with few of the population born outside of the UK. Although a quarter of the population are without qualifications, the deprivation Index is low at -2.7. The average incidence admission rate for this cluster is 206 per 100,000 all ages. The third cluster can be described as 'Other Urban', and typical of all the Midland and Northern cities of England. They are characterised by a higher proportion of people with limited long standing illness; those of working age who are permanently off sick; of those without qualifications and of those aged 16–24 who are unemployed. The deprivation Index is above average at 1.2, and the admission rate is high at 288 per 100,000 all ages. A regression analysis with overall rate as the dependent variable shows how these various indicators come together to predict incidence (Table 5 ). For every one percent increase in the 16–24 unemployment rate, the hospital admission rate for head injury increases by 17.4 per 100,000 all ages. For every one percent increase in those permanently sick of working age, the rate increases by 16.0 per 100,000 all ages. For every one percent increase in lone parent families the rate increase by 11.0 per 100,000 all ages. In contrast for every one percent increase in the use of public transport to go to work, the rate decreases by 4.6 per 100,000 all ages. With an adjusted R 2 of 0.698, the model explains a large proportion of the variation in admission rates for a head injury (F 36.77; p < 0.01; and with an acceptable pattern of residuals [Figure 1 ]). The most important variable (highest beta) was use of public transport to work. These significant predictors were then entered into another regression model, along with all possible two-way interactions. The latter were entered in a stepwise fashion, but none gained statistical significance. Table 5 Indicators of admission to hospital with a head injury: All ages 2001–2. β is increase (decrease) in rate per 100,000 admissions for each % increase of indicator. Indicator β 95% CI for β % Unemployed within age of 16–24 17.431 5.707 to 29.154 % Permanently sick of working age 16.019 6.226 to 25.773 % Lone parent households 11.012 2.892 to 19.132 % Using public transport for work -4.563 -6.204 to -2.923 % Without qualifications -7.781 -10.741 to -4.821 Figure 1 Normal probability plot of regression with Rate per 100,000 as dependent variable. The model was then validated upon the 2002–3 data at the PCT level (Table 6 ). A similar model emerged with adjusted R 2 of 0.579, again explaining a large proportion of the variation in admission at the PCT level (F70.078; p < 0.01). On this occasion the proportion of households without a car was added to the previous set of predictors. Once again, the proportion travelling to work by public transport was the most important variable. Table 6 Indicators of admission to hospital with a head injury: All ages 2002–3. β is increase (decrease) in rate per 100,000 admissions for each % increase of indicator. Indicator β 95% CI for β % Unemployed within age of 16–24 7.171 0.630 to 13.711 % Permanently sick of working age 10.772 4.900 to 16.645 % Lone parent households 11.034 6.018 to 16.051 % Using public transport for work -4.412 -5.396 to -3.427 % Without qualifications -5.214 -7.032 to -3.396 % No car 4.359 2.982 to 5.735 Discussion Head injury is common; there were 112,718 recorded admissions for English residents during the year April 2001 to March 2002, giving a hospitalised incidence rate of 229.4 per 100,000 all ages. Remaining the same for 2002–3 this incidence is similar to that of stroke although the latter is experienced in a predominately older population [ 18 , 19 ]. However, head injury affects a predominately younger population and carries with it a high potential economic impact. For example, based on these latest incidence figures and published evidence from other studies, we can estimate that about 4700 of those admitted in any given year, and who are considered to be economically active (aged 16–74) and in employment at the time of their injury would be unable to return to their work at 6 weeks [ 20 , 21 ]. Admission rates for England were found to vary by a factor of 4.6 between both health authorities and PCT's. The rates may slightly underestimate the true incidence as those residents of England treated elsewhere are not included. Also, under-reporting may depress incidence to an unknown extent [ 4 , 22 ] and, if underreporting varies by locality, will further contribute to variation. Standardised admission policies for those presenting with a head injury were not agreed at the time that these data were collected and this may also contribute to variation in incidence. The publication of the NICE guidelines for the early management of head injury may reduce any variation attributable to differing admission policies [ 23 ]. Half of all health authorities in England, and slightly more PCT's (55%) had an incidence rate which fell below or above the England average by at least 20%. This has important implications for planning local services in response to head injury. Clearly using the overall England incidence rate is unlikely to be helpful. This variation also causes problems in extrapolating the results from research undertaken in a single locality. Estimates of the number of people disabled by head injury, presented as a rate (e.g. 150 per 100000), may have little relation to reality at the local level. The disability rate is conditional upon the local incidence where the study was undertaken. Consequently it is important that research findings should be presented as proportions of the local base incidence if they are to be of any use for planning elsewhere. The extrapolations given above on return to work are based on this approach. Nevertheless such extrapolation assumes equal case mix (e.g. similar mechanisms of injury) which is also unlikely. Case mix will, to a certain extent, be a reflection of local socio-economic factors, particularly associated with sub-populations displaying chronic conditions, including alcohol misuse, shown to have significant impact on outcome [ 20 , 21 , 24 , 25 ]. The extent of deprivation has also previously been associated with higher incidence of head injury as well as general workload for primary care [ 26 , 27 ]. The data presented here tell a slightly different story. Although Camden and East London health authorities are above the English average for incidence, and have the highest levels of deprivation, and likewise Tower Hamlets and City and Hackney PCT's, the overall correlation between deprivation and incidence is low (0.21). It is the rate of 16–24 unemployment that contributes to higher incidence, and those of working age who report themselves as permanently unable to work because of ill health. Set against these 'lifestyle factors', other factors mediate the incidence level. Thus London, of all cities in England, with the highest localities of deprivation, but with its extensive public transport system, displays a lower overall incidence rate. These findings are consistent for both years examined. However, it is unclear if, at all, reductions in the number of Accident and Emergency beds in the capital (generally from a higher base than elsewhere in the country), and the consequent reduced capacity to admit and observe, may also have contributed to this lower incidence. In contrast, the very high incidence of admission for children who are resident in the area of North Manchester PCT may be associated with the accessibility to the children's hospital located in that area. Indeed, local service and residential patterns, for example, large residential or nursing homes located with a PCT's boundary, may have significant impact on incidence (as expressed by place of residence) at the very local level. Where public transport is not used for journeys to work, incidence is much higher and combined with higher unemployment rates, and other significant indicators associated with 'lifestyle indicators', high incidence rates are observed in the cities and other urban communities in the Midlands and Northern England, including Teeside and Mersyside. As demographic, social and economic factors appear to account for half the variation observed in hospitalised admission rates, one implication is that incidence can be modified at the macro policy level. For example, ceteris paribus , we might expect to see the London incidence fall as a consequence of congestion charging and associated increase in the use of public transport. Policies targeted at reducing unemployment amongst the 16–24 year old age group may also be expected to reduce incidence. Also, a continuing emphasis on prevention is clearly needed, both for the young and the old, to reduce their very high incidence rates. Some technical limitations are worth mentioning. The likelihood distance measure used in the two-step cluster analysis assumes that variables in the cluster model are independent. Further, each continuous variable is assumed to have a normal (Gaussian) distribution, although in practice the technique is robust to violation of these assumptions. In fact, reassuringly, the results of the linear regression model do suggest independent main effects for several of the key variables and, importantly, the absence of interaction effects. Also it is unknown how, if at all, the slight variation in population denominators used to calculate incidence and the socio-economic indicators for the 2001–2 data could influence these results. However, replication upon the 2002–3 data at the PCT level suggests a robust model. Due to censoring of data of elderly admissions at the PCT level we were not able to confidently include the proportion of females admitted in our validation model, and thus we do not know if variation in male-female ratios at the local level was also a contributing factor to variance in incidence. It is also worth restating that the accuracy of case ascertainment, and the coding of head injuries upon admission to hospital is known to underestimate the true incidence [ 4 , 22 ] but how much this varies across localities, and its consequent potential to bias these results, is unknown. Finally, it is possible that a few patients are double counted in that those transferred will be seen as a new admission into a different hospital, thus leading to an unknown over-estimate of incidence. However, these are likely to be those with the most severe injuries admitted initially to hospitals without neurosurgery, and numbers are likely to be small. Conclusion Incidence of head injury in England is high, similar to stroke if just admissions are considered, and the data show considerable variability at the local level. Given that most of the estimates of the potential impact of head injury rely on studies undertaken in a single locality [ 21 , 28 , 29 ] those planning for rehabilitation and other services must take care to identify the proportion of those with a head injury that experience the sequelae under scrutiny (e.g disability; job loss), together with the case mix, and not just rely on headline rates which are conditional upon local incidence. To assist in planning for services, data at the local level can now readily be obtained from on-line data sets from the Department of Health. At the national level they can be downloaded on-line at: While we have as yet little understanding of how case mix is related to, or interacts with socio-economic factors to mediate medium or longer term-outcomes, the association of such factors with incidence suggests that economic and social policies, for example in the development of prevention programmes, and in encouraging the use of public transport for journeys to work, may have a significant impact on reducing the incidence of head injury. Competing interests The author(s) declare that they have no competing interests. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Hospitalised Incidence rate for head injury in England in 2001–2. Estimated rate per 100,000; for those aged 0–15; 16–74; and 75 years and over, and in total. Estimates for England, and extant Regions and Health Authorities at the time. Click here for file Additional File 2 Incidence of admission to hospital for head injury for England, and each Primary Care Trust (PCT) in England 2002–3; Estimated rate per 100,000; for those aged 0–15; 16–74; and 75 years and over, and in total. Estimates for England, and PCT's. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC554988.xml
553981	Evaluating the role of quality assessment of primary studies in systematic reviews of cancer practice guidelines	Background The purpose of this study was to evaluate the role of study quality assessment of primary studies in cancer practice guidelines. Methods Reliable and valid study quality assessment scales were sought and applied to published reports of trials included in systematic reviews of cancer guidelines. Sensitivity analyses were performed to evaluate the relationship between quality scores and pooled odds ratios (OR) for mortality and need for blood transfusion. Results Results found that that whether trials were classified as high or low quality depended on the scale used to assess them. Although the results of the sensitivity analyses found some variation in the ORs observed, the confidence intervals (CIs) of the pooled effects from each of the analyses of high quality trials overlapped with the CI of the pooled odds of all trials. Quality score was not predictive of pooled ORs studied here. Conclusions Had sensitivity analyses based on study quality been conducted prospectively, it is highly unlikely that different conclusions would have been found or that different clinical recommendations would have emerged in the guidelines.	Background Quality assessment of trials included in systematic reviews of evidence is a resource intensive and scientifically controversial endeavour. On the one hand, the routine use of quality assessment in the development of systematic reviews is encouraged by the Evidence-based Practice Center Program of the Agency for Healthcare Research & Quality (AHRQ) and the Cochrane Collaboration, two well respected groups that coordinate a substantial number of systematic reviews [ 1 - 3 ]. Indeed, West et al released a 2002 evidence report sponsored by the AHRQ comparing and contrasting various systems to rate the strength and quality of research evidence to assist in these activities [ 4 ]. Furthermore, many journal editors consider it important to include an assessment of study quality in reports featuring meta-analyses [ 3 ]. The concept of incorporating study quality assessment into systematic review methodology has also found empirical support. There is evidence that studies of lower methodological quality tend to report larger treatment effects than high quality studies [ 5 - 7 ]. For example, Moher and his colleagues found a 34% greater estimate of treatment effect for low quality versus high quality trials and a 37% greater estimate of treatment effect for inadequately concealed versus adequately concealed trials associated with reviews addressing a variety of clinical conditions [ 5 ]. Similar bias was found by Schultz and his colleagues [ 6 ] in their analysis of trials included in the Cochrane Collaboration's Childbirth and Pregnancy reviews. In addition, Colditz and colleagues found that nonrandomized and open studies were more likely to produce positive treatment effects than randomized and double-blinded studies [ 7 ]. Although this seminal work yields compelling results, these findings are not universal and the issue is not without detractors [ 8 - 14 ]. Some studies have found no reliable relationship between quality score and effect size [ 10 - 12 ] and another has found that low study quality was associated with diminished effect sizes [ 13 ]. Further, Juni et al [ 14 ] found the relationship between study quality and effect size depended on the scale used in the assessment. Together these results suggest the study quality issue is controversial and that the merits of this methodological step in systematic review requires thoughtful analysis. Indeed, West et al conclude with recommendations advocating for research dedicated to comparing quality rating systems and the role of quality assessment within individual clinical contexts and for studies targeted at determining specific quality factors that make a difference in final quality scores [ 4 ]. The Practice Guidelines Initiative of the Cancer Care Ontario's Program in Evidence-based Care (PEBC) uses the Guidelines Development Cycle to create cancer practice guidelines comprised of a systematic review of the research literature, an interpretation and consensus of the evidence by members of the guideline development team, clinical recommendations informed by the evidence, and an external review process by Ontario clinicians [ 15 - 19 ]. We face the challenge of balancing scientific rigour and the timely production of guideline documents in an environment defined by limited financial and human resources. Hence, we try to approach our methodological decisions with a critical scientific and practical eye. We took note of the growing controversy in the study quality assessment literature and conducted an evaluation, reported below, to evaluate the benefits of assessing the quality of each study included in our systematic reviews. Our overall objective was to decide whether to augment our current practice of simply describing study characteristics to also incorporate study quality assessment as a routine formal component of the guideline development methodology. The evaluation was conducted in three steps, each of which was designed to address three specific issues: 1. What valid and reliable quality assessment instrument would be most appropriate for our context? 2. How is study quality currently being used in published systematic reviews of cancer trials and what is the relationship between effect size and study quality in this disease area? 3. What impact would study quality assessment have on the clinical recommendations made in evidence-based practice guidelines developed by the PEBC? Methods Search for a valid and reliable quality assessment tool for the PEBC context For a comprehensive review of the strengths and weaknesses of quality assessment instruments, readers are referred to the 2002 West et al. evidence report commissioned by the AHRQ [ 4 ]. For our study, which began before the release of this report, we used components of Moher et al's definition of study quality that are related to internal validity (i.e., design, conduct and analysis) [ 20 ] and updated his 1992 published reports of check lists and scales used to measure the phenomenon [ 21 ]. We searched the Medline database using the following search strategy: (quality adj rat:).tw OR (quality adj assess:).tw. OR (quality adj scale:).tw. OR (quality adj checklist:).tw. AND randomized controlled trials.sh. OR clinical trial:.tw. OR random:.tw. Reference lists of reviews were scanned for additional citations. Systematic review of the oncology literature on study quality To locate systematic reviews on oncology topics, the strategy suggested by Moher et al for finding systematic reviews [ 3 ] was combined with the terms "neoplasms.sh. OR cancer.tw. OR carcinoma.tw" to search the Medline, CINAHL, and Cancerlit databases. To ascertain if the authors assessed the quality of the studies included in the systematic reviews, the search was narrowed to include the terms [(quality adj rat:).tw OR (quality adj assess:).tw. OR (quality adj scale:).tw. OR (quality adj checklist:).tw. OR (study adj quality).tw.]. Textwords were used to search the Cochrane Library for systematic reviews on oncology topics. Systematic reviews that included analyses exploring the relationship between study quality (using any assessment instrument, not just validated tools that met our criteria as described above) and effect size were examined. Because survival following cancer treatment is commonly used as the primary outcome variable in our practice guidelines, this variable was selected as the primary outcome measure of interest. Impact of study quality on PEBC practice guidelines The validated scales were applied by two methodologists (MJ and MC) to studies reported in any our practice guidelines that included a pooled analysis based on at least ten randomized trials related to the main guideline question. Intraclass correlation coefficients with 95% confidence intervals (CI) were calculated using a random sample of the RCTs to assess inter-rater reliability, one coefficient calculated for each of the scales used. Because of budgetary limitations for staff time, the analysis was conducted on 18 randomly selected studies rather on the whole group of articles. This is a methodological limitation as fewer studies result in larger confidence intervals and less precise estimates. This may account for the difference in reliability ratings we found for the Sindu scale compared to published norms (see Table 1 ). Table 1 Quality assessment tools with published validity and reliability data – information reported by the scale developers Instrument Description Item Generation Validity Inter-Rater Reliability Application in Study Jadad et al (22) number of items: 3 or 6 scoring for 3 item: 0, 1, or 2 for two items 0 or 1 for 1 item range 0 to 5 scoring for 6 item: 0, 1, or 2 for two items 0 or 1 for 4 items range 0 to 8 cut offs not specified for either version consensus among 6 judges (pain management &/or instrument development) pretested with 3 raters on 13 clinical trial reports 3 groups of pain studies were identified a priori: • studies rated as excellent by experts (n = 7); overall score = 3.4 • studies rated as poor by experts (n = 6); overall score = 0.7 • a random selection of RCTs from a MEDLINE search (n = 23); overall score = 2.7 ICC = 0.66 (95% CI: 0.53, 0.79) 14 raters 3-item and 6-item versions in their entirety Sindhu et al (24) number of items: 53 scoring: items weighted from 1 to 10 range 0 – 100 cut offs not specified 8-member Delphi panel (clinicians and methodologists) Compared with the Chalmers scale for five studies of non-pharmacologic nursing interventions for pain management; r = 0.94 (Pearson) 11 studies considered for a published meta-analysis on hypertension were evaluated: • 9 included studies scored 73–93%; • 2 rejected studies scored below 70% ICC = 0.93 2 raters 12 of 53 items eliminated because of difficulty applying to cancer studies 41 items related to 12 domains range 0 – 79.5 Downs & Black (25) number of items: 27 scoring 0 or 1 for 25 items 1 or 2 for 1 item 0 to 5 for 1 item cut offs not specified based on epidemiological principles, reviews of study designs and existing checklists pilot tested by 2 epidemiologists rating 20 studies Compared with the Standards of Reporting Trials Group checklist scale for 10 RCTs on surgery for stress incontinence; r = 0.90 (Spearman) Spearman r = 0.75 2 raters used 13 internal validity items only items not related to our definition of quality eliminated (10 reporting items, 3 external validity items, 1 power item) range 1 to 13 RCT, randomized controlled trial; ICC, intraclass correlation coefficient; CI, confidence interval To assess the impact of study quality on effect sizes, sensitivity analyses were conducted for the meta-analysis from each guideline report. For each scale, studies were divided into two groups (low quality and high quality) based on total quality score. Where the scale developer suggested a cut-off point for low versus high quality, this was used. Where no cut point was specified, the observed median study quality score was used as the dividing point between low and high quality. Meta-analyses were repeated with the high quality studies. Because there would never be a situation in which guideline developers would consider low quality studies only, a meta-analysis using this sample of the studies was not conducted. Results Valid and reliable quality assessment tools for our context Four scales meeting our criteria were found; two instruments, Jadad et al [ 22 ] and Cho & Bero [ 23 ], were originally uncovered in the Moher review [ 21 ] and two instruments, Sindhu et al [ 24 ] and Downs & Black [ 25 ], were uncovered in our update of this review. While none of these scales were developed in the oncology setting, they all purport to be generic assessment tools that measure the quality of specific study designs regardless of clinical condition reflected in the design. The procedures undertaken to create the instruments followed appropriate methodological processes for questionnaire design. In addition, while a number of additional scales and checklists emerged from our search, validity and reliability data were not reported. Because our practice guidelines are based primarily on evidence from randomized trials, we decided to reserve to employ the scales that focused specifically on RCTs. As such, the Cho & Bero scale, which is applicable to a range of study designs, was not employed here but will be considered at a later date when we have a portfolio of diverse study designs. The characteristics of the instruments included in our study are summarized in Table 1 and 2 and detailed descriptions and comparisons can be found in West et al. [ 4 ]. Table 2 Quality assessment tools – comparison of key quality constructs Jadad Sindhu Downs & Black Randomization Max.= 2 points out of total score of 5 1. Was the study described as randomized? (1 point for yes) Give an additional point if the method to generate the sequence of randomization was described and it was appropriate. Deduct a point if it was inappropriate. Max. = 10 points out of total score of 100 2. Have the patients been randomly allocated to treatment groups? (1 point for yes) If yes: i) Is the method of randomization explicitly detailed? (1.5 points) ii) Is it valid? i.e. Are there any threats to internal validity re: designation of subjects to groups? (2.5 points) iii) Is patient consent sought prior to randomization? (2.5 points) iv) Is it secure and 'blind' to the assessors? (2.5 points) Max. = 2 points out of total score of 13 23. Were the study subjects randomised to intervention groups? Studies which state that subjects were randomised should be answered yes except where method of randomisation would not ensure random allocation. For example alternate allocation would score no... (1 point for yes) 24. Was the randomised intervention assignment concealed from both patients and health care staff until recruitment was complete and irrevocable? (1 point) Blinding Max. = 2 points out of total score of 5 2. Was the study described as double blind? (1 point) Give an additional point if the method of double blinding was described and it was appropriate. Deduct a point if it was inappropriate. Max. = 5 points out of total score of 100 9. Is the assessment blind? a) If yes, who is blinded: i) patients? (2 points) ii) therapist/carer? (2 points) iii)assessor/data collector? (1 point) b) If no, i) are reasons given as to why assessment is not blind? (2 points) ii) Is there discussion of bias resulting from non-blind assessment? (3 points) Max. = 2 points out of total score of 13 14. Was an attempt made to blind study subjects to the intervention they have received? (1 point) 15. Was an attempt made to blind those measuring the main outcomes of the intervention? (1 point) Withdrawals and dropouts Intention to treat analyasis Max.= 1 point out of total score of 5 3. Was there a description of withdrawals and dropouts? 1 point) Max. = 12 points out of total score of 100 6. Has an 'intention-to-treat analysis been performed? i.e. everyone randomized is retained in the study; everyone randomized is included in the final analysis; and no selective dropouts. (8 points) b) if not, is it clear what was done, its justification and impact on bias? (8 points) 11. Loss to follow-up a) (<) 20% loss to follow up (2 points) b) <10% loss to follow-up (2 points) Max. = 2 points out of total score of 13 25. Was there adequate adjustment for confounding in the analyses from which the main findings were drawn? The questions should be answered no for trials if: the main conclusions of the study were based on analyses of treatment rather than intention to treat;.... (1 point) 26. Were losses of patients to follow-up taken into account? If the number of patients lost to follow-up are not reported, the question should be answered unable to determine. If the proportion lost to follow-up was too small to affect the main findings, the question should be answered yes? (1 point) Appropriate statistical analysis no items Max. = 6 points out of total score of 100 7. Statistical analysis a) Is the analysis appropriate/specific to the hypothesis and to the data? (1 point) b) Is the analysis adequately described? (1 point) c) Do the statistical assumptions hold? (1 point) d) Are adequate summary statistics provided at: i) baseline? (0.5 point) ii) outcome? (05. point) e) Is the overall significance level reported protected against inflation due to multiple testing? (1 point) f) If confounders exist, are they adjusted for via multivariate techniques even if differences between groups are not significant? (1 point) Max. = 4 points out of total score of 13 16. If an of the results of the study were based in 'data dredging', was this made clear? (1 point) 17. Do the analyses adjust for different lengths of follow-up of patients? (1 point) 18. Were the statistical tests used to assess the main outcomes appropriate? (1 point) 25. Was there adequate adjustment for confounding in the analyses from which the main findings were drawn? (1 point) Compliance with treatment no items Max. = 4 points out of total score of 100 14. Has patient compliance been assessed? (4 points) Max. = 1 point out of total score of 13 19. Was compliance with the interventions reliable? (1 point) Outcome Measures no items Max. = 14 points out of total score of 100 3. Measurement of outcomes a) Is the form of measurement stated? (3 points) b) Has an attempt been made to validate the measures? (3 points) c) Has an attempt been made to test the reliability of the measures? (2 points) d) Is the outcome objective as compared to subjective? (2 points) 12. Outcomes a) How many outcomes are used (1/2 point for each, to a max. of 2) b) Are they relevant? (1 point) c) Are they independent? (1 point) Max. = 1 point out of total score of 13 20. Were the main outcome measures used accurate (valid and reliable)? (1 point) The relationship between study quality and effect size in the oncology literature The literature review located 32 published systematic reviews on oncology-related topics that included some measure of study quality. Five of the reviews examined changes in pooled estimates of effect size of mortality rates when meta-analysis was restricted to high-quality randomized trials [ 26 - 30 ]. As shown in Table 3 , four of the five reviews found somewhat larger effects (i.e., larger differences between experimental and control groups) with high-quality trials compared to all trials [ 26 - 29 ]. With one exception, the statistical relevance of the differences between the groups (i.e., significant differences or no significant differences) remained the same regardless of the number of trials included. Specifically, two of the reviews did not detect a statistically significant difference in survival between groups when all studies were included or when the meta-analysis was restricted to high-quality studies [ 26 , 29 ]. For one data set, the meta-analysis was repeated with study quality ratings used as weights [ 29 ]; there was still no significant difference between experimental and control groups. Two analyses detected significant differences between experimental and control treatments with analysis of all trials and when the analysis was restricted to high-quality trials [ 27 , 28 ]. In the fifth review, a significant difference between experimental and control interventions was detected when all trials were synthesized that became only marginally significant (p < .07) when the meta-analysis was adjusted for study quality [ 30 ]. Table 3 Systematic reviews of randomized oncology trials with sensitivity analysis exploring the relationship between study quality scores and effect sizes for mortality Systematic Review Interventions Outcome Quality Scale Definition of High Quality Effect Size (95% confidence interval) All Studies (# studies) High Quality (# studies) McAlister et al, 1998 (26) allogenic blood transfusion versus autologous or leucocyte-depleted allogenic blood during cancer surgery relative risk of death* Jadad (22) score ≥3 out of 5 RR, 0.94 (0.76 to 1.16) (n = 5) RR, 0.84 (0.47 to 1.52) (n = 2) Caubet et al, 1997 (27) nonsteroidal anti-androgens (plus LHRH or orchiectomy) versus LHRH or orchiectomy alone for advanced prostate cancer relative risk of death* Chalmers (31) score ≥50 % of total possible score RR, 0.81 (0.70 to 0.94) (n = 13) RR, 0.78 (0.66 to 0.92) (n = 4) Dube et al, 1997 (28) adjuvant chemotherapy versus control for colorectal cancer odds ratio for death* Chalmers (31) score >50 % of total possible score OR, 0.82 (0.77 to 0.89) (n = 29) OR, 0.77 (0.71 to 0.85) (n = 14) Detsky et al, 1992 (29) total parenteral nutrition versus control in cancer patients undergoing chemotherapy odds ratio for survival** Chalmers (31) score >42 % of total possible score; quality score also used as a weighting factor in meta analysis OR, 0.74 (0.42 to 1.3) (n = 8) OR, 0.69 (0.38 to 1.3) (n = 2) weighted OR, 0.61 (0.23 to 1.6) Klein et al, 1986 (30) total parenteral nutrition versus control in cancer patients undergoing surgery odds ratio for operative death* Developed specifically for the systematic review quality score used as a weighting factor in meta analysis OR, 0.44 (0.21 to 0.90, p = 0.02) (n = 10) weighted OR not reported but p = 0.07 after weighting for study quality LHRH, luteinizing hormone-releasing hormone; RR, relative risk; OR, odds ratio RR or OR <1.0 indicates fewer deaths in the experimental group than in the control group * OR <1.0 indicates more deaths in the experimental group than in the control group Impact of study quality on PEBC practice guidelines Three of the PEBC practice guidelines included at least 10 RCTs in their systematic reviews of the evidence and were eligible for inclusion in this evaluation [ 31 - 33 ]: concomitant chemotherapy and radiotherapy in squamous cell head and neck cancer (18 trials) [ 31 ]; adjuvant therapy for stage II colon cancer following complete resection (11 trials) [ 32 ]; and neoadjuvant chemotherapy in locally advanced squamous cell carcinoma of the head and neck (23 trials) [ 33 ]. For the latter guideline [ 33 ], data could not be reliably reconstructed and is not discussed further. At the conclusion of our study, we identified a fourth practice guideline which originally did not meet our 10 RCT inclusion criteria, but later did so after it was updated. The guideline focused on the role of erythropoietin (EPO) in the management of cancer patients with non-hematologic malignancies [ 34 ]. Unlike the chemotherapy trials included in the practice guidelines described above, which were not placebo-controlled and where the primary outcome was death, one-third of the EPO trials were double blind and all used the need for blood transfusion as the primary outcome. Although by the time this practice guideline emerged as eligible we had identified a preferred scale (see below), we chose to include it here and apply only the preferred scale as a demonstration of its use on a report that had differing characteristics than the chemotherapy topics covered. Inter-rater reliability Intraclass correlation coefficients used to established inter-rater reliability were 0.71 for the 3-item Jadad scale (95% CI, 0.38 to 0.88), 0.80 for the 6-item Jadad scale (95% CI, 0.54 to 0.92), 0.62 for the Sindhu scale (95% CI, 0.24 to 0.84), and 0.63 for the Downs & Black Scale (95% CI, 0.25 to 0.84). Disagreements were resolved by consensus. Where consensus could not be reached, a third rater (MB) assessed the items and provided the tiebreaker score. Application of quality scales to primary studies informing practice guidelines While the total quality scores emerging from each of the different scales did all significantly correlate with one another (range r = .35 to r = .73), there was considerable variation in the classification of studies as high quality or low quality as a function of the scale that was applied (Table 4 ). For example, of the 11 comparisons from 11 trials comprising the stage II colon cancer review, the application of the Jadad 3-item, the Jadad 6-item, the Sindhu, and Downs & Black scales yielded 0, 8, 9, and 6 of these as high quality, respectively. The 6 studies categorized as high quality using the Downs & Black tool were also categorized as high quality when the Jadad 6-item and Sindhu scales were applied. Similarly, the 8 studies categorized as high by the Jadad 6-item were also categorized as high quality by the Sindhu scale. Table 4 Meta-analysis of all trials and high-quality trials from evidence-based practice guidelines Practice Guideline Colon Cancer Adjuvant chemotherapy Outcome: Mortality (32) Head & Neck Concomitant therapy Outcome: Mortality (31) Systemic Therapy erythropoietin Outcome: need for blood transfusion (34) All Studies # studies (# comparisons) 11 (11) 18 (20) 15 (15) OR (CI) 0.82 (0.62 – 1.09) 0.62 (0.54 – 0.72) 0.57 (0.47–0.70) High Quality Studies By Assessment Scale Criteria Jadad (3-item) range = 0–5 criteria >2 # studies (# comparisons) 0 2 (2) not applied OR (CI) - 0.54 (0.34 – 0.86) Jadad (6-item) range = 0–8 criteria >3 # studies (# comparisons) 8 (8) 13 (14) 9 (9) OR (CI) 0.88 (0.69–1.13) 0.53 (0.44 – 0.64) 0.57 (0.44–0.72) Sindhu (41-item) range = 0–79.5 criteria > 44.8 # studies (# comparisons) 9 (9) 11 (12) not applied OR (CI) 0.86 (0.68–1.09) 0.62 (0.49 – 0.79) Downs & Black (13-item) range = 0–13 criteria > 7 # studies (# comparisons) 6 (6) 12 (14) not applied OR (CI) 0.86 (0.59–1.25) 0.56 (0.46 – 0.68) OR, odds ratio; CI, 95% confidence interval The 20 comparisons from the 18 trials included in the head and neck concomitant therapy systematic review yielded 2, 14, 12 and 14 high quality studies, respectively, when the Jadad 3-item, the Jadad 6-item, the Sindhu and the Downs & Black scales was used. Although both Jadad 6-item and Downs & Black scales both assessed 14 comparisons to be from high quality studies, only 11 of these 14 studies were the same. For the 12 comparisons from studies categorized as high quality with the Sindhu scale, 10 of these were also rated high quality by both the Jadad 6-item and the Downs & Black scales, the other 2 were rated as high quality by the Jadad 6-item scale only. There was 1 comparison from a study rated as high quality by the Jadad 6-item scale only and two from studies rated as high quality by the Downs & Black scale only. Impact on pooled estimates of outcome measures Mortality data (i.e., numbers of deaths and number of patients randomized for each allocation group, abstracted from published trial reports) used for the meta-analysis included in the guideline reports were available for two guidelines and need for blood transfusion data were available for the third [ 31 , 32 , 34 ]. For each guideline, the pooled odds ratio based on only the high-quality trials was compared with the odds ratio from meta-analysis of all trials that had been included originally in the review (Table 4 ). For the first guideline [ 31 ], there was a significant survival benefit for concomitant chemotherapy and radiotherapy compared with radiotherapy alone for squamous cell head and neck cancer in the meta-analyses that included all studies and the meta-analyses restricted to high quality studies, regardless of quality appraisal tool used. Although the effect size was larger for meta-analysis of high-quality RCTs than for all RCTs (irrespective of quality scale used), the confidence intervals between the two calculations overlapped and the overall conclusions and the recommendations informed by the meta-analysis would have been the same. For the second guideline [ 32 ], no survival benefit was detected for adjuvant chemotherapy compared to standard therapy for stage II colon cancer in the meta-analysis of all the studies or the high quality studies, again, regardless of quality appraisal tool used. Although the meta-analysis of the high study quality studies was associated with smaller effect sizes than the calculation including all of the studies, the confidence intervals overlapped and the conclusions and the recommendations would have remained the same. Only the 6-item Jadad scale was applied to the studies of the EPO guideline and the data were pooled to calculate an overall risk ratio for blood transfusion [ 34 ]. The risk ratio for all 15 trials was 0.57 (95% CI, 0.47 to 0.70); for nine trials that scored more than three out of eight on the 6-item Jadad scale, the risk ratio was also 0.57 (95% CI, 0.44 to 0.72) (see Table 4 ). Conclusions Several conclusions can be drawn from this study and review of the literature. First, there are established methods for assessing the quality of randomized controlled trials in which data on adequate reliability and validity were available. West et al uncovered 32 scales, check lists and component systems concerned with evaluating RCTs [ 4 ]; more than the four strategies we applied here. Although most (87%) of the instruments found by West included quality domains for which there is an empirical basis, most failed to report the use rigorous methods in their development and most failed to report data regarding reliability and validity, criteria we set for our study. Interestingly, West et al did not include the Jadad 6-item in their analysis [ 4 ], although the Jadad 3-item, Downs & Black, and Sindhu tools were reported. Although all of the scales we used have established reliability and validity estimates, we found that the number of trials categorized as high quality or low quality depended specifically on the scale that was applied. For the head and neck cancer systematic review, the number comparisons from high quality studies ranged from 2 (when the Jadad 3-item scale was applied) to 14 (when the Jadad 6-item or Downs & Black scales were applied). The range for the colon cancer review was 0 to 9. There was also considerable variability regarding the specific quality category in which each trial was placed. These finding are consistent with those of Juni et al [ 14 ] and suggest caution should be applied if the intent of quality rating scales is to restrict the number of studies considered in the systematic review; clearly the choice of scale will have a significant impact regarding what studies are eligible. The problem of identifying to which quality category, high or low, studies should be placed is exacerbated by the lack of clear cut-off criteria identified by the instrument developers. This poses a significant methodological limitation to the utility of these instruments. In our study, we chose the median score as the cut-off criteria in situations where none was reported. However, it would be useful for researchers of these tools to continue the development work to create the evidence-base from which valid criteria can be established. The lack of consistency of study classification from one scale to the next and the lack of clear cut-off criteria for users to employ when measuring quality of studies, presents a challenge to guideline developers when they need to make choice about which instrument they ought to adopt if the choose to adopt an instrument at all. Rather than clear evidence driving our decisions, we considered other features of the instrument in our decision making. Of the rating scales we examined, our preferred choice would be the Jadad 6-item instrument. In contrast to the others considered in this report, this instrument is relatively easy to implement and interpret and good inter-rater reliability was established. Further, although the 3-item version of the Jadad scale is most commonly used, we found the original 6-item version to be more relevant in our clinical context as it provides greater variation in scores. In the cancer discipline, few trials are placebo-controlled and treatment allocation tends to be poorly reported. In contrast to the pain trials which were profiled in the development phase of the Jadad instrument, the majority of the items in the 3-item version (randomization and blinding items) yield no variation in scores in our context and are, therefore, not useful to discriminate among cancer trials. The 6-item version of the scale more aptly differentiates quality across studies and includes more quality domains for which an empirical basis has been established [ 4 ]. Another conclusion that can be drawn from this study is that effect size can be related to study quality but that the nature of the relationship in one clinical area may not generalize to another clinical area. Some of the original work examining the role of study quality reinforces the need to be mindful of the variation among studies included in systematic review [ 5 - 7 ]. However, when we examined five published reviews that had conducted sensitivity analyses on pooled mortality data from RCTs, four of these found that larger effect sizes were associated with high-quality studies, not lower quality trials as has been convention, and the absence or presence of statistical differences between the two allocation groups remained constant. One of challenges in examining this work is that the number of high quality studies is limited; there is a reduction in power that subjects the point estimates to bias. Nonetheless, the potential bias of study design and quality requires thoughtful consideration within a given clinical field. We conducted sensitivity analysis on the systematic reviews comprising the guidelines developed by the PEBC. Only four systematic reviews among 36 eligible practice guidelines included more than 10 trials with data appropriate for pooling; three from which we could extract data. Although there was some variation in the odds ratios observed, the confidence intervals of the pooled effects from each of the analyses of high quality trials overlapped with the confidence intervals of the pooled odds with all of the trials. In no case would the conclusions based on these results be affected by restricting the meta-analysis to only high quality studies; the recommendations remained the same. Had sensitivity analysis based on study quality been conducted prospectively, it is highly unlikely that different conclusions would have been drawn from the systematic review or that different clinical practice guidelines would have been formulated. Together, these findings lead to our final conclusion that measuring study quality did not translate into altered conclusions from a systematic review in the oncology domain for the outcomes we used here. Thus, at this time we have decided that measuring study quality using a numerical assessment scale for the purposes of sensitivity analysis will not be a routine part of our guideline development program. We will, however, encourage guideline developers to describe the variation among studies and to point out methodologic flaws. In addition, it will be important for us to repeat this study looking at other outcome measures, such as quality of life and adverse effects, as they become more routinely reported in primary cancer research and incorporated into our practice guidelines. Outcomes other than those studied here may be more sensitive to the issues of study quality. This study highlights a strategy that may be useful for guideline programs to utilize in making decisions regarding the methods employed in their guideline development process. It is important that scientific inquiry be maintained in studying the value and role of study quality assessment rather than accepting its role as convention. By exploring it within a specific clinical context one can identify it's most appropriate application. Competing interests The author(s) declare that they have no competing interests. Authors' contributions MB, MJ and GB conceived of the project idea and developed the protocol. MB, MJ, and MC conducted by study. SH provided statistical advice and AJ provided conceptual advice. The manuscript was drafted initially by MB and MJ. All authors contributed to the final version submitted for publication. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC553981.xml
509319	How the Brain Retrieves Forgotten Memories	null	In the 2002 thriller Memento , the protagonist, Lenny, is plagued by a crippling neurological disorder that renders him incapable of storing memories for longer than fifteen minutes. He wants nothing more than to avenge the murder of his wife, but as another character in the film tells him, “Even if you get revenge, you're not going to remember it. You're not even going to know it happened.” Lenny has anterograde amnesia, a condition typically caused by stroke or other illness. (In the film, it's caused by a blow to the head.) Most forms of amnesia—including the more common retrograde amnesia, which involves the loss of long-term memory—are caused by some type of brain injury— particularly to the hippocampus. Retrograde amnesia can arise from brain damage that interferes with memory storage, retrieval, or consolidation. And it is by studying retrograde amnesia that scientists have developed theories explaining where the brain stores memory traces and how it consolidates them into long-term memories. What ultimately causes amnesia—a failure to store memories or a failure to retrieve them—is not clear. A major challenge in resolving this question experimentally is being able to determine whether an animal has truly recovered a lost memory or has simply re-learned the task at hand. By refining a classic behavioral neuroscience experiment to test spatial learning and memory, Livia de Hoz, Stephen Martin, and Richard Morris have developed a novel protocol that distinguishes retrieval from new learning. The experiment, developed by Morris over fifteen years ago, takes advantage of the fact that rats are good swimmers but prefer life out of water. Rats are placed in a pool and trained to remember the location of an escape platform, hidden just under the surface. (The rats can't see it because the water is cloudy.) Here, de Hoz et al. trained rats to a particular platform location—their escape route—then gave them hippocampal lesions. One group received “sham” lesions, another partial lesions, and a third complete lesions. Partial lesions, the authors explain, should damage only a subset of the stored memory traces and thus weaken the rats' memory rather than completely disrupting it. The rats' postoperative memory was tested by first placing them in the pool with the platform hidden: as expected, the lesioned rats couldn't remember where it was. After a minute, the platform was raised above the water, to remind them that it existed and that they could escape by climbing onto it. The platform was revealed in the original location for half the animals and in a novel location—which could be construed as misleading information if you're a rat—for the other half. If raising the platform was facilitating re-learning, then animals capable of learning would look for the platform in the new location. But if raising the platform functioned as a reminder, then animals should gravitate toward the place they were trained to, regardless of whether the platform reminder was in the original location or a new one. And that's what happened to the rats with partial lesions. De Hoz et al. then repeated the experiments in a new environment. If the rats managed to escape in this situation, the authors explain, then the reminder treatment could be causing new learning rather than triggering recall of the original training experience. Not surprisingly, the rats with total lesions failed to learn in this new environment (or to be reminded in the previous experiment), while the control rats adapted to the new location. The group with partial lesions failed to learn. Since partially lesioned rats responded to the reminder treatment by recalling the original platform location, their initial memory failure couldn't have resulted from a storage failure. And since they did not realize they could escape from the new location, the reminder didn't induce new learning. Among the many questions these results raise is what role the hippocampus plays in memory storage and retrieval. It could be, as the authors propose, that essential components of spatial memory traces are either stored in the hippocampus or reactivated there, since only the partially lesioned rats responded to reminders. Whether that proves true, de Hoz et al. have contributed a much needed resource for investigating the neural basis of memory loss. Rats given partial hippocampal lesions after training in a watermaze are initially unable to remember the location of the escape platform	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC509319.xml
549073	Extent of exposure to environmental tobacco smoke (ETS) and its dose-response relation to respiratory health among adults	Background There is a dearth of standardized studies examining exposure to environmental tobacco smoke (ETS) and its relationship to respiratory health among adults in developing countries. Methods In 2004, the Syrian Center for Tobacco Studies (SCTS) conducted a population-based survey using stratified cluster sampling to look at issues related to environmental health of adults aged 18–65 years in Aleppo (2,500,000 inhabitants). Exposure to ETS was assessed from multiple self-reported indices combined into a composite score (maximum 22), while outcomes included both self-report (symptoms/diagnosis of asthma, bronchitis, and hay fever), and objective indices (spirometric assessment of FEV 1 and FVC). Logistic and linear regression analyses were conducted to study the relation between ETS score and studied outcomes, whereby categorical (tertiles) and continuous scores were used respectively, to evaluate the association between ETS exposure and respiratory health, and explore the dose-response relationship of the association. Results Of 2038 participants, 1118 were current non-smokers with breath CO levels ≤ 10 ppm (27.1% men, mean age 34.7 years) and were included in the current analysis. The vast majority of study participants were exposed to ETS, whereby only 3.6% had ETS score levels ≤ 2. In general, there was a significant dose-response pattern in the relationship of ETS score with symptoms of asthma, hay fever, and bronchitis, but not with diagnoses of these outcomes. The magnitude of the effect was in the range of twofold increases in the frequency of symptoms reported in the high exposure group compared to the low exposure group. Severity of specific respiratory problems, as indicated by frequency of symptoms and health care utilization for respiratory problems, was not associated with ETS exposure. Exposure to ETS was associated with impaired lung function, indicative of airflow limitation, among women only. Conclusions This study provides evidence for the alarming extent of exposure to ETS among adult non-smokers in Syria, and its dose-response relationship with respiratory symptoms of infectious and non-infectious nature. It calls for concerted efforts to increase awareness of this public health problem and to enforce regulations aimed at protecting non-smokers.	Introduction The deleterious effects of exposure to ETS on the respiratory system of adults and children is well documented [ 1 - 4 ]. Exposure to ETS not only influences respiratory health among those affected but also leads to increased health care utilization and costs because of respiratory problems [ 5 , 6 ]. For example, a recent study investigating more than 10,000 children in Germany showed that the risk of emergency department visits and hospitalizations from asthma was more than double for children exposed to 10 or more cigarettes/day compared to less exposed children [ 6 ]. ETS exposure was found to be associated with respiratory symptoms, abnormal lung functions, and increased bronchial responsiveness in children and adults [ 1 - 4 , 7 , 8 ]. Of special significance for developing countries, lower respiratory infection, the single most important cause of death for children below the age of 5 years, has been found to be associated with exposure to ETS [ 1 , 9 - 12 ]. However, with most studies of ETS exposure and respiratory health being done in developed countries, local evidence to promote clean air policies and to enforce existing policies are lacking in most of the developing world. The situation with exposure to ETS in developing countries is likely to be aggravated by the widespread of smoking, lack of restrictions regarding indoor smoking, overcrowded housing conditions, and failure of health services [ 13 - 15 ]. Cigarette smoking in Aleppo is affecting some 70% of men and 20% of women aged 30–45 years, with an average of 1.2 cigarette smoker per household [ 16 ]. Moreover, Syria as well as other countries in the Eastern Mediterranean region (EMR) are experiencing an alarming increase in the popularity of waterpipe smoking [ 17 , 18 ]. Although this form of smoking is generally considered an outdoor social practice, research done at the Syrian Center for Tobacco Studies (SCTS) shows that more serious smokers demonstrate a predominantly individual use pattern (home, and alone) [ 19 ]. As such, waterpipe smoking may be an important source of ETS due to its emissions and length of smoking bouts [ 18 , 20 ]. Assessment of exposure to ETS, therefore, needs to encompass all information relevant to the studied setting and the smoking patterns of the target population. Despite these troubling facts, there is a dearth of research examining the relationship between ETS exposure and respiratory health in developing countries, with the few available studies limited by poor outcome definition, lack of systematic exposure assessment, and inadequate control of confounding [ 21 , 22 ]. A recent review of this subject identifies a major limitation of available data being restricted to industrialized nations [ 3 ]. Generally, the use of different methodologies and markers of exposure and outcome precludes arriving at a clear picture of the relationship between ETS exposure and respiratory health. For example, not all studies have found a relationship between exposure to ETS and lung function impairment [ 23 , 24 ], some did not find a dose-response relationship [ 25 ], while others demonstrated gender-specific effects [ 24 , 26 ]. The reliance on single or historic indicators of exposure (spouse's smoking, maternal smoking during pregnancy) can lead to an imprecise estimation of exposure or recall problems [ 27 , 28 ]. Previous quantitative and qualitative research done in Aleppo has identified ETS as a potentially major health hazard in the indoor environment [ 16 , 29 ]. The current study, which is the first to assess respiratory health of adults and its relation to ETS exposure in Syria, is based on a population-based household survey (Aleppo Household Survey, AHS) done in Aleppo in 2004 utilizing multiple self-reported indicators of exposure and outcome as well as expired breath CO and spirometry. Methods Population and sampling The target population consisted of adults 18–65 years of age residing in the greater city of Aleppo (around 2,500,000 inhabitants). Detailed description of the sampling design and procedures of the AHS is reported elsewhere and illustrated in Figure 1 [ 16 ]. Briefly, stratified cluster sampling was used where residential neighborhoods of the city were stratified into two strata: formal and informal; according to the official description of the municipal registry (Figure 1 ). From each stratum, residential neighborhoods were randomly selected with probability proportional to size (PPS). Within each neighborhood, households were selected with equal probably and an adult was randomly selected from each. Figure 1 The overall sampling scheme of the Aleppo Household Survey. In the 1 st step the target population was divided into two strata, formal and informal zones (where residential areas are build illegally or on a land not designated for housing). In the next step residential neighborhoods were selected with PPS, and within selected neighborhoods households and one adult within each were selected with equal probability. The survey was conducted between May-August 2004, and the protocol and the informed consent documents were approved by the Institutional Review Boards at the University of Memphis and SCTS. Design and procedures This interviewer-administered survey involved six, 2-person, mixed gender teams of surveyors equipped with notebook computers programmed to record questionnaire responses and measurements using a custom data entry program (Delphi programming language and SQL server DBMS). The questionnaire included 8 main sections; socio-demographics, general health and disability, chronic disease, respiratory health, household members' health, environmental health, smoking, and ETS exposure. For the assessment of respiratory health and risks, the questionnaire was developed based on relevant instruments (especially the European Community Respiratory Health Survey-ECRHS, the International Study of Asthma and Allergy in Children-ISAAC, and ETS exposure assessment instrument developed by Eisner and colleagues), as well surveys done in Syria [ 29 - 35 ]. Next, the survey instrument and strategy were modified in terms of format, content, language, response categories, and recall period based on formative work conducted with key informants involved in the provision of health care as well as with residents, in addition to piloting in 20 randomly selected households [ 16 , 29 ]. After being randomly selected, all study participants underwent the detailed study interview and objective measurements; height using a sliding wall meter (Seca, Germany), body weight using digital scales (Camry, China), expired breath carbon monoxide (CO) using breath CO monitors (Vitalograph, US), and lung function tests using portable spirometers (Micro-plus, UK) according to a standard protocol (16). Exposure Data from self-reported non-smokers (both cigarettes and waterpipe), validated by breath CO levels ≤ 10 ppm (Table 1 ) [ 36 , 37 ], were analyzed for this report. ETS exposure assessment utilized responses to multiple inquiries about short- vs. long-term, indoor vs. outdoor, and cigarette vs. waterpipe exposures, as well as sensory irritation due to ETS exposure (a marker of intensity of exposure) (Table 2 ) [ 29 - 35 ]. Spouse's and parental smoking assessment included inquiries about length, intensity, and type of smoking (cigarette, waterpipe). Occupational exposure to respirable pollutants other than ETS was assessed by asking those involved in paid work whether they are exposed to dust, foams, smoke or other respirable particles at their work categorized as no exposure, mild exposure (a day or less weekly), moderate exposure (more than a day per week but not daily), and severe exposure (almost daily). Parental allergy was assessed by asking whether the respondent's parents ever suffered from respiratory or nose allergy with responses categorized into none, father, mother, or both. Table 1 Basic indicators of Aleppo Household Survey (AHS) participants (n = 2038), and non-smokers satisfying criteria for inclusion in the analysis (n = 1118) All participants n (%) Non-smokers with breath CO ≤10 ppm n (%) Age 18–29 years 736 (36.1) 450 (40.3) 30–45 years 874 (42.9) 418 (37.4) 46–65 years 428 (21.0) 250 (22.4) Gender Men 921 (45.2) 303 (27.1) Women 1117 (54.8) 815 (72.9) Religion Muslim 1938 (95.3) 1073 (96.1) Christian 82 (4.0) 37 (3.3) Other 13 (0.6) 6 (0.5) Race Arabs 1625 (79.9) 912 (81.6) Non-Arabs 409 (20.1) 205 (18.4) Education Illiterate 425 (20.9) 257 (23.0) ≤ 9 years 1131 (55.5) 578 (51.7) > 9 years 482 (23.7) 283 (25.3) mean ± SD mean ± SD Total number of people in the household 6.5 ± 3.3 6.7 ± 3.2 Adults 3.3 ± 1.8 3.5 ± 1.9 Children 3.2 ± 2.5 3.2 ± 2.5 Household density (household/rooms) 2.2 ± 1.3 2.2 ± 1.3 Total SES score 4.3 ± 2.0 4.0 ± 2.0 Table 2 Various indicators of exposure to ETS among adults non-smokers (n = 1118) in Aleppo, Syria. Non-smokers with CO≤10 ppm n (%) Spouse's smoking (cigarettes and waterpipe) 351 (43.7)* Parental smoking None 374 (33.5) Father 591 (52.9) Mother 36 (3.2) Both 117 (10.5) Number of household smokers Cigarettes ≥ 1 smoker 543 (48.6) Waterpipe ≥ 1 smoker 47 (4.2) Both ≥ 1 smoker 108 (9.7) Past year regular exposure to other's smoke 769 (68.8) Past week sensory irritation from ETS exposure Sometimes 188 (16.8) Many times 103 (9.2) Past week hours spent daily with smokers At home ≤ 1 hour/day 651 (58.2) >1 hour/day 467 (41.8) At other places ≤ 1 hour/day 795 (71.1) >1 hour/day 323 (28.9) Exposure to smoking at workplace Yes/well ventilated 205 (58.9)* Yes/poor ventilated 32 (9.2)* Average cigarettes smoked daily in the house 1–10 cig/day 438 (39.2) > 10 cig/day 281 (25.1) Average waterpipes smoked daily in the house 1–2 waterpipe/day 33 (3.0) > 2 waterpipe/day 8 (0.7) House policy regarding smoking Smoking is not allowed at all 40 (3.6) Smoking is allowed for only few guests 137 (12.3) Smoking is allowed only in special places 106 (9.5) Smoking is not restricted at all 812 (72.6) Differs between cigarettes and waterpipes 23 (2.1) * Calculated from the number of non-smokers who are currently married (n = 803), or employed (n = 348) Outcomes Past year recurrent cough and recurrent shortness of breath were defined as having 3 or more recognizable episodes of these symptoms. Those reporting recurrent cough or shortness of breath were asked to select, from a provided list, one or more options that best described their symptoms (Table 3 ). The main asthma symptom (past year wheezing/whistling in the chest) and asthma diagnosis were inquired about from all participants, while other asthma symptoms (recurrent cough accompanied by wheezing, recurrent nocturnal cough unrelated to colds, and recurrent episodic shortness of breath accompanied by wheezing) were inquired about among those reporting recurrent cough or shortness of breath. Items related to physician-diagnosed conditions included ever having a diagnosis of (asthma, chronic bronchitis, or emphysema), or the occurrence of a diagnosed condition (sinusitis, acute bronchitis, pneumonia) during the past year. Hay fever was defined conservatively based on positive responses to two questions about past year nasal allergic symptoms (episodes of sneezing, runny or blocked nose when not experiencing a cold), and the co-occurrence of itchy and watery eyes [ 38 ]. Severity of respiratory complaints was based on the number of wheezing/whistling episodes for asthma (≤ 12 and > 12), reporting more than one episode of sinusitis or acute lower respiratory tract infection, and medical care (medication use, hospital or clinic visits) for respiratory problems (Table 4 ). Medication or health facility use because of respiratory problems was broken down further by condition (asthma, pneumonia, bronchitis, etc.), but because none of these outcomes were associated with exposure to ETS in our study we used only the parent general question. Table 3 Relation between different levels of exposure to ETS and respiratory symptoms/diagnosis among adult non-smokers in Aleppo-Syria (n = 1118) ETS score * Self-reported respiratory symptoms/diagnoses middle high P Dose-response General respiratory symptoms Past year recurrent cough (≥ 3 recognizable episodes) 1.3 (0.8–1.9) 1.9 (1.2–2.9) 0.004 Past year recurrent shortness of breath (≥ 3 recognizable episodes) 1.6 (1.1–2.3) 1.7 (1.1–2.6) 0.001 Past year recurrent exertional shortness of breath that disappears after rest 1.8 (1.2–2.7) 2.0 (1.3–3.2) <0.001 Past year recurrent shortness of breath almost all the time 3.0 (1.3–6.8) 2.6 (1.1–6.3) 0.02 Symptoms/diagnosis suggestive of asthma Past year wheezing/whistling in the chest 1.4 (0.8–2.2) 1.7 (1.0–2.8) 0.05 Past year recurrent episodic dry cough accompanied by wheezing/whistling 1.9 (1.0–3.7) 1.9 (0.9–3.9) 0.05 Past year recurrent nocturnal cough, not related to colds, that wakes the subject up 1.2 (0.7–1.9) 1.9 (1.1–3.2) 0.02 Past year recurrent episodic shortness of breath accompanied by wheezing 2.2 (1.1–4.4) 1.6 (0.7–3.7) 0.06 Ever diagnosed asthma 1.8 (1.2–2.8) 1.4 (0.9–2.4) 0.1 Hay fever (nasal allergy symptoms with eye itching and watering) 0.9 (0.6–1.3) 1.5 (0.9–2.3) 0.01 Symptoms/diagnosis suggestive of chronic bronchitis Productive cough that lasts most of the winter 1.2 (0.7–2.2) 1.6 (0.8–2.9) 0.2 Recurrent shortness of breath accompanied by cough and phlegm 2.2 (1.1–4.7) 2.5 (1.1–5.6) 0.02 Ever diagnosed chronic bronchitis/emphysema 1.1 (0.5–2.2) 1.2 (0.5–2.7) 0.6 Symptoms/diagnosis suggestive of respiratory infection Past year recurrent cough accompanying upper respiratory infections (cold, flue) 1.0 (0.7–1.6) 1.5 (0.9–2.4) 0.1 Past year recurrent cough with bloody phlegm 1.1 (0.5–2.6) 1.4 (0.6–3.4) 0.7 Past year sinusitis 1.0 (0.6–1.7) 1.7 (1.0–2.9) 0.09 Past year diagnosed acute lower respiratory infection (bronchitis, pneumonia) 1.3 (0.7–2.3) 1.9 (1.1–3.6) 0.03 * Odds ratio and 95% confidence interval for the relation between ETS score tertiles (lower being referent) and respiratory symptoms/diagnosis according to multivariate logistic regression adjusted for age, gender, SES score, hay fever, parental allergy, pack-years (for ex-daily smokers), occupational exposure to respirable pollutants other than ETS Table 4 Relation between different levels of exposure to ETS and severity of respiratory problems of adult non-smokers in Aleppo-Syria ETS score * Severity of respiratory problems middle high P Dose-response Number of wheezing attacks in the past year (≤12 vs. >12) 0.6 (0.2–2.2) 1.2 (0.3–4.5) 0.6 Number of episodes of sinusitis (once vs. more than once) 0.5 (0.2–1.4) 1.2 (0.4–4.1) 0.3 Number of episodes of acute lower respiratory tract infection (once vs. more than once) 1.4 (0.4–4.6) 0.7 (0.2–2.3) 0.3 Past year doctor's or hospital visit because of respiratory problems 1.4 (0.9–2.1) 1.2 (0.7–2.0) 0.4 Past month medication use for respiratory problems 1.0 (0.5–1.7) 1.0 (0.5–1.9) 0.7 * Odds ratio and 95% confidence interval for the relation between ETS score tertiles (lower being referent) and respiratory symptoms/diagnosis according to multivariate logistic regression adjusted for age, gender, SES score, hay fever, parental allergy, pack-years (for ex-daily smokers), occupational exposure to respirable pollutants other than ETS Forced Expiratory Volume in the 1 st second (FEV 1 ) and Forced Vital Capacity (FVC) were measured for all participants according to standard guidelines [ 39 ]. We used hand-held spirometer (Micro-plus, Micro Medical, Rochester, UK), which have been shown to have good precision and reproducibility [ 40 ]. We used newly calibrated spirometers and tested them weekly by team members with known lung functions (allowing for no more than 5% variation between different spirometers). Multiple maneuvers were performed until three satisfactory ones were recorded. The best effort that did not exceed the next best by more than 5% was included in the analysis [ 41 ] (Table 5 ). Table 5 Relation between exposure to ETS and lung functions among men and women non-smokers (n = 623) in Aleppo-Syria ETS score middle high P Dose-response Men FEV 1 (ml) -46.8 (-215.4 to 121.7) 34.3 (-140.5 to 209.1) 0.3 FVC (ml) 63.9 (-167.5 to 295.3) 147.9 (-89.1 to 385.0) 0.5 FEV 1 /FVC (%) -1.2 (-3.7 to 1.6) -1.0 (-3.5 to 1.6) 0.5 Women FEV1 (ml) -87.8 (-164.8 to -10.7) -58.7 (-136.5 to 19.1) 0.3 FVC (ml) 3.1 (-103.8 to 110.1) -72.2 (-182.4 to 38.1) 0.2 FEV 1 /FVC (%) -2.0 (-3.8 to -0.3) -1.0 (-2.9 to 0.8) 0.1 * Unstandardized linear regression coefficient and 95% confidence interval for the relation between ETS score tertiles (lower being referent) and lung function tests adjusted for age, BMI, SES score, hay fever, pack years (for ex-cigarette smokers), occupational exposure, parental allergy, and interaction terms of age with height and age with weight Analysis Descriptive statistics were calculated for the overall study population and for measures of ETS exposure among non-smokers (Tables 1 , 2 ). Composite scores for socioeconomic status (SES score) and self-reported ETS exposure indices were constructed for the analysis (as illustrated in the additional file, Appendix 1). Spearman correlation coefficients were calculated to assess the relation between FEV 1 , FVC, FEV 1 /FVC and ETS score. Logistic regression was used to estimate the odds ratio (OR) and the 95% confidence interval for the relation between ETS score and respiratory symptoms adjusting for age, sex, SES score, parental allergy, occupational exposure to other respiratory pollutants, and pack years (for ex-daily smokers). Linear regression analysis was used to assess the relationship between ETS score and lung function (FEV 1 , FVC, and FEV 1 /FVC) adjusting for age, BMI, SES score, and occupational exposure to other respiratory irritants, as well as interaction terms of age with height and weight. This analysis was performed separately for men and women, since lung development and response to ETS has been shown to differ by gender [ 26 , 28 ]. In both multivariate models (logistic, linear), ETS score was first entered as a categorical variable (low; bottom tertile, middle; middle tertile, high; top tertile) for the calculation of OR for different gradients of exposure, then as continuous variable for the calculation of p for dose-response relationship. Because of the concern that ex-smokers may avoid ETS exposure and have respiratory problems (giving us a group with potentially most respiratory problems but least exposure), we repeated the analysis including only never smokers, but this did not affect the results considerably (analysis not shown). All analyses were done by SPSS 11. Results From a total of 2038 valid survey responses (45.2% men, mean age 35.3 ± 12.1, response rate 86%), 1118 (27.1% men) satisfied the inclusion criteria for the exposure-symptoms analysis (Table 1 ), and 623 (30% men) for the exposure-lung functions analysis (Table 5 ). According to ETS score (mean ± SD 8.8 ± 3.6, median 9), the vast majority of non-smokers in our population were exposed to ETS, whereby only 3.6% had levels ≤ 2 and 21.1% had levels ≤ 5 (Table 2 ). Logistic regression analysis of the relation between ETS score and self-reported symptoms/diagnosis generally shows a dose-response association with symptoms and diagnosed acute lower respiratory tract infection (acute bronchitis, pneumonia). General respiratory symptoms associated with exposure to ETS were past year recurrent cough (ORs for comparison between middle, high, with the low exposure group were 1.3 and 1.9, respectively, with p for dose response 0.004 ), past year recurrent shortness of breath (ORs 1.6 and 1.7, p = 0.001 ), past year recurrent exertional shortness of breath that disappears after rest (ORs 1.8 and 2, p < 0.001 ), past year recurrent shortness of breath almost all of the time (ORs 3 and 2.6, p = 0.02 ). Additionally, several symptoms suggestive of asthma/allergy were related to ETS exposure, including past year recurrent wheezing/whistling in the chest (ORs 1.4 and 1.7, p = 0.05 ), past year recurrent episodic dry cough accompanied by wheezing (ORs 1.9 and 1.9, p = 0.05 ), past year recurrent episodic shortness of breath accompanied by wheezing (ORs 2.2 and 1.6, p = 0.06 ), past year recurrent nocturnal cough not related to cold that wakes the subject up (ORs 1.2 and 1.9, p = 0.02 ), and past year hay fever symptoms (ORs 0.9 and 1.5, p = 0.01 ). Among symptoms suggestive of chronic bronchitis, ETS exposure was associated with recurrent shortness of breath accompanied by cough and phlegm (ORs 2.2 and 2.5, p = 0.02 ). And finally, past year episodes of acute lower respiratory infection (bronchitis, pneumonia) were associated with exposure to ETS (OR 1.3 and 1.9, p = 0.03 ) (Table 3 ). In contrast, exposure to ETS was not related to severity of respiratory complaints judged by the number of episodes (wheezing, sinusitis, acute bronchitis, pneumonia), medical care utilization for respiratory problems in general (Table 4 ), as well as medical care utilization for a specific problem (e.g. asthma, pneumonia, analysis not shown). In the univariate analysis, there was a weak inverse correlation between ETS score and FEV 1 (coefficient -0.1, p < 0.001 ), FVC (-0.1, p = 0.002 ), and %FEV1/FVC (-0.6, p = 0.1 ). Linear regression analysis between ETS exposure score and lung functions showed significant inverse associations with indices of airflow limitation (FEV 1 and FEV 1 /FVC) only in women. Women in the middle category of ETS exposure had on average 88 ml deficit in FEV 1 and 2% in FEV 1 /FVC in comparison to those in the low exposure category. This association did not show a dose-response relationship (Table 5 ). Discussion This study shows that exposure to ETS is universal among non-smoking adults in Aleppo-Syria. This exposure is associated with respiratory complaints of both infectious and non-infectious etiology in a dose-response fashion, suggesting a causal relationship. Unlike data from developed countries, however, exposure to ETS was not related to increased severity of asthma or other respiratory conditions judged by symptoms frequency and medical care utilization because of respiratory problems. ETS exposure was associated with decrements in lung functions suggestive of airflow limitation (FEV 1 and FEV 1 /FVC) among women only. Error and bias in ascertainment are always a concern in cross-sectional studies, due to imprecise or differential recall. We tried to minimize such problems by not highlighting tobacco or ETS exposure when introducing the study to participants [ 42 ], by using symptoms/diagnosis descriptors that are native to the target population, and by assessing multiple indices of both exposures and outcomes. We have some indicators that such bias was limited, including lack of associations between a diagnosis of asthma or chronic bronchitis and ETS score. Remarkably, recurrent cough with bloody phlegm, one of the potentially most startling respiratory symptoms, was not associated with ETS exposure in this study, giving further support of minimal recall bias. Our reliance on self-reported exposure is also a potential limitation, but studies have repeatedly shown that self-report is a valid measure of ETS exposure that correlates with other objective markers such as cotinine [ 43 - 46 ]. Understandably, the composite ETS score is a crude quantitative measure of ETS exposure. We opted for its use to incorporate various sources of information about exposure to ETS in order to differentiate between meaningful gradients of exposure for the purpose of the analysis. On the other hand, the diversity of information relevant to ETS exposure considered in this study, in addition to the verification of non-smoking status by breath CO measurement, and the use of multiple subjective and objective outcomes, helped delineate the exposed group and conduct a robust analysis. The widespread exposure to ETS among adults in Aleppo, suggests that it is rather hard to avoid such exposure in this environment. The vast majority of non-smokers in Aleppo are exposed to ETS both at home and outside. This exposure is sufficiently intense to cause sensory (eye and nose) irritation for a quarter of nonsmokers. In comparison, less than a quarter of non-smokers in a national sample of 43,732 adults in the US report exposure to ETS [ 47 ]. Interestingly, such spread of exposure is occurring in the face of enacted laws banning smoking in public buildings, worksites, and transportation in Syria since the nineties [ 48 ]. Our results indicate that these laws are not enforced, as about two thirds of working non-smokers report exposure to others' smoke at work. Accordingly, these results should provide solid ground for public health advocates and authorities to push for the application of policies and measures to protect non-smokers from this hazardous exposure. Although not in the realm of laws and regulations, the widespread liberal attitude towards smoking in the house suggests a general lack of awareness, or dismissal, of the health damaging effects of ETS exposure to household members. Remarkably, about three quarters of studied households do not restrict indoor smoking whatsoever, and only a small minority (3.6%) has total restriction. This shows that in societies where smoking is rather a norm, it becomes hard to employ smoking restrictions even in one's own house. Increasing public awareness of this health hazard is thus an area where public health advocacy can make a difference. In general, this study shows stronger and dose-dependent relationships of ETS exposure with general respiratory symptoms (i.e. recurrent cough or shortness of breath) than with symptoms characteristic of specific respiratory problems (asthma, bronchitis). Arguably, general symptoms are more easily identifiable as well as shared among many respiratory problems. The magnitude of difference in self-reported asthma symptoms according to exposure level is generally in the range of a twofold increase. This effect magnitude is similar to that reported in adults from 16 European countries (European Community Respiratory Health Survey, ECRHS), but lower than that reported from the Swiss Study on Air Pollution and Lung Diseases in Adults (SAPALDIA) [ 49 , 50 ]. Similar to studies from developed countries, we found a dose-response relationship between exposure to ETS and respiratory symptoms, implying a causal relationship [ 28 , 49 - 52 ]. On the other hand, unlike data from European nations (ECRHS), we found a 50% increase in hay fever symptoms for the high exposure group compared to those with low exposure. We defined hay fever according to symptom report, however, while the ECRHS inquired about suffering from allergic rhinitis or hay fever [ 49 ]. Also, among symptoms indicative of chronic bronchitis, only recurrent shortness of breath with cough and phlegm was associated with ETS exposure in our study, while in the ECRHS ETS exposure during childhood was associated with increased reporting of recurrent cough and phlegm in adulthood [ 28 ]. Since cough and phlegm are common symptoms of infectious respiratory problems, likely to be widespread in our population due to overcrowding and poor housing conditions, such symptoms can be non-specific indicators of chronic bronchitis in our setting, while shortness of breath can be more specific marker of this condition. Level of exposure to ETS in our population was not associated with severity of asthma, sinusitis, or lower respiratory tract infection. In contrast, in a study of 349 adults with asthma in the US, Eisner and colleagues found that exposure to ETS at baseline was associated with more symptom severity and emergency/hospital admissions because of asthma at 18 month followup [ 53 ]. Medical care utilization for respiratory problems is likely to be an inadequate indicator of severity in a low-income country such as Syria, where the lack of medical insurance and limited public health services render seeking private health care the last resort for most adults in this country. On the other hand, as it will be discussed later, the lack of a comparison group of non-exposed individuals may have contributed to the absence of association between ETS exposure and symptoms severity in this study. Studies of the relation between exposure to ETS and lung function have generally shown a detrimental effect of such exposure. This effect, however, was not consistent and of low magnitude generally (50–100 ml) [ 3 ]. For example, in a population-based sample of adults from the NHANES III survey in the US, exposure to ETS was associated with decreased lung functions (FEV 1 , FVC, FEV 1 /FVC) in women but not men [ 26 ]. Data from the ECRHS involving 18,922 adults from 17 European countries show that exposure to parental smoking in childhood was associated with impaired lung function [ 28 ]. The effect on lung function differed, however, according to participant's gender, parental smoking (mother, father, both), and period of exposure (during pregnancy, childhood) [ 28 ]. Our study shows that exposure to ETS is associated with decreased lung function indicative of airflow limitation (FEV 1 , and FEV 1 /FVC) in female but not male non-smokers. The reduction for the middle compared to low exposure category was in the magnitude of 88 ml for FEV 1 and 2% for FEV 1 /FVC. Although other studies have reported similar gender-specific vulnerability of women [ 26 ], it is important to emphasize the small number of male non-smokers in our sample (less than one third). We also could not elicit a dose-response in the relation between ETS exposure and airflow limitation among women, although ETS score was weakly correlated to FVE 1 in the univariate analysis. It is possible that we are dealing in our setting with levels of exposure that exceed those occurring in western societies. Indeed, because of the low magnitude of the effect of ETS exposure on lung function, studies have relied on comparisons of exposed vs. non-exposed individuals to assess this relationship [ 49 , 53 - 55 ]. In our sample, however, we had very few subjects with no or little exposure, which could have reduced the sensitivity of our analysis. Another possibility is that the relatively crude measure of ETS (ETS score) we used may better differentiate between gradients of exposure at its lower stratum than higher. Conclusions This study shows that exposure to ETS is rampant among adult non-smokers in Syria, where it is hard to escape it due to a high prevalence of smoking, household over-crowding, and lack of smoking restrictions. This exposure is leading to increased respiratory symptoms/disease of both infectious and non infectious etiologies, and is likely to have deleterious effects on respiratory function among women. In addition, the dose-response association found between exposure to ETS and respiratory symptoms point towards causal relationship. These results send a clear message to health advocates and policy makers about the spread and harmful effects of exposure to ETS in Syria and on the importance of collective efforts to educate both the public and authorities about this major health hazard and ways to effectively protect non-smokers from it. In addition to giving further support to the health hazards of ETS exposure, this study can provide guidance for future research on this issue in other developing countries. List of abbreviations • AC- air condition • AHS- Aleppo Household Survey • ECRHS- European Community Respiratory Health Survey • ETS- environmental tobacco smoke • FEV 1 - forced expiratory volume in the 1 st second • FVC- forced vital capacity • NHANES III- third National Health and Nutrition Examination Survey • OR- odds ratio • PC- personal computer. • ppm- part per million • PPS- probability proportionate to size • SAPALDIA Swiss Study on Air Pollution and Lung Diseases in Adults • SD-standard deviation • TV- television Authors' contribution W Maziak, designed the study, conducted the analysis and wrote the 1 st draft of the manuscript. KD Ward, T Eissenberg, participated in the study design and co-authored the manuscript. S Rastam and F Mzayek participated in the data management, analysis, and co-authored the manuscript. Supplementary Material Additional File 1 Appendix 1 : Composite scores for SES and ETS used in the study with the total score categorized around tertile cut off points. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC549073.xml
539231	A two-way interface between limited Systems Biology Markup Language and R	Background Systems Biology Markup Language (SBML) is gaining broad usage as a standard for representing dynamical systems as data structures. The open source statistical programming environment R is widely used by biostatisticians involved in microarray analyses. An interface between SBML and R does not exist, though one might be useful to R users interested in SBML, and SBML users interested in R. Results A model structure that parallels SBML to a limited degree is defined in R. An interface between this structure and SBML is provided through two function definitions: write.SBML() which maps this R model structure to SBML level 2, and read.SBML() which maps a limited range of SBML level 2 files back to R. A published model of purine metabolism is provided in this SBML-like format and used to test the interface. The model reproduces published time course responses before and after its mapping through SBML. Conclusions List infrastructure preexisting in R makes it well-suited for manipulating SBML models. Further developments of this SBML-R interface seem to be warranted.	Background Systems biology markup language (SBML) is a standard for representing dynamical systems of biological interest [ 1 , 2 ]. Interfaces between SBML and high level computational environments are currently being developed for Mathematica [ 3 ] and Matlab [ 4 ], but to the author's knowledge, no such efforts are being carried forth for R/S-plus. This brief paper presents the author's initial developments toward a two-way SBML-R interface. The interface is currently limited in the range of SBML input files that it can handle. For example, it only handles SBML level 2 and does not handle "Events" and "FunctionDefinitions." The interface can nevertheless be used for some models, examples [ 5 , 6 ] of which are included under "demo" in the SBMLR package [ 7 ]. This paper provides an explicit example of one approach to an SBML-R interface. It is assumed throughout that the reader is already quite familiar with both SBML [ 8 ] and R [ 9 ]. Implementation The software exists completely in R. It is comprised of four functions and is currently being distributed as a developmental package called "SBMLR" through Bioconductor [ 10 ]. The software was written subject to two constraints: 1) models expressed in SBML-like R must be exchangeable with a range of SBML models; and 2) models must be amenable to simulation in R. The first subsection that follows defines an SBML-like R model structure, the second illustrates how it can be used in simulations, and the third describes its conversions into and out of SBML. An SBML-Like Model Structure in R To facilitate mappings between SBML and R, an SBML-like list structure is defined in this subsection using the purine metabolism model of Curto et al. [ 6 ] as a specific example (Figure 1 ). In this figure and elsewhere, ellipses (...) indicate missing code not critical to current discussions; complete source codes are available through the SBMLR package [ 7 ]. The essential components of an SBML model, namely, its compartments, species and reactions, are all present in this R analog of an SBML model. In the model of Curto et al. [ 4 ], there is one compartment, be it the cell or the entire human body, and 18 species: 2 boundary conditions (bc = True) and 16 state variables (bc = False), each with an initial condition (ic) or value. Each reaction is a list that includes a reaction id, the names of species that are reactants (reacts), the names of species that are reaction rate modulators (mods), the names of species that are produced by the reaction (prods), parameter values (params), and the reaction rate law (law) function definition. In this framework, only state variables need be listed as products, boundary condition reactants can equivalently be listed as modulators, and missing terms (e.g. mods in reactions 1 and 37) are equivalent to a NULL assignment. The rate law function has as its input arguments two vectors, one carrying the concentrations of reactants and modulators (r), the other carrying reaction parameter values (p). If the body of the rate law function contains n statements, the first n -1 trivially convert input vector components into variables with the same names. The n th statement then contains the complete reaction rate law. It can occupy multiple lines, but it must be a single statement, i.e. it cannot depend on substitution variables temporarily defined in preceding statements. SBML-like Model Execution in R Model definition codes such as that given in Figure 1 , when placed in a separate file (e.g. Curto.r), can be sourced into a parent script to become globally available for simulations. For example, the purine metabolism model of Curto et al. [ 6 ] can be simulated using the execution code shown in Figure 2 . This code simulates the response to a 10-fold increase in phosphoribosylpyrophosphate (PRPP) at time t = 0 and plots the responses of inosine monophosphate (IMP) and hypoxanthine (HX) as shown in Figure 3 . Two functions called by this script are defined in the SBMLR package and shown in Figure 4 . They are, getIncidenceMatrix(), which computes the incidence/stoichiometry matrix used by the second function, fderiv(), which computes state derivatives for integration by the function lsoda() of the "odesolve" package. In getIncidenceMatrix(), the incidence matrix is generated automatically using an i loop over the rows (i.e. state variables) and a j loop over the columns (i.e. reactions). If a state is a product of a reaction, the corresponding matrix element becomes a positive integer equal to its stoichiometry [factor() converts string names to factors so that summary() can count them], and similarly for reactants, though with negative numbers entering the matrix in this case (or possibly zero, if a reactant of a reaction happens to also be a product of the same reaction). The function fderiv() creates the current species vector by overriding initial states with current states clipped to positive values, and by overriding any time varying boundary conditions defined by rules (SBML rules are not needed for the purine model, but are needed to implement other models [ 5 ]). The function fderiv() then computes the reaction rate flux vector (v) based on the current species vector (St) and multiplies it by the incidence matrix to produce the current state derivative vector (xp). The names of xp and v are reset at the end of each function call to override the problem of variables gaining new composite names from the names of their expression arguments. A Two-Way Interface between SBML and R Two functions comprise the SBML-R interface: write.SBML() converts SBML-like R models (e.g. Curto.r) into SBML models (e.g. Curto.xml), and read.SBML() converts SBML models (e.g. Curto.xml) into an SBML-like R model (e.g. CurtoX.r). A key component of these two interface functions is a locally defined recursive function named recurs(). This function converts arbitrary R expressions into arbitrary MathML expressions, and vice-versa; it is defined differently, locally, in each of the two functions. In write.SBML(), shown in Figure 5 , recurs() initially takes as its input argument the last component of the body of the kinetic rate law function definition, which is the entire rate law expression (as mentioned above, rate laws involving multiple R statements are not supported). In R, expressions are LISP like in that they contain a first element, the operator, and the remaining elements, the arguments, any of which can be an expression. If the operator is the parentheses operator, the action taken is that of a unary identity operator, and we simply skip it and move on to its argument since parentheses are not needed in MathML. Each nested call to the function recurs() sends "<apply>" and the converted operator to the output file on its way in, and a matching "</apply>" on its way out. Nested calling continues until all nodes of the expression tree are of class "name" or "numeric," i.e. when all found objects are leaves of the tree rather than "expressions" that require further parsing. Leaves are then sent to the output file bracketed by <ci> and </ci>. The second of the two SBML-R interface functions, read.SBML(), maps a limited range of SBML level 2 files (function definitions and events are not handled) into SBML-like R model files. Portions of read.SBML() are given in Figure 6 . The main difference between this function, read.SBML(), and the previous function, write.SBML(), is that here, rather than using parse() to decompose the list-of-lists structure of the model defined in R, the SBML model is instead decomposed as an XML object using xmlTreeParse() of the XML package available to R [ 11 ]. In read.SBML(), the locally defined recursive function recurs() uses an overkill of parentheses to avoid operator precedence issues. This recursive function is passed a MathML reaction rate law which it parses recursively until the leaves of the tree (the "ci") are all found. During the recursion a corresponding R expression is built as a vector of character strings which, upon exit from the last of the recursive calls, is collapsed into a single string and sent to the output file as the last line of the current rate law function definition. Results The function write.SBML() was applied to Curto.r to generate Curto.xml and the function read.SBML() was then applied to Curto.xml to generate CurtoX.r. Execution of the script given in Figure 2 with line 4 of the execution code changed to act on CurtoX.r instead of Curto.r generated the same plots as before (Figure 3 ). This shows that the R model was successfully converted into an SBML file that can be reconverted back into a properly functioning R model. The intermediate file Curto.xml was successfully validated as an SBML level 2 file [ 12 ]. The SBML file could thus be imported into visualization packages such as JDesigner [ 13 ]. Discussion If the model of Curto et al. [ 4 ] were implemented in R without any knowledge of SBML, a form that it might take is that given in Appendix B (Figure 7 ). Compared to its SBML-like counterparts, this code is more compact and easier to understand, e.g. the system's network connectivity is clearly visible. The disadvantage of such code is that it is not readily converted into SBML. Since the benefits of SBML are compelling, this disadvantage alone warrants the use of SBML-like model structures. As SBML evolves to handle a broader range of dynamical systems, it will become more and more challenging for simulation packages to handle all possible SBML models. It is envisioned here that the development of this SBML-R interface will be driven by its users, and not by the model representation capabilities of SBML, i.e. it is expected that the users of this interface will be programmers who are capable of modifying it as their needs require. Conclusions Compared to Matlab, which may be better equipped than R to simulate arbitrarily complex dynamical systems, R has the advantage of list handling infrastructure in parse() and xmlTreeParse(), and it also has the advantage of indexing by names instead of numbers. A further advantage, though not exploited here, is that R is object-oriented; in future versions of this interface, a print() method might be defined for objects of class SBMLR (i.e. models) to generate more readable renderings of models in R. Another advantage of R over Matlab is that it provides access to a much broader collection of microarray analysis tools, e.g. see Bioconductor [ 10 ]. This aspect is important for those individuals who are interested in biochemical systems analyses of microarray data [ 14 , 15 ]. For statisticians already familiar with R, there are also the obvious economies of maintaining system familiarity. Finally, perhaps the biggest advantage of R over Matlab is that it is freely available. On balance, there seems to be ample motivation for further developments of this interface between SBML and R. Availability and requirements Project name: SBMLR Project home page: Operating system(s): Windows XP Programming language: R 2.0 Other requirements: R packages: XML and ODESOLVE License: GNU GPL Any restrictions to use by non-academics: no restrictions List of abbreviations SBML = Systems Biology Markup Language; XML = extensible markup language; MathML = Mathematical Markup Language; ODE = ordinary differential equation. Authors' contributions TR is the sole contributor. Appendix A The SBMLR package is available through Bioconductor as a developmental package [ 7 ]. It has been developed and tested only under Windows XP. To install, do NOT unzip the file SBMLR.zip after downloading to a local directory, rather, within the R GUI, click packages and install from local zip . The XML package installs similarly [ 11 ]. Note that an error message from library(XML) can be resolved by copying the *.dll files of the XML package libs directory into the "C:\windows" directory. The ODESOLVE package must be installed before running simulations. This package is installed from the R GUI by clicking packages and install from CRAN . Appendix B The implementation of Curto et al.'s model shown in Figure 7 is independent of any knowledge of SBML. It is included here to illustrate what comes "naturally" when implementing a model in R, see Discussion.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC539231.xml
524260	Taking Stock of Biodiversity to Stem Its Rapid Decline	null	Far more species exist in the fossil record than inhabit Earth today. An estimated 94% of all bird species that ever lived, for example, are now extinct. So why is species extinction of urgent concern today? Though species come and go over evolutionary time, mass extinctions are relatively rare. Biologists believe they have occurred only five times, arising from relatively short-lived cataclysmic natural forces like astronomical or volcanic events. We are now on the brink of the Sixth Great Extinction, and we humans are largely to blame. For thousands of years, humans have retooled the landscape, an endeavor that has rarely coincided with the life history needs of local flora and fauna: over 150 bird species alone have vanished since 1500. As our capacity to alter the landscape has mushroomed, species have started disappearing faster than biologists can identify and document them. Mindful of this crisis, nearly 200 countries (under the Convention on Biological Diversity, or CBD) agreed to staunch the loss of biodiversity by 2010, with the European Union raising the bar to halt biodiversity loss by that time. To meet this goal, biologists need reliable metrics to monitor global trends in biodiversity. Stuart Butchart et al. describe a new model for generating such indices to measure trends in extinction risk for complete classes of organisms, starting with the world's 10,000 bird species. Their “Red List Index” measures changes in overall extinction risk over time for all bird species worldwide. Similar indices are already being developed to track other groups, including mammals and amphibians, and in the future will hopefully be developed for some plant and invertebrate groups. In 2002, the CBD proposed that efforts to monitor global trends in biodiversity start by developing indicators to evaluate trends in biodiversity components, such as ecosystems and habitats, abundance and distribution of selected species, and change in threat status of species. Butchart et al. focus on evaluating trends in changes in threat status (extinction risk), relying on categories developed by the World Conservation Union (IUCN) Red List. Species are placed in categories on the Red List ranging from extinct to “least concern,” according to criteria that take into account their population size, population trends, and range size. Thousands of scientists from around the world feed these assessments, which have been widely used to measure the degree of degradation of biodiversity. Rufous-collared Kingfisher: Deforestation threatens its survival (Photo: Jakob Wijkema) To use the Red List to track biodiversity trends over time, Butchart et al. collected data from four complete assessments of the world's birds over sixteen years, supplemented by other sources. The number of threatened and near threatened species increased from 1,664 species in 1988 to 1,990 species in 2004, but many species moved between categories. To calculate the real net increase in extinction risk for the world's birds over this time, the authors first identified reasons for these category switches to remove biases introduced by factors irrelevant to genuine changes in species status; category changes owing to better knowledge, for example, do not reflect real changes in conservation status. They also accounted for time lags between status changes, and category changes owing to delays in knowledge becoming available to Red List assessors. The authors argue that the Red List Index provides a simple measure of trends in the status of avian species worldwide, in terms of their overall extinction risk. Overall, the index shows “a steady and continuing deterioration in the threat status of the world's birds between 1988 and 2004.” Though the extinction risk has improved for some species, it has deteriorated for others, with “particularly steep declines” in recent years for Asian birds—resulting from massive deforestation in Indonesia—and for seabirds such as albatrosses and petrels, which drown on the hooks of commercial long-line fisheries. Butchart et al. argue that Red List Indices complement indicators based on population trends, because although the indices show coarser temporal resolution, they have much better geographic representation; they're based on nearly all species in a group worldwide rather than on a potentially biased subset. Both types of species-based indicators show finer ecological resolution in tracking biodiversity loss than indicators like habitat or biome trends. Thus, the Red List Index provides a baseline for tracking progress toward the 2010 target. But having a reliable indicator is only the first step. Without an international commitment to halt the advancing extinction crisis, biodiversity will continue to decrease. The United States is the only industrialized nation that has not signed on to this effort.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC524260.xml
450293	Correction: Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones	null	In PLoS Biology , volume 2, issue 6: Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones Tadashi Imanishi, Takeshi Itoh, Yutaka Suzuki, Claire O'Donovan, Satoshi Fukuchi, et al. DOI: 10.1371/journal.pbio.0020162 1. The abbreviation FLJ was expanded incorrectly (as full-length long Japan) in the abbreviations list and in the first paragraph of the Results/Discussion section. FLJ stands for full-length cDNA Japan. 2. In Table 1, the number of cDNAs and the number of library origins for two of the cDNA sources were incorrect. The correct numbers are as follows: Table 1	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC450293.xml
543576	Alterations in the expression of DEAD-box and other RNA binding proteins during HIV-1 replication	Recent results showed that certain DEAD box protein RNA helicases, DDX3 and DDX1, play an important role in the HIV infection cycle by facilitating the export of long, singly spliced or unspliced HIV RNAs from the nucleus via the CRM1-Rev pathway. Close examination of an extensive microarray expression profiling dataset obtained from cells latently infected with HIV induced to undergo lytic viral replication indicated that additional DEAD box proteins, beyond DDX3 and DDX1, exhibit differential expression during lytic HIV replication, and in latently infected cells prior to induction into active replication. This finding provides additional evidence that the involvement of DEAD box proteins and other RNA-binding proteins may play roles in active HIV replication and in the control of viral latency. Agents targeting these functions may offer new approaches to antiretroviral therapy and the therapeutic manipulation of HIV latency.	Findings The DEAD box proteins, a family of RNA helicases containing the conserved amino acid motif Asp-Glu-Ala-Asp (D-E-A-D in the single letter amino acid code), play an essential role in many aspects of cellular RNA metabolism (reviewed in [ 1 , 2 ]), including RNA transport, transcription, spliceosome function, ribosome assembly, the initiation of translation, and RNA degradation. The HIV Rev protein regulates a key aspect of the HIV replication cycle by mediating the switch between the early pattern of HIV gene expression, in which short, multiply spliced messages encoding the viral regulatory genes Tat, Rev, and Nef are exported from the nucleus, and the late pattern of viral gene expression in which larger singly spliced and unspliced messages that encode the viral structural proteins and that constitute the RNA genomes of progeny virions are exported from the nucleus [ 3 - 5 ]. Recent work, reviewed in reference [ 6 ], from the Jeang [ 7 ] and Pomerantz [ 8 ] laboratories implicate the DDX3 and DDX1 DEAD box proteins as additional critical co-factors for the Rev-mediated export of the long HIV singly spliced and unspliced mRNAs. In a directed analysis of a large data set, which describes global changes in cellular gene patterns before and after a latently infected cell line was induced into active viral replication [ 9 ], we found that genes encoding many DEAD box proteins, and other RNA and DNA binding and modification proteins, in addition to DDX3 and DDX1, showed differential regulation, suggesting that HIV replication may be associated with generalized changes in the expression of many DEAD box proteins and other RNA binding proteins. Yedavalli et al. [ 7 ] identified DDX3 in a differential display-based screen for cellular messages upregulated in the presence of HIV Tat. They found that DDX3 binds the nuclear export protein CRM1, which is essential for nuclear export mediated by the HIV Rev protein [ 10 , 11 ], shuttles between the cytoplasm and nucleus, facilitates the nuclear export of RRE-containing RNA in the presence of Rev, and advances HIV replication. Fang et al. [ 8 ] identified DDX1 in a two hybrid screen as a Rev- and RRE-binding protein that enhanced HIV replication, improved the expression of RRE-containing RNA, and modified the subcellular distribution of Rev from a predominantly nuclear to a predominantly cytoplasmic distribution. Our work on large scale expression profiling in HIV actively and latently infected cells has been guided by the hypothesis that there is one set of cellular conditions which is ideal for normal cellular growth and homeostasis, that there is another set of conditions which may be better suited to supporting viral replication, and that HIV has evolved ways of altering the host cell so as to better support viral replication. An interesting corollary of the hypothesis is that targeting the products of the differentially expressed genes may inhibit viral replication by making the host cell environment less hospitable to viral replication (reviewed in [ 12 , 13 ]). Several laboratories have conducted large scale expression profiling studies investigating changes in cellular gene expression during HIV replication [ 14 - 16 ], and have, in some cases, identified new potential targets for antiviral therapy. The observations that altering the intracellular environment by, for example, targeting kinases involved in signal transduction and cell cycle regulation inhibits HIV replication lends additional support for this hypothesis [ 17 , 18 ]. Investigators studying other viruses, for example Kaposi's sarcoma-associated herpesvirus, have also noted changes in host cell gene expression patterns that accompany infection and transformation [ 19 , 20 ], and have identified targets for potential therapeutic intervention based on those studies. In examining our data, we therefore try to note interesting and potentially targetable host cell genes that show alterations in expression during HIV replication and latency. In our earlier study, we identified cellular genes encoding proteins that constituted new targets for agents aimed at activating latently infected cells into active viral replication [ 9 ]. In our initial examination of our dataset, among the several classes of genes showing discrete, temporally-dependent changes in expression during lytic replication, we noticed that several DDX genes and genes encoding other factors involved in RNA metabolism were differentially expressed. However, prior to the work by Yedavalli et al. and Fang et al., we had no clear sense of how the differential expression of those genes might contribute to facilitating HIV replication. Those recent studies prompted us to undertake a more detailed analysis of our data. In our study, we compared RNA samples obtained from HIV latently infected cell lines prior to induction of active replication by the integrated HIV-1 provirus and following such induction with phorbol myristyl acetate (PMA). We profiled ACH-2 cells treated with PMA side by side with similarly treated HIV-1 naïve parental A3.01 cells to assess differential cellular gene expression patterns associated with HIV lytic replication. The dataset was generated from samples obtained from three independent biological replicate experiments and at least two microarray hybridizations were done for each time point from each biological replicate (for a minimum of 6 microarrays per time point). A detailed description of the cell culture, induction, RNA isolation, and microarray labeling and hybridization methods are contained in reference [ 9 ]. Following microarray data acquisition, data were analyzed using commercial (GenePix Pro software, Axon Instruments) and in-house software (microarray database system (mAdb), hosted by Center for Information Technology, NIH). Using BRB -ArrayTools , the data were subjected to statistical analyses using univariate parametric and multivariate permutation analyses, based on the one sample random variance t-statistic, where significance was based on P < 0.001 and the proportion of false discoveries was limited to 0.10 with a 90% confidence level. 1740 genes showed differential gene expression at a minimum of one timepoint during lytic replication. Hierarchical clustering analyses were performed using mAdb clustering tools, as well as Treeview . Since the data was obtained from latently infected cell lines, there may be some concern that infected primary cells may behave in a somewhat different fashion. However, the advantages of using latently infected cell lines are significant: Activation into active replication is reasonably synchronous and includes essentially all the cells, so that the signal from the cells supporting active replication is not diluted by the signal from uninfected cells or cells with virus at different stages of viral replication. Also, the signal comes only from infected cell and not from cells responding to effects from the exposure to very large numbers of defective viral particles that are in high multiplicity of infection inocula. Figure 1 shows the expression patterns for genes encoding DEAD box proteins, and other genes encoding RNA helicases and RNA binding proteins, as assigned by the gene ontology database [ 21 ]. We found that a number of DEAD-box proteins were significantly up regulated (P < 0.001) immediately following induction (0.5 hr post induction p.i). These included DDX10 , a DEAD box protein with expression in many tissues having tumorigenic activity when fused to the nucleoporin NUP98 [ 22 , 23 ], DDX21 , a DEAD-box protein originally identified as a nucleolar protein thought to be involved in ribosomal RNA metabolism[ 24 ], DDX23 , a DEAD box protein first identified in U5 SnRNPs with significant homology to the yeast Prp28p splicing factor[ 25 ], and DDX52 , a human DEAD box protein identified through its homologies to a yeast gene [ 26 ]. Figure 1 A compilation of the expression profiles of genes with known or putative involvement in RNA binding, transport or splicing before and after latently infected ACH-2 cells were induced into active replication. Panel A shows selected genes involved in RNA metabolism that were differentially expressed during active replication in the infected cells (ACH-2 cells) at 0.5–8 hr post-induction (p.i.) compared to similarly induced, parental uninfected A3.01 cells. Panel B shows gene expression profiles of a subset of DEAD-box proteins, following induction into active replication, of the genes displayed in panel A. Only genes that passed the criteria for statistical significance (P < 0.001) for the 0.5–8 hr p.i. time period (but not for other time periods) are shown. Panel C shows the expression profiles of genes encoding DDX18 and DDX39, which were up regulated during viral latency and during latency and early lytic replication. Panel D shows the expression profile of genes encoding ABC transporter proteins. The bottom of the figure shows a scale indicating the color values corresponding to the expression ratio in HIV infected/HIV uninfected cells for the differential expression of each gene shown in the figure at the different time points. Panel E is a graphical representation of the expression patterns observed in the selected DEAD-box proteins showing fold change in gene expression over corresponding controls. Other genes encoding proteins with RNA splicing/binding and RNA transport activity were also up regulated during this period, including the methylated mRNA cap binding proteins EIF4G1 [ 27 ] and NCBP1 ( CBP80 ) [ 28 ], which function in translation initiation and may also be involved in mediating nuclear export of RNA. In addition, genes encoding other proteins involved in nucleic acid-protein interactions were also upregulated, such as members of the SWI/SNF family of ATP-dependent chromatin remodeling factors involved in cell cycle control and the regulation of gene expression, SMARCA2 and SMARCA5 [ 29 , 30 ]. Another class of genes that shows differential expression during the early time period (0.5–8 hr p.i.) are the genes encoding the ATP-dependent ABC transporter proteins, which share sequence homology with members of the helicase family at the ATP binding site [ 31 ], indicating that many ATP-dependent processes may be targeted by early viral replication steps, not only as a means to facilitate viral RNA transport but also as a mechanism to shut off or divert cellular functions requiring ATP hydrolysis. Our findings that several DEAD box protein genes are upregulated during HIV replication lend support to the published finding that two cellular DEAD-box proteins, including one (DDX3) that was identified in a broad-based screen for differentially expressed genes [ 7 ], may be important mediators for Rev-mediated RNA export. Our data also show that several other RNA binding proteins are differentially regulated during HIV-1 replication, suggesting that there may be a general involvement of these classes of genes in the HIV replication cycle, that the involvement is not limited only to DDX3 and DDX1. The additional DEAD box family members and other proteins involved in RNA metabolism may be interesting candidates for further mechanistic studies on HIV replication. For example, EIF4G1 has been shown to interact with CBP80 (NCBP1) [ 32 ], as well as with EIF4A [ 33 ], an RNA helicase with a DEAD-box motif in its sequence. The binding of EIF4G1 to EIF4A is essential for the proper function of EIF4A as an RNA helicase [ 34 ]. In our study, genes encoding EIF4G1 and CBP80 were differentially expressed during early lytic replication. Further study of the interactions of EIF4G1, CBP80 and EIF4A1 may thus be important in elucidating Rev function and viral RNA export, as well as the synthesis of viral proteins. While some of the differentially expressed DEAD box proteins, beyond DDX1 and DDX3, may play a part in Rev-dependent viral RNA export from the nucleus, it is also possible that the broad induction of the expression of DEAD box protein-encoding genes and genes encoding other RNA binding factors may indicate that such gene products are involved in other aspects of HIV replication. These aspects of HIV replication could involve activities in which the DEAD box proteins have already been implicated, such as transcription, spliceosome assembly, and translation. In our recent publication [ 9 ], we showed that several host cell genes were differentially expressed in latently infected cell lines, even before induction of the integrated virus into active replication. In an approach analogous to our hypotheses concerning the involvement of cellular genes in active viral replication, we showed that targeting the products of some cellular genes differentially expressed in the latently infected cells could activate viral replication, ejecting the virus from latency. In our examination of the DEAD box proteins, we noted that two genes encoding DEAD box proteins, DDX18, a DEAD-box protein induced by Myc and Max [ 35 ] and DDX39, (or URH49), a DEAD-box protein induced by growth stimulation or protein synthesis inhibition thought to be involved in splicing and nuclear export, with homology to the yeast Sub2p protein [ 36 ], were differentially expressed during viral latency (DDX39, DDX18) and at early times (DDX18) after induction into active replication. Since some DEAD box proteins are important for viral RNA nuclear export and active viral replication, it may be reasonable to consider that other members of this family could have natural inhibitory activity for HIV replication, such as that seen with mutated DDX3 proteins [ 7 ]. Accordingly, certain DEAD box proteins may have roles in maintaining HIV latency. If this reasoning is correct, then the selective targeting of such DEAD box factors might offer another means of ending HIV latency and for depleting latent HIV reservoirs. The DEAD box proteins and other RNA helicases may therefore represent important cellular factors that can be manipulated to alter viral replication in several therapeutically useful ways. Cellular genes may be differentially expressed during viral replication for many different reasons. Differential expression of cellular genes may conceivably occur because of viral actions on the host cell designed to optimize the cell for viral replication, because of cellular responses to infection aimed at inhibiting viral infection, or may be fundamentally unrelated to key aspects of viral replication. However, the findings that several DEAD box protein genes are differentially expressed during HIV replication, together with the recently published observations that two DEAD box genes, DDX3 and DDX1, exhibit differential expression during HIV replication and have important functions in HIV replication lend additional credence to the hypothesis that a careful, large scale study of differentially expressed cellular genes can provide insights into host cell factors involved in viral replication and pathogenesis. Future studies may reveal additional human co-factors for HIV replication. These cellular co-factors many represent important new therapeutic targets. Competing Interests The authors declare that there are no competing interests. Authors' Contributions VK designed and performed the experimental work and the data analysis. SZ directed and coordinated the study and participated in the data analysis. VK and SZ wrote the manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC543576.xml
552309	Effects of inhaled corticosteroids on sputum cell counts in stable chronic obstructive pulmonary disease: a systematic review and a meta-analysis	Background Whether inhaled corticosteroids suppress airway inflammation in chronic obstructive pulmonary disease (COPD) remains controversial. We sought to determine the effects of inhaled corticosteroids on sputum indices of inflammation in stable COPD. Methods We searched MEDLINE, EMBASE, CINAHL, and the Cochrane Databases for randomized, controlled clinical trials that used induced sputum to evaluate the effect of inhaled corticosteroids in stable COPD. For each chosen study, we calculated the mean differences in the concentrations of sputum cells before and after treatment in both intervention and control groups. These values were then converted into standardized mean differences to accommodate the differences in patient selection, clinical treatment, and biochemical procedures that were employed across original studies. If significant heterogeneity was present (p < 0.10), then a random effects model was used to pool the original data. In the absence of significant heterogeneity, a fixed effects model was used. Results We identified six original studies that met the inclusion criteria (N = 162 participants). In studies with higher cumulative dose (≥ 60 mg) or longer duration of therapy (≥ 6 weeks), inhaled corticosteroids were uniformly effective in reducing the total cell, neutrophil, and lymphocyte counts. In contrast, studies with lower cumulative dose (< 60 mg) or shorter duration of therapy (< 6 weeks) did not demonstrate a favorable effect of inhaled corticosteroids on these sputum indices. Conclusions Our study suggests that prolonged therapy with inhaled corticosteroids is effective in reducing airway inflammation in stable COPD.	Background Chronic obstructive pulmonary disease (COPD) is characterized by prominent airway inflammation [ 1 , 2 ]. The intensity of the inflammation strongly correlates with disease severity [ 3 , 4 ] and increases even further during exacerbations [ 5 ]. Moreover, increased expression of inflammatory markers in the sputum is associated with increased risk of exacerbations [ 6 ]. The attenuation of the inflammatory process, on the other hand, is associated with improvements in lung function and airway hyperresponsiveness in COPD [ 7 ]. It is possible therefore that the inflammatory process is an integral component in COPD pathogenesis and may represent an important therapeutic target in improving the health status and outcomes of COPD patients [ 1 , 2 ]. One potential therapy for down-regulating the inflammatory process in the airways is through the use of corticosteroids, which are potent but non-specific anti-inflammatory agents. Some in vitro studies have demonstrated that inhaled corticosteroids can modulate certain aspects of the inflammatory cascade in COPD [ 8 , 9 ]; however, other studies have shown less favorable results [ 10 , 11 ]. Despite this uncertainty, large clinical trials have shown that these medications reduce clinically relevant exacerbations by ~30% and improve health status of patients with moderate to severe disease [ 12 ]; their withdrawal, on the other hand, leads to increased risk of exacerbations and worsening of health status [ 13 ]. Since airway inflammation is associated with exacerbations [ 6 ] and since inhaled corticosteroids reduce exacerbations [ 12 ], they may also have salutary effect on airway inflammation in COPD. However, to date, the clinical studies, which have addressed this issue, have been small in size and scope and may not have had sufficient statistical power (on their own) to detect subtle but important effect of these medications on inflammatory indices in the airways. Additionally, there may be important methodologic differences between the positive and negative studies that could potentially explain the discrepancy. We, therefore, conducted a systematic review and a meta-analysis to determine whether inhaled corticosteroids do or do not suppress airway inflammation in patients with stable COPD and to explore the potential causes for the heterogeneity in reports. Methods Search for relevant studies MEDLINE (1966–2004), EMBASE (1980–2004), CINAHL (1982–2004), and the Cochrane Databases were searched for randomized, controlled clinical trials that used induced sputum to evaluate the effect of inhaled steroids on airway inflammation in stable COPD. The search was restricted on articles published in the English language, using human participants. Subject headings included disease-specific search terms (COPD, lung diseases, pulmonary diseases, airway obstruction, obstructive pulmonary disease, chronic obstructive pulmonary disease, bronchitis, emphysema, pulmonary emphysema, or mediastinal emphysema), drug-specific search terms (glucocorticosteroids, corticosteroids, beclomethasone, budesonide, fluticasone, or triamcinolone), and laboratory method-specific search terms (biopsy, bronchoalveolar lavage, or sputum). We also scanned the bibliographies and reference lists of retrieved articles to supplement the electronic searches. We contacted the primary authors for additional data and/or clarification of data. Study selection and data abstraction The primary objective of this meta-analysis was to compare the changes in sputum inflammatory indices among stable COPD patients before and after treatment with inhaled corticosteroids, using the control group in each individual studies as the referent. We chose sputum as the primary source of the analysis because there was a marked scarcity of quality studies which had evaluated the effect of inhaled corticosteroids from bronchoalveolar lavage fluid or tissue biopsy specimens. The inflammatory indices included total cell, neutrophil, macrophage, eosinophil, lymphocyte, and epithelial cell counts and interleukin (IL)-8 levels. Since the actions of oral corticosteroids may differ from those of inhaled corticosteroids, we excluded studies that evaluated the effects of oral corticosteroids on sputum inflammatory indices. From each selected article, two investigators (WQG, DDS) abstracted the following baseline information: the source of data, study design, inclusion and exclusion criteria, concomitant drugs, demographics of study participants including sample size, age, sex, current smoking status, pack-years of smoking history, predicted forced expiratory volume in one second (FEV 1 ), the ratio of FEV 1 to forced vital capacity (FVC), percent predicted reversibility with inhaled bronchodilator, the specific brand of inhaled corticosteroids and the dose as well as the duration of therapy. Cumulative dose of inhaled corticosteroids was calculated by multiplying the average daily dose by the total days of treatment. All formulations were converted to beclomethasone equivalent based on the recommendations from the Canadian Asthma Consensus Report [ 14 ]. Any questions or discrepancies were resolved through iteration and consensus. Statistical methods To accommodate any differences in patient selection, clinical treatment, and biochemical procedures that were employed across the original studies, we converted the absolute mean differences in the concentrations of the inflammatory cells between the intervention and control groups into standardized mean differences. For each study, standardized mean difference was derived by dividing the mean change in the inflammatory cell concentration at follow-up visit from the baseline visit between intervention and control groups by a pooled standard deviation of the mean change [ 15 , 16 ]. A negative standardized mean difference indicated that the participants assigned to inhaled corticosteroids had lower cell counts compared with placebo at the end of the study phase; whereas a positive number denoted increased cell count relative to the control group. For each inflammatory cell, we tested the heterogeneity of results across the studies, using a Cochran Q test. If significant heterogeneity was present (p < 0.10), then a random effects model was used. In the absence of significant heterogeneity, a fixed effects model was used [ 16 ]. We also evaluated the potential modifying effect of cumulative dose and the duration of therapy of the trials. We reasoned that trials that had higher cumulative dose (or longer duration of therapy) defined as greater or equal to the median cumulative dose (or duration of therapy) of all the trials included in this meta-analysis would be more "positive" than those that used lower doses (or were shorter in duration). All analyses were conducted using Review Manager version 4.2 (Revman; The Cochrane Collaboration, Oxford, England) and were two-tailed in nature. Results A summary of the search strategy is shown in Figure 1 . The original search yielded 155 and 63 citations in MEDLINE and EMBASE, respectively. CINAHL and the Cochrane Databases did not contribute to the search results. The abstracts of these articles were selected and reviewed. Of these, 21 articles were retrieved for a detailed review. We excluded the study from Loppow and colleagues [ 17 ] because it included 6 patients with a positive skin prick test against at least one common airborne allergen and 4 patients who had FEV 1 /FVC > 0.7. We excluded additional 14 articles because of other reasons (Figure 1 ). This process left 6 original studies meeting the inclusion and exclusion criteria, which were used for the analyses [ 7 , 18 - 22 ]. The baseline information concerning the study designs is summarized in Table 1 . The relevant demographic data are summarized in Table 2 . All 162 patients were current or ex-smokers with post-bronchodilator FEV 1 <70 % predicted, FEV 1 to FVC ratio <0.7, and reversibility with bronchodilator of <15%. The medications used included budesonide, beclomethasone dipropionate, and fluticasone propionate. The study period of these trials ranged from 2 to 12 weeks. Figure 1 Study selection process Table 1 Baseline information on original studies included in the meta-analysis. Source Setting Design Inclusion Criteria Exclusion Criteria Concomitant drugs Withdrawal Sputum specimen Sugiura et al 2003 [7] NR Randomized, placebo-controlled parallel design. FEV 1 /FVC < 0.7; all patients wereex-smokers who had stopped smoking for at least 1 year beforethe study. A history of perennial allergic rhinitis; positiveallergen skin prick tests and RAST assay; a history of periodicwheezing; an improvement in FEV 1 of more than 12 % predicted oran absolute increase of 200 ml after inhalation of 200 μg salbutamol; had bronchial or respiratory tract infectionsin the month preceding the study; had taken systemic steroids in the 2 monthsbefore the study or inhaled steroids in the month beforethe study. NR None NR Keatings et al 1997 [18] Outpatient clinics in different hospitals Randomized, single-blind, crossover design with 3–7 day run in period. The clinical part of the study was single-blind, but all differential cell counting and assayswere carried out in a double blind fashion. FEV 1 /FVC < 0.7; FEV 1 < 70% predicted; reversibility with inhaled albuterol of <10% of predicted FEV 1 ; smoking history of at least 10 pack-years; negative results on skin prick testing to four common aeroallergens. Patients who had taken inhaled or oral steroids or who had suffered an exacerbation of their airway disease in the previous 6 weeks, or patients with any history of asthma or variability in symptoms were excluded. Albuterol was allowed. 2 subjects NR Culpitt et al 1999 [19] Outpatient clinic Randomized, double-blind, placebo-controlled crossover design with a run-in period of 2 weeks. FEV 1 /FVC < 0.7; postbronchodilat or FEV 1 <85% predicted; reversibility with inhaled β 2 -agonist of <15% of predicted FEV 1 ; smoking history of at least 20 pack-years. Patients who had taken inhaled or oral steroids or who had suffered an exacerbation of their airway disease in the previous 6 weeks, or patients with any history of asthma or atopy or variability in symptoms were excluded. Three subjects had concomitant treatment with albuterol (200 μg twice a day) and ipratropium bromide (40 μg twice a day), one subject with albuterol (200 μg as needed) alone. 12 subjects Samples were considered adequate for analysis if there was < 50% squamous cell contamination. Confalonieri 1998 [20] Outpatient clinic Randomised, controlled, open study. The clinical parts of the study was open, but all differential cell counting was carried out in a double blind fashion. FEV 1 /FVC <88% of predicted in men and <89% in women; all patients were current smokers. Patients who had taken inhaled or oral steroids or had suffered a respiratory tract infection in the previous three months were excluded. None of the patients was taking theophyllines or long acting β 2 agonists. None Samples were discarded if viability levels were 50% or less, or squamous contamination was 20% or more. An overall differential cell count on 500 nucleated non-squamous cells was performed by two examiners and results reported as mean of the two counts. Mirici et al 2001 [21] Outpatient clinic Randomized, double-blind, placebo-controlled parallel design. FEV 1 < 70% predicted; no self-reported asthma; reversibility with inhaled terbutaline of <15% of predicted FEV 1 ; current smokers. Long-term treatment with oral or inhaled steroids within 6 months of study entry; A respiratory tract infection in previous 3 months; pregnancy or lactation, or presence of other serious systemic diseases. β 2 – agonists of all kinds, theophylline, and mucolytics were allowed. 10 subjects Samples were discarded if viabilitylevels were 50% or less, or squamous contamination was 20% or more Yildiz et al 2000 [22] Outpatient clinic Randomized, placebo-controlled parallel design with a run-in period of 2 weeks. FEV 1 /FVC < 0.7; FEV 1 < 70% predicted; reversibility with inhaled albuterol of <10% of predicted; smoking history of at least 10 pack-years. Patients with any history of asthma or variability in symptoms, and patients who had taken inhaled or oral steroids or had suffered a respiratory tract infection or exacerbation in the previous 6 weeks were excluded. All of the patients continued to inhale both salbutamol and ipatropium bromide. In 9 patients, sustained release theophylline was also administered. None NR Abbreviations: FEV 1 , forced expiratory volume in 1 second; FVC, forced vital capacity; NR, not reported. Table 2 The characteristics of COPD patients at baseline. Source Number of Patients Age (year) Men (%) Current Smokers (%) Pack-years FEV 1 (% predicted) Ratio (%) Reversibility (% predicted) Drug Dose (mg/day) Duration (weeks) Cumulative dose (mg) # Sugiura [7] 18 ‡ 70(7) 89 0* NR 1.2(0.4) † <70 <12 Beclomethasone 0.8 4 22.4 Keatings [18] 26 45–78 60 46 >10 35.1(4.7) <70 <10 Budesonide 1.6 2 28.0 Culpitt [19] 26 43–73 62 69 >20 49.5(16.6) <70 <15 Fluticasone 1.0 4 56.0 Confalonieri [20] 34 58 (5) 59 100 NR 59.7(37.1) 67 (5) NR Beclomethasone 1.5 8 84.0 Mirici [21] 40 53(10) 75 100 26.5 (16.1) 62.0(7.4) NR <15 Budesonide 0.8 12 84.0 Yildiz [22] 18 64(7) 78 89 52.0 (23.4) 44.5(2.7) 57 (3) <10 Fluticasone 1.5 8 168.0 † FEV 1 , liter; ‡ 6 patients in control group * All subjects were ex-smokers and stopped smoking for at least 1 year. # Cumulative dose = daily dose × days × adjusted factor for beclomethasone equivalence [14]. Continuous variables are presented as mean (SD) Abbreviations: FEV 1 , forced expiratory volume in 1 second; FVC, forced vital capacity; ratio, the ratio of FEV 1 to FVC; NR, not reported/not calculable. After treatment with inhaled corticosteroids, the total cell counts decreased. Overall, the standardized mean difference between steroid and control groups was -0.43 units (95% confidence interval, CI, -0.75 to -0.11), indicating that inhaled corticosteroids had a favorable effect in reducing total count compared with controls (test for heterogeneity, p = 0.35) (Figure 2 ). Importantly, the total cumulative dose of inhaled corticosteroids calculated on the basis of mean daily dose and duration of therapy made a material difference to the results. In the studies in which patients were exposed to 60 mg or greater of beclomethasone or its equivalent for the duration of the trial, inhaled corticosteroids were effective in reducing the total sputum cell count (-0.68 units; 95% CI, -1.11 to -0.26). In contrast, trials with cumulative dose of < 60 mg did not demonstrate a favorable effect of inhaled corticosteroids on this sputum index (-0.11 units; 95% CI, -0.58 to 0.37). All of the trials with the higher cumulative dose had exposed the trial participants to inhaled corticosteroids for at least 6 weeks; whereas, the trials with the lower cumulative dose was uniformly less than 6 weeks in duration. Figure 2 Effect of inhaled corticosteroids on total cell counts in the sputum of stable COPD patients Inhaled corticosteroids had a salutary effect on neutrophil counts in the sputum. As compared with the control group, the standardized mean difference in those treated with inhaled corticosteroids was -2.16 units (95% CI, -3.81 to -0.50; test for heterogeneity, p < 0.001) (Figure 3 ). Similar to the findings on the total cell count, trials with a cumulative dose of ≥ 60 mg of beclomethasone (or at least 6 weeks of therapy) demonstrated a significant effect of these medications on sputum neutrophil count (-4.27 units; 95% CI, -6.87 to -1.66); whereas, trials with cumulative dose of < 60 mg (or less than 6 weeks of therapy) failed to demonstrate a beneficial effect (-0.26 units; 95% CI, -0.74 to 0.22). Figure 3 Effect of inhaled corticosteroids on neutrophils in the sputum of stable COPD patients Inhaled corticosteroids also reduced the lymphocyte counts in the sputum (standardized mean difference, -0.39 units, 95% CI, -0.74 to -0.05; test for heterogeneity, p = 0.58) (Figure 4 ). Trials with cumulative dose of ≥ 60 mg (or at least 6 weeks of therapy) demonstrated a significant effect (standardized mean difference, -0.59 units; 95% CI, -1.01 to -0.17); whereas, trials with cumulative dose < 60 mg (or less than 6 weeks of therapy) failed to demonstrate a salutary effect on this endpoint (standardized mean difference, 0.02; 95% CI, -0.59 to 0.62). Figure 4 Effect of inhaled corticosteroids on lymphocytes in the sputum of stable COPD patients These medications were also effective in reducing epithelial cell counts compared with the controls (standardized mean difference, -0.51 units, 95% CI, -0.98 to -0.05; test for heterogeneity, p = 0.20) (Figure 5 ). There was an insignificant trend towards reducing eosinophil counts in the sputum with inhaled corticosteroid therapy (standardized mean difference, -0.28 units, 95% CI, -0.62 to 0.07; test for heterogeneity, p = 0.22) (Figure 6 ). Inhaled corticosteroids did not appear to have any significant effect on macrophage concentrations in the sputum (standardized mean difference, -0.02 units, 95% CI, -0.34 to 0.29; test for heterogeneity, p = 0.65) (Figure 7 ). Inhaled corticosteroids did not have significant effect on sputum IL-8 levels (standardized mean difference, -0.22 units; 95% CI, -0.77 to 0.32; test for heterogeneity, p = 0.84). Figure 5 Effect of inhaled corticosteroids on epithelial cells in the sputum of stable COPD patients Figure 6 Effect of inhaled corticosteroids on eosinophils in the sputum of stable COPD patients Figure 7 Effect of inhaled corticosteroids on macrophages in the sputum of stable COPD patients To evaluate whether the magnitude of the reduction in the inflammatory cells was modified by the absolute levels of the inflammatory cells in the sputum at baseline, we performed a stratified analysis based on the total cell counts at baseline (see Table 3 ). No significant patterns were observed with any of the cell lines suggesting that baseline "cell load" in the sputum was not a predictor of response to inhaled corticosteroids. Table 3 Total and differential cell counts at baseline and the standard mean difference (SMD) in cell counts between intervention group and placebo group after treatment. Source Total cells Neutrophils Lymphocytes Eosinophils Macrophages Number (× 10 4 /mL) SMD (95% CI) Number (× 10 4 /mL) SMD (95% CI) Number (× 10 4 /mL) SMD (95% CI) Number (× 10 4 /mL) SMD (95% CI) Number (× 10 4 /mL) SMD (95% CI) Yildiz [22] 350.0 -0.6 (-1.6 to 0.4) 260.0 -2.2 (-3.4 to -1.0) 3.5 -0.5 (-1.4 to 0.5) 7.0 -1.1 (-2.1 to -0.1) 80.0 0.2 (-0.5 to 0.9) Confalonieri [20] 219.0 -0.4 (-1.1 to 0.3) 158.8 -3.4 (-4.5 to -2.3) 6.6 -0.5 (-1.2 to 0.2) 6.2 -0.6 (-1.3 to 0.1) 45.0 -0.3 (-0.9 to 0.3) Mirici [21] 196.5 -1.0 (-1.7 to -0.3) 146.5 -7.5 (-9.3 to -5.6) 1.6 -0.7 (-1.4 to -0.1) 1.6 0.2 (-0.4 to 0.8) 38.2 0.5 (-0.5 to 1.5) Sugiura [7] 165.7 0.2 (-0.8 to 1.2) 102.9 0.1 (-0.9 to 1.1) 6.1 0.04 (-0.9 to 1.0) 4.5 -0.2 (-1.2 to 0.8) 52.0 -0.3 (-1.1 to 0.5) Culpitt [19] 165.0 -0.3 (-1.1 to 0.5) 145.0 -0.4 (-1.2 to 0.4) NR NR NR NR 25.0 -0.2 (-0.9 to 0.6) Keatings [18] 6.3* -0.1 (-0.9 to 0.7) 4.3* -0.4 (-1.1 to 0.4) 6.0* 0.0 (-0.7 to 0.8) 0.2* -0.2 (-1.0 to 0.6) 1.8* 0.2 (-0.7 to 1.2) Pooled Summary -0.4 (-0.8 to -0.1) -2.2 (-3.8 to -0.5) -0.4 (-0.7 to -0.1) -0.3 (-0.6 to 0.1) -0.02 (-0.3 to 0.3) * cell count/ml Abbreviations: NR, not reported/not calculable. After treatment with inhaled steroids, lung function improved slightly but neither the improvement in FEV 1 nor FVC reached statistical significance. For predicted FEV 1 , the overall standardized mean difference was 0.26 units, 95% CI, -0.06 to 0.57 (test for heterogeneity, p = 0.62) (Figure 8 ). For predicted FVC, the overall standardized mean difference was 0.31 units; 95% CI, -0.09 to 0.70 (test for heterogeneity, p = 0.23). Figure 8 Effect of inhaled corticosteroids on FEV1 1 % predicted in stable COPD patients. Abbreviation: FEV 1 , forced expiratory volume in one second Discussion By combining data across the clinical studies, we increased statistical power to demonstrate a salutary effect of moderate to high doses of inhaled corticosteroids on some inflammatory indices in the sputum of patients with stable COPD. Over a short term, these medications reduced neutrophil, lymphocyte and epithelial cell counts in the sputum of stable COPD patients. They had smaller (and insignificant) effect on sputum eosinophils and IL-8. They had little effect on sputum macrophages. Although the magnitudes of these reductions were relatively small, they may explain why inhaled corticosteroids decrease cough and sputum production [ 23 ], reduce exacerbations [ 24 ], and hospitalizations [ 25 ]. We also found that duration of therapy and total cumulative dose, which are related constructs, made a material difference to the overall results. Short trials (less than 6 weeks in duration) were uniformly "negative"; while longer term trials (at least 6 weeks of therapy) were mostly positive. Similarly, trials that exposed the patients to higher cumulative dose were more "positive" than those that exposed patients to lower dose. This suggests that duration of therapy and total cumulative doses may be important determinants of the effect of inhaled corticosteroids on airway inflammation. Although corticosteroids delay neutrophil apoptosis and may increase neutrophil survival [ 11 , 26 ], they also have significant inhibitory action on neutrophil performance. Likely through the annexin-I (lipocortin-1) pathways, for instance, corticosteroids interfere with neutrophil chemotaxis, adhesion, transmigration, oxidative bursts, and phagocytosis, thereby down-regulating the overall inflammatory cascade [ 9 , 27 ]. Indeed, Llewellyn-Jones and co-workers [ 28 ] showed that 4 weeks of inhaled fluticasone therapy can significantly reduce sputum chemotactic activity for neutrophils and increase its elastase inhibitory capacity in patients with well-characterized COPD. These data suggest that inhaled corticosteroids can reduce recruitment and/or adhesion of neutrophils to the airways of COPD patients, thereby lowering the overall concentration of these cells in COPD airways. Superficially, the present data on sputum eosinophils appear to be inconsistent with the known effect of corticosteroids in general on eosinophils. Many experiments have shown that eosinophils are exquisitely sensitive to corticosteroids [ 29 , 30 ]. The current data, however, suggest otherwise. Several studies have demonstrated that among COPD patients with irreversible airflow obstruction (as was the case for a majority of patients enrolled in the original studies contained in this meta-analysis), eosinophils are present in relatively small quantities in the sputum of such patients [ 10 , 31 ]. In most COPD patients, eosinophils account for less than 2% of the total cells in the sputum. This could have introduced a "floor" bias wherein the overall signal to the noise ratio for eosinophils may have been too small to detect subtle but important effect of inhaled corticosteroids on these cells. Although by combining data from these published studies we increased the power of the present analysis to detect salient changes in the inflammatory indices of the sputum, we may still have had insufficient power for analyses of cells with a relatively small signal. Our analysis may also have had insufficient power to assess the effects of inhaled corticosteroids on FEV 1 . Although there was a trend towards improvement, we did not find a statistically significant effect of inhaled corticosteroids on FEV 1 . Larger randomized trials have demonstrated, however, that inhaled corticosteroids significantly improve FEV 1 over the first three to six months of therapy [ 25 , 32 - 34 ], suggesting that for certain endpoints our present analysis still lacked sufficient power. Therefore, the "negative" associations must be interpreted cautiously. It is also important to note that none of the studies included in the present review evaluated the effects of inhaled corticosteroids on the function or performance of inflammatory cells in the airway. Thus, we can not discount the possibility that these medications could have salutary effects on the functional performance of these cells. In the present review, we did not include randomized studies that used bronchoalveolar lavage (BAL) or bronchial biopsies to measure inflammatory cells in the airways. However, in one study, Balbi and colleagues [ 35 ] observed significant reductions in the total number of cells, neutrophil counts, IL-8, and myeloperoxidase levels in the BAL fluid of COPD patients after 6 weeks of inhaled beclomethasone therapy. A similar finding was observed and reported by Thompson and coworkers [ 36 ]. In another experiment, Hattotuwa at al [ 23 ] randomly treated a group of COPD patients with 3 months of inhaled fluticasone propionate (1 mg/d) or placebo. The group that received fluticasone had significantly fewer mast cells in the subepithelial layer as well as a reduced ratio of CD8 to CD4 positive cells in the epithelial layer than those treated with placebo. Most importantly, the fluticasone group had significant improvements in cough and sputum scores and decreased use of reliever medications and experienced fewer exacerbations than did the placebo group [ 23 ]. Verhoeven et al [ 37 ] evaluated 23 patients with COPD and randomly treated 10 patients to fluticasone (1 mg/d) and the remainder to placebo. After 6 months, fluticasone treatment resulted in a significant reduction in the number of MBP and CD68 positive cells in the lamina propria and reduced tryptase levels in the epithelium. In addition, there was a trend towards fewer CD3, CD4 CD68 positive cells in epithelium of the group treated with fluticasone compared with the group treated with placebo [ 37 ]. The results from the BAL and bronchial biopsy studies largely support data from the sputum studies and are consistent with the notion that inhaled corticosteroids reduce airway inflammation in COPD. We also did not include studies that used systemic corticosteroids. Barcyk and colleagues [ 38 ] have reported that oral prednisone therapy (0.5 mg/kg/d) for 2 weeks significantly reduced myeloperoxidase levels in the sputum of COPD patients. Brightling and colleagues [ 39 ] showed that 2 weeks of oral prednisone therapy resulted in fewer eosinophils in the sputum of COPD patients. Similar findings were reported by Fujimoto and colleagues [ 40 ]. These data suggest that oral prednisone can reduce certain components of airway inflammation (e.g. eosinophils) in COPD; however, most of the studies were very short in duration, which makes it difficult to compare these data against those studies that used inhaled corticosteroids. Although in the present review, we could not adequately determine the effects of tobacco smoke exposure on the relationship between inhaled corticosteroids and airway inflammation, there is a growing body of evidence to suggest that active smoking may attenuate the effectiveness of corticosteroids in suppressing airway inflammation. Active smoking increases oxidative stress and up-regulates the production of various pro-inflammatory cytokines including Il-6, IL-8, IL-1β and monocyte chemoattractant protein-1 in airways, which may through a series of complex pathways lead to a state of steroid resistance [ 41 ]. Additionally, cigarette smoke may reduce histone deacetylase activity and its expression in alveolar macrophages, making these cells relatively resistant to corticosteroids since one of the principal targets of corticosteroid action is by switching off gene expression of inflammatory genes through the recruitment of histone deacetylases [ 41 ]. Therefore smoking cessation remains the single most important intervention in COPD management. Inhaled corticosteroids should be considered as a possible adjunctive therapy in patients who remain symptomatic despite smoking cessation. There are certain limitations with the present analysis. First, although we used stringent entry criteria in order to minimize the heterogeneity in the research methods employed by each of the selected study, there were still some variations in the study design, the exposure medications, and the target population across the original studies. However, the differences in the characteristics of the studies were relatively small and unlikely to have materially affected the overall findings of the current review. We also contacted the primary authors to clarify any ambiguities or to obtain additional data, where necessary, to further minimize the "noise" inherent to meta-analyses. Moreover, to accommodate various differences in the methodology of data collection and laboratory techniques employed across the original studies, we converted the individual data into standardized mean estimates, which enhanced the comparability of data across the original studies. Second, it is possible that corticosteroid therapy could have affected the volume of sputum recovery, decreasing the total sputum cell counts in those patients exposed to this therapy. To mitigate this possibility, the cell counts were expressed as cells per volume of sputum recovered. Conclusions In summary, the present meta-analysis suggests that inhaled corticosteroids when used for longer than 6 weeks can significantly reduce neutrophil counts and other inflammatory indices in the sputum of patients with stable COPD. Large randomized controlled trials are needed in the future to confirm these early findings and to determine whether these salutary effects persist longer than 3 to 4 months of therapy. Abbreviations COPD chronic obstructive pulmonary disease FEV 1 forced expiratory volume in 1 second FVC forced vital capacity SD standard deviationIL-8 interleukin-8 Competing interests DDS and SFP have received honoraria for speaking engagements from GlaxoSmithKline (GSK) & AstraZeneca, and have received consultation fees and research funding from GSK. However, no part of this work was financed by these companies. This work was funded by Canada Research Chair and a Michael Smith/St. Paul's Hospital Foundation Professorship in COPD. Authors' contributions All the authors contributed to the design and implementation of the study. Data analyses were performed by WQG and DDS. All authors contributed to the write-up of the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC552309.xml
551614	An in vitro evaluation of standard rotational thromboelastography in monitoring of effects of recombinant factor VIIa on coagulopathy induced by hydroxy ethyl starch	Background Rotational thromboelastography (ROTEG) has been proposed as a monitoring tool that can be used to monitor treatment of hemophilia with recombinant factor VIIa (rFVIIa). In these studies special non-standard reagents were used as activators of the coagulation. The aim of this study was to evaluate if standard ROTEG analysis could be used for monitoring of effects of recombinant factor VIIa (rFVIIa) on Hydroxy Ethyl Starch-induced dilutional coagulopathy. Methods The study was performed in vitro on healthy volunteers. Prothrombin time (PT) and ROTEG analysis were performed after dilution with 33% hydroxy ethyl starch and also after addition of rFVIIa to the diluted blood. Results PT was impaired with INR changing from 0.9 before dilution to 1.2 after dilution while addition of rFVIIa to diluted blood lead to an overcorrection of the PT to an International Normalized Ratio (INR) value of 0.6 (p = 0.01). ROTEG activated with the contact activator ellagic acid was impaired by hemodilution (p = 0.01) while addition of rFVIIa had no further effects. ROTEG activated with tissue factor (TF) was also impaired by hemodilution (p = 0.01) while addition of rFVIIa lead to further impairment of the coagulation (p = 0.01). Conclusions The parameters affected in the ROTEG analysis were Clot Formation Time and Amplitude after 15 minutes while the Clotting Time was unaffected. We believe these effects to be due to methodological problems when using standard activators of the coagulation in the ROTEG analysis in combination with rFVIIa.	Background Patients undergoing massive hemorrhage experience dilutional coagulopathy with crystalloid and/or colloid resuscitation. If hemorrhage progresses, packed red cells (RBC) are transfused together with crystalloids and/or colloids. Regarding the coagulation this is not optimal, and the patients often develop a dilutional coagulopathy, sometimes worsened by hypothermia. In addition a coagulopathy caused by the administration of dextrane or hydroxy ethyl starch (HES) may be induced. The common approach to this is transfusion of fresh frozen plasma (FFP) and platelets, but bleeding might continue, with often fatal outcome. Prophylactic use of fresh frozen plasma (FFP) or platelet transfusion is not proven beneficial to prevent hemorrhage in massively transfused patients[ 1 ]. Hemorrhage, complicated by the development of coagulopathy, is therefore still the major cause of death in trauma patients arriving alive in the hospital.[ 2 , 3 ]. A novel approach to treat these patients is the use of recombinant factor VIIa (rFVIIa) to improve the coagulation.[ 4 ]. Recently there have been several case publications of successful treatment of coagulopathic trauma patients and surgical patients. [ 5 - 7 ]. Dilutional coagulopathy can be detected by the use of thromboelastographic measurements [ 8 - 11 ]. Rotational thromboelastography (ROTEG) is a recent development of thromboelastography. ROTEG gives a viscoelastic measurement of clot strength in whole blood. It is presented as a graph representing clot strength during the build-up of a clot (figures 1 and 2 ). From the graph several variables describing different parts of the coagulation process are derived and measured numerically. It thus gives a more comprehensive picture of the coagulation than standard tests, but is on the other hand less validated and standardised than the more common coagulation tests. An advantage of the method is that it can be used as a point-of-care analysis. One condition to be fulfilled if it should be used as a point-of-care method is that the commercially available kits can be used for analysis and that special preparations and/or dilutions should not be necessary, as that would require the skills and competencies of a full laboratory. Figure 1 The figure shows the 3 representative tracings of the EXTEG analysis from one of the participants in the study. Above the normal tracing with a short CT and CFT is shown. It can be seen that the clot strength is rapidly increasing after initiation of the clotting. In the middle the tracing after hemodilution with HES is found. It can be seen that the CFT is prolonged and that the strength of the clot is increasing slower. Below the tracing after hemodilution and addition of rFVIIa is found. The clotting is then severely impaired, the clot strength is increasing slower and the maximum strength is also severely impaired. Figure 2 The figure shows the result of a ROTEG analysis. Time is represented on the X axis and clot strength on the Y axis. The clot strength is arbitrarily measured in mm where maximum clot strength is 100 mm. The Clotting Time (CT) is the time from initiation of the analysis until the clot strength is 2 mm. The Clot Formation Time (CFT) is the time from clot strength 2 mm until clot strength 20 mm. A15 is the clot strength at 15 minutes. Previous studies have shown that hemodilution with HES impairs the coagulation already at a dilution of 33% while crystalloid hemodilution does not appear to give a readily detectable impairment of the coagulation until the level of hemodilution reaches 50% [ 10 , 12 ]. In this study we have investigated if ROTEG can be used with the commercially available kits to monitor effects of rFVIIa and if rFVIIa is able to improve the coagulopathy caused by hemodilution with HES. Methods The local ethics committee of the University of Lund approved this study on healthy volunteers. Volunteers were not allowed to take any medication 14 days prior to the study. Informed consent was obtained from the participants and a total of eight were recruited. All participants had an indwelling intravenous catheter placed into the brachiocephalic vein, from which the blood samples were drawn with sterile disposable 5-ml syringes (Luer; Codan Medical Aps, Rödby, Denmark). A first 5-ml blood sample was discarded before every blood sample for the experiment described below. No tourniquet was used on the arm when samples were drawn. Dilution of the blood samples was performed with HES 130/0.4 (Voluven ® , Fresenius Co., Bad Homburg, Germany), the HES preparation found to cause the least pronounced coagulopathy after hemodilution [ 12 ]. Three different preparations of blood were examined. The first preparation contained 5 ml of undiluted blood (normal). The second preparation contained 3.3 ml of blood and 1.7 ml of HES thereby achieving a 33% dilution (dilution). The third preparation contained 3.3 ml of blood, 1.7 ml of HES and 50μl of rFVIIa at the concentration 0.12 μg/μl (dilution + rFVIIa). The latter concentration of rFVIIa was equivalent to the concentration achieved when the dose 90 μg/kg body weight is administered in vivo. 90 μg/kg is the recommended dose in hemophilia and well within the range suggested for treatment of acute hemorrhage in non hemophilia patients.[ 7 , 13 ]. The reason for the chosen dilution (33%) was that it is a clinically relevant dilution that is readily achieved during resuscitation of a patient. Before initiating this study we have also tested 50% dilution in a single person and the results were similar to the results with 33% dilution, but more pronounced. We then decided to study the 33% dilution systematically. Further on, this dilution did not induce unphysiologic changes in Ca or pH as tested in a pilot volunteer. The dilution was performed in a polypropylene test tube and the tube was gently turned to mix the blood with the added HES and rFVIIa. The HES was warmed in a heating block (Grant Instrumentation Ltd, Cambridge, UK) to 37°C prior to hemodilution in order to avoid hypothermia as a confounding factor after dilution. The tests performed on the different preparations were hemoglobin concentration (Hb), Prothrombin time (PT) and ROTEG analysis. All tests were performed at normal body temperature (37°C). For the Hb measurements a Hemocue (HemoCue Co., Ängelholm, Sweden) was used. PT measurements were performed with a Rapidpoint Coag Analyzer (Bayer AB Diagnostics, Gothenburg, Sweden) with PT-ONE test cards. The ROTEG analyses were performed on a Rotational Thromboelastograph (ROTEG, Pentapharm, Munich, Germany) and the samples were analysed 120 seconds after the blood was drawn from the intravenous catheter. Both INTEG and EXTEG analyses were performed according to standard procedure recommended by the manufacturer. In INTEG analysis the coagulation is initiated with the addition of 20 μl of the contact activator ellagic acid (Pentapharm, Munich, Germany) to 320 μl of blood pipetted from the test tube to a reaction cup used in the ROTEG. In EXTEG analysis the coagulation is activated by the addition of 20 μl of a preparation containing tissue factor (TF) (Pentapharm, Munich, Germany) to 320 μl of blood pipetted from the test tube to the reaction cup. TF activates the coagulation through binding to Factor VIIa and this is believed to be the important interaction when in vivo coagulation occurs. The parameters obtained from the ROTEG analysis were Clotting Time (CT) reflecting the initiation of the coagulation, Clot Formation Time (CFT) reflecting the rate of clot formation once the formation is initiated and A15 describing the strength of the clot 15 minutes after initiation of the coagulation (figure 2 ). Statistical analysis was performed with initial Kruskal-Wallis test and Wilcoxon's paired test was used when the Kruskal-Wallis test indicated a significant difference. All values are given as median (range). A p value of < 0.05 was considered statistically significant. Results The insertion of venous catheters and the blood sampling were performed uneventfully. The Hb values decreased as an expected sign of dilution (table 1 ). PT values increased in the dilution group compared to normal and decreased to below normal in the dilution + rFVIIa group (table 1 ). Table 1 Effects of dilution and addition of rFVIIa on Hb and PT values. A lowering of Hb and an increase in the PT were seen as signs of dilution while the addition of rFVIIa lead to a decrease of the PT (n = 8). Normal Dilution Dilution + rFVIIa Hb (g/l) 136 (127–147) 88 (81–99)* 88 (81–99)* PT (INR) 0.9 (0.7–1.2) 1.2 (0.9–1.3) § 0.6 (0.5–0.7)* ¶ * p = 0.01 compared to normal. ¶ p = 0.01 compared to dilution. § p = 0.02 compared to normal. In neither INTEG nor EXTEG analysis we found any change in the CT between normal and dilution groups, while CFT and A15 were impaired in the dilution group (tables 2 and 3 ). There were no differences between the dilution and the dilution + rFVIIa groups when analysed with the INTEG analysis (table 2 ). However, when rFVIIa was added to the dilution a prolongation of the CFT with > 200% and an impairment of the A15 with 40% were found (table 3 and figure 1 ). Table 2 Coagulation variables as assessed by INTEG. Obvious signs of dilution are found in the CFT and the A15, while the addition of rFVIIa to the diluted blood does not affect the coagulation parameters (n = 8). Normal Dilution Dilution + rFVIIa CT (s) 93.5 (82–104) 108.5 (87–136) 97.5 (68–118) CFT (s) 85.5 (59–111) 216 (158–310)* 190.5 (167–368)* A15 (mm) 56 (52–61) 43 (36–47)* 43.5 (33–50)* * p = 0.01 compared to normal. Table 3 Coagulation variables as assessed by EXTEG. Obvious signs of dilution are found in the CFT and the A15. The addition of rFVIIa to the diluted blood leads to a prolongation of the CFT and the A15 (n = 8). Normal Dilution Dilution + rFVIIa CT (s) 51.5 (30–69) 63 (41–81) 58.5 (37–99) CFT (s) 91 (67–105) 227 (171–332)* 558.5 (308–998)* ¶ A15 (mm) 57 (53–63) 42 (33–49)* 25.5 (19–37)* ¶ * p = 0.01 compared to normal. ¶ p = 0.01 compared to dilution. Discussion Treatment of dilutional coagulopathy is challenging and the primary monitoring tools are measurement of PT, activated partial thromboplastin time (APTT), platelet count and fibrinogen [ 14 - 16 ]. These tests provide us with information regarding the activation of the coagulation process and about the absolute number of platelets. However, they do not give us information regarding the dynamic properties of blood clotting and the rate at which the clot is formed once the clotting is initiated. New monitoring methods are needed and ROTEG is a monitoring tool that could potentially be of value in these situations. To this end, it has been suggested that treatment of haemophilia patients and liver transplant patients with rFVIIa can be monitored with ROTEG where a shortening of the CT and CFT has been found in case series. [ 17 - 19 ]. In our study we found that hemodilution in vitro with HES lead to augmentation of the PT and the addition of rFVIIa results in a prompt decrease of the PT. This is in line with previous studies, which have shown that dilution with HES lead to readily detectable changes in the coagulation system[ 9 , 12 , 20 ]. Former studies have also shown a decrease or a normalisation of the PT after administration of rFVIIa[ 21 , 22 ]. In this study we found an overcorrection of the PT to values below normal range. When analysing the ROTEG parameters we found that the 33% dilution with HES resulted in a prolongation of CFT values and impairment in A15 values in accordance with previous studies[ 9 , 11 , 12 , 20 ]. The CT was not significantly prolonged even though there was a trend towards a prolongation of the CT in both INTEG and EXTEG. After rFVIIa had been added to the diluted blood, coagulation variables remained unchanged when assessed with INTEG, but were markedly affected when assessed with EXTEG. CFT and A15 reflect the dynamic interplay between platelets and fibrin polymerisation, both being disturbed by HES hemodilution as can be seen in table 2 and 3 . Addition of rFVIIa in vitro , worsening both these parameters according to EXTEG analysis, suggested that platelets or fibrinogen became dysfunctional in contrast to the clinical effect of administration of rFVIIa, where rFVIIa has been found successful in case stories of bleeding patients [ 4 , 7 , 13 , 23 ], even though these cases may not have been bleeding due to a HES induced coagulopathy. The lack of effect in the INTEG analysis when adding rFVIIa to the diluted blood is most likely due to the fact that a contact activator is used to activate the coagulation in the INTEG and therefore insensitive to rFVIIa as rFVIIa initiates coagulation through interaction with TF. The impairment of CFT and A15 in the EXTEG analysis after addition of rFVIIa to the diluted blood is harder to explain. We expected addition of rFVIIa to the diluted blood to result in an improvement of the TEG parameters measured in the EXTEG analysis. This was expected partly because TF is used as an activator of the coagulation in the EXTEG analysis and the first step in the initiation of the coagulation system is the interaction between TF and FVIIa. [ 17 , 18 , 24 ]. In our study it is likely that the coagulopathy induced was at least to some extent caused by a platelet dysfunction caused by the colloid hemodilution. As bleeding caused by Glanzmann's thrombastenia and other thrombocytopathias is frequently and successfully treated with rFVIIa, is a reason why rFVIIa potentially could be effective in the treatment of this HES-induced coagulopathy[ 25 - 28 ]. The previously reported improvements of coagulation in hemophilia and liver transplant patients as evaluated with ROTEG after administration of rFVIIa also lead us to believe that ROTEG parameters would be improved after addition of rFVIIa to the diluted blood[ 17 , 18 , 24 ]. It is however important that these studies were performed on hemophilia patients suffering from a severe deficiency of factor VIII or IX and on liver transplant patients suffering from a very complex coagulopathy. It also seems important to dilute TF extensively to detect the effects of rFVIIa on ROTEG. Dilutions of TF up to 1:17000 have been performed by the Ingerslev group in Denmark [ 18 , 24 ]. These dilutions are, however, not made with commercially available reagents that are ready to use immediately and therefore not suitable for use outside research laboratories. Conclusion In conclusion we found that 33% dilution of blood with HES 130/0.4 lead to impairment of the coagulation when evaluated with ROTEG or PT. Addition of rFVIIa lead to overcorrection of the prolonged PT. ROTEG analysis revealed that INTEG analysis was insensitive to effects of addition of rFVIIa and that EXTEG analysis was dramatically impaired by the addition of rFVIIa. It may well be that rFVIIa is not effective in improving the coagulopathy induced by HES hemodilution, but the further impairment of the coagulation seen in the EXTEG analysis is likely due to methodological problems. These problems make the commercially available EXTEG analyses inappropriate for monitoring of rFVIIa effects under circumstances of HES dilution. Competing interests The author(s) declare that they have no competing interests. Authors contributions ME contributed to the design of the study, performed the analyses and drafted the manuscript. PR contributed to the design of the study, to data interpretation and to preparing the manuscript. US contributed to the design of the study, performed the analyses and participated in manuscript preparation. All authors have read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC551614.xml
521495	Ability to perform activities of daily living is the main factor affecting quality of life in patients with dementia	Background Dementia is a chronic illness associated with a progressive loss of cognitive and intellectual abilities, such as memory, judgment and abstract thinking. The objective of this study was to assess the health utilities of patients with dementia in Europe and identify the key factors influencing their Health-Related Quality of Life (HRQol). Methods This study used cross-sectional data from the Odense study; a Danish cohort of patients aged 65–84 living in Odense, Denmark. A total of 244 patients with mild to severe dementia were interviewed together with a caregiver about their health status and activities of daily living (ADL). Alzheimer's disease was diagnosed according to the NINCDS-ADRDA criteria for probable dementia. Vascular dementia and other types of dementia were diagnosed according to the DSM-IIIR criteria. Severity of dementia was defined by score intervals on the Mini Mental State Examination score: mild (MMSE 20–30), moderate (MMSE 10–19), and severe (MMSE 0–9). Based on the ADL information, the patients' dependency level was defined as either dependent or independent. Questions from the Odense Study were mapped into each of the five dimensions of the EQ-5D in order to assess patients' HRQol. Danish EQ-5D social tariffs were used to value patients' HRQol. A regression analysis of EQ-5D values was conducted with backward selection on gender, age, severity, ADL level and setting in order to determine the main factor influencing HRQoL. Results The EQ-5D weight in patients independent upon others in ADL was 0.641 (95% CI: [0.612–0.669]), and in those dependent upon others was 0.343 (95% CI: [0.251–0.436]). Conclusion Dependency upon others to perform ADL was the main factor affecting HRQoL.	Background Dementia is a chronic illness associated with a progressive loss of cognitive and intellectual abilities, such as memory, judgment and abstract thinking. Cognitive disabilities are those that impact an individual's ability to access, process, or remember information. People with profound cognitive disability will need assistance with nearly every aspect of daily living. The most visible manifestation of dementia is the progressive inability – proportional to the severity of the disease – to perform activities of daily living (ADL) and the subsequent loss of independence [ 1 ]. Progressive deterioration in the cognitive, functional and behavioural domains eventually brings patients to the later stages of dependency and, in most cases, to institutionalisation, which is linked to an increased need in caregiver assistance [ 2 ]. A patient's level of dependency is a global measurement reflecting a certain level of severity, resource consumption and Quality-of-Life (QoL) [ 3 ]. Measuring the QoL of patients suffering from dementia can take several forms. Firstly, QoL can be measured using generic health indices like the other disease specific measures. Recently several scales have been developed and validated specifically for dementia patients such as the Quality of Life-Alzheimer's Disease (QOL-AD). Another alternative to assess QoL is to use utility measurements, which are preference-based [ 4 ]. Preference-based measures evaluate the patient's preference for a health state instead of measuring the frequency and the severity of symptoms or disabilities. In order to use quality-adjusted life years (QALYs) as outcome measures for cost-effectiveness analyses, utility-weighted measures of Health-Related Quality-of-Life (HRQoL) are required. These attribute a single number to a health state using a common unit of measure allowing comparison between different strategies [ 5 ]. In general, however, HRQoL is not as broad a concept as QoL. One of the more reliable and newer tools used to measure HRQoL in a wide range of health conditions and treatments including dementia is the EQ-5D [ 6 ]. It is a generic measure designed to complement disease specific outcome measures and health characterises on five dimensions: mobility, self-care, ability to perform usual activities, pain, and anxiety/depression. It provides a descriptive health profile and a single index value for health status and, as such, it can be used to estimate utility in pharmacoeconomic evaluations of new pharmacological treatments. Health utilities have already been measured in AD in the US and Canada using the Health Utility Index (HUI) [ 7 ]. In the UK, the EuroQol instrument has been used to investigate whether HRQol data could be obtained from proxies, such as family caregivers [ 8 ]. In France, the EuroQol instrument has been administered to patients with dementia in order to determine the feasibility, reliability, and validity of the French version of the EuroQol instrument [ 9 ]. However, health utilities are not reported from the two latter studies. Furthermore, HRQol data are needed in order to carry out cost-effectiveness analysis for a Danish setting. Based on data collected alongside an epidemiological study conducted in Odense Denmark, we attempted to assess the health utilities of patients with dementia and identify the key factors influencing their HRQoL. Methods Population Data were derived from the Odense study, an epidemiological survey in which the objective was to estimate the prevalence and incidence of dementia in Denmark [ 10 , 11 ] In this study, a total of 244 patients with dementia agreed to participate in an interview accompanied by a relative or caregiver. The study was approved by the Scientific-Ethical Committee of the Counties of Funen and Vejle, Denmark, and by the Danish Data Protection Agency. Demented patients were classified by type of dementia and by severity of dementia. Alzheimer's disease (AD) was diagnosed according to the NINCDS-ADRDA criteria for probable dementia [ 12 ]. Vascular dementia and other types of dementia were diagnosed according to the DSM-IIIR criteria [ 13 ]. Severity of dementia was diagnosed according to the Clinical Dementia Rating (CDR) scale [ 14 ] and the Mini Mental State Examination (MMSE) [ 15 ]. The complete examination programme is described in Andersen, Lolk et al, 1997 [ 10 ]. Assessments All interviews were conducted by a certified nurse in the patient's home., Patient's and caregiver's socio-economic and socio-demographic status as well patients' health status and ADL were recorded. In the event that a relative was not present during the interview, a professional caregiver verified information provided by the patient. Each interview included the following information: - sociodemographic questions (age, gender, setting). - activity of daily living (ADL) questionnaire using 7 items describing patients' ability to perform physical activities (personal care, dressing, mobility and personal toiletry) and psychosocial activities (activities in the home and hobbies inside and outside of the home). Each activity was scored using a four-point Likert scale anchored at the ends with 1 = "Unable to perform the activity" and 4 = "Perform the activity without help from others". The physical ADL ranged between 4 (worst state) and 16 (best state), while the psychosocial ADL scored between 3 (worst state) and 12 (best state) [ 16 ]. - Questions mapped into each of the five dimensions of the EQ-5D: mobility, self-care, usual activities, pain/discomfort, and anxiety/depression as presented in the Table 1 [ 17 ]. Table 1 Mapping questions from the Odense Study into the EQ-5D EQ-5D The Odense Study Mobility Mobility 1. I have no problems in walking around. • Without help from others. 2. I have some problems in walking around. • Needs some help from others. • Needs help from others. 3. I am confined to bed. • Unable to walk around without help. Personal care Personal care/dressing 1. I have no problems with self-care. • Without help from others. 2. I have some problems with self-care. • Needs some help from others. • Needs help from others. 3. I am unable to wash or dress myself. • Unable to wash or dress without help. Usual activities Hobbies in the home 1. I have no problems with performing my usual activities. • Without help from others. 2. I have some problems with performing my usual activities. • Needs some help from others. • Needs help from others. 3. I an unable to perform my usual activities. • Unable to perform hobbies in the home. Pain/discomfort Patient's assessment of own health status 1. I have no pain or discomfort. • Very good. 2. I have moderate pain or discomfort. • Good/fair. 3. I have extreme pain or discomfort. • Poor. Anxiety/depression Patient's experience of emotional problems 1. I am not anxious or depressed. • Never. 2. I am moderately anxious or depressed. • Sometimes. 3. I am extremely anxious or depressed. • Often. For the mobility dimension of the EuroQol instrument, we assumed that patients that were able to walk without assistance from others had no problems in performing this activity, whereas patients who were unable to walk unassisted were classified as confined to bed. Patients that needed help from others were classified as having some mobility problems. Two questions from the ADL instrument in the Odense Study were used to classify patients on the EuroQol personal care dimension. Patients that performed both activities without help from others were classified as having no problems on this dimension. Patients in need of help with either personal care or dressing or both were classified as having some problems. Only patients unable to wash and dress without help from others were classified as such on the EuroQol instrument. The patient's ability to carry out hobbies in the home was used as a proxy for their ability to perform usual activities. For the pain/discomfort dimension of the EuroQol instrument, it was assumed that patient assessment of their own health status covered this dimension. Therefore, if they found their health status to be very good they were classified as having no pain or discomfort. A good or fair assessment was categorised as having moderate pain or discomfort, whereas a poor assessment was assumed to correspond to extreme pain or discomfort. In the Odense Study, patients stated how often they experienced emotional problems, whereas the health state being described by the EuroQol instrument refers to the patient's health at the time of filling in the instrument. Thus, the questions in the Odense Study included an aspect of time that the EuroQol does not cover. To overcome this, it was assumed that the occurrence of emotional problems converts to the degree of anxiety or depressions. That is, patients that never experience emotional problems were assumed not to be anxious or depressed, whereas patients that sometimes or often experience problems converted to moderate or extreme anxiety or depression, respectively. The procedure of mapping returned a five-digit code, where the first digit referred to the patient's mobility level; the second to the patient's level on personal care; and so forth. This five-digit code described a health state for which we looked up the HRQoL utility weight in a table of EQ-5D tariffs. The EQ-5D tariffs take values between zero and one, where zero is the worst imaginable health status, and one is the best imaginable health status. We used Danish EQ-5D tariffs from a survey based on the time trade-off technique [ 18 ]. Patients classification Based on their cognitive and functional scores patients were classified by severity and dependency level. Severity Status The severity of a patient's dementia was defined by score intervals on the MMSE [ 15 ]. Those scoring ≥20 were considered as having mild dementia, while patients scoring between 10 and 19 were classified as having moderate dementia. Patients scoring ≤9 were classified as suffering from severe dementia. MMSE scores were not available for 30 patients (21 AD patients and 9 patients suffering from vascular dementia). In order to determine these patients degree of dementia we used the CDR score to classify them into the above three severity groups. Patients with a CDR score of 0.5 were classified as mild, patients with a CDR of 1 were classified as moderate and patients with a CDR of 2 to 3 were classified as severe [ 19 ]. Dependency Status Patients were classified by their ability to perform physical and psychosocial activities of daily living (ADL) This resulted in a classification of either dependent or independent [ 3 ]. A binary variable was based on a non-hierarchical cluster analysis [ 20 ]. Firstly, we identified possible initial seeds for the analysis. The seeds were identified from a cross table of the physical ADL score and the psychosocial ADL score. Combinations of the physical and psychosocial ADL scores with five or more observations were included in the cluster analysis as possible seeds. Secondly, we carried out the cluster analysis using the PROC FASTCLUS procedure in SAS 8.2 (SAS Institute Inc., Cary, NC, USA) in order to identify two clusters. One cluster included patients with low scores on both the physical and psychosocial ADL scales. As low scores on both instruments meant that patients required help from others in performing the activities in question, they were classified as "Dependent". The other cluster included patients with high scores on both ADL scales and they were classified as "Independent". As these were composite criteria, the characteristics of the two groups of dependency were analysed. Statistical Analysis After analysing the descriptive results of the EQ-5D scores, we performed a regression analysis of EQ-5D scores on sociodemographic and clinical characteristics. Sociodemographic characteristics included gender, age and setting (living in the community or institutionalised) and clinical characteristics took into account the level of severity (Mild, Moderate, and Severe), the type of dementia (AD, vascular or other) and ADL status (independent or dependent). The regression analysis was performed with backward selection (level 5%) in order to determine the main factors influencing QoL. Because of heteroscedasticity, we estimated the heteroscedasticity consistent covariance matrix, which was used to calculate test statistics for the coefficients. Observations with missing data were automatically excluded from the analyses. That is, observations with insufficient information to establish a EQ-5D weight, e.g. that information was lacking to determine a patient's mobility level on the EQ-5D instrument. Results Table 2 illustrates the characteristics of patients included in the Odense Study by type of dementia. Of the 244 patients, 164 (67%) suffered from Alzheimer's disease (AD) and 80 suffered from vascular or other types of dementia. On average, AD patients were 3.9 years younger (95% confidence interval: [2.5 – 5.3]) than patients suffering from vascular or other types of dementia and more AD patients lived in a nursing home (p = 0.03). The remainder of patient characteristics in Table 2 did not significantly differ between the two groups. Table 2 Background Characteristics of Patients in the Odense Study Patients suffering from Alzheimer's disease Patients suffering from vascular or other types of dementia All patients suffering from dementia Number of patients 164 (67.2%) 80 (32.8%) 244 Number of females 89 (54.3%) 33 (41.3%) 122 (50.0%) Mean (SD) age in years 79.4 (5.14) 75.5 (5.21) 78.1 (5.47) Mean (SD) MMSE score 20.6 (4.62) 21.6 (4.96) 21.0 (4.75) Severity of dementia Mild (MMSE 20–30) 91 (55.5%) 49 (61.3%) 140 (57.4%) Moderate (MMSE 10–19) 51 (31.1%) 23 (28.8%) 74 (30.4%) Severe (MMSE 0–9) 22 (13.4%) 8 (10.0%) 30 (12.3%) Mean (SD) physical ADL score 14.1 (3.08) 13.7 (2.96) 14.0 (3.04) Mean (SD) psychosocial ADL score 9.3 (2.81) 9.0 (2.39) 9.2 (2.68) Living in the community 132 (80.5%) 73 (91.3%) 205 (84.0%) Tables 3 and 4 show the results from the cluster analysis, which was used to classify patients, according to their ADL status, as either independent or dependent in performing activities of daily living. Table 3 presents cluster characteristics. Of the 244 patients with dementia, 38 (16%) were classified as dependent, and 206 (84%) were classified as independent in the performance of ADL. Dependent patients were, on average, more severely stricken and institutionalised than independent patients. Table 3 Cluster Description Dependent Independent p-value Number of patients 38 (16%) 206 (85%) Number of females 17 (45%) 105 (51%) 0.4801 Mean (SD) age in years 79.3 (5) 77.9 (5) 0.1493 Number of AD patients 24 (63%) 140 (68%) 0.5622 Mean (SD) MMSE score 16.8 (6) 21.4 (4) <0.0001 Mean (SD) physical ADL score 7.9 (2) 15.1 (1) <0.0001 Mean (SD) psychosocial ADL score 4.7 (2) 10.1 (2) <0.0001 Number of patients living in the community 16 (42%) 189 (92%) <0.0001 Table 4 Number of Patients by Dependency Status and Type of Dementia ADL status Patients suffering from Alzheimer's disease Patients suffering from vascular or other types of dementia All Dependent 24 (15%) 14 (18%) 38 (16%) Independent 140 (85%) 66 (83%) 206 (84%) All 164 (100%) 80 (100%) 244 (100%) Table 4 shows the number of patients by dependency status and type of dementia. Due to of missing data, EQ-5D values were estimated for only 211 patients upon 244 demented patients. Results of comparison of EQ-5D value in different subgroups of patients were shown in Table 5 . Patient QoL seems to decrease when severity and dependency increase as well as when patients are institutionalised. Table 5 Mean EQ-5D weights by patient subgroups N Mean SD Severity Subgroups Mild MMSE > 20 135 0.636 (0.2109) Moderate 9 < MMSE < 20 64 0.596 (0.2152) Severe MMSE < 10 12 0.486 (0.2191) Dependency Subgroups Independent 193 0.641 (0.1952) Dependent 18 0.343 0.2324) Setting subgroups Community 191 0.621 (0.2173) Institution 20 0.564 (0.1861) Table 6 shows the results of a regression analyses. The results from the full model that included all predictors, as well as results from the reduced model that included only significant predictors are presented. Table 6 Results of the Regression Analysis of EQ-5D TTO Tariff on Severity of Dementia, ADL Status and Setting (n = 211) Full model Reduced model Variable Coefficient Asymptotic standard error p-value Coefficient Asymptotic standard error p-value Constant 0.579 0.0371 <0.0001 0.641 0.0140 <0.0001 Gender (0 = male, 1 = female) 0.045 0.0266 0.0948 - - - Age 0.003 0.0029 0.3553 - - - Type of dementia (0 = Other, 1 = AD) 0.006 0.0327 0.8522 - - - Severity of dementia Moderate 0.043 0.0280 0.1258 - - - Severe -0.079 0.0534 0.1405 - - - Dependency status (0 = independent, 1 = dependent) -0.289 0.0534 <0.0001 -0.297 0.0551 <0.0001 Setting (0 = community, 1 = nursing home) 0.027 0.0368 0.4672 - - - R 2 0.1776 0.1502 Patients dependent upon others to perform activities of daily living clearly had a lower QoL than independent patients. The QoL of independent patients suffering from dementia as assessed by their capacity to perform activities of daily living was 0.641 (95% confidence interval: [0.612 – 0.669]) whereas the QoL of dependent patients for the same assessment was 0.343 (95% CI: [0.251 – 0.436]). Severity of dementia and setting has no statistically significant impact on QoL. Discussion For the first time, this study provides health utilities for patients with dementia in Denmark. This study has shown that the factor that most affects the HRQoL of a patient with dementia is their dependency status as defined based by their ability to perform activities of daily living. The type of dementia doesn't seem to have a great an influence on patient's HRQoL, and severity does not appear to discriminate significantly between health utilities. However, due to missing data – particularly among patients with severe dementia – caution must be exercised when interpreting the results. In the utility results previously measured by Neumann et al. [ 7 ] using the Health Utility Index (HUI), AD patients' utilities decreased significantly with their severity levels. However, this could be explained by the fact that the EQ-5D does not consider cognition as a separate attribute, unlike the HUI scales. Despite this difference, results obtained with the HUI and the EQ-5D instruments were within the same range. As the EQ-5D values were estimated based on mapped questions, it raises the possibility of quotation bias and goodness of fit. Also, both patients and caregivers answered questions. Yet, with AD – and especially when patients are severely demented – it is impossible to collect non-proxy measurements in the later stages. The same methodology was performed in previous evaluations without knowing the impact of the difference between caregivers' and patients' perceptions. A particular strength of this study was that all data have been collected in conjunction with an epidemiological study wherein patients with dementia had been examined carefully and dementia criteria were explicitly stated. Conclusion Measuring HRQoL is as important as measuring disease severity, progression, symptom response, cognition and behavioural disturbance when assessing the impact of disease and determining proper intervention in the treatment and management of dementia. However, HRQoL is difficult to assess in a disease such as dementia for which patients suffer from cognitive disabilities. Based on study results and as previously shown by Kurz et al. [ 3 ], dependency level greatly influences patients' HRQoL and, when viewed as a global measure, reflects a certain level of HRQoL. Determining dependency levels could be considered as an indirect evaluation of HRQol. Other studies with disease specific questionnaires such as QoL-AD are needed to confirm these findings. Abbreviations ADL Activities of Daily Living EQ-5D EuroQol – Five Dimension Scale EuroQol EuroQol Scale DSM-IIIR Diagnostic and Statistical Manual of Mental Disorders 3 rd edition HRQol Health-Related Quality of Life MMSE Mini Mental State Examination QoL Quality of Life SAS Statistical Analytical Software Authors Contributions CKA is principal author and responsible for quality control. K W-J provided mapping of the questions from the Odense Study into the five dimensions of the EQ-5D. AL and KA provided data analysis and data analyses of the Odense database. P K-S provided access to the Odense database.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC521495.xml
522822	Effect of time of administration on cholesterol-lowering by psyllium: a randomized cross-over study in normocholesterolemic or slightly hypercholesterolemic subjects	Background Reports of the use of psyllium, largely in hypercholesterolemic men, have suggested that it lowers serum cholesterol as a result of the binding of bile acids in the intestinal lumen. Widespread advertisements have claimed an association between the use of soluble fibre from psyllium seed husk and a reduced risk of coronary heart disease. Given the purported mechanism of cholesterol-lowering by psyllium, we hypothesized that there would be a greater effect when psyllium is taken with breakfast than when taken at bedtime. Secondarily, we expected to confirm a cholesterol-lowering effect of psyllium in subjects with "average" cholesterol levels. Methods Sixteen men and 47 women ranging in age from 18 to 77 years [mean 53 +/- 13] with LDL cholesterol levels that were normal or slightly elevated but acceptable for subjects at low risk of coronary artery disease were recruited from general gastroenterology and low risk lipid clinics. Following a one month dietary stabilization period, they received an average daily dose of 12.7 g of psyllium hydrophilic mucilloid, in randomized order, for 8 weeks in the morning and 8 weeks in the evening. Change from baseline was determined for serum total cholesterol, LDL, HDL and triglycerides. Results Total cholesterol for the "AM first" group at baseline, 8 and 16 weeks was 5.76, 5.77 and 5.80 mmol/L and for the "PM first" group the corresponding values were 5.47, 5.61 and 5.57 mmol/L. No effect on any lipid parameter was demonstrated for the group as a whole or in any sub-group analysis. Conclusion The timing of psyllium administration had no effect on cholesterol-lowering and, in fact, no cholesterol-lowering was observed. Conclusions regarding the effectiveness of psyllium for the prevention of heart disease in the population at large may be premature.	Background A cholesterol lowering effect has been reported for a variety of soluble dietary fibres [ 1 - 5 ]. In February, 1998, the U.S. Food and Drug Administration authorized the use, on food labels and food labelling, of health claims on the association between soluble fibre from psyllium seed husk and a reduced risk of coronary heart disease [ 6 ]. Among the suggested mechanisms by which soluble fibre lowers cholesterol is the binding of bile acids in the intestinal lumen resulting in decreased absorption and increased faecal excretion of them [ 7 - 15 ]. The ensuing bile acid depletion increases hepatic demand for the de novo synthesis of bile acids from cholesterol. This requirement is met, in part, by increased hepatic LDL receptor activity, which in turn reduces circulating LDL. The accumulation and concentration of bile in the gallbladder is a continuous process. In rats which lack a gallbladder, the biliary excretion rate of bile salts is maximal at night [ 16 ] and bile is stored in the gallbladder during an overnight fast. To the extent that the cholesterol-lowering effect of psyllium requires an interaction with bile, the magnitude of its cholesterol-lowering effect should vary with the quantity of bile in contact with a given amount of psyllium. The gallbladder empties with a meal and the total quantity of bile salt presented to the small bowel should be highest when this emptying occurs at breakfast, following a fast and the associated overnight accumulation of secreted bile. Conversely, psyllium presented to the gut following a short fast and without the full stimulus to gallbladder emptying of an associated meal should result in an encounter with a smaller amount of bile. We accordingly set out to test the hypothesis that administering psyllium with breakfast would have a significantly greater cholesterol-lowering effect than would taking a similar dose at bedtime. Since the predominance of the literature with regard to the cholesterol-lowering effect of psyllium is in individuals with hypercholesterolemia, a secondary goal of our study was to confirm the cholesterol-lowering effect of psyllium in subjects with "average" cholesterol levels. Methods Patients identified in gastroenterology practices as requiring long-term treatment with psyllium, typically for chronic constipation or the irritable bowel syndrome, were invited to participate in the study. In addition, individuals who had received dietary counselling in a lipid clinic regarding cholesterol-lowering and who subsequently had cholesterol levels deemed not to require further intervention because they met targets set out in clinical practice guidelines were invited by clinic staff to participate. Subjects were deemed ineligible if they were under age 18 years, were under active treatment for hyperlipidemia, had total cholesterol greater than 7.00 mmol/L, required alterations in dosage of medications which might have an effect on lipid levels, had had a gastrectomy, had any disease which is associated with hyperlipidemia, were receiving a bile acid binding resin, or if they did not eat breakfast regularly. The study was approved by the Conjoint Health Research Ethics Board of the University of Calgary, Faculty of Medicine. Subjects were given a description of the study indicating our interest in comparing the relative efficacy of hs versus am dosing with psyllium without indicating the specific hypothesis, and were asked to sign a consent form. Gastroenterology patients were given a high fibre diet sheet as part of their therapeutic regimen and were asked to take this on a continuing basis, beginning one month prior to the initiation of the study. The diet sheet emphasized dietary sources containing predominantly insoluble fibre. Lipid clinic patients had all received in-depth counselling regarding dietary measures for hypercholesterolemia, including a high fibre regimen, and had implemented their dietary changes at least one month before beginning the study. An unsweetened psyllium preparation, "Novo-Mucilax" [NovoPharm], providing three grams of hydrophilic mucilloid per 6.2 gram powder, and a scoop known to provide at least ten grams of psyllium were provided. Containers were numbered and weighed at the conclusion of each test interval. Subjects were randomized to initially take a scoop full of psyllium either with breakfast or at bedtime. Using a crossover design, psyllium was taken in the morning or evening for eight weeks and at the alternate time for the subsequent eight weeks. Determinations of serum total cholesterol, LDL, HDL and triglycerides were made before beginning psyllium, at eight weeks and at sixteen weeks after commencing its use. A trained dietician obtained a dietary history and patients were weighed at the beginning and at the conclusion of the study. All lipid determinations were undertaken following a 14 hour fast and analyses were done in a central laboratory. After initial data inspection based on boxplots and summary measures, cholesterol values at 8 and 16 weeks were examined using analysis of variance, taking into account treatment, period, and between and within subject effects in accordance with the cross-over design. The pattern of change from baseline to 16 weeks was evaluated using paired t-tests. Results Of 86 subjects beginning the study, 33 of those referred from the gastroenterology clinics and 30 of those referred from the lipid clinic completed it. Of those withdrawing, eight did so because they could not tolerate the psyllium or it was felt to interfere with prescribed medication, 2 had elevated lipids, 4 did not complete all the required blood work, 5 were unable to comply with the protocol because of work or lifestyle changes and 4 developed intercurrent diseases which precluded completing the protocol. The age range of subjects was 18 to 77 years, mean 53 +/- 13 years, including 16 men and 47 women. The mean dose of psyllium taken was 12.7 +/ - 2.3 g for morning dosing and 12.7 +/- 2.2 g when taken in the evenings. Values for total, LDL, HDL cholesterol and triglycerides at baseline, eight and sixteen weeks for various subgroupings are tabulated in tables 1 , 2 , 3 and 4 , respectively. Data for changes from baseline for total, LDL and HDL cholesterol and triglycerides are listed in table 5 . Changes from baseline were not significant for any parameter. Furthermore, the confidence intervals given in Table 5 show that this data essentially excludes a clinically significant psyllium effect. Table 1 Total Cholesterol in mmol/L [SD] [n] Baseline 8 wks 16 wks AM first 5.76 [0.94] 5.77 [1.01] 5.80 [0.95] PM first 5.47 [0.98] 5.61 [1.10] 5.57 [1.06] Females [47] 5.72 [0.96] 5.79 [1.07] 5.74 [0.99] Males [16] 5.29 [0.94] 5.39 [0.96] 5.48 [1.04] GI subjects [33] 5.00 [0.83] 5.06 [0.91] 5.07 [0.84] Lipid clinic Subjects [30] 6.28 [0.59] 6.39 [0.69] 6.35 [0.70] All subjects [63] 5.61 [0.96] 5.69 [1.05] 5.68 [1.00] "AM first" are subjects taking psyllium in the morning for the first 8 weeks and in the evening for the second 8 weeks. Vice versa for "PM first". Table 2 LDL CHOLESTEROL in mmol/L [SD] Baseline 8 wks 16 wks AM first 3.55 [1.01] 3.51 [1.04] 3.52 [0.94] PM first 3.39 [0.90] 3.43 [1.03] 3.45 [0.92] Females 3.55 [0.92] 3.54 [1.04] 3.52 [0.92] Males 3.22 [1.03] 3.24 [0.99] 3.39 [0.97] GI subjects 2.89 [0.81] 2.85 [0.85] 2.91 [0.72] Lipid clinic subjects 4.09 [0.66] 4.15 [0.72] 4.11 [0.69] All subjects 3.47 [0.95] 3.47 [1.03] 3.48 [0.92] Table 3 HDL CHOLESTEROL in mmol/L [SD] Baseline 8 wks 16 wks AM first 1.38 [0.29] 1.42 [0.37] 1.42 [0.33] PM first 1.38 [0.40] 1.39 [0.36] 1.36 [0.33] Females 1.41 [0.30] 1.44 [0.30] 1.42 [0.29] Males 1.28 [0.45] 1.29 [0.50] 1.30 [0.43] GI subjects 1.36 [0.39] 1.41 [0.43] 1.37 [0.38] Lipid clinic subjects 1.40 [0.30] 1.40 [0.28] 1.41 [0.27] All subjects 1.38 [0.35] 1.41 [0.36] 1.39 [0.33] Table 4 TRIGLYCERIDES in mmol/L [SD] Baseline 8 wks 16 wks AM first 1.81 [0.70] 1.84 [0.86] 1.86 [0.91] PM first 1.53 [0.52] 1.69 [0.70] 1.65 [0.61] Females 1.66 [0.61] 1.72 [0.70] 1.76 [0.71 Males 1.70 [0.68] 1.89 [0.99] 1.72 [0.94] GI subjects 1.63 [0.54] 1.71 [0.82] 1.70 [0.72] Lipid clinic subjects 1.71 [0.71] 1.82 [0.74] 1.80 [0.83] All subjects 1.67 [0.35] 1.76 [0.78] 1.75 [0.77] Table 5 Mean changes from baseline at 8 and 16 weeks [mmol/L] and 95% confidence intervals [all patients, n = 63] 8 wks 16 wks TOTAL 0.080 p = 0.26 0.068 p = 0.28 CHOLESTEROL [-0.061,0.22] [-0.056,0.19] LDL 0.002 p = 0.98 0.018 p = 0.75 CHOLESTEROL [-0.13,0.14] [-0.098,0.13] HDL 0.028 p = 0.28 0.012 p = 0.61 CHOLESTEROL [-0.023,0.08] [-0.035,0.059] TRIGLYCERIDES 0.094 p = 0.22 0.082 p = 0.22 [-0.059,0.25] [-0.050,0.21] The mean caloric intake or the intakes of fat or fibre did not change significantly during the study [table 6 ]. There was a small but significant increase in the weights of subjects, which was felt to reflect the high proportion of subjects beginning the study in the fall and the associated reduction of physical activity during the subsequent winter months. There was no apparent relationship between change in cholesterol and change in weight [correlation = .1, p = .15]. Table 6 Weight and Dietary Intake [SD] at Baseline and at 16 Weeks Baseline 16 wks WEIGHT [Kg] 74.6 [16.5] 75.4 [16.9] p = 0.0039 ENERGY [kcal] 1649 [409] 1689 [419] p = 0.29 FAT [g] 49.1 [20.3] 49.8 [20.2] p = 0.66 FIBER [g] 19.7 [7.96] 19.7 [7.28] p = 0.99 Discussion We failed to prove our hypothesis that administration of psyllium in the morning would have a greater cholesterol-lowering effect than it would in the evening. Not only was there no observable difference in lipid levels between the crossover periods but the daily ingestion of a greater daily dose than the 10.2 g of psyllium for which the FDA allows health claims to be made [ 6 ] had no effect on lipid levels in our study group. No change in any lipid parameter, including total and LDL cholesterol was observed. No difference was found when subgroup analysis was undertaken for the sex of the patients, the time of day they took their psyllium, or whether they were recruited from the gastroenterology clinic or the lipid clinic. We used a crossover design since this was the most appropriate one for the primary question being addressed; accordingly, our study did not include a control group. However, the nature of the study should not have provided any motivation for study subjects to adopt any new lifestyle or dietary changes beyond those implemented well before the introduction of psyllium. Observational data has been shown to provide valid information, which is consistent with that observed in randomized, controlled trials [ 17 , 18 ]. The nature of the intervention was in keeping with those undertaken in day-to-day clinical practice and the protocol used should, therefore, have high "clinical relevance". Failure of lipid-lowering by psyllium has also been demonstrated in twenty hypercholesterolemic children [ 19 ], in twenty-four hyperlipidemic adults [ 20 ] and in a large observational study of elderly patients taking psyllium [ 21 ]. A report of lipid-lowering therapies in hypercholesterolemic veterans showed only a 2% reduction in LDL cholesterol and a small increase in LDL/HDL ratio in patients taking psyllium, but does not provide a measure of statistical significance [ 22 ]. One study revealed no difference in total cholesterol-lowering compared to placebo, but a reduction of LDL cholesterol resulted from psyllium treatment [ 23 ]. Another demonstrated no difference in total cholesterol-lowering compared to placebo, a reduction of LDL in 11 "responders" and no change in 9 "nonresponders" [ 24 ]. A reduction of HDL cholesterol has been noted in some studies [ 25 - 27 ] and was associated with changes in LDL/HDL ratios similar to placebo treatment [ 25 , 26 ]. Published studies include few normocholesterolemic subjects. Cholesterol reduction was observed in 7 normal men [ 28 ] and in 5 of 9 subjects [ 29 ], in both studies after 3 weeks of treatment. A reduction of cholesterol levels was also observed in 12 elderly patients given psyllium for 4 months [ 30 ], while 5 normocholesterolemic subjects in another study showed no reduction after 2 to 7 months of treatment [ 31 ]. A meta-analysis of 17 studies of patients with hypercholesterolemia has suggested a small but significant cholesterol-lowering effect of psyllium [ 2 ]. All of these investigations were associated in one way or another with the product manufacturer. Additional studies have also indicated some cholesterol-lowering by psyllium in hypercholesterolemic individuals [ 32 - 37 ] or in diabetics [ 38 - 40 ]; however, much of this work is uncontrolled and some protocols have specifically excluded premenopausal women [ 33 , 38 ]. The association of cholesterol-lowering effects with psyllium may be weakened in some studies by the use of a supplement containing additional forms of soluble fibre [ 42 ] or by apparent differences in intake of calories [ 43 - 46 ], soluble fibre [ 25 ] or cholesterol [ 47 ] in control and treatment groups or periods. Several reports include only small numbers of patients and/or are of short duration. There is a strong predominance of male subjects in these publications and some protocols incorporate additional treatment interventions [ 20 , 48 ]. Several factors may contribute to the difference between our observations and those of others. A meta-analysis has demonstrated that the initial level of cholesterol was highly predictive of the subsequent reduction of cholesterol by oat bran [ 49 ]. A greater effect of psyllium in men compared to women has been suggested [ 23 , 46 ] and a diet high in soluble fibre produced less cholesterol-lowering in post menopausal women than in men [ 10 ]. Soluble fibre has a lesser effect on lipid metabolism in female than in male guinea pigs [ 50 ] and there is a sex-based difference in mechanism of action in this animal [ 51 ]. Oat bran fails to lower cholesterol in young women, in contrast to men and older women [ 52 ]. The dominance of women in our study, the "normal", or only slightly abnormal cholesterol states of our subjects and the relatively young ages of some of them may, accordingly, account for some of the variance of our observations with some of those previously reported. The small increase in the weight of subjects is believed to be have resulted from reduced physical activity. In a meta-analysis of the effect of weight reduction on lipids, predominantly through dietary change, a reduction in total cholesterol of 0.05 mmol/L and of 0.02 mmol/L in LDL cholesterol per kilogram of weight lost was identified [ 53 ]. Dietary intakes were stable throughout our study and the average weight gain of less than one kilogram is very unlikely to have raised cholesterol levels to a degree sufficient to offset a significant cholesterol lowering effect of psyllium. A small cholesterol-lowering effect of psyllium appears to occur in hypercholesterolemic individuals, at least in men and possibly postmenopausal women. The notion of a benefit accruing to the general population requires additional study. The promotion of foods containing psyllium as reducing the risk of heart disease for the population at large [ 6 ] may be premature. Additional study is required and this should be undertaken in a manner that is free from concern regarding the possibility of publication bias which Brown L, Rosner B, Willett WW and Sacks FM have raised [ 2 ]. Conclusion The timing of psyllium administration had no effect on cholesterol-lowering and, in fact, no cholesterol-lowering was observed. Conclusions regarding the effectiveness of psyllium for the prevention of heart disease in the population at large may be premature. Competing interests The authors declare that they have no competing interests. Authors' contributions GVR carried out the study design, data review and writing. EAS carried out the study design and data review. RB carried out the study design, data review and statistical analysis. ALE carried out the study design and data review.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC522822.xml
524506	Moderate exercise may attenuate some aspects of immunosenescence	Background Immunosenescence is related to the deterioration of many immune functions, which may be manifested in increased susceptibility to infection, cancer, and autoimmunity. Lifestyle factors, such as diet or physical activity, may influence the senescence of the immune system. It is widely accepted that moderate physical activity may cause beneficial effects for physical and psychological health as well as for the immune system activity in aged people. Methods Thirty elderly women aged 62 to 86 were subjected to a two-years authorized physical activity program. Peripheral blood lymphocytes distribution and the production of cytokines involved in the immune response development and regulation (IL-2, IL-4 and IFN-γ) were investigated. The same parameters were evaluated in two control groups of women: a sedentary group of 12 elderly women selected for the second round of the physical activity program and in a group of 20 sedentary young women. Flow cytometry methods were used for the examination of surface markers on peripheral blood lymphocytes and intracellular cytokines expression. Results The distribution of the main lymphocytes subpopulations in the peripheral blood of elderly women did not show changes after long-term moderate physical training. The percentage of lymphocytes expressing intracellular IL-2 was higher in the group of women attending 2-years physical activity program than in the control group of elderly sedentary women, and it was similar to the value estimated in the group of young sedentary women. There was no difference in the intracellular expression of IL-4 and IFN-γ between the active and elderly sedentary women. Conclusions Our results suggest that moderate, long-term physical activity in elderly women may increase the production of IL-2, an important regulator of the immune response. This may help ameliorate immunosenescence in these women.	Background The decline in immune function associated with ageing increases the risk for infectious diseases, autoimmune disorders and tumors occurrence. The scientific interest of many laboratories is focused on the ageing of the immune system and on agents, which may retardate this process. The most frequently mentioned features of immunosenescence characterized by lymphocytes surface markers expression are: shift from naive CD45 RA + to memory CD45 RO + T cells, increased level of CD4 + (mainly in peripheral tissues), increased level of NK cells, and decreased level of CD8 + and B cells [ 1 - 4 ]. Changes in the distribution of T cell subpopulations in the blood are considered rather not significant (with the exception of naïve/memory T cells). However data are still inconsistent and may be caused by the criteria used to select healthy old individuals and by differences between human populations depending on race or geographical region [ 5 - 8 ]. Changes in cytokine production are manifested by the shift from Th1 to Th2-type cytokine production and increased level of proinflammatory cytokines (TNF-α, IL-1β, IL-6) [ 9 - 12 ]. The decrease of T cells responsiveness to in vitro stimulation may result from the reduction of IL-2 secretion and IL-2R α chain expression [ 13 ]. The most influenced by advancing age is the cell-mediated immunity, which is directly attributed to age-associated involution of thymus gland [ 14 , 15 ]. Recently, the interest of many groups and medical centers is focused on the benefits of regular exercise by older people. These benefits include enhanced cardiovascular fitness, retention of muscle mass, reduction of risk factors associated with many life-threatening diseases. There is still poor documentation of the possibility to alter the activity of the immune system as a consequence of exercise in older population. It has been shown that natural immunity is strongly influenced by physical exercise [ 16 , 17 ]. There is some evidence that the plasma levels of various, mainly pro-inflammatory cytokines increase in response to strenuous exercise [ 18 ]. A new area of research points on the relationship between certain lifestyle factors (diet, physical activity) and immune senescence. The group of 30 elderly women was selected to participate in the program of physical activity supervised by the Academy of Physical Education in Warsaw (Poland). The purpose of this program was to evaluate the modulation of physiological and psychological parameters by moderate long-term training. The beneficial results of moderate training on different body functions created the idea to check selected parameters, which characterize the activity of the immune system. Blood lymphocyte subpopulations distribution and intracellular expression of cytokines, which are central regulators of the immune responses (IL-2, IL-4 and IFN-γ), in activated blood lymphocytes have been examined. The aim of these investigations was to answer the main question related to the effect of exercise in older people: can long-term, moderate exercise, attenuate changes attributed to aged immune system? Comparative studies have been performed between the group of elderly exercising women and two control groups: elderly women selected for the new round of physical activity program and young sedentary women. Methods Characteristics of the group The group investigated consisted of 30 women aged 62 to 86 (mean age 73.2) with no contraindication of consistent workout found. This group of surveyed women had never attended regular organized exercise classes previously. The group attended 2-years authorized physical activity program aimed at taking into account the basic requirements for health regarding prophylaxis of diseases of the circulatory and respiratory systems and the kinetic system. The exercises were done twice a week for 50 minutes during 10 months of the year. The annual cycle has been concluded with a two-week trip with a profile of recreation and tourism. Each workout session was similar in nature with equal time for each exercise. The initial part (10 minutes) consisted of exercises preparing the body for physical exertion simply called "warm up" preferred to be done in higher positions. The main part (30 minutes) of different intensity consisted of: a/ workout while marching and standing – aimed at improving the condition of the circulatory and respiratory system, b/ workout in low positions – in squat, on hands and knees, lying on your side, in prone position – which intended to strengthen the kinetic system. The exercises finished with a 5 minutes "cool down" and relaxation exercises done in low positions such as lying and squat positions. The general rate of exercises was stimulated with music in character and rhythm, which was adapted to the age and abilities of those who participated in exercises and it, was from 100 to 120 bpm. The intensity of exercises was regulated additionally by the number of repetitions of particular exercises and series, the integration of the particular movements into the entire unit or set, the choice and the method of performing breathing exercises. During the exercises the heart rate was monitored and registered by a Polar Sport Tester. The pulse rate was not higher than 80% of the maximum load of exertion (100% of maximum load of effort during exercises = 200-age, according to the Worms rule). The work rate intensity was 60–80% of the maximum load. Constant observation of the attendants and systematic medical examination allowed the individual immediate modifications of the load of exertion while the women were exercising. Psychophysical self-control and self-evaluation had been installed among the participants since the very first workout session based on: heart rate (this was observed before, during and after exercising), frame of mind estimation, and subjective grading of the level of difficulty of the exercises according to the Borg Scale. Participants of the exercises had been under permanent medical control. No health contraindications for attending the exercises were found during the two years observation of the participants in the workout classes. The beneficial results of the workout program have been discovered in the research works concerning: 1/ motor activity competence, 2/ mobility of joints, 3/ physical efficiency 4/ body component, 5/ post-urography, 6/ nourishment, 7/ internal evaluation, 8/ laboratory works, 9/ orthopedics, 10/ bone density evaluation, 11/ psychology [ 19 , 20 ]. The analysis of peripheral blood lymphocyte subpopulations and expression of intracellular cytokines were performed after 2-years physical activity program. As a control we used the group of selected 12 women (aged 60–72) starting the new round of the program (Ctrl A), and the group of 20 sedentary young healthy women 20–40 years old (Ctrl B). Preparation of cells Blood was collected from donors at 08.30 – 10.00 am. PBMCs were isolated from freshly drawn heparinized blood by Lymphocyte Separation Medium (GIBCO BRL) centrifugation. Mononuclear cells from the interface were collected and washed with PBS. Peripheral blood lymphocyte subpopulations distribution Blood lymphocyte subpopulations were assessed by surface markers expression. For immunofluorescence staining of human lymphocytes the Lysed Whole Blood Method was used according to Becton-Dickinson protocol. Standard set of monoclonal antibodies against surface antigens was used: Simultest™ CD3/CD8, Simultest™ CD3/CD4, Simultest™ CD3/CD19, Simultest™ CD3/CD16CD56, Simultest™ Leucogate™ (CD45/CD14) and Simultest™ Control γ 1 /γ 2a (all reagents from Becton-Dickinson). The results are given as % of positively stained cells in the sample. Identification of lymphocytes expressing intracellular cytokines PBMCs were suspended in RPMI 1640 medium with glutamax II supplemented with 1 mM sodium pyruvate, 5 × 10 -5 M 2-mercaptoethanol, 20 mM HEPES and 5 mcg/mL gentamicin (GIBCO BRL). For the induction of cytokines synthesis peripheral lymphocytes were activated by PMA and calcium ionophore A 23187 (Sigma) at concentrations 50 ng/mL and 250 ng/mL, respectively. Approximately 3 × 10 6 cells in culture medium were placed in 24-well tissue culture plates and incubated with stimulants for 14 hours at 37°C, 5% CO 2 . Monensin was used as the protein transport inhibitor and was added to the cultures for the last 5 hours incubation. Completed the incubation, the cells were harvested and tested for viability by trypan blue exclusion. For intracellular cytokines staining the IC Screen™ Intracellular Staining Kits for human IL-2, IL-4 and IFN-γ were used (Biosource Int.). Intracellular staining was performed according to the Biosource protocol. Positively stained cells were analyzed by CellQuest™ software on FACSCalibur (Becton-Dickinson). Results are presented as the percentage of cells expressing intracellular cytokines in total lymphocytes population. Statistical analysis Data have been expressed as arithmetical mean and SD. Analysis of variance (ANOVA) was used to determine significant differences between control and experiments. Significance was determined at P < = 0.05. Results Phenotypes examination of peripheral blood lymphocytes The percentages of lymphocytes subpopulations characteristic for exercised women were presented separately for 16 women up to 70 years old, and 14 exercised women over 70 (Fig. 1 ). The decrease in mature CD3 + T cells with age is not a continuous process. The percentage of CD3 + lymphocytes is rather constant until the seventh decade and decreases after this period (21, 22). The CD3 level was the purpose to present phenotypes distribution separately for women over/up 70. The distribution of the main lymphocyte subpopulations did not change among the groups. Typical changes, however not statistically significant, in lymphocyte subsets distribution in peripheral blood of women over 70 years old have been observed: decrease of the percentage of CD3 + and CD8 + cells, and increase in the percentage of NK cells (CD16 + CD56 + ). Figure 1 Distribution of peripheral blood lymphocyte subpopulations. Ctrl A – elderly women starting with the physical activity program. After the program (up to 70) – up to 70 years old women after the physical activity program. After the program (over 70) – over 70 years old women after the physical activity program Expression of intracellular IL-2, IL-4 and IFN-γ The percentages of in vitro activated peripheral blood lymphocytes expressing intracellular IL-2 of older exercised vs. young sedentary women were similar. Both percentages were significantly higher compared to the value obtained for older women starting the physical activity program (Fig. 2 ). The percentages of lymphocytes positive for IL-4 were significantly higher in older vs. young women independently on physical training. Changes in the percentages of intracellular IFN-γ expressing lymphocytes were not observed. Representative results are also presented on density plots (Fig. 3 ). Figure 2 Intracellular cytokines expression in activated peripheral blood lymphocytes. Ctrl A – elderly women starting with the physical activity program. After the program – elderly exercised women. Ctrl B – young, sedentary women. Statistically significant data from Ctrl A are denoted by black stars. Statistically significant data from Ctrl B are denoted by black triangles. Figure 3 Intracellular cytokines expression in activated peripheral blood lymphocytes. A/ Ctrl A – elderly women starting with the physical activity program, B/ elderly exercised women, C/ Ctrl B – sedentary, young women. FL2 – lymphocytes positive for intracellular cytokines. Lymphocytes with intracellular cytokine expression are situated in the upper left (UL) part of the quadrant. The exercise intensity basing on maximum heart rate and Borg scale The exercise intensity was evaluated basing on heart rate, assuming that 80% of effort possibilities of exercising individuals (acc. to the formula 100% = 200-age) were not exceeded. When the exercise pulse was measured during training session it was stated that the average value of HR max amounted to 105,5 +/- 6,7 bpm (90–116 bpm). At the same time the participants of training group evaluated their effort by the subjective method according to the Borg scale. The average value of this evaluation was 11,2 +/- 1,1 points (9–13 points) so it means that the participants estimated their effort as fairly light. According to Borg the results of the subjective evaluation (in points) multiplied by 10 should correspond with the heart rate measured during the effort. Thus, the exercise intensity evaluated basing on the average value of HR max (105,5 bpm) was similar to the subjective evaluation of an effort acc. to the Borg scale (11,2 points × 10 = 112 bpm). Discussion Moderate physical activity may have several beneficial effects for physical and psychological health and for immune system activity in young and aged individuals. Strenuously performed exercise may cause harmful effects. The balance between beneficial and undesirable effects might be of great importance especially in older people. Exercise may mobilize NK cells, which may have originated from the liver [ 23 ]. Moderate endurance training results in increasing capacity to generate IFN-γ, but repeated exhausting exercise tends rather to down regulation of this cytokine [ 24 ]. Results of experiments performed on animal model revealed that exercised vs. control rats had greater numbers of leukocytes in the thymus, axial, and inguinal nodes. The percentage of CD4 + lymphocytes increased after exercise in the thymus, spleen and blood [ 25 ]. Results of our study did not show changes in the peripheral blood lymphocyte subpopulations distribution in the group of elderly exercised women. Cytokines generate signals required for the communication among cells of the immune system. IL-2, IL-4 and IFN-γ are multifunctional cytokines involved in the development and effectors functions regulation of T, B and NK cells [ 26 - 28 ]. The results of our study demonstrated that the percentages of lymphocytes expressing intracellular IL-4 were higher in both groups of older women (exercised and Ctrl A) than in the group of young sedentary woman (Ctrl B). One possible explanation is the higher level of memory lymphocytes in elderly [ 29 ]. The percentage of IFN-γ positive cells did not differ significantly among groups. The percentage of lymphocytes expressing intracellular IL-2 in the group of exercised women is higher from the value obtained for older sedentary women and similar to the value characteristic for young women. Normally the level of IL-2 estimated by ELISA decreased with ageing [ 13 ]. Increase in IFN-γ production and decrease in IL-2 production have been observed in old mice [ 30 ]. IL-2 is a multifunctional cytokine essential for T cells development in the thymus and for growth in the periphery. It is involved in the maintenance of lymphocyte homeostasis [ 31 ]. IL-2 deficiency may lead/facilitate multiorgan inflammation and the formation of autoantibodies of various specificities [ 32 ]. The exercise-induced increase of the percentage of IL-2 expressing lymphocytes may sustain the activity of NK cells and the generation of cytotoxic lymphocytes. In elderly humans changes in T memory versus T naïve cells is accompanied by diminished activity in IL-2 synthesis and elevated production of IL-4 and IFN-γ. It may be possible that the effect of moderate physical activity modulate the reactivity of T cells and other cells of the immune system despite physiological changes related to ageing. Recent data demonstrate that in vitro treatment of lymphocytes from old subjects with IL-2 reversed the impaired production of type-1 cytokines, restored the proliferative response of T cells and rescued from increased apoptosis [ 33 ]. Thus the increase in the percentage of lymphocytes expressing intracellular IL-2 may normalize the immune function dependent on this cytokine. Adequate IL-2 secretion is essential in naive CD4 + T cells proliferation in response to T cell receptor (TCR) stimulation and generation of effectors from naïve T lymphocytes pool [ 34 ]. Additionally, an antigen-independent process of ageing of T cells occurs because of lowered IL-2 production [ 35 ]. Thus, increased IL-2 production as the effect of moderate exercise may contribute in the restoration of naïve T cells pool. The results of investigations performed in other laboratories showed that peripheral blood lymphocytes isolated from active elderly demonstrated higher proliferative response to polyclonal mitogens and higher rates of IL-2, IFN-γ, IL-4 production than elderly sedentary subjects [ 36 ]. Experiments on animal models showed that Con A-activated splenocytes from exercised mice produced higher rate of IL-2 [ 37 ]. The results of experiments performed with exercised old mice demonstrated the increase of the naïve to memory T cell ration [ 38 ]. Our results demonstrate that moderate fitness training may have the potential to increase immune reactions in vivo in elderly women by modulating the expression of cytokines, which normally are depressed with age. Conclusions Moderate, long-term physical activity may modulate the synthesis of certain cytokines, which are important regulators of the immune response, and may ameliorate the status of the immune system in senescence. This amelioration may result in an enhancement of quality of life of older people. List of abbreviations IL – interleukin Competing interests The authors declare that they have no competing interests. Authors' Contributions EK is the author of the physical activity program and conceived of the idea for the study. PS and ND designed the study. ND drafted the manuscript and analyzed the data related to the immune system. EK and PS described the physical training program and data related to Borg scale. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC524506.xml
524512	On modeling locus heterogeneity using mixture distributions	Background Locus heterogeneity poses a major difficulty in mapping genes that influence complex genetic traits. A widely used approach to deal with this problem involves modeling linkage data in terms of finite mixture distributions. In its simplest setup, also known as the admixture approach, a single parameter is used to model the probability that the disease-causing gene of a family is linked to a reference marker. This parameter is usually interpreted as the overall proportion of linked families. Results In this article, we address two issues regarding the admixture approach. First, we tackle the question of whether the single parameter of linked proportion is well defined in general. By formulating the likelihood under a classification scheme based on distributions, we show that such a parameter is meaningful only when a certain well-characterized condition is met. Second, we study a condition given in the literature for validating the admixture approach. A counter example is constructed to illustrate that the condition does not necessarily lead to valid estimates. Conclusions Estimators from the admixture approach may be inconsistent. This holds even if a condition given in the literature to validate the approach is satisfied.	Background Mapping disease genes influencing complex genetic traits is far more difficult than mapping genes underlying Mendelian traits. One of the difficulties is due to locus heterogeneity, in which the disease in different families (or in different individuals within a family) is caused by different loci or non-hereditary factors. Finite mixture distributions have been proposed to model linkage data in the presence of locus heterogeneity [ 1 ], also known as admixture modeling. It is currently a widely used approach for testing heterogeneity and/or linkage [ 2 ]. In its simplest setup with two parameters, each independent data unit (usually a single family) is assumed to be either linked or unlinked. Here a family is said to be linked if its disease-causing gene is linked to a reference marker. In the two-parameter setup, all linked families are assumed to have the same linkage parameter, the recombination fraction θ . The heterogeneity is modeled by a single parameter ( α ) that denotes the probability that a family is linked. This parameter is interpreted by many researchers as the overall proportion of linked families. Despite the popularity of the admixture approach, a number of authors have pointed out limitations with the approach, albeit reaching different conclusions on its practical values. For example, one of the assumptions of the admixture approach is that there is only inter-family, but no intra-family, heterogeneity. Goldin [ 3 ] and Durner et al. [ 4 ] carried out simulation studies to show that violation of this assumption does not necessarily lead to a loss of power for detecting linkage. Another inherent assumption of admixture approach is that the genetic models at the linked and unlinked loci are the same. Vieland and Logue [ 5 ] showed that violation of this assumption leads to asymptotically biased parameter estimates. They showed that in this situation, the parameter α does not even measure the proportion of linked families within the sample, contrary to popular belief. Similar conclusions were obtained in the simulation study by Pal and Greenberg [ 6 ], in which the authors simulated data under various two-locus heterogeneity models, genetic parameters, ascertainment schemes, and phenocopy frequencies to see their effect on α . Nevertheless, these studies as well as some other simulation studies (see [ 7 ] and references therein) support the use of admixture approach as a robust tool for testing linkage in the presence of heterogeneity. Other researchers take a different view, pointing to potentially severe biases of the estimates obtained from the admixture approach (e.g., [ 8 , 9 ]). Whittemore and Halpern [ 9 ] provided comprehensive discussion on the genetic assumptions underlying the admixture approach. One of the foci was on whether the admixture parameter ( α ) is meaningful when certain assumptions are violated. Janssen et al. [ 8 ] showed, through examples, that estimates of α and θ can be severely biased when the distribution of informativeness in the linked families is not roughly the same as that in the unlinked families, under some measure of informativeness for linkage studies. They used a measure called Effective Number of Informative Meioses (EFNIM) to assess the informativeness of a family. It is based on the Expected LOD (ELOD) score of a family. They argued that, if the Linked and Unlinked Families are nearly Equally Distributed (LUFED) in terms of EFNIM, then the admixture procedure should provide satisfactory, i.e., nearly unbiased, results. In this article, we consider the same problems discussed in Janssen et al. [ 8 ] and Whittemore and Halpern [ 9 ], but from a more statistical perspective in terms of formulating the likelihood based on finite mixture distributions. In the usual formulation of finite mixture modeling for statistical inference, the data in the sample are independent and identically distributed (iid). The distribution of each of these data points is a finite mixture of several component distributions, with known or unknown mixing proportions, called weights [ 10 , 11 ]. In linkage analysis with family data, unless all families follow the same distributions both under linkage and no linkage (i.e., all families having the same component distributions), the usual admixture approach does not reflect the correct likelihood function (or, equivalently, the heterogeneity LOD function). In most genetic studies using family data, this condition is violated as data usually come from families of various structures, sizes and complexities. That is, the families are not iid; they are independent but not necessarily identically distributed. Therefore, the admixture modeling with a single heterogeneity parameter is under parameterized. So, a natural question is what is its implication on the estimators of the parameters. Are they consistent? Under what condition does consistency hold? We investigate these questions by formulating a more general likelihood that assigns a separate heterogeneity parameter to each class of families following the same distribution. Then using this formulation, we show that the estimators from the admixture formulation are consistent only if a certain condition holds. Furthermore, we study the argument by Janssen et al. [ 8 ] on the LUFED condition for obtaining nearly unbiased estimates. We show that LUFED is a necessary condition for obtaining consistent estimates. However, as we illustrate through counter examples, this condition is not sufficient to guarantee consistency. In fact, for certain data satisfying LUFED, the asymptotic estimates can be far from their true values. Results Formulation of expected log-likelihood Suppose that data from independent families in the sample are classified into T types such that families in the same type follow the same distribution. That is, families are classified into the same type based on their "structures" that lead to the specification of their distributions. Here we use the term structure in a broad sense that includes not only the pedigree structure but also any other available information about the pedigree (such as knowledge of phase) that contributes to the distribution from which the family is generated. Note that structure does not include any phenotypic or genotypic data of a family. Families with the same structure but different phenotypic and/or genotypic data are simply different realizations from the same distribution and hence are iid, and are classified into the same type. In the most general setup, with g potential linkage scenarios, e.g., linked or unlinked ( g = 2) in the simplest setting, the probability distribution, f t * , for a realization ( y t ) from type t , t = 1,…, T , can be expressed in terms of its component distributions, f ti , i = 1,…, g [ 10 ]: where the mixing parameters satisfy 0 ≤ α ti ≤ 1 ( i = 1,…, g ), , and θ is the linkage parameter vector, including various recombination fractions. To simplify our presentation without compromising our objectives, we assume that the disease under study is caused either by a locus linked to the reference marker with a recombination fraction θ or by a locus unlinked to the reference marker. Further, we assume that the genetic (trait) models at the linked and unlinked loci are the same. So, the linkage parameter vector is θ = { θ , 1/2}, and the probability distribution is simplified to f t * ( y t ; { θ ,1/2}) = α t f t ( y t ; θ ) + (1 - α t ) f t ( y t ; 1/2), where α t is the probability that a type t family is linked. Note that the two distributions under the mixture have the same parametric form but differ in their linkage parameter values, being θ or 1/2. Also, note that a single α parameter is used for all families in the same type. Let a denote the true proportion of linked families in the entire sample. Let p t , t = 1,…, T , be the true proportion of type t families in the linked group. Thus p = ( p 1 ,…, p T ) is the distribution of types among the linked families. Recall that all families in a given type have the same distribution, i.e., same likelihood for any value of the recombination fraction. Similarly, define q = ( q 1 ,…, q T ) to be the distribution of family types among the unlinked group. Then the probability that a family is of type t is s t = ap t + (1 - a ) q t , and the probability that a type t family is linked is thus . Furthermore, let r denote the true common recombination fraction for the linked families. The likelihood contribution for estimating the parameters { α t , θ } by a type t family with data y t is L t ( α t , θ \| y t ) = α t f t ( y t ; θ ) + (1 - α t ) f t ( y t ; 1/2). (1) Then the expected contribution to the log-likelihood from a family of type t can be expressed as Note that r and are the true underlying quantities, whereas θ and α = ( α 1 ,…, α T ) are the parameters to be estimated from the data. Combining the expected log-likelihoods, ELL t , t = 1,…, T , we can form the total expected log-likelihood from all family types as follows: Since the above formulation of likelihood is correct under the stated assumptions, it can be shown that ELL( α 0 , r ) ≥ ELL( α , θ ), for any ( α , θ ), following Stuart and Ord [ 12 ]. This implies that this formulation of likelihood is guaranteed to provide consistent estimators of ( α , θ ). Note that α is a vector of nuisance parameters; only θ is the true parameter of interest. We note that the above likelihood, although similar to the one presented in Vieland and Logue [ 5 ], is different from it in two respects: First, their likelihood (and hence log of likelihood ratio, referred to as 2T-HLOD in their paper) allows for different trait models at the linked and unlinked loci (as they wanted to see the effect of incorrectly assuming same trait models at the two loci) while these two are assumed to be the same in (1). Second, 2T-HLOD involves different α parameters for families with different structures as well as different phenotypic data. So, families with the same structure have different α parameters if they have different phenotypic data. On the other hand, in (1) families with the same structure (and hence their data coming from the same distribution as elucidated at the beginning of this section) share a common α parameter. Since the issue addressed by Vieland and Logue [ 5 ] is different from ours and is based on different assumptions, the two likelihood formulations are not directly comparable. The two-parameter setup In this subsection, we show that the two-parameter setup leads to correct likelihood formulation only when p = q , that is, the distribution of family types among the linked families is the same as that among the unlinked families. In this setup, all α t 's are set to be equal to a common parameter, α , the overall proportion of linked families. Hence, the total expected log-likelihood is with respect to the recombination fraction θ and a single proportion parameter α : The expected log-likelihood ratio between the two sets of parameter values, ( a , r ) and ( α , θ ), is where When p t = q t for each t , and by Jensen's inequality [ 13 ] In other words, when p = q , ELL( a , r ) ≥ ELL( α , θ ), for any ( α , θ ), implying that = a and = r are the maximum likelihood estimates of α and θ . However, as shown in the examples in the next section, when p ≠ q , ELL( a , r ) may be smaller than ELL( α , θ ) for some parameter values ( α , θ ). That is, the maximum likelihood estimates of α and θ may not be consistent because the likelihood model is incorrectly specified (under-parameterized). In order for the two-parameter formulation to be correct, the following constraints should be placed on the makeup of the families: which implies that p = q given the additional constraints that and . Notice that p = q is stronger than the LUFED condition. Equal distributions of types in the linked and unlinked families according to the classification scheme based on distributions of the data implies equal distributions of informativeness in the same two groups. Therefore, LUFED is a necessary condition for the admixture approach. We also note that p = q is not necessarily satisfied for every dataset (see Discussion). In general, in a parameterization that demands an estimate for the overall proportion of linked families, α , the relationship between α and α t is as follows: where u t and v t are also unknown parameters (with constraints ∑ u t = 1 and ∑ v t = 1) that need to be estimated. Note that u t and v t are the parameters to be estimated from the data and are not necessarily the same as p t and q t , respectively, as the latter are the true values of the former. We have shown earlier that the maximum likelihood estimates for α t exist. However, one cannot solve the equations in (3) to obtain a corresponding estimate for α . This is because there are only T + 2 equations (including the two constraints) but 2 T + 1 unknown parameters, and thus not all parameters are identifiable when T ≥ 2. Hence, one may not be able to find a meaningful estimate of the overall proportion of linked families, contrary to popular desire for such an estimate. This is consistent with the finding of Whittemore and Halpern [ 9 ], although the conclusions are reached from two different perspectives (see Discussion). Further investigation of LUFED condition We have already shown that LUFED is a necessary condition for obtaining consistent estimators using the admixture approach. In this section, we investigate, through a contrived dataset, whether LUFED is also a sufficient condition for achieving satisfactory results, as contended by Janssen et al. [ 8 ]. We use ELOD, a popular measure of informativeness [ 2 ], for family type classification in terms of their informativeness for linkage studies. This measure is the basis for the EFNIM criterion of Janssen et al. ([ 14 , 8 ]); see these two references for detailed description of EFNIM. In our contrived dataset, the linked group consists of two types of families: Phase Known (PK) double backcross families in proportion p 1 , and Phase Unknown (PU) families in proportion p 2 , where p 1 + p 2 = 1. The unlinked group is also composed of PK and PU families, but in proportions q 1 and q 2 , respectively, where q 1 + q 2 = 1. We choose to work with the PK and PU families as the PK families were used by Janssen et al. [ 8 ] to evaluate the admixture approach, and more generally, these family types are frequently used to evaluate exact properties of linkage analysis methods [ 2 ]. We assume that there are m children in all PK families and m + 1 children in all PU families. As these two types of families have different distributions [ 2 ], we have two family types ( T = 2) under the classification scheme according to distributions. Now, let us consider the ELOD of each family. The ELOD of a PK family with m children is: where, as before, θ is the parameter for recombination fraction while r is the true recombination fraction. Similarly, one can find the ELOD of a PU family with m + 1 children: For r close to 0, it can be seen that ELOD PK ( θ ) and ELOD PU ( θ ) are both approximately m log 10 2, when they are evaluated at θ = r [ 2 ]. Hence, both PK and PU families, irrespective of their linked or unlinked status, are of one common type according to the ELOD criterion for classification. Thus the data satisfy Janssen et al.'s [ 8 ] LUFED condition. Note that the ELOD criterion for classification (and hence the LUFED condition) is based on homogeneity LOD scores to evaluate the informativeness of a family for linkage study and thus does not involve α parameters. It is worth noting that this dataset may serve as an example for understanding classification based on distributions and family realizations that make up a family type. The phase information as well as the number of offspring are part of the family structure, which is the basis of classification according to distributions. However, phenotypes and genotypes do not configure into this classification scheme. For example, the PK data type consists of families with several phenotypes, i.e., various combinations of recombinant/non-recombinant ( m /0, m - 1/1,..., 1/ m - 1, 0/ m ) offspring. To illustrate our results, we consider two specific settings for the distributions of families in the linked and unlinked groups under the classification scheme according to distributions: (a) p = (0.9, 0.1), q = (0.1, 0.9); and (b) p = (0.3, 0.7), q = (0.4, 0.6). For each of these two settings, we take m = 3 children in PK families and m + 1 = 4 children in PU families. In both settings, the overall proportion of linked families, a , is 0.5, and the true (small) recombination fraction, r , is 0.02. So, as discussed above, the distribution of informativeness among the linked families, under the ELOD measure, is the same as that among the unlinked families. The two pictures in Figure 1 show the contour plots of the expected log-likelihood under the two settings, for the two-parameter admixture formulation as given in (2). We see that the values of the parameters ( α , θ ) where the maximum expected log-likelihood occurs are not the same as their true values. So the estimates are inconsistent. The extent of this inconsistency depends on several factors, including how close p is to q . As seen in Figure 1 (a) , where p differs greatly from q , the parameter estimates are far away from the true values. On the other hand, in Figure 1 (b) , where p is close to q , the estimates are not too far from the truth. In fact, the estimates are expected to converge to the true parameter values when the difference between p and q approaches 0 . Figure 1 Contour plots of the expected log-likelihood for the two-parameter model: (a) p 1 = 0.9, p 2 = 0.1, q 1 = 0.1, q 2 = 0.9; (b) p 1 = 0.3, p 2 = 0.7, q 1 = 0.4, q 2 = 0.6. The numbers in each plot are various levels of the expected log-likelihood. The expected log-likelihoods at the parameter combinations represented by a curve are the same. Discussion This article addresses two questions. Is the single parameter representing the overall proportion of linked families in the admixture approach well defined? Can one validate the admixture approach if the distributions of linkage information in the linked and unlinked families are roughly the same according to a certain measure of informativeness? A simple situation, where the disease is caused either by a linked or an unlinked locus following the same genetic model, suffices for our purpose. That is, although issues such as age-dependent penetrances, locus-dependent models, and intra-family heterogeneity are very important in analyzing data in the presence of locus heterogeneity, they are not within the scope of this article. The first question of interest was addressed in Whittemore and Halpern [ 9 ], where they characterized the genetic conditions under which the parameter for overall linked proportion is meaningful. In contrast, we consider the same question from a more statistical perspective by characterizing statistical conditions under which the linked-proportion parameter is well defined and consistent estimate can be obtained. Our basic argument is built upon the realization that an implicit assumption in the usual admixture approach can be badly violated. The admixture approach assumes that, implicitly in the way its likelihood is formulated, all families follow the same distribution. However, data from different families may follow different distributions, defining various types of data. Our results show that, if the distributions of the family types, classified by the distributions of the data, are the same for both the linked and unlinked families, i.e., p = q , then the parameter is meaningful, and a consistent estimate exists. Otherwise, the desire for an estimate of such a parameter is ill-conceived. Note that the condition p = q may not be satisfied in practice, even asymptotically, because linked and unlinked groups may have other differences such as different fertility levels, age of disease onset, disease severity, etc, that may indirectly lead to different family structures (and hence different distributions) in the two groups. Although the problem considered in Vieland and Logue [ 5 ] is different from what we consider, as they focus on violation of different implicit assumptions of this approach, from a broader perspective, their as well as our conclusions demonstrate the inconsistency of estimators obtained from the admixture approach. The second question stems from our curiosity on an argument made by Janssen et al. [ 8 ] for the admixture approach. We show, through counter examples with a contrived dataset, that the answer to the question is no under the ELOD measure for informativeness. The difference between the estimates and the true values of the parameters, even with infinite amount of data, can be large, if one would carry out the analysis under the admixture approach. This should serve as a warning against complacency when the LUFED condition is met. We do realize that, with phase known and phase unknown data, these examples can be quite extreme in human genetic studies, although such data arise frequently in experimental crosses. In situations where data from more general family structures are available, the effect of violation of the admixture approach assumptions may be much smaller. The criteria for classification of families deserve further clarification because it is the heart of the problem. We discuss two criteria for classification, one according to the distributions of the data, and the other according to a measure of informativeness of the data for linkage studies (leading to the LUFED condition). The classification scheme based on the distribution criterion leads to a necessary and sufficient condition for validating the admixture approach. This result is a by-product of our theoretical development for finding an answer to the first question. The classification scheme based on the informativeness criterion, on the other hand, leads to the conclusion that LUFED is only a necessary, but not a sufficient, condition for the admixture approach, contrary to Janssen et al.'s [ 8 ] contention. Finally, we note that although the general formulation of likelihood based on forming groups of families with the same distribution gives consistent estimators, its practical utility seems to be limited. This is mainly due to the difficulty of classifying families according to distributions, in most applications, except for simple situations such as those involving only the PK and PU families. The general likelihood formulation is used here as a vehicle to further the understanding of potential problems in using the two-parameter admixture approach. A practical solution that embeds the correct likelihood formulation in a Bayesian framework is being pursued in a separate study. Authors' contributions Both authors were involved in all aspects of this research project. Both authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC524512.xml
546397	Knowledge discovery in databases of biomechanical variables: application to the sit to stand motor task	Background The interpretation of data obtained in a movement analysis laboratory is a crucial issue in clinical contexts. Collection of such data in large databases might encourage the use of modern techniques of data mining to discover additional knowledge with automated methods. In order to maximise the size of the database, simple and low-cost experimental set-ups are preferable. The aim of this study was to extract knowledge inherent in the sit-to-stand task as performed by healthy adults, by searching relationships among measured and estimated biomechanical quantities. An automated method was applied to a large amount of data stored in a database. The sit-to-stand motor task was already shown to be adequate for determining the level of individual motor ability. Methods The technique of search for association rules was chosen to discover patterns as part of a Knowledge Discovery in Databases (KDD) process applied to a sit-to-stand motor task observed with a simple experimental set-up and analysed by means of a minimum measured input model. Selected parameters and variables of a database containing data from 110 healthy adults, of both genders and of a large range of age, performing the task were considered in the analysis. Results A set of rules and definitions were found characterising the patterns shared by the investigated subjects. Time events of the task turned out to be highly interdependent at least in their average values, showing a high level of repeatability of the timing of the performance of the task. Conclusions The distinctive patterns of the sit-to-stand task found in this study, associated to those that could be found in similar studies focusing on subjects with pathologies, could be used as a reference for the functional evaluation of specific subjects performing the sit-to-stand motor task.	Background In the last decade quantitative movement analysis has been increasingly used in clinical contexts [ 1 ]. This analysis makes use of fairly complex instrumentation and of models of the musculo-skeletal system. It provides a great amount of information, such as space and time characteristics of the motor task analysed, joint and segment kinematics and kinetics and electromyographic patterns of muscular recruitment. An integrated analysis of measured and estimated biomechanical quantities allows for the description of the subject performance, for the discrimination among different motor strategies and, therefore, it supports the clinical decision-making process [ 2 ]. Modern complex instrumentation and models, such as stereophotogrammetric systems and multi-segment models of the human body, provide a thorough and faithful description of the subject's movement at a local level (e.g. joints kinematics), to be used at its best as a support to the functional assessment of subsystems of the locomotor apparatus (e.g. joint function) [ 3 ]. However, the large amount of measured information is not paralleled by the capability of such information of supporting the assessment of the overall subject's mobility [ 4 ]. Simpler experimental set-ups and models may be more appropriate to functionally assess a subject performing a specific motor task [ 5 ]. In recent years, clinical tests have been devised aimed at quantitatively assessing the level of a subject's activity limitation based on simple and encumbrance-free experimental set-ups associated with mechanical models of the musculo-skeletal system. These models are designed to be associated with both the subject and the specific task being performed [ 6 ]. In this context, Minimum Measured Input Models (MMIM) have been proposed and proven to offer effective insights into the motor task execution [ 7 ]. Simplified, and therefore low-cost, experimental set-ups facilitate the gathering of data both locally (a shorter examination time is needed) and in multi-centre contexts (more laboratories can afford the necessary experimental set-up), allowing the collection of a great quantity of data which may be sent to a single data repository. However, even simplified experimental setups and models may provide a large amount of biomechanical data that requires considerable human efforts to be interpreted [ 8 ]. In fact, traditional methods of data analysis for the extraction of knowledge rely on a direct analysis, which is usually demanding and time-consuming, and on the interpretation of an experienced analyst [ 9 ]. Such analysis becomes hardly applicable when dealing with data collected multi-centrically. The aim of this study was to extract knowledge regarding the execution of a specific motor task. The term "knowledge" refers here to any relationship among attributes associated with the phenomenon under analysis. These relationships can be intended as causal and, therefore, suitable for the interpretation endeavours, or at least as tools for evidencing the presence of a repeatable pattern of variables. The declared goal was pursued by searching relationships among large amounts of biomechanical quantities by using an automatic method. Some data mining techniques ( data mining is a step of a process called Knowledge Discovery in Databases (KDD)) lend themselves to be effectively used in this context since they may reveal meaningful patterns and data structures from massive databases [ 10 , 11 ]. A specific data mining technique was applied to the data yielded by the analysis of sit-to-stand (STS) trials performed by healthy adults and carried out using the above-mentioned MMIM approach. The STS motor task was chosen because it has been shown to be adequate for determining the level of subject-specific motor ability [ 12 ]. In addition, the data provided by MMIMs were shown to be powerful overall descriptors of motor tasks. A group of unrestricted age and gender healthy adults was used with the goal of discovering knowledge inherent to the way healthy adults perform the selected motor task. In order to properly frame this study, a summary description of the MMIM approach and an overview of the KDD process are reported. Methods A MMIM applied to the STS task – The TIP model A MMIM is a model of a portion of the musculoskeletal system that includes the invariant aspects of both the modelled mechanical system and the motor task being performed. Therefore, a MMIM requires a minimum amount of measurements and provides a physiology-related description of the motor task [ 4 ]. In analysing the STS, only measurements from a single force platform are needed. The task is divided in two time phases: before- and after-seat-off (BSO and ASO). In each time phase a Telescopic Inverted Pendulum (TIP) model is applied. A TIP is characterised by a fixed base of support and by a massless link joining the base of support of the moving portion of the body to its centre of mass (CM). The link can elongate, controlled by a linear actuator (LA), and can rotate around its base of support, controlled by two actuators acting in the sagittal (SA) and frontal (FA) plane, respectively. The kinematics of, and the dynamic actions on, the CM of the modelled portion of the body involved in the movement are needed as model inputs. The outputs of the TIPs are the kinematic and kinetic variables associated with the actuators. During BSO the TIP is applied to the upper part of the body with its base of support positioned on the chair, while during ASO the TIP is applied to the whole body and its base of support is located at the ankles. In order to apply the TIP model in each phase, subject specific and experimental set-up parameters are set. A list of TIP parameters and TIP output variables may be [ 7 ] collected into a database. The KDD process The KDD process was introduced in order to provide a framework in which data-miners could work in a logical and sequential way, considering all the research aspects from the data acquisition to the information extraction [ 13 , 14 ]. An iterative five phase process may be adopted (Figure 1 ) [ 15 ]. Figure 1 A scheme of the KDD process. Input data are initially selected and target data are isolated. Pre-processing and transformation are performed to ensure the database reliability. Data mining is the core analysis. The knowledge discovery process ends with the interpretation of the results. Initially, the domain understanding, the parameter selection, and the goal definition need to be set. A subset of interest of the stored dataset can then be isolated. Pre-processing is performed to reduce noise and fill possible gaps in the target dataset. Elimination of outliers, corrections of wrong elements in the database and reduction of dimensionality are crucial transformations to reach an adequate level of suitability of the database. Data mining "is a well-defined procedure that takes data as input and produces output in the form of models or patterns" [ 16 ] and is the core of the KDD process. It is used with different aims such as Exploratory Data Analysis (EDA) [ 17 ], Descriptive Modelling [ 18 ], Predictive Modelling such as Classification and Regression [ 19 ], Retrieval by Content [ 20 ] and Discovering Patterns or Rules [ 21 ]. Innovative techniques for the data mining have been introduced to be used either in conjunction with or in alternative to traditional statistical methods for two main reasons. First, while classical statistics is applied to data collected according to a specific goal of the analyst, data mining methods are applied to data already collected and aim at finding unknown relationships among them. Secondly, data mining allows to infer general rules with adequate approximation, even if the amount of data available is not as large as that generally required by inferential statistics [ 16 ]. The selection of the data mining technique is based on the specific analysis. Prediction, clustering, classification and research of association rules are the most common tasks and each of them may be accomplished with various algorithms. Finally, data interpretation helps the user in managing and understanding the results: visualisations (clustering) or extraction of symbolic rules are common ways of evaluating the discovered knowledge. The search for association rules The technique of research of association rules, which aims at finding the most recurrent patterns in a database, was selected for the data mining. Given a database D of experimental trials T , each experimental trial is a record of D and is made of a set X of literals called items. An item represents a specific value of an attribute of a table of D, and a record can be represented as an attribute (i.e. an output variable or a model parameter) together with its value [ 21 ]. The problem may be defined as follows. Let I = {i 1 , i 2 ,...., i m } a set of items of D therefore, T can be seen as a group of items such that T I . An association rule can be defined as a logical implication: X Y where X I is the antecedent of the rule, Y I is the consequent and X ∩ Y = . A rule X Y , over a set of trials T , has a confidence c if c % of the trials in T containing X , also include Y . The same rule in the same context has a support s if s % of the trials in T contain X Y . The confidence of a rule X Y can be calculated from the support of the antecedent X and the support of the union of the antecedent X and the consequent Y : Confidence is an index of the validity of a rule. A high confidence means that there is a strong relationship between X and Y in the sense that the presence of a pattern X in a trial implies, with a high probability, the presence of Y in the same trial. Given a set of trials T , finding "interesting" association rules in T is the problem of generating all the rules whose both support and confidence are greater than a set threshold ( minimum support and minimum confidence ). The extracted rules were reported in the following format: A → B [ c % ] where the first item was the antecedent of the rule, the item which followed was the consequent, while the value indicated in square brackets was the confidence. The implication was intended to be valid only one way, from the left to the right. In case of validity of both directions, "definitions" were obtained: A ←→ B [ c-min % ] composed by two rules having the two items both as antecedent and consequent whose confidence c-min was the lowest of the two c values associated with the one way rules. The search for association rules followed the path illustrated in Figure 1 . Initially, data were read from the sources and displayed, allowing for a straightforward selection of the dataset of interest. Next, the subset of data were prepared to the analysis by eliminating possible outliers and filling possible gaps in the database due to incorrect applications of the model. Typically, data preparation is the most time consuming phase of the KDD, but is also highly crucial since the effectiveness of a data mining analysis relies on the consistency of the database. Since the theory of association rules was formulated to deal with qualitative attributes [ 22 ] characterised by a limited number of scores, the virtually infinite values of quantitative attributes were assigned to a limited amount of intervals identified by progressive numbers. Such discretisation process [ 23 ] for each attribute A , generated a variable number n of partitions ( A i_n ; i = 1,.., n ). The first partition A 1_n included the lowest values of A and the last partition A n_n included highest values of A . Items (i.e. the attribute associated with a relevant discretised value) similar to the qualitative items could thus be generated (Figure 2 ). Figure 2 Example of a discretisation process of a quantitative attribute. Grey areas represent the different partitions, i.e. the items. Vertical lines represent the values of the quantitative attribute. Self organising maps (SOM) were used to cluster the values of the attributes. SOMs are widely known as a powerful clustering tool [ 24 ] and could overcome the disadvantages related to other unsupervised approaches as the equal frequency intervals or the equal interval width techniques. [ 25 ]. The latter methods, imposing an equal number of points belonging to each interval or, similarly, each interval having a pre-determined length, may generate meaningless or even empty intervals. SOMs were chosen and purposely implemented to properly isolate residual outliers [ 26 ] from the distribution of values of an attribute, to correctly define the clouds of values and to automatically set the optimal number of intervals [ 27 ]. Values and/or intervals were mapped in a discrete domain as integer numbers and items were created. In this way, the database became a set of itemsets. The search of frequent itemsets was performed on the selected dataset. Itemsets with support greater than the minimum support were found. The set of itemsets that appeared frequently in the transactions of the database was then identified. This step of the process was the most demanding in terms of processing time and computer memory occupation. The search for association rules was accomplished by using the APRIORI algorithm [ 10 ] which was shown to perform better then other common algorithms such as AIS [ 21 ] and SETM [ 28 ]. The APRIORI algorithm iterated the two following steps: • building of a candidate set Ck of itemsets, counting their occurrences; • defining "large itemsets" Lk on the basis of support constraints. In figure 3 , the main steps of the algorithm are illustrated. Figure 3 The Apriori algorithm applied to the database under analysis. The two phases of the Apriori algorithm are highlighted. The first, referred as "join step" phase, aimed at the generation of the candidate itemsets C k built starting from L k-1 , the frequent itemset of the previous phase. In the second phase the C k itemsets underwent to a "pruning" procedure that selected the frequent itemsets L k on the base of the support check. Each frequent itemset generated a set of rules and each rule was scored by its confidence. Only rules whose confidence was higher than the minimum confidence reached the following phase. The selected association rules represented the knowledge extracted from the database expressed in a quasi natural language that the user could interpret. Efforts were made toward a clustered representation of the set of rules to increase readability and interpretability of information. A software project for the data mining phase was purposely designed and implemented as follows: the software received as input all data from the database and returned a text file containing a list of the discovered association rules and all the possible unified rules and definitions derived from the entire dataset. Support and confidence thresholds were set to 35% and 85%, respectively. Considering that in a rule more than one consequent can be found [ 29 ], a maximum number of consequent items had to be set. In this analysis such number was experimentally set to 4 to avoid the presence of meaningless items in the resulting rules. Materials Healthy adult volunteers (N = 110), both males and females between 22 and 87, participated in the study, executing a total of more than 1100 trials. They were initially asked to sit on a seat. The height of the seat was set at a value equal to the subject's tibial plateau height [ 30 ]. Subjects could choose the distance of their feet from the seat and had to keep them parallel at a distance equal to that measured between iliac anterior superior spines. Both footprints were then drawn on the floor, ensuring that the subject's feet were in the same position during all the trials. In addition, medio-lateral and antero-posterior coordinates of selected foot points were measured. Anthropometric parameters, such as the body mass and the length of the lower limb segments, were also obtained. Subjects were asked to rise from the seat at the preferred speed after an audio start signal and look at a frontal one metre distant fixed point at the height of 80% of their eyes' height, maintaining the orthostatic posture until the stop signal. Arms were kept crossed on the chest during the trial to avoid that arm swing could affect CM movements. Ground reaction forces were measured using a six component Bertec force platform (0.4 m*0.6 m), positioned under both the seat and the subject's feet. Data were collected at a sampling rate of 100 Hz and pre-processed with an internally developed Labview ® software (National Instruments Inc.). First, force platform signals were digitally low-pass filtered (second order Butterworth filter 15 Hz cut-off frequency). Data were then fed into the TIP model, which yielded the kinematic and kinetic time functions (displacement, velocity, force/couple and power) of the LA and SA. FA variables were not analysed since their contribution to the motor strategy was considered negligible, given the sagittal symmetry of the STS motor task. From these functions a subset of kinematic and kinetic variables (KK-set) was extracted including time events of the task (normalised with respect to the duration of the whole task, see caption of Table 1 for the complete list of variables) and together with experimental set-up and subject specific parameters were stored in a Microsoft Access database, and loaded using a Windows ODBC interface [ 31 ]. The resulting database contained a total of more than 52,000 items. The number of analysed attributes was set to 47, as listed in Table 1 . Table 1 The 47 attributes analysed. They included subject initial conditions (ankle and thigh angles) and experimental setup/anthropometric parameters (seat height, thigh length, foot length, TIP1 hinge and malleoli coordinates), KK-set variables and important time instants. The KK-set was made of displacements ( Disp ), velocities ( Vel ), forces or couples and powers referred to the two LA and SA actuators. So referred to seat-off. In addition, ML, AP and V referred to the medio-lateral, antero-posterior and vertical directions. Finally, the attributes labelled with an initial "T" represented the instant of occurrence of the corresponding quantity (e.g. the attribute MaxLAVelASO referred to the maximum value of LA velocity after the seat-off and the attribute TMaxLAVelASO represented the corresponding instant of occurrence). Anthropometric and Experimental set-up Attributes Kinematic and Kinetic Attributes Time-Attributes RightAnkleAngle MaxSADispBSO Duration LeftAnkleAngle MaxSAVelBSO APRightMalleolusCoord MaxSACoupleBSO TMaxSADispBSO APLeft MalleolusCoord MaxSAPowerBSO TMaxSAVelBSO APHingeCoord SADispSo TMaxSAForceBSO MLRightMalleolusCoord SAVelSo TMaxSAPowerBSO MLLeftMalleolusCoord SACoupleSo MLHinge Coord SAPowerSo t So VRightMalleolusCoord MaxSADispASO VLeftMalleolusCoord MaxSAVelASO TMaxSADispASO VHingeCoord MaxSACoupleASO TMaxSAVelASO SeatHeight MaxSAPowerASO TMaxSAForceASO ThighLength MaxLADispASO TMaxSAPowerASO ShankLength MaxLAVelASO TMaxLADispASO FootLength MaxLAForceASO TMaxLAVelASO RightThighAngle MaxLAPowerASO TMaxLAForceASO LeftThighAngle TMaxLAPowerASO Results Various rules and definitions were found. Among them, some referred to obvious relationships such as those related to symmetry between right and left coordinates, others related a single item of a temporal parameter ( TMaxSADispASO 3_3 ) as a consequent of the following kinematic and kinetic items: a) MaxSADispBSO 3_6 and MaxSAPowerBSO 1_6 , before seat-off; b) SAVelSo 3_6 , at seat-off; c) MaxLADispASO 3,4_6 , MaxLAForceASO 4_5 , MaxSADispASO 3_6 and MaxSAVelASO 2,3_5 , after seat-off; and of the following time events: Duration 1_6 , TMaxSADispBSO 3_6 , TMaxSAVelBSO 3_6 , TMaxSACoupleBSO 2_6 , TMaxSAPowerBSO 2_6 , TMaxLADispASO 5_6 and TMaxSAPowerASO 3_5 . The attribute TMaxSADispASO was the attribute with the lowest number of partitions (three partitions) and its last partition included about 90% of the observations. The discovered definitions and rules that could not be easily predicted are illustrated in Figure 4 using a cluster representation, which highlights inner and crossed relationships among items of each phase of the task; values of confidence are reported in the figure caption. Figure 4 Graphic cluster representation of both the rules and the definitions found in the study. The first ones, marked with a single-ended arrow, were found to have a confidence ranging from 86% to 96%. The second ones, marked with a double-ended arrow, both presented a confidence of 95%. Involved items are positioned according to the STS time subdivision (BSO and ASO phases and seat-off timing). The definitions related exclusively time instant items: • TMaxSADispBSO 3_6 ←→ t so3_6 [95 %], • TMaxSAAngVelASO 3_5 ←→ TMaxSAPowerASO 3_5 [95 %], The first definitions related the 'average' time of occurrence of maximum sagittal displacement during BSO (partition 3 of 6) to 'average' values of t So (partition 3 of 6). The second definition associated the time instant of maximum sagittal velocity to that of maximum power, both after the seat-off. Moreover, meaningful rules were found that involved as consequent the item MaxSAPowerBSO 1_6 . This item showed relationships, with a value of confidence varying between 86% and 96%, with the following kinematic and kinetic items: MaxSACoupleBSO 2_6 , MaxSADispBSO 3_6 , MaxLAForceASO 4_5 , and MaxSAVelASO 2_5 ; and the following temporal items: TMaxSACoupleBSO 2_6 , TMaxSAPowerBSO 1_6 , TMaxSAPowerASO 3_5 and SAVelSo 3_6 . The partitions corresponding to the attributes involved in both rules and definitions, their support and their range of variability expressed in the relevant units of measurement (UoM), are reported in Table 2 . Table 2 Items involved in the discovered rules and definitions, their support and their range of values. Item Support (%) Range UoM Duration 1_6 45.0 1.01 1.61 s MaxSADispBSO 3_6 41.3 29 36 deg MaxSACoupleBSO 2_6 35.5 0.06 0.09 Nm kg -1 m -1 MaxSAPowerBSO 1_6 81.5 0.00 0.08 Wkg -1 m -1 SAVelSo 3_6 40.1 0.58 0.77 rad s -1 MaxSADispASO 3_6 59.7 -3 14 deg MaxSAVelASO 2,3_5 84.7 0.44 1.32 rad s -1 MaxLADispASO 3,4_6 87.2 45.4 54.5 % of TIP2 final length MaxLAForceASO 4_5 43.9 10.85 11.94 N kg -1 TMaxSADispBSO 3_6 36.7 39.2 48.0 % of duration TMaxSAVelBSO 3_6 37.9 34.6 42.1 % of duration TMaxSACoupleBSO 2_6 47.5 10.3 15.1 % of duration TMaxSAPowerBSO 1,2_6 92.1 20.5 26.3 % of duration t So3_6 35.4 46.9 55.9 % of duration TMaxSADispASO 3_3 89.2 87.1 99.9 % of duration TMaxSAVelASO 3_5 44.6 42.0 54 % of duration TMaxSAPowerASO 3_5 45.4 42.2 54.4 % of duration TMaxLADispASO 5_6 36.4 90.8 96.3 % of duration The items reported in Table 2 belong to a subset of 18 attributes of the original 47. Only a limited number of items was involved in the discovered rules. Discussion The data mining analysis allowed for the discovery of both definitions and rules relating various items obtained from a MMIM analysis of the STS motor task. The most obvious and/or expected relationships, such as those related to the symmetry between right and left coordinates, also noticeable by a visual examination of the task as performed by the investigated subject, were included in the set of discovered rules and definitions. The finding of such relationships provided elements to confirm the validity of the data mining analysis. The set of rules found that related the temporal item TMaxSADispASO 3_3 to various temporal, kinematic and kinetic items needs a further analysis to be interpreted. In fact, the attribute TMaxSADispASO was mapped in only three partitions and most of its observations were concentrated in the last partition. This circumstance rendered highly probable the presence of rules relating the item TMaxSADispASO 3_3 to those items of the various attributes with support higher than 35%. Therefore, these rules were used to highlight items involved with a considerable support and therefore the usefulness of such rules was deemed limited. In general, when interpreting a rule/definition found, the analyst should be aware not only of both its confidence and the support of the items forming it, but also of the number of partitions in which the attributes involved in the rule/definition were divided. The fewer are the partitions used for a quantitative attribute, the higher is the probability of finding rules/definitions unsuitable for drawing specific patterns. This is particularly true when most of the observations fall in a single partition of the attribute. Conversely, some of the rules and definitions discovered by the data mining analysis highlighted relationships that could not be easily predicted otherwise. The two definitions reported in the results section, that related time instant items, indicated that specific 'average' timings (items belonging to central partitions of the corresponding attribute) of the sit-stand task were closely related. This finding is consistent with those present in the literature [ 32 , 33 ]. In particular referring to the second definition reported, since power is the product of moment and angular velocity, the definition that associates 'average' values of the instant of maximum sagittal velocity to those of maximum power after the seat-off could be predicted. Very interestingly, the relationships of the item MaxSAPowerBSO 1_6 with several KK-set items showed the importance of the SA in the execution of the task. In fact, almost all rules relating KK-set items to the MaxSAPowerBSO 1_6 regarded the sagittal actuator. When the maximum SA power during BSO occurred early in the task, its value was among the lowest ( TMaxSAPowerBSO 1_6 → MaxSAPowerBSO 1_6 ). Low values of SA maximum power during BSO also occurred in combination with low-to-medium couple values ( MaxSACoupleBSO 2_6 → MaxSAPowerBSO 1_6 ) and early in the phase ( TMaxSACoupleBSO 2_6 → MaxSAPowerBSO 1_6 ). The latter rules showed that before seat-off kinetic variables of the main actuator are strongly related to each other and their timing. Given a value of one of them, a limited range of values is to be expected for the others. Moreover, 'average' SA velocity at seat-off was found to be present in combination with low maximum SA power at BSO ( SAVelSo 3_6 → MaxSAPowerBSO 1_6 ) showing that relatively high speeds at seat-off could be reached even when the power exerted before seat-off was low. The presence of low value partitions in the rules may suggest that most healthy adults tend to use the least amount of energy necessary to complete the first phase of the task, showing an effective strategy of reduction of the energy expenditure [ 34 ]. A validation of this hypothesis could be obtained in a rehabilitative context, by studying databases containing data of samples of different populations (i.e. healthy subjects versus subjects with a specific motor functional limitation). The rules relating the low maximum SA power during BSO to variables occurring during ASO allowed for BSO-ASO crossed inferences. When medium-to-high maximum LA force during the elevation of the centre of mass toward the standing position was found, a low maximum SA power was generated by the SA before seat-off ( MaxLAForceASO 4_6 → MaxSAPowerBSO 1_6 ). Moreover, consistent with the relationship to the SA velocity at seat-off, low maximum power of the SA during BSO occurred in combination with low-to-medium maximum velocity values during ASO ( MaxSAVelASO 2_6 → MaxSAPowerBSO 1_6 ) showing that after seat-off a low-to-medium SA velocity can be reached and kept during the remaining part of the task, even when a low power is exerted before seat-off. Finally, average timing of occurrence of maximum SA power after seat-off implied a low maximum SA power before seat-off ( TMaxSAPowerASO 3_5 → MaxSAPowerBSO 1_6 ) showing that, when the task is performed with an 'average' distribution of the time instants, the power exerted before seat-off is at its lowest values. The results' representation of Figure 4 could be used as the main outcome of the knowledge discovery process to be used by the analyst as a reference for the examined population. In the case of the present study, the patterns found are representative of the most common characteristics of the way healthy adults, of both genders and in a wide age range, perform the sit-to-stand task. Any deviation from these patterns found in a healthy adult could be considered as an uncommon characteristic. The patterns resulting from the analysis of a database containing a subgroup of the subjects examined in the present study (i.e. female subjects or subjects over the age of 65) could be considered as specific of the selected subgroup. Similarly, if the analysis is applied to a database of subjects affected by a specific pathology then the resulting patterns would characterise that population of subjects. The comparison of those patterns and the patterns found in the present study would highlight how differently the two groups perform the task. In perspective, from a rehabilitation standpoint, the output of data mining analyses applied to various groups of subjects performing various tasks could be used as a reference tool to evaluate the performance of subject under examination and, therefore, her/his level of mobility. Conclusions The study focused on finding the most frequent patterns of biomechanical variables and parameters obtained from dynamometric measurements of healthy subjects performing the sit-to-stand motor task. Data collected in a large database underwent a knowledge discovery process. The size of the database is strongly related to the simplicity of the data acquisition procedures. Simple and less expensive experimental set-ups allow the gathering of more data and in more locations than high-cost experimental set-ups and procedures. Data acquired from force platforms, processed with specific biomechanical models, represent a favourable condition to apply knowledge discovery processes effectively. In this study, data from volunteers in a large age range and of both genders were analysed in order to extract the most common patterns of healthy people performing the task. The results of the knowledge discovery process showed that sit-to-stand time events were strongly interdependent. Low maximum sagittal power values before seat-off were strongly related to numerous parameters both before and after seat-off, highlighting, among other characteristics, that most often a low power before seat-off is related to a regular occurrence of time instants and to low-to-medium sagittal speed from seat-off to the end of the task. The patterns found may be considered as typical rules of the sit-to-stand motor task and could constitute the basis for comparisons of patterns characteristic of different groups. The knowledge acquired in this study is the first step in the direction of developing a robust clinical tool to evaluate subject mobility.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC546397.xml
535351	Functional interdependence between septin and actin cytoskeleton	Background Septin2 is a member of a highly conserved GTPase family found in fungi and animals. Septins have been implicated in a diversity of cellular processes including cytokinesis, formation of diffusion barriers and vesicle trafficking. Septin2 partially co-localises with actin bundles in mammalian interphase cells and Septin2-filamentmorphology depends upon an intact actin cytoskeleton. How this interaction is regulated is not known. Moreover, evidence that Septin2 is remodelled or redistributed in response to other changes in actin organisation is lacking. Results Septin2 filaments are associated with actin fibres, but Septin2 is not associated with actin at the leading edge of moving cells or in ruffles where actin is highly dynamic. Rather, Septin2 is spatially segregated from these active areas and forms O- and C-shaped structures, similar to those previously observed after latrunculin treatment. FRAP experiments showed that all assemblies formed by Septin2 are highly dynamic with a constant exchange of Septin2 in and out of these structures, and that this property is independent of actin. A combination of RNAi experiments and expression of truncated forms of Septin2 showed that Septin2 plays a significant role in stabilising or maintaining actin bundles. Conclusion We show that Septin2 can form dynamic structures with differing morphologies in living cells, and that these morphologies are dependent on the functional state of the actin cytoskeleton. Our data provide a link between the different morphological states of Septin2 and functions of Septin2 in actin-dynamics, and are consistent with the model proposed by Kinoshita and colleagues, that Septin2 filaments play a role in stabilisation of actin stress fibres thus preventing actin turnover.	Background Septins are a conserved family of GTPases implicated in various cellular processes. Septin-requiring processes include cytokinesis, polarity establishment, cell cycle checkpoints and formation of a diffusion barrier in yeast [ 1 ], as well as cytokinesis, vesicle trafficking and exocytosis in mammalian cells [ 2 - 4 ]. In humans, 12 septin genes have been found so far, many of which also undergo alternative splicing generating dozens of polypeptides [ 5 ]. Septins can be isolated from cytosol as hetero-polymeric complexes, which have the ability to polymerize and assemble into higher-order structures in vitro [ 6 , 7 ]. How the polymerisation is regulated and how such higher order assemblies contribute to septin function in vivo is far from clear. Septin2 (formerly known as Nedd5) is the best-characterised member of the septin family so far. It is ubiquitously expressed and belongs to the acidic subgroup of the septin family consisting of a short N-terminus, a conserved GTPase domain and a coiled coil structure at the C-terminus [ 8 ]. Septin2 forms a complex together with Septin6 and Septin7 in vitro [ 9 ] and also co-localises with these septins in vivo [ 10 ]. Kinoshita and colleagues showed that Septin2 is required for cytokinesis [ 11 ]. Microinjection of an anti-Septin2 antibody interfered with cell division resulting in bi-nucleated cells. How Septin2 functions during cytokinesis is unclear. However, its localisation to the contractile ring and midbody structure in the cleavage furrow during late stages of mitosis is consistent with a functional role of Septin2 in limiting diffusion of membrane proteins across the cleavage furrow [ 12 , 13 ]. In interphase cells Septin2 co-localises with actin bundles, and disruption of the actin cytoskeleton by latrunculin or cytochalasin perturbs Septin2 distribution, inducing curved or circular Septin2-containing assemblies [ 9 , 14 ]. Reduction in Septin2 expression level in cells results in attenuation of actin fibres, implying a functional inter-relationship between actin and Septin2. An in vitro bundling assay showed that the interaction between actin bundles and recombinant Septin2/Septin6/Septin7 can be mediated by the bundling protein anillin [ 4 ]. Although these in vitro results suggest a mechanism to account for the recruitment of Septin2 to the actin contractile ring during cytokinesis, it is unlikely that anillin has the same function in interphase cells. Anillin is sequestered in the nucleus in interphase cells [ 15 ] and the functional significance of the co-localisation between actin and Septin2 in this phase of the cell cycle is still elusive. More generally, although artificial perturbation of actin cytoskeleton can cause re-arrangement of Septin2 in cells it is not clear that this phenomenon is ever replicated during normal cell function. Here we characterise the mutual inter-dependence of the actin cytoskeleton and Septin2 using a range of in vivo approaches. Both knock down of Septin2 expression and a specific Septin2 truncation mutant resulted in loss of visible actin fibres or bundles. Expression of dominant-negative mutants of the actin-regulating Rho-GTPases RhoA, Rac1 and CDC42 caused re-organisation of both cortical actin and Septin2. Significantly, in ruffling and migrating cells we observed wholesale redistribution of Septin2 into ring-like structures with morphology and dynamics highly similar to those previously observed upon depolymerisation of actin filaments with latrunculin. We propose that Septin2 is required for actin bundling, and that global re-organisation of the actin cytoskeleton in migrating or ruffling cells triggers concomitant re-organisation of Septin2 into a distinct functional state. Results Septin2 and actin define interdependent systems We sought to understand the functional relationship between actin cytoskeleton and Septin2 in vivo . As a first step, indirect immunofluorescence was used to characterise the distribution of these molecules in NRK cells. In interphase cells Septin2 had a filamentous and granular appearance, and co-localised with actin bundles [ 9 ]. The interaction with actin bundles, however, was not uniform along the entire length of the actin; Septin2 rather partially decorated the bundle [ 11 ]. Septin2 did not co-localise with vinculin, a marker for focal adhesions (Fig. 1B ). As previously shown in 3T3 cells [ 11 ], disruption of actin filaments upon Latrunculin B treatment resulted in a striking loss of linear Septin2 staining and rearrangement of Septin2 into O- and C-shaped rings (Fig. 1C ). These Septin2 rings were sometimes found together with actin-containing particles, but ring-like structures devoid of actin were also visible (arrows in 1C). Thus we could observe the dependence of Septin2 morphology on actin polymerisation described previously [ 9 ]. This dependence suggested that septin- and actin-cytoskeletal networks could be functionally inter-dependent. Some members of the septin family also interact with microtubules [ 16 ]. For Septin2, however, we were not able to establish such an association (Fig. 1D ). In regions of the cell where Septin2 co-localised with tubulin was always actin present as well (Fig. 1E ). Nocodazole treatment, which disrupts microtubules, slightly attenuated Septin2 filaments, but did not have a big effect on overall Septin2 organisation (Fig. 1F ). Figure 1 Septin2 interacts with actin, but not with tubulin. (A) Immunofluorescence of endogenous Septin2 (red) and actin (green, stained with phallacidin) in interphase NRK cells. Note the typical filamentous-granular organisation of Septin2 and its co-localisation with actin. (B) Septin2 (red) does not colocalise with endogenous vinculin (green), a marker for focal adhesions. (C) Staining of Septin2 (red) and actin (green) after disruption of the actin cytoskeleton. NRK cells were treated with Latrunculin B for 30 min. Septin2 now forms rings. (D) Septin2 (red) and tubulin (green) mainly have distinct distributions in NRK cells. (E) Enlarged image of the boxed region in D. Regions with overlapping Septin2 (red) and tubulin (blue) staining always contain actin (green) as well (arrow). (F) Cells were treated with nocodazole for 30 min. Microtubule organisation (green) is disturbed, but Septin2 (red) distribution is largely unaffected. All images show a single confocal section. Bars, 10 μm. In order to test directly the requirement for Septin2 function in maintaining the actin cytoskeleton, we performed RNAi to knock down Septin2 expression in cells. NRK and Hela cells were transfected separately with two different siRNAs based on the Septin2 sequence. Both siRNAs produced equivalent effects, and control siRNAs produced none of the effects described below. 48 hrs after transfection cell morphology was severely compromised and the overall amount of cells was reduced to 43 ± 11.7% of control levels. 70–90% of the remaining cells showed a decreased Septin2 staining by immunofluorescence. There was no indication of selective arrest of the cell cycle at mitosis or cytokinesis, implying that Septin2 is required for correct cell morphology and adhesion or viability during interphase. Western blot analysis of whole cell homogenate revealed a 60% reduction of Septin2 level (expressed as a percentage of the Endoplasmic Reticulum Protein Erp72, so as to control for reduced cell number; Fig 2C ). The most interesting observation was the effect on the actin cytoskeleton. Overall actin level (% of Erp72) was reduced to 45% of control levels (Fig. 2C ), and in cells where Septin2 staining was diminished actin staining was markedly reduced as well (Fig. 2A ). In fact, actin bundles were no longer present in these cells and the weak residual actin staining was restricted to the cell periphery. In contrast, microtubule organisation was not affected upon knocking down Septin2 levels by siRNA (Fig. 2B ) and the amount of tubulin present in cells was unchanged (Fig. 2C ). Figure 2 Knock down of Septin2 expression abolishes actin organisation. NRK cells were transfected with siRNA targeting Septin2. (A) Immunofluorescence 48 hrs after transfection. Cells with reduced level of Septin2 (red) have no actin bundles (red) (0). (B) Tubulin distribution (green) is unaffected in cells showing a decreased Septin2 staining (red) (0). (C) 48 hrs after transfection with siRNA NRK cells were lysed and immunoblotted for Septin2, actin, tubulin and Erp72. The amounts of Septin2, actin and tubulin were quantified and normalized to the corresponding amount of Erp72 to account for reduction in cell number upon siRNA treatment. co, control; Erp72, Endoplasmic Reticulum Protein, member of the protein disulfide isomerase family. Similar results were obtained with Hela cells. A single confocal section is shown. Bars, 20 μm. The loss of actin fibres in cells with reduced Septin2 expression could either be a consequence of reduced actin protein levels or could reflect a more direct role for Septin2 function in maintaining actin fibres. Given that Septin2 forms complexes with other septins and, potentially, further proteins, we reasoned that in the latter case over-expression of Septin2 might be sufficient to sequester or perturb factors required for actin bundling. Over-expression of full length YFP-Septin2 (YFP-Sept2-PB/G/CC) resulted in the formation of large polymeric structures (Fig. 3B ). Untagged full length Septin2 also formed those structures when over-expressed, indicating that their formation was not dependent on the presence of YFP in the fusion protein (Fig. 3C ). Induction of these anomalous structures correlated directly with a marked decrease in actin bundles (Fig. 3C ). In order to ascertain which region of Septin2 is required for this effect various truncated YFP-Septin2 constructs were produced, containing different domains of the protein [ 17 ]. NRK cells transfected with YFP-Septin2-PB/G, a construct containing the N-terminus and the GTPase domain but lacking the coiled-coil domain (Fig. 3A ), showed a more or less punctate distribution (Fig. 3D ). In comparison to full length YFP-Septin2, no filaments aligned with actin stress fibres were visible, but actin organisation was not affected by over-expression of this construct. In marked contrast, a construct containing only the N-terminal polybasic domain (YFP-Septin2-PB) induced loss of actin bundles, and an apparent reduction in total actin staining to approximately 70% of control levels. YFP-Septin2-PB localized as a cytosolic haze. Staining with Septin2 antibodies revealed that endogenous Septin2 has a normal distribution in YFP-Septin2-PB-expressing cells. A third construct that lacks just the polybasic domain (YFP-Septin2-G/CC) did not affect actin bundles and had a more punctate distribution. Constructs fused to a myc-tag or untagged truncated versions of Septin2 showed the same distributions. Taken together, these experiments show that over-expression of Septin2 is sufficient to ablate actin bundles. However, the large polymeric structures induced by Septin2 over-expression are not themselves required for this effect as a construct containing only the polybasic region of Septin2 is sufficient to induce the same effect on actin bundles whilst having a diffuse cytoplasmic localisation. The finding that the polybasic region plus GTPase domain construct did not have the same effects hints at a regulatory role for the GTPase domain. Figure 3 Characterisation of truncated YFP-Septin2 constructs. (A) Schematic presentation of full length YFP-Septin2 (YFP-Sept2-PB/G/CC) and truncated YFP-Septin2 constructs. (B) In some cells overexpression of full length YFP-Sept2-PB/G/CC induced the formation of higher-ordered YFP-Septin2 containing-structures with differing morphology (C) Untagged full length Sept2-PB/G/CC is also capable of forming these structures and actin filaments are attenuated in those cells. Full length Sept2-PB/G/CC was detected with anti-Septin2-antibody. (D) Distribution of YFP-Septin2 constructs in NRK cells and their effect on the actin cytoskeleton. Cells were transfected with either full length YFP-Sept2-PB/G/CC or the truncated YFP-Septin2 constructs and stained for actin with phallacidin. Only YFP-Sept2-PB, the construct lacking the GTPase domain and the coiled coil domain affects actin organisation. The distributions of YFP-Sept2-PB/G, the construct without the coiled-coil domain, and YFP-Sept2-G/CC, which lacks the polybasic region, are different to full length YFP-Sept2-PB/G/CC, but there is no effect on actin. Images show a single confocal section. Bars. 10 μm. Another way of determining the extent of interdependence between actin and Septin2 is to perturb actin organisation by interfering with regulators of actin organization. Rho GTPases are such regulators and have diverse effects on the actin filament system [ 18 , 19 ]. RhoA, Rac1 and Cdc42 are the best-characterised family members, and each controls the formation of a distinct actin-containing structure. We studied the effects of the GDP-locked form of RhoA, Rac1 and Cdc 42 on Septin2 distribution. NRK cells were transfected with myc-tagged dominant-negative mutants of these GTPases and cells were stained for actin and Septin2. Although all mutants altered the appearance of the actin cytoskeleton and also affected Septin2 organisation, their effects on these two systems were quite diverse (Fig. 4 ). In cells expressing the GDP-locked form of Rac1 central actin bundles were attenuated or missing and the formation of thick actin bundles in the cell periphery was induced (Fig 4A ). Septin2 was no longer associated with these bundles. The filamentous organisation of Septin2 was fragmented resulting in the formation of rings similar to those seen after latrunculin treatment (inset in 4A). The Cdc42-mutant, although increasing the prominence of actin bundles, had only a slight effect on Septin2. Septin2 still exhibited the filamentous-granular appearance but with more pronounced filaments (Fig. 4B ). Cells transfected with the dominant-negative mutant of RhoA showed the most severe phenotype. The majority of the cells lost their ability to attach to the surface of the coverslip, which is explained by RhoA having a role in regulation the formation of stress fibres and focal adhesions, necessary prerequisites for proper cell attachment [ 20 ]. RhoA-mutant expressing cells that were still left on the coverslip had a disrupted actin and Septin2 organisation, with a reduction in both actin filaments and septin filaments (Fig. 4C ). All these experiments again clearly show the mutual dependence and regulation of septin and actin filaments. Figure 4 Effects of Rho-GTPases on actin and Septin2 organisation. NRK cells were transfected with GDP-locked forms of myc-Rac1 (A), myc-CDC42 (B) or myc-RhoA (C) and stained for Septin2 and actin. GTPases were detected with a monoclonal anti myc-antibody. (A) Cells expressing the myc-Rac1 mutant lack actin bundles and Septin2 organisation is disrupted. Instead of filaments, Septin2 forms rings (inset, inset was taken from another cell transfected with Rac1-mutant). (B) myc-CDC42-GDP induces the formation of thick actin bundles but has only little effect on Septin2 appearance. (C) Transfection with dominant-negativ myc-RhoA causes the detachment of most of the cells. Cells left on the coverslip have diminished actin filaments and disrupted Septin2 organisation. Images are single confocal sections. Bars, 10 μm. Septin2 and actin have distinct distributions in moving and ruffling cells Both actin depolymerisation and expression of Rho-family-GTPase mutants are non-physiological perturbations and functional significance of resultant changes in Septin2 distribution was unclear. We therefore sought a more physiological situation where parallel re-organisation of actin and Septin2 could be assayed. In NRK cells migrating into an experimentally wounded monolayer actin is concentrated in the leading-edge lamellipodia and ruffles characteristic of many moving cells (Fig. 5A ) [ 21 ]. Septin2, however, was not detectable in these ruffling lamellipodia at the leading edge. It was segregated from actin and was localised in the cell body (Fig. 5A ). The appearance of Septin2 in the cell body of ruffling cells was distinct to normal cells and very variable. Instead of filaments, Septin2 formed arc-shaped structures, O-and C-shaped rings and circles with diameters ranging from 0.6–1.4 μ (Fig. 5B , arrows), and punctae. The ring-like structures were not at all associated with actin, but were similar to rings observed after disrupting the actin cytoskeleton upon latrunculin treatment (Fig. 1C ). Lamellipodial extensions and ruffling can readily be induced in NRK cells by growing on lysine-coated coverslips (Fig. 5C ). As one would predict, actin and Rac1, a GTPase known to be involved in formation of lamellipodia, are readily detectable in the ruffles [ 22 ]. Septin2 was exclusively localized in the cell body, and was found in O-and C-shaped rings and circles as in migrating cells (Fig. 5D,5E ). These results imply that although Septin2 is associated with actin bundles and plays an important role in regulating these structures, it is not associated with actin in situations where actin organisation is more dynamic. Septin2 that does not interact with actin forms variable curled and ring-like structures morphologically highly similar to those induced by actin depolymerisation. Figure 5 Septin2 is not associated with actin in moving or ruffling cells. (A) DIC image of moving NRK cells in an experimentally wounded monolayer. Note the leading edges of the cells contain actin (green), but Septin2 (red) is completely missing. (B) Septin2 forms ring-like structures in the body of moving cells comparable to rings observed after latrunculin treatment (compare Fig. 1C). (C) In ruffling cells growing on lysine-coated coverslips Septin2 (red) is detectable in the cell body, whereas actin (green) is concentrated in ruffles. (D) Ruffles are also positive for endogenous Rac1 (green), but Septin2 (red) is clearly missing. (E) In the cell body of ruffling cells Septin2 (red) forms O- and C-shaped ring-like structures, which are not associated with actin (green, stained with phallacidin). Bars, 5 μm. The filaments and rings formed by Septin2 in vivo are highly dynamic To gain more insight into Septin2 dynamics we performed fluorescence recovery after photobleaching (FRAP). In a FRAP experiment fluorescent molecules are irreversibly bleached in a small area of the cell (= region of interest, ROI). Subsequent diffusion of surrounding non-bleached molecules into the bleached area leads to recovery and is monitored over time. We used full length YFP-Septin2 for bleaching experiments and investigated the dynamics of GFP-actin in parallel (Clontech). YFP-Septin2 had the same distribution as endogenous Septin2 in interphase and dividing cells and it did not alter the organisation of endogenous Septin2 [ 12 ]. First we checked whether Septin2 filaments, which are associated with actin stress fibres in normal NRK cells, are dynamic structures. Photobleaching of a part of the filament showed a recovery of fluorescence over time (Fig. 6B ). There was no difference in the dynamics of recovery in the middle or at the end of the Septin2 filament. Although maximal recovery was only about 63% ± 18.74 of the initial fluorescence, the original structure of the Septin2 filament was clearly reformed. Figure 6 Septin2 forms highly dynamic filaments and rings. (A) YFP-Septin2 has the same distribution like endogenous Septin2 and it does not alter the organisation of endogenous Septin2. Bar, 20 μm (B) Photobleach experiment. NRK cells were transfected with either YFP-Septin2 or GFP-actin. Part of the filaments was bleached and recovery monitored over time. The ratio between mean fluorescence intensity of the prebleached box and the mean fluorescence of the whole cell was normalised to the prebleach ratio and expressed as a function of time (Materials and Methods). The graph shows representative recovery curves, which were fitted to a single exponential curve (solid lines) to calculate t D s and amount of recovery. n = 5. Images show a single confocal section of YFP-Septin2 filaments in NRK cells. Bar, 4 μm. (C) Images of a time-laps movie of YFP-Septin2 in the cell body of ruffling NRK-cells. Arrows indicate the formation cycle of a ring. Bar, 2 μm. (D,E) Photobleach experiments of YFP-Septin2 rings (also see additional file 1 for lysine/ruffling cells and additional file 2 for latrunculin). NRK cells growing on lysine-coated coverslips to induce ruffles were transfected with YFP-Septin2. Rings formed in the cell body were bleached and recovery monitored over time. For bleaching of Septin2 rings upon latrunculin treatment, normal NRK cells were transfected with YFP-Septin2 and treated with Latrunculin B for 20–25 Min before photobleaching. The data were analysed as in B to calculate t D s of recovery (D) and amount of recovery (E). n = 5. Images show a single confocal section of YFP-Septin2 rings in ruffling NRK cells. (F) Quantitative comparison of outer diameters of Septin2 rings in ruffling cells (lysine, empty circles) and upon latrunculin treatment (filled circles). Measurements were done on images of cells transfected with YFP-Septin2. Next we looked at the dynamics of the Septin2 positive ring-like structures formed in the cell body of ruffling cells. Time-lapse movies of these cells revealed a very active behaviour of Septin2 and a relationship between Septin2 filaments and rings. For example, as shown in Fig. 6C starting with an S-shaped filament half of a ring was formed. After 110 s an O-shaped structure with a diameter of 1.45 μm was seen which then further condensed into a smaller ring (Fig 6C , 190 s). The diameter of this ring was 0.7 μm and the morphology was comparable to rings observed after latrunculin treatment (Fig. 1C ) and perturbation of actin/Septin2 organisation by Rac1-mutant (Fig. 4A ). Instead of remaining in this structure the ring opened again and formed two half rings which were 1.6 μm apart from each other (Fig. 6C , 250 s). The two halves then came together again, this time forming a C-shaped structure (Fig. 6C , 440 s). Many examples of this cycle of opening and closing were observed and sometimes a disappearing and appearing of a ring-like structure was also detected. FRAP experiments of these ring structures in ruffling cells (grown on lysine-coated coverslips) confirmed that constant exchange of YFP-Septin2 into and out of these structures occurs. Halftimes for recovery (ranging from 10 s to 73.8 s) and amount of recovery (45%-86%), however, were very variable (Fig. 6D,6E . Additional files 1 and 2 ). Sometimes the recovered structure was distinct to the initially bleached structure in terms of position and morphology (see also Additional File 2 ). Next we investigated whether Septin2 rings induced by latrunculin have equivalent properties to those seen in ruffling or migrating cells. Septin2 rings formed upon latrunculin treatment also recovered after photobleaching, with comparable dynamics (half-time for recovery was 8.5 s ± 4.5, total amount of recovery 58.3% ± 3.6; Fig. 6D,6E , movie3). Careful morphological comparison of ring structures in ruffling cells and in latrunculin treated cells again revealed considerable similarity. Rings formed upon latrunculin treatment had a slightly more uniform outer diameter (0.74 ± 0.14 μm) but were similar in size to ring-like structures in ruffling cells (0.5 μm-1.4 μm; Fig. 6F ). In summary, Septin2 can participate in large-scale macromolecular assemblies with differing morphologies in response to differing physiological situations and remodelling of the actin cytoskeleton. In all instances these assemblies are highly dynamic with constant exchange of Septin2 in and out. Discussion Previous experiments have shown that Septin2 partially co-localises with actin fibres, that actin can affect Septin2 polymerisation in vitro , that depletion of Septin2 perturbs the morphology of actin bundles, and that depolymerisation of actin fibres can cause changes in Septin2 morphology. In this study we provide additional direct evidence for the in vivo significance of actin and Septin2 interaction. Thus we show for the first time that Septin2 expression is required for maintenance of normal actin protein levels, that over-expression of a truncated version of Septin2 causes loss of visible actin bundling without perturbing the distribution of endogenous Septin2, and that the circular and ring-shaped Septin2 structures induced by actin de-polymerisation are also found in physiological situations where the actin cytoskeleton is radically remodeled. Partial co-localisation between Septin2 and the tubulin network has also been reported [ 10 ]. Our immunofluorescence data, however, argue, that the interaction between Septin2 and microtubules is different from the Septin2-actin interplay and is not crucial for microtubule integrity. Although Septin2 distribution is slightly affected upon nocodazole treatment, this effect is less severe and not comparable with the disruption of Septin2 organisation upon latrunculin treatment (Fig. 1 ). Moreover, neither knock-down of Septin2 expression upon siRNA nor overexpression of truncated Septin2 constructs affected microtubule organisation (Fig. 2 , data not shown). Since in regions of the cell where Septin2 co-localises with tubulin we always find actin as well, we do not think that there is a direct interaction between Septin2 and tubulin, as has been shown for other septins [ 16 , 23 ]. The distinct distribution of Septin9, which is associated with tubulin and is clearly affected upon nocodazole treatment, and Septin2 in dividing cells also suggests that the co-localisation seen between Septin2 and tubulin is not functionally significant [ 16 ]. Septin2 and actin distributions, however, are clearly highly interdependent. Overexpression of GDP-locked form of RhoA, Cdc42 and Rac1 highlights this interplay (Fig. 4 ). At this point we do not know how these GTPases act in this context, whether it is via modulating actin dynamics and/or controlling Septin2 dynamics. It is known that the Cdc42 effectors Borg1, 2 and 3 can bind to Septins in vitro and in vivo [ 7 , 10 ]. Endogenous Septin6 and Septin7, which form a complex with Septin2, can be immunoprecipitated by an anti-Borg3 antibody and expression of Borg interferes with normal septin distribution. Full-length myc-Borg3 induces the formation of long and thick septin fibres and Cdc42 negatively regulates this effect by inhibiting the binding of Borg3 to septins. These data suggest that the formation of thick Septin2 filaments and actin filaments we see upon expression of the dominant-negative Cdc42 mutant is controlled by a regulatory mechanism, which modulates Septin2 function directly and not only via regulating actin organisation. In our experiments comparing truncated versions of Septin2 in cells we could show that the GTPase domain of Septin2 is sufficient to prevent the loss of actin bundles induced by expression of the polybasic region alone. The exact role of septin GTPase activity is still unclear [ 17 ]. Using recombinant septins, Sheffield and colleagues [ 7 ] demonstrated that although pre-assembled Septin6/Septin7/Septin2-filaments show only a slow GTPase activity, GTP-hydrolysis occurs during formation of heterodimers, a process before the assembling of filamentous complexes. Microinjection of non-hydrolyzable GTP (GTPγS) in cells disrupted fibrous distribution of Septin2 suggesting that the fibrous distribution of Septin2 requires GTP-hydrolysis [ 11 ]. In contrast, there is no evidence that GTPase-activity is necessary for assembly of septin filaments in curved bundles and ring-like structures in vitro [ 9 ]. So far we do not know how Septin2 interacts with actin bundling proteins and how it gets recruited to actin filaments in interphase cells. We do not know whether the reduction in actin expression levels induced by septin2 RNAi is a cause or an effect of the observed loss of actin bundles. Identification of binding partners for Septin2 will be necessary to elucidate the mechanisms underlying the property of Septin2 to stabilise actin bundles. It has been shown in vitro that Septin2 is capable of forming ring-like structures and spirals in an actin-independent fashion [ 9 ]. Here, we provide in vivo characterisation of this intrinsic property of Septin2. Moving cells and ruffling cells are the first cell systems described so far where the actin-independent distribution of Septin2 in O- and C-shaped rings has been studied in a physiological context, without interfering with cell viability and function. These model systems allow us to draw two firm conclusions: 1. That Septin2 is not associated with actin in regions where highly dynamic actin is not organised in fibres and is being constantly remodeled, and indeed Septin2 is actually efficiently excluded from these regions (e.g. at the leading edge of moving cells and in ruffles): 2. That Septin2 when not associated with actin forms rings and ring-like structures instead of filaments (Figs 5 , 6 ). This is in agreement with in vitro data obtained from recombinant septin complexes showing their tendency to self-assemble into rings and spirals [ 9 ]. In contrast, however, to these in vitro structures, which are highly stable, the in vivo assemblies are highly dynamic (movies 1–3). Bleaching experiments clearly showed the constant exchange of Septin2 in and out of the assemblies as well as in and out of actin-dependent Septin2 filaments. The function of actin-independent Septin2 assemblies, however, is still elusive. Since they seem to be freely localised in the cytosol and are not associated with membrane structures (unpublished observations), they might represent storage containers of Septin2. The dynamic behaviour of Septin2 explains how cells can adjust Septin2 function to different needs. The identification of further proteins involved in this process (e.g. GAPs, GEFs, bundling proteins) is necessary for molecular characterisation of the interplay between Septin2 and actin. Conclusions Our data provide a link between the different morphological states of Septin2 and functions of Septin2 in actin-dynamics, and confirm the physiological relevance of the model proposed by Kinoshita and colleagues [ 9 ], that Septin2 filaments play a role in stabilisation of actin stress fibres thus preventing actin turnover. Methods Antibodies and constructs Anti-Septin2 polyclonal antibody was a gift from M. Kinoshita. Anti-human vinculin monoclonal antibody (clone hVIN-1), anti-c-myc monoclonal antibody (clone 9E10) and anti-α-Tubulin monoclonal antibody (clone DM1a) were all from Sigma. Anti-beta actin monoclonal antibody (AC-15) for immunoblotting was from abcam, BODIPY ® FL phallacidin and Alexa Fluor ® 568 phalloidin for immunofluoresence were obtained from Molecular Probes. Anti-Endoplasmic Reticulum Protein 72 (Anti-Erp72) was from Calbiochem. Myc-constructs of GDP-locked RhoA, Rac1 and CDC42 and the anti-rac monoclonal antibody were kindly provided by H. Mellor. pEGFP-actin was obtained from Clontech. Secondary antibodies used were as follows: Donkey Anti-Rabbit IgG Cy™3 conjugated (Jackson Immuno Research Lab.), Donkey Anti-Mouse IgG Cy™5 conjugated (Jackson Immuno Research Lab.) and Alexa Fluor ® 488 goat anti-mouse IgG 1 (Molecular Probes) for immunofluorescence. IRDye 800 donkey anti-rabbit IgG (Rockland) and Alexa Fluor ® 680 goat anti-mouse IgG (Molecular Probes) for immunoblotting. Cloning of YFP-Septin2-constructs To generate full length and truncated YFP-Septin2-constructs, PCRs of Septin2 image clone (Clone Id: 548005, from HGMP, Hinxton/UK) were performed using the following primers: 5'-GCGCTCGAGTGTCTAAGCAACAGCCAAC (sense) and 5'-ATCCCGG GTTACACGTGGTGCCCGAGAGC (antisense) for full length YFP-Septin2-PB/G/CC (nucleotides 1–1081); 5'-GCGCTCGAGTGTCTAAGCAACAGCCAAC (sense) and 5'-CGCCCCGGGTTAGCCTCTCTTGAGTCTCTC (antisense) for YFP-Septin2-PB/G (nucleotides 1–922); 5'-GCGCTCGAGTGTCTAA GCAACAGCCAAC (sense) and 5'-CGCCCCGGGTTACACCATCAGTGTGAACTC (antisense) for YFP-Septin2-PB (nucleotides 1–127); 5'-GCGCTCGAGAGTTCACACTGATGGTGGT (sense) and 5'-ATCCCGGGTTACACGTGGTGCCCGAGAGC (antisense) for YFP-Septin2-G/CC (nucleotides 113–1081). The fragments were cloned in pEYFP-C1 digested with Xho1 and Xma1. To generate untagged full length Septin2 5'-GCGCTCGAGATGTCTAAGCAACA GCCAAC (sense) and 5'-ATCCCGG GTTACACGTGGTGCCCGAGAGC (antisense) were used in the PCR reaction. The fragment was cloned in SNAG4M cut with Xho1 and Xma1. All clones were confirmed by DNA sequencing. Cell culture, drug treatment, plasmid transfections, immunofluorescence NRK cells were grown using standard techniques in DMEM, 10% FCS, 1% Penicillin at 10%CO 2 . To induce the formation of ruffles in NRK cells coverslips were coated with 0.01% poly-D-lysine for 5 min, washed with PBS and air dried before cells were seeded. A wound assay was used to investigate distribution of Septin2 in moving cells. Briefly, NRK cells were grown until confluency and a wound was scratched in the monolayer using a tip. Two hours later cells were fixed and processed for immunofluorescence. To disrupt the actin cytoskeleton or microtubules cells were incubated for 30 min in DMEM containing 150 nM Latrunculin B (Molecular Probes) or 10 μM nocodazole (Sigma), respectively. Plasmid transfections were carried out with Fugene6 (Roche). For immunofluorescence cells were fixed with 2% Formaldehyd in either PBS or microtubule-stabilisation buffer (0.1 M Pipes, pH 6.9, 2 mM EGTA, 2 mM MgCl 2 , 4% PEG 8000) for 20 min, permeabilised with 0.2% saponin/10%FCS in either PBS or microtubule-stabilisation buffer for 10 min and subsequently incubated with primary and secondary antibodies. RNA interference (RNAi) with small interfering RNA (siRNA) and immunoblotting Two siRNAs with the following sequences: 5'-AAAGGACATGAATAAAGACCA (sense), and 5'-AAGTGAATATTGTGCCTGTCA (sense) were chosen to target nucleotides 939–957 and 521–539 of Septin2, respectively, and were supplied by Dharmacon Research Inc. siRNAs were annealed to make siRNA duplexes according to the manufacture's protocol and transfections were carried out using Oligofectamine (Invitrogen). To check for specificity of knock down we used siRNA duplexes targeting caveolin 1 [ 24 ] and rhomboid (gift from A. McQuibban). 48 hrs after transfection cells were processed for immunofluorescence or immunoblotting. For immunoblotting cells were washed with PBS, scratched of in sample buffer (2% SDS, 80 mM Tris/HCl pH 6.8, 10% Glycerin, 0.01% bromphenolblue, 5% β-Mercaptoethanol) and sonicated. The samples were boiled at 80°C for 3 min, centrifuged and the supernatant was loaded onto the gel followed by Western blotting. Immunoreactive bands were detected by the Infrared Imaging System Odyssey (Li-Cor Biosciences). Microscopy and photobleaching All images and movies were obtained using BioRad Radiance and Zeiss LSM510 confocal microscopes equipped with standard filter sets and laser lines for the detection of YFP, Cy2, Alexa Fluor 488, BODIPY, Cy3 and Cy5. Live cell microscopy was carried out at 35°C in imaging medium (DMEM without phenol red, 10%FCS, 50 mM HEPES pH 7.2). Photobleaching experiments were performed with the confocal zoom set to 3 and the confocal pinhole set to 2–4 Airy units. Bleaching of actin or Septin2 filaments was carried out with a 40X 1.3 NA objective lens. A box of interest was bleached using 35 scans with the 488 and 514 laser line at full laser power. For photobleaching of ring structures a 63X 1.4 objective lens was used and a ring of interest was bleached using 25 scans with the 514 laser line at full laser power. Pre- and post-bleach images were monitored at low laser intensity. Fluorescence recovery in the bleached region and the overall fluorescence in the whole cell during the time series were quantified using the Zeiss LSM software. After subtracting the background (= mean fluorescence intensity in the bleached region after bleach) the ratio between mean fluorescence intensity of the bleached region and the mean fluorescence of the whole cell was expressed as a percentage of the pre-bleach ratio of these values. These normalised data were fitted to a single exponential curve using the PRISM software (GraphPad Software Inc., San Diego) to derive amount of recovery and characteristic diffusion time t D , which indicates the time at which half of the fluorescence has recovered. List of abbreviations used GFP, green fluorescent protein; YFP, yellow fluorescent protein; siRNA, small interference RNA; RNAi, RNA interference; ERP72, endoplasmic reticulum protein 72; FRAP, fluorescence recovery after photobleaching; ROI, region of interest; GAP, GTPase activating protein; GEF, GTP exchange factor. Authors' contributions KS carried out all the experimental work and drafted the manuscript. BJN conceived of the study, participated in its design and edited the manuscript. Supplementary Material Additional File 1 Photobleaching of YFP-Septin2 ring-like structures formed in the cell body of ruffling cells. Ruffling NRK cells were transfected with YFP-Septin2. The YFP-Septin2 ring in the middle was photobleached and recovery monitored over time. Movie covers 200 s (also see Fig. 6D ). Click here for file Additional File 2 Photobleaching of YFP-Septin2 rings upon latrunculin treatment. NRK cells transfected with YFP-Septin2 were treated with Latrunculin B for 20–25 min before photobleaching. A ring in the upper right part of the cell was photobleached and recovery monitored over time. Note recovery seems to occur from a structure in the background. Movie covers 200 s. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC535351.xml
534098	PhyME: A probabilistic algorithm for finding motifs in sets of orthologous sequences	Background This paper addresses the problem of discovering transcription factor binding sites in heterogeneous sequence data, which includes regulatory sequences of one or more genes, as well as their orthologs in other species. Results We propose an algorithm that integrates two important aspects of a motif's significance – overrepresentation and cross-species conservation – into one probabilistic score. The algorithm allows the input orthologous sequences to be related by any user-specified phylogenetic tree. It is based on the Expectation-Maximization technique, and scales well with the number of species and the length of input sequences. We evaluate the algorithm on synthetic data, and also present results for data sets from yeast, fly, and human. Conclusions The results demonstrate that the new approach improves motif discovery by exploiting multiple species information.	Background The discovery of novel transcription factor binding sites in regulatory sequences of genes has been an important scientific challenge for some years now. Computational approaches to this problem have come in two flavors. One class of methods looks for overrepresented motifs in sequences that are believed to contain several binding sites for the same factor (such as promoters of co-regulated genes) [ 1 - 6 ]. The second class of methods identifies motifs that are significantly conserved in orthologous sequences, e.g., promoters of the same gene in different species [ 7 , 8 ]. These two approaches have been applied to their respective kinds of data sets, with moderate success. However, with new genomes being sequenced regularly, motif-finding applications today often present heterogeneous sequence data that includes promoters/enhancers of multiple co-regulated genes in one species, as well as their orthologs in other species. This paper presents a probabilistic algorithm, called "PhyME" ( Phy logenetic M otif E licitation), for ab-initio detection of binding site motifs in such heterogeneous sequences. PhyME integrates two different axes of information in evaluating a candidate motif's significance. One axis is that of overrepresentation , which depends on the number of occurrences of the motif in each species. The other axis is the level of conservation of each motif instance across the species. A real motif that is not sufficiently significant along any one axis may turn out to be significant when both axes are considered simultaneously, leading to increased sensitivity and specificity of the integrated approach. Given the regulatory regions of potentially co-regulated genes along with their orthologs from other species, PhyME uses an Expectation-Maximization (E-M) algorithm to search for the motif that best explains the data. When evaluating a motif, its orthologous occurrences are assumed related to each other by a probabilistic model of evolution that takes into account the varying phylogenetic distances among the species. (The species may be related by any user-specified phylogenetic tree.) Each E-M iteration scales linearly with the total length of the input sequences and also with the number of species. The algorithm can also handle cases where the heterogeneous data is incomplete , i.e., where the orthologous regulatory regions are missing from some species. This capability makes it particularly suitable for applications that include data from incomplete genomes, or where orthology information is incomplete. An important feature of PhyME is that it allows motifs to occur in (evolutionarily) conserved as well as unconserved regions in orthologous promoters, treating the two kinds of occurrences differently when scoring a motif. It does not require each binding site occurrence in one promoter to have an orthologous occurrence in any or all other species. As a result, PhyME affords some flexibility in terms of the evolutionary distances spanned by the input sequences. For instance, using a distantly related ortholog will help pinpoint motifs located in conserved regions but will not hamper the discovery of motifs absent from that ortholog. Comparison with previous work Traditionally, motif finding algorithms have treated input sequences as being independently generated, and searched for statistically overrepresented motifs in them. These algorithms [ 1 - 6 ] do not have the notion of sequence orthology built into them, and are therefore typically run on sequences from the same species. PhyME has an obvious advantage over them, since it takes motif conservation into account. (Henceforth, conservation of motifs will be assumed to mean conservation across species.) Another class of motif-finding methods take as input sets of orthologous sequences, either aligned [ 8 ] or unaligned [ 7 ] and search for well-conserved motifs. These methods however, unlike PhyME, do not exploit the other important aspect of a motif's significance – that of overrepresentation. Some algorithms [ 9 , 10 ] take as input a heterogeneous pool of co-regulated and orthologous promoters, and find overrepresented motifs after treating all sequences (including orthologous ones) as independent. However, this "homogenizing" strategy has its disadvantage, since it treats orthologous (and hence, directly related) motif occurrences as statistically independent observations. PhyME, on the other hand, respects the distinction between orthologous and co-regulated motif occurrences. There are algorithms that attempt to handle the two axes of information by a two-step approach. For instance, Cliften et al . [ 11 ] and Kellis et al . [ 12 ] find a set of highly conserved motifs (in yeast promoters) in the first step, and then extract overrepresented ones from this set, in a second step. The algorithm CompareProspector [ 13 ] takes a Gibbs-sampling approach to find overrepresented motifs but biases the search in regions conserved across species. Conversely, one may identify overrepresented motifs in the first step, and then isolate evolutionarily conserved ones among these [ 14 ]. In either case, a motif that is relatively weak by either criterion alone, but strong when considering both, may be missed out. PhyME's integrated approach to the heterogeneous data problem addresses this issue. Admittedly, the methods of Cliften et al . and Kellis et al . have a broader range of applications, since these are genome-wide searches for motifs. A recent algorithm called orthoMEME (Prakash et al . [ 15 ]) tackles the heterogeneous data problem by using Expectation-Maximization to search the space of motifs and the space of motif alignments (orthology) simultaneously. Each motif occurrence is assumed to have an orthologous copy in the other species, that could be located anywhere in the corresponding promoter. This is in contrast to PhyME's approach, where orthologous motif occurrences are restricted to pre-aligned regions of the promoters. This restriction comes with the advantage that PhyME scales better with the number of species than does orthoMEME. This is a significant advantage in practice, since the orthoMEME implementation is able to handle only two species data, whereas we have experimented with PhyME on orthologs from up to six species. Moreover, PhyME also allows non-conserved occurrences (those residing outside aligned regions), or occurrences that are conserved in some species and missing in others. Requiring that all motif occurrences come in orthologous sets may be justified for very closely related species, but for more diverged pairs of species (e.g., D. melanogaster and D. pseudoobscura ) the promoters are known to have a mix of conserved and unconserved binding sites [ 16 ]. PhyME therefore gains an advantage by looking at both kinds of occurrences. However, orthoMEME's phylogenetic model is more powerful than that of PhyME and can handle a greater range of motif variation than PhyME can. Our approach is most similar to the algorithm PhyloGibbs (Siddharthan et al . [ 17 ]), the main differences being that PhyloGibbs (i) uses a Gibbs-sampling approach and (ii) assumes a star topology for the phylogeny, whereas PhyME uses an E-M approach and can handle arbitrary tree topologies. Thus PhyME has a broader domain of applicability in terms of the phylogenetic relationships among input sequences. It may therefore be preferable over PhyloGibbs when the phylogeny is far removed from a star, e.g., in a scenario where a pair of close species is included along with another pair of closely-related species, but the two pairs are greatly diverged from each other. On the other hand, an advantage of using PhyloGibbs is that multiple motifs (for different transcription factors) may be searched in parallel. The algorithm EMnEM (Moses et al . [ 18 ]) uses E-M and a phylogenetic model to find motifs, much like PhyME does, except that the former assumes that the input sequences are completely aligned. This assumption may be unsuitable for species at relatively large evolutionary distances, e.g., human and mouse, or D. melanogaster and D. pseudoobscura . Therefore, PhyME can handle a broader range of species divergence in its input. Another important difference between EMnEM and PhyME is the probabilistic model that each uses to model evolution. While EMnEM is implemented to use the Jukes-Cantor model [ 19 ], PhyME uses a more realistic model that incorporates binding site specificities. Thus, in calculating the joint likelihood of aligned motif occurrences, the EMnEM implementation does not use the fact that the effective mutation probability of an ancestral base to some base β depends on the fitness of a binding site with β at that position. The evolutionary model used in PhyME reflects this dependence, and the incorporation of the model into an Expectation-Maximization framework is one of the main technical contributions of our work. The Results section includes a preliminary comparison of PhyME's performance with that of EMnEM, orthoMEME and PhyloGibbs, on real data. The algorithm PhyloCon (Wang and Stormo [ 20 ]) extends the greedy algorithm of CONSENSUS (Hertz et al . [ 2 ]) to incorporate multiple species data. However, it treats all orthologous sequences uniformly, ignoring the fact that different species may be at different relative distances from each other. As such, it may be more suitable to use PhyME in cases where the phylogeny is far removed from a star topology of uniform branch length. Also, the PhyloCon algorithm proceeds by first identifying several local multiple alignments in orthologous sequences and then searching for common patterns (motifs) among these multiple alignments. As a result, it may miss motif occurrences that are not well-conserved (or are completely missing) in orthologous sequences. An advantage of PhyloCon is that it does not require the motif length to be input, and instead reports motifs of varying lengths. Results In this section, we first present the new algorithm, and then describe its evaluation on synthetic data, as well as biological data sets from various organisms. Algorithm Suppose that the input includes n different promoters (e.g., from co-regulated genes), and for each promoter there are sequences for K species. ( K may be different for different promoters.) PhyME requires that there be one designated "reference species" σ r in the input, for which there is sequence data corresponding to each of the n promoters. We shall describe PhyME's algorithm for the special case n = 1, though allowing multiple motif instances in this one sequence. The extension to n > 1 is trivial, and omitted here for simplicity. Thus, the input consists of a set of sequences S = { S 1 , S 2 ,..., S K }, where S i is the orthologous sequence from species i , and one of the S i 's comes from species σ r . The input also includes the motif length l , and the phylogenetic tree Ψ over the K species, with neutral point mutation rates (probabilities) along each branch. The output is a position weight matrix (PWM) representing the discovered motif, and its score. PhyME first partially aligns the input sequences and identifies contiguous regions ("blocks") in each that are highly conserved in . It then inputs all the sequences, along with the locations of the conserved blocks, to the core motif-finding algorithm. Alignment of sequences In this pre-processing step, PhyME computes the regions of high local similarity between and each of the other S i . The assumption is that such regions are of common evolutionary origin, and any sequence outside them is independently evolved. PhyME runs the LAGAN alignment program of Brudno et al . [ 21 ] on each sequence pair ( , S i \| i ≠ σ r ), and extracts all ungapped aligned blocks of a certain minimum size (of the order of the motif's length) and percent identity, to serve as the blocks of common origin. This is illustrated in Figure 1a , which shows orthologous promoters from three species, σ 1 (the reference species), σ 2 and σ 3 . An example of a block is region BC in σ 1 , aligned with region UV in σ 3 . Note how blocks can overlap in the reference species (BC overlaps with KL). The input is now reorganized into two kinds of sequences: 1. The sequence from the reference species, with aligned blocks of the other species "hanging off" it. (In Figure 1b , this is shown as the sequence AJ, with blocks MN, OP, QR of σ 2 and blocks UV, WX of σ 3 aligned with corresponding blocks in σ 1 .) Thus, any position in this sequence either has a single base from the reference species, or has an alignment of bases from multiple species, one of which is the reference species. This entire construct is called the "reference sequence". 2. Any subsequence not from the reference species, and bracketed by blocks on both sides. (e.g., regions NO, PQ, VW in Figure 1b .) The terminal sequences in the non-reference species, which are to the left of the left-most block and to the right of the right-most block, may be optionally included, as per the user's specification. PhyME fits the parameters of a probabilistic model on the reference and bracketed sequences simultaneously, and the desired motif comes out as a by-product of this training procedure, which is described next. Hidden Markov Model The probabilistic process that is assumed to generate sequences is described by a very simple Hidden Markov Model (HMM). For the moment, let us assume that the sequence S being generated is entirely from one species, with no aligned positions. The HMM parameters include a "motif weight matrix" W m of length l , and a "background weight matrix" W b of length 1. (The ( k, j ) th entry of a weight matrix is the probability of emitting the base j at position k of the sequence being sampled from the matrix.) At each step, the generative process of the HMM chooses either W m or W b according to their transition probabilities p m = p and p b = 1 - p respectively, where p is a model parameter. A sequence is then sampled from the chosen weight matrix, and appended at the end of the sequence S created so far. The process then proceeds to the next step. It stops when the length of S reaches its known length L . The series of motifs chosen in the successive steps of the process is called a "parse" of the sequence. The model parameters θ , which include W m , W b , and p , associate a well-defined probability Pr ( S , T \| θ ) with each parse T of the sequence S . The probability that S was generated by an HMM with parameters θ is then given by Pr ( S \| θ ) = Σ T Pr ( S , T \| θ ). Let Pr ( S \| θ b ) be the probability of generating S by using only W b . For a given θ , we define This log-likelihood ratio is the function optimized by PhyME – the parameters W m and p are trained so as to maximize F ( S , θ ). (The background weight matrix W b is not trained during this maximization, rather it is pre-computed from S , or optionally from specified background sequence, by measuring nucleotide frequencies.) The value of the objective function for a set of independent sequences is the sum of its values for the sequences taken separately. This additive property allows easy extension of the parameter training procedure to the general case of multiple sequences ( n > 1). The objective function maximized then is , the set S now including, for each of the n input promoters, the "reference sequence" as well as all "bracketed sequences" (defined earlier) as separate elements. An important aspect of computing F ( S , θ ) is the subsequence probability Pr ( s \| W ). This is the probability of generating a subsequence s of length l , (length of W ), when sampling from weight matrix W ; so , where s = s 1 s 2 ... s l , and W kj is the probability of sampling base j at the k th position of W . This formula applies when subsequence s has a single base at each position. However, we need to adapt this formula to the case where one or more positions in subsequence s may be an alignment of orthologous bases from multiple species. In this general case, we can write s as ψ 1 ψ 2 ... ψ l , where each ψ k is either a single base, or an alignment of orthologous bases at a single position of the reference sequence. The subsequence probability Pr ( s \| W ) can then be computed as , where Pr e ( ψ \| W , k ) denotes the probability of observing ψ at position k when sampling from W . Let the vector ψ = ( s 1 , s 2 , ... s K ), where s σ is the nucleotide from species σ in the single-base alignment ψ . If the s σ were independent , we could write . However, the s σ 's occur in an alignment ( ψ ), meaning that this assumption of independence is obviously untenable. Thus we need an expression for Pr e ( ψ \| W , k ) that explicitly takes the phylogenetic relationships among the species (given by the phylogenetic tree Ψ) into account. We present such an expression in the next section, and we shall thereafter return to the topic of maximizing the function F ( S , θ ). Evolutionary model This section describes the probabilistic evolutionary model that PhyME uses to incorporate phylogenetic relationships in the computation of the term Pr e ( ψ \| W , k ) mentioned above. It was first proposed in Sinha et al . [ 22 ] to model binding site evolution, and applied successfully on the two fly genomes. The model makes the crucial assumption that all positions in a binding site evolve independently, at equal rates, and the probability of fixation of a mutation α → β at position k is proportional to the weight matrix entry of β at that position. If we further assume, for simplicity of exposition, that the phylogenetic tree Ψ has a star topology, then the model assumptions give us (from Sinha et al . [ 22 ]; also see Methods.) where s σ is the nucleotide from species σ in alignment ψ , δ xy = 1 if x = y and 0 otherwise, and μ σ is the neutral mutation probability between the ancestor and the species σ . For the position k , one "creates" a base α in the ancestor with frequency W k σ , and each such base is either passed unchanged to the species σ (probability 1 - μ σ ) or mutated in species σ with probability μ σ and a new base selected with a frequency defined again by W . In the general case, when Ψ does not have a star topology, Formula (1) can be written in a recursive manner. (See Methods.) Expectation Maximization The function F ( S , θ ) that is maximized by PhyME measures how much more likely it is that S was generated using the motif weight matrix, than without it. Naturally, a PWM that maximizes this score is the motif that best explains the data. PhyME tries to find such a motif by training the parameters ( W m , p ) of the HMM, using the Baum-Welch algorithm [ 23 ], which iteratively converges to a locally optimum θ using Expectation Maximization (E-M). Let , for i ∈ { m , b } be the expected number of times the HMM plants motif W i in generating the sequence(s), the expectation being over all parses. Similarly, let be the expected number of times that the nucleotide alignment ψ is sampled at the k th position of the motif W m . and are expected values of hidden variables of the HMM. These averages are computed during the "E-step" in each iteration, using dynamic programming (the Forward-Backward algorithm, [ 23 ]). In the "M-step", two kinds of updates are made, using the values of , computed in the E-step. The parameter p is updated according to . The motif weight matrix W m is updated by solving, for each column k of the matrix, the following set of five simultaneous equations, in variables u β ( β ∈ Σ) and λ . The derivation of the update formulas is somewhat involved, and is described in the Methods section. The equations are solved using Newton's method, and the solution value of u β is used to update the ( k , β ) th entry of W m , according to W mkβ = e uβ . Newton's method involves computation of the first and second partial derivatives of log Pr e ( ψ \| W m , k ), as described in Methods. In practice, we found that Newton's method always converges from a single initial condition, and the convergence almost always happens within 3–5 iterations. The time complexity of (each E-M iteration in) PhyME is O( LKl ), where L is the length of the sequences, K is the number of species, and l is the length of the motif desired. (See Methods for details.) Results on synthetic data We first present the results of running PhyME on synthetic data. The experimental framework is largely borrowed from Wang and Stormo [ 20 ]. In each experiment, 5 "ancestral" sequences, each of length 600 bp, are created at random, and 20 "binding sites", each of length 8, are "planted" at randomly chosen locations in these sequences. The sites are chosen such that the weight matrix formed by them has a relative entropy of R . Each ancestral sequence is then "evolved" by point mutations to create K additional "orthologous" copies, assuming a star topology (with K leaves) and a common "background mutation rate" μ b along each branch. (No insertions or deletions were included in this simulated evolution, for simplicity.) The motif instances are subjected to a common "motif mutation rate" μ m , which is the probability of mutation of any position in a motif. The ancestral set of sequences is then removed and the remaining K orthologous sets are input to the motif discovery algorithm, with one arbitrarily designated the reference species. The algorithm is made to report 3 different motifs, thereby making some allowance for false positives, especially when R is low. For each reported motif, its 20 best occurrences in the reference species are compared with the planted occurrences, to give a score ranging between 0 and 1. (1 represents the best possible performance; see the Methods section for details.) The score for the best of the 3 reported motifs is the "performance score" of the algorithm. The three algorithms being compared are PhyME, MEME [ 1 ], and GIBBS (Wadsworth Gibbs sampler) [ 24 ]. PhyME was run with an evolutionary tree with a star topology, the mutation rate along each branch being μ b . MEME and GIBBS were run on the entire data set pooled together, ignoring the orthology of sequences. Figure 2 shows the effect of varying K on performance scores of the algorithms. Note that the performance of PhyME, while similar to MEME and GIBBS for K = 1, improves relative to them as K increases. The absolute performance score of GIBBS (and of MEME, to some extent) deteriorates with increasing K . With more orthologous sequences, conserved stretches of background sequence may distract the algorithm from the motif occurrences. PhyME, with the additional knowledge of orthology, is able to pick out the motif better. Figure 3 shows the effect of varying the mutation rates. The background mutation rate μ b was varied from 0.2 to 0.5 and the motif mutation rate μ m was varied between 0.1 and μ b - 0.1). As per expectation, the performance of each algorithm improved with decreasing μ m (for a fixed μ b ). Interestingly, as μ b decreases, the performance of PhyME for μ m = 0.1 first improves and then falls down. The initial improvement is because the alignment step is able to find more conserved blocks with diminishing background mutation rate. However, when the latter approaches the motif mutation rate, the distinction (in cross-species conservation) between motif and background becomes weaker, hence performance goes down. In another set of experiments, we examined the effect of the alignment step on the performance. Sequences were created with K = 2, μ b = 0.3, μ m = 0.1 and R = 12. After the alignment step of PhyME (in which the entire sequence was aligned as one conserved block), we artificially "unpaired" some number n of the planted orthologous motif occurrences, i.e., the alignment was modified so that these n pairs fell outside conserved blocks. This was followed by the usual motif-finding step, and the entire procedure was repeated for various values of n . We find a gradual degradation in performance as PhyME moves from maximum utilization of motif orthology ( n = 0, no unpaired motifs) to minimum ( n = 20, no motif pairs considered orthologous). (Figure 4 .) We also evaluated the effect of mis-estimates of the neutral mutation rates on performance. PhyME was run on random sequences created with experiment parameters K = 3, μ b = 0.3, μ m = 0.1 and R ∈ {11, 12, 13}, and in different runs, the value of μ b input to PhyME ranged from 0.1 (underestimate) to 0.5 (overestimate). We observed that underestimates of μ b resulted in significantly greater performance degradation than overestimates of equal magnitude. (Data not shown.) For instance, using μ b = 0.4 instead of the true value of 0.3 made no difference to the performance, whereas using μ b = 0.2 resulted in 15 – 50% decline. Results on biological data In the following sections, we present results of running PhyME on real data sets from yeast, fly and human. The results are compared to MEME (run by pooling orthologous sequences together), orthoMEME [ 15 ], PhyloGibbs [ 17 ], and EMnEM [ 18 ]. The latter three programs address the heterogeneous data problem directly, just as PhyME does. Another program that solves the same problem is PhyloCon [ 20 ]. PhyloCon was not evaluated in our study because we did not have a clear method to post-process its output to extract a specified number of top-scoring motifs that are non-redundant. (Our evaluations described below use the top three motifs reported by each program.) Yeast data sets We first present some examples in yeast, where sequence data from four species, S. cerevisiae , S. mikatae , S. kudriavzevii and S. bayanus was used. We performed motif-finding (with PhyME, MEME, orthoMEME, PhyloGibbs and EMnEM) on some regulons from the SCPD [ 25 ] database, which catalogues sets of co-regulated genes. For each regulon, the top η motif occurrences in S. cerevisiae reported by the algorithm ( η being the number of known sites in S. cerevisiae ) were examined for "matches" to the known weight matrix for that regulon's motif. (See Methods for details.) The number of matches was the performance score of the algorithm. We counted matches to the weight matrix, rather than to known sites, so that a reported motif occurrence that is very similar to the known motif (but not annotated as a site by SCPD) will not be counted as a false positive. Each algorithm reported 3 motifs (with η occurrences for each motif), and the results are for the best scoring motif, thereby making some allowance for false positives, such as simple repeats. Even though PhyME is implemented to handle arbitrary phylogenies, for efficiency it was run with a phylogenetic tree with a star topology, having S. cerevisiae at the center and the mutation rate along the branches of S. mikatae , S. kudriavzevii , and S. bayanus being (0.25, 0.3, 0.35) respectively. These values are based on average substitution rate per base in the corresponding pairs of species. (A more accurate tree can be derived from the work of Kellis et al . [ 12 ].) For multiple species data, MEME was run by pooling all sequences together. OrthoMEME was run only for the case K = 2 (i.e., on sequence from the two species S. cerevisiae and S. mikatae ), since its current implementation can only handle two species data. The other four programs were run for K = 1, 2, 3, 4, in separate executions. See Methods for details on how orthoMEME, PhyloGibbs and EMnEM were run. Figure 5 plots the performance scores for regulons RAP1, MIG1, CAR1, PHO4 and MCM1, which show interesting results. Note how the performance of PhyME improves with K for RAP1 and MIG1. For CAR1, both PhyME and MEME improve from K = 1 to K = 2, and then deteriorate for higher K , but PhyME at K = 3 is still better than at K = 1. For PHO4, PhyME's performance first goes up (for K = 2) and then dips below the K = 1 level, whereas MEME shows best performance at K = 4. For MCM1, PhyME scores well (over 90% matches) for K ∈ {1,3,4}, whereas MEME's performance degrades for K > 1. Thus, these examples illustrate that PhyME's approach can lead to improved motif discovery in multiple species data, and also that there may be situations when more orthologous sequences distract either algorithm from the true motif. For regulons CSRE, GCN4, MAT α 2, URS1H, REB1, and PDR3, the performance score was high (typically over 80% matches) and largely invariant of the number of species. Figure 5 also reports the scores of PhyloGibbs. This program has similar scores as PhyME on CAR1, PHO4 and MCM1. (It did not execute to completion for K = 3, 4 in MCM1.) PhyME has better scores on MIG1 and RAP1, though PhyloGibbs' scores on RAP1 with a different choice of parameters ("-G 0.7", see Methods) were similar to PhyME. (Data not shown.) We also report the scores of EMnEM in Figure 5 . (The program did not execute to completion in CAR1.) This program performs well for K = 2 (comparable to the best scores in the data sets RAP1, PHO4 and MCM1). For K = 3, 4 also, EMnEM scores are comparable to PhyloGibbs. PhyME typically performs better than EMnEM (with K = 3, 4) for RAP1, MIG1, MCM1, and comparably for PHO4. We find the scores of orthoMEME, as reported in Figure 5 , to be lower than those of PhyME (for K = 2). However, we observed that in all five regulons reported, orthoMEME reported fewer than η occurrences in S. cerevisiae per motif. This is because orthoMEME was run in the "zoops" mode (zero or one motif occurrence per sequence), since the "tcm" mode (any number of occurrences per promoter) does not perform well. Thus, with the total number of predictions being fewer than η , orthoMEME's scores are expected to be lower than other programs even for the same level of specificity. We suggest caution in comparing PhyME's scores directly with those of the other programs, since we lacked expertise in choosing optimal parameter settings for them. This is particularly true for EMnEM, which has several parameters for modeling the evolution of motifs, and we lacked experience in setting these parameters optimally. We clearly have more expertise at using PhyME than the other programs, and this makes the comparisons biased. Our goal in these experiments was to provide some examples of how multiple species data can be exploited to improve performance, rather than assessing the different motif finding programs available. A proper comparative assessment of these programs has to address several challenges not addressed here. Such a task was undertaken for several motif finding programs, in the work of Tompa et al . (unpublished). A similar assessment of the motif-finding programs in the context of the heterogeneous data problem is an important topic for future work. Fly data sets Next, we present results from fruitfly, where data from two species, D. melanogaster and D. pseudoobscura , is available. Nine different enhancers were chosen – enhancers eve1 , eve2 , eve5 , ftz3 ', gtposterior , hairy2 , hairy34 , and run1 have binding sites for the Kr transcription factor, and btdhead has Bcd sites. Well-defined weight matrices are available for both Kr and Bcd [ 26 ]. For each enhancer, the top η motif occurrences (in D. melanogaster ) reported by the algorithm ( η being the number of "strong" occurrences of the known weight matrix in D. melanogaster – see Methods) were examined, and the number of matches was the performance score of the algorithm. Six different motif-finding strategies were tested separately – (i) MEME_1 (MEME on single species) (ii) MEME_2 (MEME on both species pooled together), (iii) PhyloGibbs, (iv) orthoMEME, (v) EMnEM and (vi) PHYME (PhyME on both species, with μ = 0.5). Each strategy was required to report occurrences only in D. melanogaster . (See Methods for details of how orthoMEME, PhyloGibbs and EMnEM were run.) Figure 6 compares the scores of the different strategies for all nine enhancers. Note that either PHYME or MEME_2 performs better than MEME_1 for seven of the nine enhancers, and worse only for one ( ftz3 '), thereby making the case for using two species data. Moreover, on btdhead , gtposterior and hairy2 , PhyME performs significantly better than MEME_2, demonstrating the advantage of using orthology information. Similarly, we find PhyME to perform better than PhyloGibbs on gtposterior , hairy2 and hairy34 . EMnEM performs well in these data sets, scoring comparably to PhyME or PhyloGibbs, except on btdhead , eve2 , and gtposterior , where both PhyME and PhyloGibbs perform better, and hairy2 , where PhyME alone performs better. OrthoMEME was run in the "tcm" mode, since the "zoops" mode is not appropriate here, with several putative sites to be found in each promoter. However, orthoMEME tends not to perform well in the "tcm" mode in general, and in our tests also, its scores were poor on most of the enhancers. We thus find that PhyME is preferable to orthoMEME for cases where we expect several motif occurrences per sequence. As in the yeast data sets, the comparison of scores between PhyME and the other programs should be interpreted with caution, since we lacked expertise in choosing optimal parameter settings for the other programs. Human data sets Finally, we present results of running PhyME on two data sets from human, where orthology with mouse and rat was utilized. These data sets were chosen because all of 15 different motif-finding programs tested in an assessment project (Tompa et al ., unpublished) failed to report the correct motif in them. The first set corresponds to the transcription factor SP1, a zinc-finger protein. The heterogeneous sequence data included 35 human promoters (of length 2 Kbp each), of which four have orthologous promoters from mouse and rat, 20 from mouse only, 4 from rat only, and 7 have no orthologs. Each of the human promoters is known to have at least one functional Sp1 binding site, with a total of 76 known sites overall. Figure 7a shows a "sequence logo" representation [ 27 ] of an alignment of these known sites. PhyME was run to find motifs of length 7, using the phylogenetic tree shown in Figure 7c . The second motif reported by PhyME (Figure 7b ) is almost identical to the known SP1 weight matrix. The top 27 instances (in human promoters) of this motif included 16 that overlapped with known binding sites. We also ran MEME on the heterogeneous data set (pooling orthologous sequences together), and the second motif reported was a good match to SP1. However, of its 41 instances reported in human promoters, only 9 were overlapping with known sites. Moreover, when MEME was run on human promoters alone, none of the top three motifs matched the SP1 motif. Thus, PhyME showed a clear performance improvement over MEME, both in the single species run, and when the orthologous sequences were pooled together. The second data set used in our tests corresponds to the leucine zipper transcription factor c-Jun. The heterogeneous data set included 500 bp promoters for 11 human genes targeted by c-Jun, as well as orthologs from mouse and rat for 3 genes, from mouse only for 5 genes, and from rat only for the remaining three genes. PhyME was run exactly as in the previous data set. The known binding sites of c-Jun (in human) were aligned to produce a weight matrix that is shown in Figure 8a . The third ranked motif reported by PhyME is shown in Figure 8b , and we can see that its last five positions are similar to the first five positions of the known weight matrix. Of the top 13 instances of the discovered motif, 4 overlap with known binding sites of c-Jun, whereas a maximum of 9 could have been obtained. (Of the 11 known sites, 9 are in the 500 bp upstream regions used in our analysis.) We also ran MEME on the heterogeneous data set (using the pooling strategy), and none of the three best motifs reported by MEME matched the c-Jun motif. Thus, both the human data sets tested demonstrate how PhyME can improve motif discovery in typical motif finding scenarios by exploiting heterogeneous sequence data properly. Discussion Issues in algorithm design Alignment step In the alignment step, PhyME extracts blocks of high sequence similarity between the reference species and each of the other species. Motif occurrences in such locally conserved regions are deemed orthologous, an assumption well-justified by traditional interpretations of sequence alignment. Conversely, all orthologous motif occurrences are assumed to be aligned in such blocks. This assumption is not always true since there may be orthologous motif occurrences not aligned by the alignment program, but it heavily constrains the space of orthologous motif occurrences, implying greater efficiency of the search algorithm. Moreover, the assumption does not mean that "true" orthologous occurrences in unaligned regions are ignored – they are merely treated as independent occurrences. Our experiments on synthetic data (see Results) demonstrate that the performance is not very sensitive to the correct alignment of all orthologous motif pairs. The blocks computed in the alignment step have to be with respect to the reference species, but the alignment itself need not be done in a pairwise manner. A multiple alignment of all sequences may be computed (e.g., with M-LAGAN [ 21 ], using the input phylogenetic tree Ψ) and blocks between and each of the other S i may then be extracted. (The alignment step is implemented as a separate tool in PhyME, making it easy to switch to such alternative schemes.) Furthermore, the implementation may be modified in the future to drop the requirement of a reference species, since this requirement is not crucial to the motif finding step of PhyME. For instance, the alignment step may utilize the "Threaded Block Alignment" (TBA) program of Blanchette et al . [ 28 ], which completely circumvents the notion of a reference species in multiple alignments. Once the blocks of high sequence conservation have been identified, a possible strategy is to restrict attention to motif occurrences in these blocks, assuming that all functional binding sites must be evolutionarily conserved. However, this assumption is not true even for as closely related species as D. melanogaster and D. pseudoobscura , separated by about 25–30 Myrs. An empirical study [ 16 ] on these two species revealed that a good fraction (35–40%) of occurrences of relevant motifs occur outside of locally conserved contexts, and should therefore be taken into account when discovering motifs. Motif Finding In the probabilistic process that is assumed to generate sequences, the transition probability does not depend on the previous choice(s) made during the process, meaning that the HMM is of zeroth order, nor on the position in the sequence, meaning that any information about spatial distribution of motifs is ignored. The model, unlike that of MEME, does not fragment the sequence into all l -length words to be treated independently. Rather, it parses the sequence into a series of non-overlapping occurrences of the motif and background. The evolutionary model described by Formula 1 applies only to phylogenies having a star topology. The general case of arbitrary tree topology is described in Methods. In Formula 1, if μ σ is small (as for very closely related species), then finding different bases in orthologous positions has low probability Pr e ( ψ \| W , k ), even if their frequency in W is the same. This mirrors the intuition that mutations in locally conserved regions of closely related species are evidence against a binding site residing there. For largely diverged species (i.e., if μ σ ~1, ∀ σ ), Pr e ( ψ \| W , k ) reduces to the product of the individual bases' probabilities. It is worth emphasizing here that the weight matrix W being searched by the algorithm is assumed to be unchanged over the entire phylogenetic tree (including the ancestor). The neutral mutation rates (probabilities) along each branch of Ψ are input by the user and not trained during E-M. Training them on input data may cause overfitting, producing values that are largely inconsistent with the known evolutionary distances. The work of Moses et al . [ 18 ] studies this issue, and finds that it is more important to use correct phylogenetic relationships, e.g., an appropriate evolutionary tree, than to use accurate mutation rates. Note that the evolutionary model used by PhyME comes into play only in Equations 2 as the term Pr e ( ψ \| W m , k ). Other models of evolution, e.g., F81 [ 29 ], can be incorporated into PhyME by simply using the appropriate formulation of this term, as long as the derivatives of log Pr e ( ψ \| W m , k ) can be computed efficiently. Conclusions We have developed a new algorithm, PhyME, that detects motifs in heterogeneous sequence data by integrating two important aspects of a motif's significance – overrepresentation and cross-species comparison – into one probabilistic score. We have evaluated different aspects of the algorithm on synthetic data, and demonstrated on some biological data sets that the new approach improves motif detection. Methods The evolutionary model The evolutionary model makes the following assumptions: (i) Nucleotides in an aligned position are evolved from a common ancestor. (ii) The weight matrix applies to the common ancestor and to all descendants, a reasonable assumption given the propensity of DNA binding domains of proteins to evolve slower than cis-regulatory modules. (iii) All positions evolve independently, at equal rates, and the probability of fixation of a mutation α → β at position k is proportional to the weight matrix entry of β at that position. Suppose we are given a phylogenetic tree Ψ, with the species { σ 1 , σ 2 , .... σ K } at the leaves. Let the vector ψ = ( s 1 , s 2 , ... s K ), where s σ is the nucleotide from species σ in the (single position) alignment ψ . The term Pr e ( ψ \| W , k ) denotes the probability of observing ψ at position k when sampling from weight matrix W . For each node j of the tree, except the root, let μ j be the probability of a base in the parent species of j having mutated (under neutral evolution) in species j . Also, let ψ j be the vector formed by elements of ψ that correspond to leaf nodes descended from node j . Let C ( j ) denote the set of children of node j and let r be the root of the tree. Then, we can write (using the model assumptions): where f ( ψ j , α ) denotes the probability of observing ψ j given that the base at the parent of j is α . For a leaf node σ , this can be written as , from the model assumptions. ( δ ij = 1 if i = j , and 0 otherwise.) For an internal node j (except root r ), the expression is : For the special case where Ψ has a star topology, Equation 4 reduces to Equation 1. Training parameters in a HMM Given a sequence S and a set of position weight matrices { W i }, the objective function to be maximized is F ( S , θ ) = log( Pr ( S \| θ )/ Pr ( S \| θ b )), where Pr ( S \| θ ) is the probability of generating the sequence S using the parameters θ , and θ b represents the parameter values that only allow the background motif W b to be used by the HMM. The sequence S can be written as ψ 1 ψ 2 ... ψ L , where each ψ i is either a single base or an alignment of orthologous bases at a single position. θ includes the weight matrices W i and their transition probabilities p i . Since Pr ( S \| θ b ) depends only on W b , which is assumed constant, we shall outline how to maximize log Pr ( S \| θ ), following the description in [ 23 ]. A parse of the sequence S in terms of the W i (i.e., the series of motifs chosen in the successive steps of the generative probabilistic process) is denoted by T . We thus have The maximization is iterative, with the t th iteration computing a model θ t + 1 that improves the objective function from the current model θ t . In classical E-M fashion, let us define a function Q ( θ \| θ t ) as It is easily shown that log Pr ( S \| θ ) - log Pr ( S \| θ t ) ≥ Q ( θ \| θ t ) - Q ( θ t \| θ t ). Thus, if we maximize Q ( θ \| θ t ) over all θ , we shall always improve upon log Pr ( S \| θ t ), or remain there if the local maximum has been reached. Let A i ( T , S ) be the number of times W i occurs in the parse T of S . Also, let E ikψ ( T , S ) denote the number of times that the alignment ψ is emitted (sampled) at the k th position of the matrix W i , in parse T of S . Let l i denote the length of W i . Then we have which gives us Note that the only the first term in this expression depends on p i , and only the second term depends on W i . Hence, we maximize each of these terms independently, with respect to the appropriate free parameters. We first maximize the term Note that is the average number of occurrences of W i in S over all parses T . Thus the term to maximize is , and this is maximized when Next, we maximize the second term: Again, note that is the average number of times that the alignment ψ is sampled at the k th position of the matrix W i while generating S , the average being over all parses T . Thus, the term to maximize is . We first note that in our case, there is a single weight matrix W m to be trained. Hence, we need to maximize Q with respect to W m . We can do this maximization with respect to each column k independently. Let W kβ denote the ( k , β ) th entry of W m . Thus, for each k = 1 ... l , we need to maximize Q with respect to W kβ ( β ∈ Σ), with the constraint Σ β W kβ = 1. Using Lagrangian multiplier λ , the objective function becomes Q + λ (Σ β W kβ - 1). Transforming to log variables u β = log W kβ to ensure that the W kβ remain positive during optimization, we then have the following necessary conditions for optimality (in addition to the constraint ) : We therefore have a system of five equations (including the constraint) in the variables u β (∀ β ∈ Σ and λ . Denoting the vector of these five variables by u , we solve this system of equations using Newton's iterative method. Let us write the above system of equations as F ( u ) = 0 , where F ( u ) = [[ f β ], f λ ], with f β being the left side of Equation 9, and . Newton's method uses the update relation: Δ u = -( J ( u )) -1 F ( u ) where Δ u is the change in u in the current iteration and J is the Jacobian matrix of F . The important terms in the computation of F and J are the first and second partial derivatives of log Pr e ( ψ \| W m , k ) with respect to the u β variables. For this purpose, we need to compute Pr e ( ψ \| W m , k ) and its first and second partial derivatives. Computation of Pr e ( ψ \| W m , k ) uses the formulas 4 and 5. The partial derivatives can be computed recursively (over the tree Ψ) by using the chain rule of differentiation. These recursive computations are implemented in a bottom-up manner, so as to avoid redundant computations. Newton's method uses F and J to iteratively compute new values of u , until convergence. The Jacobian matrix J in our case is not positive definite, hence Newton's method is not guaranteed to converge. However, in practice, we found the method to always converge from a single initial seed. Upon convergence, the log variables u β are transformed back to W kβ = e uβ . The procedure is repeated for each k = 1 ... l , and W m is then updated with the new values. This update, along with that given by Equation 8, is used iteratively to improve F ( S \| θ ) until the local maximum is reached, as indicated by a very small change in its value. Time complexity The E-step computes , and , for k = 1 … l , ∀ ψ . The Forward-Backward algorithm is run once, in O ( LKl ) time, where L is the total length of the input sequences, K is the number of species, and l is the length of the motif W m . (This time complexity assumes that nodes in the phylogenetic tree have a fixed maximum degree.) Thereafter, , are computed in O ( L ) time, and all the are computed in one scan of the input, expending O ( Ll ) time. The M-step runs Newton's method to solve a system of equations, once for each column of W m . Each run of Newton's method goes through a small number (3–5) of iterations. Each iteration computes the first and second partial derivatives of log Pr e ( ψ \| W m , k ) Each of these derivatives can be computed in O ( K ) time, where K is the number of species (since \| ψ \| ≤ K ) Hence, F and J can be computed in O ( LK ) time, where L is the total length of the sequence. Hence, Newton's method takes O ( LK ) time, and is run l times, for an overall time complexity of O ( LKl ) for the M-step. Thus, the running time of (each E-M iteration in) PhyME scales linearly with the length of the sequences, the length of the motif desired, and the number of species. Implementation details PhyME is implemented in C++ for Linux, and the source code will be made freely available at . The current implementation runs in a few minutes (on a workstation) for typical applications with total sequence length ~10000 bp, 2–4 species, and motif length of ~10. PhyME uses the LAGAN alignment tool of Brudno et al . [ 21 ] for the alignment step. After alignment, the ungapped blocks extracted are required to be at least 10 bp long, and have at least 70% identity. PhyME is implemented to handle an arbitrary phylogenetic tree Ψ relating the input species. The E-M algorithm is guaranteed to converge only to a local optimum. To address this problem, the motif-finding step is executed a fixed number of times, each time using a randomly chosen substring of the input sequence as the "seed" to initialize W m , and truncating the E-M procedure after a small number of iterations. The seed with greatest score F ( S , θ ) among these runs is then used to run the E-M to convergence and the trained motif is reported, along with all its instances with posterior probability above a certain threshold. To find more motifs, PhyME masks out the central base of each of these instances. Optionally, the user may specify nsites, the expected number of occurrences of each of the desired motifs. In such a case, PhyME turns off training of the parameter p , and uses a fixed value computed from nsites. Similarly, an option maxsites specifies the maximum number of occurrences expected. PhyME considers occurrences on both strands by introducing a new weight matrix W r , and an associated transition probability p r , in the HMM parameters. The weight matrix is constrained to be the reverse complement of W m . The model has a fixed bias of planting the motif in one orientation versus the other, and this bias is trained from the data. PhyME also has the option of capturing local correlations in background nucleotide composition. To implement a κ th order Markov background, PhyME uses a special background weight matrix that is of length 1 but uses the knowledge of the previous κ bases generated to determine the emission probabilities of the next base. Performance score in experiments with synthetic data We use the following score for measuring the performance of a motif-finding algorithm on synthetic data. Let S = { S 1 , S 2 , ... S n } be the set of n input sequences. For any motif m , let I mi be the set of positions in sequence S i that are occupied by an occurrence of m . We know the occurrences of the planted motif m k , and are evaluating the motif m r reported by an algorithm. The performance score Φ is defined as follows: In other words, it is the number of positions, over all sequences, where occurrences of the known and reported motifs overlap, divided by the total number of positions at which the known or the reported motif occurs. Note that if the reported occurrences exactly concur with the known occurrences, the score is 1, and when the reported and known occurrences have no position in common, it is 0. Details of experiments with biological data sets Yeast The genes regulated by each transcription factor are listed in SCPD. For each such "regulon", the known sites and the known weight matrix were extracted from SCPD. Also, 800 bp long upstream sequences of the genes in each regulon were extracted (for S. cerevisiae ) from the RSA-Tools web site [ 30 ]. Orthologous promoters in the other yeast species were obtained from Cliften et al . [ 11 ]. Let η be the number of known binding sites in S. cerevisiae . The input to the motif finding algorithm consisted of the sequences from S. cerevisiae and their orthologs from one or more of the other species, depending on K . (In addition to S. cerevisiae , we used S. mikatae for K = 2, S. mikatae and S. kudriavzevii for K = 3, and S. mikatae , S. kudriavzevii and S. bayanus for K = 4.) The length of the motif was also input to each program. Each algorithm was made to report 3 motifs, and for each motif, the top η reported occurrences in S. cerevisiae were examined. For each such occurrence, the logarithm of the probability of sampling it from the known weight matrix was computed, and a z -score of this logarithm was obtained. If the z -score was above 3, the occurrence was called a "match". To allow for slight offsets in the reported motif, each reported occurrence was padded with 3 bp of its context, on either side. PhyME was run with the maxsites option set to η , and MEME was run with the same option set to ηK . We also experimented with running MEME with the nsites parameter set to ηK . OrthoMEME was run with a zeroth order Markov background, in the "zoops" mode, with expected number of sites between 0.8* η ("minsites") and 1.2* η ("maxsites"). PhyloGibbs was run with mutation probability 0.7 ("-G 0.3") for all species, and was asked to report three motifs (three "colors") each with 1.5 × η occurrences ("-I") initially. A 3 rd order Markov background ("-N 3") trained on the full complement of yeast promoters was used, as with PhyME and MEME. The "loose align" option ("-D 1") and the "stop after anneal" option ("-X") were used. These options were suggested by an author of PhyloGibbs (Rahul Siddharthan, personal communication). We experimented with a different value for the mutation probability ("-G 0.7"), with no improvement, except in the RAP1 regulon. EMnEM was run with default parameters, the motif length being input through the "-w" parameter. Phylogenetic trees were derived from each input promoter, using the fastDNAML software of Olsen et al . [ 31 ]. The alignments were done using the MLAGAN program of Brudno et al . [ 21 ]. In separate runs, we also tried non-default values of the parameters "-p" (relative rate of motif to background; default 0.5, also tried 0.25) and "-m" (evolutionary model; default Jukes-Cantor, also tried F81). The expected number of instances of each motif per sequence ("-e") was set to η / n and η / n + 1 in separate runs, where n is the number of input promoters. For each data set, and for each value of K , we took the best scoring choice of parameters. This was done to give some advantage to EMnEM, since we lacked expertise in choosing optimal parameter values. Fly The locations of cis-regulatory modules involved in body-patterning of the early embryo in D. melanogaster were obtained from [ 26 ], and their sequences were extracted from BDGP [ 32 ]. The evaluation procedure was identical to that in yeast, with the following difference. Since there is no complete list of verified sites in the enhancers, we first scanned the sequences (in D. melanogaster ) with the known weight matrix, and counted matches, by the same measure as above. This count was the value of η used in the experiment. An extra complication in the fly data is caused by the fact that each enhancer typically contains sites for multiple transcription factors. We restricted our tests to the factors Kr and Bcd, because their weight matrices are of better quality than others. Moreover, for each enhancer, we chose to test with the transcription factor with most putative sites (matches to its weight matrix). OrthoMEME was run as in the yeast data sets (see above), except that the "tcm" mode was used now. PhyloGibbs was also run as in the yeast data sets, except that we used a mutation probability of 0.5 ("-G 0.5"), and a 2 nd order Markov background ("-N 2"), trained on non-coding regions in fly. We also experimented with a higher value of the mutation probability, and tried specifying the initial number of occurrences per motif ("-I") differently, with no clear improvement. EMnEM was run with the Jukes-Cantor evolutionary model ("-m 0") and with the relative rate of motif to background ("-p") set to 0.5 and 0.25 in separate runs. The expected number of motifs was set to η and 1.5 × η in separate runs. The best performing choice of parameters was used for each data set. Human The genes comprising each regulon were obtained from TRANSFAC [ 33 ]. Mouse and rat orthology information for human genes was obtained from Homologene [ 34 ]. Human, mouse and rat promoters were obtained from the UCSC Genome Browser [ 35 ]. Authors' contributions All authors participated in initial discussions leading to the key idea of using Expectation-Maximization and a phylogenetic model to search in a weight-matrix space. SS designed the algorithm details, derived the E-M calculations, implemented and tested the program, and drafted the manuscript. All authors contributed to, read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC534098.xml
544594	Retinol improves bovine embryonic development in vitro	Retinoids are recognized as important regulators of vertebrate development, cell differentiation, and tissue function. Previous studies, performed both in vivo and in vitro, indicate that retinoids influence several reproductive events, including follicular development, oocyte maturation and early embryonic development. The present study evaluated in vitro effects of retinol addition to media containing maturing bovine oocytes and developing embryos in both a low oxygen atmosphere (7%) and under atmospheric oxygen conditions (20%). In the first experiment, abbatoir collected bovine oocytes were matured in the presence or absence of varying concentrations of retinol. After a 22–24 hour maturation period the oocytes were fertilized, denuded 18 hours later and cultured in a modified synthetic oviductal fluid (mSOF) in a humidified atmosphere at 38.5 degrees C, 5% CO2, 7% O2 and 88% N2. Cleavage rates did not differ among control and retinol-treated oocytes in all three experiments. Addition of 5 micromolar retinol to the maturation medium (IVM) tended (p < 0.07) to increase blastocyst formation (blastocyst/putative zygote; 26.1% +/- 2.2%) compared to the controls (21.9% +/- 1.9%). Further analysis revealed when blastocyst development rates fell below 20% in the control groups, 5 micromolar retinol treatment dramatically improved embryonic development, measured by blastocyst/putative zygote rate (14.4 +/- 2.1 vs 23.7 +/- 2.5; p < 0.02). The 5 micomolar retinol treatment also enhanced the blastocyst/cleaved rate by nearly 10% (23.7% vs 34.6%; p < 0.02). In the second and third experiments addition of 5 micromolar retinol to the embryo culture medium (IVC) under low oxygen conditions did not significantly improve cleavage or blastocyst rates, but 5 micromolar retinol significantly increased blastocyst development under 20% O2 conditions (p < 0.001). These studies demonstrate that supplementation of 5 micromolar retinol to the maturation medium may improve embryonic development of bovine oocytes indicated by their increased blastocyst rate. A significant improvement in the blastocyst development with the 5 micromolar retinol treatment under atmospheric conditions suggests a beneficial antioxidant effect during embryo culture.	Background Vitamin A is essential for reproduction, and deficiencies and excesses may result in embryonic loss and/or congenital defects [ 1 ]. Retinol (vitamin A alcohol) is the parent vitamin A compound and metabolites, analogs, and derivatives are known collectively as retinoids. Results from several studies, in a variety of species, have indicated that retinoid administration may function in very early events associated with reproductive success, including follicular development, oocyte maturation and early embryonic development. Retinol concentration in bovine follicular fluid was shown to be an indicator of follicular quality and was highest in healthy follicles, lowest in atretic follicles and highly correlated with estradiol concentrations [ 2 , 3 ]. Retinol or β-carotene administration has been shown to prevent fetal resorption in rats [ 4 ], increase the number of births in rabbits [ 5 ], and increase litter size in swine [ 6 ]. Retinol administration to ewes, in combination with superovulation followed by natural service was shown to improve the competence of resultant 1–4 cell and morula stage embryos, collected from the oviduct and uterus, respectively, to develop to the blastocyst stage when cultured in vitro [ 7 ]. In cattle, retinol injection improved the estimated quality of embryos collected from superovulated animals but did not increase the number recovered [ 8 ]. Retinol is transported systemically and intercellularly bound to retinol-binding protein (RBP). Cellular retinol-binding proteins (CRBP) and cellular retinoic acid-binding proteins function in intracellular vitamin A transport, metabolism and homeostasis [ 9 ]. All- trans and 9- cis retinoic acid (RA) are natural cellular metabolites of retinol and mediate biological activity through interaction with nuclear retinoic acid receptors (RAR) and retinoid X receptors (RXR), respectively. Ligand-bound RARs and RXRs influence transcription by interacting with response elements in the promoter regions of retinoid-regulated genes [ 10 ]. Within the ovary, RBP and CRBP are expressed in thecal and granulosa cells, and facilitate the transport of retinol from the blood into developing follicles [ 3 ]. Concentrations of RBP, and its ligand retinol, are highest in the follicular fluid of large preovulatory bovine ovarian follicles, compared to smaller and/or atretic follicles [ 3 ]. RBP synthesis and secretion increase in the oviduct and uterus coincident with the transport of the egg or embryo into these organs [ 11 , 12 ]. The cumulus oocyte complex (COC) may be a target for retinol, since the cells that nurture and communicate with the oocyte, contain transcripts and protein for several RARs and RXRs, RBP and retinaldehyde-2 dehydrogenase (RALDH-2) a metabolizing enzyme [ 13 ]. Bovine oocytes and embryos from the 2-cell to hatched blastocyst stage, also express transcripts for several RARs, RXRs, RBP and RALDH-2, and the inner cell mass and trophectoderm of blastocysts express immunoreactive protein for RAR and RXR [ 14 ]. It has been shown recently that addition of 9- cis RA to in vitro oocyte maturation medium affects trophectoderm differentiation, total cell number and inner cell mass-trophoblast cell ratios, following fertilization in cattle oocytes [ 15 , 16 ]. Together, these studies suggest that the reproductive tract delivers retinol to the oocyte and early embryo which possess key elements of retinoid metabolizing and signaling mechanisms; thus, influencing gene expression, differentiation, and development. The mechanism by which retinol or retinoic acid administration influences oocyte maturation and positively impacts early embryonic development is not known and is the subject of much investigation. Retinoic acid may influence oocyte maturation through its effects on FSH or LH receptor expression as demonstrated in porcine [ 17 ] and rat [ 18 ] granulosa cells. Alternatively, it has been suggested that retinoic acid may increase mRNA quality and processing during maturation mediated by increases in polyadenylation [ 19 ]. Expression of several growth factors is influenced by RA [ 20 ]. Midkine [ 16 ], a member of the heparin-binding growth/differentiation family, is induced by RA and has been shown to improve bovine oocyte and embryonic developmental competence [ 21 ]. In addition, retinoids may promote development through participation in an endogenous oxidative-stress protection mechanism [ 22 ]. In the present study, we investigated the effects of retinol administration to in vitro matured oocytes, and cultured bovine embryos under atmospheric O 2 and reduced O 2 conditions. Results suggest beneficial effects of retinol administration during maturation especially to less competent oocytes, and improved development of embryos cultured under atmospheric oxygen conditions, indicating protection from oxidative stress. Materials and Methods Reagents and Media All chemicals were purchased from Sigma Chemical Company, St. Louis, MO unless otherwise noted. Bovine oocyte collection medium (OCM) was composed of modified M199, 4.2 mM NaHCO 3 , 12 mM HEPES, and supplemented with 2 mM glutamine, 2% fetal bovine serum (FBS, BioWhittaker, Baltimore, MD), and penicillin/streptomycin (Specialty Media, Phillipsburg, NJ). Oocyte maturation medium (OMM) consisted of bicarbonate buffered TCM-199 supplemented with 50 μg/mL of gentamycin, purchased from Specialty Media, 5 μg/mL of FSH purchased from Vetrepharm Canada, Inc. (Ontario, Canada), 0.3 μg/mL of luteinizing hormone (LH) that was generously provided by the USDA, Beltsville, MD, 10% FBS, 0.2 μM sodium pyruvate and 2 mM glutamine. Modified Tyrode's Albumin Lactate Pyruvate (TALP) media used in sperm preparation (SP-TALP), removal of cumulus cells from oocytes (HEPES-TALP) and in vitro fertilization (IVF-TALP) were prepared as described by Parrish et al. [ 29 ]. In vitro culture (IVC) medium was a modified synthetic oviductal fluid (mSOF) [ 30 ] supplemented with 3 mg/mL of BSA, 0.6 mM sodium pyruvate, 2% (v/v) BME essential amino acids, 1% (v/v) MEM non-essential amino acids, and 100 μg/mL of penicillin and streptomycin. All- trans retinol was dissolved in 100% ethanol, appropriate dilutions made, and aliquots were stored at -80°C until use. Retinol was prepared fresh each month and checked on a spectrophotometer for accuracy. The concentration of ethanol during maturation or culture was less than 0.1%. Collection and in vitro maturation (IVM) of oocytes Ovaries from mature, cycling cattle were obtained from an abbatoir and pooled. Cumulus oocyte complexes (COCs) were quickly harvested by slicing follicles (2–5 mm) with a sterile surgical blade, and collecting them in OCM. Intact COCs with homogeneous ooplasm and two or more layers of cumulus cells were selected, washed, and approximately 50 were transferred to 500 μl of pre-equilibrated OMM, and matured for 22–23 hours in a 38.5°C incubator with an atmosphere of 5.0% CO 2 , ambient air, and saturated humidity. In vitro fertilization (IVF) Fertilization (Day 0) was performed with combined semen from two bulls of proven fertility prepared according to the method by Parrish and coworkers [ 29 ]. Briefly, spermatozoa were washed in a discontinuous Percoll gradient (45%/90%) by depositing semen on top of the Percoll layers and centrifuged for 15 minutes at 960 g . The pellet was removed and resuspended in SP-TALP and centrifuged for 8 minutes at 460 g . After removal of the supernatant, the sperm sample was reconstituted in 500 μL of IVF-TALP for a final concentration of 1 × 10 6 spermatozoa/mL. The plate was incubated for 22 hours at 38.5°C in an atmosphere of 5.0% CO 2 and ambient air with saturated humidity. In vitro culture (IVC) Approximately 18 hours after fertilization putative zygotes were denuded of cumulus cells by vortexing in 500 μl of HEPES-TALP for four minutes (Day 1). Putative zygotes (approximately 35–40) were cultured in 500 μL of mSOF for eight days in a 38.5°C incubator in an atmosphere of 5% CO 2 , 7% O 2 and 88% N 2 (first and second experiments) with saturated humidity. The mSOF medium was changed every 48 hours. Cleavage was assessed on Day 3 and blastocyst rate was calculated on Day 8. Experimental Design In the first experiment maturation medium alone was supplemented with all- trans retinol (0, 1.0, 5.0, or 10.0 μM) and embryos were allowed to develop under low oxygen conditions. In the second experiment all- trans retinol was added only to embryo culture medium (0, 1.0, 2.0, 5.0, or 10μM) on days 1, 3, 5, and 7, and the embryos developed in a low oxygen atmosphere. In the third experiment embryos were cultured under atmospheric oxygen conditions (air and 5% CO 2 ) and all- trans retinol (0 or 5μM) was added to embryo culture medium on days 1, 3, 5, and 7. Data Analysis Data were analyzed as an incomplete block design (experiments 1, 2, and 3), or a randomized block design (experiment 4), blocked on plate using mixed model procedures of SAS [ 31 ]. At least six replicates were completed for each experiment. Fisher's protected least significant differences were used for separating least square differences for experiments 1, 2, 3, and a two-tailed Student's T-test was performed on data from experiment 4. Least square means ± S.E.M. are expressed as the proportion of putative zygotes. All data were subjected to a normality test (Shapiro-Wilk, > 0.90) and were found to be normally distributed. Results In the first experiment addition of 5μM retinol during IVM tended to improve (p < 0.07) embryonic development to the blastocyst stage, compared to controls (Table 1 ). The control blastocyst rate was 21.9% compared to 26.1% in 5μM retinol. Addition of 1μM retinol to the maturation medium did not appear to affect embryonic development compared to controls. Retinol (10μM) increased blastocyst development, although not significantly. Cleavage rates did not differ among the four maturation treatments. Table 1 Effect of all- trans retinol addition to bovine oocyte maturation medium (mean ± S. E. M.). Embryos were cultured under low oxygen conditions. Retinol concentration (μM) Putative zygote (n) Cleavage Blastocyst/ putative zygote Blastocyst/ cleaved 0 1095 66.7 ± 2.7 21.9 ± 1.9 a 32.8 ± 2.2 a 1.0 464 65.5 ± 3.9 20.4 ± 2.6 a 31.7 ± 3.1 a 5.0 1069 68.3 ± 3.2 26.1 ± 2.2 b 37.1 ± 2.5 b 10.0 508 70.1 ± 3.9 24.2 ± 2.7 ab 33.8 ± 3.1 ab Values are listed as percentages. ab Means in the same column with different superscripts approach significance (p < 0.07). Further analysis of the maturation data (Table 2 ) revealed that when development to the blastocyst stage of controls was below 20%, 5μM retinol dramatically improved (p < 0.02) embryo development (14.4 % vs. 23.7%). When expressed as blastocyst/cleaved the 5μM retinol treatment also showed a significant improvement in blastocyst development (p < 0.02). Neither 1μM nor 10μM retinol treatment improved embryonic development when compared to those controls that did not achieve a 20% blastocyst rate. Table 2 Effect of all- trans retinol addition to bovine oocyte maturation medium on embryo development among replicate groups where less than 20% of control embryos reached the blastocyst stage (mean ± S. E. M.). Retinol concentration (μM) Putative zygote (n) Cleavage Blastocyst/ putative zygote Blastocyst/ cleaved 0 516 62.7 ± 3.9 14.4 ± 2.1 a 23.7 ± 2.6 a 1.0 185 60.0 ± 6.0 15.9 ± 3.4 ab 26.1 ± 4.2 ab 5.0 530 65.9 ± 4.6 23.7 ± 2.5 b 34.6 ± 3.1 b 10.0 183 63.3 ± 6.7 17.6 ± 3.8 ab 26.7 ± 4.6 ab Values are listed as percentages. ab Means in the same column with different superscripts were significantly different (p < 0.02). Further experiments were conducted during IVC under both low and atmospheric oxygen tensions. Under low oxygen conditions concentrations of 1, 2, and 5μM retinol were not statistically different from controls, and 10μM was deleterious (Table 3 ). Preliminary dose-response studies were performed under atmospheric conditions (data not shown), and additional experiments were continued with the 5μM retinol treatment. Under atmospheric oxygen conditions the 5μM concentration significantly improved blastocyst development compared to controls (p < 0.001) (Table 4 ). Cleavage rates did not differ significantly among embryos treated with and without retinol during culture under low or high oxygen (Tables 3 & 4 ). Fertilization rates did not differ significantly among all experiments (data not shown). Table 3 Effect of all- trans retinol addition to the culture medium (mean ± S. E. M.). Embryos were cultured under low oxygen conditions. Retinol concentration (μM) Putative zygote (n) Cleavage Blastocyst/ putative zygote Blastocyst/ cleaved 0 567 86.1 ± 2.5 26.5 ± 2.4 a 30.7 ± 2.6 a 1.0 312 84.7 ± 3.2 27.1 ± 3.2 ab 32.1 ± 3.5 a 2.0 414 85.3 ± 2.9 28.8 ± 2.8 a 34.1 ± 3.1 a 5.0 303 80.8 ± 3.2 20.2 ± 3.2 a 25.4 ± 3.5 a 10.0 388 81.2 ± 3.0 13.5 ± 3.0 c 16.2 ± 3.3 b Values are listed as percentages. abc Means in the same column with different superscripts were significantly different (p < 0.05). Table 4 Effect of all- trans retinol addition to the culture medium (mean ± S. E. M.). Embryos were cultured under atmospheric oxygen conditions. Retinol concentration (μM) Putative zygote (n) Cleavage Blastocyst/ putative zygote Blastocyst/ cleaved 0 400 73 ± 4.6 14 ± 2.3 a 19.2 ± 3.2 a 5.0 400 74 ± 1.5 28.8 ± 3.0 b 38.9 ± 3.9 b Values are listed as percentages. ab Means in the same column with different superscripts were significantly different (p < 0.001). Discussion In the present study, over 3000 bovine oocytes were used to evaluate effects of retinol supplementation during IVM and IVC on embryonic development to the blastocyst stage. Retinol administration during the maturation period alone resulted in concentration-dependent effects. Whereas the presence of 1μM retinol had no effect on development, 5μM retinol tended to improve blastocyst rate of development, at the p < 0.07 level, compared to controls. At a concentration of 10μM, retinol did not significantly improve embryo development compared to controls. In preliminary studies, higher concentrations (100μM) were observed to be cytotoxic (data not shown). Similarly, exposure of bovine oocytes to low concentrations of 9- cis retinoic acid was shown to improve subsequent blastocyst development but high concentrations were detrimental [ 16 ]. A more striking effect on embryonic development (p < 0.02) was observed by supplementation of 5 μM retinol to groups of oocytes with reduced developmental competence in which development of control oocytes to blastocyst was less than 20%. These results indicate that retinol supplementation during maturation may not benefit oocytes competent to progress, but rather, it improves the viability of oocytes that are developmentally challenged. In support of this, we have shown previously that retinol supplementation during maturation improves developmental competence of bovine oocytes compromised by heat stress [ 32 ]. Since most transcription in the oocyte occurs prior to maturation during preovulatory development, in vitro culture deprives oocytes of much of this activity. Meiotic inhibitors have been used as a potential means of investigating regulation of oocyte transcription and mRNA processing in vitro [ 33 ]. Treatment of cumulus-enclosed oocytes with 9-cis RA during meiotic arrest was observed to improve cortical granule migration, increase subsequent blastocyst development and increase total cell number [ 34 ]. Gomez and co-workers [ 19 ] suggested that retinoid administration may improve mRNA quality based on the observation that 9-cis RA increased poly-(A) mRNA content in meiotically arrested oocytes. Poly-(A) mRNA content of oocytes treated with 9-cis RA or ethanol vehicle was greater in matured oocytes than in oocytes prematured in the presence of 9-cis RA and then matured. Retinol supplementation of embryo culture medium dramatically improved development to the blastocyst stage (p < 0.001) when cultured in an atmosphere of approximately 20% O 2 (air and 5% CO 2 ) but not in an atmosphere of low O 2 (7% O 2 , 5% CO 2 and 88% N 2 ). The present study, and all previous in vitro studies demonstrating a positive effect of retinoid administered during maturation, were performed in an atmosphere of approximately 20% O 2 [ 15 , 16 , 34 ], a practice common to most laboratories. Together, these data indicate that retinoids may protect embryos from oxidative damage, which has been identified as a leading cause of embryonic wastage, especially in vitro [ 22 ]. Mammalian cells, including the oocyte and those of the early embryo, have evolved several mechanisms to protect against ROS damage and maintain appropriate balances in REDOX reactions. Antioxidants present in the oocyte, embryo and/or its environment include vitamins A (retinol), C and E, pyruvate, glutathione (GSH), hypotaurine, taurine, and cysteamine [ 22 ]. Antioxidant enzymes produced by oocytes and embryos include, copper, zinc superoxide dismutase (Cu, Zn-SOD), manganese-SOD (Mn-SOD), glutathione peroxidase (GPX), glutamyl cysteine synthase (GCS), glutathione reductase (GR), catalase and others [ 22 ]. Lonergan and co-workers [ 36 ] have shown that expression of several antioxidant enzymes are up-regulated during in vitro oocyte maturation compared to in vivo maturation indicating that the former environment creates oxidative stress and oocytes respond by activating internal defense mechanisms. Addition of antioxidants to culture medium or culture of embryos in an atmosphere of reduced O 2 has been demonstrated to be beneficial to in vitro survival of embryos from a variety of species [ 22 ]. Retinoids participate in a biological antioxidant network, and have been implicated as important regulators of redox signaling pathways [ 23 , 24 ]. Carotenoids and retinol can quench single oxygen molecules and interact with other antioxidant compounds [ 23 ]. Retinoic acid has been shown to protect against oxidative stress-induced apoptosis by inhibition of the c-jun N-terminal kinase (JNK) activator protein 1 (AP-1) pathway in glomerular [ 26 ] and mesangial cells [ 37 ]. In addition, anti-apoptotic effects of RA were mediated by both nuclear receptor-dependent and independent pathways [ 37 ]. Retinoids may also protect against oxidative damage by maintaining adequate endogenous levels of antioxidant compounds and enzymes. Glutathione is the major non-protein sulphydryl compound found in mammalian cells responsible for strong basal ROS scavenging activity [ 35 ]. Maintenance of adequate GSH levels is essential for oocyte maturation, fertilization and embryonic development [ 22 ]. Retinoic acid inhibited staurosporine-induced GSH depletion in neuronal cells, preventing oxidative damage and apoptosis [ 25 ]. A retinoic acid response element (RARE) has been identified in the promoter region of a specific isoform of glutathione S-transferase-pi (GSTp) in glioblastoma cells [ 38 ] and GPX2 [ 28 ], an enzyme necessary for the conversion and utilization of GSH. RA has also been shown to significantly increase survival, reduce ROS content and increase protein levels of Cu-Zn SOD and Mn-SOD in neuronal cells treated with staurosporine [ 27 ]. Recently, microarray analysis revealed that three genes which encode enzymes involved in GSH synthesis and utilization were RXRα-target genes in mouse liver [ 39 ]. The same study showed that in hepatocytes of RXRα-deficient mice there was a significant reduction in GSH synthesis rate and GSH content [ 39 ]. Together, these data provide strong evidence that in several cell systems, retinoids support and improve endogenous antioxidant defense mechanisms. Conclusions Results from the present study indicate that retinol administration during in vitro maturation particularly improved embryonic development in those oocytes that may have been developmentally compromised. Moreover, retinol addition during in vitro culture, under atmospheric conditions, also improved embryonic development compared to those embryos incubated in a 7% oxygen atmosphere. The mechanisms by which retinoids affect the developmental capacity of oocytes and early embryos may include modulation of expression of growth factors and other developmental genes, improving mRNA quality, and direct and/or indirect affects on antioxidant defense mechanisms. Authors' contributions TL Performed the experiments under low oxygen conditions, helped to coordinate experiments, and drafted the manuscript. DE Performed the experiments under high oxygen conditions and helped to coordinate experiments. JE Coordinated and assisted in the experimental design. JG Conceived of and coordinated the experiments and drafted the manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544594.xml
554977	Interrupted or continuous slowly absorbable sutures – Design of a multi-centre randomised trial to evaluate abdominal closure techniques INSECT-Trial [ISRCTN24023541]	Background 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. Design 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. Conclusion 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.	Background 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 [ 1 ]. A major problem after median laparotomy remains the adequate technique of abdominal fascia closure. In prospective studies the incidence of incisional hernias varies from 9% to 20% [ 2 , 3 ]. Wound infection, obesity and suture closure technique are addressed as major risk factors for the development of an incisonal hernia [ 4 , 5 ]. Whereas 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) [ 6 - 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 [ 5 ]. None of the prior studies comparing rapidly absorbable braided materials with interrupted sutures versus slowly absorbable monofilament suture materials in a continuous technique were able to determine a definite superiority for one technique. Possible reasons may be small numbers of patients in each group or short follow-up [ 9 - 12 ]. We 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 [ 11 ] the superiority for one closure method has not been definitely proven. The randomisation procedure will be done stratified for participating centers. The planned sample size is at least 600 patients with a follow up period of three years. INSECT 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. Design Trial organization 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. Coordination The trial is coordinated by the SDGC, which is responsible for overall trial management, trial registration (International Standard Randomised Controlled Trial Number (ISRCTN 24023541), ), database management, quality assurance including monitoring, reporting and for the scientific program of all trial related meetings. Investigators 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. Adverse events committee 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. Burst abdomen, pulmonary infection and wound infection are secondary endpoints, but are also defined as Adverse Events (definitions see table 1 ). Burst abdomen and postoperative pulmonary infection will even be always a Serious Adverse Event. The term 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. Table 1 Definition of early onset and late complications Complication Definition Burst abdomen Postoperatively missing continuity of the abdominal fascia in combination with a wound dehiscence with consecutive relapse operation. Wound infection Redness, wound dehiscence with secretion either of putrid or caliginous, smelly fluid or requiring antibiotic treatment or surgical intervention. Postoperative pulmonary complication Infection of the lung with either evidence of increased infection parameters (CRP > 2 mg/dl and/or leukocytes> 10 0000/ml) which are not caused by a different pathologic process or evidence of pulmonary infiltration in the chest x-ray, requiring antibiotic therapy. Incisional hernia Postoperative evidence of a fascia dehiscence after completed superficial wound healing with or without prolapse of abdominal organs, confirmed by abdominal ultrasound. Analysis of safety related data is performed with respect to frequency of: • Serious Adverse Events and Adverse Events stratified by body-system • Adverse Events stratified by severity • Adverse Events stratified by causality. Study material supply Study materials for all centres will be acquired by the BBD Aesculap company. Each type of suture material derives from a single batch to eliminate material inconsistencies. All materials are delivered to the participating centres by local representatives of the sponsor who also guarantee local first-level-support. On-site monitoring During recruitment of patients each centre is monitored on site according to good clinical practice (GCP) 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. Ethics, Informed Consent and Safety 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. Patient selection 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 2 . Table 2 Eligibility Criteria Inclusion criteria Exclusion criteria • Age equal or greater than 18 years • Expected survival time more than 12 months • Patients undergoing primary and elective median laparotomy (patients with prior laparoscopy or abdominal operation via paramedian incision (e.g. appendectomy) may be included in the trial) • BMI < 35 • Expected length of incision > 15 cm • Patient must be able to give informed consent • Patient has given informed consent • Peritonitis • Emergency surgery • Participation in another intervention-trial with interference of intervention and outcome of this study • Coagulopathy A group of disorders of the blood clotting (coagulation) system in which bleeding is prolonged and excessive with abnormal values in the blood laboratory. • Severe psychiatric or neurologic diseases • Lack of compliance • Drug- and/or alcohol-abuse according to local standards • Current immunosuppressive therapy (more than 40 mg of a corticoid per day or azathioprin) • Chemotherapy within 2 weeks before operation • Radiotherapy of the abdomen completed longer than 8 weeks before operation • Inability to follow the instructions given by the investigator or the telephone interviewer (insufficient command of language, dementia, lack of time) • Lack of informed consent Study objectives 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 [ 13 ]. The ranking by the surgeon is done once for each surgeon before the operation. Patients are completing the ranking twice: first at inclusion and second at their discharge, to investigate if the patient's initial perspective changes within the hospital stay. Those various outcome parameters will be evaluated as part of an additional scientific project to build up a basis for further relevant questions in abdominal wall closure. Randomisation and surgical technique A 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 (material as described above). 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 1 ). Neither a subcutaneous closure nor a subcutaneous drainage is to be inserted. Skin closure is done with skin clips. Measurement of the length of scar in centimeters (cm) is performed. A detailed description of the required surgical technique is given in the INSECT-study protocol enhanced by images and sketches as well as video material used during the investigator meeting and provided to all centres and investigators. Figure 1 Principles of continuous abdominal wall closure. A Anchorage of the suture cranially/caudally outside the incision B Intersection of the loops in the middle of the incision C Knotting of each loop Images reprinted with courtesy of Mrs. B. Wiehn, BBD Aesculap, Tuttlingen Germany In an investigator meeting before trial initiation (March 5 th – 6 th , 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. Blinding The patient is blinded for the technique of abdominal wall closure as the randomisation is performed intra-operatively. The patient will remain blinded until the assessment of the primary end-point at one year post-operatively. If feasible within the infrastructure of the participating centre, observers should be independent and not involved in completing the CRF and would thus also be blinded for the used technique. Follow up Patients are observed for 30 days postoperatively for early onset complications defined as secondary endpoints such re-admittance for burst abdomen. One year after the operation the primary end-point will be assessed by documenting the incidence of incisional hernia with a physical examination and ultrasound of the abdominal wall. Follow up is completed 3 years after the primary operation to register any long-term complications of the used method for abdominal wall closure and to document any late-occurring hernias (see table 3 for detailed follow up). Table 3 Flow Chart INSECT-Trial Visit 1 (=Screening) 2 (OP) 3 (day 2 post OP) 4 (day of discharge) 5 (6 months +/- 1 month post OP) 6 (12 months +/- 1 month post OP) 7 (three years after operation) Extra-visit (secondary endpoint, AE or SAE) Past medical history X Informed consent X Physical examination including the personal data X X X Basic study-related examination I (for each secondary endpoint, AE, SAE) X (ranking of the patient before operation) X (ranking of the surgeon) X X (ranking of the patient after operation) X X (ranking of the patient after operation) X Basic study-related examination II X Ultrasound of the abdominal wall X X X Lung function test X X Medication X X X X Past medical history: past medical history, past surgical history, indication for operation, diabetes, renal insufficiency, smoking, lung disease Personal data: gender, date of birth, height in cm, weight in kg Basic study related examination I: physical examination for evaluation of all secondary endpoints including adverse events/serious adverse events where appropriate Basic study related examination II: telephone visit of patient and/or practitioner and physical re-examination if necessary in case of unclear abdominal wall status Physical examination: vital signs (blood pressure systolic/diastolic in mmHg, heartrate in /min), prior abdominal incisions, rectus diastasis Ultrasound: standard abdominal wall investigation (if hernia present: length and width in cm) Lung function test: FEV, vital capacity % Data management and quality assurance Investigators enter data directly in paper-based case report form (CRF). 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 considerations and sample size estimation 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. To enable multiple comparisons in this three-armed study the closed testing procedure will be used[ 14 ]. All three treatment groups are considered separately without assuming any pre-specified monotonic trend among groups. All testing is done two-sided. The three elementary hypotheses address pairwise comparisons of the incisional hernia rates R 1 , R 2 , and R 3 . These hypotheses H 12 : R 1 = R 2 , H 13 : R 1 = R 3 , and H 23 : R 2 = R 3 , are all contained in the global null hypothesis H 123 : R 1 = R 2 = R 3 . 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." [ 15 ]. The calculation of sample size is based on literature data, as summarised most recently by van 't Riet et al [ 11 ]. With interrupted suturing with an absorbable material, such as Vicryl ® , incisional hernias were seen in about 13% of patients. This number is primarily based on the four-armed trial by Wissing et al. [ 16 ]. With the use of slowly absorbable materials, such as PDS ® , 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 [ 17 ]. 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 ® groups over the first postoperative year. Smaller differences are also unlikely to be of clinical relevance. Assuming annual hernia incidences of 13% and 4% in at least two of the groups, a raw sample size of 172 patients per group can be calculated with a two-sided alpha of 0.05 and a beta of 0.20 (employing Fisher's exact test). We used the PS power and sample size program of Dupont and Plummer (Version 1.0.17) for sample size estimation (freely available at: ). 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 (Figure 2 ) [ 18 ]. Figure 2 INSECT-Trial according to CONSORT (Moher et al. Lancet 2001) In the primary intention-to-treat analysis, testing will start with the global null hypothesis H 123 : R 1 = R 2 = R 3 . Only if this global test is significant at the 0.05 level, the three elementary hypotheses H 12 : R 1 = R 2 , H 13 : R 1 = R 3 , and H 23 : R 2 = R 3 will be tested next. These pairwise comparisons (Vicryl ® versus PDS ® , Vicryl ® versus MonoPlus ® , and PDS ® versus MonoPlus ® ) 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. To control for possible differences with regard to surgical procedures (colorectal vs. gastric or pancreatic operations), 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. Secondary endpoints, demographic and other variables 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 (ordinal or nominal) 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. Closing of the clinical database and follow up database 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. Current status and planning 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 5 th and 6 th , 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. Discussion The 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 [ 19 , 20 ]. This mentioned transparency increases reliability and validity of the results when a detailed description of the experiment is published prior to the conduction. The assumption how many patients should be screened to include the sufficient number of patients based on the sample size calculation needs to be transparent in order to evaluate whether results from this trial are transferable to daily practice. Therefore a flow chart according to the CONSORT statement should be included in study protocols (Figure 1 ). However, many important details of a randomised surgical trial are interesting to the reader and can not be published together with the final results. It also seems out of the question that medical journals that surgical trials should be internationally registered [ 21 ]. INSECT 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 [ 6 , 7 , 11 ]. Therefore, the technique and materials for abdominal wall closure are still determined by local material supply and surgical tradition. These preconditions and a detailed literature research gave us reason to design a trial comparing three standardised common techniques of abdominal fascia closure in abdominal surgery. Several 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 [ 11 ]. Two different suture techniques and materials became clear to show the best results in abdominal fascia closure and revealed the lowest incidence for incisional hernias: slowly absorbable continuous (or running) sutures and absorbable interrupted sutures. Aside from those two groups of suture techniques (continuous versus interrupted sutures) we included a third group using the same technique as one of the other groups (continuous suture) but using a different suture material (monofilament suture with longitudinal elasticity) which is supplied by a different company. INSECT depicts the surgical reality and variety of abdominal wall closure as it compares not only different suture techniques but also several suture materials offered by different companies. Therefore, the results of INSECT will be relevant for the majority of surgeons as the materials and techniques used in this trial are widely accepted in daily use. An 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 [ 4 ]. Because 50–70% of incisional hernias will occur within one year after operation we selected the frequency of incisional hernias at one year postoperatively as the primary end-point of our study. All other relevant objectives such as wound infection, pain, pulmonary impairment, etc. were included as secondary endpoints into the trial design. Additionally a qualitative analysis for surgeons and patients was planned to evaluate the importance of different outcome variables between surgeons and patients. A 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. Conclusion 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 [ 6 , 22 ]. Most surgeons rather use particular techniques because they are trained in than using the most evidence-based technique. The Study Centre of the German Surgical Society (SDGC) was founded 2003 in order to ensure that surgical techniques are more evidence based and not simply the result of a surgical dogma. This is the first multi-centre trial designed and conducted by the SDGC after a special Study Group was founded. We are aware of the difficulties and problems of performing large surgical multi-centre trials and, therefore, we decided to start with a trial examining a fundamental, "simple" and daily performed surgical technique. As the abdominal fascia closure is largely based on tradition rather than evidence, the results of the INSECT trial will help to create more evidence and to guide surgeons to a critical review of their surgical routine. Abbreviations AWC Abdominal wall closure CRF Case Report Form GCP Good Clinical Practice ICH International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use IEC Independent Ethics Committee ITT Intention-to-treat-analysis PDS ® Polydioxanone (absorbable suture material) RCT Randomised Controlled Trial SDGC Study Centre of the German Surgical Society Competing interests The author(s) declare that they have no competing interests. Authors' contributions 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. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC554977.xml
554787	The relationships between depression and other outcomes of chronic illness caregiving	Background Many caregivers with chronically ill relatives suffer from depression. However, the relationship of depression to other outcomes of chronic caregiving remains unclear. This study tested a hypothesized model which proposed that hours of care, stressful life events, social support, age and gender would predict caregivers' outcomes through perceived caregiver stress. Depression was expected to mediate the relationship between perceived stress and outcomes of chronic caregiving (physical function, self-esteem, and marital satisfaction). Methods The sample for this secondary data analysis consisted of 236 and 271 subjects from the Americans' Changing Lives, Wave 1, 1986, and Wave 2, 1989, data sets. Measures were constructed from the original study. Structural equation modeling was used to test the hypothesized model, and an exploratory structural modeling method, specification search, was used to develop a data-derived model. Cross-validation was used to verify the paths among variables. Results Hours of care, age, and gender predicted caregivers' outcomes directly or through perceived caregiver stress (p < .01). Depression mediated the relationship between perceived stress and psychological outcomes and explained 40% and 11% of the variance in self-esteem and marital satisfaction, respectively. Conclusion Depression predicted psychological outcomes. Whether depression predicts physical health outcomes needs to be further explored.	Background It is estimated that 31–55% of caregivers of chronically ill elderly relatives experience depression [ 1 ], and depression is likely to be one of the first [ 2 ] and most enduring psychological outcomes for caregivers [ 3 , 4 ]. Caregivers' depression scores have been found to be substantially higher than those of the general population [ 1 , 5 , 6 ], and higher levels of caregiving stress have been related to greater depression [ 7 , 8 ] and to more depressive symptoms in caregivers [ 9 ]. Factors that may be related to stress and depression in caregivers include hours of care, stressful life events, social support, age, and gender. Studies have found that hours of care were significantly related to caregivers' anxiety/depression and somatic symptoms [ 10 , 11 ], and to their emotional and physical strain [ 12 ]. Though stressful life events have been associated with both psychological wellbeing [ 13 , 14 ] and physical symptoms among the general population [ 14 ], no studies have examined stressful life events in combination with caregiving stress. Stressful life events might have additional impact on caregivers' health other than chronic caregiving. Social support may enhance the ability of the individual to cope with events or change the individual's cognitive appraisal of events [ 15 ]. Quayhagen and Quayhagen found that caregivers who reported needing more social support had lower well-being scores than other caregivers [ 16 ]. In other studies, low social support predicted higher perceived burden [ 7 ], and adverse social contacts were associated with increased stress [ 17 ]. Age has been shown to have indirect effects on depression through its influence on perceived stress, the coping process, and perceived efficacy [ 18 ]. Younger caregivers experience more distress than older caregivers [ 19 , 20 ], and they express more subjective burden than older caregivers [ 21 ]. Gender has also been shown to have effects on depression. Female caregivers report more distress [ 1 , 22 ] and higher psychiatric morbidity [ 5 ] than male caregivers. Tsai et al. have suggested that stress and depression are emotional aspects of coping mechanisms and depression is the outcome of perceived caregiver stress [ 23 ]. Stress has in turn been shown to be the strongest predictor of depression in caregivers [ 24 ]. Though depression has been associated with caregivers' physical health [ 25 , 26 ], the data on physical health are less consistent than on psychological health. Some studies have found that caregivers had poorer self-reported health than non-caregivers [ 27 - 29 ], more chronic illnesses [ 30 ], and lower immune function [ 31 ]; and they used more health care services and took more prescriptions [ 27 ]. Convinsky et al. reported that depression was associated with physical function dependence. Caregivers with functional dependence has 2.53-fold chance to be depressed as compared to those who with functional independence [ 32 ]. Other studies, however, have found that caregivers did not use more medical services [ 33 ] or rate their physical health as less satisfactory than the general population [ 6 ]. Further, as Schulz, Visintainer and Willamson point out, even though some studies have suggested possible effects of caregiving on physical health, the evidence is confounded by sampling bias, inadequacy of measurements, and subjective appraisals [ 34 ]. Although the associations between depression and self-esteem and marital satisfaction have been examined extensively, only a few studies have been conducted in the context of caregiving. Caregivers have been shown to have lower self-esteem [ 35 ], and this has been associated with depressive symptoms [ 10 ]. Caregivers who had higher self-esteem experienced less depression [ 36 ]. In one study, depressed caregivers were more likely to experience less marital satisfaction [ 37 ]. Also, high levels of marital conflict were associated with high levels of depression in adult daughter caregivers [ 38 ]. Finally, spousal caregivers reporting low marital cohesion and satisfaction had more depressive symptoms [ 39 ]. A recently developed Theory of Caregiver Stress [ 23 ] based on theoretical propositions from the Roy adaptation model [ 40 ] suggests that depression is the mediator between perceived stress and self-esteem and marital satisfaction. However, the relationships of depression to other outcomes of caregiving, such as physical function, self-esteem and marital satisfaction, remain unclear. The research reported here therefore explored these relationships. We proposed that hours of care would be the primary source of caregiver stress. Stressful life events, social support, age, and gender were antecedent variables and expected to influence caregivers' outcomes through caregiver stress. Depression was conceptualized as a mediator between caregiver stress and other outcomes of chronic caregiving (physical function, self-esteem, and marital satisfaction). Thus, a high level of stress was expected to lead to a high level of depression, which in turn would result in lower levels of physical function, self-esteem, and marital satisfaction. Methods Sample Data for the study were obtained from the Americans' Changing Lives (ACL) Survey: Wave 1, 1986 (N = 3,617), and Wave 2, 1989 (N = 2,867) [ 41 ]. The ACL collected longitudinal data on subjects aged 25 years and over in the continental United States. Individuals residing in group homes or institutions were not included. The survey used multistage-stratified probability sampling, with Blacks and elderly (60 years and older) oversampled. We used Wave 2 data to test hypotheses and build a data-derived model. Wave 1 data were then used for model validation. Only individuals with experience in caregiving to a chronically ill aged relative were included in the analyses reported here. This reduced the number of cases available for study to 335 from Wave 1 and 271 from Wave 2. The two samples were not completely independent because 99 cases were included in both waves; therefore, to ensure the independence of samples, these 99 cases were dropped from Wave 1. The final samples from Wave 1 and Wave 2 were thus 236 and 271, respectively. Measures Since the study was a secondary data analysis, indicators of the study variables (hours of care, age, gender, social support, stressful life events, perceived stress, depression, physical function, self-esteem and marital satisfaction) were selected from the Americans' Changing Lives Survey questionnaires, based on face validity. That is, the questions selected gave the appearance of measuring the content of interest. Exploratory factor analysis, confirmatory factor analysis, and internal consistency tests were then conducted to confirm the underlying structures of established scales and develop outcome measures for the current study. Hours of care were the total hours estimated by the caregiver in the past year, categorized as less than 20 hours, 20 to 39 hours, 40–79 hours, 80–159 hours, and 160 hours or more. Providing more hours of care was expected to indicate more burden of caregiving. Stressful life events were measured by a 12-item checklist of negative or undesirable events, such as being robbed or burglarized, losing a job, being physically attacked, or experiencing the death of spouse, death of a parent, death of a close friend/relative, serious illness, life-threatening illness/accident, divorce/separation, serious financial problem, death of children, and other such events. Respondents were asked to report whether they had experienced any of these events within the past 2 years. A simple score, the stressful life events index, was created by summing the number of events reported by each respondent. A high score reflected more stressful life events. Social support was measured by two items: friends/relatives' love and care, and their willingness to listen. Alphas reliabilities were .73 and .74 for Wave 1 and Wave 2, respectively. Higher scores indicated greater support from friends/relatives. Demographic data included age, defined as the chronological age of the caregiver, and gender, coded as biological sex identity. Perceived caregiver stress was measured by one item asking how much stress the caregiver felt about caring for or arranging care for the elderly relative. Responses were on a 5-point scale ranging from not stressful to very stressful; a higher score reflected more perceived stress. Other studies have shown that stress was associated psychosocial well-being, such as depression [ 42 , 43 ]; in this study the correlation between perceived caregiver stress and depression was .25 (p < .001). Depression was measured by the 11-item Center for Epidemiological Studies Depression (CES-D) scale [ 44 ], which assesses mood and level of overall functioning in the last 7 days. The CES-D was originally developed as a 20-item unidimensional scale. The shorter 11-item CES-D version contains items on feeling depressed, restless, happy, lonely and sad; feeling that people dislike me; people are unfriendly; I enjoy life (reverse scored); I have a poor appetite; cannot keep going; and everything is an effort. The items are rated on a 3-point scale from "hardly ever" to "most of the time." Higher scores indicate higher levels of depression. Based on exploratory factor analysis, three factors of the CES-D scale – depressed and positive mood, somatic symptoms and interpersonal relations – were identified as indicators of the latent variable, depression Physical function was defined as consisting of functional health, number of chronic illnesses, and self-rated health. Functional health was measured by asking the caregiver whether the caregiver was bedbound, and whether the caregiver had difficulty bathing, climbing stairs, walking, or doing heavy housework, and the degree of difficulty of these tasks. Higher scores reflected a higher level of physical function. The number of chronic illnesses was the sum of the following: arthritis or rheumatism, lung disease, hypertension, heart disease, diabetes, cancer, circulation problems, stroke, fracture, and urinary incontinence. A low score on this measure indicated high physical function. Self-rated health was measured by a single item that asked caregivers to rate their own health on a 4-point scale ranging from poor to excellent. A high score reflected high physical function. The caregiver's self-esteem was measured by five items: "I take a positive attitude toward self," "I am no good at all," "I see myself as a failure," "I have the feeling of being pushed around in life," and "I perceive myself able to solve problems." These items were measured on a 4-point scale ranging from strongly agree to strongly disagree. A higher score indicated higher self-esteem. Marital satisfaction was also measured by five items: "Overall satisfaction with relationship," "love and affection expressed from spouse or significant other," "spouse treats me well," "thinking about divorce or separation," and "things happened that I can never forget." Higher scores indicated more marital satisfaction. Cronbach's alphas for all measures were above the acceptable criterion of .70 in both waves except for self-esteem in Wave 1. However, that measure was on the margin of acceptance, at .68. Since Cronbach's alpha is a conservative estimate of internal consistency [ 45 ], the self-esteem index was retained. Analytic procedure Univariate and bivariate analyses were used to examine the descriptive findings. To test the appropriateness of the indicators for each latent variable in both waves, the following procedures were used. First, a single indicator was extracted when applicable (e.g., for social support, self-esteem, and marital satisfaction), and summary scale scores were used as single indicators. This strategy was used to reduce the number of parameter estimations in a complex model; it is considered appropriate when individual factor item loadings in a specific scale are high [ 46 ]. Second, for all latent variables with single indicators (i.e., hours of care, stressful life events, social support, age, gender, perceived caregiver stress, self-esteem, and marital satisfaction), the measurements were assumed to be perfect (with 0% error). This conservative estimation was made since increasing measurement errors would induce artificial correlations among the latent variables in the measurement model. Thus, a full factorial loading of 1.0 was assumed for all single indicators in the subsequent latent variables. For latent variables with multiple indicators (i.e., depression and physical health), one factor loading was arbitrarily set to 1.0 to test the relative contribution of the factors. Error variances were not allowed to correlate, but all the latent variables were allowed to correlate with each other. The confirmatory factors analysis indicated that all factor loadings were above 0.4 and significant (p < .01), and they accounted for at least 16% of the true score variance [ 47 ]. The only exception was the "interpersonal" factor in depression, with a factor loading of 0.39. Although it was slightly below the required value of 0.4, it was included because it is a well established measure of depression. The factor loading and measurement error for each indicator are shown in Table 1 . Table 1 Standardized factor loadings and measurement error variances for the measurement model predicting caregiver stress Latent variable Indicators Factor loading (Measurement error) Hours of care Hours of care 1.00 a (.00) b Stressful life events Number of stressful life events 1.00 a (.00) b Social support Friend/relatives positive support 1.00 a (.00) b Age Age 1.00 a (.00) b Gender Gender 1.00 a (.00) b Perceived stress Perceived caregiver stress 1.00 a (.00) b Depression CES-D Depressed & positive mood .75 a (.44) CES-D Somatic symptoms .74 (.45) CES-D Interpersonal .39 (.84) Physical function Functional health .55 a (.70) Numbers of chronic illness .65 (.57) Self-rated health .77 (.41) Self-esteem Self esteem/mastery index 1.00 a (.00) b Marital satisfaction Marital satisfaction index 1.00 a (.00) b Factors and measurement errors were from the completely standardized solution. All factor loadings and measurement errors were significant at .01 level in the preliminary measurement model. a Parameter was fixed to 1.0 in the unstandardized solution. b Parameter was fixed to 0 in the unstandardized solution. Table 2 Sample characteristics and comparisons by waves Wave 1 Wave 2 Measure n (236) % M (SD) n (271) % M (SD) p a Hours of care <20 hours 38 16.1 30 11.1 .45 20–39 hours 28 11.9 39 14.4 40–79 hours 35 14.8 48 17.7 80–159 hours 35 14.8 42 15.5 ≥160 hours 100 42.4 112 41.3 Stressful life events .57 (.69) .50 (.64) .13 Social support 7.65 (1.95) 7.91 (1.77) .12 Age, in years 53.56 (16.36) 53.41 (14.43) .91 Gender Male 85 36.0 86 31.7 .31 Female 151 64.0 185 68.3 Perceived caregiver stress Not at all stressful 46 19.5 57 21.0 .75 Not too stressful 62 26.3 78 28.8 Somewhat stressful 77 32.6 76 28.0 Quite stressful 26 11.0 35 12.9 Very stressful 25 10.6 25 9.2 Physical function 1) Functional health Most severe impairment 7 3.0 9 3.3 .87 Moderately severe impairment 19 8.1 17 6.3 Least severe impairment 18 7.6 23 8.5 No impairment 192 81.4 222 81.9 2) Number of chronic illnesses 1.26 (1.28) 1.37 (1.34) .36 3) Self-rated health Excellent 34 14.4 42 15.5 .95 Very good 88 37.3 95 35.1 Good 65 27.5 79 29.2 Fair 38 16.1 45 16.6 Poor 11 4.7 10 3.7 Self-esteem 16.02 (3.11) 16.84 (2.86) .00 Marital satisfaction b -0.12 (3.66) 0.05 (3.61) .67 Depression 16.00 (4.14) 15.15 (3.87) .02 a Statistical significance is determined either by t-test or chi-square. b Score of marital satisfaction is standardized. A covariance matrix derived from data in the Wave 2 sample was analyzed as input data in the process of model testing. Hypothesized models were tested using the maximal likelihood procedure in the LISREL statistics program. The model tests used absolute goodness-of-fit indices (Chi-square [χ 2 ], the goodness-of-fit index [GFI], and the adjusted goodness-of-fit [AGFI]) and comparative fit indices (change in Chi-square [Δ χ 2 ], the relative noncentral index [RNI] and the relative normed fit index [RNFI]). Values of GFI, AGFI, RNI, and RNFI between 0.90 and 1.00 were considered to indicate a good fit between the model and the data [ 48 ]. An exploratory structural modeling method, specification search [ 49 - 51 ], was then used to develop the data-derived model for the Wave 2 sample. The specification search procedure removed all invalid paths in the hypothesized model and added plausible paths suggested by the modification index. Cross-validation was performed to verify that this data-derived model was valid and stable across samples. In this procedure, the data-derived model was cross-validated by the Wave 1 sample, with both Wave 1 and Wave 2 data sets as input files at the same time. Results Descriptive findings The characteristics of the two wave samples are summarized in Table 2 . Less than half the caregivers spent more than 160 hours per year taking care of their chronically ill relative (42.4%, Wave 1 and 41.3%, Wave 2). About half reported no stressful life events in the past 2 years (51.7%, Wave 1 and 57.6%, Wave 2); 38% in Wave 1 and 35% in Wave 2 reported one stressful life event in the past 2 years, and 10.1% and 7.4% in Waves 1 and 2, respectively, reported two or more such events. There was no significant difference in social support between the two samples, although mean scores on social support were slightly higher in Wave 2 (M [SD] = 7.91 [1.77]) than in Wave 1(M [SD] = 7.65 [1.95). The average age was 53 in both waves. Sixty-four percent of the caregivers in Wave 1 and 68% of those in Wave 2 were female. Respondents in both waves rated perceived caregiver stress similarly: 21.6% and 22.1% in Waves 1 and 2, respectively, reported "quite and very" stressful experiences, while over 45% in both waves reported that their experiences were not at all stressful or not too stressful. Caregivers' physical function and marital satisfaction were also similar in both waves. Approximately 81% of the caregivers in each wave reported no functional impairment. Only approximately 20% of caregivers rated their health fair or poor, and the averages numbers of chronic illnesses were 1.26 and 1.37 for Waves 1 and 2, respectively. Scores on caregivers' marital satisfaction were -. 12 and .05 for Waves 1 and 2, respectively; the difference was not significant. However, caregivers in Wave 1 had significantly lower self-esteem scores than those in Wave 2 (16.02 vs. 16.84, p < 0.01). Depression also differed significantly (p < 0.05). Respondents in Wave 1 reported more depression than those in Wave 2, with mean scores on the 11-item CES-D of 16.00 (SD = 4.14) and 15.15 (SD = 3.87) for Waves 1 and 2, respectively. The hypothesized model When the hypothesized model was tested to determine whether depression mediated the relationship between perceived caregiver stress and caregiving outcomes (physical function, self-esteem, and marital satisfaction), the statistics showed a moderate fit between the model and the data (χ 2 = 237.22; d.f. = 73; GFI = .89; AGFI = .84; RNFI = .85). More hours of care and female gender predicted greater caregiver stress, accounting for 7% of the variance in stress, as shown in Figure 1 . Greater perceived caregiver stress was associated with higher depression. Higher levels of depression in turn predicted poorer physical function, lower self-esteem, and lower marital satisfaction, accounting for 40%, 40%, and 15% of the variance in physical health, self-esteem, and marital satisfaction, respectively. Depression served as a mediator between perceived caregiver stress and caregivers' physical function, self-esteem, and marital satisfaction. Figure 1 The hypothesized model The data-derived model Since the hypothesized model fit the data only moderately well, a data-derived model was built to compare with the hypothesized model. After all the insignificant paths in the hypothesized model had been dropped, paths were added at each step to improve the goodness-of-fit statistics based on the modification index and pre-set assumptions. The resulting data-derived model had a good fit with the data (χ 2 = 147.73; d.f. = 74; p = .00; GFI = .93; AGFI = .90; RNFI = 1.00), close to that of the measurement model, with an insignificant difference (Δ χ 2 = 33.58; Δ d.f. = 34; p = ns). The strength and direction of the relationships among the latent variables are shown by the standardized coefficients in Figure 2 . The data-derived model accounted for 6%, 10%, 57%, 40%, and 11% of the variance in perceived caregiver stress, depression, physical function, self-esteem, and marital satisfaction, respectively. Hours of care were predicted by age: older caregivers provided more hours of care than younger caregivers. Social support was predicted by gender and depression: males and depressed caregivers tended to have less social support. Being a female caregiver and giving more hours of care made the caregiver more susceptible to perceived stress. Having less caregiver stress and fewer stressful life events reduced the chances of depression. Older age and higher levels of depression tended to result in poorer physical function. Greater depression was also associated with less self-esteem and less marital satisfaction. The data-derived model confirmed that perceived stress mediated the relationships between hours of care, gender, and depression, while depression was the mediator between perceived stress and other outcome variables (physical function, self-esteem, and marital satisfaction). Figure 2 The data-derived model Cross-validation In order to test the robustness of the paths across the samples, the data-derived model was cross-validated by Wave 1 sample. The results are summarized in Table 3 . The validating process showed the data-derived model was not confirmed only in Step 2 when the paths between endogenous variables were constrained (p < .01). This step showed that some paths between endogenous variables were not confirmed by Wave 1. To investigate the differences between Wave 1 and Wave 2, especially in the Beta linkage, each path was examined individually. The results showed that the differences came from the links between stressful life events and depression, and depression and physical function. That is, the cross-validation procedure confirmed that the data-derived model was stable across two waves of data, except in two paths (stressful life events to depression, and depression to physical function). These two paths need to be further examined. Table 3 Summary of cross-validation for the data-derived model Comparison to previous model Steps and Purpose χ 2 df p χ 2 diff df diff p Step 0 Factor loadings, path coefficients, factor variance, and covariance were all set to be inequality across group 369.14 148 .00 Step 1 Constrain factor loadings 376.21 152 .00 7.07 4 n.s. Step 2 Constrain factor loadings, and the paths between endogenous variables (beta linkages) 390.92 159 .00 14.71 7 <.01 Step 3 Constrain factor loadings, the paths between endogenous variables (beta linkages), and the paths between exogenous variables and endogenous variables (gamma linkages) 396.04 163 .00 5.12 4 n.s. Step 4 Constrain factor loadings, path coefficients, and error variance 401.39 169 .00 5.35 6 n.s. Step 5 Constrain factor loadings, path coefficients, error variance, and factor variance 413.63 179 .00 12.24 10 n.s. Discussion The hypothesized model postulated that hours of care would be the most important factor in perceived caregiver stress, and other factors would include stressful life events, social support, age, and gender. Higher perceived caregiver stress was expected to result in more depression, which in turn would lead to poorer health function, lower self-esteem, and lower marital satisfaction. These expectations were only partially supported by the data. The data-derived model suggested that age had an indirect effect on perceived caregiver stress, through hours of care. Stone et al. found that older caregivers tended to assume the role of primary caregiver in attending to their chronically ill relatives [ 28 ]. Thus, it is likely that the older caregivers in this sample assumed more hours of care than young caregivers, resulting in more caregiver stress. Age was also found to predict physical function. George and Gwyther noted that spouse caregivers were more susceptible to diminished physical function than other caregivers and this was probably caused by their older age [ 33 ]. Stressful life events failed to predict perceived caregiver stress; instead, stressful life events predicted caregivers' outcomes through depression. One explanation for this might be that stressful life events and caregiver stress work independently in predicting caregivers' outcomes. One other study found that stressful life events did not influence health outcomes through perceived stress; rather, they were a confounding factor in predicting health [ 52 ]. And Stone et al. reported that stressful life events led directly to adverse health outcomes instead of being mediated by perceived stress [ 53 ]. Hours of care and perceived caregiver stress were expected to play important roles in caregiver outcomes. However, while hours of care predicted caregiver stress, hours of care was not the only nor the most important determining factor. Further, perceived caregiver stress explained only a small amount of the variance in depression. This is consistent with Pruchno et al's finding that caregiving had little impact on depression or the physical health of the caregiver [ 25 ]. The present findings support the view that caregivers of chronically ill relatives adapt to the demands of the situation and stabilize or even improve over time [ 54 ]. It is possible that the chronic nature of the recipient's illness enables the caregiver to adjust to persistent needs and reestablish a balanced life over a period of providing care. Viewed from this perspective, hours of care should not be expected to have a major effect on perceived caregiver stress. It can also be argued that caregivers confront many problems other than caregiving burden, and the impact of chronic caregiving may be diluted by competing daily stressors or stressful life events. Thus, the relationship between hours of care and perceived caregiver stress may not be as clear in long-term caregiving as in short-term caregiving to an acutely ill relative. Unexpectedly, in this study social support had no impact on perceived caregiver stress; but this is not unprecedented: similar results were reported by Lawton et al. [ 8 ]. We used friend/relative positive support as the indicator of social support; however, some aspects of social support may be more important than others in reducing caregiver stress. It is also possible that the measure used here was not sensitive enough to detect actual social support. Research has shown that low social support makes people more vulnerable to depression, and that has been clearly demonstrated for the elderly [ 55 - 58 ]. However, in our study, depression predicted social support rather than the other way around. Depressed persons may withdraw from some aspects of life, including their social network, especially friends and non-nuclear family relatives. The data-derived model showed that, as expected, depression mediated the relationship between perceived caregiver stress and self-esteem and marital satisfaction. However, the relationship between depression and physical function was not confirmed by the cross-validation. This is inconsistent with the findings of Pruchno et al. [ 25 ] and Zanetti et al. [ 26 ]. One possible explanation is that in this sample, depression and physical function may both have been outcomes of important factors that were not included in the study. Clearly, the relationship between depression and physical function needs to be further examined. The study was limited to the variables in the original Americans' Changing Lives survey, constricting our choices in operationalizing constructs. Further, the study was cross-sectional and consequently was limited in testing the causal relationships depicted in the model. Although the findings provide preliminary evidence of causal relations among the variables, better examination of causality will require longitudinal data. Conclusion In spite of its limitations, the study shows the importance of psychological mediators in the care of a chronically ill relative. The question of how caregivers manage to avoid adverse outcomes or why some caregivers are at risk for adverse outcomes can be answered in part by understanding the role of depression. Clearly, to avoid adverse outcomes, clinical interventions should target caregivers who are experiencing depression. List of abbreviations used Americans' Changing Lives: ACL Center for Epidemiological Studies Depression: CES-D The goodness-of-fit index: GFI The adjusted goodness-of-fit: AGF The relative noncentral index: RNI The relative normed fit index: RNFI Declaration of competing interests The author(s) declare that they have no competing interests. Authors' contributions Author 1, PT, developed the research proposal, carried out the data analysis, interpreted the data and participated in the sequence alignment of the manuscript. Author 2, MMJ, participated in developing the research proposal and interpreting the data. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC554787.xml
554778	The Sinbad retrotransposon from the genome of the human blood fluke, Schistosoma mansoni, and the distribution of related Pao-like elements	Background Of the major families of long terminal repeat (LTR) retrotransposons, the Pao / BEL family is probably the least well studied. It is becoming apparent that numerous LTR retrotransposons and other mobile genetic elements have colonized the genome of the human blood fluke, Schistosoma mansoni . Results A proviral form of Sinbad , a new LTR retrotransposon, was identified in the genome of S. mansoni . Phylogenetic analysis indicated that Sinbad belongs to one of five discreet subfamilies of Pao/BEL like elements. BLAST searches of whole genomes and EST databases indicated that members of this clade occurred in species of the Insecta, Nematoda, Echinodermata and Chordata, as well as Platyhelminthes, but were absent from all plants, fungi and lower eukaryotes examined. Among the deuterostomes examined, only aquatic species harbored these types of elements. All four species of nematode examined were positive for Sinbad sequences, although among insect and vertebrate genomes, some were positive and some negative. The full length, consensus Sinbad retrotransposon was 6,287 bp long and was flanked at its 5'- and 3'-ends by identical LTRs of 386 bp. Sinbad displayed a triple Cys-His RNA binding motif characteristic of Gag of Pao / BEL -like elements, followed by the enzymatic domains of protease, reverse transcriptase (RT), RNAseH, and integrase, in that order. A phylogenetic tree of deduced RT sequences from 26 elements revealed that Sinbad was most closely related to an unnamed element from the zebrafish Danio rerio and to Saci-1 , also from S. mansoni . It was also closely related to Pao from Bombyx mori and to Ninja of Drosophila simulans . Sinbad was only distantly related to the other schistosome LTR retrotransposons Boudicca , Gulliver , Saci-2 , Saci-3 , and Fugitive , which are gypsy -like. Southern hybridization and bioinformatics analyses indicated that there were about 50 copies of Sinbad in the S. mansoni genome. The presence of ESTs representing transcripts of Sinbad in numerous developmental stages of S. mansoni along with the identical 5'- and 3'-LTR sequences suggests that Sinbad is an active retrotransposon. Conclusion Sinbad is a Pao/BEL type retrotransposon from the genome of S. mansoni . The Pao/BEL group appears to be comprised of at least five discrete subfamilies, which tend to cluster with host species phylogeny. Pao/BEL type elements appear to have colonized only the genomes of the Animalia. The distribution of these elements in the Ecdysozoa, Deuterostomia, and Lophotrochozoa is discontinuous, suggesting horizontal transmission and/or efficient elimination of Pao -like mobile genetic elements from some genomes.	Background Schistosoma mansoni , the African blood fluke and etiological agent of intestinal schistosomiasis, is endemic in numerous countries in Africa, the Middle East, the Caribbean and northeastern South America. The life cycle of S. mansoni involves parasitism of both humans and aquatic snails of the genus Biomphalaria . Cercariae, the infectious larvae, emerge from the snails into lakes and fresh water streams, where they initiate human infection by direct penetration of the skin. Within the infected person, the worms develop into male and female adults within the portal system blood vessels and mesenteric veins of the intestines. Eggs released from the female parasite into the blood traverse the intestinal wall and are passed out with the feces. Among the tropical diseases, schistosomiasis ranks second only to malaria in terms of morbidity and mortality [ 1 ] and has proved refractory to control by the more conventional public health approaches. No vaccine is yet available. Mobile genetic elements (MGEs) represent a major force driving the evolution of eukaryotic genomes [ 2 - 4 ] and play an important role in the establishment of genome size [ 5 ]. One of the major categories of MGEs is the long terminal repeat (LTR) retrotransposable element, i.e. the LTR retrotransposons and the retroviruses [ 6 ]. These elements are of interest for their potential for horizontal transmission, as well as their ability to shed light on phylogenies of their host organisms when solely vertically transmitted. The genomes of schistosomes, blood flukes of the phylum Platyhelminthes, are estimated at ~270 megabase pairs (MB) per haploid genome [ 7 ], arrayed on seven pairs of autosomes and one pair of sex chromosomes [ 8 , 9 ]. Both the evolution and size of this genome may be highly influenced by mobile genetic elements. Indeed, more than half of the schistosome genome appears to be composed of, or derived from, repetitive sequences, to a large extent from retrotransposable elements [ 10 , 12 ]. Mobile genetic elements colonizing the genome of S. mansoni are of interest both for their potential in developing tools for schistosome transgenesis and for their influence on the evolution and structure of the schistosome genome [ 13 , 14 ]. Previously characterized schistosome MGEs include SINE-like retroposons [ 15 , 16 ], long terminal repeat (LTR) retrotransposons [ 12 , 17 , 18 ], non-LTR retrotransposons [ 10 , 11 ], and DNA transposons related to bacterial IS1016 insertion sequences [ 19 ]. Boudicca , the first LTR retrotransposon characterized from the genome of S. mansoni [ 20 ] belongs to the gypsy -like retrotransposons, one of three highly divergent groups of LTR retrotransposons: the Gypsy/Ty3 group, the Copia/Ty1 group and the Pao/BEL group [ 21 ]. Although active replication of schistosome retrotransposons has not been established, transcripts encoding reverse transcriptase (RT) and endonuclease are detectable [ 10 , 11 , 22 ], as is RT activity in parasite extracts [ 23 ], suggesting that at least some of these elements are actively mobile within the genome. Indeed, actively replicating MGEs have been described from other platyhelminths as RNA intermediates [ 24 ] and DNA transposons [ 25 , 26 ]. Furthermore, the schistosome retrotransposons characterized so far are highly represented within the genome with copy numbers of up to 10,000 [ 10 , 20 ]. It has been suggested that the Pao -like elements exhibit a host range limited to insects and nematodes [ 27 ]. More recently, however, Pao -like sequences have been reported from vertebrates including the teleost fishes Takifugu rubripes and Danio rerio [ 28 ]. Here we have characterized a new Pao -like element from the genome of S. mansoni , which we have named Sinbad after the mariner-explorer Sinbad from the classical Persian/Arabic tales of the "1001 Arabian Nights" (e.g., [ 29 ]). (Sinbad roved through near Eastern countries where schistosomiasis remains endemic even today [ 30 ].) Further, we investigated the phylogenetic distribution of Pao -like elements related to Sinbad and report that there is a discontinuous distribution of these elements throughout the Ecdysozoa, Deuterostomia, and Lophotrochozoa that suggests horizontal transmission and/or efficient elimination of Pao -like mobile genetic elements from some host genomes. Results A LTR retrotransposon in BAC 33-N-3 BLAST analysis indicated the presence in BAC 30-H-16 of a reverse transcriptase (RT)-encoding sequence with identity to Pao and other Pao -like retrotransposons including Ninja and MAX (not shown). Using a probe based on an RT encoding segment of the end sequence of BAC 30-H-16, we identified 14 positive clones in the S. mansoni BAC library [ 31 ]. DotPlot analysis of a 7,531 bp portion of one of the positive BACs, 33-N-3, revealed the presence of two identical, direct repeat sequences of 386 bp separated by ~5.5 kb of intervening sequence, suggesting the presence of an LTR retrotransposon of 6,287 bp in length. This dot matrix is presented in Figure 1 , with a map predicting the size and general domain structure of the new element provided below the matrix (both matrix and map share the same size scale). The direct repeats appeared to be LTRs, and included the promoter initiation motifs CAAT (positions 347–350) and TATA (positions 111–114 and 216–219), transcriptional signals for RNA polymerase II. The LTRs begin with TGT and end with TCA. These motifs (TGN/NCA), known as the direct inverted repeats (DIR), are common to LTRs of many retrotransposons and retroviruses [ 32 ]. BLAST searches of GenBank revealed that this retrotransposon closely resembled the elements Pao and Ninja , followed by other Pao/BEL type retrotransposons. We have termed this new retrotransposon Sinbad . The coding region between the two LTRs of Sinbad was disrupted by several stop and frameshift mutations (as has been seen in many other retrotransposons (e.g., see Ref. [ 32 ]), although the reverse transcriptase, retroviral protease, and gag -like domains of Sinbad were clearly evident. The sequence of the copy of Sinbad from BAC 33-N-3 has been assigned GenBank accession AY506538. Figure 1 Pustell DNA matrix (DotPlot) of 7,531 bp of Schistosoma mansoni genomic DNA sequenced from BAC clone 33-N-3, revealing two identical 386 bp repeat sequences flanking 5,515 bp of unique sequence. A schematic representation of the 6,287 bp retrotransposon encoded by the sequence is shown below the matrix, with the positions of the LTRs and several domains (CHB, Cys-His Box; PR, protease; RT, reverse transcriptase; RH, RNaseH; IN, integrase) labeled. The position of the probe employed in library screening and Southern hybridization is indicated by a red box above this schematic representation. Both matrix and schematic are to scale. Pao -like nucleoprotein, protease and reverse transcriptase Inspection of the region downstream of the 5'-LTR of Sinbad revealed the presence of an ORF encoding retroviral gag and pol -like proteins. A multiple sequence alignment of some of the key structural and enzymatic domains is presented in Figure 2 , with the Sinbad sequence and orthologous regions from Pao , Roo , BEL , MAX and Ninja . The Cys-His box is a highly conserved cysteine and histidine based motif of the nucleocapsid protein (part of the gag polyprotein) of retroviruses and retroviral like elements [ 33 ]. Whereas many other retroviral and retrotransposon families exhibit Cys-His boxes based on a single or double motif of three cysteine and one histidine residues, Pao -like elements are characterized by a distinctive triple Cys-His box [ 21 , 27 ], with zinc finger motifs of C X2 C X3-4 H X4 C , C X2 C X2-4 H X4-5 C , and C X2-4 C X3 H X4 H . Sinbad also exhibits the latter type, hallmark triple Cys-His box motif (Fig. 2 , panel A), although neither Sinbad nor Pao shows a doublet HH in the middle of the third zinc finger motif, another characteristic of this group of retrotransposons [ 32 ]. Notably, Tas , a Pao/BEL like element from Ascaris lumbricoides does not share this characteristic triple Cys-His box [ 34 ], and though Suzu from Takifugu rubripes exhibits a triple Cys-His box, its third zinc finger motif exhibits the structure C X4 C X6 HH X3 C [ 28 ]. As illustrated in Figure 2 , panel B, Sinbad exhibited a protease domain motif AL L D S GS -X98- LIG C D , typical of the LLD X G and LIG protease motifs conserved in Pao -like retrotransposons [ 27 ]. The usual active site tripeptide motif in retroviral aspartic proteases is DTG, with a full conserved sequence of LLDTG, complemented by another site, a highly conserved G preceded by two hydrophobic residues, often I or L, which loops around to interact with the LLDTG [ 35 ]. Whereas the Gypsy -like and Copia -like elements exhibit DTG at the active site, Sinbad has DSG, as do two other Pao -like elements, Roo and MAX . Other Pao -like elements have even more divergent catalytic domains: DCG for Kamikaze , GDG for Yamato , and DNG for Moose [ 27 ]. Since only Thr and Ser include the alcohol groups required for catalysis [ 35 ], the non-DT/SG motifs, including the DDG and DEG of Pao and Ninja likely represent inactivating mutations in non-functional copies of the retrotransposons. Figure 2 Multiple sequence alignments of key domains of the nucleocapsid protein and protease of the Sinbad retrotransposon and related elements. A. Amino acid alignment of the Cys-His box region of the nucleocapsid protein of Sinbad and five other Pao -like elements. Sinbad shares the triple Cys-His box motif of these elements (underlined). B. Amino acid alignment of the protease domain of Sinbad and five other Pao -like elements. Sinbad shares the LLD X G + LIG protease motifs conserved in Pao -like elements (underlined). Identical and chemically similar residues are boxed and shaded. Nucleotides 2761 to 3375 of the Sinbad sequence from BAC 33-N-3 encoded a RT domain, a conceptual translation of which was aligned with the RT domain from six other elements, Pao , Ninja , Roo , BEL , Max , and Saci-1 . A frameshift apparent in the ORF was resolved by inserting a N at the frameshift site, position 2761. The seven blocks of conserved RT residues of Pao -like elements, as modified by Abe et al. [ 27 ] from the blocks described by Xiong et al. [ 21 ], are annotated in green in the alignment (Figure 3 ). The Pao -like retrotransposons presented in Figure 3 all exhibited the RT active site motif YV/MDD, in block 5, a motif conserved in the RT of many other retrotransposons, including the gypsy family [ 32 ]. Figure 3 Multiple sequence alignment of deduced amino acid residues of the reverse transcriptase (RT) domain of Sinbad and six other Pao -like elements. Numbered blocks delineated by green brackets correspond to the seven conserved blocks of RT residues as described by Xiong et al. [21]. Identical and chemically similar residues are boxed and shaded. RNAse H and Integrase of Sinbad An RNaseH domain spanning ~300 amino acid residues was located carboxyl to RT, in which the conserved active site motif DAS was apparent [see Additional file 1 ]. At its COOH-terminus, the Sinbad pol included an integrase (IN) domain of ~260 amino acids in length. Integrase mediates integration of a DNA copy of the viral genome into the host chromosome. Integrase is composed of three domains, the amino-terminal zinc binding domain, a central catalytic domain, and a carboxyl terminal domain that is a non-specific DNA binding domain [ 36 ]. A multiple sequence alignment of the IN zinc binding and central catalytic (DDE) domain of several informative BEL/Pao -like retrotransposons including MAX , Saci-1 , Pao , Ninja , Roo , Suzu , BEL , and Tas as well as Sinbad is presented in Figure 4 . All three domains were apparent in the Sinbad sequence. The NH 2 -terminal zinc-finger region of Sinbad included two conserved Cys residues and one His residue characteristic of other zinc finger motifs of IN (Figure 4 ). A second His expected here was replaced by Asn in this copy of Sinbad. The catalytic active site DDE motif of Sinbad 's integrase displayed the residue spacing of D(62)D(49)E. The IN of non- Pao/BEL retrotransposable elements, for example, Copia , exhibit a DD(35)E motif [ 36 ]. However, the IN of BEL / Pao like elements is unusual in that there is an expanded number of residues between the second D and E conserved residues, with DD(45)E for Pao and DD(53)E for BEL . Sinbad conformed to this BEL / Pao -like paradigm with a spacing of DD(49)E. Saci-1 , also from S. mansoni , shows DD(49)E, although the IN domain of these two elements exhibited only 52% identity. The carboxy terminal domain of IN of Sinbad extended about 135 amino acids beyond the E residue of the catalytic domain [see Additional file 1 ]. Figure 4 Multiple sequence alignment of deduced amino acid residues of the integrase (IN) domain of Sinbad from Schistosoma mansoni and eight other Pao -BEL family retrotransposons. The position of the active site residues are indicated with asterisks above and bold face letters (D, D or E) below, as are the key Cys (C) and His (H) residues of the zinc-finger motif. Identical and chemically similar residues are boxed and shaded. As noted, the IN of Sinbad exhibited identity to Saci-1 from S. mansoni , and indeed these Pao -like retrotransposons from S. mansoni share substantial identity in deduced amino acid sequence and in structural organization [ 37 ]. This similarity extended to several other domains including the Triple Cys-His box region of Gag, 32% identical (23/71, Fig. 2A ); PR,32% identical (36/111, Fig. 2B ); and RT, 45% identical (106/236, Fig. 3 ). Whereas these levels of sequence identity confirmed a close relationship between Sinbad and Saci-1 , they also demonstrated that Sinbad and Saci-1 are distinct retrotransposons. Finally, Sinbad did not appear to encode an envelope protein, the retroviral gene product necessary for extracellular existence and infection [ 38 ]. Sinbad , a new Pao / BEL clade retrotransposon, is closely related to Pao and Ninja The RT domain of Sinbad was aligned with that of 19 Pao/BEL retrotransposon family elements, and with RT from informative Gypsy -like elements, from HIV-1, and Copia using ClustalW. Bootstrapped trees were then assembled using the neighbor joining method and Njplot. Copia was employed as the outgroup to root the tree. The phylogenetic tree confirmed that Sinbad belonged to the Pao / BEL family of LTR retrotransposons (Figure 5 ), and revealed that its two closest relatives were the Saci-1 element from S. mansoni and an unnamed element from D. rerio , the zebrafish (BK005570). Sinbad also grouped closely with Pao and Ninja . Sinbad is clearly distinct from the Gypsy -like retrotransposons, including Gulliver of Schistosoma japonicum and Boudicca of S . mansoni . Sinbad is also clearly distinct from HIV-1, representative of vertebrate retroviruses, and from Copia , representative of the Ty1/Copia group of LTR retrotransposons. Among the 20 BEL / Pao family elements represented in the tree, it was possible to distinguish several subfamilies. First, the outlying subfamily was a clade including Suzu (from T. rubripes ) and an unnamed element from zebrafish. These are the only two elements that we have observed in this subfamily, and both occur in fish genomes. The other two branches of these retrotransposons include Pao , on the one hand, and BEL on the other. Moreover, two subfamilies of elements were apparent within each of the Pao and BEL branches. For the Pao branch, one sub-family included Pao (from B. mori ), ninja (from D. simulans ) and an unnamed element from Anopheles gambiae (XP_3092181). These subfamily elements were all from insect genomes. The other subfamily included Sinbad , Saci-1 and the D. rerio element BK005570; this subfamily has elements from schistosomes (Phylum Platyhelminthes) and fish. On the BEL branch of the tree, the first subfamily includes elements solely from nematode genomes – Tas ( A. lumbricoides ), several Cer elements from C. elegans , and an unnamed element from C. briggsae (BK005572). The other branch included BEL itself (from D. melanogaster ), Kamikaze ( B. mori ), MAX ( D. melanogaster ) and Moose from A. gambiae . Members of this fifth subfamily occurred only in insect genomes. Figure 5 Phylogenetic tree based on Clustal X alignments of the reverse transcriptase domains of several Pao -like and non- Pao -like elements, drawn using the neighbor joining algorithm. The names of elements, followed by host species names, in parentheses, are provided. Size bar reflects phylogenetic divergence in genetic distance units. Bootstrap values were drawn from 1,000 trials. In addition, a phylogram of IN sequences was assembled from 14 Pao / BEL family retrotransposons. The tree displayed the same general topography of branches as the RT-based phylogram and supported our suggestion that there are (at least) five discrete sub-families of BEL - Pao family retrotransposons: Tas -like, BEL -like, Pao -like, Sinbad/Saci-1 -like, and Suzu -like (not shown; tree available from corresponding author). In similar fashion to the RT based tree, Sinbad and Saci-1 were closely related to each other and to the IN from the unnamed Pao -element from zebrafish (BK005571). Copies of Sinbad interspersed throughout the schistosome genome Southern hybridization analysis of S. mansoni gDNA, S. japonicum gDNA and BAC 33-N-3 confirmed the presence of Sinbad in the S. mansoni genome but indicated it was absent from the genome of the related schistosome, S. japonicum (Figure 6 ). Bam H I was expected to cut three times within Sinbad , whereas Hin d III, which cleaves the BAC 30-H-16 copy of Sinbad , was not predicted to cut within the sequence of the BAC 33-N-3 copy. The probe did not contain restriction sites for Bam H I or Hin d III. The hybridization signals from the two S. mansoni gDNA lanes ( Hin d III or Bam H I digested) were strong and dispersed, with a band of ~2.6 kb in the Hin d III digest. The smeared pattern of hybridization indicated that a number of copies of Sinbad were interspersed throughout the genome of S. mansoni rather than being localized at a discrete locus. By contrast, the probe did not hybridize to the gDNA of S. japonicum . Additional blots with larger amounts (30 μg) of S. japonicum gDNA, digestion with Bam H I instead of Hin d III, and exposure of the film for longer periods failed to yield any signal from S. japonicum gDNA (not shown), indicating that Sinbad was absent from this schistosome species. Strong hybridization signals were evident in the positive control lanes of digests of BAC 33-N-3. Densitometric analysis of the hybridization signals indicated the presence of 50 to 60 copies of Sinbad per S. mansoni haploid genome, based on four separate estimates comparing the signal in each of the genomic DNA lanes to the signal in each of the 33-N-3 BAC lanes (comparison of lane 1 with lane 4, comparison of lane 2 with lane 5, comparison of lane 1 with lane 5, and comparison of lane 2 with lane 4). (These estimates assumed that BAC 33-N-3 included only one copy of Sinbad .) Figure 6 Southern hybridization of Schistosoma mansoni and S. japonicum genomic DNAs, and S. mansoni BAC clone 33-N-3 BAC DNA to a Sinbad retrotransposon-specific gene probe. Lane 1, S. mansoni DNA (30 μg) digested with Hin d III; lane 2, S. mansoni DNA (30 μg) digested with Bam H I; lane 3, S. japonicum DNA (20 μg) digested with Hin d III; lane 4: BAC 33-N-3 (0.8 μg) digested with Hin d III; and lane 5, BAC 33-N-3 (0.8 μg) digested with Bam H I. Molecular size standards in kilobase pairs (kb) are indicated at the left. Copy number was estimated by two additional methods. First, upon screening the 23,808 clones of the BAC library of Le Paslier et al. [ 31 ] that represents a ~8-fold coverage of the haploid S. mansoni genome, approximately 0.7% to 1.0% of the clones were positive, indicating a copy number for Sinbad of ~20 to 30 copies (not shown). Second, the bioinformatics approach of Copeland et al. [ 20 ] was used to compare these estimates with reference copy number estimates of other mobile genetic elements and genes reported previously. BLASTn searches were undertaken using the nucleotide sequences of these reference genes and the complete sequence of Sinbad (Table 1 ). Because the construction of the BAC library involved partial digestion of the genomic DNA with Hin d III [ 31 ], genes without Hin d III sites will be underrepresented in the BAC end sequences. Accordingly, since sequenced BAC ends from this library constitute a large proportion of the genomic S. mansoni sequences in the public domain, we used only genes containing Hin d III sites as reference sequences. As shown in Table 1 , the number of hits for Sinbad , 38, was higher than the number of hits for the single-copy cathepsin D gene (0 hits) but lower than that for the multiple-copy 28S ribosomal RNA gene (157 hits) (~100 copies; Ref. [ 7 ]) and for three high copy number retrotransposons Boudicca (100 hits, 1,000–10,000 reported copies), SR2 (102 hits, 1,000–10,000 copies), and SR1 (104 hits, 200–2,000 reported copies). In overview, all three methods were in reasonably close agreement, and together they indicated that approximately 50 (range ~20–100) copies of Sinbad reside in the genome of S. mansoni . Based on copy numbers estimated for other schistosome retrotransposons (see [ 13 ]), we consider that Sinbad is not a high copy number element. Table 1 Estimation of gene copy number of the Sinbad LTR retrotransposon in the genome of Schistosoma mansoni . Gene GenBank Accession Length (bp) Number of hits (Expect 0.000001) Reported copy number Key references Cathepsin D, Intron 4 AY309267 (nt 3213–4849) 3926 0 1 66 Sinbad AY506538 6288 38 ~50 This study 28S rRNA Z46503 1694 157 100 67 Boudicca AY662653 5858 100 1,000–10,000 20 SR2 AF025672 3913 102 1,000–10,000 62 SR1 U66331 2337 104 200–2,000 61 Saci-2 BK004069 4946 107 85–850* 37 Saci-1 BK004068 5980 133 70–700* 37 *estimated solely by the gene index bioinformatics approach of DeMarco et al. [37], whereas the other copy numbers listed here were determined by hybridization and/or other analyses. Sinbad -like elements transcribed in developmental stages of S. mansoni BLASTn analyses were undertaken using the full length of Sinbad as the query sequence and the GenBank EST database of non-human, non-mouse sequences. The database includes more than 130,000 EST sequences from six developmental stages of S. mansoni – egg, miracidium, cercaria, germball (= sporocyst), schistosomulum, and mixed sex adults [ 39 , 40 ]. Significant hits were found to ESTs from all of these six developmental stages. Of these, the hits with highest similarity to Sinbad , CD111741, CD060185, CD163413, CD062550, CD156994, and CD156946, exhibited contiguous ORFs spanning each EST without frameshifts or stop mutations. Positive ESTs spanning most or all of the LTR, gag , PR, RT, RH and/or IN regions were located in most of these six developmental stages, indicating that Sinbad -like elements are actively transcribed in all or most developmental stages of S. mansoni . Discontinuous distribution of Sinbad -like elements In order to examine the phylogenetic distribution of Sinbad -like retrotransposons, we examined numerous complete and partial genomes, including prokaryotes, plants, fungi, animals, and lower eukaryotes [ 41 ]. The genomes were searched using tBLASTn with the amino acid sequence corresponding to the region of Sinbad spanning from the Cys-His box to the conserved protease catalytic domain (bp 1588–2236) [see Additional file 1 ] as the query. To minimize the likelihood of spurious positives, we lowered the E-value for significance from 10 to 0.001; this corresponded to a bit score of 40 or above. Although it is more stringent than that of the BLAST default, this cutoff point was employed because it is permissive enough to detect both Sinbad -like elements and members of the Pao/BEL family at large. No significant hits were found in any of the plant, fungal, or protist genomes examined, or in the 275 bacterial and 21 archaean genomes searched. All of the nematodes examined were positive. Of the insects, the other branch of the Ecdysozoa, Drosophila melanogaster and Anopheles gambiae contained Sinbad -like elements, whereas Drosophila pseudoobscura and Apis mellifera did not. Of the vertebrates, Danio rerio and Takifugu rubripes contained Sinbad -like sequences, whereas Homo sapiens , Mus musculus , Rattus norvegicus, Canis familiaris, Sus scrofa, Gallus gallus , and Bos taurus did not. Interestingly, although most higher chordates examined were free of Sinbad -like elements, the tunicates Ciona intestinalis and Ciona savigny , were positive for over 100 hits of sequences highly similar to the Sinbad search sequence (up to an E-value of 4e -22 ). In addition, the echinoderm Strongylocentrotus purpuratus , a non-chordate deuterostome, was positive for the Sinbad search sequence. These findings are summarized in a tree-of-life style illustration, based on the tree presented in Pennisi [ 42 ], and drawn in the style of the taxonomic relationship diagrams used at NCBI [ 43 ]. (This diagram is not a phylogram, and displays broad relationships among major taxa only; although relationships are in correct branching order, branch lengths are not to scale.) Genomes with regions of significant similarity to Sinbad are marked with a "+" symbol and those without are indicated with a "-" symbol. The results of a search of dbEST corroborated and expanded these findings, revealing nine non- Schistosoma organisms with Sinbad -like sequences: C. intestinalis , Molgula tectiformis (tunicate), S. purpuratus , D. melanogaster , Bombyx mori , Salmo salar , Xenopus laevis , and Trichinella spiralis . E-values and accession numbers for the top match for each organism are provided in Table 2 . Table 2 Organisms other than schistosomes with significant EST matches to Sinbad . Organism Accession number BLAST score (bits) Expect value Ciona intestinalis (tunicate) BW308116 89 9 e -17 Molgula tectiformis (tunicate) AU283942 87 5 e -16 Srongylocentrotus purpuratus (purple sea urchin) CD295138 83 7 e -15 Drosophila melanogaster (fruit fly) BI583252 75 2 e -12 Bombyx mori (silk worm) CK529741 59 1 e -7 Salmo salar (Atlantic salmon) CB500934 58 2 e -7 Xenopus laevis (African clawed frog) BJ073921 45 0.002 Trichinella spiralis (parasitic nematode) BG520200 45 0.002 Discussion Sinbad – a novel Pao / BEL family LTR retrotransposon from the genome of S. mansoni Although several LTR retrotransposons have been characterized previously from the genome of S. mansoni , including Boudicca , Saci-1 , Saci-2 , Saci-3 and the fugitive [ 17 , 20 , 37 ], the Sinbad retrotransposon characterized here is a novel retrotransposon and it is discrete from these other elements. Sequence identity, structure, and phylogenetic relationships indicate that Sinbad is a member of the Pao / BEL family of retrotransposons. The hallmark structures included a triple Cys-His box zinc finger domain in the Gag polyprotein, protease with the active site tripeptide DSG, RT domain that included a YVDD active site motif, RNAseH with DAS at the active site, and an integrase domain with a DD(49)E spacing of the active site aspartic acid and glutamic acid residues. The YVDD motif of RT, a version of the F/YXDD consensus motif of Gypsy -like LTR retrotransposons, is shared by Pao and BEL . Bowen and McDonald [ 32 ] reported that the Cer7-Cer12 series of elements from C. elegans displayed YVDN at this site. Whether the Asn could replace Asp as the carboxy-residue of this conserved tetrapeptide with retention of enzyme activity remains to be determined by biochemical analysis, although mutation of either aspartate in YXDD of retroviral RT (HIV-1 or Moloney murine leukemia virus) inactivates the polymerase [see [ 44 ]]. The LTRs of Sinbad in BAC 33-N-3 are identical in sequence, and appeared to contain a putative promoter for initiation of transcription by RNA polymerase II. Along with conservation of most residues contributing to the active sites of the retrotransposon enzyme domains, these structural characteristics suggested that Sinbad is active or had been transpositionally active in the recent past. Several other features also indicated that Sinbad is transpositionally active. Numerous transcripts spanning enzymatic domains and LTRs of Sinbad , from at least six developmental stages of S. mansoni , have been sequenced [ 40 ], and of these, the ESTs most closely resembling Sinbad are composed entirely of contiguous open reading frames, suggesting non-mutated copies. On the other hand, potentially inactivating mutations, including stop codons and frameshifts, suggested that the BAC 33-N-3 copy of Sinbad was incapable of autonomous retrotransposition. If active copies are present, functional proteins coded by these copies could have been used in the recent past to mobilize the 33-N-3 Sinbad copy in trans , as recorded for other retrotransposons [ 45 - 47 ], explaining the presence of identical LTRs. Indeed, Frame et al. [ 28 ] noted that mutated copies framed by similar LTRs are common in BEL like elements in C. elegans , implying recent transposition. The LTRs of Sinbad , at 386 bp in length, were substantially shorter than those of Saci-1 , ~840 bp [ 37 ], but longer than those of Gypsy -like LTR retrotransposons from schistosomes, the fugitive , Gulliver and Boudicca . Whereas Sinbad and Saci-1 are clearly closely related, dissimilar LTRs and the low amino acid identity of the most highly conserved domains (35 to 52%) confirmed they are distinct retrotransposons. Sinbad can be added to the catalog of mobile genetic elements characterized from the schistosome genome, where retrotransposons appear to have proliferated and flourished and contributed significantly to its relatively large size (270 MB; ~14,000 protein-encoding genes) [ 13 , 20 , 40 ]. The colonization of the genome of S. mansoni by Sinbad and Saci-1 and that of S. japonicum by the related Tiao element [ 48 ] represents the first demonstration of infection of a Lophotrochozoan taxon by Pao / BEL family LTR retrotransposons. The presence of Sinbad , Saci-1 , and Tiao in two species of Schistosoma suggests that an ancestral schistosome was already host to the ancestors of these elements. (Though Tiao is a Pao/BEL family retrotransposon, and is therefore predicted to be detected in low-stringency BLAST searches, as in Figure 7 , the absence of a positive signal on the genomic Southern hybridization suggests that it is not particularly closely related to Sinbad .) Figure 7 Phylogenetic illustration of species and higher taxa for which data are available concerning Pao -like elements. Species for which genomes have been sequenced and are available for whole genome BLAST searches in GenBank are enclosed in ovals. These genomes were tBLASTn searched using a deduced amino acid sequence from Sinbad (from the Cys-His Box through the protease domain) as the search sequence. Genomes with sequences significantly similar (E ≤ 0.001) to Sinbad are identified by a green "+" symbol, and those negative for Sinbad -like sequences with a red "-" symbol. Other species, with not yet fully sequenced genomes, shown to include Pao -like sequences (through EST searches or other means) are shown in smaller font and unenclosed, and are also marked with a green "+". This diagram is based on a tree of life style diagram in Pennisi [42] and reflects broad relationships between taxonomic groups only. It is not a phylogram – stem lengths do not represent phylogenetic distances. A Sinbad / Saci-1 subfamily of Pao-BEL like LTR retrotransposons Whereas the sequence and deduced structure of the three signature Pao -like elements, Pao from the silk moth B. mori , Tas from the human roundworm Ascaris lumbricoides and BEL from D. melanogaster have been known for about a decade, the Pao / BEL family is not as well understood or apparently as widespread as the other two major families of LTR retrotransposons, the Copia / Ty1 and the Gypsy / Ty3 families. However, at least three branches of the Pao / BEL family have become apparent – branches represented by Pao , BEL , and Suzu (from T. rubripes ) [ 27 , 28 , 32 , 49 - 51 ]. Using the new sequence information from Sinbad , and some related elements, we have been able to investigate the intra-family relations of the Pao / BEL elements more thoroughly. Our findings, based on phylogeny of RT, and supplemented by phylogeny of IN, indicated the presence of at least five sub-families of Pao / BEL elements. The majority of the sub-families may have a restricted host range; the Tas subfamily occurred only in nematodes (these elements may be endogenous retroviruses because they appear to include env genes), the BEL subfamily only in insects, the Pao subfamily only in insects, and the Suzu subfamily only in fishes. By contrast, the Sinbad/Saci-1 subfamily is known from schistosomes and zebrafish. Phylogenetic range of Sinbad -like retrotransposons The Pao / BEL retrotransposons are known only from animals, a less extensive distribution than those of the Copia / Ty1 or Gypsy / Ty3 groups that include elements known from fungi and/or plants as well as animals. The ostensible absence of these elements from prokaryotes, lower eukaryotes, fungi and plants suggests that ancestral Pao -like elements appeared after the differentiation of the Animalia. Though the number of sequenced entire genomes of animals is small, the distribution of Pao / BEL LTR retrotransposons within these few genomes displays a topography that we would not expect to be the result solely of vertical transmission alone (Fig. 7 ). Sinbad -like sequences were found in D. melanogaster , but not in D. pseudoobscura , nor in A. mellifera , even though close relatives are found in other insects such as B. mori and A. gambiae , and even in species as phylogenetically distant as D. rerio (a fish) and S. mansoni (a platyhelminth). Further, the distribution among chordates is enigmatic. Of the vertebrate whole genomes searched, only two, T. rubripes and D. rerio , were positive for Sinbad like elements. The human, mouse, rat, cow, chicken, pig and dog genomes were devoid of Sinbad -like matches. Since the genomes of lower chordates and a non-chordate deuterostome were positive for Sinbad -like sequences, progressive radiation would be expected to give rise to similar sequences in these vertebrates. Feschotte [ 19 ] reported a similarly patchy distribution for the Merlin DNA transposons; Merlin like elements are abundant, for example, in anopheline mosquitoes but are absent from D. melanogaster , D. pseudoobscura , and A. mellifera . Also, they are present in some vertebrate genomes but not others. Merlin -like elements are also present in schistosome chromosomes. This type of distribution suggests that either the vertical lineage of the elements has been curtailed by the extinction of these elements from several genomes, or that horizontal transmission has taken place. Genomes need to restrain the uncontrolled proliferation of mobile genetic elements, especially retrotransposons, and indeed some eliminate mobile sequences more efficiently than others [ 5 , 52 ]. Goodwin and Poulter [ 53 ] have shown that Ngaro elements have been lost from certain genomes, as evidenced by the presence of small, corrupt fragments serving as fossil sequences. Similarly, especially in view of the low number of Sinbad copies, Pao -like elements may have followed a course of progressive radiation followed by elimination from the Sinbad -negative genomes. However, if this were the case with Pao -like elements, relic sequences could be expected in at least some of the Sinbad -negative genomes. Their absence from mammalian and avian genomes favors the alternative explanation, that the current range reflects horizontal transmission. What might have been the origin of the Pao / BEL radiation within the Animalia? Felder et al. [ 34 ] suggested that a common ancestor of Tas and Pao may have undergone a horizontal transmission event between the Insecta and Nematoda, followed by the eventual differentiation of these elements, including the gain or loss of env . Of the sub-families of Pao / BEL elements apparent in the RT-based phylogram (Figure 5 ), the Tas subfamily includes retrotransposons with an envelope encoding gene (specifically Tas from A. lumbricoides and Cer7 from C. elegans ). The acquisition of an envelope protein by an ancestral Tas or Tas -like element would have enabled its extracellular existence and facilitated its horizontal transmission and infection of other hosts [ 38 ]. Interestingly, the deuterostomes bearing Sinbad -like sequences included a sea urchin, tunicates, pufferfish, zebrafish, the Atlantic salmon, and the African clawed frog X. laevis (Figure 7 ). These are aquatic species and, moreover, all are known from coastal or brackish waters at the interface of freshwater and marine systems. The secondary hosts of S. mansoni , snails of the pulmonate genus Biomphalaria , are also aquatic, as are the larval (miracidium and cercaria) stages of S. mansoni which enter and exit the snail. It will be of interest to determine whether or not Pao -like elements are present in this snail host, from which numerous RT-encoding sequences already have been reported [ 54 ]. Also of potential relevance is that the genomes of both X. laevis and S. mansoni contain Pao -like elements and that X. laevis is the secondary host of the trematode parasite Tylodelphys xenopi [ 55 ], a fluke closely related to the human schistosomes. Both T. xenopi and another human schistosome, Schistosoma haematobium , use snails of the genus Bulinus as intermediate hosts. An aquatic lifestyle is an obvious relationship that links all of the deuterostome hosts of Sinbad -like elements. This aquatic, in comparison to a terrestrial, existence may have facilitated transmission of infectious particles of the Tas -like ancestors of Pao , Tas , BEL , Suzu , Sinbad , and relatives. Alternatively, schistosomes may have acquired a Tas - element directly from Ascaris lumbricoides , an exceedingly common human parasite and the host of Tas . A. lumbricoides occurs in the intestines of infected people, as do schistosome eggs, so direct transmission of a mobile genetic element from roundworm to schistosome could have been facilitated by their physical proximity within the human intestines. Conclusion A Pao / BEL like LTR retrotransposon named Sinbad is interspersed within the genome of the blood fluke, S. mansoni . About 50 copies of this element appear to reside in the S. mansoni genome. Analyses of the phylogenetic distribution of Pao / BEL -like retrotransposons indicated that Pao / BEL -like elements are present only within phyla of the Animalia, and not in prokaryotes, fungi or plants. Further, the analyses indicated that there are at least five discrete sub-families of the Pao / BEL clade of LTR retrotransposons, and that the distribution of these retrotransposons among the Ecdysozoa, Lophotrochozoa and deuterosomes has been influenced by horizontal as well as vertical transmission. Methods Screening the bacterial artificial chromosome library Le Paslier et al. [ 31 ] described the construction and characterization of a bacterial artificial chromosome (BAC) library of the Schistosoma mansoni genome. The library, constructed in the plasmid vector pBeloBac11 with genomic DNA (gDNA) from cercariae of a Puerto Rican strain of S. mansoni partially digested with Hin d III, consists of 23,808 clones, about 21,000 of which are estimated to contain inserts ranging from 120 to 170 kb, providing ~8-fold coverage of the schistosome genome. Numerous BAC end sequences determined from randomly selected clones from this library are in the public domain. Inspection of the end sequence of BAC clone number 30-H-16 indicated identity with Pao -like LTR retrotransposons (not shown). Because the retrotransposon sequence was located at the end of the BAC, the clone was unlikely to contain the entire Pao -like element. Given that retrotransposons can be expected to be present in multiple copies in the host genome, we screened the library with a probe based on the end of BAC 30-H-16 in order to locate an entire copy of the retrotransposon. The gene probe was obtained by PCR amplification of a fragment of BAC 30-H-16 using the primers 5'-CGCGGATCCAAGAGAAAAACCTTGATAGAC and 5'-CCGGAATTCCTGTCGAAGATAAAAGAGC, was cloned into pBluescript and its identity confirmed by sequencing (Accession AY871176). This probe spanned residues 2457 to 2823 of the BAC 33-N-3 copy of the new retrotransposon (see below). The cloned insert was labeled with digoxygenin (DIG) and employed to screen the BAC library, as described [ 20 ], represented as high-density clone arrays on nylon membranes. Positive clones were cultured as described [ 31 ] and the presence of sequences with identity to the novel retrotransposon in the positive clones was confirmed by PCR (primers as above) or by colony hybridizations [ 56 ] to the DIG labeled probe. One positive clone, BAC 33-N-3, was investigated further by sequence analysis. BAC plasmid DNA was isolated from bacterial cultures using the PhasePrep BAC DNA purification system (Sigma). Analysis of the insert of 33-N-3 was accomplished after subcloning Bam H1 fragments of the BAC into pNEB 193 (New England Biolabs, MA), sequencing the inserts of the sub-clones, and also by direct sequencing of BAC 33-N-3. Automated nucleotide sequencing, using ABI BigDye Terminator chemistry (ABI, Foster City, CA) and an ABI Prism 3100 sequencer, was undertaken using primers specific for the probe and subsequently with gene specific primers at Tulane University and at Davis Sequencing (Davis, CA). Sequence analysis and alignments Contigs of the sequences were assembled using SeqMan (DNAstar, Inc., Madison, WI). Repeat sequences were identified with a Pustell style dot matrix [ 57 ] using the DotPlot3 program (Ramin Nakisa, Imperial College, London, UK) [see [ 58 ]] and the Pustell DNA Matrix function in MacVector (Accelrys). Amino acid alignments were accomplished with MacVector and ClustalW [ 59 ] using sequences from GenBank or using conceptual translations of nucleic acid sequences. Open reading frames were located and conceptually translated using MacVector. Sequences of the following retrotransposons were used in the multiple sequence alignments based on gag , protease, and reverse transcriptase: Ninja , T31674; Pao , S33901; MAX , CAD32253; Roo , AAN87269; BEL , AAB03640; and Saci-1 , BK004068. Sequences of the following retrotransposons were used in the multiple sequence alignment based on Integrase: Saci-1 , DAA04498; Pao , S33901; Ninja , T31674; Roo , AAN87269; Suzu , AF537216, BEL , AAB03640, Tas , Z29712, and MAX , CAD32253. Parasite DNAs, Southern hybridization, densitometric estimation of copy number Genomic DNAs of cercariae of a Puerto Rican strain of S. mansoni and of adults of a Chinese (Anhui Province) strain of S. japonicum were extracted using the AquaPure Genomic DNA Purification system (Bio-Rad, Hercules, CA). S. mansoni gDNA (30 μg/lane) and 33-N-3 BAC DNA (800 ng) were digested with Hin d III and Bam H I restriction enzymes, and S. japonicum gDNA (20 μg/lane) was digested with Hin d III. Digested gDNA and BAC DNA were size fractionated by electrophoresis through a 0.8% agarose gel, transferred to a nylon membrane (Zeta-Probe GT, Bio-Rad) by capillary action [ 60 ], and UV-light cross-linked to the membrane. Southern hybridization analysis to the DIG-labeled probe (above) was performed as described [ 20 ]. Chemiluminescent signals were detected using X-ray film (Fuji). Densitometric analysis of Southern hybridization signals was accomplished using the Versa-Doc gel documentation system (Bio-Rad) and Quantity-One software (Bio-Rad). Densitometry values for signals evident in the gDNA and BAC DNA lanes were used to estimate the copy number for the new retrotransposon, Sinbad , according to the formula [( A / B ) × C ]/ E = F . This formula was derived from two equations: ( A / B ) × C = D and D / E = F , where A was the number of copies of Sinbad in the BAC 33-N-3 lane, B was the density volume of the 33-N-3 lane in units of optical density per mm 2 , C was the density volume of the S. mansoni genomic DNA lanes in units of optical density per mm 2 , D was the total number of copies of Sinbad per genomic DNA lane, E is the number of haploid genomes in the gDNA lane, and F represented the copy number of Sinbad per haploid S. mansoni genome. The insert of 33-N-3 was estimated to be 145 kb in length and assumed to contain only a single copy of the retrotransposon. Other copy number estimations In addition to the densitometry-based estimate, estimates of the copy number of the Sinbad retrotransposon also were obtained by a comparative bioinformatics approach [ 20 ] wherein BLAST analysis of the bacterial artificial chromosome (BAC) -end database of S. mansoni genomic sequences targeted more well-characterized retrotransposable elements from S. mansoni for which copy numbers had been reported. These included the Boudicca LTR retrotransposon [ 20 ] and the non-LTR retrotransposons SR1 and SR2 [ 61 , 62 ]. The NCBI database was searched by BLAST using the sequences of these mobile genetic elements and some other genes of S. mansoni , all of which included at least one Hin d III site. Specifically, the Advanced BLAST function was used, set to search only the S. mansoni sequences in the GSS database (Limit by Entrez Query: <Schistosoma mansoni[organism]>), and with the E value at 0.000001. The E value (Expect value) reflects the probability of obtaining a match purely by chance. Scores at or below this stringent cutoff E value of 10 -6 were counted as positive. This exceptionally stringent cutoff value was used to minimize the chance of counting other Pao -like elements in the total copy number of Sinbad . Since the formula for E is based not only on the bit scores of the local alignment of each pair of sequences, but also on the lengths of the subject and query [see [ 63 ]], no additional correction was made for the length of the query sequence. Phylogenetic analysis of Pao -like elements Sequences for phylogenetic analysis comparing the RT region of several different retrotransposons were prepared by trimming sequences from the large single polyprotein of each retrotransposon to just the conserved domains of RT (see [ 21 , 27 ]). Pol sequences presented in Xiong et al. [ 21 ] and Abe et al. [ 27 ] were trimmed exactly to the stretch of sequence shown by these authors to represent the RT domain. Other elements were aligned with these sequences and likewise trimmed to obtain just the RT domain. For some elements, nucleotide sequences were analyzed for open reading frames and translated before being trimmed to include just the 7 conserved blocks of the RT domain. Alignments were accomplished using Clustal X [ 64 ], after which bootstrapped trees (1,000 repetitions) were prepared using the neighbor joining method [ 65 ] and drawn with Njplot. The accession numbers for sequences included in the phylogenetic analysis are as follows: Ty3 , S53577; Tas : Z29712; Suzu , AF537216; Sinbad , AY506538 (an N was inserted at position 2761 to a resolve a frameshift and generate a single ORF) Saci-1 , DAA04498; Roo , AAN87269; Ninja , T31674; Moose , AF060859; Max , CAD32253; Kamikaze , AB042120; HIV-1, P04585; Gypsy , GNFFG1; Gulliver , AF243513; Copia , OFFCP; BEL , AAB03640; Cer7, AAB63932, Cer8, CAB04994, Cer9, CAB1647, and Cer11, AAA82437, two uncharacterized Anopheles gambiae retrotransposons, XP_309281 and XM_308737, an uncharacterized Caenorhabditis briggsae retrotransposon, AC084491, and two uncharacterized Danio rerio retrotransposons, BX537152 and BX005079 [see Additional file 2 ]. Two additional sequences were either not in the database or were composites made to reconstruct sequences more closely resembling non-mutated forms of the retrotransposons. The sequence representing Pao was a reconstruction prepared by Abe et al. [ 27 ], from accession numbers S33901, AB042118, and AB042119; the sequence representing Boudicca was a composite of translated cDNA sequences introduced in Copeland et al. [ 22 ], AY308018, AY308019, AY308021 and AY308022 [see Additional file 2 ]. Screening entire or partial genomes for Sinbad A panel of fully or partially sequenced entire genomes was searched by BLAST for elements exhibiting sequence similarity to Sinbad . The deduced amino acid sequence encoding the region from the Cys-His Box through to the protease domain (encoded by nucleotides 106 to 753 of Sinbad [Y506538]) was employed as the query to search each genome individually using tBLASTn. The genomes searched in this way were as follows: Homo sapiens, Mus musculus, Rattus norvegicus, Takifugu rubripes, Danio rerio, Bos taurus, Gallus gallus, Sus scrofa, Canis familiaris, Anopheles gambiae, Apis mellifera, Drosophila melanogaster, Drosophila pseudoobscura, Brugia malayi, Caenorhabditis elegans, Caenorhabditis briggsae, Strongylocentrotus purpuratus, Ciona intestinalis, Ciona savigny, Giardia lamblia, Plasmodium falciparum, Plasmodium yoelii, Plasmodium berghei, Cryptosporidium parvum, Eimeria tenella, Theileria annulata, Toxoplasma gondii, Dictyostelium discoideum, Entamoeba histolytica, Leishmania major, Trypanosoma brucei, Trypanosoma cruzi, Arabidopsis thaliana, Avena sativa, Glycine max, Hordeum vulgare, Oryza sativa, Triticum aestivum, Zea mays, Lycopersicon esculentum, Schizosaccharomyces pombe, Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces bayanus, Saccharomyces castelli, Saccharomyces kluyveri, Saccharomyces kudriavzevii, Neurospora crassa, Magnaporthe grisea, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus terreus, Candida albicans, Coccidioides posadasii, Gibberella zeae, Coprinopsis cinerea, Cryptococcus neoformans, Ustilago maydis and Encephalitozoan cuniculi . In addition, 275 eubacterial and 21 Archaean genomes were searched [see Additional file 3 ]. Genomes with matches with E values less than 0.001 (corresponding approximately to bit scores greater than 40) were considered positive for Sinbad -like elements. GenBank accession numbers Sequences of the Sinbad LTR retrotransposon have been assigned accession numbers AY506537, AY506538, AY645721, AAT66412, and AY871176. Other sequences introduced here been assigned GenBank Third Party Annotation accession numbers; BK005570 ( Danio rerio ), BK005571 ( D. rerio ), BK005572 ( Caenorhabditis briggsae ), BK005573 ( Anopheles gambiae ), BK005574 ( D. rerio ). Abbreviations MGE, mobile genetic element; ORF, open reading frame; EST, expressed sequence tag; gDNA, genomic DNA; LTR, long terminal repeat; RT, reverse transcriptase; PR, protease; IN, Integrase; CHB, Cys-His box; BAC, bacterial artificial chromosome; MB, megabase pairs Authors' contributions CSC carried out the sequence analyses, sequence alignments, phylogenetic studies, other bioinformatics analyses, and Southern hybridizations, participated in cloning, sequencing and design of the study, and, together with PJB, drafted the manuscript. VHM and MEM participated in cloning and sequencing. BHK contributed to the design of experiments and analyses. PJB participated in the design and coordination of the study, and drafting the manuscript. All authors read and approved the final version of the manuscript. Supplementary Material Additional File 1 "Annotated Sinbad sequence". Nucleotide and deduced amino acid sequence of the entire Sinbad retrotransposon in BAC clone 33-N-3. Hallmark features of the retrotransposon are identified in colored highlights as described in the key at the bottom of the figure. Click here for file Additional File 2 "RT domain sequences of new and consensus elements used in the phylogenetic analysis". Deduced amino acid sequences of the RT domains used in the phylogenetic analysis from newly characterized elements, uncharacterized elements found within genome survey sequences, and elements for which consensus sequences were used. Accession numbers for the source sequences of each element are listed, as well as references where applicable. Click here for file Additional File 3 "Prokaryotic genomes negative for Sinbad like elements" Table of prokaryotic genomes indicated by whole genome analysis to be devoid of Sinbad like elements. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC554778.xml
544580	Neuroblastoma and pre-B lymphoma cells share expression of key transcription factors but display tissue restricted target gene expression	Background Transcription factors are frequently involved in the process of cellular transformation, and many malignancies are characterized by a distinct genetic event affecting a specific transcription factor. This probably reflects a tissue specific ability of transcription factors to contribute to the generation of cancer but very little is known about the precise mechanisms that governs these restricted effects. Methods To investigate this selectivity in target gene activation we compared the overall gene expression patterns by micro-array analysis and expression of target genes for the transcription factor EBF in lymphoma and neuroblastoma cells by RT-PCR. The presence of transcription factors in the different model cell lines was further investigated by EMSA analysis. Results In pre-B cells mb-1 and CD19 are regulate by EBF-1 in collaboration with Pax-5 and E-proteins. We here show that neuroblastoma cells express these three, for B cell development crucial transcription factors, but nevertheless fail to express detectable levels of their known target genes. Expression of mb-1 could, however, be induced in neuroblastoma cells after disruption of the chromatin structure by treatment with 5-azacytidine and Trichostatin A. Conclusion These data suggest that transcription factors are able to selectively activate target genes in different tissues and that chromatin structure plays a key role in the regulation of this activity.	Background The complex process of tumor development often involves changes in the transcription regulatory networks. In human cancer, genetic changes involving the transcription factor p53 gene is particularly common and the gene is found mutated in cancers originating from numerous cell types. This factor is, however, broadly expressed and directly involved in cell cycle regulation and apoptosis explaining the common involvement of the protein in tumor development. Many malignancies are characterized by specific chromosomal translocations that frequently affects the expression or structure of transcription factors with a more tissue specific expression pattern, often with an important function during development [ 1 ]. Examples of this can be found within the hematopoetic system where for instance translocations of Tal-1 is associated with T cell leukemia's [ 2 ] while modified BCL-6 [ 3 ] or c-myc [ 4 ] is associated with B cell non-Hodgkin's lymphomas. The close correlation with a specific tumor type and pathology to a specific transcription factor modification could well be explained by differential expression patterns and accessibility of the gene for translocation events. Another possibility could be that the action of the transcription factor is context dependent and therefore the ability of the modified protein to contribute to tumor development depends on the cell in which it arises. To investigate mechanisms involved in lineage specific gene regulation and transcription factor target gene selection in tumor cells we have compared transcription factor expression in neuroblastoma and pre-B lymphoma cells. This revealed that both these highly divergent tumor types expressed the transcription factor EBF [ 5 ] that has been contributed a central role in B cell development [ 6 ]. The protein is a helix-loop-helix family member [ 7 , 8 ] essential for B-lymphopoesis in mice [ 6 ] where it has been shown to regulate a large number of pre-B cell restricted genes including the surrogate light chains [ 9 ], CD19 [ 10 ] and the signal transduction proteins Igα ( mb-1 ) [ 7 , 11 ] and Igβ ( B29 ) [ 12 ]. The EBF-1 protein is highly conserved between human and mouse [ 13 ] and it also appears as if the target gene spectra has been conserved between species even though the primary promoter sequences of these genes has diverged [ 13 , 14 ]. Pre-B cells express exclusively EBF-1 [ 15 ] while neuroblastoma cells express other family members including EBF-2 and -3 [ 5 ]. There are limited information about EBF target genes in neuroblastoma cells but binding sites for EBF proteins were identified in the promoters controlling the expression of the neuron restricted Chromogranin A ( CGA ) and SCG10 genes [ 5 ]. In the present study we were interested in the mechanisms of tissue specific target gene activation by comparing transcription factor function and gene expression patterns in neuroblastomas and pre-B cells. We here report that even though neuroblastoma cells express both Pax-5 and E-proteins, both suggested to be crucial co-activators for EBF target genes in pre-B cells, the cells do not express the pre-B cell restricted target genes mb-1 and CD19 . The expression of the mb-1 gene could, however, be activated by treatment of the neuroblastoma cells with chromatin disrupting agents. This suggests that chromatin structure is a key component in the regulation of transcription factor function, by restricting the accessibility of target genes, possibly contributing to the apparent link between a transcription factor and specific malignancies. Methods Cell culture HeLa, THP-1, KM3 and Nalm6 cells were grown in RPMI 1640 medium supplemented with 7.5% fetal calf serum (FCS), 10 mM HEPES, 2 mM pyruvate and 50 μg/ml gentamicin (complete RPMI media) (Life Technologies) at 37°C and 5% CO 2 . SH-SY5Y, IMR-32 and SK-N-BE(2)c neuroblastoma cells were cultured in Eagle's Minimum Essential Medium (MEM) with 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin in an atmosphere of 5% CO 2 at 37°C (Life Technologies). KCN-69n and LA-N-1 neuroblastoma cells were cultured in RPMI 1640 medium supplemented with FCS and antibiotics as above. The SK-N-BE(2)c cells were differentiated with 10 μM all- trans -retinoic acid (RA) in MEM containing 10% FCS for 0, 2, 8, 24, and 96 hours. 5-azacytidine (5-azaC) treatments were performed on SK-N-BE(2)c and SH-SY5Y cells at a concentration of 400 nM. After 64 h, fresh medium containing 50 nM Trichostatin A (TSA) was added. The cells were harvested in PBS after 24 h. RT-PCR analysis RNA was prepared from cells using Trizol (Life Technology) and cDNA was generated by annealing 1 μg of total RNA to 0,5 μg of random hexamers in 10 μl DEPC-treated water. Reverse transcriptase reactions were performed with 200 units of SuperScript Reverse Transcriptase (Life Technologies) in the manufacturers' buffer supplemented with 0,5 mM dNTP, 10 mM DTT and 20 units RNase inhibitor (Boeringer Mannheim, Bromma, Sweden) in a total volume of 20 μl, at 37°C for 1 hr. One-twentieth of the RT reactions were used in the PCR assays. PCR reactions were performed with 1 unit of Taq-polymerase (Life Technologies) in the manufacturers' buffer supplemented with 0.2 mM dNTP, in a total volume of 25 μl. GADPH was amplified by 25 cycles (94°C, 30 s, 55°C, 30 s and 72°C, 30 s) while 30 cycles were used to amplify CGA, SCG10, mb-1 and CD19 message (94°C, 30 s, 61°C, 30 s, 72°C 30 s). Primers were added to a final concentration of 1 mM. PCR products were blotted onto Hybond N + nylon membranes (Amersham) using capillary blotting with 0.4 M NaOH. Membranes were pre-hybridized in 5X Denhardt's, 6XSSC, 0.1% SDS and 50 μg/ml Salmon Sperm DNA, at 57°C for 90 minutes and hybridized with γ[ 32 P] labeled oligonucleotide for 12 hours at 57°C in the same solution. Membranes were washed at room temperature 2 times in 2XSSC supplemented with 0.1% SDS for 15 minutes. Oligonucleotides used for RT-PCR were: GADPH sense; 5'-CCACCCATGGCAAATTCCATGGCA; GADPH antisense; 5'-TCTAGACGGCAGGTCAGGTCCACC; CGA sense: 5'-GAAGATGAACTCTCAGAGGTTC CGA antisense: 5'-GGATCTCCTTGTAGCCAAGGCTCG CD19 sense: 5'-AGTCATTGCTGAGCCTAGAGCTG CD19 antisense: 5'-CTCGGAGTCCTCCTCACTGTCAG mb-1 sense; 5'-CCAGCATCATTGATGGTGAGCC mb-1 antisense: 5'-GACATCTCCTATGTTGAGGCTGC mb-1 hybridization; 5'-CCCGCACAATAGCAGCAACAACGCCAACGT SCG10 sense; 5'-ATGCTGTCACTGATCTGCTCTTGC SCG10 antisense; 5'-CAGGTTGAACTGTCTGGCTGAAG EMSA DNA probes were labeled with γ[ 32 P] ATP by incubation with T4 polynucleotide kinase (Roche Molecular Biochemicals), annealed and purified on a 5% polyacrylamide Tris-borate-EDTA (TBE) gel. Nuclear extract [ 16 ], or in vitro transcribed-translated protein, was incubated with labeled probe (20,000 cpm, 3 fmol) for 30 min at room temperature in binding buffer (10 mM HEPES pH [7.9], 70 mM KCl, 1 mM Dithiothreitol, 1 mM EDTA, 2.5 mM MgCl 2 , 1 mM ZnCl 2 , 5% Glycerol) with 0.75 μg Poly(dI/dC) (Amersham Pharmacia). Antibodies (anti Pax-5 SC-1974, anti Pu.1 SC-352, anti actin SC-1616, ets1/2 SC-275 and anti myc SC-764 all from Santa Cruz Biotech and anti E2-2 from Pharmingen) were added 10 min before the addition of the DNA probe. The samples were separated on a 6% polyacrylamide TBE gel, which was dried and subjected to autoradiography. Oligonucleotides used for EMSA were the following: mb-1 sense 5'-AGCCACCTCTCAGGGGAATTGTGG; mb-1 antisense 5'-CCACAATTCCCCTGAGAGGTGGCT; CD19 -BSAP sense 5'-GCAGACACCCATGGTTGAGTGCCCTCCAGG; CD19 -BSAP antisense 5'-CCTGGAGGGCACTCAACCATGGGTGTCTGC; μ E5 sense: 5'-GGCCAGAACACCTGCAGACG; μ E5 antisense: 5'-CGTCTGCAGGTGTTCTGGCC; Oct binding site sense 5'-CATCTCAAGTGATTTGCATCGCATGAGACG; Oct binding site antisense 5'-CGTCTCATGCGATGCAAATCACTTGAGATC; Lambda B (Pu.1 site) sense: 5'-GAAAAAGAGAAATAAAAGGAAGTGAAACCA AG; Lambda B antisense: 5'-CTTGGTTTCACTTCCTTTTATTTCTCTTTTTC; CRE (ATF5 site) sense: TCA TGG TAA AAA TGA CGT CAT GGT AAT TA CRE antisense: TAA TTA CCA TGA CGT CAT TTT TAC CAT GA cDNA micro-array analysis RNA from Nalm6, SK-N-BE(2)c, SH-SY5Y, KM3, THP-1 and HeLa cells was extracted using Trizol™ (Invitrogen, Carlsbad, California). A common RNA control obtained by mixing a variety of cell lines was used for all hybridizations. RNA was concentrated to 50 μg total RNA (25 μg sample RNA and 25 μg control RNA) to generate aminoallyl-modified cDNA. Sample cDNA was labeled with Cy3-dCTP and control RNA was labeled with Cy5-dCTP using CyScribe Post-Labeling Kit (Amersham Pharmacia Biosciences). A hybridization solution was made by combining labeled cDNA with 20 μl Cot-1 DNA (1 mg/ml), 3 μl Poly dA (4 mg/ml) and 1.5 μl yeast t-RNA (4 mg/ml), dry down by speed-vac and resuspended in 40 μl Pronto! Universal Hybridization Solution™ (Pronto!™ Universal Microarray Reagent System, Corning). The hybridization solution was added to a pre-hybridized microarray slide (DNA microarrays were obtained from the SWEGENE DNA Microarray Resource Center, Lund University). The arrays were hybridized at 42°C for 18 hrs, washed according to the manufacturers recommendations (Pronto!™ Universal Microarray Reagent System, Corning), dried by centrifugation and scanned on Agilent microarray scanner. Scans were analyzed using GenePix Pro versions 4.0.1.9 and 4.1.1.4. BioArray Software Environment (BASE) (Saal et al. Genome Biology 2002, 3(8):software0003.1–0003.6). The settings for the analysis presented were, Background Correlation: Mean FG – Mean BG, Spot filter: (Raw) SNR ch1 mean > = 2, (Raw) SNR ch2 mean > = 2, (Raw) Flags = 0, (Raw) Spot diameter > = 40, Normalization: Lowess, Reporter filter: in # of assays = 16, Analysis: Hierarchical clustering (reporter) Results Neuroblastoma and lymphoma cell lines display similarities in overall gene expression patterns but not of known EBF target genes Knowing that neuroblastoma and pre-B cells share the expression of EBF [ 5 , 13 , 14 ], we wanted to investigate the potential similarities in overall gene expression patters in these two types of tumor cells. To this end we used cDNA micro-array analysis with material from two pre-B cell lines (Nalm6 and KM3), one EBF high and one EBF low expressing neuroblastoma cell line (SK-N-BE(2)c and SH-SY5Y) [ 5 ] (Data not shown, 13) one epithelial cell line (HeLa) and one myeloid cell line (THP-1). The level of specific mRNAs in the samples were compared to that obtained by a pooled reference RNA on cDNA gene chip (Appendix A). This allowed us to reliably investigate variations in expression of 2 162 genes in our cell lines and to group the different samples based on overall similarity in gene expression patterns (Figure 1 ). This suggested that the pre-B and neuroblastoma cell lines clustered to the same half of the expression tree even though THP-1 is a hematopoetic cell line. It also appeared as if while the two pre-B cell lines were rather similar the two neuroblastoma lines displayed larger discrepancies in overall expression patterns. It should be noted that the SK-N-BE(2)c cell line carries an amplified N-myc gene, which in the clinic is correlated to adverse outcome of the disease, while the SH-SY5Y cell line carry an N-myc gene in germ line configuration [ 18 ]. The finding that these two different types of tumors did display a degree of similarity in overall gene expression patterns opened the possibility that the previously identified EBF target genes would be expressed in both cell types. To investigate this possibility we extracted RNA from pre-B cell lines as well as neuroblastoma cells and investigated the expression of the pre-B cell EBF target genes CD19 [ 19 ] and mb-1 [ 13 ] as well as the potential neuroblastoma EBF target genes SCG10 and CGA [ 5 ] by RT-PCR (Figure 2 ). This indicated that while the neuroblastoma cells expressed SCG10 and CGA message they did not express either CD19 or mb-1 message. The opposite pattern was observed in the pre-B cell lines where CD19 and mb-1 but not SCG10 or CGA message could be detected. This show that even though neuroblastoma and pre-B lymphoma cells to some extent share gene expression patterns, they do not appear to share the expression of specific EBF target genes. Neuroblastoma and pre-B lymphoma cells share the expression of the transcription factor Pax-5 Previous work has shown that both the mb-1 and CD19 genes are genetic targets for both EBF and the paired domain protein Pax-5 (BSAP) [ 13 , 19 - 21 ]. Thus, one possibility to explain lineage-restricted expression of these genes could be selective expression of Pax-5 in the Pre-B cells. To investigate this possibility we performed EMSA analysis with nuclear extracts from two human pre-B cell lines (Nalm6 and KM3) and two neuroblastoma cell lines (SH-SY5Y and SK-BE(2)c) (Figure 3A ). As probes we used a consensus Oct binding site and the Pax-5 binding site from the human CD19 promoter [ 20 ]. These experiments revealed that not only the pre-B cell lines, but also the neuroblastoma cell lines expressed proteins able to interact with the CD19 Pax-5 binding site. No such binding activity was detected in extracts from epithelial cells (Data not shown). To verify that the binding activity was due to the presence of Pax-5 we performed a super-shift experiment using a Pax-5 specific antibody, a control antibody or no antibody in the binding reaction (Figure 4 ). While the control antibody did not affect formation of the complex the Pax-5 antibody resulted in a reduced DNA binding and also the appearance of a weak super-shifted band using either pre-B (Data not shown) or neuroblastoma nuclear extracts. These data confirmed that the DNA/protein complex observed in extracts from the neuroblastoma cells was composed of Pax-5 protein. Pax-5 protein could also be detected in the neuroblastoma cell lines IMR-32, KCN-69n and LA-N-1 (Figure 4 ). Since the binding activity of several transcriptions factors such as E-proteins and EBF appear to be modulated during the induced differentiation of neuroblastoma cells [ 5 , 22 - 24 ] we wanted to investigate if this was the case also for Pax-5. To this end, SK-BE(2)c cells were treated with retinoic acid and proteins were extracted at 2, 8, 24, 48, 72 and 96 hours after stimulation. The induction of differentiation was assayed by morphological change and dendrite outgrowth of the stimulated cells. We then analyzed the amount of Oct protein as well as of Pax-5 proteins at the different time points after stimulation by EMSA (Figure 4 ). This indicated that by using the octamer binding activity as a reference there were no major alterations in Pax-5 DNA binding in the course of SK-BE(2)c differentiation. Thus, several neuroblastoma cell lines express both EBF proteins and Pax-5, two genes known to induce expression of the mb-1 and CD19 genes in pre-B cells, but neuroblastoma cells nevertheless fail to express these two target genes. Pre-B lymphoma and neuroblastoma cell lines display differential expression of E-, Ets and ATF proteins In addition to provide information of overall relationships between different types of tumors, the micro-array analysis also yield preliminary information about differential gene expression patterns of individual genes (Appendix A). The data obtained in our experiments suggested differential expression of genes such as myb, CBFβ and HMG 1, 3 and 4 proteins with high expression restricted to the pre-B cells while Bcl-6 appeared to be over-expressed specifically in neuroblastoma cells (Appendix A). To further investigate other potential differences and similarities between pre-B and neuroblastoma cells with regard to expression of gene regulatory proteins we extracted expression data regarding additional genes encoding transcription factors from our data set (Figure 4 ). This analysis indicated that even though the expression of several factors appeared similar, there were large discrepancies in the mRNA expression of the E-protein E47 and the ATF5 protein. These genes were expressed to a high level in lymphoma cells but to a lower level in the neuroblastoma cells. To investigate this further we analyzed the expression of E-proteins by EMSA using a binding site from the mouse Immunoglobulin heavy chain intron enhancer as probe. This suggested that while one prominent complex was formed in the pre-B cell lines, the SK-N-BE(2)c cells did not contain any significant amounts of μE5 binding activity (Figure 5A ). Several complexes were formed using nuclear extracts from SH-SY5Y neuroblastoma cells, but the migration in the gel was different from that of the complexes observed in pre-B cells. Investigating the μE5 binding activity in additional neuroblastoma cell lines suggested that nuclear extracts from IMR-32 cells gave rise to a rapidly migrating complex while extract from KCN-69n cells contained proteins forming two different complexes. This suggests that there might exist differences in E-protein expression in different neuroblastoma cell lines. To further investigate the E-box binding activities in SH-SY5Y cells as compared to Nalm6 cells we performed super-shift assays using antibodies against E2-2, E47 or c-myc (Figure 5B ). The major complexes formed by nuclear extracts from the pre-B cell lines could be super-shifted either by the addition of anti E2-2 or anti E47 antibody while the complex formed in SH-SY5Y extracts reacted only on the addition of anti E2-2 antisera. None of the complexes in any of the cell lines were affected by the addition of either the c-myc or the actin antibody. This indicates that even though E2-2 is a major component in the formation of μE5 binding activity in the pre-B cells, it appears to form complex together with E47. This was not seen in the SH-SY5Y cells suggesting that there exist a distinct difference in the composition of E-box binding complexes in the two cell types. The micro-array analysis did also suggest a difference in the expression of activator of transcription 5 (ATF5/ATF7/ATFX) [ 25 , 26 ]. ATF5 is a transcription factor of the basic-leucinezipper family that has been suggested to modulate neurogenesis [ 27 ] making it potentially interesting in the biology of the neuroblastoma cells. This protein binds to c-AMP responsive element (CRE) [ 25 , 26 ] hence, we performed EMSA analysis using oligonucleotide with a CRE binding site and nuclear extracts from either pre-B cell lines or neuroblastoma cells (Figure 6A ). Both the pre-B cells and the neuroblastoma cells generated two major complexes (C1, C2) migrating with apparently the same mobility as compared to the Oct proteins. The relative abundance of these two complexes did however differ since the pre-B cells contained larger relative amounts of the slowly migrating complex (C1). This suggests that there are differences in the expression of CRE binding proteins in pre-B and neuroblastoma cells lines investigated. EBF has also been suggested to share pre-B cell restricted target genes with Ets proteins and one factor belonging to this family of proteins suggested to contribute to B lineage identity is the Ets protein Pu.1 [ 28 , 29 ]. In order to investigate the DNA binding activities in neuroblastoma and pre-B lymphoma we incubated a PU.1 binding site (λB) from the mouse Immunoglobulin λ enhancer with nuclear extracts from the two cell types (Figure 7 ). This showed a complex pattern of DNA binding activities but only the pre-B cells contained a complex reacting to the addition of anti Pu.1 anti-sera. Thus, we conclude, that even though neuroblastoma and lymphoma co-express several potent transcription factors there are also specific differences. Thus, even though both cell types express EBF, the protein would have to act in a different transcription factor context in the two cell types. Chromatin structure participates directly in the regulation of tissue specific gene expression patterns These experiments suggested that there indeed are specific differences in transcription factor expression between the two cell types, but also that they share expression of several key regulatory molecules. This indicates that additional components of gene regulation may be of crucial importance for target gene selection. One potential barrier to gene activation could be the chromatin structure, that is, the epigenetic setting of the cells directs the transcription factors to their tissue specific target genes. To investigate this possibility we analyzed the expression of the mb-1 gene in neuroblastoma cells after disruption of the chromatin structure by culturing the cells in the presence of the DNA de-methylating agent 5-azaC and the histone deacetylase inhibitor TSA. RT-PCR analysis suggested that while the untreated control cells did not express any detectable amount of mb-1 transcripts the 5-azaC/TSA treated cells expressed significant amounts of mb-1 transcripts (Figure 7 ). To obtain an approximation of the obtained expression levels we compared the amount of mb-1 message in the treated neuroblastoma cells to that of a serially diluted cDNA from Nalm6 pre-B cells. This suggested that while the expression obtained in SK-N-BE(2)c cells treated with 5-azaC/TSA was about one twentieth of that observed in a pre-B cell, the SH-SY5Y cells were induced to express levels comparable to that observed in the pre-B cells. In contrast we were unable to observe an induction of CD19 expression (Data not shown). Thus, we conclude that the lineage specific regulation of the mb-1 gene may be directly dependent on chromatin status, while expression of other pre-B cell specific EBF target genes, such as CD19 , must be explained by other mechanisms. Discussion We here report that neuroblastoma and pre-B lymphoma cells share the expression of transcription factors believed to be essential for normal B cell development while the expression of their B lineage target genes remains tissue specific. The finding that neuroblastoma cells expressed Pax-5 was somewhat surprising even though this factor has been shown to be involved in midbrain development. Mice carrying a homologous disruption of the Pax-5 gene lack both B-lymphoid cells and a fully developed central nervous system [ 30 , 31 ]. In the B cell compartment it also appears as if Pax-5 is crucial to lock the cell in the B-lymphoid developmental pathway. That is, even though pro-B cells from Pax-5 deficient mice express a whole set of early B cell markers [ 30 , 31 ], they can, in contrast to normal pro-B cells, be differentiated into other hematopoetic lineages [ 32 , 33 ]. Thus, Pax-5 appears to be essential for lineage fidelity in addition to be crucial for the completion of the B lymphoid development pathway. The former has been attributed to an ability of Pax-5 to inhibit the expression of cytokine receptors like M-CSF [ 32 ] and also of Notch-1 [ 34 ]. Interestingly, Notch signaling plays a key role in fate selection of neuronal cells and we have previously shown that over expression of a constitutively active form of Notch-1 inhibits induced differentiation of neuroblastoma cells [ 23 ]. It is currently not clear whether a link between Pax-5 and Notch1 expression is at hand in neuronal cells, but it is noteworthy that neuroblastoma cells express relatively high levels of Pax-5. Elevated expression levels of Pax-5 has been detected in two other tumors of neuronal origin, medulloblastoma [ 35 ] and astrocytoma [ 36 ] and in vitro data [ 37 ] as well as observed translocations in human B lineage tumors [ 38 - 40 ] indicate that Pax-5 have oncogenic properties. In contrast to the stable expression of Pax-5 there appeared to be large differences in E-protein expression in the neuroblastoma cell lines investigated. In neuronal tissues a number of tissue specific bHLH proteins have been defined. Importantly, these pro-neuronal bHLH proteins require heterodimerization with E-proteins for the formation of DNA binding complexes. In the developing sympathetic nervous system HASH-1 is transiently expressed and is of pivotal importance for the formation of the autonomic and olfactory nervous system, neuroendocrine cells of the lung and specific regions of the telencephalon (reviewed in [ 41 ]). We have previously shown that a majority of the neuroblastomas express HASH-1, supporting the notion that the tumor is of embryonal origin [ 22 ]. We also showed that E2-2 is the preferential binding partner of the pro-neuronal bHLH protein HASH-1 in SH-SY5Y neuroblastoma cells [ 42 ], and the EMSA results presented in this paper, showing that E2-2 is the main E-protein binding a μE5-E-box, corroborate this observation. Some findings indicate that E2-2 has specific and important functions in neuronal tissues [ 43 , 44 ]. The gene is expressed at substantial level in brain compared to most other tissues [ 44 ] and E2-2 has been shown to bind and regulate several neuronal/neuroendocrine promoters, such as brain-specific FGF-1.B [ 44 ]. Several lines of investigation has suggested that E-proteins are redundant in B cell development and that the apparent need of E2A protein in B cell development is due to lack of sufficient doses of functional E-proteins [ 43 , 45 ]. This notion is substantiated by the finding that E2A deficiency can be rescued by expression of another E-protein, Heb [ 46 ], and that all these proteins appear able to activate the pre-B cell restricted λ5 promoter in synergy with EBF [ 47 ]. Thus, based on these findings it is unlikely that the E-protein composition would have any dramatic impact on EBFs ability to activate target genes in neuroblastoma. Our data also suggest that the chromatin structure of DNA is able to modulate the function of transcription factors in a dramatic manner. In the case of the mouse mb-1 promoter is has been shown that methylation of specific C residues in the promoter prevents the complex formation between Pax-5 and Ets proteins, thereby reducing functional activity of the promoter [ 48 ]. Both the Ets and Pax-5 binding sites are conserved between mouse and human and similar mechanisms acting on the human promoter may well account for the observations in this report [ 49 ]. It is also noteworthy that we obtained a much higher level of mb-1 transcription in the SH-SY5Y cells then in the SK-N-BE(2)c cells (Figure 7 ), a finding that could be expected due to that the SH-SY5Y cells expressed high levels of BSAP and E-proteins while SK-N-BE(2)c cells appeared to express somewhat lower levels of BSAP and no detectable amounts of μE5 binding E-proteins (Figure 3A and 5A ). However, we were unable to observe any activation of CD19 gene even after treatment with demethylating agents suggesting that gene regulation is exerted at several different levels or that the human CD19 gene is highly dependent also of other factors, not present in the neuroblastoma cells. In this study we disrupt chromatin structure by treating the cells with a combination of a methylation inhibitor and a HDAC inhibitor. The processes of DNA methylation and histone acetylation are intimately connected. It has been known for a long time that histone deacetylase inhibitors, by reactivating gene expression, can inhibit growth and/or survival of cancer cells. Even though the precise mechanisms behind the effect of these drugs are largely unknown several of them are now evaluated in clinical trials. Furthermore, the observations of abnormal DNA methylation patterns in malignant cells are becoming increasingly interesting, as new information of the link between gene activation and methylation status increase. One dilemma, however, resides in the issue if a gene is demethylated due to activation or activated due to demethylation. In our specific case the latter would be the most likely explanation. We cannot however exclude that our observations are due to the induction of other unknown proteins. The selective activation of mb-1 and the presence of all the known crucial transcription factors in the neuroblastoma cells would, however, be in line with de-methylation being the primary event. The shared expression of a whole set of transcription factors in such diverse malignancies as lymphoma and neuroblastoma may also be of importance for our view on the relationships between different tumor types. That is, even though these tumors are highly divergent and show large differences in the clinical outcome, there may be genetic links that can be used for drug targeting thus reducing the complexity of cancer treatment dramatically. Conclusion These data suggest that transcription factors are able to selectively activate target genes in different tissues and that chromatin structure plays a key role in the regulation of this activity. Competing interests The author(s) declare that they have no competing interests. Authors' contributions Anna Lagergren has performed the EMSAs, the micro-array experiments and the PCR experiments under the supervision of Mikael Sigvardsson while Christina Manetopoulus has performed the cell culture work as well as 5-aza-Cytidine treatments and RNA purifications under the supervision of Håkan Axelson. All contributors have been involved in the writing of the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 The data shows hierarchical clustering of the microarray data displaying differential gene expression patterns of individual genes in the different cell lines used in this study. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544580.xml
520748	The FK506 binding protein 13 kDa (FKBP13) interacts with the C-chain of complement C1q	Background The pharmacological action of specific immunosuppressants is mediated by immunophilins. While cyclosporin A binds to cyclophilins, FK506/tacrolimus, rapamycin, and others bind to FK506 binding proteins (FKBPs). Different physiological actions of immunophilins were described but their genuine function, however, remains elusive and is still under investigation. A yeast two-hybrid screen was performed using the FK506 binding protein 13 kDa (FKBP13) as a bait and a fetal liver expression library as a prey. Results The C-chain of complement C1q (C1q-C) was detected to interact with FKBP13 in the yeast two-hybrid system and in a protein complementation assay. Neither FKBP12, FKBP25, FKBP52 nor the unrelated immunophilin CypA did react with C1q-C in the yeast system stressing the specificity of the interaction. Binding of C1q-C to FKBP13 could not be prevented in the presence of FK506, demonstrating that possibly other regions than the binding pocket of the drug are responsible for the interaction of the two proteins. Conclusion It is concluded that exclusively FKBP13 but no other FKBPs tested so far interact with the C-chain of complement C1q in the two different assays and further work will be initiated to investigate the physiological relevance of the interaction.	Background The pharmacological action of cyclosporin A and FK506/tacrolimus is mediated by cytosolic immunophilins namely cyclophilin A and the FK506 binding protein 12 kDa (FKBP12). The drug immunophilin complex binds to and inhibits the protein phosphatase calcineurin thus preventing signal transduction in activated T cells [ 1 ]. FKBPs are a family of proteins that were found from procaryotes to humans. At least 10 human FKBPs are known and can be found in every tissue so far analyzed. Some FKBPs bind to and "stabilize" intracellular receptors. For example, FKBP12 and FKBP12.6 interact with the ryanodine Ca 2+ channels RyR1 and RyR2, respectively [ 2 , 3 ]. FKBP52 is part of the steroid receptor complex [ 4 ]. Another FKBP, FKBP13, is localized in the endoplasmic reticulum. FKBP13 mRNA is up-regulated in the presence of unfolded proteins, e.g. after tunicamycin treatment and heat shock and it is regarded to act as a chaperone [ 5 ]. On the other hand FKBP13 was shown to interact specifically with single proteins like a homologue of the erythrocyte membrane cytoskeletal protein 4.1 and a FKBP associated protein 48 kDa [ 6 , 7 ]. Finally, FKBP25 was described to be a nuclear protein and the transcription factor YY1 or histone deacetylases (HDAC1 and HDAC2) specifically interact with this FKBP family member [ 8 , 9 ]. The phylogenetically ancient complement system composed of more than 30 proteins is part of the immunogenic system including a cascade of interacting proteins called C1 to C9. Activation finally leads to lysis of marked cells. C1, the first protein in this cascade, is composed of one C1q and two C1r and C1s proteins. While C1q binds the Fc regions of two IgGs or one IgM molecule, C1r and C1s are known to activate other components of the pathway, namely C4 and C2. C1q contains 18 polypeptide chains (six A-, six B-, and six C-chains) and is composed of six globular heads linked via six collagen like stalks to a fibril-like central region [ 10 ]. Each of the globular heads is formed by association of the C-terminal region of an A-, B-, and C-chain. In addition C1q seems to play an important role as an immunmodulator in its own and has pathophysiological impact. C1q deficient individuals develop a systemic lupus erythematodes (SLE)-like disease [ 11 ] and C1q deficient mice show elevated auto-antibody titres and develop glomerulonephritis and renal damage probably because of accumulation of apoptotic bodies [ 12 ]. As C1q can bind to a variety of pathologically relevant targets in an antibody-independent manner it might directly activate cellular functions. Indeed, several receptors for C1q have been described. C1qRp is a putative receptor for phagocytosis enhancement by monocytes [ 13 ]. The rodent homologue of C1qRp is tightly regulated during development [ 14 ]. C1q was shown to bind to cell lines expressing the complement receptor 1 (CR1) in a saturable manner [ 15 ]. A receptor for the collagenous domains of C1q has been purified and was shown to be idenical to calreticulin [ 16 ]. Finally, a binding protein for the globular head of C1q (gC1qbp) was isolated [ 17 ]. In this work interaction of the C-chain of complement C1q (C1q-C) with a member of the FK506 binding protein (FKBP) family was detected and the specificity of the interaction was further investigated. Results Yeast two-hybrid screen The physiological function of immunophilins in endocrine systems is rarely characterized. A commercially available fetal human liver cDNA expression library was used to identify proteins that interact with the immunophilin FKBP13 which is localized in the endoplasmic reticulum. Plasmids encoding FKBP13 without signal peptide fused to the LexA-DBD (pBTM-FKBP13w/oS) and the liver cDNA library were sequentially transformed into yeast L40. Ten million yeast double transformants (corresponding to 3.5 × 10 6 independent clones) were screened and selected for histidine prototrophy. Among 216 colonies isolated as His + , 21 were found to display beta-galactosidase (β-gal) activity. Plasmids were isolated from each of these clones and were used for retransformation of yeast L40 pretransformed with pBTM-FKBP13w/oS. Six clones remained positive (clone A3, B10, B31, B55, C8, C30) and turned out to code for five different cDNAs after sequence analysis. One of the clones (A3) representing the cDNA of the C-chain of complement C1 corresponding to amino acids 122 to 217 was selected for the present study. The interaction of a second clone with FKBPs was described elsewhere [ 18 ]. Interaction of full length C-chain of C1q with FKBP13 and with other FKBPs FKBPs share a common binding motif for the immunosuppressant FK506. To check the specificity of the FKBP13/C1q-C interaction, yeast L40 was cotransformed with a plasmid coding for the full length C1q-C-Gal4AD hybrid protein lacking a 26 amino acids signal peptide (pGAD-C1q-Cw/oS) and different plasmids each of them coding for FKBP12, FKBP13w/oS, FKBP25, and FKBP52 fused in frame to the DBD of LexA (pBTM-FKBP12, 13w/oS, 25, and 52). Cotransformants were checked for histidine prototrophy and β-gal activity. Only FKBP13 interacted with the C1q-Cw/oS protein in the yeast two-hybrid system whereas FKBP12, 25, and 52 failed in this respect. Interaction of C1q-C with cyclophilin A Cyclophilins are the second family of immunophilins. To check for cross reactivity, binding of C1q-Cw/oS to the prototype of cyclophilins, cyclophilin A (CypA), was investigated in the same system. Yeast L40 was cotransformed with a plasmid expressing LexA-CypA hybrids (pBTM-CypA) and pGAD-C1q-Cw/oS and was tested for β-gal activity and histidine prototrophy. C1q-Cw/oS and CypA failed to interact in the yeast two-hybrid system. Interaction of C1q-C with FKBP13 in a protein complementation assay In order to verify the results obtained with the yeast two-hybrid screen, the GST pull-down approach was used first. As the expression of C1q-Cw/oS either in a coupled reticulocyte lysate system or in bacteria failed, the interaction of C1q-Cw/oS with FKBP13w/oS was analyzed in a protein complementation assay. INS-1 cells were cotransformed with a plasmid coding for FKBP13w/oS fused in frame via a 15 amino acid peptide linker to aa 198 to 287 of β-lactamase (pCDNA-FKBP13w/oS-Bla2) and a plasmid coding for β-lactamase aa 21 to 196 fused in frame via a 15 amino acid peptide linker to C1q-Cw/oS (pCMVSPORT-Bla[1]M182TlinkCENP-A). As shown in Fig. 1 , cotransformation of the hybrid-proteins resulted in β-lactamase activity; the interaction of C1q-Cw/oS with FKBP13w/oS was confirmed by using this second assay. Figure 1 Interaction of C1q-C and FKBP13 in the protein complementation assay. INS-1 cells were transfected with pCMVSPORT-Bla[1]M182TlinkBla[2] (control), or were cotransfected with pCDNA3.1Zeo+/F [I]M182T-15-Zip and pCDNA3.1Zeo+/Zip-15-F[2] (Zip+Zip), or were cotransfected with pCMVSPORT-Bla[1]M182TlinkC1q-Cw/oS and with pCDNA-FKBP13linkBla[2] (13+C1q-C). As a negative control cells were transfected with pCDNA-FKBP13linkBla[2] (sham). After lysis of the cells, β-lactamase activity was measured by hydrolysis rates of nitrocefin. The insert resembles the time dependent effect. The absorbances at 490 nm after 90 min of three independent experiments (mean ± S.E.M.) are shown. The control was normalized to 100 %. Interaction of C1q-C with FKBP13 in the presence of FK506 The crystal structure of FK506/FKBP complexes predicts that the recognition site in the endogenous ligand(s) equivalent to FK506 would best be emulated by a Iso-Pro or Leu-Pro motif [ 19 ]. FK506 might mimic the binding motif of C1q-C to FKBP13. For this reason, the influence of the drug on the interaction of C1q-Cw/oS with FKBP13w/oS was investigated in the yeast two-hybrid system. Cotransformants of yeast L40 with pGAD-C1q-Cw/oS and pBTM-FKBP13w/oS were incubated in the presence or absence of FK506. Cells were collected and measured for β-gal activity. As shown in figure 2 , FK506 does not decrease the interaction of C1q-C with FKBP13, but rather seems to increase the affinity of C1q-C to the FKBP at low micromolar concentrations. The interaction of C1q-C with FKBP13 could not be suppressed, even in the presence of 100 μM FK506. Figure 2 Influence of FK506 on the interaction of C1q-C with FKBP13. Yeast L40 was cotransformed with pBTM-FKBP13w/oS and with pGAD-C1q-Cw/oS. Cotransformants were incubated in the presence or absence of FK506 and analyzed as described in material and methods. Shown are the results of 4–8 independent experiments ± S.E.M.. The control (absence of FK506) was defined as 100 %; the solvent ethanol was without influence. Discussion The C-chain of complement C1q interacts with FKBP13 A yeast two-hybrid screen was carried out using the FK506 binding protein 13 kDa (FKBP13) as a bait and a human liver expression library as a prey. The screen revealed the C-chain of complement C1q (C1q-C) to interact with FKBP13. Three other FKBPs, namely FKBP12, FKBP25 and FKBP52 failed to interfere with C1q-C under same conditions indicating specificity for the positive result in the yeast system. The interaction of C1q-C with FKBP13 was proved by using additionally a protein complementation assay (PCA), demonstrating that the interaction of C1q-C with the FKBP13 was not an artifact in the yeast two-hybrid system. The interaction of C1q-C with FKBP13 in the PCA seems to be comparable to the interaction of the two GCN4 leucine zippers (Zip) which were used as a positive control. The intensity of the interaction might depend on the efficiency of the cell transfection, cell density, cell growth, and other factors but hydrolysis rates of nitrocefin as a marker of the interaction are clearly different from the negative control which can be best seen after 270 min of incubation (Fig. 1 , insert). The verification of the interaction in a classical GST pull-down assay was used elsewhere [ 18 ] but failed in this work as the rather small C1q-C protein could not be produced and labelled in vitro (data not shown). Immunoprecipitation of the interacting proteins was not planned since a C1q-C antibody is not available. The interaction, therefore, of C1q-C with different FKBPs was demonstrated in two independent approaches albeit the presence of an additional cellular factor necessary for the interaction can not be excluded with these cellular assays. The C-chain of complement C1q does not interact with cyclophilin A Immunophilins belong to the class of peptidyl prolyl isomerases (E.C. 5.2.1.8). They can be divided into FKBPs that bind for example FK506/tacrolimus or rapamycin and cyclophilins that mediate the pharmacological action of cyclosporin A (CsA). Cyclophilin A (CypA) can be regarded as the prototype of the cyclophilins. Unlike the aforementioned FKBP13, the immunophilin CypA does not interfere with C1q-C in the yeast two-hybrid system underlining the specificity of the interaction of C1q-C with FKBP13. FK506 fails to negatively influence the FKBP13 C1q-C interaction The immunosuppressive drug FK506 is not able to decrease the affinity of C1q-C to FKBP13 in the yeast two-hybrid system even when used in a concentration of 100 micromolar. The common binding motif of FKBPs for FK506 is, therefore, less likely to be uniquely involved in the C1q-C FKBP13 interaction. On the other hand, at low micromolar concentrations FK506 seems to „strengthen" the interaction of C1q-C with FKBP13. Possibly, C1q-C, when produced artificially in yeast, binds to yeast FKBP as well and has to be liberated from these interfering positions by FK506, then contributing to the interaction with the FKBP13-AD hybrid protein. An excess of FK506 should further diminish the C1q-C FKBP13 interaction but the drug cannot be tested in the milimolar range because of its toxicity. The influence of FK506 on the interaction of the two proteins was not investigated in a mammalian cell line because the influence in the yeast system is more likely comparable to other investigations [e.g. [ 7 , 18 ]]. Additionally the immunosuppressive agent is expected to be far toxic in the micromolar range when used in a mammalian cell system. Finally, a negative effect of the immunosuppressive drug on the C1q-C FKBP13 complex could not be demonstrated albeit the involvement of the FK506 binding pocket in the interaction cannot be excluded. Conclusions Using two different experimental approaches, the C-chain of complement C1q was shown to interact with FKBP13. FKBP13, initially used as the bait for the yeast two-hybrid screen in this work, is regarded as an endoplasmic reticulum (ER) protein. It might act as a chaperone but specifically interacts with other proteins like a homologue of the erythrocyte membrane cytoskleletal protein 4.1 or the FKBP associated protein 48 (FAP48), too. FKBP13 was detected in membrane preparations from erythrocytes as well, demonstrating that its expression and maintenance is not restricted to the ER [ 6 ]. The C-chain of complement C1q is part of the C1q molecule that is composed of six A-, six B-, and six C-chains, respectively. Interaction of FKBP13 with the A- or B-chain or with the entire C1q molecule has not been investigated yet. The carboxy-terminal part of the C-chain is sufficient to interact with FKBP13 in the yeast two-hybrid system. If the entire C1q molecule interacts with FKBP13, interaction of its globular head with the immunophilin will be likely therefore, as the globular head is is formed by association of the C-terminal regions of an A-, B-, and C-chain. On the other hand, FKBP13 might act as a specific chaperone, triggering C1q-C in the endoplasmic reticulum and preventing the complement protein to interact with other intracellular proteins. Further work will be necessary to elucidate the physiological significance of the interaction of the two proteins and to investigate whether FKBP13 plays an important role in the complement system, too. Methods Expression vectors The cDNA encoding FKBP13 amino acids 16–142 was amplified from pBluescript-FKBP13 (a gift from Dr. S. Burakoff, Dana-Faber Cancer Institute, Boston, MA) using specific primers CGCCGGAATTCATGCTGAGCGCCGTG and CGGCTGGATCCGAACAGTCTGGTC. The cDNA encoding full-length FKBP25 was amplified from pBluescript-FKBP25 (a gift from Dr. S. Burakoff, Dana-Faber Cancer Institute, Boston, MA) using the specific primers CGCGCGAATTCAACA GTCTGGTCCCTGATG and GGCGTAGGATCCGGGGTTGACTCCGGGGGC. The cDNA encoding full-length cDNA of CypA was amplified from I.M.A.G.E clone 5264185 (ResGen, Huntsville, AL) by using the specific primers GGTCCGGAATTC ATGGTCAACCCCACCGTGTTC and GGCAGCTGGATCCACAAGTCA AAC TTATTCGAG. After restriction digest, cDNAs were fused to the DNA binding domain of LexA by inserting the EcoRI/BamHI fragments into pBTM116 [ 20 ] to give pBTM-FKBP13w/oS, pBTM-FKBP25, and pBTM-CypA, respectively. pBTM-FKBP12 and pBTM-FKBP52 (a gift from Dr. B. Chambraud, INSERM U488, Bicêtre, France) are described elsewhere [ 7 ]. Full lenght cDNA of human C1q-C lacking a 26 amino acids signal peptide was amplified from I.M.A.G.E clone AL568589 (ResGen, Huntsville, AL) using the specific primer CACGGAATTCAA GCCAACACAGGCTGCTAC and primer M13 reverse. After restriction digest the cDNA fragment was fused in frame to the GAL4AD by inserting the EcoRI/NotI fragment into pGAD1318 to give pGAD-C1q-Cw/oS. The two vectors pCDNA3.1Zeo+/F [I]M182T-15-Zip and pCDNA3.1Zeo+/Zip-15-F[2], a gift from Dr. S.W. Michnick, Montreal, were used as a positive control in the protein complementation assay and are described elsewhere [ 21 ]. The cDNA encoding the TEM-1 β-lactamase gene amino acids 21 to 196 (Bla[1]M182T) was amplified from pGEM11Zf(+) using the primer pairs (i) TTGGGCACCATGGACCCAGAAACG CTGGTGAAAG and GTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACA GGCGACGAGGTGTC and (ii) GCGGCGAAGCTTCAATTGGGCACCATGGAC CCAGAAACG and ACCACCGGATCCGCCAGTTAATAGTTTGCGCAACG. Note that a point mutation leading to M182T was inserted thatway [ 22 ]. The cDNA encoding the TEM-1 β-lactamase gene amino acids 198 to 287 and an amino-terminal 15 amino acid linker (linkBla[2]) was amplified from pGEM11Zf(+) sequentially using the primer pairs (i) GGTGGTGGTAGTCGAATTCTACTTACTCTAGCT TCCCGGC and CCAGCTCTCGAGTTACCAATGCTTAATCAGTGAGGCACC (primerBla2XhoI) and (ii) CTGGCGGATCCGGTGGTGGTGGTAGTGGTGGTG GTGGTAGTGGTGGTGGTGGTAGTCGAATTCTAC and primer Bla2XhoI. After restriction digest cDNA of FKBP13 (from pBTMFKBP13w/oS) was fused into the EcoRI/BamHI site of pCDNA3.1 (Invitrogen, Groningen, The Netherlands) and the linkBla[2] fragment was fused into the BamHI/XhoI site of this plasmid to give pCDNA-FKBP13linkBla[2]. After restriction digest Bla[1]M182T and linkBla[2] fragments were sequentially fused into the MunI/BamHI and BamHI/XhoI site of pCMVSPORT6 (Invitrogen, Groningen, The Netherlands) to give pCMVSPORT-Bla[1]M182TlinkBla[2]. After restriction digest cDNA of C1q-C lacking the signal peptide (from pGAD-C1q-Cw/oS) was inserted into the EcoRI/XhoI site of pCMVSPORT-Bla[1]M182TlinkBla[2] to give pCMVSPORT-Bla[1]M182TlinkC1q-Cw/oS. All inserts were verified by sequencing. Two-hybrid screen The human fetal liver MATCHMAKER cDNA library (Clontech, Palo Alto, CA) was a gift from Dr. C. Sorg (Institute of Experimental Dermatology, Münster, Germany). The yeast reporter strain L40, containing two reporter genes, HIS3 and LacZ , was sequentially transformed with pBTM-FKBP13w/oS and the cDNA library using the lithium acetate method [ 23 ]. Double transformants were plated on Minimal SD Base containing the -Leu/-Trp/-His DO supplement (Clontech, Palo Alto, CA). The plates were incubated at 30°C for 5 days. His + colonies were patched and assayed for β-gal activity. Positive clones, inserted into the pACT2 vector, were rescued and tested for specificity by retransformation into yeast L40 with pBTM-FKBP13w/oS. Different clones, that expressed detectable β-gal activity within four hours, were used for further analysis. All assays of β-gal activity and histidine prototrophy were performed according to standard procedures [ 24 ]. Protein complementation assay The protein complementation assay (PCA) was performed as described elsewhere [ 21 ]. Briefly, 1 × 10 6 INS-1 cells, an insulinoma cell line [ 25 ], were cotransformed with 1 μg of the plasmids pCDNA-FKBP13linkBla[2] and with 1 μg pCMVSPORT-Bla[1]M182TlinkCENP-A, or with pCMVSPORT-Bla[1]M182TlinkBla[2] alone, or with the positive control plasmids using the FuGENE 6 transfection reagent (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. After two days, cells were washed with PBS and harvested into 100 μl of 100 mM phosphate buffer, pH 7.0 and lysed by three freeze-thaw cycles. After centrifugation at 13,000 g for 4 min, the supernatant was used to check for β-lactamase activity. In a 96-well microtiter plate each well was loaded with 80 μl of the phosphate buffer, 80 μl dejonized water, 20 μl of phosphate buffer containing 1 mM nitrocefin (EMD Biosciences, San Diego, CA, freshly prepared from a 10 mM stock solution in DMSO) and 20 μl of each supernatant. The absorbance at 496 nm was measured time-dependently in a platereader (Spectra Max 340, Molecular Devices, Sunnyvale, CA). Data after 90 min of incubation were used to calculate the absorbance as percent of control. Authors' contributions The authors contributed equally to this work	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC520748.xml
514604	3 dimensional modelling of early human brain development using optical projection tomography	Background As development proceeds the human embryo attains an ever more complex three dimensional (3D) structure. Analyzing the gene expression patterns that underlie these changes and interpreting their significance depends on identifying the anatomical structures to which they map and following these patterns in developing 3D structures over time. The difficulty of this task greatly increases as more gene expression patterns are added, particularly in organs with complex 3D structures such as the brain. Optical Projection Tomography (OPT) is a new technology which has been developed for rapidly generating digital 3D models of intact specimens. We have assessed the resolution of unstained neuronal structures within a Carnegie Stage (CS)17 OPT model and tested its use as a framework onto which anatomical structures can be defined and gene expression data mapped. Results Resolution of the OPT models was assessed by comparison of digital sections with physical sections stained, either with haematoxylin and eosin (H&E) or by immunocytochemistry for GAP43 or PAX6, to identify specific anatomical features. Despite the 3D models being of unstained tissue, peripheral nervous system structures from the trigeminal ganglion (~300 μm by ~150 μm) to the rootlets of cranial nerve XII (~20 μm in diameter) were clearly identifiable, as were structures in the developing neural tube such as the zona limitans intrathalamica (core is ~30 μm thick). Fourteen anatomical domains have been identified and visualised within the CS17 model. Two 3D gene expression domains, known to be defined by Pax6 expression in the mouse, were clearly visible when PAX6 data from 2D sections were mapped to the CS17 model. The feasibility of applying the OPT technology to all stages from CS12 to CS23, which encompasses the major period of organogenesis for the human developing central nervous system, was successfully demonstrated. Conclusion In the CS17 model considerable detail is visible within the developing nervous system at a minimum resolution of ~20 μm and 3D anatomical and gene expression domains can be defined and visualised successfully. The OPT models and accompanying technologies for manipulating them provide a powerful approach to visualising and analysing gene expression and morphology during early human brain development.	Background Brain development, particularly in human, involves complex changes in shape and structure over time. During a period of approximately 4 weeks (from 26 to 56 days of development; Carnegie stages CS12 to CS23) the major subregions of the human brain are established and development proceeds from a simple neuroepithelial tube to a highly complex three dimensional (3D) structure [ 1 ]. For many years it has been recognized that three dimensional models are an important aid to interpreting these developmental changes. In the past, these have been based on labour intensive methods for reconstructing representations of physical sections (e.g. Born reconstructions [ 2 ]) or, more recently, on computer-based methods, although these are still labour intensive [ 3 ]. Non-invasive techniques have also been used and these have advantages of speed and lack of sectioning artifacts and, for example with MRI, the ability to generate in vivo images and good quality images for larger specimens [ 4 ]. Recently, a new, rapid and non-invasive 3D modelling method, Optical Projection Tomography (OPT; [ 5 , 6 ]), has become available, and we have used this method to generate computer-based 3D models from intact early human developmental specimens. OPT has the advantage over MRI in that detailed models can be produced from small samples. With MRI, low signal-to noise ratios make it more difficult to obtain high quality data from embryos younger than CS17 [ 7 ]. MRI is likely to be useful for specimens larger than CS23 where the size of the specimen and the density of the tissue are too great to allow penetration of the light. The OPT models are visualised and manipulated using MAPaint, a suite of software programmes developed as part of the Edinburgh Mouse Atlas Project ( [ 8 - 10 ]). The software allows any OPT model to be digitally sectioned in any plane and several different planes can be viewed simultaneously. These planes can be selected at any arbitrary viewing orientation and position through the model. In addition anatomical regions can be defined and manually "painted", allowing the user to interactively assess developing anatomy. OPT reconstructions were made of human embryos for a number of different stages of development. In all cases, reconstructions were made from autofluorescent imaging – in other words, the intrinsic fluorescence from the fixed specimens was used as the basis for the histological contrast seen in the 3D model. One of the models (CS17; approx. 41 days of development) was examined in detail in order to test the resolving power of the OPT technology on these unstained embryos, in relation to structures in the developing nervous system and to determine the feasibility of using the model as a framework for mapping gene expression patterns. Digital OPT sections were compared with corresponding histological sections stained in 3 different ways: standard haematoxylin and eosin stain to visualise cell nuclei, cytoplasm and connective tissue [ 11 ] and immunocytochemistry to detect GAP43 or PAX6 expression. H&E staining distinguishes amongst the ventricular, intermediate and mantle layers in the central nervous system and cranial nerves and ganglia are clearly identifiable. Growth-associated-protein 43 (GAP43) is expressed in growing dendrites and axons [ 12 ] and is expressed in the peripheral nervous system and developing tracts of the central nervous system. A number of genes have been identified, particularly in mouse and chick, that are involved in the specification of different brain regions (e.g. reviewed in [ 13 ] and [ 14 ]). Such gene expression patterns can be used to identify specific brain regions and compare their relative extent in different species (reviewed in [ 15 ] and [ 16 ]). PAX6 expression is well characterized as defining several regions and boundaries in the developing mouse brain [ 17 , 18 ] and [ 19 ]. The effectiveness of using 2D section data to generate 3D expression domains was tested by examining two boundary regions in the CS17 model, the zona limitans intrathalamica (between the dorsal and ventral thalamus) and the midbrain-diencephalon boundary. Results and Discussion An OPT model was generated from an intact, unstained embryo which had been staged according to the Carnegie staging protocol [ 20 ] modified for use with individual embryos rather than in comparisons of many embryos simultaneously [ 21 , 22 ]. The embryo was staged as CS17, which is approximately 41 days of development. The developing central nervous system (CNS) is clearly visible even in the external view of the CS17 model (Figure 1 and the accompanying movie, additional file 1 1). Differences in autofluorescence within the CNS and among different organs are also apparent. There is very little detail in the developing liver, for example, compared to the CNS. Blood vessels, dorsal root ganglia and developing vertebrae are clearly visible. Figure 1 CS17 OPT model (a). still shot from movie of 3D OPT model of a CS17 human embryo (approximately 41 days of development). bv, blood vessel; drg, dorsal root ganglion; h, heart; H, hindbrain; l, liver; T, telencephalon; v, vertebrae. (b; Additional file 1) Mpeg movie of 3D CS17 OPT model. Following OPT the embryo was embedded in paraffin wax and sectioned using standard methods and then every fifth section was either stained with haematoxylin and eosin or immunocytochemically with antibodies against GAP43 or PAX6. The actual plane of sectioning was identified in the OPT model by manipulating the model in MAPaint. This permitted a matched series of digital and physical sections to be compared (examples are shown in Figure 2 ) in order to assess the resolution of internal structures within the CS17 OPT model. Different features are highlighted by the different staining techniques and some examples of these are also shown in Figure 2 . The cranial nerve ganglia stain partially with GAP43 (e.g., Xg) and in the H&E sections (e.g., Vg) and are clearly visible in the OPT model (Figure 2a and 2g ). Cranial nerves also stain for GAP43 (e.g., III) but are less visible in both the H&E section and the OPT model. Surprisingly the rootlets of cranial nerve XII do show up clearly in the OPT model (Figure 2a ). In the CNS, the ventricular layer can be clearly distinguished as more darkly staining in the H&E sections and it is also visible as a darker layer in the OPT model (Figure 2a,2d and 2g ). The core of the zona limitans intrathalamica (zli) is seen as a pale region in all three sections ( 2g,2h,2i ). The GAP43 stained fibres in the floor of the hindbrain ( 2e ) show clearly as a pale area in the OPT model ( 2d ). As was visible in figure 1 , blood vessels and dorsal root ganglia show up very clearly in the OPT model ( 2a and 2g ). Fluid filled spaces are also clearly visible in the OPT model, such as the diencephalic, midbrain and hindbrain ventricles and the otic vesicle ( 2a and 2d ). Some artefactual changes in shape seen in the physical sections (for example, a collapse of the fourth ventricle roof [r in fig. 2f ]) are not present in the OPT model (compare 2d and 2f ). The size of a variety of structures was measured in the H&E and/or GAP43 stained sections in order to determine the resolution in the CS17 model. When OPT is performed on specimens in which specific cells have been fluorescently-labelled, then these small structures can be detected (unpublished data). However, in cases where the specimen contains no tissue-specific dyes, a resolution of 5 to 10 micron has previously been found [ 5 ]. At the magnification used to generate this CS17 model, the smallest clearly measurable structures are the rootlets of the XII cranial nerve which are approximately 20 μm in diameter (Figure 2a ). Figure 2 Comparison of digital OPT sections with histology sections from the same embryo Digital OPT sections of the CS17 model (a, d and g), viewed using MAPaint software, compared with sections stained using antibodies against GAP43 (b, e and h), and Haematoxylin and Eosin stained sections (c, f and i). In b, e and h, expression is demonstrated by the brown chromagen. Structures of 20 μm in diameter (for example the hypoglossal rootlets) are clearly identifiable, as are the differences amongst a variety of developing tissues. bv, blood vessel; D, diencephalon; drg, dorsal root ganglion (~150 μm); fp, floor plate; H, hindbrain; III, oculomotor nerve; M, midbrain; nh, neurohypophysis (~200 μm by ~50 μm); ov, otic vesicle; r, collapsed roof of 4th ventricle; sc, spinal cord; Vg, trigeminal ganglion (~300 μm by ~150 μm); vl, ventricular layer; X, vagus nerve; Xg, vagus ganglion; XIIroot, hypoglossal rootlets (~20 μm); zli; zona limitans intrathalamica (~100 μm, core is ~30 μm). Scale bars = 200 μm MAPaint, a UNIX-based software suite, allows the OPT models to be digitally sectioned in any plane and several planes to be viewed simultaneously. This is illustrated for the CS17 model in Figure 3a ( additional file 2 ) which moves through a series of digital transverse sections with the corresponding position of each section shown on a sagittal section, followed by a series of sagittal sections and the corresponding position on a transverse section. The CS17 model is shown with some painted anatomical domains and Figure 3b shows a snapshot of all fourteen 3D domains with the position of the two transverse sections shown in Figure 3c indicated in white. The anatomical domains were painted in one plane and then checked and refined by corrections introduced in orthogonal planes. The ease of manipulation of the model means that the best digital plane (normally orthogonal to any boundary or set of them) can be selected for each anatomical region being painted. The authors who did these tracings actually found the experience intellectually rewarding, since it amounted to being able to check instantly any difficult point upon a collection of identical specimens sectioned in many different planes. Doubts that often remain unresolved could be resolved very convincingly. Figure 3 Painted anatomical domains. Fourteen regions of the central nervous system in the CS17 specimen have been defined and painted. Forebrain , red (secondary prosencephalon), dark orange (prosomere 3 including ventral thalamus), light orange (prosomere 2 including dorsal thalamus) and yellow (prosomere 1 including pretectum); midbrain , light green; hindbrain ; isthmus, dark green; various shades of blue and purple indicate rhombomeres 1–6 and the caudal medulla oblongata; spinal cord , dark red. (a; Additional file 2) In the Mpeg movie sagittal and transverse views of the painted model are shown, together with a representation of the 3D domains. The model is first sectioned in the transverse plane. This section plane has been matched to that of the histology sections shown in fig 2. As the section is moved through the model the corresponding position is displayed in the 3D box, and by a line on the sagittal section. The model is then moved through the sagittal plane, and the position shown by a line on the transverse section. A snapshot of the fourteen 3D anatomical domains (b), and two examples of painted sections that intersect several anatomic domains (i.e., are topologically nearly horizontal to the reconstructed transverse boundaries) (c). The position of the two digital transverse sections is indicated by white lines on the 3D view. As described in the Methods section, and in the legend to Figure 2 , paraffin sections were immunostained with either anti-GAP43 or anti-PAX6 antibodies at approximately 50 μm intervals throughout the head of the CS17 specimen and the data were captured and mapped to the CS17 model. Figure 4a illustrates a sagittal section through the model with digital GAP43 expression (in red) which has been generated from the data thresholded from transverse sections, an example of which is shown in Figure 4b . There is an unexpected apparent region of no GAP43 expression in the hindbrain on the sagittal section (Fig 4a , arrow). However, the ability to relate the sagittal and transverse sections in the model makes clear that the lack of expression is due to the sagittal section being "cut" obliquely and the section is passing through the floor plate region of the hindbrain at that point, where there is no GAP43 expression (arrowed in Figure 4b ). A sagittal section with digital PAX6 expression shown in green also illustrates the lack of PAX6 expression in the same floor plate region in the hindbrain (Figure 4c ). Comparing Figure 4a and 4c it can be seen that the boundary of the PAX6 expression in the caudal diencephalon is approximately the same boundary as that of GAP43 which stains the posterior commissure, just caudal to which is the boundary between the pretectum, the most caudal region of the developing diencephalon [ 23 , 24 ] and the midbrain. The lack of PAX6 and GAP43 expression in the region just caudal to the posterior commissure is shown (Figure 4d ; upper 2 panels) while the expression of both GAP43 and PAX6 in the pretectum is shown in the lower two panels of figure 4d . The 3D PAX6 domain identifies both the diencephalon-midbrain boundary and the negative intrathalamic boundary or zli (as shown in Figure 5a,5b and the accompanying movie ( Additional file 3 ). Additional limits of PAX6 expression visible in Figure 5b correspond to the basal telencephalon (the striatopallidal boundary) and the alar-basal boundary across the entire diencephalon [ 25 , 23 , 24 ]. Separate areas of PAX6 expression appear in the hindbrain (Figs. 5a,5b ) Figure 4 CS17 OPT model showing 3D gene expression domains. (a) Digital sagittal section through the CS17 OPT model, with the GAP43 gene expression domain shown in red. The plane of this sagittal section is shown by a line on the corresponding transverse section in (b). The GAP43-negative region in the hindbrain floor plate is shown on both sections by an arrow. (c) The same digital sagittal section, with PAX6 gene expression displayed in green. (d) High power images of GAP43 and PAX6 expression near the diencephalon/midbrain boundary. The upper two panels correspond to the rostral midbrain, where there is no expression of GAP43 or PAX6. The lower two panels correspond to the caudal diencephalon, in the region of the posterior commissure. Here both genes are expressed (brown chromagen). Figure 5 3 dimensional gene expression domains. A surface rendered model of the 3D expression pattern of PAX6. Separate gene expression domains in the forebrain and hindbrain are shown in green. For reference the neural tube has been painted pale grey and the eye dark grey. The diencephalon/midbrain (D/M) boundary, the absence of staining in the zona limitans intrathalamica (zli), plus the forebrain alar-basal boundary and the striatopallidal boundary in the basal telencephalon can be seen by viewing the 3D model at various angles (a, frontal and b, lateral). (c; Additional file 3) Mpeg movie of the PAX6 expression domain. The period from CS12 (approximately 26 days of development) to CS23 (approximately 56 days of development) is important because during this time all the major regions of the developing brain are established [ 26 , 1 ] and most major congenital abnormalities can arise. Major changes take place in the size, shape and complexity of the developing brain during this time and we tested the feasibility of generating OPT models throughout this period. There is a more than 60 fold increase in volume between the CS12 embryo and the CS23 head, however, OPT models have been generated successfully throughout these stages. Figure 6 shows snapshots of models at each of the stages from CS12 to CS23. These are not to scale because each model is generated at the maximum magnification possible, which varies according to the size of the specimen. The changes in shape and complexity are clearly seen even in these static images. We currently have 54 OPT models, including a male and female at each stage CS12-CS20 and CS22. At CS17 we have twelve different OPT models and have assessed their natural variability for 3 specific neurodevelopmental features (development of the choroid plexus in the lateral ventricles, the zli and the floor plate in the hindbrain). At this stage, there was little variation in the features assessed (data not shown). Movies of all these models can be viewed at our website ( [ 27 ]), which includes a database of gene expression. The models are too large to display fully or to manipulate online, but a selection of the full models are available on CD on request and a new viewer, the Java Atlas Viewer, has been developed which enables the models to be viewed and manipulated on many platforms (Burton, Feng, Hill and Baldock, paper in preparation). See additional files 4 and 5 for the request form and Academic use licence agreement. Figure 6 OPT models of CS12 to CS23. Still shots of the left lateral side of 3 dimensional OPT models of human embryos spanning the major period of organogenesis (CS12-CS23). The developmental stage (e.g. CS12), specimen number (e.g. N285) and karyotype for each model are given underneath. The movies for all of these models can be viewed at [27]. The full models for all stages are available on request. Conclusions Many structures within the developing nervous system can be identified in the CS17 OPT model with a minimum defined resolution of approximately 20 μm. The CS17 model also acted as a framework onto which anatomical domains were easily painted and gene expression patterns mapped. OPT models have been successfully generated from CS12 to CS23, and these models will provide a means of analyzing and relating changes in anatomy and gene expression both within individual developmental stages and across developmental time. In the long-term, our aim is to link the 3D models to an anatomical database and embed both of them within a custom-designed gene expression database in order to create an Electronic Atlas of the Developing Human Brain ( [ 27 ]). Methods Embryo collection Human embryos were collected from termination of pregnancy material, with appropriate written consent, approval from the Newcastle and North Tyneside NHS Health Authority Joint Ethics Committee and following national guidelines [ 28 ]. Embryos were collected into cold PBS, separated from surrounding tissue and fixed overnight in 4% paraformaldehyde at 4°C before short-term storage at 4°C in 70 % ethanol. Placental tissue was sampled for karyotype analysis prior to fixation of the embryo tissue. OPT Intact specimens were rehydrated through a graded series of ethanol and embedded in a block of 1% low melting point agarose. They were then dehydrated through a graded series of methanol before being cleared using a mixture of benzyl alcohol and benzyl benzoate. 400 digital images were then captured while the now almost transparent specimens were rotated in a full circle, with 0.9° steps between each image. The signal corresponded to the weak autofluorescence originating from the paraformaldehyde-fixed tissue and was detected using a wideband FITC filter with excitation at 465–500 nm and emission from 515–560 nm. The images were then assembled to recreate the 3D shape of the embryo, using modified tomography algorithms [ 5 ]. Post OPT processing and histology After OPT scanning the CS17 embryo N365 was rehydrated through a graded series of methanol and was then removed from the agarose block by incubation in a 0.29 M sucrose solution at 55°C. It was then processed for paraffin wax embedding and 5 μm microtome sections were cut. Every 5 th section was stained with haematoxylin and eosin, following standard procedures. Immunohistochemistry The remaining N365 sections were used for immunohistochemistry. Alternate one-in-five section series were stained with antibodies against GAP43 (GAP-7B10, Sigma), or PAX6 (PRB-278P, Covance) using standard techniques. The reaction was visualised with diamino benzidine and the sections lightly counterstained with toluidine blue. Gene Expression Mapping Images of the stained sections were captured through a ×2.5 objective (as viewed down the microscope at 25× magnification) using the Zeiss Axiovision system. For the PAX6 and GAP43 data, a modified warping interface of the MAPaint software was used to match each stained, physical section to the corresponding digital OPT section. Correspondences between the physical (source) and digital (target) images were identified and manually tie-pointed. The source image was then transformed to the shape of the target section, and the image transformation saved. The interface uses interactive thresholding to extract the expression signal from the source image and then applies the image transformation to map this signal into the space of the 3D OPT model. This was carried out for approximately 190 sections through the head of the CS17 embryo until the full 3D expression pattern was built up for each of the GAP43 and PAX6 data sets. Authors' contributions JK reconstructed the OPT data, carried out the immunocytochemistry, mapped the GAP43 gene expression patterns to the CS17 model and drafted the manuscript. MS optimised the OPT methodology for human specimens, scanned the embryos, carried out the sectioning and mapped the PAX6 gene expression. JS invented the OPT technique and established the methodology in Newcastle. LP, MdlT and JLF identified and painted anatomical regions of the CS17 neural tube. GF developed the Java Atlas Viewer. RB designed/authored the MAPaint software and with DD heads the Edinburgh Mouse Atlas Project. TS, SCR and SL co-ordinate and oversee the Human Developmental Biology Resource which provided the human material. SL and TS conceived of the study and participated in its design and coordination. All coauthors participated in drafting or refining the text. List of abbreviations 3D-three dimensional 4D-four dimensional bv-blood vessel CNS-central nervous system CS-Carnegie Stage D-diencephalon D/M-midbrain/ diencephalon boundary drg-dorsal root ganglion EADHB-Electronic Atlas of the Developing Human Brain fp-floor plate GAP43-Growth Associated Protein 43 h-heart H&E-haematoxylin and eosin H-hindbrain III-oculomotor nerve l-liver M-midbrain nh-neurohypophysis OPT-optical projection tomography ov-otic vesicle r-collapsed roof of 4 th ventricle sc-spinal cord T-telencephalon v-developing vertebrae Vg-trigeminal ganglion X-Vagus nerve Xg-Vagus ganglion XIIroot-hypoglossal rootlets Zli-zona limitans intrathalamica Supplementary Material Additional File 1 CS17 OPT model.mpg. Mpeg movie of 3D CS17 OPT model. Click here for file Additional File 2 CS17 painted anatomy.mpg. Mpeg movie of anatomical domains painted on the CS17 model. Sagittal and transverse views of the painted model are shown, together with a representation of the 3D domains. Click here for file Additional File 3 PAX6 3D expression.mpg. Mpeg movie of the 3D PAX6 expression domain. Click here for file Additional File 4 JAtlasViewer request form.pdf Click here for file Additional File 5 Academic Licence Agreement.pdf Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC514604.xml
549517	LDL receptor expression on T lymphocytes in old patients with Down syndrome	Background In Down syndrome patients several metabolic abnormalities have been reported, some involving the lipid metabolism. The level of LDL in plasma is the major determinant of the risk of vascular disease. There appear to be no studies on the LDL receptor in Down syndrome patients. Methods Flow cytometric methods for measuring the LDL receptor in peripheral blood mononuclear cells (PBMC) can identify patients with hypercholesterolemia. We applied this method in 19 old patients with Down syndrome and 23 healthy controls. Results Down syndrome patients had high levels of triglycerides and low levels of HDL, and high levels of CRP. We also found a down-regulation of LDL receptor expression. Conclusions Down syndrome patients show no increase in the frequency of cardiovascular disease. The low incidence in cardiovascular disease despite the low level of HDL, high levels of CRP and reduction of LDL receptor expression lead to the conclusion that either these are not risk factors in these patients or that other risks factors – not yet identified – are considerably lower.	Introduction Several studies have discussed the psychological and intellectual problems, immunological deficiencies, and early aging of Down syndrome (DS) patients. Several metabolic abnormalities have been reported, some involving the lipid metabolism [ 1 ]. Apart from some contradictory studies in the past, there are only few investigations of the cholesterol fractions in DS patients. Therefore, it must be concluded that the low prevalence of coronary artery disease in individuals with DS cannot be explained by their cholesterol fractions. Mortality statistics of these patients showed practically no deaths due to advanced atherosclerosis [ 2 ], and similarly, pathological studies have detected no increase in atherosclerosis – or even a complete absence of atherosclerotic changes [ 3 ]. In children [ 4 ] and also adolescents [ 5 ] with DS low levels of high-density lipoprotein (HDL) have been reported and recently, we have learned much about the vasoprotective HDL cholesterol [ 6 ]. Anyway DS remains a disease in which atherosclerosis is rare [ 7 ]. Measurements of LDL receptor expression are also necessary to fully characterize the functional status of the low-density lipoprotein (LDL) pathway which substantially influences LDL levels in plasma, and its discovery constituted a major biological advance by providing molecular explanations of hypercholesterolemia. The plasma LDL level is the major determinant of the risk of vascular disease. We analyzed, also, C reactive protein (CRP), a cardiovascular risk factors coded by genes lying on Chromosome 21. Flow cytometric methods for measurement of LDL receptor on peripheral blood mononuclear cells (PBMC) may be used to identify patients with familial hypercholesterolemia [ 8 ]. Data in uremic patients suggest that a defect in LDL receptor function in PBMC may be due to a decrease in LDL receptor expression, which could contribute to the aberrant lipoprotein metabolism [ 9 ]. We therefore investigated LDL receptor expression on uninduced PBMC, particularly T lymphocytes because they express more LDL receptors than monocytes [ 10 ]. Since the progression of atherosclerosis is age-dependent, LDL receptor interactions are important in lipid plaque formation and T cells are present in early atherosclerotic lesions, interacting with LDL through the LDL receptor [ 11 ], we studied LDL expression on T lymphocytes in a group of old patients with DS. Methods Blood samples were drawn from 19 old DS patients (male, average age 55 years) and 23 healthy individuals (male, average age 55 years) without dyslipidemia or any family history of coronary heart disease, no smokers or drunkers, with a Body Mass Index (BMI) < 25. Lipid measurements are given in Table 1 . Plasma C reactive protein (CRP) concentration form DS and control was evaluated by LANIA (Latex Agglutination Nephelometric Immunoassay) technique (Biolatex, Spain). Samples were diluted 1:36 and results were calculated automatically by IMMAGE system. The minimum detectable concentration was 0.4 mg/dl. Table 1 Cholesterol fractions in old patients with Down syndrome and healthy subjects. Means ± SD. Healthy subjects Down syndrome Total cholesterol 150 ± 19.64 152 ± 28.79 Triglycerides 55.9 ± 21.46 104.5 ± 50.2 HDL-cholesterol 48.4 ± 10.5 40.6 ± 4.24 LDL-cholesterol 88.3 ± 17.2 89 ± 24.4 None had been treated with lipid-lowering drugs before blood sampling. This study was conducted in accordance with the Declaration of Helsinki, 1975, amended in 1983. Blood, collected in tubes containing EDTA, was cooled to 20°C and diluted 1:1 with Hank's buffered saline solution (HBSS, Biochrome, Biospa, Milan, Italy). PBMCs were prepared under sterile conditions, using Ficoll-Hypaque (Pharmacia Biotech, Milan, Italy) and diluted blood was layered in a centrifuge tube and centrifuged for 40 min at 400 g , 20°C. The interface containing the PBMCs was isolated, and the cells were washed three times in HBSS and resuspended in RPMI-1640 (Biochrome, Biospa, Milan, Italy) with L-glutamine (290 mg/L), penicillin (100,000 U/L), streptomycin (100 mg/L) and 100 mL/L human lipoprotein-deficient serum (HLPDS) to a final concentration of 10 6 cells/mL. Tissue culture flasks were placed in ice-water for 60 min in the dark to reduce cell adhesion. PBMCs were removed by flushing with ice-cold HBSS (4°C) and washed twice in ice-cold HBSS with 20 mL/L HLPDS. The cell number was adjusted to 0.3 × 10 6 cells/mL, and 100-μL aliquots of cell suspension were pipetted into polypropylene tubes and placed in ice-water. Cells were incubated with 1.5 μg of monoclonal mouse anti-human LDL receptor-specific antibody, clone C7 (Amersham Life Science, Milan, Italy), for 30 min in the dark at 4°C. After this the cells were washed twice in ice-cold HBSS with 20 mL/L HLPDS, and incubated with 3 μL of fluorescein isothiocyanate (FITC, Dako Cytomation, Milan, Italy) for 30 min in the dark at 4°C. Cells were then incubated with 1 μL of R-phycoerythrin (RPE)-conjugated monoclonal antibody CD3-RPE or IgG 1 isotype-RPE for T lymphocytes. The flow cytometry measurements were done in a FACScan flow cytometer (Becton Dickinson, Milan, Italy) equipped with a 15 mW, 488 nm, air-cooled argon laser and linked to a computer with CellQuest software. Forward scatter (FSC) and side scatter (SSC) were adjusted to exclude debris and dead cells. FITC emission was measured at 530 nm (FL1) and RPE emission at 585 nm (FL2); compensation was set using FITC-conjugated C7 (C7 FITC)-labeled cells (FL2-FL1) and CD3-RPE-labeled cells (FL1-FL2). Means were compared by the unpaired t -test or one-way analysis of variance (ANOVA). Data are presented as means ± SD. Differences were considered statistically significant at p < 0.05. Results Table 1 shows cholesterol fractions of DS patients and healthy controls. DS total cholesterol and LDL did not differ from controls ( p = 0.8 and p = 0.9 respectively). Blood levels of CRP were higher in DS than in controls, as illustrated in Figure 1 (Controls 1.3 ± 0.3; DS = 5.7 ± 4.6 mg/L, p < 0.01). A regression analysis of data shows non relationship among CRP and cholesterol-related molecule levels. Figure 1 Levels of C reactive protein (CRP) in plasma from children with DS and age matched controls. Data are presented as mean ± S.D. Triglycerides were higher, and HDL lower in DS patients ( p < 0.01 and p < 0.05). Our data also show that the expression of LDL receptor on T lymphocytes was down-regulated in DS patients (Table 2 and Figure 2 ). Table 2 Mean fluorescence intensity (MFI, %) of LDL receptor expression in healthy subjects and old patients with Down syndrome. Means ± SD. Healthy subjects Down syndrome MFI (%) 196.76 ± 20.54 139.87 ± 13.32 Figure 2 Mean fluorescence intensity (MFI) of LDL receptor expression in healthy subiects (bold line) and Down syndrome old subjects (fine line). Mean intensity fluorescence (MIF) of LDL receptor expression was significantly different in DS patients and healthy controls ( p < 0.0001) (Table 2 ). Discussion Patients with DS who reach adolescence nowadays have a nearly normal life expectancy thanks to better medical care. When they die at a later age, cardiovascular diseases are less common than in the general population and they have even been proposed as "an atheroma-free model" [ 3 ]. Our results concerning cholesterol fractions suggest that DS patients should have a cardiovascular disease risk, if conclusions valid for the general population can be transferred to this category of patients. Although serum lipoprotein profiles cannot explain the lower prevalence of cardiovascular disease in individuals with DS, our triglyceride and HDL findings are in line with published figures. Similar findings were reported by other authors but in young patients [ 4 , 5 ]. Multiple factors are responsible for atherosclerosis, such as dietary habits but still the unexplained decline of LDL receptor expression with aging contributes importantly to borderline-high levels and cannot be ignored. For example the loss of estrogen-stimulated LDL receptor synthesis after menopause is an important contributor to elevated cholesterol in postmenopausal women. In addition, several genetic defects inherited singly appear to be causes of moderate hypercholesterolemia [ 10 , 12 ]. Generally defects of LDL receptor expression are associated with a high risk of premature atherosclerosis. In the elderly LDL receptor uptake is unexpectedly increased, and LDL receptor regulation and expression and serum LDL composition seem abnormal. There may also be alterations to the lipid metabolism of immune system cells during aging [ 13 ]. In our series, a reduction in LDL receptor expression was not correlated with high LDL serum levels or total cholesterol. Moreover, lipophospholipid (LPC) is generated by hydrolysis of phosphatidylcholine which is present in LDL; LPC may promote the start of an immune response and atherosclerosis may be the most extreme demonstration of this immune regulation pathway [ 14 ]. LPC may be a potent super-regulator of T-cell activation by inflammation at sites of tissue damage and in the early stages of atherosclerosis. Interestingly, DS patients show no increase in their frequency of cardiovascular disease. These conditions may be explained by mild immune defects in the syndrome, mainly involving macrophages and/or T H 1 lymphocytes responses [ 15 ]. Alternatively, a over expression of atherosclerotic protective factors -yet unknown- maight be present in Down syndrome. As we reported earlier, the low incidence of cardiovascular disease in these patients and the high-risk factor of oxidatively modified LDL (oxLDL) [ 16 ] with – in this study – the low level of HDL, high levels of CRP and reduction of LDL receptor expression, lead to the conclusion that in this group of "healthy old" DS subjects, classical biochemical risk factors for atherosclerosis have been detected but risks, probably, are considerably lower. List of abbreviations Down syndrome (DS); high-density lipoprotein (HDL); C reactive protein (CRP); Body Mass Index (BMI); low-density lipoprotein (LDL); peripheral blood mononuclear cells (PBMC); lipophospholipid (LPC). Competing interest The author(s) declare that they have no competing interests.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC549517.xml
524248	Perception, Action, and Roelofs Effect: A Mere Illusion of Dissociation	A prominent and influential hypothesis of vision suggests the existence of two separate visual systems within the brain, one creating our perception of the world and another guiding our actions within it. The induced Roelofs effect has been described as providing strong evidence for this perception/action dissociation: When a small visual target is surrounded by a large frame positioned so that the frame's center is offset from the observer's midline, the perceived location of the target is shifted in the direction opposite the frame's offset. In spite of this perceptual mislocalization, however, the observer can accurately guide movements to the target location. Thus, perception is prone to the illusion while actions seem immune. Here we demonstrate that the Roelofs illusion is caused by a frame-induced transient distortion of the observer's apparent midline. We further demonstrate that actions guided to targets within this same distorted egocentric reference frame are fully expected to be accurate, since the errors of target localization will exactly cancel the errors of motor guidance. These findings provide a mechanistic explanation for the various perceptual and motor effects of the induced Roelofs illusion without requiring the existence of separate neural systems for perception and action. Given this, the behavioral dissociation that accompanies the Roelofs effect cannot be considered evidence of a dissociation of perception and action. This indicates a general need to re-evaluate the broad class of evidence purported to support this hypothesized dissociation.	Introduction Several anatomical, neurophysiological, clinical, and behavioral investigations of human subjects and nonhuman primates have provided evidence for two separate and dissociable cortical systems for visual processing. One of these systems—the ventral stream—resides in a swath of cortex that extends in an anteroventral direction from primary visual cortex to the temporal lobe. The second system resides in a dorsal stream that roughly extends from primary visual cortex to the parietal lobe. These ventral and dorsal systems were originally thought to be dedicated to the visual processing required to determine an object's identity and location, respectively ( Ungerleider and Mishkin 1982 ). However, a more recent hypothesis suggests that both streams process information concerning object properties and locations, but that they do so for different purposes. In this revised model of the visual system ( Milner and Goodale 1995 ), the ventral stream is presumably responsible for the formation of perceptual/cognitive representations of objects and events in the world, whereas the dorsal stream is responsible for guiding sensorimotor actions in response to those objects and events. Much of the evidence for separate perception and action systems has come from behavioral studies of normal subjects. The general logic of these behavioral paradigms (e.g., Bridgeman et al. 1981 , 1997 ; Aglioti et al. 1995 ; Daprati and Gentilucci 1997 ; Goodale and Murphy 1997 ; Haffenden and Goodale 1998 ) is as follows: An illusory stimulus is presented to the subject, who is required to report some characteristic of the stimulus (location, size, orientation, etc.) using perceptual (e.g., verbally compare the test object to some reference object) or sensorimotor means (e.g., make a movement to reach toward or grasp the test object). As a general finding, it seems that perceptual reports are more prone to illusions than are sensorimotor responses, suggesting that the systems are dissociable not only in terms of their cortical pathways, but also in terms of their processing capabilities. As a specific example of this type of evidence, Bridgeman et al. (1997) tested the ability of subjects to indicate the location of a small visual target presented within an illuminated frame that was offset left or right from the subject's midline plane ( Figure 1 A). When asked to perceptually compare the location of the target with respect to an array of possible locations learned earlier, subjects reported the target to be in a location that was shifted in a direction opposite that of the frame—a perceptual phenomenon known as the induced Roelofs effect ( Bridgeman et al. 1997 ; see also Roelofs 1935 ). In contrast, subjects could accurately guide the hand to the target's location, indicating that sensorimotor localization was immune to the illusion. These findings were cited as strong evidence for the existence of two distinct, differently abled visual systems for perception and action. However, Bridgeman et al. (1997) further demonstrated that not all actions were immune to the illusion; in particular, sensorimotor responses were prone to the induced Roelofs effect when subjects were required to point to the remembered location of the target after a delay of 4 s. As suggested by Bridgeman et al. (1997) , this delayed sensorimotor effect could possibly be explained by a sensorimotor system that lacks a memory of its own and therefore must rely on the memory of the illusion-prone perceptual system to determine the goal of a movement directed toward a remembered target. Figure 1 Schematic of the Induced Roelofs Effect (A) Example visual display (not drawn to scale) comprising a target (red circle) and a frame offset to the subject's left. Gray circles (unseen by subjects) represent the remembered positions of the items within the comparison array, centered on the subject's midline. (B) One possible mechanism for the inaccurate perceptual report of the target location, based on an illusory rightward shift of the perceived target location (green circle). (C) An alternative mechanism for the inaccurate perceptual report, based on a leftward shift of the memorized location of the comparison array (blue circles). Either mechanism (B or C) would result in the subject reporting the target to occupy the remembered location of item 4 in the comparison array. Although these findings are compatible with the hypothesis of two separate visual systems that are differentially affected by Roelofs illusion, it is important to consider the possibility of an alternative explanation. While it is true that both perceptual and sensorimotor measures within the task of Bridgeman et al. (1997) assayed the subjects' abilities to determine the location of the target, the two measures did so in markedly different ways. For the sensorimotor task, subjects could complete the task knowing only the location of the target within a body-centered or egocentric reference frame. In contrast, the perceptual task required the subjects to compare the location of the target relative to the locations of the items within the remembered comparison array. Given this, errors in the perceptual report could be due to either a misrepresentation of the target's position ( Figure 1 B) or a misrepresentation of the position of the remembered comparison array ( Figure 1 C). Whereas Bridgeman et al. (1997) concluded that the target is perceptually mislocalized in a direction opposite the frame offset, an inaccurate perceptual report could equally be due to a memory of the comparison array that is shifted in the same direction as the frame. The studies presented here were designed to test this alternative hypothesis for the mechanism underlying the induced Roelofs effect, and to critically examine the apparent dissociation of perception and action related to the illusion. We first replicate the findings of Bridgeman et al. (1997) , using saccadic eye movements rather than a pointing task. We then test subjects' memory for the comparison array and show that it is biased by the offset frame in a way that can completely account for the perceptual illusion. A subsequent experiment demonstrates that this distortion of remembered visual space occurs when the brain, faced with an impoverished visual environment, incorrectly uses the location of the frame as a cue to establish an egocentric reference map whose origin (the apparent midline) is transiently biased toward the direction of the frame. Furthermore, movements guided within this same distorted reference map are shown to be accurate, given that the errors of target localization will be cancelled by subsequent errors of motor guidance. Thus, the perceptual and sensorimotor effects of the Roelofs illusion can be mechanistically explained without requiring the existence of separate neural processing streams for perception and action. Results/Discussion Perceptual and Sensorimotor Effects of the Illusion We first sought to replicate the findings of Bridgeman et al. (1997) by testing subjects' abilities to indicate the locations of targets presented within the context of a centered frame or one displaced 5° left or right of the midline. Subjects provided a perceptual report of each target location by comparing it to an array of five possible target locations (−4°, −2°, 0°, 2°, and 4° from the subject's midline, at eye level) learned during an earlier training session. As had been demonstrated previously ( Bridgeman et al. 1997 ), the displaced frame did cause a mislocalization of the target, whether the subject responded immediately after the offset of the target and frame ( Figure 2 A, solid line; Table 1 ) or after a 4-s delay period during which the subject was in complete darkness ( Figure 2 A, dashed line; Table 1 ). The size of this illusion was quantified by subtracting the magnitude of the localization bias caused by a right-shifted frame from that caused by a left-shifted frame, resulting in an effect size of 1.47° ± 0.32° (mean ± SEM) across all subjects for immediate responses and 1.37° ± 0.30° for delayed responses. Figure 2 Perceptual and Sensorimotor Roelofs Effects (A) Effect of frame location on immediate (solid line) and delayed (dashed line) perceptual judgments of target location, with a significant main effect of frame offset but no frame × delay interaction ( Table 1 ); error bars represent the standard error of the mean localization errors for each subject. (See also Figure S1 A for a time line of the task events, Figure S1 B for a plot of the Roelofs effect for each of the individual target locations, and Figure S1 C for plots of the Roelofs effect within individual subjects.) (B) Effect of frame offset on immediate (solid line) and delayed (dashed line) saccadic eye movements, with a significant main effect of frame offset and a significant frame × delay interaction. When tested separately, the main effect of frame offset was not significant for immediate responses, but was significant for delayed responses ( Table 1 ; see also Figure S2 ). Table 1 Significance of Effects a Main effect: Frame duration b Interaction: Frame offset × Frame duration NA, not applicable n.s., not significant A second group of subjects was instructed to make open-loop saccadic eye movements to the target location. Saccades performed immediately after the frame and target were extinguished showed no significant effect of frame position (effect size = −0.01° ± 0.13°, Figure 2 B, solid line; Table 1 ). This finding replicated the general pattern of sensorimotor responses described by Bridgeman et al. (1997) and extended them by demonstrating that immediate saccadic eye movements, like pointing movements of the hand, can be guided accurately to targets that are perceptually mislocalized. However, when subjects were required to withhold this sensorimotor response during a 4-s delay period, the eventual saccadic eye movement did reflect a small but significant Roelofs effect (effect size = 0.60° ± 0.26°, Figure 2 B, dashed line; Table 1 ). Again, this delayed sensorimotor Roelofs effect replicated the findings of Bridgeman et al. (1997) . A Mislocalization of the Target or of the Comparison Array? To test the hypothesis that the perceptual Roelofs effect can be explained by a memory of the comparison array that is shifted in the direction of the frame, subjects were asked to indicate the remembered locations of the five items within the array of possible targets that was learned in complete darkness during the earlier training session. In experimental trials, a centered or offset frame was presented near the time that an auditory cue instructed the subject to make a saccadic eye movement to one of the remembered locations. If an offset frame caused a distortion in the memory of the reference array, the accuracy of the saccadic responses would be affected. This was indeed the case, with targets mislocalized in the same direction as the displacement of the offset frame (effect size = −1.61° ± 0.32°, Figure 3 A, solid line; Table 1 ). Since this pattern of mislocalization for remembered targets was in the opposite direction of the normal Roelofs effect reported by Bridgeman et al. (1997) and replicated above (see Figure 2 A), we refer to it as an inverse Roelofs effect for remembered space. This finding provides strong evidence that the perceptual errors associated with the normal Roelofs effect are most parsimoniously explained by the subject's comparison of the target location with a distorted memory of the comparison array. As a further test of this hypothesis, it is possible to use the pattern of mislocalizations evident with the inverse Roelofs effect for remembered space to predict a subject's perceptual report when comparing a target location with the inaccurately remembered comparison array. The resultant prediction for the perceptual Roelofs effect very closely matched the measured Roelofs effect (see Figure 3 B), with a predicted effect size (1.61°) that did not significantly differ from the measured effect size (1.47° ± 0.32°). Thus, the inverse Roelofs effect for remembered space effectively accounts for the mislocalizations that occurred when subjects provided perceptual reports of the locations of targets presented within the context of an offset frame. Figure 3 Inverse Roelofs Effect for Remembered Space (A) An inverse Roelofs effect for immediate (solid line) and delayed (dashed line) sensorimotor responses toward remembered reference array locations, with a significant main effect of frame offset and a significant frame × delay interaction. When tested separately, the main effect of frame offset was significant for both immediate and delayed responses ( Table 1 ; see also Figure S3 ). (B) The inverse Roelofs effect for remembered space can be used to predict the pattern of the Roelofs effect for targets presented within an offset frame. For example, a frame offset to the right would cause the remembered comparison array to be mislocalized as being shifted approximately 1° to the right (from Figure 3 A, solid line); a target presented at the center location of the comparison array (i.e., at the objective midline) would therefore be reported to lie approximately 1° to the left of the remembered center location. Computed in this way for all target and frame locations, the predicted Roelofs effect (gray lines and data points) closely matched the measured Roelofs effect for the perceptual judgment (black lines and data points, from Figure 2 A, solid line), with a predicted effect size (1.61°) that did not significantly differ from the measured effect (1.47° ± 0.32°; t[9] = 0.44, n.s.). Furthermore, the measured Roelofs effect did not differ from the predicted effect for any individual frame position (left frame: t[9] = 0.39, n.s.; center frame: t[9] = 0.01, n.s.; right frame: t[9] = 0.36, n.s.). Distortion of the Apparent Midline Although the inverse Roelofs effect for remembered space provides an explanation for the perceptual mislocalization that occurs in the presence of an offset frame, the mechanism whereby the offset frame is capable of distorting remembered space remains to be explained. The locations of the items within the comparison array were learned in complete darkness and therefore could only be localized in egocentric coordinates, perhaps with respect to the subject's apparent midline ( Mergner et al. 2001 ). Under normal conditions, the center of the visual field would serve as an accurate indicator of straight-ahead. However, the impoverished visual environment of the present experiment contained only the large rectangular frame, which might have served to attract the apparent midline in the direction of the frame's offset ( Werner et al. 1953 ; Brosgole 1968 ; Brecher et al. 1972 ; Dassonville et al. 2004 ), dragging the spatial memory of the comparison array with it. To directly test the hypothesis that the offset frame in the current context is capable of biasing the apparent midline, subjects were asked to perform a version of the task in which they were simply asked to “look straight ahead” immediately after the presentation of a centered or offset frame. Subjects' reports of “straight-ahead” were indeed found to be affected by the presence of the frame, with the movements biased in the same direction as the offset frame (effect size = −1.08° ± 0.14°, with a negative value once again reflecting an effect in the direction opposite the normal induced Roelofs effect; Figure 4 , solid line; Table 1 ). Figure 4 Inverse Roelofs Effect for the Apparent Midline An inverse Roelofs effect for immediate (solid line) and delayed (dashed line) sensorimotor responses toward the apparent midline, with a significant main effect of frame offset and a significant frame × delay interaction. When tested separately, the main effect of frame offset was significant for both immediate and delayed responses ( Table 1 ; see also Figure S4 ). These findings can also explain the absence of errors seen with immediate sensorimotor responses, if one assumes that the movements are guided within the same distorted frame of reference that is used to encode the target location. For example, a target presented at the subject's true midline in the presence of a left-shifted frame would be encoded by the brain as having been located a small distance to the right of the apparent midline (which itself has been pulled leftward by the frame; Figure 5 A). If the corresponding sensorimotor response is guided within this same distorted reference frame, the eye or hand would be expected to move to a location just to the right of the distorted apparent midline ( Figure 5 B). In essence, the error in target localization would be exactly cancelled by the error in motor guidance, resulting in an accurate response. Thus, an accurate sensorimotor response is fully expected when the target and response are encoded within the same distorted map of space. Figure 5 The Biased-Midline Hypothesis (A) A depiction of the manner in which a target (red circle), located directly in front of the subject, would be perceived as being a small distance to the right of the subject's apparent midline (dotted line), which has itself been biased to the left in the presence of the left-shifted frame. (B) An immediate open-loop sensorimotor response (pointing movement, as shown here, or saccade begun immediately after the target and frame are extinguished) would be accurate if the goal of the movement were encoded in the same distorted reference frame (that is, a small distance to the right of the distorted apparent midline). (C) With the frame and target extinguished during an imposed delay, the apparent midline would drift back to veridical (gray arrows), dragging the remembered location of the target (gray circle) with it. A subsequent sensorimotor response aimed at the remembered target (located a small distance to the right of the now-veridical apparent midline) would result in a delayed sensorimotor Roelofs effect. Transient Effects of the Illusion In contrast to the stable Roelofs effect with delayed perceptual responses (see Figure 2 A, dashed line), we found that the inverse Roelofs effect for remembered space was diminished (to an effect size = −0.87° ± 0.28°) when a 4-s delay was imposed between the frame presentation and the saccade to a remembered item in the comparison array (see Figure 3 A, dashed line). Similarly, the effects of an offset frame on the apparent midline diminished during a delay imposed after the frame was extinguished (to an effect size = −0.56° ± 0.15°; see Figure 4 , dashed line). These findings demonstrate that the distortions of the apparent midline and remembered space are transient, decreasing over time when the offset frame is no longer visible. However, even after a delay of 4 s, responses were still somewhat biased by the preceding frame, indicating either an extended time course during which the effects of the frame dissipate or a hysteresis that prevents the apparent midline from becoming fully veridical in the absence of visual input. The transient nature of the apparent midline distortion can also provide an explanation for the increase in Roelofs effect seen when a delayed saccade is made to a target presented within the offset frame (see Figure 2 B, dashed line). As an example, let us once again assume a target presented at the subject's true midline, in the presence of a left-shifted frame. During the imposed delay, it is reasonable to assume that the memory of the target's location would be encoded with respect to the distorted apparent midline ( Mergner et al. 2001 )—in our example, the target would be remembered as being a small distance to the right of the apparent midline, which has been pulled leftward by the frame (see Figure 5 A). After the frame is removed and its distorting influences diminish, the apparent midline would drift back toward its veridical orientation under the influence of vestibular ( Fischer and Kornmueller 1930 ; Morant 1959 ) and proprioceptive ( Karnath 1999 ) inputs, dragging the remembered target location with it. The delayed response would then be directed to this incorrectly remembered location, just to the right of the newly corrected apparent midline (see Figure 5 C). If this account were true, one would expect the normal Roelofs effect for sensorimotor responses to increase during a delay by an amount comparable to the decrease in the inverse Roelofs effect for delayed movements directed to the apparent midline or items in the remembered comparison array. Indeed, the current studies found the sensorimotor Roelofs effect to increase 0.61° during the imposed delay (that is, from −0.01° to 0.60°; see Figure 2 B, solid versus dashed lines), while the inverse Roelofs effect decreased 0.74° for movements to items in the comparison array (see Figure 3 A, solid versus dashed lines) or 0.52° for movements to indicate the apparent midline (see Figure 4 , solid versus dashed lines). In contrast, one would not expect an imposed delay to have any effect on a perceptual report of target location, since the relative relationship of the remembered target and the remembered comparison array would remain unchanged as the apparent midline returned to veridical. To more closely examine the hypothesis that the increase in the Roelofs effect for delayed sensorimotor responses is due to a drift of the apparent midline back to veridical after the frame is extinguished, a group of subjects performed a version of the delayed saccade task in an experiment in which, on half of the trials, the frame continued to be visible during the 4-s delay period, disappearing only when the subject received the verbal cue to respond. In the other half of the trials, the frame and target were extinguished simultaneously, with the subject sitting in complete darkness during the delay. When the frame was absent during the delay, the eventual response was significantly affected by frame position ( Figure 6 , dashed line; Table 1 ), replicating the results from our previous delayed saccade task (see Figure 2 B, dashed line). In contrast, for those trials in which the frame was present during the delay, no effect of frame position was evident (see Figure 6 , dotted line; Table 1 ). Thus, it seems that the continued presence of the frame maintains the apparent midline in a biased orientation, such that the errors in target encoding are cancelled by the errors of motor guidance even after a delay. Since the transient nature of the Roelofs effect is specifically not a function of the delay from target presentation to response (but rather depends on the delay from frame offset), these findings argue against the hypothesis of Bridgeman et al. (1997) that the transience reflects a lack of memory for target location within a system that guides the sensorimotor responses. Figure 6 Effect of the Frame during the Delay Period Effect of frame offset on delayed saccadic eye movements, for trials in which the frame was either extinguished at the start of the delay period (brief frame, dashed line), or was present throughout the delay (extended frame, dotted line). There was a significant frame × delay interaction; when tested separately, the main effect of frame offset was not significant for the extended frame duration, but was significant for the brief duration, replicating the results shown in Figure 2 B, dashed line ( Table 1 ; see also Figure S5 ). Reevaluating the Need for Separate Perception and Action Systems While the present findings provide an alternative explanation for the behavioral dissociation of Roelofs illusion, it could still be argued that they do not completely rule out the possibility of separate systems for perception and action. For example, it could be that there does exist a context-independent “action” system whose only function is to guide movements aimed immediately and directly toward a currently visible target, and a “perceptual” system capable of guiding all other movements (e.g., movements to remembered targets, to mirror-image locations of currently visible targets [ Dassonville et al. 2004 ], or to indicate straight-ahead, all of which reflect the errors associated with Roelofs effect). If this were true, then it would be useful to contrast the capabilities of the action system with those of the perceptual system under equivalent conditions (i.e., for movements guided immediately and directly to currently defined targets). To do this, we designed an experiment in which subjects were asked to make saccadic eye movements to targets that were defined purely through the use of contextual cues that could serve as targets only for the presumed perceptual system, if the action system truly operates in a context-independent fashion. Specifically, stimuli consisted of three corners (and two sides) of a rectangle, with the target location defined as the missing corner ( Figure 7 A). These stimuli were then presented within a large rectangular frame that was centered or offset from the subject's midline. The pattern of mislocalizations seen with these allocentrically defined targets was identical to that seen with real targets (compare Figure 7 B to Figure 2 B), with no Roelofs effect evident for immediate responses ( Figure 7 B, solid line; Table 1 ), but a significant effect evident for responses delayed by 4 s ( Figure 7 B, dashed line; Table 1 ). Thus, if separate action and perception systems do exist, it would seem that they are not differently abled with regard to Roelofs effect after all. Instead, both would be capable of guiding immediate movements accurately in spite of the Roelofs effect, with the action system simply immune to the Roelofs distortions, while the perceptual system would be required to guide movements within the same distorted reference frame as the target is encoded, so that the errors cancel, as described above. While it is technically possible that the brain would maintain two such redundant systems for guiding movements, it seems improbable. Instead, a more parsimonious explanation for the behavioral dissociation that accompanies Roelofs effect is provided by the brain's use of a single reference frame whose origin (the apparent midline) is transiently distorted by the presence of an offset frame for both perceptual judgments and sensorimotor responses. Figure 7 A Roelofs Effect for Allocentrically Defined Targets (A) Visual display used to define allocentric targets; subjects were instructed to move the eyes to the missing corner of the partial rectangle (gray circle, not seen by subject). During experimental trials, this stimulus array was presented within a large rectangular frame that was either centered or offset left or right of the subject's midline. (B) Effect of frame offset on immediate (solid line) and delayed (dashed line) sensorimotor responses to targets defined allocentrically, with a significant main effect of frame offset and a significant frame × delay interaction. When tested separately, the main effect of frame offset was not significant for immediate responses, but was significant for delayed responses ( Table 1 ; see also Figure S6 ) Further Evidence for the Use of Contextual Information in Motor Control As is the case for many illusions, the distortion of visual space associated with Roelofs illusion would seem to be a by-product of the brain's use of contextual cues that—under normal circumstances—would provide additional information that could allow for the creation of a more accurate neural representation of the world. After all, most perceptual judgments and movements are made within the context of a well-lit, highly structured visual scene, the center of which would normally provide an accurate indicator of straight-ahead. Given this, it would be somewhat surprising if the beneficial information that is provided by contextual cues under normal circumstances were used for perception but not for motor control. Indeed, many previous investigations have demonstrated that contextual cues do affect the guidance of movements, even those directed to currently visible targets. For example, the accuracy and kinematics of open-loop pointing movements are greatly affected by the presence of a small distractor ( Howard and Tipper 1997 ; Tipper et al. 1997 ; Gangitano et al. 1998 ) or a well-lit, highly structured visual scene ( Foley 1975 ; Conti and Beaubaton 1980 ; Blouin et al. 1993 ; Toni et al. 1996 ). This has also been demonstrated using paradigms in which the visual representation of target location is first distorted by altering the relationship between actual eye position and the brain's representation of eye position (e.g., by paralyzing the extraocular muscles with curare [ Matin et al. 1982 ], stretching them [ Stark and Bridgeman 1983 ], fatiguing them [ Shebilske 1984 ], or vibrating them [ Velay et al. 1994 ]). Although these distortions of represented eye position have been shown to cause errors in open-loop pointing movements aimed at targets presented in otherwise complete darkness, the presence of a highly structured visual scene significantly reduces the magnitude of these errors. Contextual cues are also used by the oculomotor system to minimize the errors of saccadic eye movements directed toward targets presented near the time of a preceding saccade ( Honda 1993 , 1999 ; Dassonville et al. 1995 ). Furthermore, several other studies have clearly demonstrated that illusion-causing contextual cues can affect the dynamic characteristics of pointing and grasping movements ( Smeets and Brenner 1995 ; Brenner and Smeets 1996 ; Gentilucci et al. 1997 ; van Donkelaar 1999 ; Jackson and Shaw 2000 ; Westwood et al. 2001 ; Bartelt and Darling 2002 ) and the accuracy of eye movements ( Festinger et al. 1968 ; Binsted and Elliott 1999 ; Both et al. 2003 ; McCarley et al. 2003 ; Sheliga and Miles 2003 ). Additional Evidence against a Simple Perception/Action Dissociation Although previous authors have suggested that the perceptual effects of Roelofs illusion could be explained by a distortion of the apparent midline and egocentric reference frame ( Werner et al. 1953 ; Brosgole 1968 ; Brecher et al. 1972 ; Dassonville and Bala 2004 ; Dassonville et al. 2004 ), we have demonstrated here that this same transient distortion can also provide a full, precise, and mechanistic explanation of the immediate and delayed sensorimotor effects of Roelofs illusion. By extending this hypothesis to include dynamic visual displays, a similar mechanism can also be used to explain the behavioral dissociation seen with illusions of induced motion ( Bridgeman et al. 1981 ; Wong and Mack 1981 ). Most important, this hypothesis accounts for both phenomena without relying on an assumption of separate neural maps of space for perception and action. Given this, the behavioral dissociation evident with these illusions cannot be used as evidence that exclusively supports the existence of a perception/action dissociation in visual processing. Of course, several other behavioral studies can still be pointed to as evidence for a perception/action dissociation in visual processing. However, many of these studies have recently come under intense scrutiny, with some researchers failing to replicate previously reported dissociations once important control conditions were included ( Honda 1990 ; Dassonville et al. 1992 ; Pavani et al. 1999 ; Franz et al. 2000 ; Franz 2003 ). Other researchers have proposed alternative explanations for obvious behavioral dissociations by pointing out that the perception and action tasks differed along other dimensions as well (e.g., semantic versus pragmatic requirements [ Jeannerod 1997 ], relative versus absolute judgments [ Vishton et al. 1999 ], allocentric versus egocentric reference frames [ Bruno 2001 ], and size versus position judgments [ Smeets and Brenner 2001 ]). Similarly, in studies that have purported to demonstrate a perception/action dissociation in patients with dorsal and ventral lesions, it can be argued that the behavioral tests used to characterize the deficits also suffered from these same confounds. For example, Dijkerman et al. (1998) found that a patient (DF) with a ventral lesion was impaired in a task that required her to reach for and grasp an object by placing her fingers in two or three circular holes whose locations were varied from trial to trial. Although these findings led Dijkerman et al. (1998) to conclude that movements like these must be controlled by a ventral perceptual system that happens to operate within an allocentric reference frame, it is also possible to interpret these data as suggesting that the lesion simply caused a specific deficit in allocentric encoding rather than a general deficit in perception. This same patient has also been found to be impaired in perceiving the visual pitch of a plane tilted from vertical, even though that same plane causes a distortion of her perception of vertical eye level, just as it does in healthy subjects ( Servos et al. 1995 ). Thus, although DF demonstrates a dissociation in her ability to use information concerning visual pitch, it is a dissociation of two perceptual measures and specifically not a dissociation of perception and action. While it seems clear that there do exist at least some examples of dissociations in the accuracy of various behavioral responses from normal subjects and patients, a great deal of evidence now suggests that these dissociations cannot simply be attributed to separate systems for perception and action. Indeed, the dorsal and ventral processing streams are both composed of a myriad of functionally distinct and highly interconnected visual processing areas, each with its own mechanisms for representing various aspects of the visual world. Each visuomotor task would undoubtedly rely on the processing capabilities of a subset of these areas, with different tasks relying on different subsets depending on their precise requirements. With this in mind, it seems overly simplistic to consider visuomotor behavior as being derived from the function of only one of two distinct processing streams. Rather, it is more plausible that flexible functional networks would form among the areas required to play a role in the completion of the task at hand. The characteristics of the behavioral performance would then be dependent on the representational idiosyncrasies of those areas involved ( McGraw et al. 2003 ). Several previous studies of the perceptual and motor effects associated with various visual illusions have indicated an apparent dissociation of visual pathways for perception and action, with perception generally found to be prone to illusions to which actions are immune. These conclusions, however, are not without controversy; as described above, several other studies have questioned the perception/action dissociation attributed to many illusions, after contrary evidence or alternative explanations were produced. A notable exception to this has been the induced Roelofs effect, where the presumption of a perception/action dissociation has remained unquestioned since it was originally proposed ( Bridgeman et al. 1997 ). The results presented here, however, point to a brain mechanism for spatial localization that can fully and precisely explain the behavioral dissociation of the induced Roelofs effect without requiring the existence of separate neural systems for perception and action. The visual image of a large frame, whose center is offset left or right of an observer's midline, was demonstrated to cause a transient distortion of an observer's egocentric reference frame by biasing the apparent midline. Within this distorted reference frame, objects are perceived to be located in a direction shifted opposite that of the frame offset. The fact that movements of the eyes and hands can be accurately directed to this misperceived target location can be explained by a cancellation of errors that occurs when the movement is guided within the same distorted reference frame. Thus, these findings indicate that both perceptual judgments and motor responses are based upon either a single map of space or separate maps that are equally prone to the distortion caused by the Roelofs effect. Materials and Methods Subjects In each experiment, ten subjects (undergraduate students of the University of Oregon) provided informed consent to participate and were compensated with either course credit or a small monetary payment, as per a protocol approved by the University of Oregon Committee for the Protection of Human Subjects/Institutional Review Board. Visual display Subjects were placed in a completely darkened room and presented with a visual display that was back-projected (Cine7 projector, Barco, Kuurne, Belgium) onto a screen measuring 128 × 96 cm, positioned 122 cm from the eyes. Visual targets were small (0.35° of visual angle, 100-ms duration) red spots, located −4°, −2°, 0°, 2°, or 4° from the subject's midline, at eye level. During experimental trials, targets were presented within a large red unfilled frame (21° horizontal × 8.5° vertical, 1° thickness; 1,000-ms duration) that was either centered with respect to the subject's midline or shifted 5° left or right of the midline. All visual images were presented on a black background, with the high contrast of the Barco Cine7 projector preventing subjects from seeing the edges of the screen. Eye movement monitoring Head and binocular eye positions were monitored at 250 Hz with an eye tracker (Eyelink; SensoMotoric Instruments, Needham, Massachusetts, United States) that allowed head-free measurement of gaze (precision = 0.01°); however, target placement was such that head movements contributed to only a small fraction of the total gaze displacement on any trial. To start each session (and as necessary throughout each session), eye-tracker calibration was performed using a 3 × 3 grid of targets spaced 13.5° apart in the horizontal dimension and 10.5° apart in the vertical dimension. For each subject, the average fixation error across the nine calibration targets was required to be 1° or less before beginning the subsequent practice and experimental trials; thus, absolute tracking errors within this calibration field were at most 1° in magnitude and were typically only 0.5°. In addition, subjects began each experimental trial by directing the eyes to a fixation point and pressing the space bar of a keyboard to indicate readiness. Upon this signal, the eye-tracker computer performed an adjustment of the calibration to correct for any drift that had occurred since the onset of the previous trial; that is, the calibration was adjusted so that the signal of eye position matched the known location of the fixation point. In experiments requiring eye movement responses, the gaze signals from the two eyes were averaged to yield a single representation of gaze direction as the dependent variable. Behavioral tasks Each experiment was preceded by a set of practice trials (36–71 trials) in which subjects performed the appropriate task (see below) in the absence of the large Roelofs-inducing rectangular frame. Feedback was provided at the end of each practice trial to assist subjects in improving their performance. For all experiments, feedback included the illumination of a small circle at the target location. For the experiment in which subjects provided a button press to report the perceived target identity, a computer-generated voice also provided auditory feedback to indicate the correct target identity. For all experiments in which subjects indicated the target position with a saccadic eye movement, feedback following each practice trial also included the illumination of a small square to indicate the final gaze position; subjects were instructed to use the feedback in an attempt to minimize the distance between the target and final gaze positions. No feedback concerning response accuracy was ever provided during the experimental trials. To test the perceptual effects of Roelofs illusion (see Figure 2 A; see also Figure S1 A), subjects were first trained to recognize targets presented in each of the five possible target locations. Each trial began with a fixation point (centered horizontally, 8.5° above eye level) that was extinguished 1150–1650 ms before the onset of the target (100-ms duration). A computer-generated voice (“Respond,” presented just before or 4 s after the target) provided subjects with a temporal cue to press one of five keys on the computer keyboard to indicate the identity of the target based on its perceived location (using the right hand, thumb = extreme left target, little finger = extreme right target, etc.). Throughout each trial, subjects were required to maintain gaze within an invisible window of 4° centered on the fixation point, even after the fixation point was removed. At the end of each practice trial, the target was displayed again to provide visual feedback, and the computer-generated voice provided a verbal indication of the target's actual location. Subsequent experimental trials had a similar time course, except for the inclusion of a large frame (1,000-ms duration) that was illuminated 900 ms before target onset (frame and target were extinguished simultaneously). Subjects in this and all other tasks were explicitly instructed to ignore the presence of the large frame when making their judgments of target location. Similar trials were used to test the sensorimotor effects of Roelofs illusion (see Figure 2 B; see also Figure S2 A), with the exception that subjects were instructed to make a saccadic eye movement to the target location after being cued to “Respond” by the computer-generated voice. After making any necessary eye movements to fixate the remembered target location, subjects ended each trial by pressing the “Enter” key on the keyboard (this final gaze position was used as the subject's indication of target location). Subjects in this version of the task were never informed that there were only five possible target locations. To test the effects of Roelofs illusion on remembered visual space (see Figure 3 A; see also Figure S3 A), subjects were instructed to make eye movements to the remembered locations of the five possible targets. During practice trials in which no frames were presented, the visual target was replaced with a computer-generated voice providing the identity of the target location (“One” = extreme left target, “Five” = extreme right target, etc.). After the computer-generated cue to “Respond” (presented just before or 4 s after the cue for target identity), subjects moved their eyes to the remembered location of the target and ended the trial by pressing the “Enter” key. Feedback during practice trials was provided in the way of an illumination of the correct target location and a small square indicating the final gaze position for that trial. In subsequent experimental trials, the large rectangular frame was presented 900 ms before the onset of the auditory target-identity cue, and no feedback was provided. To test the effects of Roelofs illusion on the apparent midline (see Figure 4 ; see also Figure S4 A), subjects were instructed to make eye movements to look straight-ahead when cued to “Respond” by the computer-generated voice. To prevent the fixation point from providing information about the actual midline in this version of the task, the horizontal position of the fixation point was varied randomly from trial to trial (−3°, −1°, 1°, or 3° from midline). To determine whether the delayed sensorimotor Roelofs effect was modulated by the presence of the frame during the imposed delay period (see Figure 6 ; see also Figure S5 A), all trials contained an imposed delay of 4 s between target presentation and the subsequent computer-generated “Respond” command. In half of the trials, the rectangular frame (1-s duration) was extinguished simultaneous with the target (these trials exactly replicated those used to originally measure the delayed sensorimotor Roelofs effect; see Figure 2 B, dashed line). In the remaining trials, the frame (5-s duration) remained illuminated until the onset of the computer-generated “Respond” command. To test the effects of Roelofs illusion on saccades directed toward targets defined allocentrically (see Figure 7 ; see also Figure S6 A), stimuli consisted of three small circles (connected by two thin lines), indicating three of the four corners of a small rectangle whose size and orientation was varied randomly from trial to trial. The partial rectangle was positioned so that the missing corner was located −4°, −2°, 0°, 2°, or 4° from the subject's midline, at eye level. Subjects were instructed to move their eyes to the location of the missing corner when cued to “Respond.” To accommodate the larger space required to define the target location allocentrically, the Roelofs-inducing frame was enlarged (28° horizontal × 14° vertical, 1° thickness) in this version of the experiment. Statistical analysis In each experiment, the signed magnitudes of localization errors (i.e., the difference between the actual and reported locations of the target in the horizontal dimension, with six repetitions for each trial type) were analyzed with a full-factorial analysis of variance, in a 5 (target location) × 3 (frame position) × 2 (response delay) design (see Figures 2 , 3 , and 7), a 5 (target location) × 3 (frame position) × 2 (frame duration) design (see Figure 6 ), or a 4 (fixation location) × 3 (frame position) × 2 (response delay) design (see Figure 4 ). In the present analyses, only the effects of frame position, frame duration, and response delay are considered. The effect of target location has been explored elsewhere ( Dassonville and Bala 2004 ). Supporting Information Figure S1 Time Line and Results for the Perceptual Roelofs Effect (A) Time line of task events for immediate (black) and delayed (gray) perceptual judgments of target location. Note that in this and all other experiments, feedback was presented only during practice trials, and the frame was presented only during experimental trials. (B) Effect of frame location on immediate (solid line) and delayed (dashed line) perceptual judgments of target location for each of five target locations. (C) Effect of frame location on immediate (solid line) and delayed (dashed line) perceptual judgments of target location for each of ten subjects. (579 KB TIF). Click here for additional data file. Figure S2 Time Line and Results for the Sensorimotor Roelofs Effect (A) Time line of task events for immediate (black) and delayed (gray) sensorimotor responses. (B) Effect of frame offset on immediate (solid line) and delayed (dashed line) sensorimotor responses for each of five target locations. (C) Effect of frame offset on immediate (solid line) and delayed (dashed line) sensorimotor responses for each of ten subjects. (1.1 MB TIF). Click here for additional data file. Figure S3 Time Line and Results for the Inverse Roelofs Effect on Remembered Space (A) Time line of task events for immediate (black) and delayed (gray) sensorimotor responses toward remembered reference-array locations. (B) An inverse Roelofs effect for immediate (solid line) and delayed (dashed line) sensorimotor responses toward remembered reference-array locations, for each of five target locations. The nonlinear effect of target location has been addressed elsewhere ( Dassonville and Bala 2004 ). (C) An inverse Roelofs effect for immediate (solid line) and delayed (dashed line) sensorimotor responses toward remembered reference array locations, for each of ten subjects. (2.5 MB TIF). Click here for additional data file. Figure S4 Time Line and Results for the Inverse Roelofs Effect on the Apparent Midline (A) Time line of task events for immediate (black) and delayed (gray) sensorimotor responses toward the apparent midline. (B) An inverse Roelofs effect for immediate (solid line) and delayed (dashed line) sensorimotor responses toward the apparent midline, for each of ten subjects. (856 KB TIF). Click here for additional data file. Figure S5 Time Line and Results for Testing the Effects of Frame Duration (A) Time line of task events for delayed sensorimotor responses, for trials in which the frame was either extinguished at the start of the delay period (brief frame, black) or was present throughout the delay (extended frame, gray). (B) Effect of frame offset on delayed sensorimotor responses, for trials in which the frame was either extinguished at the start of the delay period (brief frame, dashed line), or was present throughout the delay (extended frame, dotted line), for each of five target locations. (C) Effect of frame offset on delayed sensorimotor responses, for trials in which the frame was either extinguished at the start of the delay period (brief frame, dashed line) or was present throughout the delay (extended frame, dotted line), for each of ten subjects. (4.7 MB TIF). Click here for additional data file. Figure S6 Time Line and Results for the Roelofs Effect on Allocentrically Defined Targets (A) Time line of task events for immediate (black) and delayed (gray) sensorimotor responses to targets defined allocentrically. (B) Effect of frame offset on immediate (solid line) and delayed (dashed line) sensorimotor responses to targets defined allocentrically, for each of five target locations. (C) Effect of frame offset on immediate (solid line) and delayed (dashed line) sensorimotor responses to targets defined allocentrically, for each of ten subjects. (1.8 MB TIF). Click here for additional data file.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC524248.xml
535345	"Tied together like a woven hat:" Protective pathways to Alaska native sobriety	Background The People Awakening Project (1RO1 AA 11446-03) had two purposes, completed in Phase I and Phase II of the project. The purpose of Phase I was to complete a qualitative study; the research objective was discovery oriented with the specific aim of identification of protective and recovery factors in Alaska Native sobriety. Results were used to develop a heuristic model of protective and recovery factors, and measures based on these factors. The research objective of Phase II was to pilot these measures and provide initial validity data. Methods Phase I utilized a life history methodology. People Awakening interviewed a convenience sample of 101 Alaska Natives who had either recovered from alcoholism (n = 58) or never had a drinking problem (n = 43). This later group included both lifetime abstainers (LAs) and non-problem drinkers (NPs). Life histories were transcribed and analyzed using grounded theory and consensual data analytic procedures within a participatory action research framework. Analyses were utilized to generate heuristic models of protection and recovery from alcohol abuse among Alaska Natives. Results Analyses generated a heuristic model of protective factors from alcohol abuse. The resulting multilevel and multi-factorial model describes interactive and reciprocal influences of (a) individual, family, and community characteristics; (b) trauma and the individual and contextual response to trauma, (c) experimental substance use and the person's social environment; and (d) reflective processes associated with a turning point, or a life decision regarding sobriety. The importance of cultural factors mediating all these protective processes is emphasized. For NPs, the resilience process drew from personal stores of self-confidence, self-efficacy, and self-mastery that derived from ability to successfully maneuver within stressful or potentially traumatizing environments. In contrast, for many LAs, efficacy was instead described in more socially embedded terms better understood as communal mastery. One style of mastery is more associated with individualistic orientations, the other with more collectivistic. Future research is needed regarding the generalizeability of this group difference. Conclusions Results suggest that preventative interventions should focus on intervening simultaneously at the community, family, and individual levels to build resilience and protective factors at each level. Of particular importance is the building of reflexivity along with other cognitive processes that allow the individual to think through problems and to reach a life decision to not abuse alcohol.	Background Many American Indian and Alaska Native people experience problems with alcohol abuse that lead to social, psychological, and physical problems [ 1 - 3 ]. Unfortunately, little is known about American Indian or Alaska Native people who live sober and healthy lives. This paper presents initial findings from the People Awakening Project (PA), a collaborative study involving the Alaska Native community, and Native and non-Native university researchers. The goal of PA was to provide an Alaska Native understanding of the sobriety process. In earlier work, we provided a detailed description of PA's focus on cultural and spiritual understandings of sobriety [ 4 ], and its use of participatory research methodologies with Alaska Natives [ 5 ]. Sobriety in the addiction literature is generally defined as total abstinence following a period of alcohol abuse and/or dependence. However, many Alaska Natives also consider life-long abstinence, as well as non-abusive or moderate use of alcohol, as examples of a sober lifestyle. PA has adopted this broader definition of sobriety. Recent research on resilience identifies and describes protective factors that moderate risk and adverse environmental circumstances; this work has relevance to understanding the sobriety process [ 6 - 10 ]. Resilience is "a capacity that develops over time in the context of person-environment interactions" [ 11 ] (p. 517). Protective factors are those attributes that contribute to this capacity, and include those "individual characteristics or environmental conditions that help children and youth resist or otherwise counteract the stress to which they were exposed. They delay, suppress, or neutralize negative outcomes" [ 12 ] (p. 4). Protective factors can be grouped according to three broadly conceived categories [ 13 - 15 ]: (a) internal or dispositional attributes of the individual, such as sociability, intelligence, social competence, and internal locus of control; (b) familial attributes, such as warmth and closeness of affectional ties, and level of active emotional support within the family network; and, (c) contextual factors, such as social support, and characteristics of school, work and church settings. Because protective factors include personality traits and family, community, and environmental characteristics, it is difficult to compile a universal list of factors appropriate to all groups of people in very diverse contexts, especially when the nature or the composition of those categories includes diverse cultural dimensions [ 16 ]. For example, self-efficacy is a commonly cited protective factor [ 13 , 14 , 17 ], but few studies describe the nature of self-efficacy and how it works to protect American Indians or Alaska Natives. Hobfoll, Jackson, Hobfoll, Pierce, and Young [ 18 ] expanded our understanding of how efficacy may differ in a collectivist culture. A measure of communal mastery developed for the Hobfoll et al. study, but not a standard self-efficacy measure [ 19 ], predicted lower depressive mood and anger among American Indian women in stressful situations. Research among other ethnically diverse populations, including work with indigenous people in Kauai [ 15 ], Asian-Americans [ 20 ], and culturally-diverse inner city populations [ 21 , 22 ] similarly highlight the importance of cultural factors in the understanding of protective processes. Triadic Influence theory (TI) [ 23 ] provides a multi-level, multi-factorial model for understanding protective factors in sobriety that both integrates constructs from other theories on alcohol use and abuse, and provides a conceptual framework for interventions [ 24 ]. However, Petraitis, Flay, and Miller [ 25 ] noted that there has been limited research on protective factors within a TI framework associated with race and ethnicity. The limited existing research on the role of cultural factors within protective processes from substance abuse among American Indians and Alaska Natives has focused on cultural identity processes and has yielded mixed findings. Beauvais and Oetting's [ 26 ] review of research suggested high levels of cultural identification function as a protective factor from substance abuse among American Indian adolescents, and Schinke et al. [ 27 ]found bicultural skills training an effective preventive intervention against substance abuse for this population. However, other studies of cultural identity and substance abuse have found no relation [ 28 ], or a positive relationship for women [ 29 ]. Oetting, Donnermeryer, Trimble, and Beauvais [ 30 ] concluded that simple relationships between cultural identification and substance abuse are unlikely to be found given four potentially overlapping considerations. First, members of an ethnic group vary on level of cultural identification, which may effect conformity to substance use norms. Second, substance abuse may originate from norms socialized in the subculture and differ from those of the larger ethnic group. Third, cultural identification and substance use norms may differ in different contexts. Fourth, cultural identification may originate from primary socialization sources that are different than drug use norms. Instead of attempting to study cultural factors through measurement of identification with Alaska Native culture, the narrative form of the qualitative study reported in this paper allows for an alternative approach involving the generation of hypotheses on ways in which specific culturally mediated processes are conceptualized as protective by the members of the culture themselves. In summary, there is a need for research that examines the resilience experience of Alaska Natives who lead sober lives, and in particular, for research that includes an examination of the role of cultural factors in the protective process. In order to provide the rich description necessary to understand the range of experience and cultural processes of Alaska Natives who never drank abusively or who have recovered, qualitative methodologies are used. The goal of this study is to generate a theoretical model [ 31 ] of protection grounded in the experience of Alaska Native people that could inform the development of culturally anchored prevention approaches. Aligned with this goal, in this article we focus on Alaska Native pathways to the sobriety outcomes of abstinence and nonproblem alcohol use. Our analysis of the recovery group in this study is therefore restricted to identification of unique attributes within the abstinent and nonproblem drinking group not found among the recovery group. Future research will explore Alaska Native pathways of recovery from alcohol abuse. Methods Sample A purposive sampling procedure was used. Selection criteria were established by the PA Coordinating Council, a statewide group consisting of Alaska Native community leaders, individuals involved with grassroots Alaska Native sobriety movement efforts, and Alaska Native substance abuse services providers, who functioned as co-researchers in the participatory methodology. The Council distinguished three groups of interest: (1) lifetime abstainers (LAs) defined as individuals who have never drank more than two drinks per year, (2) non-problem drinkers (NPs) who report drinking alcohol with no problem and score less than 12 on the lifetime total consequences score of the Drinkers Inventory of Consequences for Alaska Natives (DrInC-AN)-a culturally adapted version of the Drinkers Inventory of Consequences (30), and (3) five years or greater of sobriety (5+) who identified themselves as recovered after a serious problem with alcohol, scored greater than 12 on the DrInC-AN lifetime total consequences score, and reported abstinence for at least five years. The project goal for Phase I was to select 36 participants with equal representation from the five Alaska Native tribal groups-Aleut/Alutiq, Athabascan, Inupiaq, Tlingit/Haida/Tsimshian, and Yup'ik/Cup'ik,-balanced by gender, age, and sobriety group status, and to oversample 12 additional interviews from the Yup'ik because Phase II measurement development would focus on this group. PA utilized nomination and snowball procedures to identify potential participants. Age representation was categorized into three age groups: 21 to 30, 30 to 55, and 56 and over. These age ranges were selected by the Council as indicative of culturally significant age ranges, marking indigenous age transitions from young adulthood to middle adulthood to elder. The Council selected these three sobriety categories to maximize our ability to discover potential protective factors as well as recovery factors, together which would define broadly resilience factors used by Alaska Natives in dealing with alcohol. Consultants from the respective tribal communities, the regional non-profit corporations, area health service providers, and other Native political organizations nominated individuals for participation, who then nominated others. Additionally, radio shows, advertisements, and newspaper articles solicited volunteers. This yielded 152 volunteers. Because our Council indicated it would be culturally inappropriate to not interview people following their offer to tell their life story to the project, PA offered interviews to all volunteers, and 101 completed the entire interview process. The results presented here analyze 37 long life history interviews and 14 briefer interviews on sobriety experiences. These participants were distributed across tribal group affiliation (Aleut/Alutiq-6, Athabascan-7, Inupiaq-6, Tlingit/Haida/Tsimshian-6, Yup'ik/Cup'ik-26), and the three sobriety types: LA - 10, NP - 19, and 5+ - 22, with proportional representation of the long life histories by gender and age in each sobriety category. In addition to over-sampling from the Yup'ik cultural group for life history interviews, 14 Yup'ik briefer interviews are included in this analysis in order to maximize the generalizeability of the findings to this cultural group, as the next phases of PA involve the development of measurement instruments and preventative interventions in regions of Alaska that include a Yup'ik majority. Sixty-two percent of participants spoke English as a first language and 48% their indigenous language. Eighty-two percent had been married at one time, with the average length of marriage being 10 years. At the time of the interviews 57% remained married. Participants' immediate families averaged 3 children. Participant incomes ranged from below $10,000 to over $100,000 per annum with the mean at $46,800. Most participants had graduated from high school (84%) and education ranged from no school to doctoral degrees. Of those who had recovered from alcohol abuse/dependence, mean years of sobriety was 17.5 years. Procedures PA was approved by the Institutional Review Board at the University of Alaska Fairbanks prior to participant enrollment. Nominees were contacted initially by phone, the purpose and structure of the interviews was described, and participation invited. Preference for location of interview, gender of interviewer, indigenous language or English interviewer, and interviewer that they knew or did not know was established. Interviewers were trained in the interview protocol, including protection of human participant procedures, prior to this contact. Life history interviews followed an open-ended for long life histories (LLH) or semi-structured format for brief life stories (BLS). The mean for LLH was 173.5 minutes (SD = 87.5), median was 159.5, and mode was 141.9. For BLS the mean was 119.5 minutes (SD = 49.5), median was 110, and mode was 106.5. Range for LLH were 20 to 452 minutes and for BLS were 45 to 272 minutes. The interview protocol elicited lifespan information with a focus on what the person considered most important in their process of sobriety. The intent was to garner rich detail about each person's life story. Briefer interviews were semi-structured. Questions addressed specific issues including the role of culture, spirituality, role models, parenting, and the methods of coping that individuals utilized to either not abuse alcohol or to recover. However, it is important to note that Alaska Native narrative patterns [ 32 ] at times overrode the distinction between these interview types and participants often responded to both formats similarly in time duration and style of discourse. Many participants tended to respond to either question format with a narrative, and did not distinguish more structured questions from less structured ones, e.g. "When did you first drink and what was your experience like?" in contrast to, "Tell me about your life in as much detail as possible from whatever point that you wish?" would often be answered in the same way and expanded upon equally. Our sense was that older participants in particular would often respond to either type of question by telling their entire life story. Additionally, we noted the length of the interviews also often varied by the experience of the interviewer and/or how the interviewer responded to the content of the interviews. For example, some interviewers felt it was best to close off interviews that began to bring out too much emotional material, whereas others with more clinical experience were more comfortable in moving through emotional material, framing and containing it, and then move on to other material. Interviews were recorded digitally using mini-disk recorders. At interview conclusion, participants completed a demographic questionnaire and the DrInC-AN, Analysis Our analytic approach combined elements of grounded theory analysis [ 31 ] with recent methodological advances in team-based coding and analysis [ 33 ] and consensual qualitative data analysis [ 34 ]. Interviews were verbatim transcribed, reviewed by the interviewer, then, in the case of the life history interviews, the transcript was mailed to and reviewed by the participant for accuracy, additions, or changes. The following describes the analytic process from which a heuristic model of protective factors in Alaska Native sobriety emerged. Although the analytic structure is presented in stages for exposition of its elements, the analysis in practice functioned in an iterative process through multiple passes through stages, involving continual reassessment of inferences and analyses. Step 1: Memoing Each analysis team member memoed the recordings of assigned interviews while also making additions and corrections to the transcripts for fidelity to the recorded interview. Memoing entailed three steps: (1) open coding identify possible codes, (2) connecting codes through overarching themes, and (3) documenting how codes and themes fit possible theories of protection. Team members then read all memos. Additionally, some of the team members shared their memos with the participant to gather feedback on the accuracy of their perceptions. Changes to the coding and analysis were made to reflect the perceptions of the participant. Most participants made no changes to the transcripts or small changes to the transcripts. A small number made changes by adding material or deciding to delete material, e.g. a number of individuals dropped names of people that were in the interview. A few added material that they had remembered. We gave the participants their verbatim transcripts (with all pauses, false starts, "ahs", etc.) and discovered participants were often embarrassed by their unedited nature. We learned immediately we needed to explain the nature of the transcription process and its intent, and that their interviews would not be published in such a form (participants' interview transcripts were confidential, but several participants expressed a cultural value in their desire to have their interviews made available to others who may be struggling with alcohol themselves and find them helpful). An initial set of codes and overarching themes or domains under which the codes clustered was identified and then systematized in an initial draft coding manual. Step 2: Open coding and coding manual development Two research team members continued to read and open-code interviews. The team met periodically with Gerald V. Mohatt, Principal Investigator, who also coded a number of transcripts, to discuss coding discrepancies and refine coding rules. The goal at this stage was inclusive not exclusive, and to add as many codes as possible; therefore, we did not limit ideas. We spent much time operationalizing definitions in order to ensure that each code was clearly distinguished from others and could be reliably scored using the codebook criteria. This was done through hours of discussion, with final agreement regarding the definition of each code arrived at between the PI, the research Project Director, and at least one of the Co-Investigators or research assistants. This process resulted in 220 separate codes organized under 25 hierarchical domains. Coding reliability was enhanced in the revised coding manual through development of definitions for each code, along with examples of the code in use and decision rules where appropriate. Step 3: Coding/content analysis and codebook refinement The research team trained coders to code using AnSWR software [ 35 ] and content analyze the remaining transcripts. Inter-coder reliability between coders was assessed on every seventh transcript. What represents adequate inter-coder reliability in qualitative research continues to provoke divergent viewpoints in the literature. Miles and Huberman [ 36 ](p. 64) suggest that final inter-coder agreement in qualitative data analysis should approach or exceed 90%, though Stein[ 37 ] recently published a study where she used less than 80% agreement. Moreover, simple proportions do not account for the possibility that coders might agree due to chance, which is a function of the frequency or infrequency with which a code appears [ 38 ] and therefore provide a biased over-estimate of the true level of agreement. To correct for this, we used the kappa statistic [ 39 ]. Carey, Morgan, & Oxtoby [ 40 ] judged that a kappa less than .90 indicated a problem with agreement in the way a code was being used in qualitative research. However, insistence upon very high levels of reliability can also have the effect of diminishing validity [ 41 ], and this is a particular concern in discovery-based research such as that of the present study. Therefore, we adopted minimum criteria for the 25 hierarchical categories of kappa .90 or greater, and coding of the 220 lower level categories of no less than .60. Kappas ranged from .60 to .81 for all lower level categories, and all hierarchical categories were at .90 or above. The team continued to reconcile divergences in coding, refine coding categories, open code, and revise the codebook. Previously coded transcripts were recoded, using the revised codebook. Step 4: Cultural auditing The team submitted a sample of transcripts to the PA Coordinating Council as part of a cultural auditing procedure. The co-researcher role of this Council, which included members of all five Alaska Native tribal groups interviewed by the project is described elsewhere [ 5 ]. The Council collectively open-coded five transcripts from participants selected from all three sobriety groups. Council members coded the transcript of a participant from their own cultural group. The Council convened to discuss their coding and address specific research team questions; such as, have we identified and labelled the codes appropriately. This cultural auditing process moved the team forward in understanding the narratives from a more culturally grounded perspective. For example, Council members understood "being a role model" within the context of the cultural value of contributing to the good of the family or community, and not merely in terms of individual achievement. The Council also indicated that we should add codes such as shame, praise, and pride to our coding system, and elaborated on their definitions. An overall comparison of the coding and domains generated by the Council with those of the research team displayed high levels of consistency, along with selected important divergences which were discussed to mutual understanding, then adopted by the research coding team. Step 5: Generating theories through a consensual analytic process Team members next identified how coded segments clustered and interacted, generating potential theories on protective factors through comparison of the life histories of LAs and NPs to 5+ individuals. The team discussed multiple theories, and reconciled potential theories to case histories of non-agreement through revision or abandonment of the theory. Step 6: Developing and refining a theoretical pathway to sobriety Team discussions were summarized and synthesized by the principal investigator into competing models. The team reread transcripts, discussed and refined models, converging on one model that best fit the majority of transcripts, which was then presented to the PA Coordinating Council. The Council added refinements and culturally grounded elaborations to this model. Step 7: Doubling back The team re-read transcripts and reassessed the model, refining and elaborating elements until consensus that the full set of transcripts supported the model. As part of this process the team enlisted the Cuiliat Group of Yup'ik speakers, who were our cultural consultants, and would also assist us in the Phase II measurement development. Translating each of the protective factors into Yup'ik forced us to clarify definitions and ensured that they differentiated culturally specific dimensions of each protective factor. For example, from this process the importance of collective group factors became clearer. Methods for Verification In qualitative research, the analogue for validity in quantitative research is often termed credibility , which can be defined through (1) the confidence that can be placed in the data and analysis [ 42 , 43 ], (2) how well the conclusions from the data analysis are grounded and supported in the data [ 44 ], and (3) the degree to which the descriptions and analyses provide an understanding of the experience studied [ 45 ]. In this study, several methods [ 36 ] were used to enhance the credibility of the findings: prolonged engagement with the participants resulting in rich, thick description; initial memoing of each narrative prior to coding; confirmation of the narrative and its transcription, and of the memoing, through checks with the study participants; team data coding with ongoing reliability checks and refinement of the coding system; triangulation through the use of multiple data sources and multiple co-researcher perspectives; negative case analysis, or the examination of events and perceptions that did not fit emerging themes; cultural auditing of the coding and interpretative process; and team-based consensual analytic processes. Examples of triangulation included sending transcripts and memoing to the participant,, discussion of the memoing and transcripts with the Council, and the parallel discussions within the research team, which provided three typically converging perspectives on the analysis, along with recognition and discussion of discrepancies whenever they occurred, to the point of mutual understanding, and resolution and agreement. Depending upon the specific theme that was divergent, action could involve reworking of the coding theme to make it more congruent, dropping the theme as an unreliable code,, or addition of a new theme that was not seen by the research coding team, but was identified by others who analyzed the transcripts. Given the multiple cultural perspectives, this provided rich, deep, and inclusive coding categories allowing for the generation of multiple hypotheses regarding themes and the connections between them in the life stories. Generalizability The research aim of the PA study was discovery-based, and not proof through hypothesis testing and falsification. Our objective was to characterize the types of protective factors utilized within this purposive sample, and not to generalize to all Alaska Natives or American Indians. Our goal was to generate a heuristic theory that would suggest testable hypotheses that could later be investigated in a larger, population based study, using measures developed in Phase II. We also hoped to offer ideas to services programs regarding variables that they could test for effectiveness in prevention or treatment. Results Using the above process we first identified a set of factors protective from alcohol abuse. We use the direct words of participants to illustrate each to allow the reader to move through the process in a manner similar to the research team. Each protective factor in the model is translated into Yup'ik, the indigenous language of the group we plan to collaborate with on an intervention program. The complete Heuristic Model of Alaska Native Protective Pathways can be found in Figure 1 . The mode represents a culture specific mapping of protective processes and as such, is presented in a format that allows for hypothesis testing using quantitative methods. The model is theoretical and heuristic in nature, and shows postulated relationships between factors consistent with Triadic Influence Theory, rather than empirically supported causal factors. We describe below each protective factor, along with its relationship to the model and function. Figure 1 Heuristic Model of Alaska Native Protective Pathways Key . CC (community characteristics) Yuut cayarait includes the way the community organizes family, school, and community activity, and enforces alcohol policy and the drinking status of the community, CC includes role models, opportunities, limits, and safe places. FE (family environment) Ilakelriit cayarait includes family functioning in such areas as cohesion, conflict, recreation outlets, moral-spiritual focus, and home organization. Factors included parent-child relationship, affection and praise, transmission of expectations, safety and protection from harm and models of sobriety. IC (individual characteristics) Yuum Ayuqucia are belief in self (communal and self-mastery), wanting to contribute to others and Ellanqaq (Yup'ik mindfulness and awareness. SE (social environment) Yuuyaraq includes role models and social support from extended family, peers, and other adults outside of immediate, nuclear family. TR (trauma) Akngirneq includes sexual abuse, domestic violence, and death of loved ones. It includes being a victim and observing others being a victim. An individual's perception of trauma is critical, as is the meaning they attach to their experience and how they respond to it. ESU (experimental substance use) Meqerraaryaurtellemni are early experiences with substances, including alcohol, prior to the establishment of use patterns or abstinence. TO (thinking it over) Umyuangcallemni involves reflecting on one's experience and developing a personal life narrative. TP (turning point) Ayuqucinellemni comes out of this reflective process and leads to a decision about how the person will use alcohol. Community Characteristics (CC) Yuut cayarait . Participants described the context of the community that protected them during childhood and provided a sense of security. As one participant indicated, "I guess, my life as a child was pretty much sheltered...so, as the expression goes, the village was my oyster then." Protective communities possessed role models for the proactive caring of others that exemplified a sense of a collective responsibility for the care of children, or, as another participant described this, "That's also what I remember is people taking care of us even if we're not their children, they looked after us, and they corrected us." Participants described how protective communities provided both opportunities to learn and alternatives to drinking. One young man described how the community school gave opportunities to travel, engage in sports, debate, and engage in student leadership that gave him ideas about college and careers. Opportunities were also often contextualized in ways the community helped children through important culturally defined transitional rites in the development of adult roles: "They still do this community sponsored moose hunt. They go out and they go hunting for the moose and for a lot of young men that is the time that they have the rite of passage. This is their first moose. And in the beginning when it started out it was just the men, just boys were allowed to go. And it evolved into a community wide project and it does include girls. And the whole community is involved because they'll go and they'll come back in and they'll have a big potluck and it's the rite of passage for he who caught his first moose. Everybody gets to participate. He gets to provide for his community, you know for the first time and that is something that he can do." One of the most important community protective factors related to how the community established limits. While some individuals discussed the local option laws that allow some communities to vote to regulate or ban alcohol, a larger number discussed how significant individuals in the community took a personal stand to protect children from alcohol-related harm. What was fascinating was in which community characteristics were frequently embedded within the context of the family, and occurred within the interface between the family and the community. A vivid instance of this is described by a middle-aged woman recalling her childhood: "When I first was aware of somebody drinking, I was already nine years old. And I never saw anybody drunk before. ...And my father stood up, and he said no; he just let him turn around and he walked out with him. And then I heard him out there, 'Don't you ever come in my house like that.' We asked my mom, what is wrong with that man? And she would never tell us; she would say in due time you will know. In your own time, you will know." Here we see the individual actions within a family as an important component within a community-wide expectation regarding the setting of limits upon alcoholic behavior, reciprocally mirroring and contributing to a community standard. Participants reported how they were exposed in childhood to adults that abused alcohol. Protective communities had safe places children could go to that prevented them from becoming victims of alcohol-related violence. Most often the safe place was with a close relative, but it could include a friend, teacher, or member of the clergy. As one participant described: "I like the way my grandma took care of me when I was small. Her house was always clean, everything smelled good. It was always a safe place to go to. And I have realized after I got my own place and became an adult, that my home, to other people, was always a safe place to go to." Family Characteristics (FC) , Ilakelriit Cayarat . In the words of one participant: "In the Native community families are tied together in a certain way that they're close. And it doesn't matter who you are, we're tied together like a woven hat." This interdependence of family and community highlights both the kinship and collectivist [ 46 ] nature of Alaska Native communities. The most fundamental of the protective family factors described by participants was the nature of the caregiver relationship: An affection and praise that included important culture specific elements providing children a sense of being valued appears in the following narrative: "And I remember my grandparents bringing us to other elders' homes, just to introduce us to them, because our grandparents were proud of us, and they wanted to share us with the elders in the community. So they brought us to the elders and let us visit with them. I remember when we started hunting and fishing, we got a lot of praise, and even more praise than today, from our relatives and elders. You know, if an elder found out that you caught your first rabbit or your first moose, everybody praised you for that. And it helped to build up the esteem." Another quality of the caregiver relationship was a sense of being treated as special , as very important to the family. One participant noted: "So I grew up to be pretty special, only because I was the only girl of my family. My older brothers took very good care of me. They treated me well." Others who avoided alcohol problems in their lives recalled being told they were to become healers or shamans, or would have similar important roles in the community, and were encouraged to live in a way that prepared them for this role. Families also provided safety/protection from harm . In addition to simply providing a place of safety, caregivers also established limits and enforced them for the good of children. One narrative related the importance of modeling values through the power of both words and action: "I would put the kids to bed and make, you know, put them to bed and make sure those people that were there, some of them I would kick them out and other ones, a lot of times I would let them go, say 'Go drink somewhere else. This is not the place to drink."' Participants who never developed a drinking problem also described models of sobriety in the family who taught them explicitly about how to deal with alcohol: "So my Dad was a non-drinker. And he said when I was eight year old he say, he sat me down, and he told me he said, my son being the oldest in the family, he said, there is something that I want you to do for me. And he said, I want you to carry a torch for me, a torch that you would say that all of my life I wouldn't drink and I wouldn't smoke. He say I took his word for it and he say, I want you to do the same for me. Carry that torch for me. And I guess that's the biggest thing you know that right there and then I thought okay." Protective families also actively engaged in transmission of the expectations they had for their children: "We were a poor family as any village people. But things were happy when we were growing up, and our Mom very seldom went out to work so she was home with us a lot. And my dad would talk to the boys about what's expected of them when they grew up, and how to take their place in the community or in their tribal relatives, how everything worked together. So that's how we all grew up." Many of these protective factors mirror each as interdependent community/family systems that protected children from exposure to alcohol abuse and alcohol related violence. Individual Characteristics (IC) Yuum Ayuqucia . Protected individuals displayed a set of characteristics that included a preference towards a cognitive style of thinking through reflectively about what one will or will not do. This reflective style allowed self-control around alcohol use and decisions to immerse oneself in activities that avoid or are incompatible with alcohol use: "But, like I said, it hasn't bothered me – drinking hasn't bothered me. I don't know if it will. In my head – in my mind, it never will. I'm – I'm a positive person and that's the way I like to live my life, is live positively and things go smoother that way. But, living a Yup'ik life, just in general, doing all the traditional activities that we do on a daily – day-to-day basis here in the village, this keeps me away, makes me not think about it." Participants describe this reflective process as part of a collectivist, other-centered orientation specific to Alaska Native cultures. One participant talked about wanting to be a role model : "And I had made a choice when I was ten or eleven to not drink alcohol, to remain sober and to show my brother, my sister that there is something different to do besides drinking and alcohol." The sense of responsibility within a kinship network led to a desire to give to others – contribute : "I think he [father] meant that I was going to help people sort out their lives, help them to understand, that you know, be a good listener for them, and counsel them when they need it, or at least let them know they have tools to help themselves. In order to give and contribute one must have a fundamental sense of one's own capacity, a belief in self , as a competent individual. One participant describes: "Like I mentioned, my parents, from as far back as even both of us can remember, I have always been an adult to them. I have always talked to them. Even like when I was ten years old, I talked to them like I was an adult, meaning I listened to them, I didn't talk about silly things. But we were able to converse, and so they treated me like an adult...that gave me the choice to do what I wanted and also to make the decision not to drink." Some participants described a sense of mastery as knowing and caring for oneself and one's capacity to endure. In the words of one Alaska Native person: "My mother taught me too much to love myself. I've always felt I was a very strong person. I have been able to put up with a lot of shit." However, important differences in mastery emerged between NPs and LAs. NPs often described a sense of efficacy and self-actualization focused more on self-confidence and independence than responsibility to the family and community. One traditional Yup'ik elder NP described how he took the initiative in his socio-cultural education. "Yes I learned on my own. Whenever I am going to construct something I would look at it from all sides and memorize it. When I was about to construct a large boat fashioned after one that is manufactured, I looked at a finished one from all sides and then I constructed it without anyone guiding me. I was not given a lot of advice by anyone." In contrast, for LAs, efficacy was described in more socially embedded terms better labeled as communal mastery [ 18 , 47 ], or a sense that one masters situations best by joining with others. In this way, several of the life stories describe a socialization process within interconnected collectivist community and family structures that foster becoming aware of how one's actions affect others, described as an awareness of consequences : ellangneq . Ellangneq is a Yup'ik concept, but similar elements appeared throughout many of the narratives across all the Alaska Native cultural groups. The child learns that reciprocity exists between individual actions, and the good of the community and family: control over one's own actions can affect others positively. Ellangneq is this culturally valued awareness of the consequences of one's individual actions upon the whole. This special type of awareness is incompatible with intoxication; intoxication only reduces awareness and the ability to control oneself and one's own life, thereby engendering potentially negative reciprocal effects on family, community, and others. In the words of a Yup'ik LA, "But at that time I had already decided for myself that I wasn't going to drink. Part of that had to do with getting out into the woods. And that was part of my reason for refusal. Why would you want to go out and drink and kind of get out of your mind, loose mental control? You know I had so much fun doing the things I wanted to so I wanted to be aware of what I was doing." Elaborating the Protective Process Community and family protective characteristics lowered exposure to alcohol and alcohol-related trauma, or moderated the negative impact of traumatic experiences. They also fostered individual protective characteristics such as sense of mastery, awareness ( Ellangneq ), and a sense of responsibility to family and community. Nearly half who never drank abusively describe directly experiencing or frequently observing significant trauma during childhood. Trauma and/or trauma exposure (TR), Akngirenq , included the death of loved ones or other unexpected and intense loss, witnessing domestic violence, or the experience of child abuse including sexual abuse. The pathway of participants who did not use alcohol as a coping response to trauma was facilitated by the protective community, family, and individual characteristics identified in the model, along with the youth's social environment , (SE) Yuuyaraq , including the presence of healthy, non-alcohol abusing role models and social support for lifestyles free of alcohol abuse from extended family, peers, and other adults outside of the immediate, nuclear family. Social environment is a subset of community characteristics specific to the time in youth when experimental substance use (ESU), Meqerraaryaurtellemni , begins, that functions as a support during periods of ESU or in times of crisis such as the experience of trauma. A male who had experienced significant family trauma described this: "I have a Russian Orthodox priest who's going to wed us in a civil ceremony. And I asked him when I was 15, 'If I ever get married, will you marry me?' He is also somebody who was a mentor for me as a kid.... I think that he was there for me at the right time. Especially, I think, and I probably don't remember a lot of things that happened at that age, but I knew that there was somebody who I could look to." A period of ESU was quite common in the narratives; a majority of NPs and several LAs engaged in ESU. This typically occurred in early or mid-adolescence, after which the decision to drink responsibly or not drink was made. Consistent with a worldview imbued with concepts allied with that of Ellangneq , NPs and in particular many LAs who tried alcohol decided in youth after ESU, or after the experience of significant alcohol-related trauma, that the consequences of alcohol did not fit with how they wanted to affect others. Though even in the presence of multiple family, community, and individual protective factors, children would often still engage in a period of ESU, the outcome among NPs and LAs who experienced these protective factors was a conscious decision, a turning point (TP) Ayuqucinellemn , that virtually all identified as a pivotal event in their narratives, to either not continue to use alcohol or not use it in a manner that led to abuse. This turning point typically occurred as part of a reflective process of thinking over (TO), Umyuangcallemni , one's personal experience with alcohol. As one NP described: "Later on after I graduated from high school I still knew I didn't want to be a drunk or you know, get drunk or look all ugly and do stupid stuff. (...) I didn't want to not know what I was going through. I wanted to be totally aware of my every live moment and I wanted to be in control of everything that I was doing. And so I think that's when my responsible drinking started." Through this process of thinking over and turning point, LAs and NPs composed a personal life narrative in which they were in charge of their lives. Figure 1 shows community, family, and individual characteristics reciprocally influencing each other. Strong, cohesive communities support the development of healthy families; together these institutions provide the networks of social support that develop a set of individual characteristics that enhance resilience. Strong and positive communities and familial relationships also decrease the likelihood of alcohol-related trauma exposure. They additionally are part of the development of a social environment from which individuals can seek support or resources if trauma is experienced. This occurs in part through development of individual characteristics that enhance the likelihood of a response to trauma or ESU experience that involves thinking over (TO) the experience and the broad and reciprocal consequences of one's actions. This reflective process (TO) facilitates a turning point (TP) in LA and NP outcomes, resulting in a decision to not abuse alcohol, in affirmation of a life goal of contribution to family and community. Discussion We present here a multifactorial and multilevel model for the understanding of the sobriety process of Alaska Natives that lead a life free of alcohol abuse. The model was generated through a participatory action research process, elements of which can be adapted for work with American Indian and other ethnic minority communities. Cultural factors emerged central to an understanding of the sobriety process of Alaska Natives demonstrating the importance of culture as proximal variable [ 48 ] in research that seeks to understand sobriety and alcohol abuse with American Indians and Alaska Natives. The resulting heuristic model for Alaska Native protective pathways is an indigenous explanatory model [ 49 ] describing how culturally mediated protective factors interact in complex ways. However, it is also consistent with Triadic Theory of Influence [ 23 ] assertions that substance abuse in adolescents is best explained by the interaction of community, family, and individual level variables. The model suggests that community and family build a wider social environment that both supports the youth and interacts with individual factors in the decision to not abuse alcohol following a period of ESU. The mechanism that appears to facilitate the turning point of a sobriety decision is Ellangneq , a sense of awareness, mindfulness, and the reciprocity of action developed through the teaching of parents, extended family, and community. Ellangneq can be understood as a manifestation of an interdependent [ 50 ], constitutive [ 51 ], or expanded sense of self [ 52 ] found among many Alaska Native and other non-western people that links the individual to a collective, tribal context [ 46 ]. Individuals who are socialized within such a context are allocentric [ 46 ], with a heightened sensitivity to the effects of their behavior on the whole, and drawing strength from the whole. Ellangneq becomes operative through the actions of family and community. Many researchers have found that a significant relationship with at least one parent is a critical variable in protective outcomes [ 53 ], though substitute caregivers can also be of great importance early in the child's life [ 9 , 11 , 54 ]. This emerged as an important factor in this Alaska Native sample as well; however, the mutual influences of a supportive extended family and community also contributed importantly to resilience. Also important were ways in which security, safety, pride, and affection were experienced through the parent-child interaction, and how the family related to other caregivers to enhance the community network of caregiving. One important difference distinguished the NP and LA sobriety groups in this sample. For NPs, the resilience process drew from personal stores of self-confidence, self-efficacy, and self-mastery that derived from ability to successfully maneuver within stressful or potentially traumatizing environments [ 55 ]. In contrast, for many LAs, efficacy was described in more socially embedded terms of communal mastery [ 18 , 47 ]. One style of mastery is more associated with individualistic orientations, the other with more collectivistic. Future research is needed regarding the generalizability of the group difference in this finding. Nonetheless, the finding highlights important differences between Alaska Native individuals regarding the processes underlying the decision to not abuse alcohol. This finding is of importance both for future research, and in planning interventions for Alaska Native people. The fact that this important difference reflects culturally mediated processes also suggests the decision is itself mediated by variables such as acculturation and cultural identity. Indeed, cultural factors surfaced repeatedly as important components in an understanding of how social influences within a community and family context functioned as salient protective factors in sobriety for Alaska Natives. As Triandis [ 46 ] remarked, "Culture is to society what memory is to the person" (p. 511). In our Alaska Native participants' narratives, cultural processes emerged as much more than immersion in activities, social grouping, or self-perception, imbuing structure and meaning to all aspects of their thoughts and behavior. Even basic components of cultural processes, such as a person's identification with their Alaska Native culture, emerged as complex, situational, and multidimensional, echoing previous critiques of cultural identity research with American Indians and Alaska Natives [ 56 ]. Conclusions This study presents a heuristic model of Alaska Native pathways to sobriety. What is significant about the model is that it emerged from in-depth study of the experience of Alaska Natives, rather than that of other groups. The model moves current research in the direction of developing a culturally and contextually based explanatory model [ 49 ] or emic model [57] of Alaska Native sobriety, because it comes out of the life histories of Alaska Natives and a collaborative analysis process that included Native and non-Native researchers, the community of concern, and the participants themselves, as co-researchers [ 5 ]. Tests of hypotheses and path analytic models generated by the heuristic model, and design and investigation of the efficacy of prevention programs based upon the model are important future steps for research. In addition, the findings of this study offer perspectives on the resilience and the sobriety process of indigenous people and more precisely contextualize elements of the Triadic Theory of Influence within one indigenous group. This initial analysis of the PA data set provides as many questions as answers for our understanding of the sobriety process of Alaska Natives. We hope that as the answers become more clearly defined, those pathways to recovery and resilience walked by the research participants become more known to those in need. The seeds of resilience form a sense of the family and community, a desire to make a difference as one Alutiq elder acknowledges: "We're not here tomorrow. Got to leave a few tracks around, right? I want to. So my grandkids could say, well, I remember when grandma used to – you know. You know you feel like, hey, you want to be able to leave some kind of memory." Competing interests The author(s) declare that they have no competing interests. Authors' contributions GVM is PI for the research. He led the analysis and interpretation, and completed many of the interviews. SMR completed much of the text analysis, wrote the first draft, and edited many drafts. LT interviewed most of the Tlingit participants, participated in all analysis and interpretation, and edited the paper. JA was a collaborating investigator involved in all aspects of data gathering, analysis, and interpretation. He edited each draft and significantly contributed to the final draft. KH was also a collaborating investigator and edited a number of the drafts. The People Awakening Team completed many of the interviews and assisted with the analysis and interpretation of the data. CH was research coordinator for PA and assisted in model development, analysis, interpretation of data, and editing.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC535345.xml
521683	No major association between TGFBR1*6A and prostate cancer	Prostate cancer is the most commonly diagnosed cancer in men and one of the leading causes of cancer deaths. There is strong genetic evidence indicating that a large proportion of prostate cancers are caused by heritable factors but the search for prostate cancer susceptibility genes has thus far remained elusive. TGFBR1 6A, a common hypomorphic variant of the type I Transforming Growth Factor Beta receptor, is emerging as a tumor susceptibility allele that predisposes to the development of breast, colon and ovarian cancer. The association with prostate cancer has not yet been explored. A total of 907 cases and controls from New York City were genotyped to test the hypothesis that TGFBR1 6A may contribute to the development of prostate cancer. TGFBR1 6A allelic frequency among cases (0.086) was slightly higher than among controls (0.080) but the differences in TGFBR1 6A genotype distribution between cases and controls did not reach statistical significance (p = 0.67). Our data suggest that TGFBR1 *6A does not contribute to the development of prostate cancer.	Background Transforming Growth Factor Beta (TGF-ß) is one of the most potent inhibitor of cell growth [ 1 ]. Almost all cancer cells loose the ability to be growth inhibited by TGF-ß, which makes loss of TGF-ß growth inhibition a hallmark of cancer development [ 2 ]. TGFBR1 6A is a common variant of the type I TGF-ß receptor, TGFBR1 [ 3 ]. TGFBR1 6A (6A) has a deletion of three GCG triplets coding for alanine within a nine alanine (9A) repeat sequence of TGFBR1 (9A) exon 1, resulting in a six alanine (6A) repeat sequence. The 9-bp deletion that differentiates 6A from 9A is located within the predicted signal sequence cleavage region. In vitro studies have demonstrated that TGFBR16A responds less effectively than TGFBR1 to TGF-ß growth inhibitory signals [ 4 , 5 ]. The additional findings of an overrepresentation of TGFBR1 6A heterozygotes and homozygotes among patients with a diagnosis of cancer as compared with the general population led us to postulate that TGFBR1 6A might act as a tumor susceptibility allele [ 5 ]. Two recent meta-analyses show that TGFBR1 6A carriers may have an increased risk of breast, colon and ovarian cancer [ 6 , 7 ]. To test the hypothesis that TGFBR1 6A may contribute to the development of prostate cancer, we conducted a case control study of patients with biopsy verified prostate cancer cases and geographically and ethnic-status matched controls. Results A total of 907 cases and controls were genotyped for TGFBR1 6A. The mean age of cases was significantly higher than controls (p < 0.01) but there were no differences in ethnic status between the two groups. There were 59 TGFBR1 6A heterozygotes and three TGFBR1 6A homozygotes among cases, 62 TGFBR1 6A heterozygotes and 1 TGFBR1 6A homozygote among controls. TGFBR1 6A allelic frequency among cases (0.086) was slightly higher than among controls (0.080) but the differences in TGFBR1 6A genotype distribution between cases and controls did not reach statistical significance (p = 0.67) (Table 1 ). Effect estimates from conditional logistic regression were similar (OR 1.01, 95% CI 0.29–3.52) to those from unconditional logistic regression (OR 0.96, 95% CI 0.56–1.64). Analyses restricted to subjects aged 40 years and above controlling age on a continuous scale produced essentially the same effect estimates (OR 0.94, 95% CI 0.56–1.60). To examine the possibility that TGFBR1 6A is associated with early onset prostate cancer, we determined the prostate cancer risk for individuals above and below the age of 55. Among younger patients with prostate cancer we found that 13 of 59 were TGFBR1 6A carriers yielding an allelic frequency of 0.119, one of the highest TGFBR1 6A allelic frequency ever reported. Only 45 of 367 controls in the same age range were TGFBR1 6A carriers yielding an allelic frequency of 0.063, which is similar to the TGFBR1 6A allelic frequency found among 3,451 healthy controls from Europe and the U.S. [ 7 ]. The association between carrier status of TGFBR1 6A and prostate cancer in younger age group was significant after adjustment for race (OR 2.13, 95% CI 1.06–4.27) but was not significant after adjustment for race and age strata within groups (OR 2.11, 95% CI 0.98–4.57) (Table 2 ). While in the older age group we did not observe a significant association either (OR 0.57, 95% CI 0.30–1.10), the test for multiplicative interaction between age and carrier status of TGFBR1 6A was significant (p = 0.01). Table 1 Distribution of Age, Ethnicity, and TGFBR1 Genotypes and Adjusted Odds Ratios of Prostate Cancer by TGFBR1 Genotype Status Cases (N = 442) Controls (N = 465) P-value 1 Adjusted OR (95% CI) 2 N % N % TGFBR1 genotype 9A/9A 380 86.0 402 86.5 0.67 1.00 (ref) 9A/6A 59 13.4 62 13.3 0.96 (0.56–1.64) 6A/6A 3 0.6 1 0.2 Age 20–40 1 0.2 205 44.1 <0.01 41–60 126 28.5 204 43.9 61–80 308 69.7 55 11.8 80+ 7 1.6 1 0.2 Race White 396 89.6 415 89.3 1.00 Black 26 5.9 29 6.3 Hispanic 8 1.8 8 1.7 Asian 2 0.5 2 0.4 Other 1 0.2 2 0.4 Unknown 9 2.0 9 1.9 1 p -value for Chi-Square or Fisher's Exact Test (comparing proportions) 2 OR was adjusted for age strata and race, based on dominant model. Table 2 Adjusted Odds Ratios of prostate cancer by age groups (> 55, <= 55 years old) Age group/ Genotypes Cases N (% within age strata) Controls N (% within age strata) OR (95% CI) 1 OR (95% CI) 2 P for testing multiplicative interaction Age = 55 9A/9A 46 (78.0) 322 (87.7) 1.00 1.00 9A/6A or 6A/6A 13 (22.0) 45 (12.3) 2.13 (1.06–4.27) 2.11 (0.98–4.57) Age > 55 0.01 9A/9A 334 (87.2) 80 (81.6) 1.00 1.00 9A/6A or 6A/6A 49 (12.8) 18 (18.4) 0.64 (0.36–1.17) 0.57 (0.30–1.10) 1 OR was adjusted for race. 2 OR was adjusted for race and age strata within age groups Discussion Prostate cancer is the most common cancer and the second most common cause of cancer death among U.S. men [ 8 ]. A similar pattern is observed throughout the western world. There is strong epidemiologic evidence indicating that a large proportion of prostate cancers are caused by heritable factors. The most convincing data is a study of 44,788 Scandinavian twins showing that 42% of prostate cancer cancers may be caused by shared genes [ 9 ]. Despite intense efforts led by several research teams, the search for prostate cancer susceptibility genes has thus far remained elusive. Recent studies suggest that carriers of deleterious mutations of the BRCA2 gene have an increased prostate cancer risk [ 10 ]. However, given the low prevalence of deleterious BRCA2 mutations in the general population, it is unlikely to account for a significant proportion of prostate cancer cases. Approximately 14% of the general population carries at least one copy of TGFBR1 6A, which makes it the most common candidate tumor susceptibility allele reported to date. While there is growing evidence that TGFBR1 6A predisposes to the development of breast, colon and ovarian cancer, our data do not suggest that it predisposes to the development of prostate cancer. We have previously shown that TGFBR1 6A homozygotes have an O.R of 2.69 and 2.02 for ovarian and colon cancer, respectively. The present study has the power to detect an O.R. for prostate cancer of 1.70 or higher and therefore rules out a major association between TGFBR1 6A and prostate cancer. However, it does not exclude a smaller O.R., which might have clinical relevance given the high TGFBR1 6A allelic frequency in the general population. It is possible that age differences in cases and controls affected the allele frequencies observed. If the TGFBR1 6A allele predisposes to a lethal malignancy such as prostate cancer, however, its frequency could be higher, not lower, in a younger cohort. Thus, the younger mean age of controls could result in a bias toward the null hypothesis, resulting in a stronger association than that observed. The intriguing findings of a high TGFBR1 6A allelic frequency among prostate cancer cases diagnosed before the age of 55 have to be cautiously interpreted given the fact that this group only included 46 patients. We have previously shown that TGFBR1 6A is not associated with an increased risk of bladder cancer [ 6 ]. Our results suggest that the association between TGFBR1 6A and prostate cancer is at best very weak but further studies are needed to formally exclude an association with early onset prostate cancer. Methods DNA was extracted from lymphocytes of blood specimens from 465 consecutive individuals diagnosed with adenocarcinoma of the prostate who received care at the outpatient urology clinic at Memorial Sloan-Kettering Cancer Center from April 2000 to September 2002. The blood samples were collected following completion of diagnostic studies. They were unselected for age or family history. Clinical and pathological records were reviewed to confirm the diagnosis of prostate cancer in all subjects. Once pathological diagnosis of prostate cancer was confirmed, the age of diagnosis was recorded, and all other identifying links were destroyed. The study design and anonymization method were approved by the Memorial Sloan-Kettering Cancer Center Institutional Review Board. A population of 465 healthy male controls aged 20 to 87 years with well-defined ethnic background who had donated blood for various reasons (predominantly pre-natal screening for non-cancer disease) constituted the control group. Controls were matched to the cases on ethnicity and were from the same geographic locations as the prostate cancer cases. None of the controls had any personal history of cancer at the time of blood donation. This was ascertained by a questionnaire completed by each control. Exact age information was not available for 205 controls since it was not collected prospectively but the age range (20 to 40) was known. All personal identifiers were permanently removed from both cases and controls. DNA was extracted by standard technique using the Qiagen DNA extraction kit. The PCR primers used were 5'-CCA CAG GCG GTG GCG GCG CGA TG-3' in the forward direction and 5'-CGT CGC CCC CGG GAF CAG CGC CGC-3' in the reverse direction. A standard solution was prepared using the Clontech Advantage ® GC rich kit (BD-Biosciences Clontech, Palo Alto, CA). The PCR reaction mixture included 20 ng of genomic DNA in a 10-µL reaction volume and the following concentration of other reagents: primers (0.25 µM each), 1X GC genomic PCR reaction buffer, 1.625 mM Mg 2+ , 0.2 mM dNTPs and 0.16 µL of Advantage-GC genomic polymerase mix. Polymerase chain reaction cycling conditions consisted of an initial denaturation period of 3 minutes at 94°C, then 35 cycles of denaturation for 30 seconds at 94°C and annealing/extension for 2 minutes at 72°C, followed by a final extension step of 5 minutes at 72°C. Quality controls were run on a 2% agarose gel. The ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) was used for data acquisition. A peak at 115 base pairs corresponded to TGFBR1 allele, whereas a peak at 107 base pairs corresponded to the TGFBR1 6A variant. The rare equivocal results were confirmed by cloning of the PCR product followed by automated sequencing. Samples were read by two independent investigators unaware of the case /control status. Ten percent of samples were randomly selected and run for quality assurance. Concordance rate was 100%. Statistical analysis Distributions of TGFBR1 genotypes, age, and ethnicity were compared between cases and controls using Fisher's exact tests. To test the hypothesis that the hypomorphic TGFBR1 6A gene is related to an increased prostate cancer risk, adjusted odds ratios of prostate cancer were estimated using both conditional and unconditional logistic regression models. Both models were run since the matched controls of cases with missing genotypes had to be excluded in the conditional models but could be included in the unconditional models. Adjusted odds ratios of prostate cancer were estimated comparing carriers of TGFBR1 6A versus non-carriers under dominant models. Potential confounders such as age (in four strata) and ethnicity were controlled in the analysis. Whether the effects of TGFBR1 6A on prostate cancer differ by age was evaluated by stratified analysis and tests for multiplicative interaction. A small p value indicates that interaction of age and gene is statistically significant on the multiplicative level. For the unconditional models, sensitivity analysis was conducted to evaluate the impact of the fact that the exact age of some controls with age 20–40 years is unknown (N = 126). With 442 cases and 465 controls, the power to detect an OR of 1.7 and 2 in the present study was 0.86 and 0.98, respectively, based on a two-tailed test at the 0.05 significance level. Authors' contributions All authors made substantial contributions to this paper, including conceiving of the ideas, discussion and writing. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC521683.xml
314477	RNAi Therapeutics: How Likely, How Soon?	RNA interference (RNAi) is used in the lab to silence virtually any gene. Previously, antisense and ribozymes were successful in the lab, but have been disappointing in the clinic. Will RNAi succeed where these two have not?	RNA interference (RNAi) has been called “one of the most has exciting discoveries in biology in the last couple decades,” and since it was first recognized by Andrew Fire et al. in 1998, it has quickly become one of the most powerful and indispensable tools in the molecular biologist's toolkit. Using short double-stranded RNA (dsRNA) molecules, RNAi can selectively silence essentially any gene in the genome. It is an ancient mechanism of gene regulation, found in eukaryotes as diverse as yeast and mammals, and probably plays a central role in controlling gene expression in all eukaryotes. In the lab, RNAi is routinely used to reveal the genetic secrets of development, intracellular signaling, cancer, infection, and a full range of other phenomena. But can the phenomenon hailed by the journal Science as the “Breakthrough of the Year” in 2002 break out of the lab and lead to novel therapies as well? Pharmaceutical giants are hoping so, and several biotech companies have bet their futures on it, but not everyone is sanguine about the future of RNAi therapy. At the heart of its promise as a powerful therapeutic drug lies the exquisite selectivity of RNAi: like the fabled “magic bullet,” an RNAi sequence seeks out and destroys its target without affecting other genes. The clinical applications appear endless: any gene whose expression contributes to disease is a potential target, from viral genes to oncogenes to genes responsible for heart disease, Alzheimer's disease, diabetes, and more. But for all its promise, RNAi therapy is a long way from entering the clinic. While it is a proven wunderkind in the lab, to date no tests have been done in humans, and only the most modest and circumscribed successes have been demonstrated in animals. The road to clinical success is littered with great ideas that have come a cropper along the way, including two other RNA-based therapies, antisense and ribozymes, both of which showed promise at the bench but have largely stumbled before reaching the bedside. Is RNAi also likely to fall short? Or is it different enough to make this third try the charm? Clinical Naïveté, Mysterious Mechanisms To be a successful drug, a molecule must overcome a long set of hurdles. First, it must be able to be manufactured at reasonable cost and administered safely and conveniently. Then, and even more importantly, it must be stable enough to reach its target cells before it is degraded or excreted; it must get into those cells, link up with its intracellular target, and exert its effect; and it must exert enough of an effect to improve the health of the person taking it. And, finally, it must do all this without causing significant toxic effects in either target or nontarget tissues. No matter how good a compound looks in the lab, if it fails to clear any one of these hurdles, it is useless as a drug. For RNA-based therapies, manufacture has been seen as a soluble problem, while delivery, stability, and potency have been the most significant obstacles. “There was a lot of clinical naïveté” in the early days of antisense and ribozymes, according to Nassim Usman, Vice President for Research and Development at Sirna Therapeutics in Boulder, Colorado. “Compounds were pushed into the clinic prematurely.” Sirna began as the biotech startup Ribozyme Pharmaceuticals, which tried to develop ribozymes to treat several conditions, including hepatitis C. A ribozyme is an RNA molecule whose sequence and structure allow it to cleave specific target RNA molecules (see Figure 1 ). “The initial results with hepatitis C were not that inspiring,” says Usman, because the molecule they used had low potency and a short half-life once in the body. Despite “enormous doses,” the viral load was not significantly affected. “It just didn't have the characteristics to be a drug,” he says. No ribozyme has yet been approved for use by the United States Food and Drug Administration (FDA). Figure 1 Ribozymes A ribozyme binds to a specific mRNA, cleaves it, and thus prevents it from functioning. Similarly, despite much initial enthusiasm, attempts to develop antisense drugs have been largely disappointing. Antisense is a single strand of RNA or DNA, complementary to a target messenger RNA (mRNA) sequence; by pairing up with it, the antisense strand prevents translation of the mRNA (see Figure 2 ). At least that was the theory, and early clinical results seemed to support the theory: antisense drugs effectively reduced tumor sizes in anticancer trials and viral loads in antiviral trials. But closer inspection revealed these results were largely due to an increase in production of interferons by the immune system in response to high doses of the foreign RNA, rather than to specific silencing of any target genes. (The relatively high proportion of C–G sequences in antisense mimics bacterial and viral genes, thus triggering the immune response.) When the antisense dose was lowered to prevent the interferon response, the clinical benefit largely disappeared as well. Thus, rather than being a highly specific therapy, antisense seemed to be a general immune system booster. Figure 2 Antisense Antisense DNA or RNA binds to a specific mRNA and prevents it from being translated into protein. But as long as patients were getting better, does it matter what the mechanism was? “It doesn't matter if you are a patient, but it does matter if you are trying to develop the next drug,” says Cy Stein, Associate Professor of Medicine and Pharmacology at Columbia University College of Physicians and Surgeons in New York City. Stein has researched antisense for more than a decade. “If you get the mechanism wrong, you're not going to be able to do it.” To date, only one antisense drug has received FDA approval. Vitravene, from Isis Pharmaceuticals in Carlsbad, California, is used to treat cytomegalovirus infections in the eye for patients with HIV. Vitravene is actually a DNA antisense drug, which binds to viral DNA, though, says Usman, “it's unclear whether it actually works by an antisense mechanism.” Stein expresses a similar skepticism about the mechanism of a second antisense drug, Genasense. Genasense is a DNA-based treatment that targets Bcl-2, a protein expressed in high levels in cancer cells, which is thought to protect them from standard chemotherapy. The FDA is currently reviewing an application for Genasense, based on promising results in the treatment of malignant melanoma. RNAi: A Natural Alternative Growing disillusionment with antisense and ribozymes coincided with the discovery of RNAi and the realization that it was a far more potent way to silence gene expression. RNAi uses short dsRNA molecules whose sequence matches that of the gene of interest. Once in a cell, a dsRNA molecule is cleaved into segments approximately 22 nucleotides long, called short interfering RNAs (siRNAs) (see Figure 3 ). siRNAs become bound to the RNA-induced silencing complex (RISC), which then also binds any matching mRNA sequence. Once this occurs, the mRNA is degraded, effectively silencing the gene from which it came. (Details of the structure and function of the RISC are still largely unknown, but it is thought to act as a true enzyme complex, requiring only one or several siRNA molecules to degrade many times that number of matching mRNAs.) Figure 3 RNAi RNAi is a recently described naturally occurring process in which small RNA molecules activate a cellular process that results in the destruction of a specific mRNA. The extraordinary selectivity of RNAi, combined with its potency—in theory, only a few dsRNAs are needed per cell—quickly made it the tool of choice for functional genomics (determining what a gene product does and with what other products it interacts) and for drug target discovery and validation. By “knocking down” a gene with RNAi and determining how a cell responds, a researcher can, in the course of only a few days, develop significant insight into the function of the gene and determine whether reducing its expression is likely to be therapeutically useful. But does RNAi have a better chance to succeed as a drug than antisense or ribozymes? “The fundamental difference favoring RNAi is that we're harnessing an endogenous, natural pathway,” says Nagesh Mahanthappa, Director of Corporate Development at Alnylam Pharmaceuticals in Cambridge, Massachusetts, the second of two major biotech company developing RNAi-based therapy. The exploitation of a pre-existing mechanism, he says, is the main reason RNAi is orders of magnitude more potent than either of the other two types of RNA strategies. Delivery, Delivery, Delivery More potent in the test tube, at least. But stability and delivery are also the major obstacles to successful RNAi therapy, obstacles that are intrinsic to the biochemical nature of RNA itself, as well as the body's defenses against infection with foreign nucleotides. “For the strongest reasons, you can't get away from this,” says Stein. “The problem is that a charged oligonucleotide will not pass through a lipid layer,” which it must do in order to enter a cell. John Rossi, Director of the Department of Molecular Biology at City of Hope Hospital in Duarte, California, who has worked on RNA-based therapies for 15 years, concurs. “The cell doesn't want to take up RNA,” he says, which makes evolutionary sense, since extracellular RNA usually signifies a viral infection. Injected into the bloodstream, unmodified RNA is rapidly excreted by the kidneys or degraded by enzymes. To solve the problem of cell penetration, most researchers have either complexed the RNA with a lipid or modified the RNA's phosphate backbone to minimize its charge. Mahanthappa thinks the complexing approach is unlikely to be a simple solution, since drug approval would require independent testing of the lipid delivery system as well. Instead, Alnylam is pursuing backbone modification. “Some minimal modification is going to be necessary” to increase cell uptake and to improve stability in the blood stream, Mahanthappa says. “What we have learned from the antisense field is that even without other delivery strategies, when you administer RNA at sufficient doses, if it's stable, it gets taken up by cells.” “Anything that can be done to increase half-life in circulation would improve delivery,” says Judy Lieberman, a Senior Investigator at the Center for Blood Research and Associate Professor of Pediatrics at Harvard Medical School in Cambridge, Massachusetts. But that may not be the only problem, she cautions. Lieberman's lab recently demonstrated the ability of RNAi to silence expression of the Fas gene in mice, protecting them from fulminant hepatitis. Fas triggers apoptosis, or programmed cell death, in response to a variety of cell insults. In her experiment, Lieberman delivered the RNA by high-pressure injection into the tail. The RNA got to the liver, silenced Fas, and protected the mice from hepatitis. However, a significant fraction of animals died of heart failure, brought on because the injection volume was about 20% of the mouse blood volume. Such a delivery scheme simply will not work in humans. “Delivery to the cell is still an obstacle,” Lieberman explains. “Unless you really focus on how to solve that problem, you're not going to get very far.” Unanswered Questions Even assuming delivery problems can be solved, other questions remain, including that of whether therapeutic levels of RNAi may be toxic. Mahanthappa says, “The conservative answer is we just don't know. The more aggressive answer is there's no reason to think so.” Rossi isn't so sure. “The target of interest may be in normal cells as well as cancer cells,” he says. “That's where you get toxicity.” But if small RNAs can be delivered to target cells efficiently and without significant toxicity, will they be effective medicines? Usman of Sirna is confident they will be. “If you can get it there, and if it's in one piece, there no doubt in our minds that it will work,” he says. To date, numerous experiments in animal models suggest RNAi can downregulate a variety of target genes effectively. However, there are still two unanswered questions about whether that will translate into effective therapy. The first is whether RNAi's exquisite specificity is really an advantage beyond the bench. “It's unclear whether highly specific drugs give you a big therapeutic effect,” says Cy Stein. For instance, he says, “most active antitumor medicines have multiple mechanisms of action. The more specific you make it, the less robust the therapeutic activity is likely to be.” Rossi agrees: “Overspecificity has never worked,” he says. The second question is what effect an excess of RNA from outside the cell will have on the normal function of the RISC, the complex at the heart of the RNAi mechanism. The number of RISCs in the cell is unknown, and one concern is that the amount of RNA needed to have a therapeutic effect may occupy all the available complexes. “We are usurping a natural cell system that is there for some other purpose, for knocking out endogenous gene function,” says Rossi. With the introduction of foreign RNA, will the system continue to perform its normal function as well, or will it become saturated? “That's the big black box in the field,” he says. Looking Ahead to the Clinic Despite the questions and unsolved problems, Sirna, Alnylam, and several other companies are moving ahead to develop RNAi therapy; indeed, some outstanding questions are probably only likely to be answered in the process of therapeutic development. The first applications are likely to be in cancer (targeting out-of-control oncogenes) or viral infection (targeting viral genes). To avoid some of the problems of delivery, initial trials may deliver the RNA by direct injection into the target tissue (for a tumor, for instance) or ex vivo, treating white blood cells infected with HIV, for example. Having spent a decade trying to develop ribozymes, says Usman, Sirna is prepared for the rough road it faces. “We haven't solved all the problems, but we know how to proceed to work through them. It's no surprise to us—we've seen this movie before.” Usman expects Sirna to file an Investigational New Drug Application with the FDA by the end of 2004 and to have a human clinical trial in progress in 2005. “Without a doubt, there will be an RNAi-based drug within ten years,” Usman predicts. Stein isn't so sure and thinks that too much is still to be learned about RNAi and the body's reaction to it to be confident that RNA-based therapies will ultimately be successful. “The whole field was founded on the belief it was rational, but there are huge gaps in our knowledge, and so you need a bit of luck to be successful,” he says. “I think people are surprised at how complicated it is, but why should it be any other way? It's an intellectual conceit to think that nature is simple.”	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC314477.xml
439782	Synaptotagmin VII Restricts Fusion Pore Expansion during Lysosomal Exocytosis	Synaptotagmin is considered a calcium-dependent trigger for regulated exocytosis. We examined the role of synaptotagmin VII (Syt VII) in the calcium-dependent exocytosis of individual lysosomes in wild-type ( WT ) and Syt VII knockout ( KO ) mouse embryonic fibroblasts (MEFs) using total internal reflection fluorescence microscopy. In WT MEFs, most lysosomes only partially released their contents, their membrane proteins did not diffuse into the plasma membrane, and inner diameters of their fusion pores were smaller than 30 nm. In Syt VII KO MEFs, not only was lysosomal exocytosis triggered by calcium, but all of these restrictions on fusion were also removed. These observations indicate that Syt VII does not function as the calcium-dependent trigger for lysosomal exocytosis. Instead, it restricts the kinetics and extent of calcium-dependent lysosomal fusion.	Introduction Exocytosis allows cells to transport membrane-impermeable macromolecules outside without compromising the integrity of the plasma membrane. The proteins that form the conserved machinery for constitutive and regulated exocytosis have been identified ( Sollner and Rothman 1996 ), and calcium has been identified as the most common trigger for regulated exocytosis ( Burgoyne and Morgan 1998 ; Jaiswal 2001 ). However, there is not yet a consensus on the calcium-responsive components involved in this process. It has been suggested that multiple Ca 2+ -binding proteins with distinct properties could act as the trigger for membrane fusion ( Burgoyne and Morgan 1998 ). Evidence supporting the role of synaptotagmin I (Syt I) as the Ca 2+ -dependent trigger for synaptic vesicle fusion in several organisms has led to the belief that the members of the synaptotagmin family act as ubiquitous calcium-dependent triggers for exocytosis ( Brose et al. 1992 ; Geppert et al. 1994 ; Littleton and Bellen 1995 ). While Syt I is the most well-studied member of this family, there are at least 15 different synaptotagmin isoforms with differing affinities for calcium and phospholipid and different cellular localization ( Chapman 2002 ; Fukuda 2003 ). Some members of synaptotagmin family (including Syt I) have also been found to regulate endocytosis and even negatively regulate Ca 2+ -dependent exocytosis ( Jorgensen et al. 1995 ; Martin et al. 1995 ; Morimoto et al. 1995 ; Baram et al. 1999 ; Tucker and Chapman 2002 ). Thus, the role of synaptotagmin family members as Ca 2+ -dependent triggers for exocytosis is still an open question. We have previously identified that in nonprofessional secretory cells calcium preferentially triggers exocytosis of lysosomes ( Jaiswal et al. 2002 ). A variety of agents that result in calcium increase, including membrane damage, trypanosome invasion , calcium ionophores, or the IP3 agonists thrombin or bombesin, trigger lysosomal exocytosis ( Rodriguez et al. 1997 ; Caler et al. 2000 , 2001 ; Ayala et al. 2001 ; Reddy et al. 2001 ; Jaiswal et al. 2002 ). However, the molecular machinery that regulates this calcium-triggered lysosomal exocytosis has remained elusive. Syt VII is the synaptotagmin isoform present on lysosomes ( Martinez et al. 2000 ). It is expressed in most tissues and is present in organisms ranging from nematodes to humans ( Fukuda et al. 2002 ). Syt VII is involved in processes requiring lysosomal exocytosis, namely, release of lysosomal enzymes, repair of membrane rupture, and trypanosome invasion ( Martinez et al. 2000 ; Caler et al. 2001 ; Reddy et al. 2001 ). Further, the recent demonstration that cells from Syt VII knockout ( KO ) mice are compromised in these functions supports a role of Syt VII in regulating lysosomal exocytosis ( Chakrabarti et al. 2003 ). To understand how Syt VII regulates lysosomal fusion, we used total internal reflection fluorescence microscopy (TIR-FM) and studied the behavior of individual lysosomes following calcium increase in mouse embryonic fibroblasts (MEFs) from wild-type ( WT ) and Syt VII KO mice. Results To monitor the fate of exocytic lysosomes in MEFs, we labeled their lumen using fluorescent dextran (FITC–dextran). Treating MEFs with calcium ionophore A23187 or the IP3 agonist bombesin or thrombin caused lysosomal exocytosis ( Figure 1 A and 1 B). Fusion of a FITC–dextran-loaded lysosome was indicated by a transient increase followed by a decrease in its fluorescence ( Figure 1 A– 1 C). The increase in fluorescence was due to a combination of two factors: (a) movement of the lysosome closer to the coverslip, which results in better excitation of its cargo by the evanescent wave; (b) opening of the fusion pore, which results in dissipation of the acidic pH of the lysosomes, resulting in dequenching of the fluorescence of FITC–dextran. The rapid decrease in fluorescence was due to the diffusion of lumenal cargo away from the site of fusion ( Figure 1 A– 1 C). In some of the exocytosing lysosomes, the lumenal fluorescence decreased down to baseline, indicating that they completely released their lumenal cargo ( Figure 1 A). The fluorescence of other lysosomes did not decrease down to baseline at the site of fusion ( Figure 1 B). Thus, these lysosomes only partially released their contents upon fusion. To resolve whether partial release represented a very slow diffusion of lumenal content or an opening of the fusion pore that was transient, we observed the lysosomes for longer periods. During partial release, the lumenal fluorescence decreased rapidly within the first second ( Figure 1 B), but remained relatively constant afterwards, decreasing only at the rate of photobleaching ( t 1/2 for FITC in our setup is 28.5 s). Absence of any subsequent decrease in its fluorescence, even over longer periods, indicated that cessation of release was the result of closure of the fusion pore prior to complete release of the lumenal cargo. Quantitation of the lumenal contents retained in all exocytosed lysosomes analyzed in the WT MEFs revealed that only 21% completely released their lumenal content ( Figure 1 D, gray bar). The percentage of lysosomes in individual WT MEFs that only partially released their lumenal cargo of 70 kDa dextran in response to A23187-induced increase in cellular Ca 2+ was 65.3% ( n = 7 cells) ( Figure 1 E, black bars; Table 1 ; Video S1 ). A comparable fraction of lysosomes, respectively, underwent partial release when calcium increase was triggered by the IP3 agonists thrombin (66.3) or bombesin (69.5), ( Figure 1 E; Table 1 ). Figure 1 Fate of Lumenal Content during Lysosomal Fusion MEFs were incubated for 2 h with 70 kDa FITC–dextran followed by more than 3 h in dextran-free media to chase the dextrans into the lysosomes. These cells were then treated with calcium ionophore (A23187) to trigger exocytosis of lysosomes. (A and B) The middle panels are images of lyosomes undergoing complete (A) and partial (B) exocytosis. Intensity plots for the regions in these images marked by dotted circles are shown in the lower panel. The top panel shows a schematic representation of these different stages. (C) Schematic fluorescence intensity plots for lysosomes undergoing partial (red) or complete (green) fusion. Owing to the exponential decay of the evanescent field (blue; top panels in [A] and [B]) away from the coverslip, a lysosome that is more than 150 nm from the cell membrane (black line in top panels in [A] and [B]) is not fluorescent. As this lysosome moves closer (labeled as “entry into evanescent field”), its fluorescence intensity increases. Since the lumen of lysosome is acidic, it quenches FITC fluorescence. As soon as the fusion pore is formed, the lysosomal lumen is rapidly alkalinized resulting in an increase of FITC–dextran fluorescence (“pore opening”). Following the pore opening, the dextran is released and it diffuses away from the site of the fusion, causing the lumenal fluorescence to decrease (“release”). (D) A histogram of the fraction of lumenal contents released by exocytosing lysosomes. Upon ionophore-triggered fusion, 21% of all lysosomes analyzed in WT MEFs ( n = 47; gray bars) and 45% of all in Syt VII KO MEFs ( n = 51; white bars) completely released their lumenal content. (E and F) To monitor the nature of lysosomal fusion in individual WT MEFs (E) and Syt VII KO MEFs (F), calcium was increased using ionophore ( WT , n = 7 cells; KO , n = 9 cells) as well as the IP3 agonists bombesin ( WT , n = 6 cells; KO , n = 9 cells) and thrombin ( WT , n = 5 cells; KO , n = 7 cells). Irrespective of the means, increase in calcium led to most lysosomes to fuse partially in WT MEFs (E) and completely in Syt VII KO MEFs (F). The error bars represent SEM. Table 1 Nature of Release of Lysosomal Lumenal Content Lysosomes in MEFs were labeled with 70 kDa FITC–dextran as described in Figure 1. Increase in cytosolic calcium in response to treatment with calcium ionophore or with the IP3 agonist bombesin or thrombin led to either partial or complete release of 70 kDa FITC–dextran. The table describes the fraction of lysosomes that showed these in a few WT and SytVII KO MEFs Found at In order to determine the fate of the membrane proteins during Ca 2+ -triggered lysosomal exocytosis, we simultaneously imaged the lysosomal lumenal (TRITC–dextran) and membrane (CD63–GFP) cargoes in WT MEFs. We observed that the membrane proteins were delivered to the plasma membrane during complete release of lumenal content ( Figure 2 A), but not when the lumenal contents were released partially ( Figure 2 B). Even when the lysosomal membrane proteins were delivered to the plasma membrane, their diffusion into plasma membrane was restricted ( Figure 2 A and 2 C; Table 2 ). This is unlike the fate of membrane proteins during exocytosis of biosynthetic vesicles ( Schmoranzer et al. 2000 ; Kreitzer et al. 2003 ; Schmoranzer and Simon 2003 ) or recycling endosomes ( Lampson et al. 2001 ), where following its delivery to plasma membrane, the vesicular membrane protein diffuses away completely from the site of fusion. Thus, exocytosis of lysosomes in the WT MEF is different from other exocytic events in two manners. First, the majority of exocytic lysosomes only partially release their lumenal cargo with no release of membrane proteins. Second, even when the lumenal cargo is completely released, the membrane proteins delivered to the plasma membrane do not diffuse freely, but are retained into punctae at the site of fusion. Figure 2 Fate of Membrane Protein during Lysosomal Fusion Lysosomal membranes in MEFs were labeled by transfecting cells with a vector encoding a CD63–GFP fusion protein, and expression was allowed for 48 h. For simultaneous labeling of lysosomal membrane and lumen, the CD63–GFP transfected cells were labeled with 70 kDa TRITC–dextran as described in Figure 1 . (A) Following ionophore-induced calcium increase in WT MEFs, when the TRITC–dextran was released completely (left), CD63–GFP (right) was delivered to the plasma membrane, but it remained in multiple puncta near the site of fusion rather than diffuse away. The panels are pseudocolor surface plots, with the x and y axis representing the coordinates and the z axis representing the fluorescence intensity of individual pixels. (B) In the event of partial release of TRITC–dextran (top row), the CD63–GFP (bottom row) did not appear to be delivered to the plasma membrane. The lower panel shows the plot of fluorescent intensity of lumenal and membrane label (within the dotted circle) of the lysosome shown in (B). (C and D) Analysis of CD63–GFP-labeled lysosomes in WT MEFs (C) and in Syt VII KO MEFs (D) indicates that while CD63–GFP is retained in puncta in the WT MEFs, it diffuses freely in the plasma membrane in the SytVII KO MEFs. The lower panel shows the total and peak intensity plots of CD63–GFP-labeled lysosome in (D). Table 2 Nature of Release of Lysosomal Membrane Protein Lysosomal membranes in MEFs were labeled with CD63-GFP as described in Figure 2 . When ionophore-induced lysosomal exocytosis resulted in delivery of CD63-GFP to plasma membrane, it either diffused away from the site of fusion or remained trapped into punctae near the site of fusion (see Figure 4B and 4D ). The table describes the fraction of lysosomes that showed these behaviors in individual cells in WT and SytVII KO MEFs Found at To test the role of Syt VII in regulating these processes and acting as a calcium-dependent trigger for lysosomal exocytosis, we carried out a similar analysis using embryonic fibroblasts from mice deficient in Syt VII (Syt VII KO MEF) ( Chakrabarti et al. 2003 ). Absence of Syt VII did not abolish calcium-dependent triggering of lysosomal fusion in response to ionophore, thrombin, or bombesin (see Figure 1 D and 1 F; Video S2 ). Upon examining individual exocytic lysosomes, we observed two significant differences between the WT and Syt VII KO MEFs. First, in individual Syt VII KO MEFs, a significantly greater (2-fold) fraction of lysosomes completely released their lumenal contents when the cellular calcium was raised using ionophore ( p = 0.001) ( Video S2 ), thrombin ( p = 0.001), or bombesin ( p = 0.003) (see Figure 1 F; see Table 1 ). Second, upon complete fusion, the membrane protein of most exocytic lysosomes in Syt VII KO MEFs diffused freely in the plane of plasma membrane (see Figure 2 D; see Table 2 ). These phenotypes indicated that the presence of Syt VII restricts complete fusion of lysosomes. To identify whether Syt VII restricts lysosomal fusion by preventing its flattening into the plasma membrane or by regulating the size of the fusion pore, we quantified the simultaneous release of dextrans of different sizes in individual lysosomes (10 kDa [Stokes radius = 2.4 nm], 70 kDa [5.8 nm], 145 kDa [8 nm], 250 kDa [10.5 nm], and 500 kDa [14.7 nm]). Lysosomes were loaded with dextrans of two different sizes, each tagged with a different fluorophore (FITC or TRITC). In WT MEFs, all the lysosomes that released the TRITC–dextran of 10 or 70 kDa also released similarly sized FITC–dextran ( Figure 3 A and 3 B). Thus, the fluorophore did not appear to affect release of the dextran. The fluorophore also had no affect on the lysosomal uptake of the dextran, as every lysosome that had the TRITC-labeled dextran also had the FITC-labeled dextran (data not shown). Further, just prior to fusion, the fluorescence of the TRITC cargo and the FITC cargo in each lysosome started to increase at the same moment ( Figure 3 ). This increase is the result of the movement of the lysosome to the plasma membrane just prior to fusion, which increases the excitation of the fluorophores (see Figure 1 A– 1 C). Thus, the TRITC and FITC cargos were not only spatially and temporally coincident in the plane of the plasma membrane, but also coincident in the plane perpendicular to the plasma membrane even in a motile lysosome. These observations rule out the possibility that the two fluorophores are present in separate lysosomes. Figure 3 Presence of Syt VII Restricts the Size of the Fusion Pore The lumen of lysosomes was loaded simultaneously using different-sized FITC- and TRITC-labeled dextran, using the approach described in Figures 1 and 2 . Representative plots shown here demonstrate the fate of both dextran populations in individual lysosomes following the increase in intracellular calcium by addition of calcium ionophore. In WT MEFs, exocytosing lysosomes that released 70 kDa TRITC–dextran also simultaneously released 70 kDa FITC–dextran (A and B), 250 kDa FITC–dextran (C and D), but not 500 kDa FITC–dextran (E and F). In Syt VII KO MEFs, lysosomes that released 70 kDa TRITC–dextran also released 500 kDa FITC–dextran (G and H). In all cases the plots represent the normalized fluorescence intensity of the region marked in the images by dotted circles. In WT MEFs, each lysosome that partially released the 10 or 70 kDa TRITC–dextran at the same time also partially released the 145 kDa (data not shown) and 250 kDa FITC–dextran ( Figure 3 C and 3 D). However, when the lysosome was simultaneously loaded with 500 kDa FITC–dextran and with 70 kDa TRITC–dextran, the smaller cargo was released and the larger cargo was not ( Figure 3 E and 3 F). This suggests the fusion pore opened large enough to release the smaller but not the larger cargo ( Figure 4 ). Furthermore, at the moment of release of the TRITC cargo, the fluorescence from the 500 kDa FITC–dextran increased to a significantly greater amount. As the FITC fluorescence is partially quenched by the acidic lumen of the lysosomes, this provided additional evidence in favor of the conclusion that the lysosome formed a fusion pore, without releasing the 500 kDa dextran. The lack of subsequent decrease in the fluorescence of the 500 kDa FITC–dextran, when the 70 kDa TRITC–dextran fluorescence decreases, indicates that the radius of the fusion pore is smaller than the size of the 500 kDa dextran (Stokes radius = 14.7 nm; diameter, 29.4 nm). We repeated these studies in Syt VII KO MEFs and found that in these cells all exocytosing lysosomes were able to fully release dextrans of all sizes, including the 500 kDa dextran (see Figure 3 G and 3 H). This suggests that the presence of Syt VII blocks complete release of lysosomal contents during exocytosis, potentially by restricting the size of the exocytic pore. Figure 4 Schematic Representation of the Fate of Lumenal and Membrane Cargo during Lysosomal Exocytosis (A) Partial fusion of the lysosomes from WT MEFs result in the release of a fraction of the smaller (70–250 kDa; red circles) dextran, but no release of the larger (500 kDa; blue circles) dextran. None of the lysosomal membrane protein (green bars) is delivered to the plasma membrane. (B) Complete fusion leads to release of both the large and the small dextrans and delivery of the membrane proteins to the plasma membrane, but the proteins aggregate in small puncta near the site of fusion. (C) Knockout of Syt VII causes larger-sized dextran to be released even during partial fusion, and the membrane protein is still not delivered to the plasma membrane. (D) During complete fusion, both sized dextrans are released completely and the membrane proteins delivered to plasma membrane are free to diffuse away from the site of fusion. Since cargo of any size would be released more rapidly through a larger pore, we independently assayed for the size of the fusion pore by measuring the time taken by exocytosing lysosomes to release their lumenal cargo. Increase in FITC–dextran fluorescence was taken as the indicator for the time of opening of the fusion pore (see Figure 1 C). We measured the time taken for the lumenal fluorescence of individual exocytic lysosomes (fusing partially or completely) to decrease to the post-fusion resting value in WT and Syt VII KO MEF (see Figure 1 C). For most lysosomes (greater than 81%) in WT MEFs, it took longer than 0.75 s for the fluorescence of the lumenal cargo to reach their post-fusion resting value (gray bars in Figure 5 A). In contrast, in Syt VII KO MEFs for most lysosomes (greater than 75%), this occurred in less than 0.75 s (white bars in Figure 5 A). The increased propensity of lysosomes to rapidly release their lumenal content was also observed when Syt VII KO cells were treated with bombesin or thrombin ( Figure 5 B). This suggests that in the absence of Syt VII, the fusion pore either opens faster, opens to a larger size, or both. While we cannot distinguish among these possibilities, they are all consistent with Syt VII restricting the expansion of the fusion pore. Figure 5 Temporal Analysis of Lysosomal Exocytosis and Fusion Pore Opening Using 70 kDa FITC–Dextran as Lumenal Marker (A) Histogram of the release time (time taken for the vesicular fluorescence to drop from peak to postfusion resting value). Release time was less than 0.75 s for more than two-thirds of lysosomes in Syt VII KO MEF ( n = 62 lysosomes), while most lysosomes (81%) in WT MEFs ( n = 56 lysosomes) released their lumenal content for more than 0.75 s. (B) Analysis of release time of lysosomes using ionophore, bombesin, and thrombin to trigger lysosomal exocytosis. Irrespective of the means of calcium increase, lysosomes in Syt VII KO MEFs released their lumenal content significantly faster ( p = 0.002, 0.01, and 0.03, respectively). (C) Histogram of the number of lysosomes exocytosing as a function of the time following calcium ionophore addition. Fluorescent dextran was used as a lumenal marker; the time axis indicates seconds elapsed following the addition of ionophore. Exocytosis initiated earlier in the Syt VII KO MEFs (white bars; n = 8 cells) compared to WT MEFs (gray bars; n = 6 cells). (D) No change was observed in the total number of lysosomes that exocytosed at the basal surface of WT or Syt VII KO MEFs when calcium was raised using ionophore or thrombin; however, compared to WT MEFs, bombesin triggered exocytosis of half as many lysosomes in the Syt VII KO MEFs (asterix represents p value < 0.02). The error bars represent SEM. To further explore potential consequences of the presence of Syt VII on lysosomal secretion, we analyzed the effect of Syt VII knockout on the time course of initiation of lysosomal exocytosis. Similar to what we observed previously with CHO cells ( Jaiswal et al. 2002 ), in WT MEFs, lysosomal exocytosis was initiated approximately 35 s after the addition of ionophore and peaked by 110 s (see Video S1 ; gray bars in Figure 5 C). In contrast, in Syt VII KO MEFs, the earliest lysosomal fusion event was observed within 7 s after the addition of ionophore, and it peaked within 40 s (see Video S2 ; white bars in Figure 5 C). As the time delay for IP3 agonist-induced calcium release varied significantly among the cells within a dish, we could not determine whether these agents have a similar affect on the latency of calcium-triggered lysosomal fusion in Syt VII KO MEFs. While all the behaviors of individual lysosomes described above were independent of the agent used to trigger calcium increase, the bulk cellular behavior, such as the total number of lysosomal fusion events that occur at the basal surface of the cell, was dependent on the agent used to increase the cellular calcium level ( Figure 5 D). While ionophore or thrombin triggered exocytosis of a similar number of lysosomes ( p = 0.46 and 0.51, respectively) in both WT and KO MEFs, there was a 2-fold decrease ( p = 0.02) when bombesin was used to raise cellular calcium in KO MEFs. Discussion It has been shown that biosynthetic vesicles, secretory granules, and synaptic vesicles can undergo partial release ( Holroyd et al. 2002 ; Aravanis et al. 2003 ; Gandhi and Stevens 2003 ; Schmoranzer and Simon 2003 ; Taraska et al. 2003 ). However, our analysis of calcium-triggered fusion of individual lysosomes has revealed several unique features of this process. In WT MEFs, partial fusion is the predominant mode of lysosomal exocytosis. Unlike partial fusion of secretory granules in PC12 cells, which results in incomplete release of the large lumenal cargo ( Holroyd et al. 2002 ; Taraska et al. 2003 ), partially exocytosing lysosomes do not release any of the large (500 kDa) lumenal cargo. Similarly, during partial release of lumenal contents, none of the lysosomal membrane protein diffuses into the plasma membrane. The complete release of lysosomal cargo is also unlike the complete release of biosynthetic vesicles, where the membrane proteins of secretory vesicles fully diffuse into the plasma membrane ( Schmoranzer and Simon 2003 ). In constrast, the lysosomal membrane proteins remain as puncta near the site of fusion. These unique features associated with lysosomal exocytosis are dependent on the presence of Syt VII. Absence of Syt VII causes most lysosomes to fuse completely and allows the membrane proteins of completely fusing lysosomes to diffuse freely into the plasma membrane. Moreover, in the presence of a functional Syt VII, opening of the fusion pore is restricted both temporally (the pores open more slowly and remain open for a short period of time) and geometrically (large lumenal cargo cannot leave and membrane proteins cannot diffuse out into the plasma membrane). Thus, our analysis reveals that Syt VII is not critical for calcium-dependent triggering of lysosomal exocytosis. This is consistent with what has been proposed earlier and observed both in vitro and in vivo for Syt I ( Popov and Poo 1993 ; Martin et al. 1995 ; Morimoto et al. 1995 ; Mahal et al. 2002 ). The effects of Syt VII on the kinetics, size, and extent of calcium-triggered exocytosis of lysosomes in WT MEF are also consistent with observations reported for Syt I: the slower dilation of fusion pores caused by overexpression of Syt I ( Wang et al. 2001 ), and a 10-fold increased frequency of asynchronous release in Syt I null cells ( Yoshihara and Littleton 2002 ). Interestingly, unlike the knockout of Syt I, which blocks the fast component of calcium-triggered exocytosis in neuronal cells ( Yoshihara and Littleton 2002 ), knockout of Syt VII did not abolish calcium-triggered exocytosis in the MEFs. This could reflect different roles played by Syt VII (inhibitor for lysosome fusion) and Syt I (calcium-dependent trigger) in calcium-dependent exocytosis. Alternatively, each synaptotagmin may play multiple roles, including being a trigger ( Geppert et al. 1994 ), an inhibitor of asynchronous release ( Yoshihara and Littleton 2002 ), or an inhibitor of fusion pore dilation ( Wang et al. 2001 ). While it remains to be determined whether other members of the synaptotagmin family can act as inhibitors of fusion, this possibility is supported by the recent observation that overexpression of Syt IV causes increased partial release of secretory granules in PC12 cells ( Wang et al. 2003 ). Involvement of Syt VII in premature closure of the fusion pore (leading to partial release) is also consistent with the proposed role of Syt I in facilitating the rapid retrieval of vesicular membrane following exocytosis ( Jorgensen et al. 1995 ). Syt VII function has been shown to be crucial for membrane repair and trypanosome invasion ( Martinez et al. 2000 ; Caler et al. 2001 ; Chakrabarti et al. 2003 ). Cells lacking a functional Syt VII show reduced membrane repair and Trypanosoma cruzi invasion ( Chakrabarti et al. 2003 ). Our observations suggest a few mechanisms by which Syt VII may contribute. Syt VII KO MEFs lose the restricted fusion of lysosomes: unlike the lysosomes of WT MEFs, the membrane-proximal lysosomes in Syt VII KO MEFs fully release their contents and deliver their membrane proteins to the surface. Since membrane of lysosomal origin has been shown to be required for healing membrane rupture and forming parasitophorous vacuoles during trypanosome invasion, it is possible that retaining the lysosomal membrane components at the site of fusion aids in both these processes. Additionally, upon treatment with bombesin, which may recapitulate the calcium signaling occurring during T. cruzi invasion ( Tardieux et al. 1994 ), total lysosomal exocytosis is decreased 2-fold in the Syt VII KO MEF. This effect is reminiscent of the approximately 2-fold decrease in lysosomal enzyme secretion observed by collagen matrix-mediated wounding of Syt VII KO MEFs ( Chakrabarti et al. 2003 ). We have previously observed that calcium predominantly triggers the exocytosis of membrane-proximal lysosomes ( Jaiswal et al. 2002 ). In the Syt VII KO MEFs, these lysosomes also showed decreased propensity for partial release. Thus, it is possible that the membrane-proximal lysosomes play a role in decreased membrane repair and trypanosome invasion observed in the Syt VII KO MEFs ( Chakrabarti et al. 2003 ). Our analysis of Syt VII function not only adds to the roles of synaptotagmin in regulating calcium-triggered exocytosis, but also provides mechanistic clues regarding how lysosomal exocytosis might regulate membrane repair and pathogen invasion. Materials and Methods Cell growth and treatments MEFs were prepared from day 13.5 embryos of WT and Syt VII-deficient mice, expressing functional ( WT MEF) or truncated Syt VII ( KO MEF), as described elsewhere ( Chakrabarti et al. 2003 ). Cells were cultured in DMEM (Cellgro, Mediatech, Washington, District of Columbia, United States) supplemented with 10% FBS (GIBCO Technologies, Carlsbad, California, United States). All experiments were done with cells between passages 1 and 3. For imaging, cells were plated for more than 24 h on sterile glass coverslips (Fisher Scientific, Hampton, New Hampshire, United States). Just before imaging, the medium was replaced with cell imaging medium (CIM) (HBSS plus 10 mM HEPES plus 1% FBS [pH 7.4]). Transient transfection of cells with CD63–GFP ( Blott et al. 2001 ) was carried out using Lipofectamine 2000 (Invitrogen Corporation, Carlsbad, California, United States) 48 h prior to imaging. For calcium ionophore and calcium agonist treatments, growth medium was replaced with CIM, the coverslip was mounted on the microscope stage maintained at 37 °C, and while the cells were being imaged using TIR-FM, agents were added to a final concentration of 10 μM A23187 ionophore, 0.2 U/ml thrombin, or 20 nM bombesin. Calcium ionophore-A23187, thrombin, bombesin, 70 kDa FITC–dextran, and 65 kDa TRITC–dextran were obtained from Sigma (Sigma Chemicals, St. Louis, Missouri, United States). All other fluorescent dextrans were obtained from Molecular Probes (Eugene, Oregon, United States) and used to load lysosomes as previously described ( Jaiswal et al. 2002 ). TIR-FM The illumination and image acquisition using TIR-FM was done as previously described ( Jaiswal et al. 2002 ). For simultaneous dual-color imaging of GFP/TRITC or FITC/TRITC, we used an emission splitter (Dual-View, Optical Insights, Santa Fe, New Mexico, United States). Cells were excited using the 488 nm line of an argon laser, containing the emission band pass filters (GFP/FITC, HQ525/50M; TRITC, HQ580LP). All filters were obtained from Chroma Technologies Corporation (Brattleboro, Vermont, United States). Images were acquired with a 12-bit cooled CCD ORCA-ER (Hamamatsu Photonics, Hamamatsu, Japan) with a resolution of 1280 × 1024 pixels (pixel size = (6.45 μm) 2 ). The camera and mechanical shutters (Uniblitz, Vincent Associates, Rochester, New York, United States) were controlled using MetaMorph (Universal Imaging, Downingtown, Pennsylvania, United States). Images were acquired at 5–10 frames/s. Images containing a region of interest of the cell were streamed to memory on a PC during acquisition and then saved to hard disk. The depth of the evanescent filed for the Apo 60× N.A. 1.45 lens (Olympus Scientific, Melville, New York, United States) was typically approximately 70–120 nm ( Schmoranzer et al. 2000 ). Image processing and quantitative analysis For dual-color video sequences, the images acquired through the emission splitter were separated, subtracted for background fluorescence, aligned within accuracy of a single pixel, and analyzed using MetaMorph. For measuring fluorescence intensity, a region was drawn around the vesicle and the peak and average intensity was measured in this region. The minimum and maximum average intensities were normalized on a scale of 0 to 1. For measurement of total number of exocytic lysosomes in a cell, fusion events were counted starting from the addition of ionophore until the cells start to lift off the coverslip (1–3 min in WT and fewer than 2 min in Syt VII KO MEFs). Supporting Information Video S1 Lysosomal exocytosis in WT MEFs Lysosomes of WT MEFs were loaded with 70 kDa FITC–dextran and the cells were treated with 10 μM calcium ionophore. The video shows a single cell 30 s after ionophore addition. The images were acquired at five frames per second, and alternate frames are displayed in the video at five frames per second. The time stamp indicates the time (mm:ss) elapsed since the start of the video. (6.8 MB AVI). Click here for additional data file. Video S2 Lysosomal exocytosis in Syt VII KO MEFs Lysosomes in Syt VII KO MEFs were loaded with 70 kDa FITC–dextran and the cells were treated with 10 μM calcium ionophore. The video shows a single cell immediately following ionophore addition. The images were acquired at five frames per second, and alternate frames are displayed in the video at five frames per second. The time stamp indicates the time (mm:ss) elapsed since the start of the video. (4.9 MB AVI). Click here for additional data file.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC439782.xml
535806	Treatment decision-making and the form of risk communication: results of a factorial survey	Background Prospective users of preventive therapies often must evaluate complex information about therapeutic risks and benefits. The purpose of this study was to evaluate the effect of relative and absolute risk information on patient decision-making in scenarios typical of health information for patients. Methods Factorial experiments within a telephone survey of the Michigan adult, non-institutionalized, English-speaking population. Average interview lasted 23 minutes. Subjects and sample design: 952 randomly selected adults within a random-digit dial sample of Michigan households. Completion rate was 54.3%. Results When presented hypothetical information regarding additional risks of breast cancer from a medication to prevent a bone disease, respondents reduced their willingness to recommend a female friend take the medication compared to the baseline rate (66.8% = yes). The decrease was significantly greater with relative risk information. Additional benefit information regarding preventing heart disease from the medication increased willingness to recommend the medication to a female friend relative to the baseline scenario, but did not differ between absolute and relative risk formats. When information about both increased risk of breast cancer and reduced risk of heart disease were provided, typical respondents appeared to make rational decisions consistent with Expected Utility Theory, but the information presentation format affected choices. Those 11% – 33% making decisions contrary to the medical indications were more likely to be Hispanic, older, more educated, smokers, and to have children in the home. Conclusions In scenarios typical of health risk information, relative risk information led respondents to make non-normative decisions that were "corrected" when the frame used absolute risk information. This population sample made generally rational decisions when presented with absolute risk information, even in the context of a telephone interview requiring remembering rates given. The lack of effect of gender and race suggests that a standard strategy of presenting absolute risk information may improve patient decision-making.	Background In contemporary U.S. society, prospective users of preventive therapies often must evaluate complex information about therapeutic risks and benefits. While health information is increasingly available, it is frequently incomplete and sometimes inaccurate [ 1 , 2 ]. For people to be active participants in health care decisions, information regarding the benefits and risks of therapies must be presented in ways that support high-quality, effective decision-making [ 3 , 4 ]. Prior research on presentation formats that are most effective, comprehensible, and likely to lead to effective decision-making suggests that a critical element is the format of risk information [ 5 ]. Mazur [ 6 ] suggests that how patients use and react to information are important dimensions for assessing patients' understanding. For example, prior studies have demonstrated the importance of the visual aspects of information presented [ 7 , 8 ] on shaping preferences and use of information. A key format difference is absolute versus relative gain or loss [ 9 , 10 ]. Absolute risk is a rate. It expresses the actual number of people experiencing an outcome, e.g., 1 in 1,000 women. Relative risk is a ratio (risk among exposed divided by the risk among the non-exposed). Thus, in relative risk terms, those who smoke are described as twice as likely as non-smokers to get lung cancer; taking medication X is described as cutting the risk of disease in half. However, relative risks do not reveal the actual rates of occurrence. Doubling a 2% risk is very different in its consequences from doubling a 40% risk. It has been previously demonstrated that relative risk presentation can produce errors. By "errors," we mean that people's perceptions of risk and benefit information does not conform to normative Expected Utility (EU) theory assumptions. Central to EU theory is the assumption that individuals who make "rational" choices should prefer an option with the highest expected utility relative to all options being considered. Prior clinical studies have found that presenting information in absolute versus relative risk formats can affect the interpretation of information about medications and personal risks of developing diseases. For example, in a sample of family practice patients, Hux and Naylor [ 10 ] found that patients were less willing to take a lipid-lowering drug when risks were described in an absolute compared to a relative risk format (42% versus 88%). Likewise, in a relative risk assessment task, Woloshin et al. [ 11 ] found that about two-thirds of a sample of women who were actually at high risk for breast cancer classified themselves as being at less risk than they really were. However, results of clinical studies may or may not be generalizable to the general population. Without cross-sectional samples, it is difficult to develop a general population profile of those most likely not to understand health information, i.e., to not use it "correctly." This study tested the effects of alternative information presentation formats (absolute risk versus relative risk formats) on medication decision-making of adults in the general population. Methods Design Data were collected as a part of a cross-sectional telephone survey of the adult, non-institutionalized resident population of Michigan. The telephone survey was part of a continuing series of quarterly surveys in Michigan known as the State of the State Survey (SOSS) conducted by Michigan State University's Institute for Public Policy and Social Research (IPPSR). 952 interviews were completed within the designated interviewing period. The margin of sampling error for the sample as a whole is ± 3.2%. The average interview lasted approximately 22.8 minutes (median = 22 minutes; standard deviation = 4.8 minutes). A more detailed description of the SOSS methodology, sample design, procedures, and content, is provided in Hembroff and Silver [ 12 ]. Sample The sampling design for the State of the State Surveys is a disproportionate stratified random sample of phone numbers across the state's regions, over-sampling the less populated regions, and the city of Detroit. Trained interviewers called the random-digit dial (RDD) sample of telephone numbers to identify those that were actually working household phone numbers. Within identified households, interviewers interviewed a single adult selected using the "next birthday" technique [ 13 ] in which the adult in the household who would have the next birthday was chosen to be the respondent. The data being reported have been weighted to adjust appropriately for the unequal probabilities of respondent selection based on the number of phone lines to the household and the number of adults within the household. Cases were also weighted to adjust for differential response rates among categories of race, gender, age and to adjust for regional disproportionate sampling. The final weighted data file very closely matches the demographic profile of Michigan's adult population. Procedures and response rate Interviews were conducted using the computer assisted telephone interviewing facility (CATI) of IPPSR's Office for Survey Research (OSR). Interviewing took place over a six week time period during June and July of 1997. The overall completion rate among eligible households was 54.3%. The calculated response rate is based on assigning final outcome dispositions codes according to Standard Definitions, the American Association of Public Opinion Research [ 14 ] guidelines for calculating response rates using Formula RR4. Presenting health chances and patient treatment decision-making The interview contained a split-ballot experiment to examine the effects of alternative ways of communicating risks. All respondents were presented with a baseline scenario. In this case, we asked respondents to ... "Suppose you had a friend who was told that she was very likely to get a bone disease that would gradually make her crippled. Suppose the doctor said that there was a medication she could take on a daily basis that would greatly reduce her chances of getting the bone disease." Respondents were asked if they would recommend that their friend take or not take the medication. Respondents were then randomly assigned to be presented one of four follow-up questions. Each follow-up question provided additional information about the hypothetical medication in terms of its benefits or risks. Half of the respondents were told that the medication would increase the patient's risk of breast cancer while the other half were told that the medication would decrease the patient's risk of heart disease. For each half, however, the information was presented in one of two randomly assigned alternative formats. Regarding a decreased risk of heart disease, respondents were told either that: "by taking the medication she would also reduce her risk of heart disease by more than half," or that "by taking the medicine she would also reduce her risk of heart disease from 1 in 200 to 1 in 500." Regarding the increased risk of breast cancer, respondents were told either that "by taking the medication she would also double her risk of breast cancer," or that "by taking the medication she would also increase her risk of breast cancer from 1 in 10,000 to 2 in 10,000." Based on the additional information received, respondents were asked what they would recommend that their friend do. Subsequently, each of these four groups was randomly divided again and given the additional information about increased risk of breast cancer or decreased risk of heart disease that they had not yet received. Then they were asked one more time to indicate what they would recommend to the friend about taking the medication. Thus, respondents were asked to indicate their treatment recommendations three separate times, under three of nine sets of conditions represented by the table below. An initial treatment recommendation was made based on the information given in condition 1, and then again based on the combinations of information represented by conditions 2, 3, 4, or 7. A third and final recommendation was made based on the combination of information represented in conditions 5, 6, 8, or 9. Comparing the results for conditions 2 through 9 to the results from the baseline in condition 1 allows an assessment of the incremental effect of each additional piece of information on the treatment recommendation. The dependent variable is the decision to recommend the medication or not. We hypothesized responses to vary by risk format and number of competing risks. For example, the number of "don't know" responses would be greater in conditions where complete information about competing risks was given. The benefits-only format would produce a majority of positive recommendations. However, we could not predict how large those proportions might be. Direction of change was expected to be positive for added benefit information, negative for more risk information, and reflecting trade-offs when risks and benefits were present. That is, we expected respondents to make a "rational" choice based on the perceived greatest expected utility. Analyses To assess whether additional risk information about breast cancer or heart disease affected decisions in conditions 2 through 9, we compared the distributions of change responses for each condition. A "non-change response" occurs when the respondent gives the same answer to the follow-up question as to the baseline question. All other patterns of answers, such as a "don't know" or a "don't take the medication response to the follow-up after giving a "take the medication" response to the baseline, or vice versa, constitute change responses. Although some error variance would be expected, analyzing the distributions of change responses across baseline decisions for the individual treatment conditions determines whether or not the experimental manipulations had any systematic effect. If the added information supports rational decision-making, the percentage of "don't knows" and recommendations against taking the medication would go down if the net effect of the additional information is further risk reduction. Conversely, if the net effect of the additional information is to increase the risk of health problems, the percentage of change responses would be greater among those who initially said "don't know" or to take the medication. The greater the magnitude in the new absolute risk, the greater should be the percentage of respondents who should change their decisions. To test for the effect of risk information format, we aggregated the respondents' decisions. We dummy-coded whether the risk information had been presented in absolute risk terms or not. We also dummy-coded whether the information was given for breast cancer (= 1) or heart disease (= 0). To test the effect of format using logistic regression, we also aggregated recommendations. A dummy variable was created to indicate whether or not the risk information for breast cancer was presented in absolute risk terms (= 1), and similarly for heart disease risk. To develop a profile of those who could not make a recommendation, we dummy-coded recommendations to the baseline scenario into those recommending either to take or to not take the medication (DONTKNOW = 0) and those who said they did not know what to recommend (= 1). To develop a profile of those who made seemingly non-rational recommendations, we dummy-coded recommendations to the baseline scenario into those recommending to take the medication (WRONG = 0) and those who recommended not to take the medication or said they did not know what to recommend (WRONG = 1). All analyses were performed using SPSS for Windows 7.5. Results A total of 952 individuals were interviewed. In the weighted datafile, 54% were female; 12% were African Americans and 3% were Other non-whites; 26% were under age 30, 40% were 30–49, 18% were 50–64, and 16% were 65 or older; and 43% had a high school education or less, 31% had some college, and 26% had a college degree or more education. The reported median household income was between $40,000 and $50,000. In terms of health indicators, 15% rated their health as only "fair" or "poor;" 14% reported engaging in no leisure time exercise; 25% were current smokers; 38% were overweight or obese; and 1% were heavy drinkers. This profile shows that the sample obtained was representative of the general population of Michigan. All demographic and clinical characteristics were within the survey's margin of sampling error for the prevalence rates for these reported by the Centers for Disease Control and Prevention (CDCP) for Michigan in 1997 except for leisure time exercise. Information-based decisions In the baseline condition, two-thirds of the respondents (66.8%) said they would recommend their friend take the medication, one in nine (11.2%) said they did not know what to recommend, and 22.0% recommended that their friend not take the medication. Table 2 shows the percentages of respondents who changed their recommendation under each of the eight additional information conditions from their baseline decision. For each column, only the percentage of respondents who changed is shown, along with the total number of cases who made each of the three possible baseline choices in that condition. As shown in Table 2 , the experimental manipulations were effective and influenced treatment decisions as predicted. The table shows that the additional information presented did significantly effect respondents' decisions in conditions 2, 3, 4 and 7. In conditions 2 and 3, with additional breast cancer risk information, initial positive or "don't know" recommendations were 4 to 6 times more likely to change with new information. In conditions 5 and 7, where new information was about reduced risk of heart disease, those who had initially recommended not taking the medication or did not know were 7 to 10 times more likely to change their decision than those who had already made positive recommendations. Similarly, in conditions 5, 6, 8, and 9, the percentage of respondents who changed their recommendation from baseline differed. That is, the information in conditions 5, 6, 8 and 9 were similarly effective at creating systematic changes in respondents' recommendations. Furthermore, those who changed a response from "don't know" at baseline, made changes consistent with the health outcome logically effected. This was true across all eight non-baseline conditions. Format of risk presentation Table 3 shows treatment recommendations for all nine conditions. Added information about reduced risk of heart disease increased the percentage of positive recommendations. The format appeared to make no difference, perhaps because the absolute risk of heart disease was roughly consistent with respondents' prior assumptions. However, the percentage of "don't knows" was greater under absolute risk conditions. Table 3 demonstrates that the added information about increased risk of breast cancer dramatically reduced the percentage of positive recommendations. When presented only in relative risk terms, the percentage of positive recommendations declined by more than two-thirds. When increased risk was presented in absolute risk terms, and risks were very low, the decrease was smaller. The difference appears to be that, although doubled, the risk of breast cancer was still very small in absolute risk terms. Table 3 shows the results for the conditions in which various combinations of additional information were given. Additional information that the medication would both double the risk of breast cancer and cut the risk of heart disease by more than half reversed decisions in the baseline condition. Virtually the same percentage of respondents chose to recommend against the medication as when only the additional breast cancer information was given. The change was much greater than when only the additional information about the reduced heart disease risk was given. On the other hand, when absolute risk for breast cancer increased and absolute risk of heart disease decreased, the percentage recommending medication was the same as the percentage recommending against medication. Both fall roughly midway between the corresponding relative risk percentages. Where the increased absolute risk of breast cancer due to the medication was given along with information about the decreased relative risk of heart disease, decisions to recommend the medication were nearly as great as in the baseline condition. However, the evidence clearly suggests respondents paid attention to both additional pieces of information. Where the very low absolute risk of breast cancer was presented, the increased risk appeared to be more acceptable in the face of the benefit in reducing the risk of heart disease. Where respondents were given information in absolute risk terms for both breast cancer and heart disease, twice as many chose to recommend the medication as did where both risks were given only in relative terms. This was only two-thirds as many as did so in the baseline condition. Compared to the baseline condition, respondents seemed to use both additional pieces of information. This is consistent with Expected Utility theory predictions, but was clearest under absolute risk conditions. Table 4 shows the overall results of risk format. The logistic regression equation predicted the correct outcome for 70% of the cases choosing to recommend not taking the medication and 68% of the cases recommending taking the medication, and 69% overall. The interaction term was significant, indicating that communicating the information in absolute risk terms significantly affected the chances that the respondents would elect to recommend taking the medication, but that the effect was different depending on the type of disease. Receiving the risk information in absolute risk terms for breast cancer significantly increased the likelihood of a positive recommendation, but was not so for heart disease. For heart disease, the information regarding the absolute risk of breast cancer appears to have suggested that the overall risk was lower than previously assumed. In this case, knowing the absolute risk made medication-induced risk more acceptable. Table 5 shows the effect of absolute risk on decisions. Controlling for demographic, geographic, health, and health risk variables did not change the results (data not shown). The logistic regression equation predicted the correct outcome for 85% of the cases choosing not to recommend taking the medication, 36% of the cases recommending medication, and 69% overall. The table (again) shows that the interaction term is significant, indicating that the effect of the risk information format for each disease differs depending on what is known about the other disease. In this case, the table indicates that communicating risk information about both heart disease and breast cancer in absolute risk terms significantly changes positive recommendations compared to scenarios in which respondents received risk information in other combinations of formats. Non-rational decisions or no decisions A third of the cross-sectional sample chose something other than to recommend taking the medication in the baseline condition. While the primary purpose of this experiment was to explore the effect of information format on treatment decisions, additional analyses were performed to better understand individuals who seem to make decisions contrary to the evidence given, or who indicated an inability/unwillingess to make a decision. The two dummy variables constructed for this purpose, DONTKNOW and WRONG were analyzed against a broad set of demographic and clinical variables, including gender, race, ethnicity, living as a couple or not, age (in categories), rural/urban dwelling, education (in 4 categories), current smoker, current heavy drinker, perceived health as only fair or poor, physical activity, overweight, employed, and avoids medical care or uses alternative sources of care. (Heavy drinker and overweight definitions were taken from the Centers for Disease Control and Prevention (CDCP) Behavioral Risk Factor Surveillance System. Table 6 indicates that those most likely to report not knowing what to do (DONTKNOW) were respondents who were not employed and those who had children under 18 living in the household. Other than these factors, none of the other variables were significant predictors. Those who were not working and those who had children at home under 18 were somewhat more likely than others to say they received most of their health information from newspapers or teachers. They were also somewhat less likely to say they received most of their information from doctors. Somewhat more than other respondents, these individuals also more frequently reported believing information in the media to be clear and not confusing. The number of cases on which this analysis is based is quite small (105 of the 937 respondents answered "don't know" to the baseline question) making the findings only suggestive. Nevertheless, it is interesting that those who were uncertain how to proceed were typically more likely to rely on newspapers and teachers rather than doctors as their major source of health information [ 17 , 18 ]. Table 6 also shows predictors of WRONG, i.e., those who gave either the "don't know" or "do not take the medication" responses. The table indicates that five variables were significant predictors. Hispanics were more likely to give the "non-rational" answer, as were those who smoke, older respondents, those with children living in the household, and those who had more education. Those who said their health was only "fair" or "poor" were actually more likely than others to give the "rational" answer. Conclusions In general, the risk presentation format appeared to influence the resulting decisions. Without knowing the absolute risks, respondents appeared to assume the risk of breast cancer was much greater than the risk of heart disease, or that the perceived likely consequences of breast cancer were significantly less acceptable than those of heart disease. The pattern of recommendations was made to avoid the greater perceived risk. When the competing risks were stated in absolute risk terms, respondents changed their recommendations appreciably. The results of the experiment strongly suggest that people's prospective preventive treatment decisions can be greatly affected by the manner in which potential risks and benefits are communicated to them. When given information regarding the absolute risks associated with a proposed treatment, individuals make remarkably "rational" decisions, clearly indicating the weighing of risks and benefits. It appears that presenting risks in absolute rather than relative risk formats may be more effective in helping respondents make decisions that are consistent with maximization of expected utility. It appears, in the scenarios devised here, this may be due to the previously documented finding that people exaggerate the risk of cancer [ 15 - 17 ]. They appear to draw on such preconceptions when phrases such as "double your risk" or "cut risk in half" are used in describing possible side effects of treatments. To some degree, exaggerated cancer risk perceptions may result from media reports, public health cancer screening campaigns, and defensive medicine protocols for screening for rare diseases [ 18 ]. Our findings seem to indicate that, in the absence of explicit information regarding the absolute risks of particular outcomes, patients interpret information about relative benefits or risks based on whatever preconceptions they may have about their chances of getting the disease in question. That is, they make reasonable use of the relative risk information if they do not know the base risk rates. However, to the extent that these preconceptions may be quite inaccurate, relative risk information may lead them to make decisions that seem irrational and inconsistent with their actual preference. Without information about the absolute risks, health information may become misleading and individuals may reach conclusions that go quite counter to what they actually think about the appropriate balance of benefits and risks of treatments. Framing the scenarios as recommendations to a friend made the decision-making relevant to both males and females. The logistic regression analyses found no significant differences in decisions based on race, gender, or other demographic characteristics in this relatively large and representative sample. This suggests that these results may represent a general population response pattern. It is clear from the results that members of the public will generally use explicit absolute risk information in quite rational ways when it is available. Consequently, providers of health information, both public and clinical, would be well-advised wherever possible to provide patients with the necessary risk/benefit information in terms of the absolute risks associated with treatment options. Making the best information readily available and comprehensible may reduce reliance on errant preconceptions and opinions. This research suggests that having absolute risk information routinely available to patients and physicians for use in making clinical decisions may be an important part of improving health care decisions. This study is unique in surveying the general public rather than clinical populations on the effects of risk formats on decision-making. However, it was limited to presenting risks only in terms of the chances of an outcome occurring. It did not address the magnitude of the outcome's impact on the individual's life or the acceptability to the individual of the relevant alternatives. Future research should address these aspects of risk as well as explicitly investigating assumptions about risks of specific diseases. Research that explicitly assesses general public risk perceptions may be valuable in determining the need for absolute risk information in specific disease contexts. Providing baseline absolute risk information in certain disease contexts may lead to improved patient treatment decision-making. Competing interests The author(s) declare that they have no competing interests. Authors' contributions LAH conceived and carried out the survey. MHR and CEW provided medical decision making context for interpretation and discussion. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC535806.xml
516775	Cloning and expression analysis of cDNAs corresponding to genes activated in cucumber showing systemic acquired resistance after BTH treatment	Background Infection of plants by necrotizing pathogens can lead to the rapid and localized induction of a complex set of defense responses resulting in a restriction of pathogen growth and spread. Subsequently, an increase of plant resistance against a broad spectrum of pathogens is observed systemically. This plant immunity is known as Systemic Acquired Resistance. To identify components of the transduction pathway, we cloned and analysed the expression pattern of several mRNAs accumulating in cucumber plants after induction of Systemic Acquired Resistance. Results We tested on cucumber different compounds known to induce systemic acquired resistance. Among these, BTH (benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester) proved to be very effective. mRNA RT-PCR differential display was used to identify mRNA sequences induced 24 hours after the application of 10 μM BTH to cucumber plants. A cDNA library constructed from cucumber plants sprayed with 10 μM BTH was screened to get corresponding full length cDNAs. Among the identified cDNAs were those coding for a putative ras-related GTP-binding protein, a putative beta-1,4- N -Acetylglucosaminyltranferase III and a putative pathogenesis related protein. The time course of accumulation of the three corresponding mRNAs was analysed by northern blotting in plants treated by BTH or in plants infected by Colletotrichum lagenarium . Conclusions The mRNA RT-PCR differential display technique allowed the identification of three genes possibly involved in Systemic Acquired Resistance in cucumber. Pathogenesis-related proteins are known to be involved in plant defence against pathogens. GTP-binding protein and N -acetylglucosaminyltranferase III have been reported to be components of signal transduction pathways in mammals and plants.	Background Infection of plants by necrotizing pathogens can lead to the induction of a complex set of defense responses resulting in a restriction of pathogen growth and spread. The infected leaves develop a hypersensitive response (HR), i.e., rapid, localized cell death occurring at the sites of pathogen entry [ 1 ]. Concomitant with the HR is the accumulation of salicylic acid and several classes of pathogenesis-related (PR) proteins, many of which exhibiting antimicrobial activity [ 2 ]. Subsequently, an enhancement of the plant defensive capacity against a broad spectrum of pathogens is observed. This resistance is expressed locally as well as in distal, uninfected tissues and can last for several weeks to months. It is known as Systemic Acquired Resistance or SAR [ 3 ]. Salicylic acid was identified as an important signal in the SAR transduction pathway. It was shown to accumulate during the onset of SAR in cucumber [ 4 ], tobacco [ 5 ], and Arabidopsis thaliana [ 6 ]. Exogenously supplied salicylic acid induces the same set of genes and resistance against the same spectrum of pathogens, as with pathogen-induced SAR [ 3 ]. Plants in which salicylic acid accumulation is prevented by over expression of a bacterial salicylate hydroxylase gene failed to develop SAR and/or exhibited increased susceptibility to pathogen infection [ 7 , 8 ]. Analysis of A. thaliana mutants has revealed that the NPR1 protein (also known as NIM1) is required for SAR induction and acts downstream of salicylic acid in the SAR pathway. Plants carrying mutations in this gene fail to express several PR genes and display enhanced susceptibility to infection [ 9 , 10 ]. The NPR1/NIM1 protein interacts with members of the TGA/OBF family of basic leucine zipper transcription factors. Some of these factors have been shown to bind to salicylic acid-responsive promoter elements of the PR-1 gene [ 11 - 13 ]. The salicylic acid induced interaction of NPR1 with TGA factors is localized in the nucleus [ 14 ], while cytoplasmic NPR1 appears to modulate crosstalk between salicylic acid- and jasmonate-dependent defense pathways [ 15 ]. Other components of the salicylic acid signaling pathway have been identified using genetic approaches in Arabidopsis thaliana [ 16 ]. They include PAD4 and EDS3 which activate EDS4, SID2 and EDS5 leading to salicylic acid accumulation [ 17 ]. Besides pathogens, SAR is also induced by exogenously applied chemicals. In addition to salicylic acid, well-known chemical inducers of PR genes include 2,6-dichloroisonicotinic acid (INA), benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) and L-α-amino butyric acid (ABA) [ 18 - 23 ]. SAR induction appears to be governed by a complex signal transduction process [ 24 ] that involves a signal originating at the site of infection or treatment, and moving throughout the plant. Chemical inducers provide tools for dissection of the signal transduction pathway that regulates the defense responses (they enter the pathway at different points). On another hand, the cucumber plant provides a good model system for studying induced disease resistance [ 25 ]. Local and systemic increases in chitinase and/or peroxidase activity have been observed in response to inoculation with necrotizing pathogens or to treatment with SA, INA and BTH [ 26 - 28 ]. The aim of the present work was to identify and clone mRNAs accumulating after induction of SAR in cucumber plants. These sequences could correspond to proteins potentially involved in the signal transduction pathway leading to SAR or to proteins responsible for the state of resistance. In a first set of experiments, potential SAR inducing chemicals were tested in order to set up a reproducible system of activation of this resistance in cucumber. The mRNA RT-PCR differential display method [ 29 ] was then used to identify genes activated during SAR induction. Several differentially expressed cDNA were identified and some of them were cloned and sequenced. Their expression was analyzed by Northern blotting. Results Chemically induced resistance in cucumber The SAR inducing activity of different chemicals has already been documented in one or several plant species [ 18 - 23 ]. To evaluate their activity on cucumber, cotyledons of two-week-old plants were infiltrated with 0.5 mM salicylic acid, 50 mM L-α-amino butyric acid, 0.78 mM 2-thiouracil, 1 mM thiamine or 20 mM BaCl 2 . Control plants were infiltrated with water. In a parallel experiment, four-week-old cucumber plants (with two true leaves) were sprayed with 5 mM salicylic acid, 50 mM L-α-amino butyric acid, 0.78 mM 2-thiouracil, 1 mM thiamine, 20 mM BaCl 2 or 1 mM BTH. Control plants were sprayed with water or left untreated. Total RNAs were extracted from treated leaves 6 h, 24 h or 48 h after treatment. Induction of PR-8 gene was assayed by Northern blotting followed by hybridization with a cucumber PR-8 cDNA probe. PR-8 cucumber gene encodes an acidic chitinase which accumulates after tobacco necrosis virus (TNV) infection or salicylic acid induction and is considered as a SAR molecular marker [ 30 ]. In infiltration experiments (results not shown), control plants showed positive hybridization signals. To check whether this activation of the PR-8 gene could be due to wounding during infiltration, the effect of wounding alone was evaluated by making a cut from the midvein to the outer edge of the cotyledons. A strong signal was obtained after wounding which could be explained by induction of the acidic chitinase itself or of an isoform. Therefore infiltration did not prove to be an appropriate method for applying SAR inducers in cucumber, because it probably induced a wound response. When applied by spraying, BTH, L-α-amino butyric acid and 2-thiouracil strongly induced PR-8 mRNA accumulation, while SA treatment led to a lower induction level (Fig. 1 ). Thiamine and BaCl 2 induced slightly PR-8 expression (results not shown). RNA from control plants did not show any hybridization signal indicating that spraying itself did not induce PR-8 expression. BTH, which is an analogue of salicylic acid, showed the strongest PR-8 induction. But cucumber plants treated with 1 mM BTH showed a reduced growth (smaller leaves area and shorter internodes). As BTH was shown to be a very efficient activator of disease resistance in different plant species [ 20 - 23 ], we tried to induce SAR in cucumber plants with lower BTH concentrations (10 μM, 30 μM and 100 μM). PR-8 transcript accumulation was quite similar for the three concentrations (see Additional file 1 ) and no adverse effect on plant growth was observed. In cucumber, SAR has been shown to be effective against anthracnose caused by Colletotrichum lagenarium and salicylic acid treatment was shown to induce resistance against this pathogen [ 31 ]. To confirm that BTH acts as a chemical inducer of SAR in cucumber, four-week-old plants were sprayed with water, 10 μM BTH, 50 μM BTH, or left untreated, 5 days prior to inoculation of the second true leaf with C. lagenarium . Two weeks after inoculation, the plants were scored for spreading lesions, i.e. lesions showing a larger size than the initial inoculated area. As shown in figure 2 and table 1 , BTH treatment resulted in decreased symptoms. Control leaves inoculated with C. lagenarium showed large spreading lesions and thus extensive fungal growth. Treatment of plants with BTH prior to C. lagenarium inoculation resulted in a markedly decreased lesion formation (Fig. 2 ). In untreated plants and water treated plants, 67 and 64 % of inoculation sites showed spreading lesions respectively (Table 1 ). In BTH treated plants, only 23 % (10 μM BTH) or 9 % (50 μM BTH) of inoculated sites developed spreading lesions (Table 1 ). To check whether BTH is able to act systemically or is transported through cucumber plants, the third leaf of four-week-old plants was wrapped with a plastic film and the plants were sprayed with H 2 O, 10 μM BTH, 50 μM BTH or left untreated. This leaf was inoculated with C. lagenarium 7 days later. The percentage of spreading lesions on third leaves from pathogen-challenged untreated or H 2 O treated cucumber plants (infected controls) were respectively 86% and 83.5%. In BTH treated plants, 38 % (10 μM BTH) or 12 % (50 μM BTH) of inoculated sites developed spreading lesions (Table 2 ). These results suggest that BTH can activate resistance in untreated leaves. Differential expression of genes in BTH induced cucumber plants Plants were sprayed with 10 μM BTH or water. Sampling was performed 24 h after treatment in order to point out early-induced differences in gene expression. RT-PCR products were separated on polyacrylamide gels. Bands that appeared on the display of RNA only from BTH treated plants, or only from water treated plants were likely to correspond to differentially expressed mRNAs. Twelve differentially expressed bands were identified (Table 3 ). Their sizes ranged between 250 and 450 bp. They corresponded to genes expressed in the BTH treated plants and not in the control ones or presenting a higher expression in the BTH treated plants compared to the control ones (Fig. 3 ). These bands were eluted from the gel, PCR amplified and cloned in the pGEM-T vector. In order to confirm the differential expression of these genes, reverse slot blot hybridizations were performed: DNA from four to ten clones obtained from each differentially displayed band was fixed on a membrane and hybridized with the corresponding reverse transcribed products from either BTH or H 2 O treated plants (Fig. 4 ). Six from the twelve fragments were confirmed to be differentially expressed: dd3, dd4, dd5, dd6, dd7 and dd11. Five bands (dd1, dd8, dd9, dd10 and dd12) were either expressed evenly between BTH and H 2 O treated plants (differential display false positives or signal masked by the expression of homologous genes). In the remaining case (dd2), no signal could be observed on the slot blots. This absence of hybridization signal could be due to a lack of sensitivity of the slot blot hybridization procedure. The dd3, dd4, dd5, dd6, dd7 and dd11 cDNA fragments were sequenced. Blast analysis did not lead to identification of any homology with known sequences. This is not surprising as mRNA differential display generates fragments corresponding to 3'untranslated regions for which homologies are difficult to find. Two of the six fragments were proved to be part of the same gene (dd5 and dd7). A cDNA library was constructed from cucumber plants sprayed with 10 μM BTH (leaves were sampled 24 h after treatment). The library was screened with the five differentially expressed fragments. Hits were obtained for four of these probes (corresponding to dd3, dd4, dd6 and dd11). Full length cDNAs corresponding to dd4, dd6 and dd11 were obtained. Homology between cloned cDNA and sequences in databases is presented in table 4 . The cDNA dd3 matched to a putative RAS-related GTP-binding protein from A. thaliana . It will be referred to as CRG (cucumber RAS-related GTP-binding protein). The cDNA dd4 matched to a translation releasing factor 2. It will be referred to as CTR (cucumber translation releasing factor 2). The cDNA dd6 matched to a putative β-1,4- N -Acetylglucosaminyltranferase III from A. thaliana . This protein was named CGT (cucumber acetylglucosaminyltransferase III). The cDNA dd11 matched to a putative pathogenesis related protein from A. thaliana and, with slightly less homology, to a pathogenesis related protein from barley induced by fungal infection [ 32 ]. It will be referred to as CPR (cucumber pathogenesis-related protein). PR-8 , CPR , CRG and CGT expression analysis To investigate the expression of PR-8 , CPR , CRG and CGT genes in cucumber, the two first leaves of four-week old plants were sprayed with 10 μM BTH. In another batch of plants, the first leaf was inoculated with C. lagenarium . Total RNA was extracted from treated leaves or upper untreated leaves at various times after treatment. Accumulation of the PR-8, CPR, CRG and CGT mRNAs was assayed by Northern blotting followed by hybridization with the corresponding cDNA probes (fig. 5 ). Expression of PR-8 gene was induced at a high level by both 10 μM BTH spraying and C. lagenarium infection in the leaves submitted to the treatment. PR-8 mRNA could be detected as soon as 5 h after BTH application and a maximum of expression could be observed after 72 h. PR-8 expression was observed as from 72 h after inoculation with C. lagenarium . No significant PR-8 mRNA accumulation could be detected in the untreated leaves from BTH sprayed plants, while in non infected leaves PR-8 expression was observed 72 h after infection by C. lagenarium ; the level of expression was still increasing 2 weeks after C. lagenarium inoculation. CPR gene expression was strongly induced in both 10 μM BTH treated and C. lagenarium inoculated leaves. CPR mRNA could be detected as soon as 5 h after BTH application and a maximum of expression could be observed after 24 h. CPR expression was observed 72 h after infection by C. lagenarium . No expression of CPR gene could be observed in untreated leaves from BTH sprayed plants, while CPR mRNA accumulation could be detected in uninfected leaves from C. lagenarium infected plants 72 h after inoculation. CGT gene showed a significant basal level of expression as it could be observed in control plants (fig. 5 ). Nevertheless the expression of CGT was increased both by spraying with 10 μM BTH (with a maximum at 24 h) and by inoculation with C. lagenarium (with a maximum at 72 h) in the leaves submitted to the treatment. A systemic induction could also be observed in untreated leaves from C. lagenarium inoculated plants (as from 72 h after infection). The expression level of the CRG gene was too low to allow us to show an induction of the expression (data not shown). Expression of the same genes was analyzed in wounded cucumber plants. A cut was made from the midvein to the outer edge of the first leaf. Total RNA was extracted from the cut leaf or the upper leaf, 24 and 48 h after wounding. An induction of the expression of PR-8 and CPR genes was only observed in the cut leaf 24 h after wounding. No expression was observed later nor in the uncut upper leaf. The CGT and CRG genes were not induced by wounding (results not shown). Expression of these genes was also analyzed in cucumber plants showing rhizobacteria-induced systemic resistance. Plants were grown in compost drenched with a Pseudomonas putida BTP1 suspension, a plant growth-promoting rhizobacteria known to promote induced systemic resistance in cucumber [ 33 ]. Total RNA was extracted from cotyledons collected three weeks after sowing. PR-8 , CPR , CGT and CRG did not show any induction of expression in these conditions (results not shown). Discussion We tested here two different techniques and several compounds to induce SAR in cucumber. The expression of PR-8 gene, which encodes an acidic chitinase from cucumber, was used as an indicator of the activation of the defense response. The accumulation of this acidic chitinase after tobacco necrosis virus infection or salicylic acid induction correlates with induced resistance [ 30 ]. Among the different compounds tested, BTH applied by spraying proved to be the stronger inducer of PR-8 mRNA accumulation. BTH sprayed at a concentration of 10 μM was still able to induce PR-8 gene expression. The protective effects of BTH on cucumber were confirmed in challenge experiments with C. lagenarium . Spraying cucumber plants with BTH (10 μM or 50 μM) allowed effective local and systemic protection against C. lagenarium . BTH was recently identified as a safe, reliable and non phytotoxic plant protection agent by scientists at Novartis. Exogenous application of BTH to tobacco, A. thaliana , wheat and cucumber has been shown to activate a number of SAR associated genes leading to enhanced plant protection against various pathogens [ 20 - 22 , 28 ]. Identifying SAR induced genes could provide clues to elucidate the signal transduction pathway leading to plant defense responses. We used RT-PCR differential display in BTH-treated cucumber plants to detect SAR associated mRNAs. We have identified four BTH-inducible genes: a putative ras-related GTP binding protein (CRG), a putative translation releasing factor 2 (CTR), a putative β-1,4- N -acetylglucosaminyltranferase III (CGT) and a putative pathogenesis related protein (CPR). Expression of PR-8 , CPR and CGT genes was shown to be induced by 10 μM BTH treatment as well as C. lagenarium infection. The response to BTH was observed earlier (5 h after treatment) than the one developed after pathogen inoculation (72 h after infection). However, systemic expression of these genes was only observed in C. lagenarium infected plants. Small GTP-binding proteins form a large family of nucleotide triphosphatases whose activity is related to the binding, hydrolysis and release of guanosine triphosphate [ 34 ]. MAP kinase cascade represents an important downstream effector pathway for RAS in most cells [ 35 ] and recent studies have shown that MAP kinases are activated in plants in response to pathogen attack and wounding [ 36 , 37 ]. Small GTP-binding proteins have been shown to be involved in important cell mechanisms like cell division, transduction of external signals across the plasma membrane, endocytosis and exocytosis, cell death in plants and establishment of plant defense reaction [ 34 , 38 - 42 ]. The β-1,4- N -Acetylglucosaminyltranferase III catalyses the addition of N -acetylglucosamine to the β-mannoside of the tri-mannose core in N -glycans, resulting in the suppression of further processing and elongation. In mammals, N -acetylglucosaminyltransferase III expression is associated with differentiation, cell adhesion and tumor progression [ 43 , 44 ]. It was shown recently to interfere with epidermal growth factor signaling and H 2 O 2 -induced activation of protein kinase C [ 45 , 46 ]. N -linked glycans were shown to be widely distributed in plants and highly expressed at the cell surface, which might suggest a putative function in cell/cell communication [ 47 ]. To our knowledge β-1,4- N -Acetylglucosaminyltranferase has never been described in the context of a plant-pathogen interaction. Several cDNAs or gene encoding enzymes involved in the biosynthesis of complex N -linked glycans have been cloned from plants. They include N -acetylglucosaminyltranferase I cDNAs from A. thaliana , potato and tobacco [ 48 ], N -acetylglucosaminyltransferase II cDNA from A. thaliana [ 49 ] and defense-related glucosyl transferase gene from tomato [ 50 ]. GTP-binding proteins and N -acetylglucosaminyltranferases are involved in important mechanisms, notably signaling pathways and therefore seem of high interest. The putative PR protein, which has been first identified in barley after fungal infection [ 32 ] has no known function. Only PR-8 and CPR genes were induced by wounding but not systemically. PR-8 and CPR genes were not induced by P. putida BTP1 treatment, which triggers induced systemic resistance (ISR) in cucumber. ISR is phenotypically similar to SAR but the mechanisms of this resistance were shown to be different: although this was not tested with P. putida BTP1, in other experimental systems ISR was shown to be dependent on NPR/NIM1 function, but does not involve salicylic acid, nor PR protein accumulation [ 51 ]. Our results correlate with those of Pieterse et al. who showed that induced systemic resistance in A. thaliana was independent of PR genes expression [ 51 ]. In the same way, ISR also seems to be independent on CGT and CRG expression, as these genes were not induced by P. putida BTP1. Conclusions Among different SAR inducers tested on cucumber, BTH proved to be very efficient. Applied by spraying, BTH induced PR-8 mRNA accumulation and allowed effective local and systemic protection against C. lagenarium . Using RT-PCR differential display, we identified four BTH-inducible genes, including a putative ras-related GTP binding protein (CRG), a translation releasing factor 2 (CTR), a putative β-1,4- N -acetylglucosaminyltranferase III (CGT) and a putative pathogenesis related protein (CPR). Genes expression studies confirmed the local induction of CGT and CPR after BTH treatment or C. lagenarium infection and their systemic induction in response to C. lagenarium infection. Moreover CPR was locally induced by wounding. The significant level of expression of CGT and CPR genes during defense responses suggests a potential role for the gene products in the SAR pathway or in the state of resistance in cucumber. Methods Organisms and growth conditions Cucumber plants ( Cucumis sativus cv. Marketer ) were grown in compost at 22°C with a 16 h light photoperiod. Cultures of Colletotrichum lagenarium were maintained on malt agar at 25 °C. Spores suspensions in water supplemented with 0.01 % tween 80 were prepared from four- to six-week-old cultures grown in petri plates. Suspensions were filtered through cheesecloth and the spore concentration determined with a hemacytometer. Chemical treatments All chemicals were obtained from Sigma except for BTH (provided by Novartis). Chemicals were dissolved in water and the pH adjusted to 6.6 with NaOH except for thiamine, BaCl 2 and BTH. Infiltration experiments: cotyledons of two-week-old plants were infiltrated through a vein until the whole leaf was impregnated with the solution. Spraying experiments: the two first true leaves of four-week-old cucumber plants were sprayed with +/- 3 ml solution/plant. The upper leaves were protected with a plastic sheet when systemic resistance was to be tested. ISR induction Prior to planting, disinfected cucumber seeds were soaked for 10 min in a Pseudomonas putida BTP1 suspension (4.10 8 CFU/ml in 0.85% NaCl). Control seeds were soaked in 0.85% NaCl. Cucumber seeds were sown in 10 cm-pots containing sterilized potting soil previously mixed with a P. putida BTP1 inoculum to a final concentration of 3.10 7 CFU/g or with an equal volume of sterile water for control plants. Cucumber were germinated at 25°C with a 16 h light photoperiod. Six and 12 days after sowing, 20 ml of a bacterial suspension at 10 8 CFU/ml were added as a drench to the roots. Control plants were watered with 20 ml of 0.85% NaCl. C. lagenarium infection Plants were placed in a humidity chamber 48 hours before infection experiments. The first true leaf was inoculated with 10 drops (10 μl) of a conidial suspension of C. lagenarium (10 6 spores/ml). In challenge experiments, inoculation was performed 5 (or 7) days after chemical treatment on the second or third leaf. Plant RNA extraction and analysis The extraction of total RNA from cucumber leaves (5 plants per treatment) was performed using a phenol/SDS method [ 52 ]. Northern hybridization of RNA fractionated by agarose-formaldehyde gel electrophoresis [ 52 ] was performed with a α- 32 P PR-8, CPR, CGT or CRG cDNA probe (Random Primed DNA labeling kit, Roche). mRNA Differential Display The mRNA differential display was performed using the differential display kit from Eurogentec (Belgium) and following the instructions of the supplier. PCR products were labeled with α- 35 S dATP, separated by electrophoresis on denaturing 6% polyacrylamide gels and visualized by autoradiography. PCR reactions showing differentially displayed bands were repeated twice on two different RNA samples in order to reduce the number of false-positives. Cloning and sequencing Differentially displayed bands were excised and eluted during 30 min in 100 μl of H 2 O followed by boiling during 10 min, then precipitated and suspended in 10 μl of H 2 O. A second PCR amplification was performed on 4 μl of DNA following the same protocol than for the differential display itself. Products were cloned in pGEM-T vector (Promega) and manually sequenced by the dideoxy sequencing method. Available public databases (i.e. EMBL) were searched for homology with our sequences using the GCG software package (Blastall, Fasta). Slot-Blot hybridisation PCR amplified DNA from four to ten clones of each differentially displayed band was blotted on a positively charged nylon membrane using a filtration manifold and following the instructions of the supplier (Hoefer). Blots were hybridized with the 32 P labeled RT-PCR corresponding products from either BTH or H 2 O treated plants. cDNA library construction and screening mRNA from cucumber plants sprayed with 10 μM BTH (leaves were sampled 24 h after treatment) were purified with the PolyATractmRNA Isolation System III from Promega. The cDNA library was performed with the SMART cDNA Library Construction Kit from Clontech following the instructions of the supplier for cDNA synthesis by primer extension. The library was screened with 32 P labeled cDNA probes. cDNA sequencing was performed at Genome Express, France. Available public databases (i.e. EMBL) were searched for homology with our sequences using the GCG software package (Blastall, Fasta). Authors' contributions CB carried out the plants treatments, the molecular biology studies and the database research and drafted the manuscript. MO supplied the pathogen and participated in the challenge and ISR induction experiments. JD and PT participated in the design and coordination of the study. All authors read and approved the manuscript. Supplementary Material Additional File 1 PR-8 mRNA accumulation in cucumber leaves in response to different concentrations of BTH . Total RNA was extracted from cucumber leaves 24 h and 48 h after spraying with water, 10 μM BTH, 30 μM BTH, 100 μM BTH or from untreated leaves (UT). RNA was fractionated by electrophoresis on agarose gel. Northern blot was probed with α- 32 P labeled PR-8 cDNA. Loading of equal amounts of RNA was confirmed by ethidium bromide (Et Br) staining. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC516775.xml
546195	Gene expression profiling of tumours derived from rasV12/E1A-transformed mouse embryonic fibroblasts to identify genes required for tumour development	Background In cancer, cellular transformation is followed by tumour development. Knowledge on the mechanisms of transformation, involving activation of proto-oncogenes and inactivation of tumour-suppressor genes has considerably improved whereas tumour development remains poorly understood. An interesting way of gaining information on tumour progression mechanisms would be to identify genes whose expression is altered during tumour formation. We used the Affymetrix-based DNA microarray technology to analyze gene expression profiles of tumours derived from ras V12 /E1A-transformed mouse embryo fibroblasts in order to identify the genes that could be involved in tumour development. Results Among the 12,000 genes analyzed in this study, only 489 showed altered expression during tumour development, 213 being up-regulated and 276 down-regulated. The genes differentially expressed are involved in a variety of cellular functions, including control of transcription, regulation of mRNA maturation and processing, regulation of protein translation, activation of interferon-induced genes, intracellular signalling, apoptosis, cell growth, angiogenesis, cytoskeleton, cell-to-cell interaction, extracellular matrix formation, metabolism and production of secretory factors. Conclusions Some of the genes identified in this work, whose expression is altered upon ras V12 /E1A transformation of MEFs, could be new cancer therapeutic targets.	Background Cellular transformation is a complex process which involves activation of proto-oncogenes and inactivation of tumour-suppressor genes [ 1 ]. After transformation, the cells can generate malignant tumours, by mechanisms only partly understood yet. It is supposed that some modifications in the pattern of gene expression will promote survival of transformed cells in situ , other modifications will favour eventual formation of metastases [ 2 ], the capacity to adapt a new microenvironment being of major importance for successful tumour development and progression [reviewed in [ 3 ]]. Therefore, identification of genes whose expression is altered during tumour formation should provide important information on the underlying molecular mechanisms. In the present work, we used the Affymetrix-based DNA microarray technology to analyze gene expression profiles of tumour-derived from ras V12 /E1A-transformed primary mouse embryonic fibroblasts (MEFs), in order to identify genes associated with tumour development. Results and Discussion The ras oncogene can transform most immortalized rodent cells to generate tumour cells, whereas transformation of primary cells requires either a cooperating oncogene or the inactivation of a tumour suppressor gene. The adenovirus E1A oncogene cooperates with ras to transform primary rodent fibroblasts [ 4 ] and injection of athymic mice with such transformed fibroblasts induces tumour development. The ras V12 /E1A model of tumour formation was used in this work to analyze genes necessary for tumour progression. This model was chosen because transformation is induced in a simple and controlled way, avoiding the difficulties of analyzing the multiple and complex transformation mechanisms observed in cellular models derived from human tumours. Mouse embryo fibroblasts (MEFs) were chosen for transformation by ras V12 /E1A to keep the model homogeneous, the host being the athymic mouse. Because non-transformed MEFs are unable to induce tumour when injected into athymic mice, we previously analyzed the change in gene expression profile induced in MEFs by ras V12 /E1A-transformation [ 5 ], the idea being that such genetic changes are, directly or indirectly, responsible for the capacity of transformed MEFs to form tumours upon injection. As a follow-up, we used in this work microarray analysis to compare expression profiles of about 12,000 genes in ras V12 /E1A-transformed MEFs and in the tumours formed after their injection into nu/nu mice. With Affymetrix microarray technology, differential expression values greater than 1.7 are likely to be significant, based on internal quality control data. We present data that use a more stringent ratio, restricting our analysis to genes overexpressed or under-expressed at least 2.0 fold in tumours, compared to the parent ras V12 /E1A-transformed fibroblasts. Most striking findings are summarized below while complete data are presented in Tables 1 and 2 (see additional files 1 and 2 ), values being the average of three separate experiments. Among the 12,000 genes analyzed in this study, only 489 (4%) showed altered expression upon tumour development. Whereas 213 were up-regulated, 276 were down-regulated. Sixty seven genes encode ESTs. For 10 genes, expression data from microarrays were confirmed (Figure 1 ) by semiquantitative RT-PCR (see Material and Methods). Figure 1 Confirmation of microarray results by sequantitative RT-PCR analysis . Total RNA was isolated from primary embryo fibroblasts (MEFs), ras V12 /E1A MEFs and ras V12 /E1A MEF-induced tumours. In these three preparations, mRNA encoding arginase 1, bone morphogenetic protein 10, cathepsin S, insulin-like growth factor binding protein 4, interferon stimulated gene 12, serum deprivation response, thrombomodulin, adenylate kinase 1, connective tissue growth factor and S100 calcium binding protein A11 were amplified by RT-PCR as described in Material and Methods section. It is noteworthy that, to form tumours, transformed cells require the vicinity of blood vessels and components of the stroma, fibroblasts and inflammatory cells. Consequently, mRNA quantified in our system will come from transformed cells growing within the tumour and from stromal cells provided by the host. For example, mRNAs for haemoglobin, selectin or immunoglobulin heavy chain (V10 family) will very probably originate from erythrocytes, endothelium and leucocytes respectively. Gene transcription, mRNA processing and translation-associated genes Down-regulation was observed for some DNA-binding proteins and transcriptional factors such as zinc finger protein 36, C3H type-like 2, forkhead box M1, T-box 14, SET and MYND domain containing 2, sine oculis-related homeobox 1 homolog (Drosophila), liver-specific bHLH-Zip transcription factor, deformed epidermal autoregulatory factor 1 homolog (Drosophila), HMG box Bromodomain (5 domains) Zinc finger C2H2 type, BTB (POZ) domain containing 14A, Jun oncogene, general transcription factor III A, MYB binding protein (P160) 1a, general transcription factor II H polypeptide 1, transcription factor Dp 1, myeloblastosis oncogene-like 2, general transcription factor IIF polypeptide 1, v-ets erythroblastosis virus E26 oncogene homolog 1 (avian), transcriptional regulator SIN3B homolog (yeast), RNA polymerase I transcription factor RRN3, Kruppel-like factor 5, general transcription factor III C 1, nucleosome assembly protein 1-like 1, general transcription factor IIB and transformation related protein 53 (p53) whereas CCR4-NOT transcription complex subunit 7, transcription factor 15, dimerization cofactor of hepatocyte nuclear factor 1 alpha (TCF1), chromobox homolog 3 (Drosophila HP1 gamma), ets variant gene 1, basic helix-loop-helix domain containing class B2, pre-B-cell colony-enhancing factor 1, homeo box C6, BTB (POZ) domain containing 1, zinc finger protein 37, enhancer of zeste homolog 1 (Drosophila) and AT rich interactive domain 3B (Bright like) were up-regulated. A number of genes involved in RNA maturation, protein translation, processing and secretion were down-regulated in tumours, such as eukaryotic translation initiation factor 4 gamma 1, ER degradation enhancer mannosidase alpha-like 1, paraspeckle protein 1, CUG triplet repeat, RNA binding protein 1, copper chaperone for superoxide dismutase, splicing factor arginine/serine-rich 2 interacting protein, ribosomal protein S6 kinase polypeptide 4, sorting nexin 14, sorting nexin 17, elongation protein 3 homolog (S. cerevisiae), eukaryotic translation initiation factor 5B, peptidylprolyl isomerase F (cyclophilin F), ubiquitin-conjugating enzyme E2S, DnaJ homolog subfamily C member 3, SEC14-like 1 (S. cerevisiae), mitochondrial ribosomal protein L18, mitochondrial isoleucine tRNA synthetase, brix domain-containing protein, proteasome 26S subunit non-ATPase 1, COP9 subunit 5, small nuclear ribonucleoprotein polypeptide A, heat shock protein 105, heat shock protein 1, ribosome-binding protein p34, splicing factor 3b subunit 1, proteasome 26S subunit non-ATPase 7, mitochondrial processing peptidase beta, translocating chain-associating membrane protein 1, mitochondrial ribosomal protein L44, mitochondrial ribosomal protein S25, mitochondrial ribosomal protein S10, eukaryotic translation initiation factor 3 subunit 1 alpha, eukaryotic translation initiation factor 1A, COP9 subunit 2, RNA-binding region (RNP1, RRM) containing 1, DNAJ domain-containing and Der1-like family protein, whereas only 7 genes from this group were up-regulated including ubiquitin specific protease 18, proteosome subunit beta type 8, heterogeneous nuclear ribonucleoprotein U, ERO1-like (S. cerevisiae), proteasome subunit beta type 10, hnRNP-associated with lethal yellow and peptidylprolyl isomerase (cyclophilin)-like 2. Altogether these results show that transcriptional factors and DNA-binding proteins involved in transcription, as well as proteins involved in RNA maturation, protein translation, processing and secretion are preferentially down regulated during tumour development, suggesting that protein synthesis is less active in tumours than in transformed cells in vitro . Interferon-induced genes It is interesting to note that 11 genes activated by interferon were found up-regulated in tumours from 3.2 to 44.6 folds. They comprise the interferon stimulated gene 12 and genes encoding the interferon-induced protein with tetratricopeptide repeats 1, the interferon-induced protein with tetratricopeptide repeats 3, the interferon regulatory factor 7, the interferon alpha-inducible protein, the interferon consensus sequence binding protein 1, the interferon-inducible GTPase, the interferon induced transmembrane protein 2, the interferon gamma induced GTPase, the interferon-g induced GTPase and the interferon gamma-inducible protein 16 Genes encoding signalling factors Expression of several genes involved in signalling was modified in tumours. Among up-regulated were genes encoding the TYRO protein tyrosine kinase binding protein, ADP-ribosylation factor-like 4, ARF-GAP RHO-GAP ankyrin repeat and pleckstrin homology domains-containing protein 3, guanosine diphosphate dissociation inhibitor 2, SET domain bifurcated 1, acid phosphatase 5, guanylate nucleotide binding protein 3, SH3-domain GRB2-like 2, rap2 interacting protein x, TGFB inducible early growth response 1, pleckstrin homology domain containing family A (phosphoinositide binding specific) member 1, A kinase (PRKA) anchor protein 8, PDZ and LIM domain 4, protein tyrosine phosphatase non-receptor type 13, calcium and integrin binding family member 2, diaphanous homolog 2 (Drosophila), SH3-domain GRB2-like 3, Janus kinase 3, dual specificity phosphatase 9, Nik related kinase, synaptojanin 2, zinc finger RAN-binding domain containing 1, inositol hexaphosphate kinase 1, spleen tyrosine kinase, ribitol kinase putative, B lymphoid kinase, DEAD box polypeptide 27, mitogen activated protein kinase kinase kinase kinase 1, tumor-associated calcium signal transducer 1 and ral guanine nucleotide dissociation stimulator. Among down-regulated during tumour development where genes encoding adenylate kinase 1, serum deprivation response, ATP-binding cassette sub-family F member 2, protein kinase C alpha, RIO kinase 1 homolog (yeast), protein phosphatase 1 regulatory (inhibitor) subunit 2, phosphotidylinositol transfer protein beta, RAN GTPase activating protein 1, v-crk sarcoma virus CT10 oncogene homolog (avian)-like, serum/glucocorticoid regulated kinase, DEAD box polypeptide 48, nucleolar GTPase, calcium/calmodulin-dependent protein kinase II delta, cellular retinoic acid binding protein I, AFG3(ATPase family gene 3)-like 1 homolog (yeast), transforming growth factor beta regulated gene 4, enabled homolog (Drosophila), Rho GDP dissociation inhibitor gamma, S100 calcium binding protein A11 (calizzarin), cornichon homolog (Drosophila), butyrate-induced transcript 1, phosphatidylinositol 3-kinase regulatory subunit polypeptide 1 (p85 alpha), mitogen activated protein kinase 1, IQ motif containing GTPase activating protein 1, protein kinase C epsilon, transducin (beta)-like 3, serine/threonine kinase 16, thymoma viral proto-oncogene 1, ATP-binding cassette sub-family B (MDR/TAP) member 7, protein phosphatase 1 regulatory (inhibitor) subunit 7, dual specificity phosphatase 1, Rho-associated coiled-coil forming kinase 2, acid phosphatase 1 soluble, PAK1 interacting protein 1, RAN binding protein 1, dual specificity phosphatase 16, RAB23 member RAS oncogene family, WD repeat domain 26, PDZ and LIM domain 1 (elfin) and mitogen activated protein kinase kinase 4. Apoptosis-related genes The ras V12 /E1A-transformed MEFs are very sensitive to apoptosis in vitro [ 6 ] whereas, on the contrary, these cells do not show signs of apoptosis in tumours as judged by microscopic analysis. A possible explanation is provided by the observation that many proapoptotic genes are down-regulated in tumours, such as growth arrest and DNA-damage-inducible 45 beta, wild-type p53-induced gene 1, Bcl2-associated X protein, programmed cell death 2, TP53 apoptosis effector, programmed cell death 6 interacting protein, apoptotic chromatin condensation inducer 1, cell division cycle and apoptosis regulator 1, large tumour suppressor 2 and caspase 7 and several antiapoptotic genes are up-regulated, including genes encoding the spermatogenesis apoptosis-related protein and BCL2/adenovirus E1B 19kDa-interacting protein 1 (NIP3). Cell growth-involved genes Another interesting point to be underscored is that expression of many cell growth-related genes was found decreased in tumours, whereas none of them was up-regulated. Among up-regulated genes were those coding for he bone morphogenetic protein 10, cytokine receptor-like factor 1, insulin-like growth factor binding protein 4, ephrin A2, schlafen 4, early growth response 2, retinoblastoma-associated factor 600, receptor tyrosine kinase-like orphan receptor 2, cyclin-dependent kinase 7 (homolog of Xenopus MO15 cdk-activating kinase), inhibitor of growth family member 4 and neoplastic progression 1 were up-regulated and connective tissue growth factor, ephrin B2, neural proliferation differentiation and control gene 1, nerve growth factor beta, Eph receptor A2, neoplastic progression 3, cyclin G1, bone morphogenetic protein receptor type 1A, cell division cycle 34 homolog (S. cerevisiae), cyclin-dependent kinase inhibitor 1A (p21), SGT1 suppressor of G2 allele of SKP1 homolog (S. cerevisiae), nuclear casein kinase and cyclin-dependent kinase substrate, G two S phase expressed protein 1, prohibitin, nucleostemin, CDK2-associated protein 1 and block of proliferation 1 This is not a surprise since transformed cells grow more rapidly in vitro than during tumour development. Angiogenesis-involved genes Some proangiogenic genes such as angiopoietin-like 4, selectin, endothelin 1, angiopoietin 2 and endothelial PAS domain protein 1 were up-regulated during tumour growth whereas the antiangiogenic factor thrombospondin 1 was found to be down regulated. Surprisingly, the proangiogenic endothelial cell growth factor 1 (platelet-derived) and the angiomotin like 2 were found down-regulated. Cytoskeleton and cell-to-cell contact genes Expression of some genes involved in cytoskeleton and cell-to-cell contact was modified in tumours. mRNAs encoding junctophilin 3, fibrillin 2, plexin A3, ARP1 actin-related protein 1 homolog A, MYOSIN-IXA homolog, myosin IB, tubulin alpha 1, cadherin 3, integrin beta 5, dynamin and stathmin-like 4 genes were up-regulated whereas mRNAs encoding MAP/microtubule affinity-regulating kinase 2, nucleoporin 54 kDa, smooth muscle cell associated protein-1, plakophilin 2, annexin A3, myosin X, ARP1 actin-related protein 1 homolog B (yeast), follistatin, golgi associated gamma adaptin ear containing ARF binding protein 2, filamin beta, vinculin, cytoskeleton-associated protein 1, annexin A11, lamin A, cortactin, pericentrin 2, gap junction membrane channel protein alpha 1, importin 4, nucleolar protein 5, exportin 7, vinculin, actinin alpha 1, nucleoporin 88, fibulin 2 and CD44 antigen were down regulated. Extracellular matrix compounds In cancer, transformed and mesenchymal cells synthesize and secrete several compounds which participate to tumour organization. In tumour cells, genes encoding the procollagen type XVIII alpha 1, Nice-4 protein homolog isoform 1, glycophorin A, collagen type V alpha 1 and procollagen type VI alpha 1 were up-regulated whereas, to our surprise, procollagen type V alpha 2 was found down regulated. Metabolic enzymes and secretory factors Finally, an interesting finding is that the majority of genes encoding enzymes involved in metabolism were down regulated in tumours whereas the majority of secretory factors or their receptors were up-regulated (see additional files 1 and 2 ) suggesting some reduction of intracellular metabolic activity and increased signal exchanges during tumour formation. Conclusion The microenvironment of the metastatic cancer cell and the interaction between these cells and the stroma play critical roles in tumour development and progression. However, the molecular mechanisms and genes involved in tumour development remain largely unidentified. In the experimental model of tumour formation used in this study, we identified 489 genes whose expression is modified during tumour formation. Among them, 213 were up-regulated and 276 were down-regulated. These genes are involved in a variety of cellular functions, including control of transcription, mRNA processing, regulation of translation, activation of some interferon-induced genes, intracellular signalling, apoptosis, cell growth, angiogenesis, cytoskeleton, cell adhesion, extracellular matrix formation, metabolism and production of secretory factors. These results can be interpreted in two ways i/ many cellular functions need to be adapted to allow successful tumour development or ii/ successful tumour formation has induced changes in gene expression. In fact, both interpretations are probably correct but the important point is that among them are found the genes involved in adaptation of the cancer cell to a new environment, which are potential targets for cancer therapy. This study therefore suggests that, after screening ~12,000 genes, the most interesting candidates for clinical applications are among the 213 genes up-regulated in the tumour. Material and Methods Transformation of MEFs by retroviral infection Primary embryo fibroblasts were isolated from 14.5 day-old SV129J mouse embryos and grown in Dulbecco's modified Eagle's medium supplemented with 10% foetal calf serum as previously described [ 7 ]. We transduced MEFs with the pBabe-ras V12 /E1A retroviral vector which expresses both the ras V12 mutated protein and the E1A oncogene to obtain transformed fibroblasts. pBabe-ras V12 /E1A plasmids were obtained from S. Lowe. Bosc 23 ecotropic packaging (10 6 ) cells were plated in a 6-well plate, incubated for 24 hr, and then transfected with PEI with 5 μg of retroviral plasmid. After 48 hr, the medium containing the virus was filtered (0.45 μm filter, Millipore) to obtain the retroviral supernatant. MEFs were plated at 2 × 10 5 cells per 35 mm dish and incubated overnight. For infection, the culture medium was replaced by an appropriate mix of the retroviral supernatant and culture medium (V/V), supplemented with 4 μg/ml polybrene (Sigma), and cells were incubated at 37°C. Transformation of MEFs by the pBabe-ras V12 /E1A retroviral vector was evaluated by examining changes in their morphological aspect, by quantifying expression of the RAS protein by western blot, by monitoring cell proliferation, colony formation in soft-agar and tumours in nude mice as previously described [ 7 ]. Tumour induction in athymic mice Suspensions of the pBabe-ras V12 /E1A transformed MEFs (10 6 /200 μl PBS) were injected subcutaneously into the flank of male 8 week-old nu/nu mice, and tumours were allowed to develop for 20 days. Tumours were removed and stored at -80°C. Microscopical analysis reveals that tumours contain about 15% of vascular and stromal cells. Microarray Total RNA from ras V12 /E1A-transformed cells and tumours from three independent experiments was isolated by Trizol (Gibco-BRL). Twenty μg of total RNA was converted to cDNA with SuperScript reverse transcriptase (Gibco-BRL), using T7-oligo-d(T) 24 as a primer. Second-strand synthesis was performed using T4 DNA polymerase and E. coli DNA ligase followed by blunt ending by T4 polynucleotide kinase. cDNA was isolated by phenol-chloroform extraction using phase lock gels (Brinkmann). cDNA was transcribed in vitro using the T7 BioArray High Yield RNA Transcript Labeling Kit (Enzo Biochem, New York, N.Y.) to produce biotinylated cRNA. Labelled cRNA was isolated using an RNeasy Mini Kit column (Qiagen). Purified cRNA was fragmented to 200–300 mer cRNA using a fragmentation buffer (100 mM potassium acetate-30 mM magnesium acetate-40 mM Tris-acetate, pH 8.1), for 35 min at 94°C. The quality of total RNA, cDNA synthesis, cRNA amplification and cRNA fragmentation was monitored by micro-capillary electrophoresis (Bioanalizer 2100, Agilent Technologies). The cRNA probes were hybridized to an MG u74Av2 Genechip (Affymetrix, Santa Clara, CA). Fifteen micrograms of fragmented cRNA was hybridized for 16 h at 45°C with constant rotation (60 rpm). Microarrays were processed in an Affymetrix GeneChip Fluidic Station 400. Staining was made with streptavidin-conjugated phycoerythrin (SAPE) followed by amplification with a biotinylated anti-streptavidin antibody and a second round of SAPE, and then scanned using an Agilent GeneArray Scanner (Agilent Technologies). The signal intensities for the β-actin and GAPDH genes were used as internal quality controls. The ratio of fluorescent intensities for the 5' and 3' ends of these housekeeping genes was <2. Scanned images were analyzed with the Microarray Suite 5.0 software (Affymetrix). Validation of gene expression profiles One microgram of total RNA from primary embryo fibroblasts, ras V12 /E1A transformed MEF and its derived tumour was subjected to PCR with reverse transcription using the One Step RT-PCR kit (Promega) according to the manufacturer's protocol. Selected RNA species were amplified using the following primers: arginase 1, sense, 5'-gaaaaggccgattcacctgag-3' and antisense, 5'-atgtggcgcattcacagtcac-3'; bone morphogenetic protein 10, sense, 5'-ggatctggacctggactcaga-3' and antisense, 5'-gaagctttctgggaattcttg-3'; cathepsin S, sense, 5'-gaagggctgcgtcactgaggt-3' and antisense, 5'-acaccgcttttgtagaagaag-3'; insulin-like growth factor binding protein 4, sense, 5'-gaaggtgtagagtagaggctc-3' and antisense, 5-ggaccagaatggggccattcc-3'; interferon stimulated gene 12, sense, 5'-ctcaacatgttgggaacactg-3' and antisense, 5-catctcctgcgtagtctgtac-3'; serum deprivation response, sense, 5'-gtctagtattataacctaacc-3' and antisense, 5-aagagtagagagttcgagccc-3'; thrombomodulin, sense, 5'-cagaaatttcaggtaaccaaa-3' and antisense, 5-tcagctcggcacgaagcacac-3'; adenylate kinase 1, sense, 5'-cactgggtgccaaggagctgt-3' and antisense, 5-ggcttcctgtgtaatgagacc-3'; connective tissue growth factor, sense, 5'-ggagtcagagccttgtctgtt-3' and antisense, 5-agtcataatcaaagaagcagc-3'; and S100 calcium binding protein A11, sense, 5'-gctgttttccaaaagtacagc-3' and antisense, 5-cgcttctgggaagtttggatg-3'. Reverse transcription was carried out 45 min at 48°C followed by 25–32 cycles of PCR, each cycle consisting in a denaturing step for 1 min at 94°C, an annealing step for 2 min at 56°C, and a polymerization step for 2 min at 72°C. PCR products were separated on a 1.0% agarose gel containing ethidium bromide and photographed under ultraviolet light. Authors' contributions SV prepared cells and retroviruses, CM carried out RNA purification and RT-PCR analysis, ELC was in charge of microarray hybridization, JCD participated in the design of the study, JLI participated in the analysis of data and wrote the manuscript. Supplementary Material Additional File 1 Genes up-regulated during tumour development . Genes found up-regulated by microarray analysis are listed, with their GenBank accession number, the over-expression factors (relative to ras V12 /E1A transformed MEFs) observed in three separate experiments. Click here for file Additional File 2 Genes down-regulated during tumour development . Genes found down-regulated by microarray analysis are listed, with their GenBank accession number, the down-regulation factors (relative to ras V12 /E1A transformed MEFs) observed in three separate experiments. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC546195.xml
545488	General practitioners' attitudes towards research in primary care: qualitative results of a cross sectional study	Background Research in General Practice requires the participation of General practitioners (GPs). In Germany there is little tradition of research in this field, and GPs are not used to be participants in research. Little is known about German GPs attitudes towards research. Therefore the aim of our study was to assess the willingness of German General Practitioners to participate in primary care research and their attitude towards research in general practice. The results should enable a more successful approach to GPs in further studies. Methods Cross sectional study using semi-structured interviews with a random sample of 76 General Practitioners who participate in the teaching of medical students at the University of Heidelberg. Results Despite little experience, over 85 % of GPs appreciated research in their field. Important reasons for scepticism about research were the gap between theoretical research and practical work of GPs and the domination of research by specialists. Main barriers for participation are clinical workload, administrative overload and the newly introduced Disease Management Programs. The highest motivation for GPs to participate in research emanates from the will to substantiate their quality of care with solid research data. Conclusions Financial incentives and personal support e.g. with study nurses are certainly necessary to establish a research culture and to overcome main barriers against participation. The most successful approach to motivate GPs to participate is to convince them that research documents their quality of care. This data may reflect the facts on which the financial resources are provided in the future health care system.	Background Compared to other European countries, Germany still has little of a research tradition in general practice. Increasingly policy-makers have realized that the continuity and the efficacy of the healthcare system have to be improved. For this a well-developed primary medical care system is needed. In recent years a number of new chairs of general practice have been established and a national funding programme was created in order to promote General Practice as an academic discipline in Germany. The University of Heidelberg, which is the oldest university in Germany, is among the beneficiaries of these developments. Despite of a well established network of teaching practices, the research group for general practice and health services research was only created in 2002 [ 1 ]. This group faces the challenge to perform studies with general practitioners who have little experience with participation in scientific research. It is known, that by specifically addressing strategies significant improvements in participation rates can be achieved [ 2 ]. The aim of the study was to investigate the willingness of GPs to participate in research and to learn about their attitude towards research in their field in general. These data should help to create successful approaches for further projects. Methods Study design We performed a cross sectional observational study collecting qualitative data. The Ethical Commission of the University of Heidelberg approved the study. Study population A random sample of 76 GPs in the area of Heidelberg was approached for the study. The GPs were selected by choosing every third of an alphabetical list of 250 practices. These GPs were associated with the university by frequently teaching students in their practices. Due to old data, in six cases the GPs did not practice any more. So finally 76 GPs were included. All of the selected GPs were in practice for more than five years. Former studies indicated that relevance of the topic has a positive predictive value for the recruitment rate. Therefore we selected a topic with a high clinical relevance in daily practice: osteoarthritis [ 3 , 4 ]. Based on this information we performed a fictitious study, aiming at improving the quality of care of patients with osteoarthritis. The GPs received an official letter from the Department of General Practice and Health Services Research. This letter contained detailed information about the relevance of the topic, the aim of the study and the possible benefit for GPs, their teams and their patients. They were also informed about the time requirement for the study, which was estimated to be 30 minutes. The allowance for participating was fixed to 50 Euro to exclude financial reasons for consent. The letter concluded with the request to fax an agreement form back to the university. Measures No letters were returned to the university because of wrong addresses. A reminder or anything similar did not follow the first letter. One week after the letter, every GP was called by the principal investigator and was asked – after giving him again information on the study – if she or he wanted to participate. This approach was chosen to get qualitative information of all approached GPs about their willingness to participate and their opinion in general. This way of data collecting has already been used in this field of research and enables not only a high rate of data response, it is also a feasible way of collecting qualitative data [ 5 ]. If the GP decided not to participate, her or his reason to do so were recorded without further discussion. Every GP, whether he denied or agreed to participate was asked about his opinions concerning research in general practice in general and the relevance of the research topic to him or her. The GPs who agreed to participate where asked to fax the sheet of agreement. Analysis We were mainly focused on qualitative information. Therefore the statements of the GPs were grouped and coded by two separate researchers and then discussed in order to agree on the selected categorisation according to the guidelines for qualitative researchers [ 6 ]. Results A total of 18 GPs (23.8%) of the approached GPs was female, 58 GPs (72.2 %) were male. Only two GPs faxed their agreement-sheet within the first week, before they were phoned and interviewed by the principal investigator. During the telephone calls 25 GPs (32.8 %) agreed to participate and promised to fax the sheet. Out of this group 5 GPs (18.5%) sent their fax during the subsequent two weeks. A total of 8 (10.5 %) faxes were returned. Five female (27.7 %) and 22 male GPs agreed (37.9 %) to participate. A total of 27 GPs agreed to participate ultimately. Table 2 shows the GPs reasons for non-participation. 24 (31.5 %) of the GPs argued they had no time, because of overwork in their practice caused by the daily routine work. The second most frequent reason named was the regular administrative workload. Seven GPs specified this argument by blaming especially the newly introduced "disease management program, DMP", founded by German sick funds for chronic illnesses like diabetes and hypertension. This program was perceived to increase the daily paperwork tremendously. Other important reasons for non-participation were disbelief that possible results can be implemented in daily work without financial incentives. GPs argued that changes, which are accompanied by any additional time effort, could only be implemented in daily practice if they receive adequate financial reimbursement. "Money sets the course", as one GP stated. Two GPs declared they had no problem in dealing with osteoarthritis and regarded also dealing patients suffering from osteoarthritis quite easy. Four GPs named participation in courses and congresses as a reason for non-participating. One GP mentioned that this kind of research is only for academic interest and helps only the career of the researcher. An other GP argued that he already feels monitored by all the data collected by health insurance and the government. Table 2 Reasons mentioned by GPs for non-participation in research n % Overwork in practice 24 31.6 Already too much paperwork / bureaucracy 13 17.1 The results might not be implemented in practice because of financial constrains 10 13.2 Overload because of "disease management program" 7 9.2 No belief in results because of the degenerative progress of the illness 5 6.6 Personal time exposure for courses, etc. 4 5.3 Private reasons 2 2.6 Adherence to an other study at the same time 2 2.6 To less connection between (theoretical) university research and practical work as a GP 2 2.6 No problem in treating arthritis patients 2 2.6 No decision 1 1.3 Feeling of being monitored 1 1.3 Only the researcher takes benefit out of this research 1 1.3 Total 76 100 As can be seen in table 3 , 85.6 % of the GPs had positive attitudes regarding research in their field. They consider it reasonable and eligible, but in most of these cases the answer was not substantiated with a further argumentation. Interestingly, answers, which were allocated to the category „makes sense because it improves the reputation of GPs and documents our quality of care", were only given by GPs who agreed to participate in the study. So this aspect seemed to be the most important motivation for an GP to take part in research. In addition, this particular group of GPs regularly added further comments regarding role of the GP in the German health care system. Important reasons for scepticism were the gap between theoretical research and practical work and the domination of research by specialists. One GP argued it would be better to spend more money on treatment than on research. Table 3 GPs' attitudes regarding research in General Practice in general n % Reasonable and eligible 54 71.1 Makes sense because it improves the reputation of GPs 11 14.5 Not sure if it makes sense ("I am not convinced"), no further explanation 3 3.9 University research and daily work in family medicine have only little in common 3 3.9 Makes no sense because research is dominated by specialists 2 2.6 Does not lead to results (without more explanation) 1 1.3 Better more money for the GPs then for research 1 1.3 Feeling of being monitored 1 1 Total 76 100 Discussion There were three main conclusions that can be drawn out of our interview results. Firstly, the research topic improving the quality of care for patients suffering from osteoarthritis was considered as highly relevant by the interviewed GPs. This is concordant to our assumptions based on epidemiological data, which led to the fictitious research topic. The same reasoning causes GPs to seek support in the daily treatment of patients with osteoarthritis. Consequently this will be subject of future research projects. Secondly, most of the GPs appreciate research in general practice, but a few were very sceptical. German GPs still don't realise it as a professional obligation as their colleagues in countries like e.g. the Netherlands or the United Kingdom, with a much longer tradition in research, do [ 7 ]. The third main result of our survey has not yet been shown in former studies. It is the fact that the willingness for participating in research emanates mainly out of the motivation to improve the reputation of family medicine in general by documenting the high quality of care with data attained in solid surveys. This may reflect the increasing self-confidence of German GPs, which are about to expend the influence in the health care system, and their awareness that an own research culture helps to enhance this. Facing decreasing financial resources in the Health care system, GPs may also be aware that a solid database documenting the quality of care will get more important for the distribution of financial resources in the near future. The revealed barriers against participating in studies mentioned in our telephone survey are in line with results from previous studies in other countries [ 8 ]. According to those former results, relevance of the research topic, reimbursement and compatibility with routine general practice work are important factors. Ideally the GPs are embedded in an existing research culture [ 7 , 9 , 10 ]. Study nurses or mentors could be an important factor to enhance GPs' preparedness to participate in General Practice research because they reduce the administrative workload for GPs and enhance the motivation to participate in research [ 8 , 10 , 11 ]. Furthermore financial incentives for participation are essential because of time constraints and overwhelming administrative work that compete with research and represent important barriers [ 8 , 11 ]. An unexpected quantitative result of this study was that being involved with the training of medical students and being linked with the University is not reflected per se in a higher motivation in participating in research. Participating rates of about 30 % are usually achieved in random postal mailings to GPs without academic affiliation [ 5 , 9 , 13 , 14 ]. Previous studies have shown that involvement in student teaching represents a positive predictive factor for participation in research, so we assumed to achieve a much higher participation rate. It appears that a well-established teaching network does not necessarily yield much benefit for research purposes [ 11 ]. Conclusions Previous studies were mainly focused on formal or external barriers for GPs against participating in research, or revealed approaches that cannot easily be transferred, as e.g. the enrollment of friendly GPs [ 15 ]. What this study adds is that there is an important target to aim at, if GPs have to be involved in research: the motivation to underline their daily work with solid data reflecting their high quality of care. With this knowledge GPs may be easier approached if they need to be motivated to participate in future projects. Aiming more on psychological targets, this approach should be transferable to other countries as well. However, researchers should be aware that beside the chance of motivating GPs, this strategy also contains a risk: GPs could be discouraged and kept away from future participation if the anticipated demonstration of their quality of care is not as obvious as expected. Competing interests The author(s) declare that they have no competing interests. Authors' contributions TR conceived and performed the study and draft the manuscript. JS participated in the study design. All authors read and approved the final manuscript. Table 1 Agreement for participation related to sex Sex n Agreement after letter Agreement during telephone call Total agreement Male 58 2 20 22 (37.9%) Female 18 0 5 5 (27.8%) Total 76 2 (2.6 %) 25 (32.8 %) 27 (35.5 %) Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC545488.xml
535812	Adoptive immunotherapy of cancer with polyclonal, 108-fold hyperexpanded, CD4+ and CD8+ T cells	T cell-mediated cancer immunotherapy is dose dependent and optimally requires participation of antigen-specific CD4 + and CD8 + T cells. Here, we isolated tumor-sensitized T cells and activated them in vitro using conditions that led to greater than 10 8 -fold numerical hyperexpansion of either the CD4 + or CD8 + subset while retaining their capacity for in vivo therapeutic efficacy. Murine tumor-draining lymph node (TDLN) cells were segregated to purify the CD62L low subset, or the CD4 + subset thereof. Cells were then propagated through multiple cycles of anti-CD3 activation with IL-2 + IL-7 for the CD8 + subset, or IL-7 + IL-23 for the CD4 + subset. A broad repertoire of TCR Vβ families was maintained throughout hyperexpansion, which was similar to the starting population. Adoptive transfer of hyper-expanded CD8 + T cells eliminated established pulmonary metastases, in an immunologically specific fashion without the requirement for adjunct IL-2. Hyper-expanded CD4 + T cells cured established tumors in intracranial or subcutaneous sites that were not susceptible to CD8 + T cells alone. Because accessibility and antigen presentation within metastases varies according to anatomic site, maintenance of a broad repertoire of both CD4 + and CD8 + T effector cells will augment the overall systemic efficacy of adoptive immunotherapy.	Introduction Cancer immunotherapy, using T lymphocytes that recognize tumor-specific antigens, holds great promise. Advantageous features include: exquisite specificity for targeted antigens, thereby sparing normal tissues, and the ability of effector T cells to traffic to tumor in all anatomic locations. Although most effector T cells are subject to activation-induced cell death (AICD), a memory response is established leading to sustained protection [ 1 ]. Despite the theoretical appeal of T cell-mediated immunotherapy, clinically relevant benefits have been documented in only a small subset of human cancer patients who present with metastatic disease [ 2 - 5 ]. Several factors contribute to the poor host immune response including defective Antigen Presenting Cell (APC) function in cancer patients, and production of immunosuppressive substances by tumors [ 6 - 8 ]. Cognizant of these features, many preclinical studies of active immunotherapy have used a vaccination/challenge scheme to avoid tumor-induced immunosuppression or have alternatively treated hosts with minimal tumor burdens. Several human clinical trials have similarly focused on hosts with minimal residual disease in order to define the magnitude and characteristics of the immune response. These studies have clearly established that immune responses are successfully generated in vaccinated cancer patients. However, the frequency of responding T cells is typically less than one percent even after multiple cycles of vaccination [ 9 - 14 ]. In contrast, the immune response to pathogens generates a tremendous amplification of reactive T cells [ 15 - 17 ]. In the clinical setting, relatively little is yet known about the magnitude of proliferation of individual precursor cells (burst size) as they mature into effector cells, or the flux between lymphoid tissue, peripheral blood, and tumor sites. This results in ambiguity about the optimal time and site to quantify the immune response. Likewise, analysis of apoptosis of effector cells is likely to be important [ 18 , 19 ]. These gaps in fundamental knowledge have made it difficult to identify components of active immunotherapy that could be enhanced to boost the aggregate immune response to a therapeutic level. Adoptive immunotherapy is another approach to cancer immunotherapy that circumvents some of the limitations of active immunotherapy. Animal tumor models have convincingly demonstrated that hosts bearing progressively growing weakly immunogenic tumors nevertheless generate sensitized T cells in TDLN [ 20 ]. Antigen-sensitization causes T cells to downregulate expression of L-selectin (CD62L) providing a convenient phenotypic marker for segregation of primed T cells from the majority of irrelevant T cells [ 21 - 23 ]. Our previous studies have demonstrated that ex vivo activation of purified CD62L low T cells from TDLNs generates potent effector CD4 + and CD8 + T cells that can mediate regression of advanced tumors in every tested anatomic location [ 24 - 26 ]. The high potency of such cells permitted brief 5-day activation and limited numerical amplification (10-fold) to supply sufficient quantities of cells for the previous mechanistic analysis of the anti-tumor response. Importantly, these experiments demonstrated that there is tight dose dependence, oftentimes with even a three-fold reduction in the number of transferred cells accounting for a difference between minimal treatment effect and complete cure. The relative efficacy of CD4 + versus CD8 + effector cells also varies considerably between pulmonary metastases and intracranial (i.c.) or subcutaneous (s.c.) tumors [ 27 ]. This indicates that maintenance of CD4 + as well as CD8 + tumor-reactive effector T cells would be required for optimal adoptive immunotherapy against disseminated metastatic disease. We investigated whether we could overcome quantitative limitations associated with active immunotherapy through extensive numerical expansion of effector cells. In a previous study, we determined that in vitro activation of tumor-sensitized L-selectin low precursors with anti-CD3 mAb and high concentrations of IL-2 (100 U/ml) induced rapid proliferation of CD8 + effector cells [ 28 ]. Adoptive transfer of such cells cured established tumors in recipients. However, these culture conditions led to maximal proliferation in 9 days with subsequent decline in cell numbers thus limiting the total expansion to approximately 10 3 -fold. In this report, we define ex vivo activation conditions that permit numerical expansion of either CD4 + or CD8 + effector T cells to greater than 10 8 -fold while retaining their high therapeutic potency and preserving a broad T cell receptor (TCR) repertoire. Materials and methods Mice and tumors Female C57BL6N (B6) mice were purchased from the biologic Testing Branch, National Cancer Institute (Frederick, MD). They were maintained in a specific pathogen-free environment according to National Institute of Health guidelines. Mice were used for experiments at 8–10 weeks of age. The MCA 205 and MCA 207 fibrosarcomas, syngeneic to B6 mice were serially passaged in vivo s.c. as described previously [ 29 ]. Preparation and culture activation of TDLN CD62L low cells Tumors were established by s.c. flank inoculation of 1.5 × 10 6 MCA 205 cells and 12 days later the TDLNs were removed and mechanically disrupted to obtain a single cell suspension. The TDLN cells were incubated with 100 μl anti-CD62L microbeads per 10 8 cells and applied to MACS columns (Miltenyi Biotech, Auburn CA) and the flow through fraction was collected. For CD4 + hyperexpansion, the CD62L low subset was depleted of CD8 + cells by MACS on day 0 and day 36 of culture activation. CD62L low cells, containing approximately 50% TCR + and 50% B220 + subsets, were suspended in complete medium (CM) and incubated for 2 days at 4 × 10 6 per well in 24 well culture plates coated with anti-CD3 (145-2C11) as previously described [ 28 ]. Activated cells were washed, counted, and suspended at 0.5 × 10 5 /ml in CM with IL-2 (4 U/ml) (Chiron Corp. Emeryville, CA), with or without rmIL-7 (10 ng/ml) or rhIL-23 (2 ng/ml) (each from R&D Systems, Minneapolis, MN) and then diluted to 10 5 /ml on day 5 of activation. On days 9 and 15, the cell concentration was adjusted to 2 × 10 5 /ml. For experiments with two cycles of anti-CD3 stimulation, T cells were incubated with immobilized anti-CD3 for 14 hrs on day 15 and used for adoptive therapy on day 23. For long-term expansion, cultures were maintained for 23 days after the initial anti-CD3 stimulation in CM with the indicated combination of IL-2 (4 U/ml), IL-7 (10 ng/ml), and IL-23 (2 ng/ml) and then were stimulated with anti-CD3 for 14 hrs on day 23 and every 7 days thereafter. IFN-γ and FACS analysis T cells were stimulated with a single cell suspension of either MCA 205 or MCA 207 tumors at a 1:1 ratio, or with immobilized anti-CD3. Brefeldin A was added after five hours of stimulation and the cells were harvested after 20 hrs and stained for intracellular IFN-γ according to the manufacturers instructions (BD Biosciences, San Diego, CA). FACS analysis was performed using FITC or PE conjugated antibodies or isotype control antibodies (BD Biosciences). RNA isolation and CDR3 size distribution analysis (TCR spectratyping) TDLN cells were lysed using TRIzol reagent (Invitrogen, Carlsbad, CA) and total RNA was reverse transcribed into cDNA using the SuperScript II RT kit (Invitrogen). cDNA was amplified using PCR with 22 different VB-specific primers paired with a hex-labeled constant region primer which spans the CDR3 region as previously described [ 30 ]. CDR3 size distribution analysis was performed by mixing 1.0 μl of hex-labeled PCR amplified cDNAs with 12.0 μl deionized formamide (Sigma) and 0.5 μl size standard (Genescan-400 ROX, ABI 310; Perkin-Elmer, Shelton, CT), heated for 2 minutes at 90°C and chilled on ice prior to analysis. Samples were applied to an ABI 310 sequencer for CDR3 size distribution analysis. Samples were determined to be oligoclonally skewed if the CDR3 size patterns failed to exhibit a Gaussian bell-shaped distribution and were dominated by one or two prominent peaks. Adoptive immunotherapy Mice were inoculated with MCA 205 or MCA 207 tumor cells (3 × 10 5 ) i.v. to establish pulmonary metastases. Subcutaneous tumors were established by inoculation of 1.5 × 10 6 cells. Intracranial tumors were established by transcranial inoculation of 10 5 tumor cells at a depth of 4 mm as previously described [ 31 ]. Mice bearing 3-day s.c. or i.c. tumors or 10-day pulmonary metastases were treated with 5 Gy nonmyeloablative total body irradiation (TBI) delivered from a 137 Cs irradiator prior to intravenous transfer of the T cells whereas mice with 3-day pulmonary tumors were not irradiated. For pulmonary tumors, mice were euthanized on day 20 post inoculation, the lungs were insufflated with India ink and the number of surface tumor nodules was enumerated using a dissecting microscope. Subcutaneous tumors were measured in two perpendicular dimensions three times per week and mice with progressive tumors were euthanized when the product of dimensions exceeded 200 mm 2 . Mice bearing intracranial tumors were monitored daily for survival or were euthanized when neurologic symptoms such as decreased grooming and decreased spontaneous movement were apparent. Statistical analysis Treatment groups consisted of five individuals. Analysis of tumor size for s.c. tumors was performed by the Mann-Whitney rank sum test. For pulmonary tumors, a t test was performed on paired samples and p < 0.05 was considered significant. Survival of mice bearing i.c. tumors was compared using the Wilcoxon rank sum test. Results Ex vivo stimulation with anti-CD3, IL-2, and IL-7 augments effector cell generation During the progressive growth of weakly immunogenic tumors an immune response, albeit sub-therapeutic, is initiated in TDLNs. In previous studies our laboratory has demonstrated that the CD62L low subset of T cells contains the tumor-reactive subset whereas the reciprocal CD62L high subset does not have any therapeutic effect and displays suppressor activity [ 1 , 24 , 32 ]. This finding is consistent with numerous studies documenting the high expression of CD62L on naïve T lymphocytes and its rapid downregulation upon antigen stimulation [ 21 - 23 ]. TDLNs were harvested from mice bearing 12-day subcutaneous MCA 205 tumors and the CD62L low subset was purified by MACS depletion of CD62L high cells. The typical yield of CD62L low cells was 1.5 × 10 6 per TDLN, which represented 7–8.5% of the initial cells. The phenotype of the total TDLN prior to MACS separation and the negatively selected CD62L low subset is demonstrated in Figure 1A . The separated cells were highly enriched for the CD62L low fraction consisting of 36% TCR + cells among which 7% were CD8 + and 22% were CD4 + . The cells were activated with immobilized anti-CD3 mAb for 48 hrs during which time aggregates of lymphoblasts developed. The activated cells were resuspended at a low density, 0.5 × 10 5 /ml, in medium supplemented with IL-2 (4 U/ml) with or without IL-7 (10 ng/ml) and the cell concentration was adjusted to 10 5 /ml on day 5 and 2 × 10 5 /ml on day 9. As demonstrated in figure 1B , there was an initial 2-fold decline in cell number during the first 48 hrs of culture, primarily though depletion of CD62L low B220 + cells. This was followed by a rapid burst of proliferation from day 2 until day 15 when the IL-2 supplemented culture peaked at 175-fold proliferation and the IL-2 + IL-7 cultures reached 1000-fold proliferation. Figure 1 Proliferation and efficacy of CD62L low TDLN cells cultured with IL-2 +/- IL-7. (A) Freshly isolated whole TDLN cells were stained for expression of TCR and CD62L (left panel). The purified CD62L low subset was stained for TCR and CD62L expression (center panel), or for CD4 and CD8 expression (right panel). (B) CD62L low TDLN cells were activated with immobilized anti-CD3 mAb for 2 days then cultured in medium with IL-2 (4 U/ml) (closed circle) or the combination of IL-2 and IL-7 (10 ng/ml) (open circle). Cells density was adjusted to 10 5 /ml on days 5 and 9 and total proliferation was calculated. (C) Mice bearing 3-day s.c tumors were treated with 5 Gy TBI then received adoptive transfer of 5 × 10 6 T cells cultured for 9 days with IL-2 alone (open circle), IL-2 + IL-7 (open triangle), or HBSS (closed circle). Each treatment group is significantly different than HBSS control ( P < 0.01). The morphology of the cells changed from lymphoblastoid to small round cells at day 15 and there was no additional proliferation. IL-7 preserved the viability of cells whereas IL-2 alone could not prevent a 8-fold numerical decline between days 15 to 30. Because the TDLN cells were initially segregated based on phenotype rather than antigen specificity and the anti-CD3 stimulation was antigen-independent, it was not known whether the enhanced proliferation achieved in the presence of IL-7 was due to preferential growth of irrelevant T cells or preservation of tumor-reactive T cells. As demonstrated in figure 1C , there was equivalent therapeutic efficacy against s.c. tumors at day 9 using T cells cultured with IL-2 alone or the combination of IL-2 and IL-7. Regression of established tumors requires efficient trafficking and the dose of 5 × 10 6 CD62L low TDLN cells is near the lowest threshold dose required to cure 3-day s.c. MCA 205 tumors [ 26 , 33 ]. Thus, the addition of IL-7 during in vitro activation augmented the total number of cells but did not substantially diminish per-cell therapeutic efficacy. Preservation of effector function after anti-CD3 re-stimulation The activated T cells were re-stimulated with anti-CD3 at the time of maximal proliferation on day 15 to determine whether additional numerical expansion could be initiated. Re-stimulated cells rapidly regained lymphoblast morphology and as anticipated nearly half of the cells underwent AICD [ 34 ]. The surviving cells underwent a 100-fold numerical expansion before achieving a growth plateau (Figure 2A ). In addition, the composition of the T cell cultures changed over time due to the more rapid intrinsic proliferative response of CD8 + T cells [ 35 ]. Although CD4 + and CD8 + T cells each proliferated in the presence of IL-2 and IL-7, by day 23 CD8 + cells comprised 86% of the culture whereas there were only 4% CD4 + cells (Figure 2B ). The re-stimulated cells cultured in IL-2 or the combination of IL-2 plus IL-7 each retained potent therapeutic efficacy against 10-day MCA 205 pulmonary metastases but not against MCA207, demonstrating retention of antigenic specificity (Figure 2C ). The hosts bearing 10-day pulmonary tumors were treated with 5 Gy TBI, which causes transient lymphopenia, prior to adoptive transfer. Moreover, hosts did not receive adjunctive IL-2. Thus, the transferred cells were able to function independently of radiosensitive host cells and were not dependent on exogenous cytokine support. Because the combination of IL-2 plus IL-7 promoted greater numerical expansion of CD8 + T cells with preservation of effector function, it was used for subsequent hyperexpansion studies. Figure 2 Restimulation of activated T cells induces additional proliferation with retention of specific anti-tumor efficacy. (A) CD62L low TDLN cells were activated with anti-CD3 mAb from day 0–2 and again for 14 hrs on day 15. T cells were cultured in the presence of IL-2 (4 U/ml) (closed circle) or IL-2 plus IL-7 (10 ng/ml) (open circle) and the total proliferation is indicated. (B) FACS analysis of activated T cells on day 23 of culture stained for CD4 and CD8. (C) Mice bearing 10-day pulmonary metastases of either MCA205 or MCA207 tumors were pre-treated with 5 Gy TBI then received adoptive transfer of 2.5 × 10 7 cells cultured with IL-2 alone, the combination of IL-2 plus IL-7, or control HBSS as indicated. Difference between the groups bearing MCA 205 treated with T cells cultured with IL-2 or IL-2 plus IL-7 and all other groups is ( P < 0.01). (D) Mice bearing 3-day s.c tumors were pre-treated with 5Gy TBI followed by injection of; HBSS (closed circles), 5 × 10 6 T cells activated for 5 days (open circles, P < 0.01), 5 × 10 6 restimulated T cells at day 23 of culture (closed triangles, P = 0.4), 1.5 × 10 7 re-stimulated T cells (open triangle, P < 0.01), or 4 × 10 7 re-stimulated T cells (closed square, P < 0.01). Number of mice showing complete regression in each treatment group of 5 mice is indicated in parentheses. The relative per-cell potency of re-stimulated cultures on day 23 was compared with the 5-day culture activation approach we have employed in previous studies. The segregated CD62L low TDLN cells were frozen and one aliquot was thawed and activated for a total of 23 days with anti-CD3 stimulation on days 0–2 and again on day 15. The second aliquot was thawed on culture day 18 of the first aliquot and stimulated with anti-CD3 for 48 hrs and then cultured with IL-2 and IL-7 for an additional 3 days. The two T cell cultures were synchronously harvested and transferred into hosts bearing 3-day s.c. tumors. As demonstrated in Figure 2D , whereas 5 × 10 6 cells activated for 5 days was curative in 5/5 mice, 5 × 10 6 cells cultured for 23 days had minimal therapeutic effect. However, a modest increase in the cell dose to 1.5 × 10 7 cells led to a significant therapeutic effect and at a dose of 4 × 10 7 cells 2/5 mice were cured. The s.c. tumor model is highly dependent on the presence of tumor-specific CD4 + T cells [ 36 , 37 ]. The relative decrease in percentage of CD4 + cells from 24% on day 5 of culture to 4% on day 23 may account for some of the differential therapeutic effects. In contrast to the modest difference in per-cell efficacy, there was nearly 1000-fold greater proliferation in the 23-day versus 5-day cultures indicating that the aggregate therapeutic effect was substantially greater following extended culture. Repetitive anti-CD3 stimulation induces hyper-expansion of CD8 + effectors There are immunologic scenarios that demonstrate exhaustion of the effector response leading to failure of immunologic control of infection or tumor [ 38 - 40 ]. By contrast, selection and extensive propagation of T cell clones indicates that T cells can undergo massive proliferation yet retain antigen-specific function. To assess whether there is an intrinsic limit to the retention of in vivo effector function of CD62L low cells, they were stimulated with anti-CD3 followed by IL-2 and IL-7 for 23 days. Starting on day 23, the T cells were activated with anti-CD3 every 7 days. The time course between the initial and subsequent anti-CD3 stimulations was chosen based on evidence that T cells undergo changes in gene expression, phenotype, and function over a twenty-day time course in the transition from naïve to memory cells [ 41 ]. As demonstrated in Figure 3A , repetitive anti-CD3 stimulation was accompanied by immediate AICD in approximately 50% of the cells followed by rapid proliferation. There was no evidence that the T cells became effete over the 50-day expansion period despite a total proliferation of 2–6 × 10 8 -fold that was consistent in three independent experiments. TCR Vβ expression was determined for freshly harvested TDLN T cells, the CD62L low subset, and activated cultures from several independent experiments (Table 1) "see additional file 1 ". As demonstrated, multiple TCR Vβ families are represented prior to and following hyperexpansion. There is a relatively low level of variability in the prevalence of multiple TCR Vβ families between experiments, especially considering that greater than 10 8 -fold total proliferation had occurred. In addition, TCR spectratype analysis for each Vβ family revealed a poyclonal rather than clonal or oligoclonal distribution (data not shown). Figure 3 CD8 + effector T cells can be hyperexpanded through repetitive anti-CD3 stimulation. (A) CD62L low TDLN cells were restimulated with anti-CD3 mAb for 14 hrs every 7 days starting on day 23 of culture and overall proliferation was measured for three independent experiments. (B) T cells were harvested on day 50 of culture and adoptively transferred to hosts bearing either MCA 205 or MCA 207 3-day pulmonary metastases ( P < 0.01 for MCA 205 tumors treated with either 6 × 10 6 or 2 × 10 7 compared with all other groups). (C) Mice bearing 3-day s.c tumors were treated with 5 Gy TBI then received adoptive transfer of the indicated number of T cells hyperexpanded for 50 days ( P = 0.06 for 4 × 10 7 cell dose) (D) Mice bearing 3-day i.c. tumors were treated with 5Gy TBI then received adoptive transfer of the indicated number of T cells hyperexpanded for 50 days. Mice were sacrificed when they developed neurologic symptoms indicating progressive tumor ( P = 0.9 for treatment groups versus control). At day 50, the cultures were >99% TCR + and CD8 + indicating preferential expansion or survival of CD8 + cells under the conditions employed. As shown in Figure 3B , adoptive transfer of 2 × 10 7 cells to hosts with 3-day MCA205 pulmonary metastases eliminated tumors and 6 × 10 6 cells was the threshold dose for complete response whereas 2 × 10 6 cells were subtherapeutic. In addition, there was no response against the antigenically distinct MCA207 tumor. In an independent experiment, the dose of T cells required to completely eliminate 3-day pulmonary metastases was 2 × 10 6 indicating some inter-experimental variability in per-cell efficacy. Because of the critical role of CD4 + T cells for therapy of s.c. or i.c. tumors it was not anticipated that the hyperexpanded CD8 + cultures would mediate complete regression of tumors at these anatomic sites. Indeed, there was substantially less efficacy against 3-day s.c.tumors. Adoptive transfer of 4 × 10 7 cells showed a trend toward response ( P = 0.061) with 1/5 mice cured in only one of two identically designed experiments (Figure 3C ). In addition, a dose of 2 × 107 cells was subtherapeutic against 3-day i.c. tumors (Figure 3D ). Despite the rapid proliferation of CD8 + T cells in vitro , there was no evidence of lymphoid hyperplasia when the mice were sacrificed to enumerate lung metastases 17 days after adoptive transfer. Moreover, there was no evidence of lymphoproliferative disease even when the hyperexpanded T cells were transferred into 5Gy TBI hosts bearing i.c. or s.c. tumors that had transient lymphodepletion of host cells. Thus, despite extensive proliferation in vitro the T cells did not demonstrate any evidence of transformation. Hyperexpanded CD4 + T effector cells mediate regression of i.c. and s.c. tumors The clinical utility of adoptive immunotherapy for patients with metastatic cancer is dependent on the ability of T cells to function at all anatomic sites of disease. To selectively activate CD4 + T cells, the CD62L low TDLN were depleted of CD8 + cells with magnetic beads prior to anti-CD3 activation and again on day 36 of culture. The CD4 + cells were activated with anti-CD3 mAb for 48 hrs and cultured in the presence of IL-7 and either IL-2 or IL-23 with anti-CD3 restimulation performed on days 26 and day 36 of culture. The rate of proliferation of CD4 + cells was similar in the presence of IL-2 or IL-23 (Figure 4A ). On day 43 of culture, cells cultured with IL-7 plus IL-2 were 87% CD4 + and 11% CD8 + , whereas cells cultured in IL-7 plus IL-23 were 98% CD4 + and less than 1% CD8 + . The T cells were adoptively transferred to hosts with 3-day s.c (Figure 4B ) or 3-day i.c. (Figure 4C ) tumors demonstrating that a dose of 3 × 10 7 cells cultured in the presence of either IL-2 or IL-23 was curative. Figure 4 Hyperexpanded CD4 + T cells mediate regression of intracranial or subcutaneous tumors. ( A) CD62L low TDLN cells were depleted of CD8 + cells prior to anti-CD3 activation and were maintained in medium with IL-2 (4 U/ml) plus IL-7 (10 ng/ml) or alternatively with IL-7 (10 ng/ml) plus IL-23 (2 ng/ml) and were restimulated for 14 hrs with anti-CD3 mAb at the indicated time points. The total proliferation with indicated losses due to AICD or re-purification of CD4 + cells is indicated. (B) Mice bearing 3-day s.c.tumors were treated with 5 Gy TBI followed by adoptive transfer of 3 × 10 7 CD4 + T cells culture activated for 43 days and tumor size was measured. On day 37, one mouse from IL-2 + IL-7 group was euthanized due to progressive tumor growth, however complete regression was observed in the remaining 4 mice (P = 0.015 versus control). Complete regression was observed in all five recipients of IL-7 + IL-23 cultured CD4 + T cells (P = 0.005 versus control). ( C ) Mice bearing 3-day intracranial tumors were treated with 5Gy TBI followed by adoptive transfer of 3 × 10 7 CD4 + T cells culture activated for 43 days. Mice were followed for survival (P < 0.01 for both treatment groups versus control). (D) Mice bearing 3-day subcutaneous tumors were treated with 5 Gy TBI followed by adoptive transfer of 4 × 10 7 CD8 + T cells hyperexpanded to greater than 10 8 -fold for 50 days, or 1.5 × 10 7 CD4 + T cells hyperexpanded to greater than 10 8 -fold for 85 days. On day 28, 4 mice from CD8 treatment group (* P = 0.39 versus control) and 2 mice from CD4 treatment group (# P = 0.019 versus control) were euthanized due to progressive tumor growth but complete tumor regression was observed in the remaining mice. The CD4 + T cells maintained in IL-7 plus IL-23 were subjected to continued repetitive anti-CD3 restimulation every 7 days starting on day 56. These conditions led to exclusive proliferation of CD4 + T resulting in 1.2 × 10 8 -fold total proliferation by day 85 of culture. Despite extensive in vitro proliferation in response to antigen-independent stimulation for 85 days, 1.5 × 10 7 CD4 cells retained efficacy against 3-day s.c.tumors, with 3/5 mice achieving complete tumor regression (P = 0.019 versus control) (Figure 4D ). As previously demonstrated, CD8 + cells synchronously cultured in the presence of IL-2 plus IL-7 for 50 days and expanded to 10 8 -fold demonstrated minimal efficacy against subcutaneous tumors (P = 0.39 versus control) with 1/5 mice achieving complete tumor regression. Hyperexpanded cultures retain IFN-γ producing cells The initial antigen priming event in vivo was driven by tumor-specific antigens but the segregation of the CD62L low subset and all subsequent in vitro activation stimuli were antigen independent. Consequently, reactivity of cultures against tumor antigens might fluctuate over time. This possibility was analyzed by quantifying the percentage of culture activated T cells that produce IFN-γ specifically when exposed to tumor in vitro . For this assay, a single cell digest of in vivo propagated tumor is used which contains MHC class II + APC as well as tumor cells and is capable of stimulating both CD4 + and CD8 + T cells. As demonstrated in Figure 5 , there was minimal spontaneous production of IFN-γ and minimal reactivity against the antigenically distinct MCA 207 tumor. By contrast, on day 8 of culture 39% of CD8 + T cells produced IFN-γ in response to MCA 205 tumor cells. This percentage of IFN-γ positive CD8 + cells decreased to 10% on day 36. CD8 + T cells that were simply maintained in culture with IL-2 and IL-7 cytokine support but without anti-CD3 restimulation did not proliferate but remained viable. Under non-proliferative conditions, the percentage of MCA 205 reactive T cells was maintained at 13% on day 36. Similarly, CD4 + T cells cultured in the presence of IL-7 plus IL-23 had 11% IFN-γ positive cells in response to MCA205 tumor with minimal spontaneous production and minimal response to MCA207 tumor digest. The in vitro assay of IFN-γ production may not be fully reflective of the in vivo capacity of T cells to mediate tumor regression because CD4 + T cells cultured with IL-2 plus IL-7 had only 2% IFN-γ positive cells despite equivalent in vivo anti-tumor efficacy. The vast majority of T cells (73–86%) produced IFN-γ in response to anti-CD3 stimulation throughout the in vitro culture activation period. Figure 5 Hyperexpanded CD4 + and CD8 + T cells produce IFN-γ in response to tumor stimulation. CD62L low TDLN cells were culture activated with anti-CD3 and IL-2 plus IL-7 for 23 days then were restimulated with anti-CD3 every 7 days. T cells were removed from culture on day 8, on day 36 for CD8 + cultures, or day 43 for CD4 + cultures. T cells were incubated without additional stimulus to determine spontaneous production of IFN-γ or with single cell digest of MCA205 or MCA207 tumors or with immobilized anti-CD3 mAb and Brefeldin A was added at 5 hrs and cells were harvested after 14 hrs. Intracellular IFN-γ was determined by FACS and the percentage of T cells is indicated. Discussion These experiments demonstrate that T cells, sensitized to tumor antigens in vivo , can be activated in vitro under conditions that promote hyperexpansion of either the CD4 + or CD8 + subset while retaining their potent therapeutic efficacy against established tumors. A notable feature of in vitro activation is that it permits selection and enrichment of a minor subset of tumor-reactive precursor cells. The mechanism of antigen sensitization of T cells through cross-priming by APC within draining LNs provides a convenient localized anatomic source that is already highly enriched. When coupled with physical segregation based on phenotypic characteristics that distinguish between antigen-stimulated versus naïve T cells, enrichment to nearly 40% of tumor-specific T cells was achieved. One important aspect of this strategy for selection and enrichment is that it does not require pre-existing knowledge of the immunogenic tumor antigens and does not require freshly acquired T cells to exhibit effector function. These conditions have relevance for many clinical situations where tumor antigens are not yet described or where unique tumor antigens may be immunodominant. Moreover, signaling defects have been observed in freshly acquired T cells from tumor-bearing hosts that might impede segregation based on functional properties [ 42 - 44 ]. Starting with a highly enriched population of T cells we were able to use a powerful, yet antigen-independent, stimulus such as anti-CD3 mAb that preserved the initial TCR repertoire diversity. Interestingly, anti-CD28 stimulation was not required for this experimental model, presumably because the T cells had already received co-stimulation during APC-mediated priming in vivo . The principal advantage to in vitro activation is that the culture conditions can be adapted to optimize proliferation of distinct subsets of responding cells. It is important to note that anti-CD3 activation in the absence of exogenous cytokine support did not lead to T cell proliferation even among CD4 + cells. The low cell density may have prevented secreted cytokines from reaching a critical threshold concentration. Moreover, IL-7, produced by non-hematopoetic cells and IL-23, produced by APC, mandated an exogenous source of these cytokines for in vitro culture activation. The combination of IL-2 and IL-7 provided rapid proliferation of CD8 + T cells and preserved their viability after completion of the initial mitogenic burst. The reason this combination was effective is that IL-7 receptor α chain is constitutively expressed on naïve and memory T cells but is downregulated on activated T cells [ 45 , 46 ]. By contrast, the IL-2 receptor α chain is reciprocally expressed on activated cells in a transient manner. Thus, the combination of these two cytokines ensured continuous mitogenic signal transduction. IL-7 is crucial for development and homeostasis of T cells and is markedly increased following lymphodepletion. Therefore, there is considerable interest in employing lymphodepletion as a strategy to augment active as well as adoptive immunotherapy [ 47 ]. Likewise, exogenous IL-2 has been administered in the context of tumor antigen vaccination as well as in nearly every clinical application of adoptive transfer to provide helper function [ 48 ]. However, in addition to their mitogenic effects on antigen-stimulated T cells, systemic production of IL-7 or systemic administration of IL-2 has effects on irrelevant T lymphocytes, other hematopoetic cells, and the vasculature. The inability to target cytokine support specifically to the relevant T cells limits the effectiveness of in vivo cytokine administration. More importantly, we have clearly documented that adjunctive IL-2 inhibits trafficking of adoptively transferred T cells into intracranial or subcutaneous tumors [ 49 ]. By contrast, cytokine stimulation can be targeted specifically to effector cells under optimal conditions in vitro without adverse systemic effects on the host. Future experiments to adjust the sequence, and concentration of supplemental cytokines using more sophisticated schedules than employed here might provide superior effector function. These experiments confirm the importance of CD4 + T cells for therapy of tumors in certain anatomic sites, such as the brain and subcutaneous tissue. The slower rate of CD4 + cell proliferation relative to CD8 + cells following the initial anti-CD3 stimulation led to their rapid marginalization in mixed cultures. However, depletion of CD8 + cells and use of cytokine combinations such as IL-7 and IL-23 favored the selective hyperexpansion of CD4 + cells that retained potent in vivo function. IL-23 is a member of the IL-12 family of cytokines and contains the IL-12 p40 subunit that transduces signals through the shared IL-12β1 chain in addition to the unique p19 subunit [ 50 , 51 ]. The IL-23 receptor is expressed on memory but not naive CD4 + cells, thus it is ideal for previously sensitized LN T cells. Myeloid cells, which are the natural source of IL-23, disappear rapidly in the in vitro cultures mandating an exogenous source. There is not a substantial amount of data on the effects of in vivo IL-23 administration, therefore its utility as a systemically administered adjuvant for T cell adoptive immunotherapy is unclear. However, the related cytokine IL-12 has substantial systemic effects that have limited its clinical use [ 52 ]. Although CD4 + T cells have been investigated as a source of helper function for CD8 + cytolytic cells our previous experiments have clearly established their stand-alone potential against MHC class II negative tumors [ 53 ]. CD4 + T cell anti-tumor function is mediated through cross-presentation of specific tumor-antigens by tumor associated APC [ 54 ]. As demonstrated, CD4 + cells cultured with IL-23 produced greater levels of INF-γ that would augment antigen presentation. Indeed, addition of tumor-reactive CD4 + cells to tumor digest increases the reactivity of the CD8 + cells. In addition to their autonomous effector functions, CD4 + cells are required to generate a functional CD8 + memory response in vivo [ 55 , 56 ]. Our recent experiments have demonstrated that adoptive transfer of effector T cells causes tumor destruction and sensitization of a secondary wave of regenerating host T cells [ 57 ]. In this regard, IL-23 stimulation of CD4 + cells might be particularly useful because it, unlike IL-12, induces production of the pro-inflammatory cytokine IL-17 [ 58 ]. Our observation that such sensitization occurred even in hosts with partial tumor regression indicates that the presence of effector CD4 + T cells and inflammatory conditions of tumor antigen acquisition by host APC are important in perpetuating the anti-tumor response. Repetitive anti-CD3 stimulation was utilized to drive hyperproliferation of T cells yet the TCR/CD3 complex also activates genetic programs required for effector function. Effector molecules such as Fas, Perforin, and IFN-γ have autoinhibitory as well as paracrine inhibitory effects during culture activation. Viewed purely in operational terms, sequential in vitro activation first under conditions that optimize T cell proliferation, then with conditions that restore effector functions immediately before adoptive transfer, would be advantageous. Future experiments will explore whether it is possible to dissociate proliferative signaling pathways from those mediating effector function through selective transient gene inactivation. The quantitative aspects of hyperexpansion are of less scientific interest but do have some practical implications. The 10 8 -fold extent of proliferation far exceeded what was required to treat the tumor models employed. Moreover, the availability of uniformly primed T cells for mechanistic studies is not numerically limited when using an inbred strain. However these experiments establish an approach to maintain polyclonality and preserve effector function despite extensive antigen-independent proliferation. As such, the quantitative aspects of hyperexpansion may have relevance to certain clinical situations where an autologous source of antigen-primed T cells may be limited and extensive host tumor burden may demand a large number of effector T cells. Supplementary Material Additional File 1 This is a table describing the percentage of cells expressing various TCR Vβ family members at three different time points. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC535812.xml
514610	Frequency of cancer in children residing in Mexico City and treated in the hospitals of the Instituto Mexicano del Seguro Social (1996–2001)	Background The objective of this article is to present the frequency of cancer in Mexican children who were treated in the hospitals of the Instituto Mexicano del Seguro Social in Mexico City (IMSS-MC) in the period 1996–2001. Methods The Registry of Cancer in Children, started in 1996 in the IMSS-MC, is an on-going, prospective register. The data from 1996 through 2001 were analyzed and the different types of cancer were grouped according to the International Classification for Cancer in Children (ICCC). From this analysis, the general and specific frequencies by age and by sex were obtained for the different groups of neoplasms. Also, the frequency of the stage of the disease that had been diagnosed in cases of children with solid tumors was obtained. Results A total of 1,702 new cases of children with cancer were registered, with the male/female ratio at 1.1/1. Leukemias had the highest frequency with 784 cases (46.1%) and, of these, acute lymphoblastic leukemias were the most prevalent with 614 cases (78.3%). Thereafter, in descending order of frequency, were tumors of the central nervous system (CNST) with 197 cases (11.6%), lymphomas with 194 cases (11.4%), germinal cell tumors with 110 cases (6.5%), and bone tumors with 97 cases (5.7%). The highest frequency of cancer was found in the group of one to four year-olds that had 627 cases (36.8%). In all the age groups, leukemias were the most frequent. In the present work, the frequency of Hodgkin's disease (~4%) was found to be lower than that (~10%) in previous studies and the frequency of tumors of the sympathetic nervous system was low (2.3%). Of those cases of solid tumors for which the stage of the disease had been determined, 66.9% were diagnosed as being Stage III or IV. Conclusions The principal cancers in the children treated in the IMSS-MC were leukemias, CNST, and lymphomas, consistent with those reported by developed countries. A 2.5-fold reduction in the frequency of Hodgkin's disease was found. Of the children, the stage of whose disease had been determined, two thirds were diagnosed as having advanced stages of the disease.	Background The frequency of malignant neoplasms in children has been found to vary among countries. For example, in children in Canada, the United States, and Europe, the three most common cancers are leukemias, tumors of the central nervous system (CNST), and lymphomas [ 1 - 3 ], whereas in children in Latin America, the order of frequencies is distinct: leukemias are still in first place, with lymphomas being more common than are CNST [ 2 , 4 - 6 ]. In other countries such as Nigeria, Malawi, and Egypt, lymphomas are the principal neoplasias [ 2 ]. The percentage of cases of each type of neoplasm in relation to the total number of cancers is also different. In the developed countries, the percentages for leukemias range between 30 and 37%; for CNST, between 18 and 27%; and for lymphomas, between 7 and 12% [ 1 - 3 ]. In Latin America, the percentages for leukemias are between 27 and 44%; of lymphomas, between 13 and 22%; and of CNST, between 10 and 19% [ 2 , 4 - 6 ]. In African countries, the percentages of lymphomas range between 30 and 64% [ 2 ]. In Asiatic countries such as Japan and China, the percentages of leukemias have been found to be between 30 and 40%; of CNST, between 12 and 20%; and of lymphomas, between 10 and 20% [ 2 ]. The study of the frequency of cancer in children not only is of interest to the clinical physician because it helps him/her to establish the pre-testing probability for a child suspected of having cancer [ 7 ], but also is of interest to the personnel in charge of the planning and programming of the medical attention for these children, as pertains to the assignment of human resources (physicians, specialized nurses, social workers, and others) and to the allotment of financial resources (centers providing medical attention, laboratories, imaging facilities, medicines, etc.) that are necessary for treating the children [ 8 ]. In Mexico, there existed data only from retrospective studies on the epidemiology of cancer in children and those studies had been carried out prior to 1993 [ 9 , 10 ]. Therefore, it was necessary to use more recent data, especially those from prospective studies in which the under-reporting of cases was reduced. The objective of this paper is to present the frequency of malignant neoplasias in the child population residing in the area served by the Instituto Mexicano del Seguro Social (IMSS-MC). The data were obtained from the Registry of Cancer in Children which was started in 1996 at, and is maintained by the Hospital de Pediatría del Centro Médico Nacional "Siglo XXI" of the Instituto Mexicano del Seguro Social in Mexico City (IMSS-MC). Methods Type of study Observational, descriptive, and prospective hospital inquiry. Population studied The Instituto Mexicano del Seguro Social (IMSS) offers medical attention to the population of workers and their families, which comprises 50% of the Mexican population [ 10 ]. The IMSS divides the population that it attends into four regions: North, South, East, and West. The IMSS-Mexico City (IMSS-MC) attends the population of the Southern region, which includes the populations in various states in the country, in addition to that of Mexico City. With the objective of avoiding a bias in the selection of the population for the present study, we included for analysis only cases from those states in which we were sure that more than 90% of the cases presented had been registered. This would be true of cases from the states that are located closest to Mexico City and of the cases in which the children who developed cancer had to be sent to Mexico City for treatment. Therefore, the cases analyzed came from Mexico City and from the following selected states: State of Mexico, Morelos, Guerrero, and Chiapas. It should be mentioned that not only the cost of treatment, but also the cost of transportation to Mexico City is covered by IMSS. This financial support provided by IMSS to families helps to ensure that cases of cancer come to the attention of IMSS-MC and are duly registered. Newly diagnosed cases of malignant neoplasias in children less than 15 years of age treated in the IMSS-MC were included in this study. In 100% of these cases, the diagnosis was confirmed by histological tests and/or by aspiration of the bone marrow. Participating facilities The IMSS-MC has two hospitals that provide medical attention to children with cancer. The Departments of Hematology and of Pediatric Oncology of both the Hospital de Pediatría of the Centro Médico Nacional "Siglo XXI" (HP) and the Hospital General del Centro Médico Nacional "La Raza" (HR) participated in this study. Both facilities have the infrastructure (competent personnel and suitable technology) needed for the precise diagnosis of a cancer. Study period Included in this study were the cases attended from 1 January 1996 to 31 December 2001. Study variables Prior to carrying out the study, a form for recording the variables of interest was designed. For this article, the variables analyzed were the following: type of neoplasia, sex of the patient, age at the time of diagnosis, and the stage of the cancer for those children with solid tumors. Procedure At each hospital, a fulltime nurse was assigned to register all new cases of cancer. Prior to collecting the data, each nurse was instructed in procedure necessary for obtaining all the different variable of the study. The nurse interviewed the parents and reviewed the clinical record of each child in order to obtain all the necessary information. The nurse was also taught how to encode and to determine the stage of cases of solid tumors. The standardization of the coding and determination of the stage of the solid tumors was done by all personnel (three physicians and two nurses) concerned with the registry; an excellent concordance was obtained (unweighted Kappa of 0.85) [ 11 ]. In addition to other duties related to the Registry, each nurse spent three days a week in the oncology and hematology departments of the hospital (HP or HR), searching for cases of children who, being suspected of having cancer, had been registered in a specific file and boarded at the hospital. After reviewing the clinical record in each file, the nurse either included the case in the study (encoding the data if the diagnosis of cancer had been confirmed) or eliminated the case if the diagnosis was not confirmed. If for any reason, the patient was discharged from the hospital and the diagnosis was undetermined, the nurse reviewed the clinical record in the clinical archive of the hospital in order to learn what the final diagnosis was. To encode the different cases of cancer, topographical and morphological coding was used. The second edition of the "International Classification of Diseases for Oncology" (ICD-O-2) was used for the cases collected from 1996 through 1999; the third edition (ICD-O-3), for the cases collected from 2000 through 2001 [ 12 , 13 ]. For determining the stage of cases of lymphomas and carcinomas, the recommendations of the American Joint Committee on Cancer (AJCC) and the International Union Against Cancer (IUAC) were used [ 14 ]. The stages for tumors of the central nervous system (CNST), neuroblastoma, retinoblastoma, renal tumors (Wilms' tumor), and those of the liver, bones, soft tissues, and germinal cells (GCT) were determined following the recommendations of the Children's Oncology Group [ 15 ]. The Child-Check Program developed by the International Agency for Research on Cancer (IARC) [ 16 ] was used to evaluate the internal consistency of the individual registries of cancer and to convert the nomenclature of ICD-O-2 to the International Classification of Childhood Cancer (ICCC) [ 17 ]. This program made crosses between different variables in order to find inconsistencies among the collected data. The crosses that were made were sex-topography, sex-histology, age-tumor type, unlikely combinations of topography-morphology, errors between date of birth and diagnosis, and duplication of cases. The result was a list of combinations, although either improbable or of low probability, that were needed for review in order to verify data or to correct data by rechecking the records of the patients. Cases of the ICD-O-3 that were not included in the Child-Check Program were classified by using other procedures and the data entered manually. Statistical analysis Cases were grouped according to the ICCC [ 17 ] that has established 12 different groups of cancer in children. From these were calculated the absolute and relative frequencies, both in general and according to sex and to age, the latter category being divided into four subgroups: under one year; one to four year-olds; five to nine year-olds; and ten to 14 year-olds. Due to the fact that the procedure for the determination of the stage of the disease was initiated in 1 Jun 1998, such determinations were made in 658 of the case of solid tumors. Therefore, the frequencies of the diagnostic states were based on this number of cases of children with solid tumors. Results A total of 1,702 cases of malignant neoplasias were analyzed. Of these, in the order of the most frequently found, were the following types of tumors: leukemias, 784 cases (46.1%); CNST, 197 cases (11.6%); lymphomas, 194 cases (11.4%); germinal cell tumors (GCT), 110 cases (6.5%); and bone tumors (BT), 97 cases (5.7%); and the remainder of the neoplasias were found in low percentages (Table 1 ). Table 1 shows that, in examining the subtypes of the different groups of neoplasias, it was found that, of the leukemias, the most frequent were the acute lymphoblastic leukemia (n = 614; 78.3%); of the lymphomas, the non-Hodgkin lymphomas [Burkitt, non Burkitt, and nonspecific together (n = 129; 66.5%)]; of the CNST, the astrocytomas (n = 97; 49.2%); of the tumors of the sympathetic nervous system (SNST), neuroblastomas and ganglioneuroblastomas together (n = 36; 92.3%); of the renal tumors, nephroblastoma [Wilms' tumor (n = 62; 87.3%)]; of hepatic tumors, hepatoblastoma (n = 26; 86.7%); of BT, osteosarcoma (n = 70; 72.2%); of the sarcomas of the soft tissues, rabdomyosarcoma and embryonic sarcoma together (n = 49; 55.1%); of the GCT, gonadal tumors (n = 80; 72.7%); and of the carcinomas, adrenocortical, malignant melanoma and skin carcinoma together (n = 9; 50.1%). The percentages of the different neoplasias showed variations according to sex and age group. These findings modified the pattern of presentation and made it different from the overall pattern. In males, over 70% of the cases consisted of the following types of tumors: leukemias (n = 412; 46.6%); lymphomas (n = 138; 15.6%); and CNST (n = 84; 9.5%). In females, 73.6% of the cases consisted of leukemias (n = 372; 45.5%); CNST (n = 113; 13.8%); GCT (n = 61; 7.5%); and lymphomas (n = 56; 6.8%). Overall, the ratio of males to females was 1.1; however, this ratio varied for the different groups of neoplasias, most notably a high of 2.5 for lymphomas and a low of 0.8 for both hepatic tumors, BT and GCT (Table 2 ). Table 2 shows that, for all age groups, leukemias had the highest frequency, ranging between 27.9 to 50.5%. In second and third place for the different age groups were the following types of tumors: under one year of age, retinoblastoma (n = 15; 17.4%) and GCT (n = 13; 15.1%); 1–4 year-olds, CNST (n = 69; 11.0%) and retinoblastoma (n = 56; 8.9%); 5–9 year-olds, lymphomas (n = 79; 16.2%) and CNST (n = 59; 12.1%); and 10–14 year-olds, lymphomas (n = 66; 13.2%) and BT (n = 65; 12.9%). With respect to the neoplasias in patients from other states in the Mexican Republic, leukemias were also found to have the highest frequency, followed by lymphomas and/or CNST, with discrete variations in the frequencies as mentioned above. It should be noted that in one state (Chiapas), although retinoblastoma was only the fourth most frequent there, its frequency (8.9%) was one of the highest (Table 3 ). In regard to the spread of the disease at the time of diagnosis for those children the stage of whose solid tumors had been determined, 89 (13.5%) were Stage I; 129 (19.6%), Stage II; 242 (36.8%), Stage III; and 198 (30.1%), Stage IV or higher. Discussion This is the first report of data, covering the six year period from 1996–2001, taken from the on-going Registry of Cancer in Children that was started in 1996 in Mexico City by the Instituto Mexicano del Seguro Social (IMSS-MC). The strategy of having placed a nurse in each of the two hospitals involved with the Registry of Children with Cancer resulted in the great majority (more than 90%) of the new cases that were treated by the two hospitals being identified and duly registered. We therefore concluded that this Registry of Cancer in Children was one of the most complete of its kind undertaken in Mexico City. Having access to the clinical records of the patients served to improve the quality of the data that was registered because, since cases were registered as soon as they were diagnosed, few cases were overlooked. The quality of the data was also enhanced by the use of the Child-Check Program, with which the possible errors, not only of registry but also of capture, were reviewed and were eliminated upon rechecking the respective clinical record in the hospital files. As mentioned in Methods, in 100% of the cases, the histopathology reports for children with solid tumors, or the reports on the aspirated bone marrow for children with leukemia, were obtained. Therefore, the data that was obtained for the children with cancer who were residents in the area covered by IMSS-MC and treated in Mexico City were the most precise that have been gathered to date in Mexico. The frequency of a disease in a population is a method of obtaining the pre-test probability that a patient has before a diagnostic test is performed [ 7 ]. It is important that this value be known, because there is a direct correlation with the positive predictive value of the test. This, in turn, is the probability that an individual whose test result was positive has of having the suspected disease [ 7 ]. That is, in the case of the children who were attended in the tertiary health care hospitals in IMSS-MC, the pre-test probability for a child whose parents sought medical attention (without taking into account his/her symptomatology) is a 43.4% chance of having some form of leukemia, an 11.1% chance of lymphoma, or a 12.6% chance of CNST. This probability increases or decreases depending on the symptomatology that the child presents, on the test requested, and on the result (positive or negative) of said test. Knowing the frequency of diseases in general and, in this case, the frequency of the different types of cancer that Mexican children present is therefore an important aid in diagnosis. The same can be said for the frequency of cancer by age and by sex. As has been mentioned, knowing the frequency of diseases serves in the estimation of administrative needs, not only with respect to personnel but also to the equipment and supplies necessary for diagnosis and treatment, and for providing medical attention in general and in particular for children with cancer [ 8 ]. Given that, from the data, 46.1% of these children develop leukemia, appropriate provision has to be made for their treatment, as well as for the children with other forms of cancer. A more clinical aspect, particular to children with cancer, that indicated the spread of their disease was the stage of the disease at the time of diagnosis. It was established that 66.9% were in stages III or IV, a finding that generally means the prognosis for the patient is not good. This datum was consistent with, and more precise than the result of the previously reported retrospective study, in which it was found that 56.4% had been diagnosed as having an advanced stage (III or IV) of the disease [ 10 ]. This fact indicates that, for Mexican children that develop a cancer, programs for integrated medical attention must be designed such that early diagnosis is a priority. Although the early diagnosis of cancer in children as a factor in a good prognosis is controversial [ 18 ], it is probable that such a program would have a great impact in Mexico. For this reason, as has been pointed out in prior studies [ 19 ], the influence of various factors such as the patient's family (educational level, socio-economic status, etc.), the type of cancer, the age of the child, the health system, and the physicians that care for the child on intake must be taken into account. Although the results obtained for the different groups of cancer in Mexican children in this study showed general agreement with data previously reported, there were some differences. Consistent with data obtained in other studies [ 9 , 10 ], it was found that leukemia, CNST, and lymphoma were the principal neoplasias and that Mexican children have one of the highest percentages of leukemias in the world (46.1% vs. 27–44%) [ 2 , 20 , 21 ]. In contrast to previous studies in Mexico, the frequency of Hodgkin's disease was notably lower, thus reducing the overall frequency of lymphoma to slightly lower than that of CNST and putting it in the range reported for developed countries (11.4% vs. 7–12%) [ 2 ]. We consider this reduction in the frequency of Hodgkin's disease a noteworthy and probably real effect, not just an artifact of the quality of the registry. The previous studies, being retrospective, had a greater tendency to under-register the number of cases and therefore result in an lower frequency than the actual value; yet, here, with a more accurate study, the frequency obtained was lower still (10% a 3.8%) (Table 4 ) [ 9 , 10 ]. However, we consider it necessary to obtain the incidence rates to confirm this and will do so for a forthcoming study in which we will present exclusively the incidence of cancer in children who are residents of Mexico City. It should be mentioned that the same phenomenon (reduction in the HD) was shown by Linet et al. [ 22 ] in their study of children in the U.S. during the period 1974 to 1995. There has been no explanation of this phenomenon because no program attempting to reduce HD in Mexico or in other parts of the world has been established. However, should an infectious agent be one of the causes in the development of HD, it is possible that the indiscriminant use of antiviral and/or antibiotics in Mexico may play a role. Perhaps future studies will be able to explain the phenomenon. In this and the two prior studies in Mexico, the frequency of CNST was found to be less than that in developed countries (18–27%). However, the lowered frequency of lymphomas found in the present study changed the hierarchical pattern of tumors, with CNST now having just edged out lymphomas for second place. We do not consider that this population has, strictly speaking, the pattern of U.S.-Canada-Europe, nor that of Latin America. We can say that the pattern of neoplasias in Mexican children was found to be a phenomenon in transition. It was interesting that the pattern of presentation found in children in Mexico City was different to that of the children that live in the northern part of the country (data not shown). The pattern in children from the north was similar to that of children in the U.S. and Canada, in that the principal neoplasias were leukemias, CNST, and lymphomas and had very similar frequencies [ 23 , 24 ]. This finding suggested the possibility that factors which cause cancer in children in the north of Mexico may be different to those causing cancer in children in Mexico City. Data for children under one year of age showed that, whereas neuroblastoma was the principal tumor for this age group in developed countries [ 1 , 3 ], in our study it was leukemias (27.9%), with the frequency of neuroblastoma being much lower than in developed countries (10.5 vs. 27.4). This finding is one that should be followed up because there are two factors that would have affect the reported number of cases and, hence the frequency: Because Mexico does not have the screening programs for detecting children with neuroblastoma that other countries do [ 25 ], it was probable that cases of this disease were not being diagnosed. Also, it is known that some of these tumors do regress spontaneously [ 26 ]. Another interesting aspect of the present study was that one of the highest frequencies of GCT (6.5%) in the world was found, a frequency similar to that for some Asiatic countries, such as Japan and Singapore (6.8%) [ 2 ]. We do not have an explanation of this finding, but studies directed toward establishing the causes of this high frequency should be carried out. Finally, it should be mentioned that one state of the Mexican Republic, Chiapas, was found to have one of the highest frequencies of retinoblastoma (8.9%), a frequency very similar to that of countries of Africa (Zimbabwe, 9.6%) and in India (Madras; 9.4%) [ 2 ]. Chiapas is one of the poorest states in Mexico and, as has been suggested, it is possible that the state of nutrition may play a role in the development of this tumor [ 27 ]. However, as for Hodgkin's Disease, it will be necessary to calculate the incidence rates to make this observation more precise. Conclusions It may be concluded that, in the children residing in Mexico City that were included in this study, the principal neoplasias were leukemias, CNST, and lymphomas, findings that were consistent with previously published data. It was found that, in comparison to previous studies in Mexico City, there was a reduction in the frequency of lymphomas and especially of Hodgkin's disease, and that, of the children with solid tumors, two thirds were diagnosed as having advanced stages (III-IV) of the disease. Competing interest None declared. Authors' contributions S J-O analyzed the data and wrote the first draft of the manuscript. G G-M registered, recorded, and analyzed the data. JM M-A, ME R-M, and MC M-G conceived and the designed the study and analyzed the data. A F-G conceived and designed the study, analyzed the data, and provided guidance to all aspects of this project. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC514610.xml
521697	Nutritional care of Danish medical inpatients: Effect on dietary intake and the occupational groups' perspectives of intervention	Background Many patients do not eat and drink sufficiently during hospitalisation. The clinical consequences of this under nutrition include lassitude, an increased risk of complications and prolonged convalescence. The aim of the study was 1) to introduce intervention targeting nutritional care for medical inpatients, 2) to investigate the effect of this intervention, and 3) to investigate the occupational groups' attitudes towards nutritional intervention and nutritional care in general. Methods The design was to determinate the extent to which the protein and energy requirements of medical inpatients were met before and after intervention. Dietary protein and energy intakes were assessed by 72-hour weighed food records. A total number of 108 medical patients at four bed sections and occupational groups in the two intervention bed sections, Aarhus University Hospital, Denmark participated. The intervention included introduction and implementation of nursing procedures targeting nutritional care during a five-month investigation period using standard food produced at the hospital. The effect of intervention for independent groups of patients were tested by one-way analysis of variance. After the intervention occupational groups were interviewed in focus groups. Results Before the intervention hospital food on average met 72% of the patients' protein requirement and 85% of their energy requirement. After intervention hospital food satisfied 85% of the protein and 103% of the energy requirements of 14 patients in one intervention section and 56% of the protein and 76% of the energy requirement of 17 patients in the other intervention section. Hospital food satisfied 61% of the protein and 75% of the energy requirement in a total of 29 controls. From the occupational groups' point of view lack of time, lack of access to food, and lack of knowledge of nutritional care for patients were identified as barriers to better integration of nutritional care into the overall care provision. Conclusion There was ample room for improving the extent to which standard hospital food satisfies patients' protein and energy requirements, but implementation of procedures addressing nutritional care were difficult, especially at bed sections with a large staff turnover.	Background Many patients do not eat and drink sufficiently during hospitalisation. Thus, 30–50% of the elderly patients are undernourished [ 1 , 2 ] and most of these patients' protein and energy requirements are not met [ 3 , 4 ]. Their muscular tissue, including their heart and respiratory muscles, is adversely affected by this situation [ 5 ] and their immune function is suppressed [ 1 , 6 ]. The clinical consequences include lassitude, difficulty in mobilising, prolonged convalescence [ 1 , 7 ] and an increased risk of pressure wounds [ 8 ], phlebitis and infections [ 9 , 10 ]. Patients often have reduced appetite, nausea or aversion towards certain types of food, which may partly explain the inadequacy of their food and liquid intake. Intervention studies have shown that by offering food or in-between meals rich in energy and protein, it is possible to increase the patient's protein and energy intake [ 11 - 14 ]. However, most of these intervention studies only use quantitative data. The present intervention study offers data, both quantitative on patients' food intake and qualitative on the occupational groups' attitudes and experiences in relation to the intervention, the food service and the nutritional care in general. These data can contribute to raise our knowledge of nutritional care in general and to identify issues crucial to an improvement of hospitalised patients' food intake in particular. The first aim of this research was to examine to which extent standard hospital food met hospitalised medical patients' protein and energy requirements. The second aim was to introduce nursing procedures focusing on the nutritional care based on the Danish nutritional recommendations for inpatients [ 15 ] to investigate the effect of this intervention on the patients' intake of protein and energy. The third aim was to explore the involved occupational groups' attitudes towards nutritional intervention and nutritional care in general. Particular attention was paid to the identification of problems possibly related to insufficient patient nutrition. Methods Setting The setting was an endocrinology ward with 49 beds and 3481 patients discharged during 2002 (divided into bed sections IA and IB) and a cardiology ward with 53 beds and 4542 patients discharged during 2002 (divided into bed sections IIA and IIB) [ 16 ]. All hospital food was produced in a central hospital kitchen and transported in heated containers to the bed sections where it was portioned out and served to the patients. Design of the study Medical patients' pre-intervention dietary protein and energy intakes were assessed by 72-hour weighed food records [ 17 ] at four bed sections (two wards) to include the appropriate number of patients. Before the intervention the bed sections at each ward was randomised to intervention or control. After a five-month intervention period, patients' dietary protein and energy intakes were assessed to evaluate the effect of intervention. After intervention the occupational groups involved in the nutritional care and the food service at the two intervention sections were interviewed in focus groups or by individual interview. Participants Both acute and referred medical patients at all ages participated. The inclusion criteria were defined as: 1) the patient was not placed on a prescribed diet, 2) the patient had no contact with, or had not previously received dietary advice from a clinical dietician, 3) the patient did not belong to an ethnic minority, and 4) the patient was hospitalised for at least five days. Patients with dementia and patients who were severely mentally or physically impaired were excluded. Typical patient diagnoses included acute or chronic lung disease (e.g. chronic obstructive lung disease, asthma, bronchitis), acute or chronic cardiovascular disease (e.g. hypertension, angina, thrombosis, apoplexy), metabolic disorders (e.g. thyrotoxicosis, osteoporosis) or infectious disease (e.g. pneumonia, cystitis). The nursing staff selected the patients meeting all the criteria. The patients received oral and written information about the investigation underlining the voluntary nature of their participation. Three or four patients from a bed section participated at the same time, providing data for the food records. The occupational groups participating in the focus group interviews were: nurses, health care support staff and nurse aides on day or evening duty from one of the two intervention sections IB and IIB (four interviews), two nurses in charge from the two intervention sections (one interview), three maids from the two intervention sections (one interview), two clinical dieticians from the two intervention sections (one interview), and one catering officer from the kitchen (one individual interview). In total 26 informants participated in eight interviews. Patients' characteristics Data on patient age, date of and diagnosis on hospitalisation, second diagnosis, oedema, dehydration, body weight on hospitalisation (if measured) were collected by the investigators from hospital records. Body temperature (if fever) was collected from the hospitals records during the 72-hour food recording. Patients' body weights were recorded twice: at hospitalisation (or when they were included in the study) and on discharge. This weighing was standardised according to time of the day, the patients dress and the scales. The changes in body weight during hospitalisation were recorded for the patients not having oedema or dehydration. Patient height was measured and body mass index (BMI, kg/m 2 ) calculated. The patient was asked about ability to chew and swallow and recorded as 'effortless', 'slight difficulty' or 'with difficulty'. On discharge they ascribed to the meals during hospitalisation was recorded as 'very important', 'of some importance', 'almost no importance' and 'no importance'. Food records The patients had their food and drink weighed for 72 hours at breakfast, lunch, afternoon coffee and supper by the investigators. The patients, relatives or staff recorded the last-meal-of-the-day and individual between-meals as estimated records. The investigators contacted the patients three to five times a day to follow up on these estimated records. The weight of the between meals provided by visitors was estimated by weighing similar food items. The food items were weighed in the form received from the kitchen. Potatoes, mashed potatoes, sauce, meat, etc. were weighed separately. Standardized menus such as stew, open sandwiches, sandwiches, etc. were weighed in full. The total weight of the food items each patient was served was weighed before serving as was plate waste after the meal. Drinks were estimated and recorded when poured into a glass or feeding cup one centimetre below the rim. Dietary intake of protein and energy 24-hour food records were checked and coded to calculate protein (gram) and energy (kJ) intake by the investigators. The calculations were based on the data from the recipes used in the hospital kitchen and the Danish Database 'Dankost 2000', which contains data from the Danish food tables [ 18 ]. Physical activity Each patient's physical activity was recorded every hour during three days and nights (72 hours) in the period of food recording. It was recorded whether the patient was 'lying asleep', 'lying awake', 'sitting', 'walking' or 'training'. For walking or training the approximate duration of activity was recorded as a fraction of an hour, and a factor of physical activity was estimated for each 24-hour period [ 15 ]. The investigators contacted the patients three to five times a day to follow up on the recording of the physical activity. Estimation of protein and energy requirements Official Danish food recommendations for institutions propose that patients with chronic diseases have 1.0–1.5 gram of protein per kilogram body weight depending on the degree of stress metabolism [ 15 ]. A factor of 1.2 gram was used as an estimate of moderate metabolic stress [ 19 , 20 ]. However, they did not allow for the underestimation of underweight and overestimation of overweight patients' requirements. The calculations were adjusted accordingly in the following way: If the BMI was below 20, the recommended requirement was calculated as 1.5 gram per kilogram bodyweight per 24 hours. If the BMI was above 30, the recommended requirement was calculated as 1.0 gram per kilogram bodyweight per 24 hours [ 21 ]. The estimated energy requirement was calculated as 'basal metabolic rate' (Harris Benedict equation [ 22 ]) x 'the factor of physical activity' x 'the factor required to increase the body weight (if the BMI was below 20)' or a 'factor of stress (if the BMI was above or equal to 20)' [ 15 , 21 ]. If the BMI was below 20, the factor 1.3 was used instead of the stress factor. The factor required to increase the body weight was an estimate of the amount of energy the patient was able to consume [ 15 , 22 ]. The stress factor was applied in patients judged to have metabolic stress because of their pathological condition. The stress factor range was 1.1–1.4 for patients with chronic lung disease, chronic heart disease and apoplexy: severe infections were given a factor of 1.3. The stress factor was determined by the temperature and was set at 1.2 at a temperature of 38°C, 1.3 at 39°C and 1.4 at 40°C. Only one factor of stress was used, and the temperature stress factor had the highest priority [ 15 ]. The mean recorded protein (g) and energy (kJ) intake was compared with the estimated protein (g) and energy requirements (kJ), and the degree (in percent) to which the patient's 24-hour requirements were met. Intervention The nurses in charge from the two intervention bed sections IB and IIB received information a) specifying to which degree the patients' protein and energy requirements were being met before intervention and b) detailing the Danish Recommendations for Hospitalised Patients [ 15 ]. In order to introduce and facilitate continuous staff monitoration of the patients' nutritional status during their hospitalisation the following intervention procedures were formulated in collaboration with the two nurses in charge. Such monitoration would allow the staff to identify patients at risk of under nourishment and would secure continuous registration, which was seen as a precondition for optimising the patients' uptake of nutrients. The procedures were formulated as one standard applying to be used to all non-diet patients admitted to bed sections IB and IIB: The patient's nutritional status is assessed on admission and during hospitalisation. As recommended by the nurses in charge three forms (A, B and C) with different purpose were made to support the staff in relation to the nutritional care. In Form A patient data related to the nutritional care were collected upon admission: height, body weight, BMI, usual body weight and changes in body weight for a defined period (if possible), oedema or dehydration, the date and by whom the patient had been informed about the food service, the result of the first assessment of the nutritional status (result from form B), a short description of 1) the patient's ability to eat and drink, and 2) of the action taken by the staff 3) the date of the next assessment of the nutritional status. The form also allowed room for the results of the next five assessments of the patient's nutritional status. The purpose of Form B was to assess the patient's nutritional status/risk score and suggesting the action the staff could take. Actions were performed according to detailed English Standards [ 23 ]. The assessment parameters were body weight for height (BMI), appetite and ability to eat. The patient was assessed at 'low risk' when BMI was normal, the appetite good and the patient fully independent. The patient was assessed at 'moderate risk' when underweight but stable, the appetite poor, and the patient needed help with feeding or had some swallowing difficulties. The patient was assessed at 'high risk' when severely under weight or actively lost weight, ate very little or have had no food for the last four meals, and was dependent on others for feeding or had severe swallowing difficulties. Form B allowed the patient's nutritional status to be recorded six times to ensure continuity of the assessment of nutritional status. A short guide to action was provided to the staff for each of the assessment categories; For the 'low risk' patient 'no action necessary, but check weight weekly'. For the patient at 'moderate risk' the action could be 'check weight weekly, encourage with eating and drinking, replace missed meals with supplements and repeat score after one week and ask medical staff to refer patient to clinical dietician if no improvement'. For the patient at 'high risk' the action was to 'focus on encouraging with eating and drinking, replace missed meals with supplements and repeat score after three to four days and ask medical staff to refer patient to clinical dietician'. In Form C the estimated record of the patient's protein and energy intake could be calculated and compared to data in the nutritional handbook describing what the food items contained of protein and energy. This handbook contained a standardised description of all meals delivered from the kitchen in household measurements (spoons, pieces, decilitre, etc.) and the estimated protein (g) and energy (kJ) content. Introducing the standard The investigators convened meetings with the nursing staff and the domestic helpers at the two intervention sections IB and IIB. The rationale of the standard was explained detailed and both oral and written instructions about the use of the forms were given. Four meetings were held in bed section IB and six in bed section IIB. At these meetings problems, ideas, etc. related to the standard and the forms were discussed and adjusted according to these discussions. The investigators contacted the staff in bed sections IB and IIB once or twice a week during the five-month investigation period to give support if wanted. The intervention at the two bed sections had no influence on the food production in the hospital. But before the intervention the kitchen produced a 'unrestricted diet' to all patients not placed on a prescribed diet, which contain about 8250 kJ and 70–80 gram of protein with about 15, 41 and 43% of energy from protein, fat and carbohydrates [ 24 ]. During the intervention period the kitchen changed the production to two different diets to meet the Danish Nutritional Recommendations for Diseased People [ 15 ]; From the kitchen the diets were introduced in the following way; one diet for the elderly and people with little appetite – the so-called 'hospital diet' – and one diet for all patients with ischemic heart disease and diabetes mellitus – the so-called 'normal diet'. The 'hospital diet' contained about 10000 kJ and 90 gram of protein with 18, 40 and 42% of energy from protein, fat and carbohydrates. The 'normal diet' contained about 9000 kJ and 80 gram of protein with 10–15, 30 and 55–60% of energy from protein, fat and carbohydrates [ 24 ]. The changes in the diets were introduced to the staff by the clinical dieticians. Besides these diets different commercial and no-commercial protein- and energy supplements, stewed fruit, soup etc. were available from the kitchen. Statistical methods The number of patients required was calculated in the following way:The clinically relevant difference between the average extent to which the patient's protein and energy requirement was met before and after the intervention was estimated to 15% [ 25 ]. Patients' dietary protein and energy intakes were estimated to lie 0–50% below their requirements (standard deviation (SD) 12.5–15.0%). A 5% significance level was chosen and the power was chosen to lie at 90%. The t-test was used to calculate an appropriate sample size for the control and intervention groups, viz. a minimum of 21 patients. The dietary protein and energy intake was calculated as a 24-hour mean (SD) for each patient and for each group (SD) of patients at each bed section. The outcome measure was the percentage degree to which the patient's actual protein and energy requirement was covered compared with his/her estimated requirement. Confidence intervals for the outcome measures were estimated. The effect of intervention for independent groups of patients were tested by one-way analysis of variance (ANOVA) using the SPSS version 9.0. The assumptions of independence, normality and identical variances were fulfilled. Analyses of covariance were described for non-comparable variables for the four patient groups after intervention. The interview in the occupational groups An interview guide was designed for each of five occupational groups: 1) nurses, health care support staff and nurse aides (four interviews), 2) charge nurses, 3) maids, 4) clinical dieticians and 5) one catering officer from the kitchen [ 26 ]. In the interview the investigator focused on the informants' actions, attitudes, experiences and reflections in relation to the intervention and nutritional care. Focus group interviews were considered the most appropriate form of data collection given the intent of the study [ 27 ]. All the 26 informants shared experience from the intervention study and from the situations where patients' meals were served. Eight focus group interviews were carried out at the hospital during the working hours in rooms familiar to the voluntary informants. The focus group interviews were tape-recorded with the permission of the informants, who were informed that they could read the transcribed interview, should they wish so. The qualitative data were analysed as a text. Ethical approval The study fulfilled the declaration of Helsinki II and was approved by the Local Scientific Ethics Committee. Results Patient characteristics Food records were completed for 48 patients before and 60 patients after intervention. Table 1 summarises the baseline characteristics of the participating patients. The patient groups were comparable with regard to BMI, stress factor and ability to chew and swallow. The average age of the medical patients was 72 ± 11 years. Before the intervention 17 patients out of 22 lost body weight. After the intervention 20 patients out of 37 lost body weight (table 1 ). Before the intervention, 56% of the patients participating in the study were weighed on admission (defined as within 48 hours from their arrival to the bed section). After intervention 52% of the patients in the control sections and 45% in the intervention sections were weighed on admission by the staff. Table 1 Summarised baseline characteristics of participating patients before and after intervention. Values are group averages (standard deviation (SD)) unless otherwise stated. Medical ward I Medical ward II Before intervention Bed section IA Status Bed section IB Status Bed section IIA Status Bed section IIB Status Number of patients (women/men) 12 (5/7) 12 (10/2) 10 (7/3) 14 (12/2) Age, years (SD) 74 (13) 68 (14) 72 (7) 70 (10) Length of stay, 24 hours (SD) 25 (21) 33 (29) 24 (18) 25 (18) BMI, kg/m 2 (SD) 26.4 (4.2) 26.6 (5.1) 26.1 (6.2) 24.7 (6.0) BMI, women, kg/m 2 (SD), 27.5 (4.2) 27.0 (5.5) 26.5 (6.8) 25.3 (6.2) BMI, men, kg/m 2 (SD), 25.7 (4.4) 24.4 (1.5) 25.2 (5.7) 21.1 (2.5) Change of body weight per 24 hours, gram (SD) (n)# 20 (100) (4) -78 (83) (7) -154 (107) (5) -6 (174) (6) Medical ward I Medical ward II After intervention Bed section IA Control Bed section IB Intervention Bed section IIA Control Bed section IIB Intervention Number of patients (women/men) 16 (9/7) 14 (10/4) 13 (9/4) 17 (7/10) Age, years (SD) 74 (12) 73 (13) 71 (9) 73 (9) Length of stay, 24 hours (SD) 26 (20) 24 (17) 14 (7) 16 (10) BMI, kg/m 2 (SD) 22.2 (6.3) 22.1 (3.7) 25.9 (7.1) 24.9 (4.9) BMI, women, kg/m 2 (SD) 21.8 (4.7) 22.1 (4.3) 26.6 (8.4) 21.9 (4.5) BMI, men, kg/m 2 (SD) 22.6 (8.0) 22.3 (2.2) 24.4 (3.6) 27.0 (4.0) Change of body weight per 24 hours, gram (SD) (n) # -72 (184) (8) 11 (108) (11) -105 (140) (8) -87 (238) (10) Number of patients receiving 'Hospital diet'/'Normal diet' 10/6 11/3 9/4 10/7 # Patients who take diuretics and patients with dehydration or oedema are excluded. Patient requirement and protein and energy intake In table 2 the average degree to which protein requirements were met before and after intervention are summarized. In table 3 the corresponding figures for energy requirements. There were no significant pre-intervention differences between the groups concerning the average degree to which their estimated protein (p = 0.918) and energy (p = 0.367) requirements were met. Table 2 Dietary intake of protein, estimated requirement of dietary protein and degree to which need for dietary protein per 24 hours was covered before and after intervention. Values are group averages (standard deviation (SD)) unless otherwise stated. Medical ward I Medical ward II Before intervention Bed section IA Status Bed section IB Status Bed section IIA Status Bed section IIB Status Dietary intake of protein in grams per 24 hours (SD) 63 (26) 56 (10) 55 (20) 59 (23) Estimated need for dietary protein in grams per 24 hours (SD) 85 (13) 79 (12) 79 (18) 84 (13) Estimated need for dietary protein covered in per cent (SD) 73 (27) 73 (15) 71 (24) 72 (30) 95 % confidence interval 56–90 63–82 54–89 55–89 Medical ward I Medical ward II After intervention Bed section IA Control Bed section IB Intervention Bed section IIA Control Bed section IIB Intervention Dietary intake of protein in grams per 24 hours (SD) 44 (20) 61 (26) 49 (15) 49 (20) Estimated need for dietary protein in grams per 24 hours (SD) 74 (12) 72 (13) 81 (17) 85 (14) Estimated need for dietary protein covered in per cent (SD) 60 (26) 85 (31) 62 (19) 56 (19) 95 % confidence interval 46–73 67–102 51–74 46–66 The results of the intervention was different at bed section IB and IIB; The intervention significantly improved the degree to which the energy and protein requirements were met among patients in intervention section IB compared with patients in the control sections IA and IIA (protein p = 0.009 and energy p = 0.010). On average, the former had an intake of 85% of their calculated protein requirement and 103% of their energy requirement. In intervention section IIB, the patients only had an intake reaching 56% of their protein and 76% of their energy requirement. These values were on average much lower than for patients in section IB and they were comparable to those obtained in the control sections IA and IIA. Analysis of co-variance for the non-comparable variables age, patient mobility, BMI, type of diet and number of bed-days showed no significant effect on the outcome measure for the degree of meeting the patients' requirement of protein and energy. The patients ability to chew and swallow, and the importance of the meals to the patients during hospitalisation were comparable in the four groups of patients before and after the intervention. In the control sections the diet met 61% of the patients' protein and 75% of their energy requirements after intervention. These levels were not significantly different from those recorded before the intervention, but 11% and 14% lower than before the kitchen changed the diets. The intervention and the occupational groups During the intervention period, the nursing staff in bed section IB used the forms for assessing the nutritional care of three patients. In intervention section IIB the forms was used assessing the nutritional care of 17 patients. The patients nutritional status/risk score were not determined otherwise. Analysis of the qualitative data from the eight interviews extracted five templates with questions relevant to an increased risk of insufficient nutritional care: 1. Divergent attitudes towards intervention. 2. Lack of flexibility during meals. 3. Lack of knowledge about nutritional care for patients. 4. Nutrition – a subordinate part of the care. 5. Lack of recognition of responsibility for nutritional care. Divergent attitudes towards intervention Analysis showed that the staff in the intervention sections had not been using the nutritional records systematically. Several nurses thought that the records were too comprehensive and overwhelming. Many mentioned that they had not had the time to learn how to use the records and they were clearly perceived as an extra workload. The nurses in charge mentioned that it was not unproblematic to burden staff with material they did not have the time or resources to read. However, a few staff members, among them two nurse students from bed section IIB, had learned how to use the records. They found that they were utilizable and easy to use. The two nurses in charge had divergent views on the usability of the intervention study. The charge nurse in bed section IIB thought that the intervention had improved their work with the patients' nutrition. The staff had previously accepted that patients would lie without eating for seven to ten days. Intervention caused the staff to use a feeding tube on threatened patients earlier than before the intervention. However, the charge nurse from bed section IB declared that the staff in her section had not shown much commitment to the intervention. The staff had not taken 'ownership' of the intervention study because the decision to participate in the project had not been a staff decision but one taken by the central management. She emphasized that the staff's attitude was rooted in the fact they had to take in new ideas and instructions all the time. Several care providers in bed section IB thought that it was a sizeable extra workload to use the records for recording patients' nutritional statuses and that this had constituted a barrier to their active participation in the process. Other nurses in bed section IB declared that they did not think that it was necessary to continuously register a patient's nutritional status. It sufficed for some nurses to use their 'clinical judgement' and on this basis monitor the patient's weight status. These nurses were not interested in any new initiatives and in tools for nutritional care. The records were not – and are still not – an integral part of the nutritional care in the intervention sections. This impacted on care continuity. The few staff members who had actually been using the records and had been able to identify patients at risk of insufficient nutrition reported that their observations had not been translated into action. Although the food records were only used to a minor extent, the staff generally agreed to the relevance of focusing on the patients' nutrition. Several nurses had not previously paid much attention to the patient's nutrition, but the intervention had made them more conscious of this issue: "I must say that after we have begun to pay attention to the diet, it has become clear to me how important it is. You have always known that it was important, but you do not really expect the patients to be undernourished when they are hospitalised" Nurse After the intervention the nurses were more conscious of their choice of food rich in energy than " before where they did not pay much attention to the fact that febrile patients constituted a special group at risk of falling into nutritional deficit". The general belief that 'fat is bad' for patients was widespread before the intervention. This belief springs from general dietary recommendations for healthy people. However, the intervention raised consciousness of the fact that public dietary recommendations may be suitable for healthy, but not for ill people. Lack of flexibility during meals The focus group interviews overall showed that the concept of 'individual nutrition' was not easily introduced in the nutritional practice at the two bed section. The staff was able to offer food five times during 12 hours during a 24-hour period. The duration of the meals was dictated by tight time schedules for maids and the hospital orderly. Lunch and dinner were often served under time pressure. Between the fixed meals, the care providers often lacked the time to offer patients various kinds of between-meals in the form of frozen, heated food. Assuming that nutritional care rests on the efforts of a committed staff, it may be claimed that the very organisation of the food service was counterproductive to individual nutritional care because the staff did not have real opportunities to offer the patients any food outside the fixed meal times. Individual nutritional care was also hampered by the fact that the kitchen ran a 24-hour nutrition schedule. This makes it difficult for the patients themselves to decide which meals they want to eat and hence to involve them in their own nutritional care. This was especially a problem for elderly nibblers. Lack of knowledge about nutritional care for the patients The clinical dieticians disseminated knowledge about nutrition to the staff in the bed sections, e.g. knowledge about a change in diet from 'unrestricted diet' to 'normal diet and 'hospital diet'. However, such knowledge dissemination was obstructed in several ways. A large staff turnover in some sections meant that such knowledge did not stay in the sections. The exchange of permanent nursing staff during a seven month period including the intervention was 9% and 46% in intervention section IB and IIB respectively (Information from the administration, Aarhus University Hospital). It was difficult for the clinical dietician to get through to the entire staff, as those who were willing to listen were those who took interest in the patients' diet: "But those we do see are those among the staff who take active interest. It's the old guard turning up" Clinical dietician Some care providers found that it was time-consuming to acquire knowledge about nutrition. Thus a member of the health care support staff mentioned that "just learning what a 'normal diet' and a 'hospital diet' is takes so much time". Along this line, several staff members, especially nursing assistants and health care support staff mentioned that it would be very useful if they had a resource agent they could ask about nutritional issues. It was possible for the care providers to refer the patients to the clinical dieticians. However, they felt that the dieticians were often so busy giving advice to referred patients that they could hardly assume a role in the daily nutritional care. The clinical dieticians, on their side, indicated that they would like the care providers to involve them more so that they could also give advice to patients who had not been referred. However, it was difficult for the clinical dieticians to be allowed to contribute: "The nurses think that they can manage the patients' nutritional situation. I think that is what they believe today. But if we were there when a question was raised, then they would use us. That's what I think" Clinical dietician Much would be gained, according to the clinical dieticians, if the staff knew that the patients' loss of weight during hospitalisation should be avoided and if such knowledge was used in the nutritional care. One nurse put forward the view that recommendations for healthy people also applied to patients. But during the intervention she expressed that she had changed her perception, but she found it difficult to manage nutritional requirements of ill patients and at the same time relate to dietary advice to healthy people, which were also used in the nutritional care of hospitalised patients. The clinical dieticians had also noted that many elderly patients were served the 'normal diet' even if they needed 'hospital diet'. These observations could signal the existence of a gap between the knowledge the nurses had and the knowledge actually needed to asses, among others, which diet suited particular patients best. The introduction of two diets caused some confusion and uncertainty among the occupational groups involved. Nutrition – a subordinate part of the care The care providers expressed an interest in the patients' nutrition, but also mentioned that they often had to ignore this aspect of care because of their tight work schedule. Some days when they had the time and the resources, they would pay more attention to the patients' nutrition, overseeing for example how much the patients were eating. But on busy days the care providers had to abstain – e.g. from offering the patients an extra portion " because it's nutrition and similar things which we must choose not to include when we are busy". Time was a limiting factor in nutritional care. The overall message was that the staff found it difficult to find time for determining the patient's height, calculate BMI, talk with the patient about losing weight, the patient's wishes for diet and his/her possible problems with eating and drinking. Time was hence both a real and an imagined barrier to recording the patients' nutritional status and to including the patients in their own nutritional care. In relation to that several care providers in section IB said that the nutritional care was a secondary priority. Hence, it was not perceived as a part of the care and treatment itself, but rather as a service " along with laundering and ironing" , as mentioned by the charge nurse in section IB. Other care providers in section IB also said that serving food and beverages for the patients was not part of their job: "You feel you are in the catering business in some way when you have to wait on the patients" Nursing assistant This would seem to suggest that for some care providers, nutritional care and the tasks such care demanded was not perceived as a natural part of their care activities. If a part of the staff defined nutritional care and the task of making sure that the patients got enough to eat as a job function outside the normal realm of their occupation this evidently constituted a barrier to an improvement in the patients' nutritional care. The patients' nutrition was hence not a priority area within the overall care work performed by the nurses and it was not an active part of the treatment. Inversely, the nurse in charge in section IIB thought that nutrition should be a first line priority to ensure that the work performed by the other occupational groups could have optimal effect. She pointed, among others, to the restoration of physical strength among stroke patients. She was aware that the work routines and the barriers to knowledge dissemination to other occupational groups was a factor limiting the speed with which changes could be implemented. The nurses in charge' attitudes to intervention and nutritional care were reflected in the attitudes of the rest of the staff. The data paradoxically showed that although nutritional care falls within the nurses' competence area, they only engaged in such care when they had the time to do so. When the nurses were busy, which they often were according to themselves, they gave lower priority to nutritional care. However, continuity of nutritional care was particularly important in nibblers according to the clinical dietician: "It is not always big science or intricate calculations; it's almost just a simple matter of remembering to serve the food to the patient" Clinical dietician Lack of recognition of responsibility for nutritional care The results suggest that the occupational groups involved in the food service had different guidelines. The assistant catering officer from the kitchen declared that she adopted a 24-hour approach to the planning of menus and distribution of energy percentages. The care providers prioritised that patients ate the meals they chose from the menus. However, this target was compromised by constraints of time and choice. The maids stuck meticulously to the diet previously decided by the nurses for each individual patient, while the care providers did not. Moreover, contrary to the clinical dieticians, some care providers thought that overweight patients should lose weight during their admission. The dietary change introduced to make some of the patients lose weight was, however, criticized by some of care providers in section IIB. They found that the change to 'normal diet' was not clear to the patients and was an expression of abuse of power, because the patients did not have any choice. The data gave no indications that the involved occupational groups shared a common goal as far as nutritional care was concerned. Inversely, the different groups had different priorities and showed neither insight nor any understanding of the professional competences of the other groups. The clinical dieticians also mentioned that a relatively high staff turnover at the bed sections ran counter to continuity of nutritional care and made it difficult to maintain a high, constant level of nutritional knowledge at the bed section derived through instruction and teaching undertaken by the clinical dieticians. Furthermore, the responsibility for the practical aspects of nutritional care could not be precisely located because many different staff groups were involved. This invariably increased the risk that responsibility was diluted, viz. that the individual care provider loses his/her sense of responsibility and overview of the situation. The staff in the bed sections did not see precise definition of responsibility as a central issue as opposed to staff outside the bed sections who would like to see a clear formal distribution of responsibility for nutritional care with a view to improving communication and procedures. "Nutritional care, distribution and orders should be given priority from above. It should not be the maids who should work for this. They are often having all the problems because the care providers have other duties they must see to; so the maids are sometimes doing as best they can; and what else can they do? But it should be a priority coming from the very top" Assistant catering officer The data suggested that dilution of responsibility was accompanied by an element of responsibility evasion. The care providers are, theoretically, responsible for the patients' nutritional care, but the maids assumed the lion's share of this responsibility in practice. The maids were employed in the maintenance section and therefore had no occupational responsibility for the patients' nutrition. However, the maids were very committed and felt responsible for the patients' nutrition. They found it difficult to accept that they had no guarantees that other staff members would take responsibility for the patients' diet when they were not at work. When the maids were having their weekends, holidays etc., the substitutes would often take over their function. The maids declared that they would be happy to take a more active role in the patients' nutritional care. Through their teaching at the bed sections, the clinical dieticians had learned that the maids in general were showing much commitment, attention and responsibility towards the issue of the patients' nutritional status. Inversely, several care providers found it difficult so see themselves take a more active role vis-à-vis nutritional care. As a measure intended to counteract the dilution and evasion of responsibility, the assistant catering officer suggested that central hospital management should issue a clear statement that the patients' nutritional status was a high priority area that deserved serious attention from all occupational groups. Such a message could also give impetus to a process of clarifying responsibilities and tasks related to nutritional care in all bed sections. Discussion Prior to intervention food ingested during hospitalisation on average met 72% of the patient' protein (table 2 ) and 85% of their energy requirement (table 3 ), and there was no significant difference between the four bed sections. But the intervention targeting the nutritional care had a significantly better effect in bed section IB than in intervention section IIB measured as the extent to which the protein and energy requirements were met. But the quantitative results revealed that the forms designed for assessing the patients nutritional status had been used only to a limited extent. This result was reflected in the results showing that the staff on admission only weighed half of the patients. The outcome of the intervention was probably influenced by the reluctance among the staff in bed section IB to implement the new guidelines, and by the large staff turnover in bed section IIB. Interestingly, the patients' intake of protein- and energy increased significant in bed section IB during the intervention. It cannot be excluded that the focus on the nutritional care coming from an investigator outside of the organization, had led to this paradox that, despite the reluctance identified among the staff, nutritional care was optimised. Table 3 Dietary intake of energy (kJ), estimated requirement for dietary energy (kJ) and degree to which requirement for energy per 24 hours was met before and after intervention. Values are group averages (standard deviation (SD)) unless otherwise stated. Medical ward I Medical ward II Before the intervention Bed section IA Status Bed section IB Status Bed section IIA Status Bed section IIB Status Dietary intake of energy, kJ/24 hours (SD) 7525 (2927) 6202 (1213) 6938 (2441) 6623 (2352) Estimated mean of need for dietary energy, kJ/ 24 hours (SD) 8244 (1418) 8177 (1396) 8331 (1533) 7782 (1055) Estimated need for dietary energy per 24 hours covered in per cent (SD) 92 (35) 77 (18) 85 (30) 85 (28) 95 % confidence interval 76–109 60–94 67–103 70–100 Medical ward I Medical ward II After the intervention Bed section IA Control Bed section IB Intervention Bed section IIA Control Bed section IIB Intervention Dietary intake of energy, kJ/24 hours (SD) 5359 (1993) 7267 (2317) 5811 (1851) 5923 (2096) Estimated mean of need for dietary energy, kJ/ 24 hours (SD) 7396 (1687) 7119 (1619) 7761 (1409) 7810 (1491) Estimated need for dietary energy per 24 hours covered in per cent (SD) 74 (30) 103 (24) 76 (22) 76 (23) 95 % confidence interval 62–87 89–116 62–89 64–89 Patients who were severely mentally or physically impaired were not included in the study of ethical reasons, although they as 'nibblers' did not receive a different form of nutritional care. So the sample is not representative for all the medical patients. If this group of patients had been included the quantitative results probably would have been lower, as described in an other Danish study [ 28 ]. The average length of the hospital stay for the patients participating in this study was 23 days. The average length for medical patients in the Aarhus County was six days [ 16 ]. This significant difference may be ascribed to the fact that patients hospitalised for less than five days were excluded in this study. On the other hand, mentally or physically impaired patients were not included. The official statistics on the length of hospital stays include a large group of patients who are long-term hospitalised. In this study 27% of the patients were hospitalised for more than four weeks. Long-term hospitalisation demands that particular attention be paid to the problem of weight loss. A 24-hour weight loss reaching 154 gram was found before the intervention, which may, indeed, be regarded as a problem during hospitalisation. The introduction of new diets made a difference both to the patients and the staff at the bed sections. Thus the 'normal diet' had a lower fat energy percentage than the other diets. This meant that patients had to consume a very sizeable diet in order to cover their energy requirement, which was rarely manageable for patients with reduced appetite. The clinical dietician mentioned that they had most frequently met patients with a poor or reduced appetite and a simultaneous need for a diet with a high nutrient density. This observation is corroborated by observations made by other Danish clinical dieticians [ 29 ]. The results of this study indicate that it is hardly appropriate to base nutritional care on recommendations intended for healthy individuals if the staff's nutritional knowledge matches that seen in the present study. The consequences seem to be a deterioration of the nutritional status in an even larger fraction of patients. The common understanding and recognition of the integration of nutritional care as part of the overall care among all occupational groups is a key prerequisite in an effort to see nutritional care as part of the care for the individual patient [ 23 , 30 , 31 ]. Another key prerequisite is that responsibility for such care is vested in real professional competence that lies with a single staff group, i.e. that it is backed by knowledge [ 32 ]. On this basis it might be possible to establish cooperation and launch a fruitful dialogue. Nutritional care fell within the competence of the nurses who were therefore able largely to determine to which extent other occupational groups were allowed to contribute with knowledge about nutrition. The clinical dieticians mentioned that they would like a more extensive dialogue with the other care providers about the patients' nutritional status, but these groups did not welcome such cooperation. One of the nurses in charge found that the intervention had made the nurses pay more attention to nutritional issues including, in particular, patients at increased risk of becoming undernourished. However, it was difficult to translate increased attention into specific nutritional care actions such as recording the patients' nutritional status upon admission by using the special food records. In a study of the relationship between nurses' competences and their knowledge about nutrition and diet in a hospital in the South of England, Lin Perry showed that there was no clear association between the nurses' attitudes, knowledge and actions, as neither knowledge nor attitudes were translated into action [ 32 ]. The study also demonstrated discrepancies between what the nurses said they were doing in relation to the patients' nutrition and what was actually documented in the patient records. Perry concluded that nursing care was frustrated by absence or inadequate knowledge among nurses about nutrition or by the failure to communicate such knowledge and a lack of common standards in general. However, the fact that the staff entertained views on the importance of the food and the food service did not imply that all groups were committed to seeing nutritional care as an element of the overall care effort. And the intervention study was an external project that was not anchored in the bed section's own staff. So that may explain the moderate reluctance to take active part in the study shown by some of the occupational groups [ 33 ]. The effect was that the nutritional care was not optimal. Some nurses gave as a reason for this situation that nutritional care was not part of the nursing care and that the time pressures induced by other tasks forced them to give lower priority to nutritional care. In the recommendations of The International Council of Nurses (ICN) the patients' nutritional status is placed second after the first dimension 'ability to breathe' [ 34 ]. This implies that a patient's nutritional status is considered an important issue in nursing theory, which is the basis of patient care. This is interesting in the light of the results of the focus group interviews presented here, because it appears that there is no agreement between the guidelines issued by the ICN and the Danish Nurses Organisation as far as the importance of nutrition and the nutritional care the patients receives during hospitalisation is concerned. Several papers in Danish and international nursing journals hence advocate that nurses assume a central role in countering patient under nourishment – a role rarely entertained by nurses today [ 32 , 35 - 37 ]. Yet, the clinical dieticians and the maids found that the nurses took into account neither their knowledge about patient nutrition in general, nor their knowledge of the individual patient's situation. Paradoxically, however, the nurses still claimed that nutritional care fell mainly within their competence. The clinical dieticians, the maids and the assistant catering officer reported poor communication between patients, nursing staff and kitchen. But the care staff did not share this view. The individual bed sections apparently did not have a clear distribution of responsibilities embracing all aspects of nutritional care. On the contrary, the data suggested that dilution of responsibility was accompanied by an element of responsibility evasion. The degree to which patients' energy and protein requirements are covered undoubtedly varies from hospital to hospital depending on the menus served and the commitment to the nutritional care shown by the care staff and management. However, any food service involves a long chain of tasks and work processes reaching from the kitchen to the patient, and the food service is essentially organised in the same way and priorities are generally the same in all Danish hospitals. It is therefore likely that the problems associated with insufficient nutritional care are of a similar nature in Danish hospitals and some European hospitals [ 38 ]. The perspective for further investigation could be a Health Technology Assessment (HTA) to evaluate the aspect of the patients, the organisation and the economy of the nutritional care of medical inpatients. Conclusion The average intake of energy and protein among hospitalised medical patients did not cover their requirements. Prior to intervention, food ingested during hospitalisation on average met 72% of the patients' protein and 85% of their energy requirement. After changing the diets from 'unrestricted diet' to 'normal diet' and 'hospital diet', the diet on average met 61% of the control patients' protein and 75% of their energy requirements. Intervention allowed a significantly better satisfaction of the patients' protein and energy requirements at one of the intervention sections using standard hospital food. However, the implementation of procedures focusing on nutritional care appeared to be difficult, especially at bed sections with a large staff turnover. Consequently, the results of the study call attention to the existence of barriers to efforts aimed at improving the nutritional care of patients. Introduction of nutritional care as part of the overall care met with barriers among the care providers. Focus group interviews identified these barriers as lack of time, lack of knowledge, lack of contact with resource agents concerning nutrition, lack of commitment, resistance towards a additional perceived workload and resistance towards providing service to the patients. Care providers who wished to provide individual nutritional care saw the very organisation of the food service as an obstacle to their freedom of action and flexibility. The effect of this was that it was difficult to accommodate individual patients' requirements. Occupational groups involved in nutritional care worked on the basis of different perceptions, had no shared target and no clear division of responsibility. Improvement of the nutritional care requires that focus be directed towards the final link in the food service chain. This study showed that nutritional care was a subordinate rather than a coordinate element in the overall care effort. The failure of coordination hinged on dimensions of organisation, knowledge and resource utilisation and it significantly affected the degree to which patients' nutritional requirements were met. An increase in the priority given to nutritional care by central hospital management and a concomitant general change in attitude towards nutritional care is needed and is probably a precondition for achieving a level of sufficient nutrition among hospitalised patients. Authors' contributions Karin O. Lassen carried out the research design, the fundraising, coordination of organisational communication, food record planning and implementation, performed the data analysis, drafted the manuscript and is the guarantor of the manuscript. Filip Kruse participated in the designing the focus group interviews, in the data analysis, and as author of the manuscript. Merete Bjerrum participated in the data analysis and as author of the manuscript. Lillian Jensen participate in the planning of the food records, performed the calculation of protein and energy intake and participate in the discussion of the manuscript. Kjeld Hermansen contribute to organisational support and discussion of research design and manuscript. All authors read and approved the final manuscript. Competing interests None declared.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC521697.xml
406406	Random Processes Underlie Most Evolutionary Changes in Gene Expression	xx	Are evolutionary changes in gene expression determined mostly by natural selection or by random forces? It's been some 150 years since Charles Darwin proposed that organisms adapt to their environment through the process of natural selection, yet the debate still rages, particularly at the molecular level. Darwinian selection was challenged in 1983 by the Kimura neutral theory of molecular evolution, which argues that the majority of differences in DNA (nucleotide) and protein (amino acid) sequences within and between species have only minor or no selective effect and that these differences arise through mostly random processes. Mutations at the nucleotide level occur randomly and regularly. Some of them survive through generations, resulting in “fixed” evolutionary changes between species. Two potential mechanisms can lead to the fixation of a particular change: natural selection, which favors changes that convey a selective advantage, and stochastic (random) events, such as genetic drift (the random fluctuations in genotype frequencies that occur from generation to generation in small populations). DNA mutations can lead to changes in gene expression levels, some of which may convey a selective advantage to an organism and therefore become fixed via natural selection. But since variation is produced at the genotype level, while selection is thought to operate largely at the phenotype level (that is, the physical manifestation of the genotype), it is reasonable to expect selection to be less apparent at the level of DNA sequence, and by extension, at the level of gene expression. Microarray technology has made it possible to systematically study expression levels of thousands of transcripts (the RNA copies of DNA that are translated into amino acid sequences) and to ask whether most changes of gene expression fixed during evolution between species result from selective or stochastic processes. To investigate this question, Philipp Khaitovich and colleagues analyzed the observed transcriptome differences among primate and mouse species as well as among various brain regions within a species. The team started out by analyzing the expression levels of some 12,000 genes in the prefrontal cortex of various primates, including humans. If evolutionary changes are caused by chance and not by natural selection, they will accumulate as a function of time rather than as a function of physical or behavioral changes in the organism. And that's what the authors found: the changes in gene expression among the species progressed linearly with time, suggesting that gene expression in primate brains evolved in large part from random processes introducing selectively neutral, or biologically insignificant, changes. According to neutral evolution theory, the same forces determine the rate of evolution both within and between species because similar random processes are at work on both levels. Consequently, genes that vary more within species should be more likely to vary between species. Comparing the expression levels of genes according to their variation within humans, the authors showed that genes with high variation among humans changed significantly faster between species than genes with low variation among humans. The authors also compared changes observed in genes to changes observed in pseudogenes (genes that over evolutionary time acquire a mutation that renders them nonfunctional) and found no significant difference between the two, suggesting again that most expression changes have no functional significance. While their analysis cannot exclude a role for natural selection, all the results are consistent with a neutral model of transcriptome evolution. This means that the majority of gene expression differences within and between species are not functional adaptations but selectively neutral and that we won't be able to explain species differences based on variation in gene expression in general. In addition to examining differences in gene expression in a particular tissue between species, the authors also discuss the evolution of different tissues within a species. The human brain is composed of regions that differ in function and histology (microscopic structure). Each of these regions acquired a functional or histological difference that separated it from its sister regions at some point in our evolutionary past. The authors show that the amount of change between regions correlates with tissue-divergence times estimated by other methods. If this finding applies for other tissues within and outside the brain, it could provide a method to reconstruct the evolution of tissues within a species.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC406406.xml
544966	Cerebrospinal Fluid Research: A new platform for dissemination of research, opinions and reviews with a common theme	Cerebrospinal Fluid Research aims to provide a specialist platform for high quality articles on all aspects of the cerebrospinal fluid (CSF), bringing together experts working in the field and promoting synthesis and dialogue. This, launch Editorial provides an overview of the field, some history, and highlights some of the journal policies.	Cerebrospinal Fluid Research is an open access, online journal that publishes manuscripts on cerebrospinal fluid (CSF) in health and disease in the broadest sense. The CSF, its composition, circulation and absorption play vital roles in normal and abnormal brain function. The fluid within the CSF system is important for normal chemical signaling, physical and chemical buffering, and for neurodevelopment. In disease states, the CSF impacts on neurodevelopmental disorders such as hydrocephalus and neural tube defects, brain inflammation, brain injury and repair, normal pressure hydrocephalus and neurodegenerative diseases such as Alzheimer's, Parkinson's and multiple sclerosis. The CSF can be used as a tool for diagnosis, through composition analysis, and as a window for drug delivery to the brain. Fluid within the brain cavities was known of in ancient times. Hippocrates in the 4 th century B.C. recorded the presence of fluid and is thought to have tapped the ventricles in a patient with hydrocephalus. Subsequently, in the 2 nd century A.D., Galen described the ventricles and his dogma that the ventricles contained a gaseous vital spirit, lasted for over 1000 years. The return to the fluid hypothesis occurred in 1543 with Vesalius who made detailed observations of the anatomy and noted the presence of a watery humor. Further studies in the 17 th and 18 th centuries by Valsalva, Haller, and Contugno elaborated and extended this knowledge. Magendie in 1825 made chemical and physiological studies on the fluid and coined the name liquide cephalo - rachidien or fluid cerebrospinal. He saw pulsatile movement and concluded the fluid was under positive pressure. Later in the 19 th century the anatomists Key and Retzius made extremely detailed studies of the cavities and the membranes of the brain, and provided a foundation for many 20 th century investigations starting with Dixon and Haliburton, 1913 [ 1 ], Dandy and Blackfan, 1914 [ 2 ] and Weed 1935 [ 3 ]. At this stage it was known that the fluid originates in the choroid plexus and circulates throughout the internal cavities and external spaces to the venous sinuses. It was also shown by dye studies that there was a 'barrier' for movement of substances between the blood and the brain (blood-brain barrier) and between the blood and CSF (blood-CSF barrier). Physiological studies began in the 1950's with numerous investigations led by Hugh Davson, the 'father' of CSF physiology [ 4 ]. It was demonstrated that the CSF was not a plasma ultrafiltrate but a secreted fluid under homeostatic control with its own unique composition for electrolytes, small non-electrolytes and proteins. Hugh Davson was a remarkable scientist who aided and encouraged many collaborating researchers to work in the field of CSF. Particular milestones in the latter half of the 20 th century include the measurement and control of CSF secretion by Pappenheimer et al [ 5 ], and uptake functions for the choroid plexus by Welch [ 6 ] and Pollay and Davson, [ 7 ], the demonstration of tight junctions at the endothelium and choroid epithelium by Brightman and Rees [ 8 ], and the effect of inhibitors on secretion by Davson and Segal [ 9 ]. Further studies have brought us to understand that the choroid plexus is regarded as a complex secretory, regulatory, and absorptive organ. The CSF is no longer considered to be only a cushion for the brain but a multifunctional organ with homeostatic, hormonal and signaling mechanisms that have important functions in health, and particularly in neurological diseases. This is an active and fast growing field containing researchers in many disciplines. However, to date, the subject area does not have a specialist platform and articles are published in a variety of different journals. We invite you to help us to bring the field under one umbrella by submitting your manuscripts online to Cerebrospinal Fluid Research [ 10 ]. The journal will provide a comprehensive medium, offering quality peer-review of manuscripts on all aspects of CSF. Cerebrospinal Fluid Research is supported by an international Editorial Board [ 11 ]. Each manuscript will be reviewed by a member of the Board or, where appropriate, allocated to external reviewers. At least two reviewers will be sought for each manuscript; a third will be approached where there is a significant difference in opinion. Reviewers can choose whether to remain anonymous. Authors will be asked to provide a list of suggested reviewers on submission of their manuscript. Cerebrospinal Fluid Research will consider the publication of research articles, reviews, commentaries, book reviews and meeting proceedings. All articles will be published immediately upon acceptance (after peer review) and listed in PubMed. The intention is to publish, as supplements, the proceedings of scientific meetings in relevant subject areas. Cerebrospinal Fluid Research's Open Access policy changes the way in which articles are published. First, all articles become freely and universally accessible online, and so an author's work can be read by anyone at no cost. Second, the authors hold copyright for their work and grant anyone the right to reproduce and disseminate the article, provided that it is correctly cited and no errors are introduced [ 12 ]. Third, a copy of the full text of each Open Access article is permanently archived in an online repository separate from the journal. Cerebrospinal Fluid Research's articles are archived in PubMed Central [ 13 ], the US National Library of Medicine's full-text repository of life science literature, and also in repositories at the University of Potsdam [ 14 ] in Germany, at INIST [ 15 ] in France and in e-Depot [ 16 ], the National Library of the Netherlands' digital archive of all electronic publications. Open Access has broad benefits for science and the general public. Most importantly, authors are assured that their work is disseminated to the widest possible audience, given that there are no barriers to access their work. In addition, authors are free to reproduce and distribute their work, for example by placing it on their institution's website. Furthermore, there is evidence that free online articles are more highly cited because of their easier availability [ 17 ] and publicly funded research will become accessible to all taxpayers (not just those with access to a library with a subscription). As such, Open Access could help to increase public interest in, and support for, research. Note that this public accessibility may become a legal requirement in the USA if the proposed Public Access to Science Act is made law [ 18 ]. Resource-poor countries (and institutions) will be able to read the same material as wealthier ones providing they have access to the internet [ 19 ]. This is an exciting opportunity to disseminate our science in the new world wide medium of electronic publishing. Cerebrospinal Fluid Research looks forward to receiving your submissions.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544966.xml
550654	Bilateral recurrent discloation of the patella associated with below knee amputation: A case report	Background Recurrent dislocation of the patella in patients with below knee amputation is a known entity. Abnormally high-riding patella (patella alta) and medial patellofemoral ligament insufficiency in these patients predisposes them to patellar instability. The established treatment of this problem is surgical realignment. Case presentation A 25 year old male patient with bilateral below knee amputation presented with bilateral recurrent dislocation of the patella while walking on knees on uneven ground. Clinical and radiographic studies showed patella alta. A simple shoe modification was used to treat this patient. Conclusions A simple shoe modification can be used to treat such a condition which is otherwise treated surgically.	Background Recurrent dislocation of the patella can follow a violent initial dislocation, but occur more often in knees with one or more underlying anatomic abnormalities that predispose the patella to dislocation or subluxation. In these knees, less trauma is needed for dislocation to occur. The underlying pathologic condition causes an abnormal excursion of the extensor mechanism over the femoral condyles. High-riding patella (patella alta) and a damaged medial patellofemoral ligament at the time of first episode of dislocation leads to such an abnormality and leads to recurrent dislocation of the patella [ 1 - 4 ]. Patella alta has been reported in patients with below knee amputation using patellar tendon bearing prosthesis. The usual treatment in these cases is surgical reconstruction. We present a case of bilateral recurrent dislocation of the patella with below knee amputation which was managed conservatively. Case presentation A 25 years old bilateral below knee amputee presented with recurrent dislocation of the patella while walking on knees in emergent situations on uneven ground without the prosthesis. Amputation was performed at the age of 15 years as a result of train accident. Since then he has been using patellar tendon bearing (PTB) type below knee prosthesis on both sides. First episode of dislocation occurred after 5 years of amputation. The patient used to walk on his knees without using the prosthesis for in-house activities on uneven ground. His patella used to dislocate whenever there was an unnoticed pressure on the medial side of the knee. On clinical examination, patella alta and positive apprehension test were noted on both sides. The ratio of patellar length to patellar tendon length was 0.8 on both sides demonstrating relative elongation of the patellar tendon. The normal ratio is 1.0 [ 1 ]. Modified shoes were given to the patient which were moulded in the inner surface around the patellar tendon and femoral condyles to provide uniform distribution of weight over a wider area. Another moulding was done on lateral side that prevented excessive movement of patella laterally [Fig. 1 ]. The patient was allowed to walk on knees after wearing these shoes [Fig. 2 ]. At 6 months follow up the patient is doing well with no recurrence. Figure 1 Close-up photograph of the shoe (arrow = moulding on the lateral side providing protective force against dislocation) Figure 2 Patient wearing modified shoes Discussion High-riding patella (known as Patella alta) leads to patellar instability [ 1 ]. In the presence of such instability, sudden laterally directed forces can lead to dislocation of the patella. The first episode of lateral dislocation of patella invariably damages the medial patellofemoral ligament (MPFL) which is the primary passive restraint to lateral patellar displacement[ 4 , 5 ]. An injured MPFL leads to further instability and both the factors combined predispose to recurrent dislocation of the patella. In below knee amputees using patellar tendon bearing (PTB) prosthesis, the prolonged, upwardly directed force against the patellar tendon gradually elongate the tendon and produce patella alta [ 2 , 3 ]. It takes a long time for such an instability to develop. The first incidence of patellar dislocation occurred after 5 years of amputation in our patient. Similar time lag was described by Mowery et.al. in his patients where the time lag was from 5–13 years [ 3 ]. The patient described here used to walk on knees for certain emergent and short mobility in-house activities e.g., going to toilet especially at night, going to kitchen etc. As most of rural houses in developing countries do not have cemented floors, walking on uneven ground cannot be prevented. Walking on knees in such conditions produces eccentric forces on the patella. Sudden laterally directed forces which might result when the knee strikes some elevated surface on medial side can lead to dislocation of patella in patients with patellar instability. Walking on knees is usually discouraged in below knee amputees as it can lead to flexion contractures of the knee joint. In cases of bilateral amputees it is not always possible for the patients to wear prosthesis on both sides in emergency situations as discussed above. Considering these points, the occasional walking on knees could not be avoided in this patient. Surgical correction of instability would not have helped much as walking on knees on uneven ground could have nullified the results of surgery. A modified well moulded shoe was given to the patient for walking on knees that prevented any eccentric forces and protected the patella against dislocation. 6 months follow-up results were excellent in this patient with no episode of dislocation on either side. The patient was very comfortable in using the shoes as it takes him only 15 seconds to wear them. Hence a simple low cost shoe modification has been used to treat a condition which is mostly treated surgically. Conclusions Recurrent dislocation of the patella in cases of below knee amputees using patellar tendon bearing prosthesis is very rare and the usual treatment is surgical realignment. To our knowledge no case of bilateral recurrent dislocation of the patella in below knee amputees has been described in the literature. A simple shoe modification can be used in difficult situations to treat such a condition. Competing interests The author(s) declare that they have no competing interests. Authors' contributions SB was the principle surgeon who planned the treatment protocol of this patient, in addition to conceptualizing and drafting the article. RK guided the designing of the shoe. PL was the prosthetist who designed and made the shoe. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC550654.xml
548147	Climate change and recent genetic flux in populations of Drosophila robusta	Background Studied since the early 1940's, chromosomal polymorphisms in the deciduous woods species Drosophila robusta have been characterized by well-defined latitudinal, longitudinal, and elevational clines, but – until at least ten years ago – stable, local population frequencies. Recent biogeographical analyses indicate that D. robusta invaded North America from southeast Asia and has persisted in eastern temperate forests for at least 20–25 my without speciating. The abundant chromosome polymorphisms found across the range of D. robusta are thus likely to be relatively ancient, having accumulated over many well known climatic cycles in North America. Sufficient long-term data are now available such that we can now gauge the rate of these evolutionary changes in natural populations due to environmental change. Results Recent local collections have revealed significant changes in the frequencies of several chromosomal forms. New data presented here extend the range of these changes to six states, three in the northeastern United States and three west of the Mississippi River. These data reinforce recent directional changes in which the frequencies of three gene arrangements have reached percentage levels typical of distant southern populations consistent with regional climatic changes. Another gene arrangement has been steadily decreasing in frequency at a number of the sites studied. Meteorological records from 1945 to 2003 indicate temperature increases at all study sites, particularly average minimum air temperatures. Conclusions Observation of parallel genetic flux suggests that these long-term temporal frequency shifts in widely disparate populations of D. robusta are evolutionary responses to environmental change. Since these chromosomes are known to be sensitive to ambient temperature, regional climatic shifts associated with global warming are likely to be responsible.	Background In recent years, numerous publications – over 50 pages of them in current listings of the bibliographic search engine Medline – have detailed changes in biological systems and organisms that appear associated with the climatic changes commonly referred to as global warming (e.g., [ 1 - 5 ], dealing with observed or impending shifts in the habitats of various organisms). Few studies, however, have documented long-term changes in the genetic structure of species populations on a regional scale, necessary for understanding the microevolutionary consequences of global change [ 6 ]. In the genus Drosophila , comprised of over 1500 species [ 7 ], only four species have received such attention. While some long term increases have been documented in the frequencies of chromosomal gene arrangements Standard ( ST ), Tree Line ( TL ), and Pike's Peak ( PP ) in Drosophila pseudoobscura in the western United States and Canada over ca 40 years [ 8 - 10 ] and of several arrangements in Drosophila melanogaster in Japan [ 11 ], little is known of the causes for these changes. Orengo and Prevosti [ 12 ] first suggested that climatic warming was responsible for long-term temporal changes in the frequencies of certain gene arrangements in European populations of D. subobscura . Later, Rodriguez-Trelles and co-workers [ 13 - 15 ] and Sole', et al . [ 16 ] convincingly demonstrated that climatic changes, including global warming, were likely the driving force of microevolutionary changes in these populations. Recently, significant changes have also been documented in the chromosomal variation of D. robusta Sturtevant, some of them possibly attributable to global warming [ 17 - 19 ]. Here we describe additional data that underscore the variety of historical changes being experienced by populations of this species. Clearly, the chromosomal polymorphisms in D. robusta are also dynamic, and when compared with the considerable geographical and experimental data available for this species collected over the last 60 years, strongly implicate regional climatic changes as a cause for these temporal frequency shifts. Results Regional patterns of climate change were revealed from ANCOVA analysis of temperature and precipitation data from 1945 – 2003. No long term tends were detected for precipitation except for a significant increase in Central Park, NY (data available from the authors). Significant long-term temperature changes were apparent at all six 2003 study sites (Fig. 1 ; plus Philadelphia, last studied in 2002) for the three available temperature indicators: average monthly minimum temperature ( MINTMP ), average monthly temperature ( AVETMP ), and average maximum monthly temperature ( MAXTMP ; all 3 ANCOVA models, P < 0.0001). AVTEMP and MAXTMP varied significantly from site to site in different years, but MINTMP significantly increased over the 58-year period (P < 0.0001) with no heterogeneity among sites. Thus, the most consistent change in climate across sites in this study was due to significant increases in minimum monthly temperatures. Figure 1 Map of the eastern United States showing the locations of collecting sites in this study. This modified map is used by permission of the University of Texas Libraries, the University of Texas at Austin. Evidence for site-specific temperature increases from 1945 – 2003 was evident for these three temperature indicators (Fig. 2 ). Five of the seven sites showed linear or curvilinear increases in MINTMP , usually starting in the 1970's and extending until 2000. In contrast, both Fayetteville and Englewood, NJ have experienced significant temporal decreases in MAXTMP (with a concurrent significant increase in MINTMP in Fayetteville). Causes for these decreases are obscure, but in New Jersey may be due to the exaggerated temperature fluctuations in the early 1990's (as well as missing data for 1992 and 1993, Fig. 2 ). Figure 2 Temperature data for each site in this study plotted from 1945 to 2003. Regression lines and equations are shown only for statistically significant trends in mean maximum monthly temperature ( MAXTMP ), mean monthly temperature ( AVTMP ), and mean minimum monthly temperature ( MINTMP ) at each site. Significance and sign of the regression coefficients are indicated (P < 0.05, * P < 0.01, * P < 0.001, ** P < 0.0001). Y axes are in degrees Centigrade. Replicate data plotted for Fayetteville and New Jersey show incomplete secondary weather station data at sites closer to the locations where flies were collected. See text for details. Concurrent with these temperature shifts, chromosome elements in populations of D. robusta show systematic temporal frequency changes (all frequency data are available from the authors). X chromosome combinations 1S and S1 show substantial frequency changes with time, with S1 doing so in a site-specific manner (Fig. 3 ). Frequencies of combination 1S have decreased over time, and are negatively correlated with increasing temperatures (Tables 1 , 2 ). Increases in S1 are only marginally correlated with temperature, but this may be due to other factors, including its low frequency in eastern populations (Fig. 3 ). Gene arrangements 2L-1 and 3R-1 have also increased in frequency since 1945 (Fig. 4 , 5 ; Tables 1 , 2 ), and the changes are significantly site-specific as indicated by the significant year × site interactions. While temporal changes in 2L-1 and 3R-1 are correlated (r = 0.419, P = 0.0016, n = 54), only those of 3R-1 are significantly correlated with increasing temperatures (Fig. 5 ; Table 2 ). Figure 3 Frequencies (in percent) of D. robusta X -chromosome arrangement combination S1 in the seven study localities, including Philadelphia [18]. When frequencies across years were statistically homogeneous, they were combined within each locality. Y axes for eastern populations reflect the much lower frequencies for S1 in that part of the species range. Table 1 ANCOVA results for temporal changes in the frequencies of several X chromosome arrangement combinations and autosomal inversions across the seven collecting sites in this study. r 2 is an estimate of the proportion of the total variance explained by the model used. X chromosome combination 1S Source df Type III SS F Value Pr > F r 2 Model 13 1.151 50.31 < 0.0001 0.942 Year 1 0.014 5.97 0.019 Site 6 0.207 14.93 < 0.0001 YearSite 6 0.199 14.35 < 0.0001 X chromosome combination S1 Source df Type III SS F Value Pr > F r 2 Model 13 2.589 50.94 < 0.0001 0.943 Year 1 0.000 0.02 0.894 Site 6 0.206 8.79 < 0.0001 YearSite 6 0.197 8.39 < 0.0001 Gene arrangement 2L-1 Source df Type III SS F Value Pr > F r 2 Model 13 1.317 20.55 < 0.0001 0.870 Year 1 0.038 7.66 0.0085 Site 6 0.215 7.25 < 0.0001 YearSite 6 0.216 7.29 < 0.0001 Gene arrangement 3R-1 Source df Type III SS F Value Pr > F r 2 Model 13 2.054 54.09 < 0.0001 0.946 Year 1 0.043 14.76 0.0004 Site 6 0.057 3.28 0.0102 YearSite 6 0.060 3.40 0.0084 Table 2 Pearson product-moment correlations between average monthly temperature and frequencies of several X chromosome arrangement combinations and autosomal arrangements for all seven localities studied. n = 54 for all tests. 1S S1 2L-1 3R-1 Average temperature for month of collection - 0.341 0.250 0.195 0.336 P = 0.012 P = 0.071 P = 0.163 P = 0.014 Figure 4 Frequencies (in percent) of D. robusta gene arrangement 2L-1 in the seven study localities, including Philadelphia [18]. See Fig. 3 for details. Figure 5 Frequencies (in percent) of D. robusta gene arrangement 3R-1 in the seven study localities, including Philadelphia [18]. See Fig. 3 for details. The most consistent historical changes have been gains in the frequency of arrangement 3R-1 with concomitant decreases of its allelic form, 3R (Figure 5 ). Coupled with the clinal tendency of 3R-1, varying between 100% in the southernmost latitudes and zero in the northernmost [ 20 ], its similar directional increases of recent years in at least four states, some far apart, and concordance with long term temperature increases indicates that 3R-1 is responding to regional climatic shifts. The frequency of arrangement 2L-1 increased directionally in most of the same localities as 3R-1 (Figure 4 ). The frequency of 2L-2, which is also more common in the south than in the north, has been decreasing steadily, almost to the point of extinction, in the four northern 2003 populations. This is probably not a temperature effect, however, as in the past 2L-2 has been irregularly distributed across the southern states, rarely rising above frequencies of 20 percent, without evidence of a latitudinal cline [ 20 - 22 ]. Like 2L-1 and 3R-1, the frequency of gene arrangement XL (the sum of data for SS , S1 , and S2 ), tends to be higher in southern populations than in northern ones [ 20 ]. Except for a few deviations in small samples, it has increased steadily – with concomitant decreases in northern arrangement XL-1 – in all the localities studied except Olivette (where it is already about 95%) and Iowa. At Englewood, for example, it rose from about 40% in 1948 to an average of 78% in 2000–2003, that of northern arrangement XL-1 falling from about 60% to about 22% in the same period. Frequencies of X chromosome combination 1S (XL-1.XR) are clearly consistent with this pattern (Tables 1 , 2 ). Discussion Studies of Drosophila inversion polymorphisms have now provided historical insights into environmental change. On different continents, temporal genetic changes correlated with increasing temperatures in populations of D. subobscura [ 12 - 16 ] and D. robusta [[ 17 , 18 ], this study] bear the imprint of large scale climate change. For D. robusta , not all temporal changes were consistent with this hypothesis. In the course of comparing plant growth in rural and urban areas in relation to ozone exposure, rural areas tended to have lower average temperatures than urban ones [ 23 ]; this may explain the smaller (or lack of) change in 3R-1 frequencies in relatively rural Fayetteville and Iowa City as compared to the more urban locales. Also, there are fewer data available for these sites collected over time spans long enough to document temporal trends. Another possibility is that the climatic changes have not been of equal intensity in all localities, as indicated by regional increases and decreases in local temperatures (Fig. 2 ) suggested by the site by year interactions for AVTMP and MAXTMP in the ANCOVA analyses. Carson [ 24 ] noted that the chromosomal variation in populations of Drosophila robusta at Olivette, Missouri over a 10-year period was characterized by "extraordinary stability," wherein "certain frequencies may shift significantly as compared with the previous year, but in every case the observed frequencies approximate some previously observed level." This pattern continued until at least 1967 (Fig. 3 , 4 , 5 ). Temperature records from this period seem rather stable before significantly increasing through 2000 ( AVTMP and MINTMP second order regression slopes are both positive; Fig 2 ). Similar stability, frequency changes of less than 10 percent, has also been recorded near Blacksburg, Virginia from 1950 to 1962 (M. Levitan, unpublished data) and in northeastern New Jersey from 1948 until at least 1975 (Fig. 3 , 4 , 5 ). By contrast, every population sampled in 2003 evidenced at least one significant change of chromosomal polymorphism frequency compared to the numbers in the same area ten or more years previously, with indication that the changes in at least three are part of similar, if not identical, directional historical processes. Therefore, documentation of temporal genetic changes in these populations requires at least 10 to 20 years of comparative data. Influences of natural selection due to ambient temperature variation on frequencies of these gene arrangements and X chromosome associations have been demonstrated in laboratory experiments [ 24 , 25 ], and inferences from latitudinal and multiple elevational clines [ 26 - 31 ]. Frequencies of arrangements XL-1 and 2L-3 are clearly associated with cooler temperatures at higher elevations and latitudes, and 2L-1 increases in frequency in the laboratory under warmer temperatures. Carriers of 2L-3 have shorter egg to adult development times expressed in cooler temperatures, explaining increases in this gene arrangement with elevation and latitude [ 26 ]. Evidence of strong natural selection maintaining earlier observed genetic stability has also come from perturbation experiments in the wild [ 20 ]. At four time periods, large numbers of flies carrying X-chromosome combinations S2 and 22 , autosomal combination 2L-1.2R-1 ( 11 ), and 3R-1 from South Carolina, Alabama, and Mississippi were released in midsummer in the Englewood, NJ woods. In other years the released flies carried XL-1, 2L-3, and 3R from Minnesota and Michigan. Despite evidence of hybridization of the introduced flies with the local population, in each case the following spring saw return to frequencies of the previous year. According to Wright [ 32 ], "The alternative to natural selection in a changing environment is, as noted by Dobzhansky [ 33 ], the emergence of superior genetic systems" in the gene arrangements that have been increasing in frequency. He envisioned this happening in one population by recombination in inversion homozygotes, with the new adaptive gene complex in one or a few flies spreading to other localities by "occasional very long dispersion by the wind." He conceded that such a hypothesis would be most satisfactory for cases where a very rare inversion suddenly started increasing, such as the rises of TL and PP of D. pseudoobscura mentioned above, since high frequencies of inversion homozygotes would undermine the structural stability of the new adaptive complex. The sudden rises of D. robusta X-chromosome combination S1 in New York and New Jersey and of S2 in Missouri could be cases in point, but it would not explain the near doubling of S1 in Arkansas from a 35% base between 2001 and 2003 nor the three most consistent directional changes, those of 3R-1, 2L-1, and XL (Figures 3 , 4 , 5 ). Indeed, the nearly simultaneous changes in places as far apart as New York and Allentown, Pennsylvania, let alone New York and Missouri, would be very unlikely to depend on chance wind-blown dispersions. Here, causes for systematic frequency changes in widespread populations can be inferred to be a result of common environmental causes given the large amount of background data available for D. robusta . When compared to the inversion frequency data collected over 60 years from more than 150 natural populations [ 20 , 21 ] and results from numerous laboratory experiments, temporal frequency shifts across the range of D. robusta strongly suggest temperature variation as a likely mechanism driving microevolutionary change [ 17 , 18 ]. Such regional frequency shifts suggest a common response to temperature fluctuations or causes correlated with them, whether due to regional climatic changes or global warming. Conclusions Although direct evidence for climatic change has been accumulating for many years, its consequences for causing evolutionary changes have only recently been observed. Chromosome polymorphisms in Drosophila species have been historically important genetic systems for understanding mechanisms of evolutionary change, and have now been studied long enough to begin revealing widespread, systematic temporal frequency shifts in response to environmental change. These polymorphisms thus represent excellent indicators of future climatic shifts. Methods There are 14 commonly encountered gene arrangements segregating in natural populations of D. robusta located on five of the six arms of the 3 major chromosomes. The "Standard" arrangements were labeled for the respective chromosome arms: XL, XR, 2L, etc. Others were named in order of their discovery, e.g., XL-1, XL-2, XR-1, 2L-1 [described and configured in [ 20 , 24 ]]. The Standard arrangement of each arm was dubbed "S," and the other arrangements are referred to by the Arabic numerals in their names [ 34 ]. A fly with karyotype XL/XL-1, XR/XR-2, for example, would be S/1, S/2 in this notation. Depending on the linkage combination of the arrangements, it is also either SS / 12 or S2 / 1S . Linkage relationships are inferred from karyotypic analyses of adult males and females [ 35 ]. Female D. robusta , unlike many other drosophilids, quickly deplete stored sperm in the absence of remating so that wild-caught females can be despermed by repeated transfers to fresh food vials and then crossed to homokaryotypic males in a controlled fashion. Karyotypes of at least 6 larvae from these test crosses were prepared in order to infer the linkage combination of X chromosome gene arrangements. Salivary gland smears from larvae derived from matings in the wild, so-called "egg sample" data, were included when collected females did not survive the desperming transfers. This report compares data obtained in 2003 to earlier summer collections at a number of geographically isolated populations going back in some cases to 1946: Olivette, a suburb of St. Louis, Missouri; woods alongside Route 4 in Englewood, New Jersey; Trexler Memorial Park on the western outskirts of Allentown, Pennsylvania; the North Woods of Central Park in New York City; Fayetteville, Arkansas; and woods along the Iowa River at Iowa City, Iowa. The significance of year-to-year differences in frequency of each chromosome or chromosome arm obtained prior to 2003 was determined by G-tests [ 36 ]. Chronologically contiguous results that proved statistically homogeneous are combined in the figures. Individual X- and second-chromosome arrangements could not be tested in this way due to problems of independence. Analysis of meteorological data Temperature and precipitation data were obtained online from the National Climatic Data Center for stations with complete records extending from 1945–2003 nearest to each site. Continuous data for Fayetteville and Englewood were not available for the closest weather stations, so records from those stations with complete records were used. Correlations between temperatures from these stations were highly significant: Drake Field and the Agricultural Experiment Station for Fayetteville (r = 0.48 – 0.90, P < 0.01), Little Falls and Ridgefield for Englewood (r = 0.84 – 0.98, P < 0.0001); see Fig. 2 . Analysis of covariance in PROC GLM [ 36 ] was used to test for overall significance of temperature trends and chromosome frequency changes across sites, and to evaluate regional patterns of temperature and precipitation change. Polynomial regression analyses of temperature and precipitation data with time were performed with PROC REG [ 37 ]. In all cases, linear or second order polynomial regression explained the most variation for a particular model. Pearson product-moment correlations between arcsin transformed chromosome frequencies and the average temperature of the month for each collection were calculated with PROC CORR [ 37 ]. Non-parametric correlation analyses produced equivalent results. We also assessed correlations with temperatures for the month prior to collection, and the average temperature of the 3 months prior to collection: none were significant. Authors' contributions ML collected the Allentown, New York, and New Jersey flies and carried out the chromosomal analyses. WJE contributed the Fayetteville flies. Both authors contributed to planning the study, analyzing the data, and writing and revising the manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC548147.xml
515308	Perception and beliefs about mental illness among adults in Karfi village, northern Nigeria	Background This study was designed to examine the knowledge, attitude and beliefs about causes, manifestations and treatment of mental illness among adults in a rural community in northern Nigeria. Methods A cross sectional study design was used. A pre-tested, semi-structured questionnaire was administered to 250 adults residing in Karfi village, northern Nigeria. Results The most common symptoms proffered by respondents as manifestations of mental illness included aggression/destructiveness (22.0%), loquaciousness (21.2%), eccentric behavior (16.1%) and wandering (13.3%). Drug misuse including alcohol, cannabis, and other street drugs was identified in 34.3% of the responses as a major cause of mental illness, followed by divine wrath/ God's will (19%), and magic/spirit possession (18.0%). About 46% of respondents preferred orthodox medical care for the mentally sick while 34% were more inclined to spiritual healing. Almost half of the respondents harbored negative feelings towards the mentally ill. Literate respondents were seven times more likely to exhibit positive feelings towards the mentally ill as compared to non-literate subjects (OR = 7.6, 95% confidence interval = 3.8–15.1). Conclusions Our study demonstrates the need for community educational programs in Nigeria aimed at demystifying mental illness. A better understanding of mental disorders among the public would allay fear and mistrust about mentally ill persons in the community as well as lessen stigmatization towards such persons.	Background Throughout the world, there is an increasing awareness of mental illness as a significant cause of morbidity [ 1 ]. This awareness has increased with the steady decline of morbidity due to nutritional disorders, communicable diseases and other forms of physical illness, especially in countries undergoing epidemiological transition [ 2 ]. Mental and behavioral disorders are common, affecting more than 25% of all people at some time during their lives [ 3 ]. They are also universal, affecting people of all countries and societies, regardless of age, gender and income. The point prevalence of mental illness in the adult population at any given time is about 10% [ 3 ]. Similarly, around 20% of all patients seen by primary health care providers have one or more mental health disorders [ 3 ]. The role of the community in the prevention and care of the mentally handicapped has now been widely acknowledged and is regarded as the most appropriate basis for the development of mental health programs. Several studies have shown that knowledge of public attitude to mental illness and its treatment is a vitally important prerequisite to the realization of successful community-based programs [ 4 - 6 ]. The recognition of mental disorder also depends on a careful evaluation of the norms, beliefs and customs within the individual's cultural environment. Furthermore, community attitude and beliefs play a role in determining help-seeking behavior and successful treatment of the mentally ill. Unarguably, ignorance and stigma prevent the mentally ill from seeking appropriate help. People tend to have strong beliefs about the mentally ill, and many of these concepts are based on prevailing local systems of belief [ 7 ]. In developing any mental health education program, the basis of such beliefs must be taken into consideration. People's beliefs regarding mental illness should not only be known, but the purpose of their beliefs should be understood. Such attitudes and beliefs about mental illness can only be studied within a cultural context. Although the knowledge and perception of mentally ill patients and their relatives regarding mental illness has been reported from southwest Nigeria [ 8 ], to date there is little research on public attitudes towards mental illness from northern Nigeria, a culturally distinct part of the country. This paper is therefore one of the first to report findings related to attitudinal research on mental illness from northern Nigeria. The 1995 Nigeria National Mental Health Policy advocates the integration of mental health promotion, treatment and rehabilitation into primary health care services (PHC). However, this goal cannot be successfully achieved without an understanding of community attitudes towards mental illness. We therefore set out to ascertain the perceptions, attitudes and beliefs of adults regarding the causes, manifestations and treatment options of mental illness in a traditional Hausa community near Kano, Nigeria. Methods Study area The study population included 250 adults residing at Karfi village, about 15 kilometers from Kano city. This typical Hausa community has 11,314 inhabitants [ 9 ]. Majority are Muslims preoccupied with farming and petty trading. About 32.0% of the populace are literate. The village has one primary health centre and several traditional healers. Referrals from the health centers are sent to Kura General Hospital and occasionally to Aminu Kano Teaching Hospital. Informed consent was obtained from respondents prior to commencement of the interviews. Study design A cross sectional descriptive study Sample size and sampling technique A multistage sampling technique was adopted. In the first stage, four wards out of the seven in the village were randomly selected. After house numbering, 250 houses were selected from the four wards using a table of random numbers. Where more than one household was found in a house, one household was selected by balloting. Finally, one adult was selected at random from each household for the interview. A final sample size of 250 adults was therefore obtained. Instrument description/ Data collection We adopted and modified a pre-existing semi-structured questionnaire [ 7 ] to evaluate the perceptions and beliefs of adults in Karfi village. The questionnaire was in three parts; the first section inquired about personal data including age, sex, ethnicity, religion, marital status, educational level and occupation; the second part elicited awareness of existence of mental illness in the community, knowledge of causal factors, manifestation of the disorder and awareness and preference of treatment options, while the third part explored the attitudes, beliefs and perception of the respondent towards the mentally ill. Attitudes such as fear, avoidance, anger, suspicion, mistrust, hostility were considered negative, whereas sympathetic attitude, willingness to care for a mentally ill relative or friend and tolerance were considered positive attitudes. The study instrument was validated using a pilot study of 10 randomly selected households in a nearby village with similar demographic characteristics (Kumbotso). Results of the pilot study were used to modify content and wording of the questionnaire. Previously trained medical undergraduates fluent in Hausa language administered the questionnaires to the sample population. Data analysis The data was analyzed using the Epi-Info ® 6.0 statistical software package (CDC Atlanta, Georgia, USA). Descriptive statistics were depicted using absolute numbers, simple percentages, range, and measures of central tendency (mean, median) as appropriate. The Chi-square test was used to test the significance of associations between categorical groups. All tests of hypothesis were two-tailed with a type 1 error rate fixed at 5%. Results In all, 250 respondents were interviewed with 167 males and 83 females giving a sex ratio of 2:1 in favor of males. Their ages ranged from 18 to 74 years. Majority (78.0%) of the respondents were aged between 25 and 60 years. The median age (± SD) for the respondents was 34.5 ± 4.6 years; 36.0 ± 2.3 years for males and 28.5 ± 1.6 years for females. The Hausa-Fulani ethnic group constituted 89% of respondents, and the rest were Yoruba 2.0%, Igbo 5.0% and other minority Nigerian tribes 4.0%. About 16.0% of respondents had primary education, 12.0% had secondary education, and 4.0% had tertiary (post-secondary school) education. Approximately 27.0% of the population sampled had no formal education. Nevertheless, 41.0% of all respondents had Quranic education. The majority of respondents (90.0%) were Muslims and the remaining 10.0% were Christians. About 10.0% were single, 80.0% were married, 6.0% were divorced and the remaining 4.0% were widowed. Forty eight per cent of the respondents were engaged in farming, 25.0% were full-time housewives, 20.0% were engaged in petty trading, 7.0% were students and the remaining 4.0% were civil servants. About 13 persons interviewed (5.2%) had a relative with mental illness, but because of the small sample size we did not separately analyze findings from this group of participants. The most common symptoms proffered by respondents as manifestations of mental illness (Table 1 ) included aggression/destructiveness (22.0%), talkativeness (21.2%), eccentric behaviour (16.1%), and wandering (13.3%). Drug misuse in form of alcohol ingestion, cannabis and other psychoactive street drugs were identified as major causes of mental illness (34.3%), followed by effect of divine wrath or God's will (18.8%), magic or spirit possession (18.0%), and accidents/trauma (11.7%) (Table 2 ). Heredity, family conflicts and financial distress/poverty were uncommon responses. Table 1 Respondents' perceived manifestations of mental disorder Manifestation No.* (%) Rank order Aggression/destructiveness 173 (22.0) 1 Talkativeness 167 (21.2) 2 Eccentric behavior 127 (16.1) 3 Wandering 105 (13.3) 4 Self-neglect 86 (10.9) 5 Nudity 56 (7.1) 6 Restlessness/anxiety 50 (6.4) 7 Insomnia 15 (1.9) 8 Loss of consciousness 8 (1.0) 9 * Multiple responses recorded. Percentages represent proportions of responses obtained. Table 2 Perceived causes of mental illness Perceived cause No.* (%) Rank Misuse of drugs † 88 (34.3) 1 Divine punishment, God's will 48 (18.8) 2 Magic, spirit possession 46 (18.0) 3 Accidents/trauma 30 (11.7) 4 Heredity 27 (10.5) 5 Family conflicts/marital disharmony 14 (5.5) 6 Financial distress/poverty 3 (1.2) 7 Multiple responses recorded. † Include street drugs and alcohol. The majority of respondents (46.0%) opted for orthodox medical care when asked about preferred source of treatment for the mentally ill. This was followed by spiritual healing (exorcism) (34.0%) and the use of traditional herbal medicines (18.0%) (Table 3 ). Table 3 Respondents' preferred treatment for mental illness Response No. (%) Orthodox Medicine 116 (46.0) Traditional Medicine 46 (18.0) Spiritual Healing 85 (34.0) Others 3 (2.0) Total 250 (100.0) Table 4 shows that majority of the respondents harbored negative feelings towards the mentally ill, mainly in the form of fear (n = 113) and avoidance (n = 81). A total of 117 respondents (46.8%) were sympathetic towards the plight of the mentally sick with female respondents showing more inclination for sympathy compared to their male counterparts. The female respondents, however, tend to be fearful and avoid the mentally sick more than their male counterparts. Table 4 Distribution of attitude towards the mentally ill by gender Attitude Male Female Total No. (%) No. (%) Fear 24 (20.8) 89 (79.2) 113 Avoidance 12 (14.4) 69 (85.6) 81 Anger 48 (96.8) 2 (3.2) 50 Suspicion 36 (90.4) 4 (9.6) 40 Hostility 32 (93.6) 3 (6.4) 35 Mistrust 28 (89.6) 4 (10.4) 32 Indifference 16 (96.0) 1 (4.0) 17 Sympathy 39 (33.6) 78 (66.4) 117 Kindness 17 (20.8) 62 (79.2) 79 Literacy status was significantly associated with the type of feeling exhibited by the participants. Literate respondents were seven times more likely to exhibit positive feelings towards the mentally ill as compared to non-literate subjects (OR = 7.6, 95% confidence interval = 3.8–15.1) (Table 5 ). Table 5 Influence of literacy level of respondents on attitude towards the mentally ill Positive attitude Negative attitude Total Odds ratio (95%CI) Literate 92 15 107 7.6 (3.8–15.1) Not literate 64 79 143 Total 156 94 250 *CI = confidence interval, p < 0.0001 Discussion Aggression/destructiveness, talkativeness, and eccentric behaviors were the most frequently mentioned perceived symptoms of mental illness by respondents. This finding suggests that one has to display behaviour that attracts public attention and is therefore socially disruptive, to be recognized as having a mental disorder. This finding is similar to that documented by White in Tanzania [ 10 ] and Asuni et al . [ 7 ] among Yoruba patients in Western Nigeria. It is notable that hallucinations and delusions that are frequently mentioned in the literature as prototypes of gross psychotic states were not mentioned by the respondents as features of mental illness, probably because such features are not as tangible as aggressive attitudes. Misuse of drugs ranked highest among the respondents as a perceived cause of mental disorders than most of the other traditional etiologies. This finding may not be unconnected with increasing use of illicit drugs among the youth in developing countries. Although drug abuse was acknowledged by Iliyasu and Last [ 11 ] in their work on mental illness in Kano, northern Nigeria as a leading cause of drug dependent psychosis, Holzinger and colleagues [ 12 ] reported that drugs and alcohol was not considered by schizophrenia patients or their relatives to be a common cause of mental illness. Divine punishment ranked second as a perceived causative factor. This response may not be unconnected with the leading response (drug misuse), as many individuals are of the belief that one evokes supernatural wrath by taking intoxicants thus leading to the development of mental illness [ 3 ]. Belief in demons as the cause of mental health problems is a well-known phenomenon in many cultures of the world [ 13 ] but in our study this factor was ranked 3 rd place by respondents (18% of the responses). Our finding is also in contrast to Adebowale and Ogunlesi [ 8 ] who found that "supernatural causes" were the most acceptable etiological factor among both mentally ill patients and their relatives in southwest Nigeria. Only 1.0% of respondents admitted that financial distress or poverty was a possible cause of mental disorder. Such a low score in the face of the present adverse socio-economic conditions prevailing in Nigeria may be explained by the Hausa cultural cum religious belief in providence and patience in the face of adversity. Other factors have also been reported by investigators as being associated with perceived causes of mental illness among patients and their relatives. Srinivasan and Thara [ 14 ] found that patient gender and education, duration of illness, the key relative's education, and the nature of relationship were associated with family beliefs about the cause of mental illness. There was a higher score on the preference for modern medical care in treating psychiatric illness. Similar changes in attitude towards the modern scientific approach regarding mental disorders was documented by Alem et al [ 15 ] in their work on mental illness in Ethiopia and by Iliyasu and Last [ 11 ]. Fear and avoidance of the mentally sick was frequent among female respondents. Traditionally, men are expected to be outwardly brave and less submissive towards aggression. The high frequency of negative attitudes among our respondents may be due to the fact that the concept of the mentally sick has an unfavourable public image. It has been shown that people may evaluate mental illness negatively, reject and discriminate against mental patients, and base their views on traditional stereotypes [ 4 , 10 , 16 ]. Literacy was found to be significantly associated with positive attitude towards the mentally sick. Similar findings were reported by Madianos et al . [ 17 ] in Greece, and by Alem et al . [ 15 ] in Ethiopia. A study on community attitudes towards the mentally ill in New Zealand also reported that those who had previous contact with the mentally ill held informed and enlightened views [ 18 ]. Interestingly, a recent Hong Kong study reported a generally negative attitude towards the mentally ill despite a fairly good knowledge of mental illness among the respondents [ 19 ]. Our results support the hypothesis by Wolff et al . [ 19 ] that negative attitudes towards the mentally ill are fuelled by a lack of knowledge. Conclusions This study demonstrates the need for educational programs aimed at demystifying mental illness. A better understanding of mental disorders among the public would allay fear and mistrust about mentally ill persons in the community as well as lessen stigmatization towards such persons. Our findings may be of utility to health policy makers in the design of community mental health education programs and community mental health services in existing primary health centers in Nigeria. Competing interests None declared. Authors' Contributions KM and IZ initiated the study, and participated in the field work. IZ and AIS did the preliminary analysis and wrote the draft manuscript. KM, AIS and IZ participated in the design of the study and performed the statistical analysis. KM, IZ, and AIS participated in its design and coordination. AMH, KM and IZ wrote the final version of the draft manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC515308.xml
529432	Resistin Response to Inflammation	null	Obesity, in particular visceral adiposity, is positively correlated with insulin resistance and type 2 diabetes. Although the link is well established in humans and in rodent models, the mechanisms involved in obesity-related insulin resistance are not clear. One possibility is that hormones secreted by adipocytes compromise peripheral insulin sensitivity, and a number of candidates for such adipocyte signals have been identified. One of them, resistin, was discovered a few years ago by Mitchell Lazar and colleagues, who showed that the protein is expressed by mouse adipocytes and regulated by a group of anti-diabetic drugs called thiazolidinediones. Several lines of evidence from functional studies in rodents suggested that resistin could be the missing mechanistic link between obesity and diabetes. The human homolog of resistin has subsequently been under intense investigation, but initial studies revealed more differences than similarities between the human and rodent proteins: human resistin is mostly expressed in macrophages, not in adipocytes, and its serum levels do not correlate as clearly with obesity, insulin resistance, or diabetes. Similarly, genetic association studies between allelic variants of the resistin gene and metabolic abnormalities have so far been inconclusive. These results prompted some of the scientists in the field who had jumped on the resistin bandwagon after the initial results in rodents to jump off again. Others, including the resistin discoverers, continue their quest to uncover resistin's role in humans, and have started to think outside the framework defined by the mouse data. Connections between obesity and inflammation Starting with the role of macrophages in inflammation and encouraged by the fact that obesity and insulin resistance are associated with markers of systemic inflammation, Lazar and colleagues examined the resistin response to inflammatory stimulators. As they report in this issue, resistin production in macrophages and serum levels in patients are significantly increased by these stimulators. This response can be blocked by the thiazolidinedione rosiglitazone and by aspirin, two drugs that have dual anti-inflammatory and insulin-sensitizing actions and antagonize the immune regulator NF-kappaB. The researchers go on to show that activation of NF-kappaB is sufficient to induce resistin expression. And NF-kappaB is necessary for the resistin response to inflammatory stimuli. Lazar and colleagues now view obesity as a state of chronic inflammation and speculate that in obese individuals inflammatory cytokines lead to elevated production of resistin by macrophages and elevated serum resistin levels, which in turn contribute to insulin resistance and diabetes. This is consistent with some studies that have found higher resistin levels in obese individuals and patients with insulin resistance and/or diabetes, but not all studies have found such differences. Jeffrey Flier, an obesity researcher who was not involved in the study, calls the article “an excellent and timely paper that demonstrates the fact that inflammatory pathways induce resistin expression and levels in human monocytes ex vivo, and in intact humans. The work appears to provide a novel link between inflammation and insulin resistance, through monocyte derived resistin.” He points out, however, that “several other factors also appear to contribute directly to insulin resistance in inflammation (e.g., cytokines themselves, without invoking resistin) so the full biologic implications of the high resistin levels for insulin resistance in humans cannot be determined from this study.” Resistin, it seems, continues to resist easy interpretations.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC529432.xml
550668	Initiation to heroin injecting among heroin users in Sydney, Australia: cross sectional survey	Background Heroin injection is associated with health and social problems including hepatitis C virus (HCV) transmission. Few studies have examined the circumstances surrounding initiation to heroin injecting, especially current users initiating others. The current study aimed to examine the age of first heroin use and injection; administration route of first heroin use; relationship to initiator; the initiation of others among a group of heroin users; and to examine these factors in relation to HCV status and risk. Method Heroin users in Sydney were recruited through needle and syringe programs, a methadone clinic and snowballing. Participants were interviewed about their own initiation to heroin use, blood-borne virus risk and knowledge, and whether they had initiated others to heroin injecting. Information on HCV status was collected via self-report. Data was analysed using univariate and multivariate statistical techniques for Normally distributed continuous and categorical data. Results The study recruited 399 heroin users, with a mean age of 31 years, 63% were male, 77% reported heroin as their primary drug and 59% were HCV positive (self-report). Mean age at first heroin use and injection was 19 and 21 years, respectively. The majority of heroin users commenced heroin use via injecting (65%), younger users (<25 years, 25–30 years) were less likely than older users (>30 years) to commence heroin use parenterally. Participants were initiated to injection mainly by friends (63%). Thirty-seven percent reported initiating others to heroin injection, but few factors were related to this behaviour. Those with longer heroin using careers were more likely to report initiating others to heroin injection, but were no more likely to have done so in the preceding 12 months. Participants who had initiated others were more likely to have shared injecting equipment (12 vs 23%), but were no more likely to be HCV positive (self-report) than those who did not. Conclusion Interventions to prevent heroin users initiating others to injecting are necessary. Peer groups may be well positioned to implement such interventions.	Background Heroin is one of the most commonly injected illicit substances worldwide [ 1 , 2 ]. Heroin injection is associated with a range of harms including poor health [ 3 , 4 ], poor psychosocial functioning [ 5 ] and increased risk of fatal and non-fatal heroin overdose [ 6 , 7 ]. Heroin injection is also a significant risk factor for blood-borne viral infections (BBVI) such as hepatitis C, hepatitis B and HIV [ 8 - 12 ]. Despite these harms little is known about initiation to heroin injecting. Hepatitis C is probably the most prevalent health infection among injecting drug users (IDU) worldwide. The prevention of hepatitis C has proven difficult; unlike hepatitis B there is currently no vaccine available and programs which have been successful in reducing HIV have had only a small impact on the HCV epidemic [ 13 , 14 ]. It has therefore increasingly been acknowledged that prevention of initiation to drug injection is likely to be one of the most effective prevention strategies for blood-borne viral transmission [ 15 ]. Heroin injecting in particular is associated with increased HCV prevalence compared to injection of amphetamine – the two most commonly injected drugs in Australia [ 16 ]. A decrease in the age of initiation to drug use, including heroin use, across birth cohorts has been documented in both Australia [ 17 , 18 ] and the United States [ 19 ]. This decrease in the age of heroin initiation has been associated with greater poly drug use, unintentional overdose and criminal behaviour, independent of years of heroin use [ 17 ]. Better understanding of the circumstances surrounding initiation to injecting heroin use is needed if appropriate interventions are to be formulated. Crofts et al . examined a number of factors surrounding initiation to injecting among a group of young, recently initiated IDUs [ 20 ]. They found that the majority of their sample had instigated the first injecting episode, but were assisted or injected by friends; only a very small proportion were injected by a family member. The research also found that more females than males reported being injected by their partner, a pattern consistent with the literature in that females are more likely than males to have an IDU sex partner and are less likely to be able to inject themselves [ 21 - 23 ]. A recent Canadian study of street youth also found that females were more likely than males to be injected by their partner or lover [ 24 ]. However, research from the United States found only 13% of IDU interviewed were initiated by their sex partner, with no difference between males and females; indeed females were more likely to be initiated by another female [ 25 ]. Little attention has been paid to the initiation of others into injecting [ 15 ]. One small study of a brief intervention to prevent initiation to injecting found that 40% of the 86 IDU interviewed had initiated a mean of two people to injecting [ 26 ]. Crofts et al . found that 47% of recently initiated IDUs had also initiated another into injecting and of those who had, few informed new initiates of BBVI risk [ 20 ]. The relationship between initiators' BBVI status and risk behaviour was not reported, so it was not possible to determine whether those engaging in risky behaviours were more or less likely to initiate others to injecting. Moreover, the study focused on young or recently initiated injectors, so the role of age and experience in initiating others to injecting was unable to be determined. Crofts and colleagues found that the majority of recently initiated IDU were aware of HIV and hepatitis B and that the viruses could be transmitted via shared injecting equipment [ 20 ]. A much smaller proportion knew of HCV, though those who were aware of the virus were also aware that it could be transmitted via shared injecting equipment [ 20 ]. However, information on serostatus or hepatitis B vaccination status was not reported, nor was injecting risk behaviours such as needle and syringe sharing. This study examined initiation to heroin injection. Specifically the study aimed to examine: 1) age of first heroin use and first heroin injection; 2) route of first heroin use; 3) relationship to initiator; and 4) the initiation of others among a group of heroin users. These factors were then examined in relation to demographic variables such as gender, ethnicity, level of education and also blood borne virus status. Methods Procedure Heroin users in Sydney were volunteers recruited through needle and syringe programs and a methadone clinic. In order to sample a range of heroin users, snowballing, facilitated by a peer interviewer, was also used. Drug users were eligible to participate if they reported the use of heroin at least once a month in the preceding six months. All participants gave informed consent to be interviewed and received AUD20 for travel expenses. Measures A structured questionnaire was administered to participants by trained interviewers. Information was sought on blood-borne virus status and knowledge, and initiation to heroin use and included questions on age of first heroin use, age of first heroin injection, relationship to initiator and the number of times participants had initiated others to injecting. Information on HCV status was collected via self-report. Self-reported HCV status has a concordance of approximately 80% for those who have been tested [ 27 , 28 ]. For the purposes of this study, self-reported HCV is relevant as risk behaviour is influenced by the belief about one's status, not actual status [ 29 ]. To determine ethnicity, participants were asked how they identified ethnically, those identified as being of Aboriginal or Torres Strait Islander decent were categorised as Indigenous, those not born in Australia and those born in Australia but who identified as belonging to another ethnic group were categorised as 'other Australian'. The study was approved by four institutional ethics committees: University of New South Wales Human Research Ethics Committee, Central Sydney Area Health Service Ethics Review Committee, South Western Sydney Area Health Service Research Ethics Committee, and South Eastern Sydney Area Health Service Research Ethics Committee. Analysis Continuous variables were assessed using t -tests and one-way analysis of variance. Linear regression was employed to test the relationship between continuous variables. The chi square (χ 2 ) statistic was used for univariate analysis of categorical data. Multiple logistic regression, using backward elimination, was used for multivariate analysis to examine independent relationships between dichotomous variables. All data were analysed using SPSS version 11.01. Results Sample characteristics The sample consisted of 399 heroin users, of whom 63% were male. The mean age of participants was 31 years (SD 8.2, range 17, 58). The majority of participants were born in Australia (78%), a small proportion of which identified as another ethnic group. Participants were categorised as either 'Australian' (67%), Indigenous (17%) or 'other-Australian' (16%). The majority (81%) of participants had attended secondary school, but only 42 (11%) participants had tertiary education. Thirty-four (9%) participants had completed primary (elementary) school only. Sixty-one percent of participants had a history of incarceration. Heroin was the primary drug used by 77% of the sample, with a median of 9.5 (range <1, 39) years of use and injecting. The majority (98%) of the sample injected heroin; 14% of the sample had been injecting heroin for three years or less and 80% for more than three years (18 cases had missing data for this variable). Those who did not inject heroin were excluded from analysis pertaining to injecting. Age of initiation to heroin use and injection The mean age at first heroin use was 19 years, (SD 6.0, range 9–43 years) and the mean age of first injection was 21 years (SD 6.3, range 13–43 years). The mean age of first heroin use and first heroin injection was similar for males (18 years for both) and females (18 and 19 years, respectively). There were no differences in terms of ethnicity for either age of first heroin use (18 years for all groups) or first heroin injection (18, 19 and 18 years for Australians, 'other Australians' and Indigenous Australians respectively). For those who completed primary school only, the mean age of first heroin use was 17 (SD 5.4) years and 19 (SD 6.0) years for those who attended secondary school or above, the difference failed to reach significance ( t 395 = -1.89, p = .0.56). Participants who completed primary school only, were a mean age of 17 (SD 4.8) years when they first injected heroin and the mean age of first injection for those who attended secondary school and above was 20 (SD 6.3) years ( t 386 = -2.46, p = .014). Linear regression was used to examine the relationship between current age and age of initiation to heroin use and injecting. There was a significant relationship between participants' current age and age of first heroin use (β = 0.41, p < .001). Similarly, current age and age of first injection were also significantly related (β = 0.39, p < .001). Route of first heroin administration Heroin was most commonly first administered by injection (65%). For the 141 participants who used non-parenteral routes of administration on initiation of heroin use, the most common method was smoking (burning and chasing), with 28% of the sample reporting initiating heroin use with this method. Only seven percent first used heroin intranasally, orally or by other means. Participants who first injected heroin and those who first used heroin non-parenterally, commenced heroin use at similar ages (19 and 20 years, respectively). Those who initiated heroin use by injection had been using heroin for more years than those who initiated heroin use non-parenterally (13 years vs 9 years respectively, t 379 = 4.36, p = 0.001). Route of administration was also associated with ethnicity: 74% of Indigenous participants commenced heroin use via injection, 69% of 'Australians' and 36% of 'other Australians' (χ 2 = 26.40, p < .001). There were no differences in the proportion of participants who commenced heroin use via injecting in terms of gender (males 64% and females 65%) or level of education (primary education 77%, secondary education 64%). Logistic regression was used to determine the factors independently associated with initiation to heroin use via injecting. Variables entered into the model were age, gender, ethnicity and age at first injection (before or after 18 years). Level of education was not entered into the model as there were too few participants with only primary school level of education who commenced heroin use via a non-parenteral route of administration. The final model was significant (χ 2 = 43.92, 4 df , p < .001) and the Hosmer and Lemeshow test indicated good fit (χ 2 = 1.5 6 df , p = .96). Gender was not a characteristic independently associated with route of first heroin injection and was removed from the model. Participants aged less than 25 years and 25–30 years were less likely than those aged 30 years or more to initiate heroin use via injecting. 'Other Australians' were also less likely to reported initiating less injecting (Table 1 ). Table 1 Characteristics of those who first injected heroin, using multivariate logistic regression Characteristic No. participants %1 st injected Adjusted odds ratio 95% CI P Age 31+ 184 77 - - 25–30 106 60 0.48 0.28–0.83 .008 ≤ 24 96 54 0.34 0.19–0.61 <.001 Ethnicity Australian 269 67 - - - ATSI 69 74 1.18 0.61–2.26 ns Other Australians 61 36 0.30 0.16–0.55 <.001 Age 1st injected ≥ 18 164 77 - - <18 234 56 0.63 0.39–1.02 .059 CI = Confidence interval Relationship to initiator Participants were usually taught to inject by a friend (63%), family member (14%) or their partner (11%). Ten percent of the sample reported 'other', which was typically self-taught. Males and females differed significantly in terms of who taught them to inject (χ 2 = 24.75, df = 2, p < .001; Table 2). Specifically, a greater proportion of females were taught to inject by their partner than were males (χ 2 = 23.96, df = 1, p < .001), while more males were taught to inject by a friend (or other) than females (χ 2 = 11.77, df = 1, p < .001). The relationship between participants and their 'initiators' did not differ according age of first injection, ethnicity or level of education across those initiated by friends, family or their partner. Initiating others Over a third (37%) of participants reported having taught someone to inject drugs and 17% had done so in the preceding 12 months. Among those who had ever taught someone to inject (n = 149), the median numbers of people taught was three (range: 1–200) and two (range: 1–50) in the preceding 12 months. Similar proportions of males and females reported teaching someone to inject ever (38% vs 37%) and in the preceding 12 months (16% vs 19%). There was also no significant difference in terms of ethnicity or mean age of those who had ever taught someone to inject ever or in the preceding 12 months. Those who had taught someone else to inject heroin had been injecting for a greater mean number of years than the remainder of the sample (13 v. 10 years; t = -3.08, df = 370, p = 0.002). However, this difference diminished for those who had recently (preceding 12 months) taught someone to inject (11 years for both groups). Blood-borne viral infections Ninety-two percent of participants reported being tested for HCV infection; 62% in the six months preceding interview and 78% in the 12 months preceding interview. The median number of weeks since the last test was 19 (range: 1–572 weeks). Two-hundred and thirty-five participants (59% of the total sample) self-reported being HCV positive. Participants who reported being HCV positive initiated heroin injecting at a younger mean age (19 years, SD 5.7) than the remainder of the sample (21 years, SD 6.8). HCV positive participants were more likely to report initiating heroin use via injecting than those who were HCV negative (72% vs 58%, χ 2 = 9.14, df = 1, p = 0.003). IDUs typically become infected with HCV early in their injecting career [ 30 ], thus analysis was stratified by years of injecting (≤ 3 years vs >3 years). The relationship remained only for those who had been injecting for more than three years (61% vs 76%, χ 2 = 7.93, df = 1, p = 0.005). There was no difference in self reported HCV status between those who had initiated someone to injecting in the preceding 12 months and those who had not. Eighty-eight percent of participants had reported being tested for HIV a median of six months prior to interview (range <1–144). Of these, seven (2% of the total sample) reported being HIV positive. One-hundred and forty-seven participants (37%) reported having been vaccinated against hepatitis B. There were no differences between those who reported being vaccinated against hepatitis B and those who had not in terms of age at first heroin injection, initial route of administration and initiating some one else to injecting in the preceding 12 months. Only a small number of participants (9%) reported having used a needle or syringe after someone else in the month preceding interview. Sharing needles and syringes was not associated with mean age at initiation to heroin injection or initial route of heroin administration. More participants who had recently initiated someone to injecting (i.e. preceding 12 months) reported sharing needles and syringes (17%) than those who had not recently initiated someone (8%), though the difference failed to reach significance (χ 2 = 3.51, df = 1, p = .061). Sharing (borrowing or lending) injection paraphrenia in the month preceding interview was reported by just over half (52%) the sample. Participants who had engaged in this behaviour were more likely to have initiated heroin use via injecting (71% vs 61%, χ 2 = 4.37, df = 1, p < .037) and to have recently initiated someone to injecting (12% vs 23%, χ 2 = 7.95, df = 1, p < .005) compared to those who not shared injection paraphernalia. There was no difference in terms of age at first heroin injection. Disscussion This study has identified factors associated with initiation to heroin injecting. The majority of heroin users commenced heroin use via injecting, though those who initiated heroin use via this method had been using heroin for a longer period than those who initiated heroin use via non-parenteral methods. Participants were initiated to injection by a range of people, mainly friends. A large proportion of study participants also reported initiating another person to heroin injection, a practice that was associated with longer heroin use careers and recent sharing of injecting equipment. Increasing attention has been paid to the role of IDUs in initiation to injecting drug use [ 15 ], though few studies have examined the prevalence of this behaviour. Crofts and colleagues postulate that the behaviour can be understood as communicable and modelled as in the case of infectious diseases [ 20 ]. Fewer participants in the current study reported initiating others to injecting than that reported by Crofts et al . (37% vs 47%) and only 17% reported doing so in the preceding 12 months. Crofts et al . sampled only young injecting drug users (i.e. 17–24 years) [ 20 ], whereas the injecting experience of participants sampled for this study ranged from less than one year to 39 years and younger participants were no more likely than older participants to report initiating others to injecting. A third of the sample initially used heroin by non-parenteral routes of administration. Participants from ethnically diverse backgrounds (i.e. born outside Australia or identify other than Australian) were more likely than other participants to have first used heroin by means other than injecting, which is consistent with other Australian research which found smoking heroin to be more common among Indochinese than Caucasian heroin users [ 31 ]. Participants who commenced heroin use via injecting were also older than those who first used a non-injecting route of heroin administration, possibly indicating an overall shift toward non-injecting routes of administration. A number of interventions aimed at reducing the incidence of transition to injecting have received attention [ 15 ]. One study found that brief interventions delivered through existing drug services can have an impact on injecting drug users' behaviour, however, the study was modest and further research is warranted [ 26 ]. The current study found a direct significant relationship between current age and age of first heroin use and first heroin injection. This result is consistent with other Australian research [ 17 , 18 ]. Nevertheless the finding is subject to bias due to 'right censoring' of the data [ 17 ]. For example, an 18 year old recruited into the study cannot have an age of initiation above 18 years, while a 30 year old can have any age of initiation up to 30 years, and be included in the study even if they commenced injecting at 25 years [for a more detailed discussion see [ 17 ]]. There are numerous explanations for this decrease in initiation to heroin use; the rapid expansion of the Australian heroin market between 1996 and 2000, where the price of heroin decreased concomitant with an increase in purity and availability from 1996 to 2000 [ 32 ], may in part explain this phenomenon, though greater examination of the structural determinants of drug use are also warranted [ 33 ]. Although the proportion of participants reporting using a needle or syringe after another person (sharing) in the preceding month was low, it is consistent with other research examining transitions to injecting [ 31 ], but lower than Australian national estimates of this behaviour [ 16 ]. It is possible that the data is subject to a social desirability bias and thus under-reported. Participants reported comparatively high levels of 'indirect' sharing (sharing injecting paraphernalia other than needles and syringes), a phenomenon recently found to significantly and independently increase the risk of HCV transmission [ 34 ]. This behaviour was reported more often by those who reported teaching someone to inject in the 12 months preceding interview. That those who are more likely to engage in this behaviour are also teaching others to inject is cause for concern, as it has the potential to perpetuate the problem of injecting related risk taking behaviour among new recruits. The majority of the sample believed themselves to be HCV positive and, not surprisingly, those who injected at a younger age were more likely to be HCV positive and to have first used heroin via injection. Though importantly, there was no difference in terms of HCV status between those who had recently taught another to inject and those who had not. Conclusions This study has confirmed that initiation to heroin use in Australia typically occurs via injection, though this is less apparent among younger heroin uses. The study has also found that more than a third of heroin users have initiated others into injecting, with close to a fifth having done so recently. Many of those who engaged in this behaviour tended to take greater injection related risks which has important implications for the transmission of blood-borne infections. A better understanding of the circumstances surrounding the initiation to heroin injection is needed. Peer-led interventions, which have been found to be effective in changing IDUs' attitudes and behaviours [ 26 ], may have a role to play in reducing the number of heroin users initiating others to injecting. Competing interests The author(s) declare that they have no competing interests. Authors' contribution C Day coordinated the study, was responsible for the statistical analysis and writing the paper. P Dietze was responsible for study design at the national level, secured funding and provided comments on the manuscript. J Ross and K Dolan supervised all aspects of the work and provided extensive comments on the manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC550668.xml
517494	Mutations in the IGF-II pathway that confer resistance to lytic reovirus infection	Background Viruses are obligate intracellular parasites and rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for understanding viral life cycles, and may provide cellular targets for new antiviral therapies. Results A gene entrapment approach was used to identify candidate cellular genes that affect reovirus infection or virus induced cell lysis. Four of the 111 genes disrupted in clones selected for resistance to infection by reovirus type 1 involved the insulin growth factor-2 (IGF-II) pathway, including: the mannose-6-phosphate/IGF2 receptor ( Igf2r ), a protease associated with insulin growth factor binding protein 5 ( Prss11 ), and the CTCF transcriptional regulator ( Ctcf ). The disruption of Ctcf , which encodes a repressor of Igf2 , was associated with enhanced Igf2 gene expression. Plasmids expressing either the IGF-II pro-hormone or IGF-II without the carboxy terminal extension (E)-peptide sequence independently conferred high levels of cellular resistance to reovirus infection. Forced IGF-II expression results in a block in virus disassembly. In addition, Ctcf disruption and forced Igf2 expression both enabled cells to proliferate in soft agar, a phenotype associated with malignant growth in vivo . Conclusion These results indicate that IGF-II, and by inference other components of the IGF-II signalling pathway, can confer resistance to lytic reovirus infection. This report represents the first use of gene entrapment to identify host factors affecting virus infection. Concomitant transformation observed in some virus resistant cells illustrates a potential mechanism of carcinogenesis associated with chronic virus infection.	Background Viruses as obligate intracellular parasites rely upon the host cell for different steps in their life cycle, including attachment, disassembly, transcription, translation, reassembly, and egress. Consequently, characterization of these cellular processes will be essential for any understanding of viral life cycles, and may provide cellular targets for new antiviral therapies. The susceptibility to virus infection varies greatly among different cell types, and virus-resistant cells frequently emerge post-infection [ 1 - 4 ]. This suggests that host cell contributions to the virus life cycle, although complex, have genetic determinants. We therefore used a genetic approach to identify cellular genes required for infection by reovirus, a small cytolytic RNA virus that replicates in the cytoplasm. A gene trap retrovirus was use to create libraries of rat intestinal epithelial (RIE-1) cell clones in which each clone contained a single gene disrupted by an integrated retrovirus. The mutant libraries were then infected with reovirus, and resistant clones were selected. We hypothesized that genes mutated by gene entrapment may confer reovirus resistance as a result of either haploinsufficiency or loss of heterozygosity and could be identified by characterizing the genes disrupted by the entrapment vector. From these experiments we have isolated 152 clones and have characterized mutations in 111 different genes, providing potential candidates required for reovirus infection and/or cell killing. Many of the disrupted genes have known or imputed functions, and several are known to function in the same or related pathways. For example, four mutations affected genes in the insulin growth factor-2 (IGF-II) pathway, including genes encoding the IGF-ll/manose-6-phosphate receptor [ 5 , 6 ] ( Igf2r , locus ID 25151), the IGF binding protein 5 protease [ 7 ] ( Prss11 , locus ID 65164, 2 clones), and CTCF ( Ctcf , locus ID 83726), a transcriptional repressor of the IGF-II gene ( Igf2 ) involved in paternal imprinting. The frequency of mutations involving the IGF-II pathway led us to investigate the role of IGF-II in reovirus infection. Clone 6B72, which contains a mutation in Ctcf [ 8 ], was found to over express Igf2 transcripts, consistent with the known role of CTCF as a transcriptional repressor of the Igf2 gene. Moreover, forced expression of IGF-II in RIE-1 cells was sufficient to confer cellular resistance to lytic reovirus infection. Enforced IGF-II expression also transformed RIE-1 cells to anchorage independent growth, a phenotype associated with malignant change. These results represent the first use of gene entrapment to identify components of host cell metabolism required for virus infection and illustrate a potential mechanism of carcinogenesis associated with chronic virus infection. Results Disruption of the CTCF gene results in cells resistant to lytic infection by reovirus Gene entrapment strategies to identify host genes required virus replication depend on methods to select for virus resistant clones present at about one in 10 4 –10 5 mutagenized cells. Unfortunately, cells persistently infected with reovirus (PI) can emerge at high frequencies (one in 10 2 –10 3 ) and are intrinsically resistant to the virus [ 9 , 10 ]. In preliminary studies, we found that hepa 1/a cells were not suitable for genetic studies due to the emergence of PI clones. However, persistently infected rat intestinal epithelial cells (RIE-1) [ 11 , 12 ] were found to require a serum survival factor and die when placed in serum free media (Figure 1 ). In vitro infection of RIE-1 cells with reovirus also appears to mirror virus replication in the rodent host [ 13 - 15 ]. Consequently, RIE-1 cells were used in the present study, and reovirus-resistant clones were selected in serum-free medium to remove PI survivors. RIE-1 cells were mutagenized by using the U3NeoSV1 gene trap shuttle vector [ 16 ] (see Methods) and the resulting libraries of mutagenized cells were infected with reovirus serotype 1/Lang at a multiplicity of infection (MOI) of 35, to select for clones resistant to lytic infection. The isolated clones did not express reoviral antigens, and did not produce infectious virus as assessed by plaque assay, suggesting these reovirus resistant clones were not PI. Figure 1 Persistently infected RIE-1 cells fail to survive in serum-free media. RIE-1 parental cells and cells persistently infected with reovirus type 1 were plated in complete medium (FBS + ) or in media in which the serum was omitted (FBS - ). Surviving cells were stained with gentian violet after 7 days. Darkly staining wells represent cell survival. Regions of genomic DNA adjacent to the U3NeoSV1 provirus in each virus resistant clone were isolated by plasmid rescue and sequenced. Altogether, of the 151 isolated clones, 62% of flanking sequences matched known or presumptive genes, and an additional 23% were represented in the public databases of expressed sequence tags (dbEST) or non-redundant sequences (nr). From the 111 clones matching known or presumptive genes, 10 genes were represented more than once. Many of the disrupted genes have known or imputed functions, and several are known to function in the same or related pathways. For example, the library included 4 independent mutations involving three genes that encoded proteins associated with the insulin growth factor-2 (IGF-II) signalling pathway, namely, IGF-ll/manose-6-phosphate receptor [ 5 , 6 ] ( Igf2r , locus ID 25151), the IGF binding protein 5 protease [ 7 ] ( Prss11 , locus ID 65164, 2 clones), and CTCF ( Ctcf , locus ID 83726), a transcriptional repressor of IGF-II involved in paternal imprinting [ 17 , 18 ]. The position of the provirus (clone 6B72) in the first intron of the Ctcf gene is shown in Figure 2 . CTCF differentially represses maternal Igf2 gene expression, whereas the imprinted paternal gene escapes repression due to methylation of CTCF binding sites [ 17 , 18 ]. Figure 2 Disruption of the Ctcf gene in 6B72 cells. DNA sequences flanking the U3NeoSV1 provirus in 6B72 cells were cloned and sequenced. The flanking sequences were identical to sequences in the rat genome, placing the provirus in the first intron of the Ctcf gene (A). Filled and open boxes indicate coding and non-coding exons, respectively. Flanking sequences 5' of the U3NeoSV1 provirus (B) include the first exon (shaded) of the gene. Igf2 transcripts induced by CTCF disruption Considering that four clones had mutations in the IGF-II pathway, the relationship between the Ctcf mutation in 6B72 cells and the virus resistant phenotype was investigated further. Levels of CTCF protein in 6B72 cells were reduced by about 50% as assessed by western blot analysis (Figure 3A ), consistent with the disruption of one allele. Diminished CTCF expression was associated with an increase in Igf2 transcripts as assessed by Northern blot hybridization (Figure 3B ). In addition, two products were amplified from 6B72 cells by RT-PCR using primers flanking the IGF-II pro-hormone coding sequence (Figure 3C ). The first product was identical to the rat IGF-II pro-hormone (pro-IGF-II) coding sequence (Accession X17012), whereas, the second contained 14 additional nucleotides generated by splicing of exon 2 to an alternative splice acceptor located 14 nucleotides upstream of the normal Igf2 exon 3 splice acceptor site. The alternative transcript is expected to encode a pro-IGF-II protein extending 11 amino acids into the E-peptide sequence ending in a stretch of 60 amino acids lacking homology with any known protein (Figure 4 ). Figure 3 CTCF and Igf2 expression in RIE-1 and 6B72 cells. Levels of CTCF protein were assessed by Western blot analysis (A), normalized to a β-actin control. Levels of Igf2 transcripts were assessed by Northern blot analysis (B) normalized to GAPDH control. Protein content, as assayed by western blot analysis and standardized to β-actin was decreased in the 6B72 cell clone to 30% of control. Reverse transcriptase PCR analysis of Igf2 transcripts (C). The RT-PCR products (arrows) were separated on a 2% agarose gel, revealing an additional transcript in the 6B72 cells. The DNA sequence of the larger RT-PCR product (D) revealed an alternatively spliced transcript ( Igf2 sv ) generated by splicing of exon 2 to a cryptic 3' splice site located 14 nucleotides upstream of exon 3. Figure 4 Alternatively spliced product contains a single nucleotide polymorphism in the igf2 coding sequence. Intron sequences incorporated into the alternatively spliced transcript (highlighted in black) alter the translational reading frame of the pro-homone downstream of the coding sequence of the processed IGF-II protein (italics and bold). The Igf2 sv PCR product also contained a G to A base substitution (underlined) that replaces alanine with threonine at codon 62 (boxed) of the mature hormone. Resistance to reovirus lytic infection results from increased Igf2 expression The stability of virus resistance in the 6B72 cell was tested by infecting RIE-1 and 6B72 cells with reovirus type 1 at a MOI of 10. There was approximately a 10 fold lower titre of reovirus obtained following infection of 6B72 cells as compared to RIE-1 cells at 24 hrs. post-infection (4.5 × 10 5 versus 5.1 × 10 6 ), and the difference was also maintained at 48 hours post-infection (data not shown). Additionally, there was a dramatic difference in the survival of 6B72 cells after being exposed to high titres of reovirus type 1 (Figure 5 ). Figure 5 IGF-II modulates reovirus resistance in RIE-1 and 6B72 cells. RIE-1 and 6B72 cells expressing either the IGF-II pro-hormone (proIGF2) or the alternatively spliced transcript (IGF2 SV , Figure 3D) were challenged with serial dilutions of reovirus type 1 (upper panel), and the surviving cells were stained with gentian violet 4 days post-infection. The multiplicity of infection (MOI) for each row is indicated. pro-IGF-II converted RIE-I cells to a reovirus resistant phenotype (RIE-1/proIGF2) but had little if any effect on already-resistant 6B72 cells (6B72/proIGF2). Plasmids expressing the alternatively spliced Igf2 transcript had no effect on RIE-1 cells (RIE-1/IGF2 SV ) but abrogated virus resistance in 6B72 cells (6B72/IGF2 SV ). The experiment was repeated 3 times. Expression of I gf2 transgenes (lower panel) was monitored by Northern blot hybridization, and the expression of Igf2 in RIE-1 cells is shown for comparison. Expression of Igf2 in 6B72 is not shown. To determine whether Igf2 confers resistance to reovirus infections, clones of RIE-1 cells over-expressing the full-length Igf2 transcript or the splice variant ( Igf2 sv ) were generated and examined for their capacity to resist lytic infection. As shown in Figure 5 expression of wild type ( Igf2 ), but not the splice variant ( Igf2 sv ) increased the resistance of RIE-1 cells to reovirus infection by over 100 fold. However, when the Igf2 sv was transfected into 6B72 cells, the ability of 6B72 cells to survive infection was abolished. Expression of the Igf2 gene in an anti-sense orientation caused no significant difference in the capacity of 6B72 cells to resist infection (data not shown). These studies suggest that increased Igf2 expression in 6B72 cells is associated with their capacity to resist reovirus infection, and that the Igf2 sv encodes a trans-dominant isoform that blocks the activity of Igf2 . The IGF-II hormone confers resistance to lytic infection Igf2 transcripts encode a pro-hormone of 180 amino acids that is processed to generate the 67-residue IGF-II protein [ 19 ]. Other proteolytic products including the 89-residue carboxy-terminal E peptide may also be biologically active [ 20 ]. The Igf2 sv PCR product also contained a single nucleotide substitution (G1393A) that results in the substitution of a threonine for alanine at position 62 of IGF-II, raising questions about Igf2 sequences that influence reovirus resistance. Vectors expressing only the 68-residue IGF-II protein both with and without the A62T change were compared for their ability to confer resistance to lytic infection by reovirus type 1 (Figure 6 ). The IGF-II expression vector converted RIE-1 cells to a virus resistant phenotype; whereas, the IGF-II 62T expression plasmid was inactive (Figure 6 ) and did not suppress the resistance of 6B72 cells (data not shown). These results indicate that virus resistance can be affected by mutations in the IGF-II coding sequence and downstream sequences, including the E-peptide, are not required. However, the trans-dominant effects of Igf2 sv apparently require alterations to the carboxy-terminus of the IGF-II pro-hormone. Figure 6 IGF-II sequences lacking the E-peptide can convert RIE-1 cells to a reovirus-resistant phenotype. Two independent clones of RIE-1 cells transfected with plasmids expressing native (IGF-II) and mutant (IGF-II A62T ) proteins without the carboxyl terminal extension-peptide (E-peptide) were challenged with serial dilutions of reovirus type 1 as described in Figure 4. Native IGF-II protected RIE-1 cells from reovirus infection (IGF-II (a) and IGF-II (b) ), while the IGF-II A62T mutant (IGF-II A62T(a) and IGF-II A62T(b) did not. Non-infected RIE-1 cells (C), and infected 6B72, and RIE-1 cells were included as controls. Expression of Igf2 transgenes (lower panel) was monitored by Northern blot hybridization, and the expression of Igf2 in RIE-1 cells is shown for comparison. The native IGF-II (small arrow) is slightly larger than the cDNA constructs (larger arrow), whereas the double-sided arrow marks the constitutively expressed GAPDH , as shown. Expression of Igf2 in 6B72 is not shown. Over expression of igf2 confers resistance to other reovirus sertotypes RIE-1 cells are intrinsically resistant to infection by reovirus type 3 [ 13 ]; therefore, experiments to assess the effects of IGF-II expression on lytic infection by reovirus type 3, were performed using murine L-cells. As shown in Figure 7 , L-cells expressing Igf2 were significantly more resistant to both reovirus serotypes than the parental cells, indicating that the ability of IGF-II to confer resistance to reovirus infection is not limited to a single cell or virus type. Figure 7 Decreased lytic infection of L-cell clones over expressing the IGF-II gene. Constitutively expressed GAPDH was used to assess loading of RNA in lanes. Survival of lytic infection was determined by infecting 10 5 L-cells or L-cells expressing the pro-IGF2 transgene with varying concentrations of reovirus type 1 (A) or type 3 (B). The multiplicity of infectious virus particles per cell (MOI) is indicated for each virus serotype. Surviving cells were visualized at 4 (A) or 5 days (B) following infection with gentian violet. Transgene expression was determined by northern blot (C). Experiments were repeated three times and a representative experiment is shown. IGF-II expressing RIE-1 cell has delayed disassembly of virus particle Previous studies have indicated that proteoytic disassembly of virus particles occurs in the lysosome and requires the participation of cathepsin B and L that are transported by the Igf2r gene [ 21 - 23 ]. Igf2 binds to Igf2r resulting in alteration in cathepsin trafficking [ 24 ]. Georgi and colleagues showed that directly fluorescentated reovirus particles dissipated their fluorescent signal coincident with disassembly of the outer capsid [ 25 ]. To determine whether over expression of igf2 would affect disassembly of virions, we examined the fate of virus during the first hours of infection. Purified reovirus type 1 virions were directly labeled with fluorescein and adsorbed to RIE-1 or 6B72 cells, and the persistence of a fluorescein signal determined at 2 hrs following virus absorption (fig 8A,8B ). The binding of virions to RIE-1 cells and 6B72 cells and initial accumulation of virus particles within cells was similar, however fluorescence was almost non-detectable in RIE-1 cells (Figure 8A ) whereas it was still present in 6B72 cells at 2 hrs (Figure 8B ). In addition, western blot analysis of virion proteins 2 hrs following attachment to cells confirmed the persistence of the σ3 protein in the 6B72 cell clone, but not RIE-1 cells (fig 8C,8D ). Therefore, disassembly of virions is altered in the 6B72 cell clone. Figure 8 6B72 cells have delayed disassembly of reovirus type 1. Fluorescein-labelled reovirus particles were absorbed to RIE-1 (A) or 6B72 cells (B). Persistent fluorescence at 2 hours was found in 6B72 cells, but not in RIE-1 cells. Non-replicating reovirus type 1, at 3 × 10 4 particles per cell, was adsorbed to RIE-1 (C) or 6B72 (D) cells at 4°C, washed and incubated at 37°C for 2 and 4 hours. Cells were lysed and the state of virus particles determined by western blot. The outer capsid proteins μ1 and σ3 are present in the 6B72 cell preparations at 2 and 4 hours, but not in the RIE-1 cells. CTCF deficient RIE cells grow in soft agar, a consequence of increased Igf2 expression Accumulating evidence suggests that enhanced IGF-II expression associated with loss of Igf2 genomic imprinting may promote tumour formation [ 26 , 27 ]. Although RIE-1 and 6B72 cells appeared to proliferate at similar rates as assessed by MTS/PMS incorporation (Data not shown), 6B72 cells that have a disrupted Ctcf gene were capable of forming colonies in soft agar (Figure 9b ). To assess the role of IGF-II in anchorage-independence, cells transfected with Igf2 and Igf2 sv were tested for their ability to grow in soft agar. RIE-1 cells transfected with Igf2 , but not Igf2 sv formed colonies in soft agar (Figure 9a ), whereas 6B72 cells transfected with Igf2 sv lost their capacity to proliferate in soft agar as did cells transfected with an anti-sense Igf2 construct (Figure 9b ). The IGF-II 62T expression plasmid had no transforming activity (Figure 9d ). On balance, transforming activities of the different Igf2 plasmids on RIE-1 and 6B72 cells as assessed by the soft agar assay were similar to their effects on reovirus resistance, although the vector expressing only IGF-II was less active in transforming RIE-1 cells to anchorage independence than vectors expressing the entire pro-hormone. Figure 9 Anchorage-independent growth phenotypes of RIE-1 cell clones. 10 5 cells were suspended in media containing 1% agarose and plated in 6 well culture dishes. RIE-1 cells acquire the ability to grow in soft agar after being transfected with a vector expressing pro-IGF2 but not the IGF2 SV splice-variant (a). The vector inserted in the Ctcf gene (6B72) confers the ability to grow in soft agar, but the phenotype is suppressed by expression of IGF2 SV (b). Clones selected for reovirus resistance with gene trap vectors inserted into the Prss11 , Igf2r and Anxa2 genes failed to grow in soft agar (c). RIE-1 cells expressing native IGF-II protein without the E-peptide grew in soft agar but the colonies were smaller (d) than produced by pro-IGF-2 (a), while the corresponding IGF-II A62T protein (E-peptide) did not transform RIE-1 cells to anchorage independence (d). Colonies were photographed (20×) after 7 days except (d) where the cells were photographed after 10 days. The capacity to proliferate in soft agar was not a property of other reovirus resistant cell clones that contained mutations in the IGF-II pathway. Figure 9 shows that cell clones with disrupted Igfr or Prss11 genes did not form colonies in soft agar (Figure 9c ). An additional reovirus resistant-clone with an insert in the Anxa2 (annexin 2) gene, associated with cytomegalovirus infection [ 28 ] and recognized to bind to the insulin and insulin growth factor receptor-1 [ 29 ], also failed to grow in soft agar (Figure 9c ). Therefore, the capacity to proliferate in soft agar was not a general property of reovirus-resistant cells, even in clones that contain mutations in the IGF-II pathway. However, L-cells also displayed enhanced ability to grow in soft agar in addition to virus resistance, following enforced Igf2 expression (Figure 10 ). Figure 10 IGF-II increases colony formation of L-cells in soft agar. Forced expression of the rat pro-Igf2 gene (pro-Igf2 c ) in L-cells increases the number of soft-agar colonies by 4 to 5 fold as compared to the L-cell parent, as shown in this representative photomicrograph at 7 days (10×). Discussion Insertional mutagenesis provides an approach to identify genes associated with selectable cellular phenotypes. We have isolated over 100 potential clones with mutations in genes that may play roles in the life cycle of reovirus. In the present study, one clone resistant to reovirus lytic infection contained a provirus inserted into the gene encoding the CTCF transcriptional regulator. CTCF binding motifs are present in many genes, including Igf2 [ 26 ], H19 [ 27 ], and Myc [ 8 ]. However, since 3 other clones selected for reovirus resistance contained mutations in the IGF-II pathway, the role of IGF-II in virus-resistance was investigated further. Reduced expression of the Ctcf gene was associated with enhanced Igf2 expression in virus-resistant cells, while forced expression of the Igf2 gene in the parental RIE-1 line was sufficient to confer resistance to lytic reovirus infection. By inference, the recovery of inserts affecting other genes in the IGF-II signalling pathway suggests that mutations in multiple genes may affect the same phenotype by acting on a common pathway. The insert in Igf2r was found to decrease the expression of the gene as assayed by northern blot analysis (data not shown). As IGF-II targets the igf2r to lysosomal degradation, mutations in the genes encoding either the receptor ( Igf2r ) or its ligand ( Igf2 ) will affect the activity of the other, and result in a reduced endosomal trafficking of hydrolases necessary for reovirus disassembly [ 24 ]. Our data indicates there is a decrease in virus disassembly in 6B72 cells, consistent with a block at this step in morphogenesis. Further studies will be required to assess if and how inserts in the Prss11 and Igf2r genes influence reovirus resistance. As the entry, disassembly, transcription, translation, and repacking of viruses share common features; we anticipate that common cellular pathways will influence infection by other virus families. Indeed, Igf2r has been implicated in herpes simplex and zoster virus infection [ 30 , 31 ]. However, the present study is the first to show a direct connection between Igf2 gene expression and resistance to lytic virus infection. By using constructs that encode the mature hormone without the E-peptide, we were able to show that forced IGF-II expression is sufficient to confer a reovirus resistant phenotype. These results differ from other studies in which reovirus replication was enhanced by treatment of RIE-1 cells with insulin. The latter effect is presumably caused by enhanced virus replication associated with cell proliferation [ 13 ]. Expression of the Igf2 gene is frequently elevated in common childhood and adult neoplasms [ 27 , 32 - 38 ] and has been associated with tumour progression and metastasis [ 39 , 40 ]. Igf2 also transformed RIE-1 cells to anchorage-independence, a phenotype that predicts the potential for malignant growth in vivo . Virus-resistant 6B72 cells also grew in soft agar, presumably as a result of enhanced IGF-II expression. Since plasmids expressing only IGF-II were less active in transforming RIE-1 cells to anchorage independence than vectors expressing the entire pro-hormone, further study is needed to determine whether the capacity of RIE-1 cells to proliferate in soft agar is enhanced by the E-peptide or other products derived from the carboxy terminus of the pro-hormone. In other studies, the E-peptide enhanced insulin secretion from β-cells [ 20 ], and may play a role in cellular transformation [ 31 ]. cDNA clones of an alternatively spliced Igf2 transcript ( Igf2 sv ), blocked the ability of IGF-II to promote reovirus resistance and anchorage independent growth in a trans-dominant manner. The protein coding sequence of Igf2 sv contains a frame shift in the E-peptide region and lacks a site [ 20 , 41 ] required for the proteolytic processing of the pro-hormone. The alternative splice site is used very infrequently [only one EST (AA259833) in dbEST was similarly spliced] and thus probably plays no physiological role. Further studies are planned to determine molecular basis for the dominant negative activity of Igf2 sv . 6B72 cells were highly resistant to reovirus infection as determined by virus yield and cell survival at different times post-infection. Virus resistance was a genetically selected trait manifested by clonally pure cell populations, could be conferred by enforced IGF-II expression, and involved decreases in virus disassembly. Although decreased virus disassembly is sufficient to explain virus resistance, we do not exclude the possibility that other mechanisms may contribute to the resistance of 6B72 cells, since any genetic selection may generate clones with multiple, independent mutations. The fact that an early step in infection (uptake/disassembly) is defective in 6B72 cells makes it very difficult to test whether downstream steps might also be affected. While the original impetus of our studies was to understand the replication cycle of intracellular pathogens that cause acute and chronic infectious diseases, the finding of cell growth phenotypes associated with virus resistance is of some interest. It has been proposed that lytic viruses may used to treat certain malignancies [ 42 - 45 ]. However, based upon our observations, such therapy may carry a risk associated with selection of virus resistant cell clones with enhanced growth/survival potential. Additionally, chronic infections contribute to the development of a number of human cancers [ 46 - 49 ]. While the carcinogenic process is not well understood, cell proliferation associated with inflammation is also thought to contribute to tumour promotion [ 50 , 51 ]. The present study illustrates how carcinogenesis could also be influenced by selection for virus-resistant cells with mutations in genes affecting cell proliferation or survival. Conclusions This is the first reported use of gene entrapment to identify host genes affecting the susceptibility of cells to virus infection. These results indicate that IGF-II, and by inference other components of the IGF-II signaling pathway, can confer high levels of resistance to lytic reovirus infection. IGF-II expression specifically blocked virus disassembly. Ctcf disruption and forced Igf2 expression both enabled cells to proliferate in soft agar, a phenotype associated with malignant growth in vivo . Therefore, these results illustrate a potential indirect mechanism of viral carcinogenesis by which cells selected to virus resistance may also have enhanced oncogenic potential. Methods Entrapment mutagenesis and selection of reovirus resistant clones To identify genes required for reovirus lytic infection, a gene trap retrovirus shuttle vector, U3NeoSV1, was used to generate mutagnized rat intestinal (RIE-1) cells [ 52 ]. RIE-1 cells were infected with the gene trap vector at a multiplicity of infection <0.1, and were selection in media containing G418 sulfate (0.7 mg/ml) (Clontech, Palo Alto, CA, USA)[ 52 ]. Twenty libraries of mutant RIE-1 cells, each consisting of 10 4 independent gene entrapment events, were generated and expanded until each mutant clone was represented by approximately 10 3 sibling cells. These cells were plated at low density and incubated in serum-free media for 3 days until they became quiescent, infected overnight with reovirus serotype 1 at a multiplicity of infection of 30 plaque forming units (pfu) per cell. The infected cells were detached with trypsin, DMEM medium containing 10% fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, UT, USA) was added and the cells were allowed to reattach. After 4–6 hours the medium was replaced with serum-free medium and the cells were incubated for several days until only a few cells remained attached to the culture flask. Cells that survived the selection were allowed to form colonies that were expanded for further analysis. Reovirus stocks and infectivity assays Reovirus type 1 (Lang) and reovirus type 3 (Dearing) were previously described [ 53 ]. A stock of reovirus that was passaged twice in L cells was purified [ 14 ] and the purified virus band was fluorescein labelled as previously described [ 25 ]. For some experiments the top component, consisting of virus particles that are devoid of genome, was used to study the entry pathway [ 54 ]. Survival of parental L- and RIE-1 cells and RIE-1 and L-cells transfected with Igf2 constructs was determined in 96-well plates seeded at 5 × 10 4 per well. On the following day, serial dilutions of reovirus type 1 or type 3 were added in 100 μl of media and cells were incubated at 37°C and 5%CO 2 for 1 hour. Cells were washed three times in PBS, and fresh media was added containing 0.1% anti-reovirus antibodies to inhibit secondary infection. Cells were incubated for 4 to 5 days, and surviving cells visualized with gentian violet. Studies were repeated a minimum of three times. To determine the titre of reovirus present in cells, cells were frozen and thawed three times and plaque assays were performed as previously described [ 14 ]. Titres of virus were repeated twice. Fluorescence microscopy For experiments involving fluorescein labeled reovirus, cells were grown on glass slides and fixed following appropriate times with 4% paraformaldehyde, dehydrated, and mounting in cytoseal acrylic resin (Stephens Scientific, Cornwall NJ, USA) to improve clarity and prevent bleaching. Fluorescence microscopy was performed using an Axiophot microscope (Carl Zeiss, Inc., Thornwood, NY, USA), with a 40×/1.3 plan Neofluar objective and fluorescein filter set. Images were captured with a low-light, cooled CCD camera (Micromax, Photometries, Inc., Tucson, AZ, USA). Identification of genes disrupted by gene entrapment To identify the gene disrupted by the vector in clones surviving reovirus infection, the shuttle-vector property of U3NeoSV1 was utilized. Regions of genomic DNA adjacent to the U3NeoSV1 provirus were cloned by plasmid rescue, and sequenced [ 52 ]. Sequencing was done using an automated sequencer (ABI 3700 DNA Analyzer, Applied Biosystems, Foster City, CA, USA), and the results obtained were compared to databases available in the public domain (BLAST nr, est, and hgts). The probability of a match to sequences in the databases occurring by chance alone varies due to interspecies conservation and the length of the match. Matches to characterized genes were considered significant if the interspecies matches had a probability score p <10 -5 and involved non-repetitive sequences. As indicated, virtually all of the genes identified had matches to murine or human gene sequences with p < 10 -10 and rat with p < 10 -20 . Igf2 expression was assessed by northern blot hybridization Total RNA was isolated from cultured cells using Trizole reagent (Gibco BRL, Gaithersburg, MD, USA). 5 μg of RNA was separated on 1.2% agarose gel, and transferred to a nitrocellose membrane. Membranes were hybridized with random prime-labeled (Strategene, Cedar Creek, TX, USA) probes corresponding to a full length of Igf2 cDNA and either glyceraldehyde dehydrogenase (GAPDH ) or β-actin cDNA. Igf2 cDNA isolation and expression Rat Igf2 cDNAs were obtained using reverse transcriptase PCR (RT-PCR). Total RNA was extracted from RIE and 6B72 cells using Trizole reagent (Life Technologies, Rockville, MD, USA). RT was performed on 1 μg of total RNA (PTC-100 programmable Thermal Controller, MJ Research. Inc, Watertown, MA, USA). A pair of primers was designed according to rat sequences: CTTCCAGGTACCAATGGGGATC (forward) and TTTGGTTCACTGATGGTTGCTG (reverse). A 500 bp DNA was amplified under following conditions: 95°C, 1 min; 40 cycles of 95°C 30 seconds, 60°C 30 seconds and 68°C 3 minutes; 68°C 10 min; 4°C. Immunoblotting analysis Cells were washed with PBS and lysed in SDS Lamelli buffer. Protein concentration was determined using the bicinchoninc acid protein assay (Sigma-Aldrich Corp., St. Louis, MO, USA). 20 μg of protein extract was loaded in each lane of a 10% SDS-PAGE and run at 100 V. Protein was transferred to a nitrocellulose membrane at 22 V overnight at 4°C. The membrane was washed three times with TBST (50 mM Tris pH 7.5, 150 mM NaCI, 0.05 % Tween 20) and then incubated in blocking buffer (TBST and 5% non fat dry milk, pH 7.5) for 1 hour at room temperature. The membrane was then incubated with anti-mouse, CTCF (1:500, BD Transduction laboratories) and β-actin (1:3000, Sigma-Aldrich Corp., St. Louis, MO, USA) in blocking buffer overnight at 4°C. Following 3 washes, the membranes were incubated with goat anti-mouse secondary antibody (1:20,000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 hour at room temperature, followed by three 15-min washings. Immune complexes were visualized by addition of chemiluminescence reagent (Renaissance, DuPont NEN, Boston, MA, USA) and the membrane was exposed to Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY, USA). Transfection Cells were cultured to semi-confluence and plasmids expressing wild type and variant IGF-II transcripts were transfected into RIE-1 and 6B72 or L-cells using SuperFect Reagent (Qiagen, Inc. Valencia, CA, USA) according to the manufacturer's protocol. After 48 hours, transfected cells were passaged, 1:10, into medium containing hygromicin B (selective medium) at a concentration determined to kill 100% of non-transfected cells (150 mg/ml for RIE-1 and 6B72 cells, 650 mg/ml for L-cells). Cells were maintained in selective medium until clones appeared. Soft agar colony forming assay Dual layers of sea plaque agarose were made with the bottom layer consisting of a 50:50 mixture of 1.6% agarose solution 1:1 and 2X medium. The bottom layer was allowed to set for 4 hours, and then a 50:25:25 solution consisting of 2X medium, 1.6% stock agarose, and 1X medium containing cells, at a final concentration of 5000 cells/ml, was vortexed in a conical tube, and 2 ml was added to each well. Following 30 minutes at room temperature to allow the upper layer to set, plates were incubated at 37°C, 5% CO 2 incubator for 7–10 days and checked for colony formation by microscopy. Analysis of viral protein expression in infected cells Cells were plated at 1.5 × 10 6 per well in 2 ml of medium in 6-well plates and allowed to sit over night. Cells were washed with phosphate-buffered saline (PBS), pH 7.4, and then infected with reovirus type 1 at the specified MOI. Virus was allowed to adsorb to cells for 1.5 hours at 4°C, washed twice with serum-free medium and incubated at 37°C and 5% CO 2 . At the indicated times, cells were scraped and lysed in Tris lysis buffer (10 mM Tris [pH 7.5], 2.5 mM MgCI 2 , 100 mM NaCI, 0.5% Triton x-100, 1 tablet Protease Inhibitor Cocktail Tablets [Roche Applied Science, Indianapolis, IN, USA] per 10 ml). After 30 min on ice, Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA, USA) were added to cell lysate samples (1:1). Protein samples were loaded in a 12% SDS-PAGE gel and run at 100 V. Protein was transferred to a nitrocellulose membrane at 100 V for 1 hour on ice. The membrane was washed three times with TBST (50 mM Tris pH 7.5, 150 mM NaCI, 0.05 % Tween 20) and then incubated in blocking buffer (TBST and 5% non fat dry milk, pH 7.5) for 1 hour at room temperature. The membranes were then incubated with rabbit anti-reovirus type 1 (1:50) and β-actin (1:3000, Sigma-Aldrich Corp., St. Louis, MO, USA) antibodies in blocking buffer overnight at 4°C. Following 3 washes in TBST, the membranes were incubated with goat anti-rabbit (for reovirus) or goat anti-mouse (β-actin) secondary antibodies (1:20,000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 hour at room temperature, followed by three 15-min washes. Immune complexes were visualized by addition of chemiluminescence reagent (Renaissance, DuPont NEN, Boston, MA, USA) and the membrane was exposed to Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY, USA). Cell Proliferation Assay RIE-1, 6B72, or Igf2 transfected RIE-1 or 6B72 cells were seeded at 5 × 10 4 per well in 96-well plates, incubated at 37°C and 5% CO 2 . At 4, 6, 18, 48 hours post plating, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium [MTS], and an electron coupling reagent (phenazine methosulfate, [PMS]) were added at 20 μl per well (CellTiter 96 Aqueous Non-Radioactiver Cell Proliferation Assay, Promega, Madison, Wl). Plates were incubated for 2 hours, and then the absorbance was determined at 490 nm. Each set of conditions was repeated in triplicate. Authors' contributions JS, ELO, and CH conducted most of the laboratory work. KSW assisted in the analysis of fluorescein-labelled virus preparations. HER provided the vectors and advice on their use. DHR discovered that persistently infected cells require serum to survive, allowing the selection of genetically resistant cell clones. HER and DHR provided funding and supervision for the research, and prepared the manuscript. All authors have read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC517494.xml
515297	Genetic structure of four socio-culturally diversified caste populations of southwest India and their affinity with related Indian and global groups	Background A large number of microsatellites have been extensively used to comprehend the genetic diversity of different global groups. This paper entails polymorphism at 15 STR in four predominant and endogamous populations representing Karnataka, located on the southwest coast of India. The populations residing in this region are believed to have received gene flow from south Indian populations and world migrants, hence, we carried out a detailed study on populations inhabiting this region to understand their genetic structure, diversity related to geography and linguistic affiliation and relatedness to other Indian and global migrant populations. Results Various statistical analyses were performed on the microsatellite data to accomplish the objectives of the paper. The heretozygosity was moderately high and similar across the loci, with low average G ST value. Iyengar and Lyngayat were placed above the regression line in the R-matrix analysis as opposed to the Gowda and Muslim. AMOVA indicated that majority of variation was confined to individuals within a population, with geographic grouping demonstrating lesser genetic differentiation as compared to linguistic clustering. D A distances show the genetic affinity among the southern populations, with Iyengar, Lyngayat and Vanniyar displaying some affinity with northern Brahmins and global migrant groups from East Asia and Europe. Conclusion The microsatellite study divulges a common ancestry for the four diverse populations of Karnataka, with the overall genetic differentiation among them being largely confined to intra-population variation. The practice of consanguineous marriages might have attributed to the relatively lower gene flow displayed by Gowda and Muslim as compared to Iyengar and Lyngayat. The various statistical analyses strongly suggest that the studied populations could not be differentiated on the basis of caste or spatial location, although, linguistic affinity was reflected among the southern populations, distinguishing them from the northern groups. Our study also indicates a heterogeneous origin for Lyngayat and Iyengar owing to their genetic proximity with southern populations and northern Brahmins. The high-ranking communities, in particular, Iyengar, Lyngayat, Vanniyar and northern Brahmins might have experienced genetic admixture from East Asian and European ethnic groups.	Background The Indian subcontinent is regarded as a natural genetic laboratory, owing to the co-existence and interaction of socio-culturally, linguistically, ethnically and genetically diversified endogamous populations in a geographical terrain. It is believed that the earliest humans leaving Africa for Eurasia might have taken a coastal route across Saudi Arabia, through Iraq, Iran, to Pakistan and finally entered India along the coastlines [ 1 ]. A second wave of migration (~10,000 years ago) brought in Proto-Dravidian Neolithic farmers from Afghanistan, who were later displaced southwards by a large influx of Indo-European speakers ~3500 years ago in to the subcontinent [ 2 , 3 ]. The origin and settlement of the Indian people still remains intriguing, fascinating scientists to explore the impact of these past and modern migrations on the genetic diversity and structure of contemporary populations [ 4 - 6 ]. Anthropologically, southern and northern populations are distinct and these differences are further substantiated by (i) the presence of Neolithic sites in this region suggests that Neolithic people of southern India came from north by land and the west-coast by sea [ 7 ], (ii) the southern megaliths resemble closely with those of the Mediterranean and western-Europe, while those from northern India are similar to megaliths found in Iran and Baluchisthan [ 8 ], and (iii) the predominance of Dravidian language in this region as opposed to their secluded occurrence in central Asia and other parts of India, suggests that the Dravidian languages might have originated within India [ 9 ]. It is, thus, of considerable genetic interest to understand the genetic structuring and relationships of southern populations. The present study was carried out on one of the largest southern states, Karnataka, positioned on the southwest coast of India, with a dwelling of about 50 million people. This expanse has been a rich source of prehistoric discoveries dating back to the Paleolithic era that are akin to those seen in Europe [ 7 ]. Karnataka has received continuous gene flow from different caste and linguistic groups residing in the adjoining areas of Maharashtra, Andhra Pradesh and Tamil Nadu [ 10 ], resulting in the congregation of a large number of diverse endogamous groups within this region. Its large coastline of about 400 Km also attracted the Portugese, Dutch and French traders, who were seeking more profitable ventures on the southern coast at large [ 2 ]. Southwest India is, thus, one of the most disparate terrains, with extensive colonization in the past and justifies an in-depth genetic study. A few studies utilizing classical markers have been carried out on southern populations [ 5 , 11 , 12 ], including few communities of Karnataka [ 13 , 14 ]. However, sound inferences relating to their genetic structuring and diversity could not be drawn due to low discriminatory power of these markers. Recently, microsatellite markers have gained immense popularity in precisely defining population structure, diversity, affinities, gene flow and other crucial aspects associated with population genetics [ 15 - 21 ] because of the relative expediency, with which a large number of loci and alleles can be typed, facilitating the accumulation of vast data sets that can be readily analyzed with an extensive array of statistical tools [ 22 , 23 ]. These markers also demonstrate high heterozygosity [ 24 ], rendering them highly suitable for carrying out the present study. Among the different caste and tribal groups inhabiting the southwest coast of India, we have selected four predominant Dravidian-speaking communities from Karnataka: Iyengar Brahmin, Lyngayat, Gowda and Muslim, they not only belong to dissimilar groups of the Indian caste hierarchy but also have varied migration histories, conferring them uniqueness and significance from a genetic perspective. The present microsatellite study primarily attempts to understand the genetic structure of the four selected populations and to determine their genetic relationship with other linguistically and ethnically similar groups of southern India and Brahmin groups of northern India. It has been suggested that that despite the linguistic homogeneity in southern India, these populations have remained genetically diversified [ 25 ]. Hence, we sought to determine the role played by geographical location and linguistic affiliation in genetically differentiating Indian populations. Also, as mentioned earlier, the western coast has witnessed colonization from different world populations, we aim to divulge the impact of these past migrations on the gene pool of the present southern populations by discerning their relationship with historically acclaimed and established migrant groups, ethnically represented by European, Hispanic, East Asian and African populations. Results Allele frequency at 15 STR was used to compute the heterozygosity (observed) for the four studied populations, which varied for each locus, and population but reflected similar values, ranging between 0.724 and 0.797 (Table 1 ). An average G ST value of 0.009 elucidates the low degree of genetic differentiation in them. However, the G ST value for the pooled Indian and global populations demonstrated a high value at 2.3% (data not shown). Genetic relationship of studied populations with other similar southern groups; Vanniyar, Gounder, Pallar and Tanjore Kallar [ 26 , 27 ], northern Brahmins belonging to Orissa [ 28 ] and Bihar [ 29 ], and four relevant global ethnic groups: European, Hispanic, African [ 30 ] and East Asian [ 31 ] was divulged by computing DA distances (Table 2 ) and represented using NJ tree (Fig. 1 ). Among the four studied populations, Iyengar, Gowda and Muslim formed a distinct cluster. Although NJ tree clearly depicts the clustering of southern populations, D A distances indicate that among these groups, Iyengar, Lyngayat and Vanniyar are more similar to the northern Brahmins (0.030). Furthermore, genetic distances emphasize the affinity of Lyngayat with Tanjore Kallar (0.029), Iyengar (0.026) and Vanniyar (0.028). Estimation of relatedness between the southern and global populations shows that all the southern communities formed a separate cluster, nevertheless, genetic distances disclose the affinity of upper caste Indian communities; Iyengar, Lyngayat, Vanniyar, Bihar and Oriya Brahmin with Europeans and East Asians. The Indian populations were most distant to Africans. Table 1 Average heterozygosity and G ST values for 15 loci in the four studied populations. OBSERVED HETEROZYGOSITY LOCUS G ST BRAHMIN LINGAYAT GOWDA MUSLIM TPOX 0.707 0.581 0.542 0.555 0.010 D3S1358 0.661 0.793 0.559 0.488 0.036 THO1 0.815 0.785 0.678 0.688 0.005 D21S11 0.876 0.857 0.779 0.733 0.005 D18S51 0.907 0.938 0.779 0.888 0.006 PENTA E 0.921 0.876 0.864 0.800 0.014 D5S818 0.692 0.724 0.525 0.733 0.007 D13S317 0.753 0.714 0.745 0.733 0.007 D7S820 0.723 0.734 0.754 0.800 0.005 D16S539 0.861 0.846 0.830 0.777 0.010 CSF1PO 0.723 0.734 0.745 0.733 0.009 PENTA D 0.815 0.755 0.741 0.933 0.006 vWA 0.784 0.734 0.779 0.688 0.002 D8S1179 0.861 0.822 0.745 0.755 0.007 FGA 0.861 0.894 0.803 0.911 0.017 Average 0.797 0.785 0.724 0.747 0.009 Table 2 D A distance matrix between ten Indian and four global groups based on allele frequency at 15 microsatellites. Pop HS NE MO CA OB IB LY GO MU BB PL VN TK GD HS NE 0.079 MO 0.049 0.123 CA 0.029 0.086 0.07 OB 0.044 0.092 0.052 0.04 IB 0.044 0.091 0.041 0.04 0.03 LY 0.047 0.096 0.047 0.04 0.03 0.026 GO 0.066 0.122 0.072 0.07 0.055 0.036 0.047 MU 0.076 0.118 0.078 0.07 0.066 0.051 0.056 0.054 BB 0.043 0.101 0.052 0.05 0.038 0.031 0.037 0.054 0.068 PL 0.061 0.11 0.067 0.07 0.064 0.05 0.056 0.075 0.077 0.063 VN 0.045 0.105 0.042 0.04 0.034 0.023 0.028 0.039 0.053 0.037 0.049 TK 0.047 0.096 0.053 0.05 0.044 0.028 0.029 0.052 0.062 0.044 0.052 0.032 GD 0.064 0.112 0.064 0.06 0.051 0.036 0.043 0.057 0.073 0.054 0.059 0.032 0.043 Abbreviations used in Table- Hispanic – HS, African – AF, Asian – AS, European – EU, OriyaBrahmin – OB, Iyengar Brahmin – IB, Lyngayat – LY, Gowda – GO, Muslim – MU, Bihar Brahmin – BB, Pallar – PL, Vanniyar – VN, Tanjore Kallar – TK, Goundar – GD. Figure 1 Neighbor-joining tree depicting the genetic relationship of Karnataka populations with related Indian and global ethnic groups based on 15 STR markers. The regression model (Fig. 2 ), of mean per locus heterozygosity against distance from centroid assumes that when a population experiences same amount of gene flow from a homogenous source, a linear relationship exists between the expected and observed heterozygosity. A change in gene flow directly affects this linear relationship. The R-matrix when applied to the Indian populations assists in understanding the influence of external gene flow and admixture among populations. The higher observed than expected heterozygosity of Iyengar and Lyngayat, placed above the theoretical regression line helps infer that these populations have received more than average external gene flow, which was also observed in Vanniyar, Pallar and Oriya Brahmin. The Gowda and Muslim groups exhibit lower than expected heterozygosity values and fall below the regression line, suggesting lesser admixture in them. Figure 2 Regression plot demonstrating the relatively higher gene flow levels in high-ranking populations of India. Abbreviations used in figure: OB-Oriya Brahmin, PL-Pallar, IB-Iyngar Brahmin, VN-Vanniyar, LY-Lyngayat, TK-Tanjorekallar, GD-Goundar, Mu-Muslim, BB-Bihar Brahmin, GO-Gowda. The microsatellite diversity computed using AMOVA revealed that the genetic variation observed in Indian populations was mainly confined to variation amongst individuals (~98%), irrespective of their geographic or linguistic grouping (Table 3 ). The geographical clustering of populations into three regions: north, southwest (Karnataka) and southeast (Tamil Nadu) demonstrated a low variance of 0.29%, p = 0.010 (Table 3a ). As compared to geographical grouping, the linguistic clustering (Indo-Caucasian and Dravidian) exhibited a noticeable increase in the molecular variance between the two groups, 0.65% (p = 0.06, Table 3b ). The genetic diversity among populations within each group remained almost similar at both levels of analysis. Table 3 Genetic differentiation of Indian populations based on AMOVA (a) Geographical grouping Groups in set 1 Source of Variation Percentage Variation Group 1 – North: Bihar and Orissa populations Among groups 0.29 Group 2 – South-west: Karnataka populations Among populations in groups 0.97 Group 3 – South-east: Tamil Nadu Populations Within populations 98.74 (b) Linguistic grouping Groups in set 2 Source of Variation Percentage Variation Group 1 – Indo-European: Orissa and Bihar Among groups 0.69 Group 2 – Dravidian: Southern populations Among populations in groups 0.94 Within populations 98.40 Discussion In recent years, population genetics has witnessed extensive use of microsatellite markers to understand and evolutionary histories of contemporary human populations [ 17 , 32 - 34 ]. Though, the populations inhabiting south India have played a major role in formation of the Indian gene pool, however, very few genetic studies have been carried out on them. The present study utilizes 15 STRs to provide comprehensive genetic information on four predominant communities inhabiting the southwest coast of India, which may significantly help in understanding the genetic composition of southern populations. Genetic structure of Karnataka populations The most distinctive feature revealed by the fifteen microsatellites was the considerable genetic homogeneity amongst the four diverse caste groups residing in southwest India. The presence of an almost similar allele frequency pattern [ 34 ], suggests that these populations might have a common ancestry or probably experienced very high gene flow during the period of their coexistence. The above finding is further supported by the low genetic differentiation of 1.0% among the studied groups irrespective of their caste and migration histories. The high heterozygosity and rii values in Lyngayat reflect the admixture and stochastic processes experienced by it. The genetic affinity of Lyngayat with other related southern caste populations, like, Iyengar, Vanniyar and Tanjore Kallar reiterates its heterogeneous past. It is noteworthy that although the southern populations exhibited higher affinity amongst each other, the high-ranking populations, like, Iyengar, Lyngayat and Vanniyar also displayed some genetic similarity to Brahmins from Bihar and Orissa, indicating that the gene pool of Iyengar and Lyngayat probably consists of genetic inputs from both southern and northern groups. However, strong conclusions cannot be drawn due to low genetic differentiation among the studied populations. Though the Gowda is known to have moved in to Karnataka from the adjoining area of Tamil Nadu, our study reveals that Gowda cluster with the studied populations and not with Tamil groups. The low hetetozygosity and high rii values of Gowda implies that it might have differentiated as a result of stochastic processes. Furthermore, the relatively lower heterozygosity and admixture levels of Gowda and Muslim might be attributed to the socio-cultural practice of consanguineous marriages in them. The Muslim group was found to be genetically similar to local populations. Regional conversions from diverse castes that occurred during the period of Islamic dominance might elucidate the more or less identical genetic relationship between Muslims and other studied groups. The microsatellite study emphasizes the genetic similarity among the Karnataka populations, with the lack of any strong caste or religious bias in them. Analysis of genetic variance AMOVA test strongly suggests that genetic diversity among the southern populations was mainly confined to intra-population variation, further emphasizing the genetic homogeneity in them. Analysis using different genetic markers corroborate with our finding that the genetic diversity in human populations can be mainly attributed to variation within populations [ 4 , 17 , 19 , 34 , 36 , 37 ]. An exploration of the genetic differentiation based on geographical grouping of populations discloses the genetic similarity among populations residing in a region. Nevertheless, the geographic affinity was comparably lesser to that observed within the two linguistic families, viz., Dravidian and Indo-European. Our finding provides evidence to the strong linguistic affinity prevailing amongst the Dravidian speaking populations and imparts them genetic distinctness from the Indo-European linguistic group. Even though prior studies have indicated that genetic clusters often correspond closely to predefined regional and linguistic groups [ 34 ], AMOVA suggests that caste system along with geographical contiguity are not ideal platforms for differentiating the analyzed Indian populations. It must, however, be acknowledged that use of less number of polymorphisms in this study might plausibly have led to the greater influence of linguistic affiliation on these populations rather than geographical proximity. Genetic affinity with global populations The genetic differentiation of the studied populations with relevant global migrant groups was estimated to be 2.3%, relatively lower than the 9% observed in another similar study [ 16 ], which had used a different set of microsatellite markers. Sampling from a confined area, as well as the use of lesser number of loci might have contributed to this apparent difference in the results. The southern populations formed a separate cluster from the world populations. Molecular studies on Indian populations using diverse markers (nuclear, mtDNA and Y-chromosome) have demonstrated that the upper caste populations have higher semblance with Europeans than Asians [ 26 ]. Intriguingly, in the present study, communities belonging to the upper strata of the Hindu caste hierarchy, i.e., Iyengar, Lyngayat, Vanniyar and northern Brahmins, displayed almost identical genetic affinity with both Europeans and East Asians. Therefore, all though it is believed that south India remained isolated and cushioned from the foreign invasions, the southern populations, especially, the high-ranking groups might have genetically admixed with migrant groups that entered via the west coast and north. Further exploration of their relationship is essential before drawing concrete conclusions. A more comprehensive picture would emerge on analysis of mtDNA and Y chromosome markers. Methods The populations The populations selected in this study comprise of three major Hindu castes-Iyengar, Lyngayat, Gowda and a Muslim community, inhabiting the southwest coastal terrain of Karnataka (11.3 – 18.45°N latitudes and 74.12 – 78.40°E longitudes). All the populations belong to the Dravidian linguistic family and are speakers of the local dialect, Kannada, but differ in caste hierarchy and socio-religious practices. Consanguineous marriages have been reported in Karnataka, with inbreeding levels of the order 0.020 to 0.033, in general [ 38 ]. Iyengar hold a high position in the Indian caste hierarchy and sporadic accounts on Brahmin, suggests that they primarily migrated from the upper Gangetic plains to southern India. Nonetheless, few bioanthropological studies have revealed that morphologically Brahmins of a geographical region are similar to the local groups. Lyngayat community was initially formed, as a religious cult by the amalgamation of people from different castes and geographical regions but later developed into a distinct community practicing strict marriage endogamy with social sub-divisions such as clans, sub-castes and sects [ 10 ]. Gowda is a low ranking agriculturist caste group that typically exhibits the Dravidian socio-cultural characteristic of consanguineous marriage. It is believed to have moved in from the adjoining area of Tamil Nadu. Muslim is a linguistically heterogeneous, complex religio-ethnic group, [ 10 ]. It is believed that the invasion of Turks, Afghans (A.D 998–1030) and Moghals during the 15 th century, introduced new genes only in northern India, suggesting that Muslims from Southern India are mainly local converts [ 3 ]. Micosatellite loci studied The 15 STR marker set analyzed in this study consists of thirteen tetra nucleotide repeat loci: D3S1358, THO1, D21S11, D18S51, D5S818, D13S317, D7S820, D16S539, CSF1PO, vWA, D8S179, TPOX, FGA and two penta nucleotide repeat loci: Penta D, Penta E. Their repeat size makes them less prone to slippage of polymerase during enzymatic amplification compared to the dinucleotide repeats, allowing unambiguous typing [ 20 ]. The 15 selected loci are situated on 13 different chromosomes, with D5S818 and CSF1PO being present on chromosome 5 and Penta D and D21S11, located on chromosome 21. The alleles across the loci are substantially unlinked, making them suitable for analyzing inter and intra-population genetic diversity. STR Typing The blood samples were collected from unrelated individuals belonging to – Iyengar (65), Lyngayat (98), Gowda (59) and Muslim (45) communities, residing in different districts of Karnataka. DNA was extracted from blood by the phenol-chloroform method [ 40 ], followed by quantitation using the QuantiBlot™ kit (Perkin-Elmer, Foster City, CA, USA). Two nanogram of the isolated DNA was used as template for the PCR amplification of the 15 STRs using the PowerPlex™16 kit (Promega Corp., Wisconsin Madison, USA). Raw data were collected with the GeneScan™ software, Ver. 3.2.1 (Applied Biosystems, Foster City, CA, USA) and typed using the PowerTyper™ 16 Macro (Promega Corp., Wisconsin Madison, USA). Statistical Analysis Allele frequencies of the 15 STR loci were calculated using the gene counting method [ 40 ]. The genetic diversity (G ST ), observed heterozygosity and pairwise genetic distances (DA) were computed using allele frequencies [ 42 ]. The DA distance is least affected by sample size and can precisely obtain correct phylogenetic trees under various evolutionary conditions [ 43 ]. Neighbor-joining trees were constructed using DA distances [ 44 ], and its robustness was established by bootstrap resampling procedures. Analysis of molecular variance (AMOVA) was performed using the Arlequin Ver. 2.00 package [ 45 ]. Two levels of analysis were performed to explore the microsatellite diversity among the four studied populations along with six other socio-culturally similar groups inhabiting different regions of India. At the first level, three geographical groups were constructed: (1) north (2) southwest: Karnataka and, (3) southeast: Tamil Nadu, to estimate the genetic variance among populations from diverse geographical regions. The second set of analysis was aimed at investigating the genetic diversity between the Dravidian and Indo-European linguistic family. To assess the gene flow experienced by these populations, the rii value, i.e., the genetic distance of a population from the centroid was calculated using the regression model [ 46 ]. This model utilizes the heterozygosity of each population and the distance from the centroid as the arithmetic mean of allele frequencies: where, r ii is the distance from the centroid, p i is the frequency of the allele in i th population and is the mean allelic frequency. List of abbreviations STR – Short Tandem Repeat AMOVA – Analysis of Molecular Variance NJ tree – Neighbor-Joining tree Authors' contributions RR carried out the molecular studies, analyzed the genetic data and drafted the manuscript. VKK participated in the design, conceiving and preparation of manuscript. Both authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC515297.xml
555738	Role of axillary sentinel lymph node biopsy in patients with pure ductal carcinoma in situ of the breast	Background Sentinel lymph node (SLN) biopsy is an effective tool for axillary staging in patients with invasive breast cancer. This procedure has been recently proposed as part of the treatment for patients with ductal carcinoma in situ (DCIS), because cases of undetected invasive foci and nodal metastases occasionally occur. However, the indications for SLN biopsy in DCIS patients are controversial. The aim of the present study was therefore to assess the incidence of SLN metastases in a series of patients with a diagnosis of pure DCIS. Methods A retrospective evaluation was made of a series of 102 patients who underwent SLN biopsy, and had a final histologic diagnosis of pure DCIS. Patients with microinvasion were excluded from the analysis. The patients were operated on in five Institutions between 1999 and 2004. Subdermal or subareolar injection of 30–50 MBq of 99 m-Tc colloidal albumin was used for SLN identification. All sentinel nodes were evaluated with serial sectioning, haematoxylin and eosin staining, and immunohistochemical analysis for cytocheratin. Results Only one patient (0.98%) was SLN positive. The primary tumour was a small micropapillary intermediate-grade DCIS and the SLN harboured a micrometastasis. At pathologic revision of the specimen, no detectable focus of microinvasion was found. Conclusion Our findings indicate that SLN metastases in pure DCIS are a very rare occurrence. SLN biopsy should not therefore be routinely performed in patients who undergo resection for DCIS. SLN mapping can be performed, as a second operation, in cases in which an invasive component is identified in the specimen. Only DCIS patients who require a mastectomy should have SLN biopsy performed at the time of breast operation, since in these cases subsequent node mapping is not feasible.	Background Ductal carcinoma in situ (DCIS) is defined as the proliferation of malignant epithelial cells in the mammary ductal system, with no evidence of invasion of the basement membrane. By definition, the disease is localized to the breast, with no spread to regional nodes or distant sites. In the pre-mammography era, DCIS was rarely diagnosed, accounting for only 1 to 2% of all breast cancers. The increasing use of screening mammography in recent years has resulted in a dramatic increase in the diagnosis of DCIS, which now accounts for roughly 20% of all mammographically detected cancers [ 1 ]. Axillary treatment in patients with a diagnosis of pure DCIS is controversial. By definition, pure DCIS is not invasive and cannot spread to the regional nodes, so axillary staging should not be indicated in DCIS patients. In practice, however, nodal metastases are found in 1 to 2% of patients with a diagnosis of pure DCIS, and this paradoxical condition usually depends on a focus of invasion being missed by the pathologist because of a sampling bias [ 2 ]. Before the introduction of sentinel lymph node (SLN) biopsy, it was widely agreed that axillary lymph node dissection (ALND) should be avoided in patients with DCIS, because of the low yield of positive findings in these cases and the significant morbidity associated with the procedure [ 3 ]. The introduction of SLN biopsy as a minimally invasive tool for staging the axilla led to a renewed interest in axillary staging for these patients, for two reasons. First, this procedure is far less invasive than ALND and has minimal morbidity and complications, so the cost/benefit ratio may be positive even if the likelihood of finding metastatic nodes is low. Second, SLN biopsy allows more accurate axillary staging than ALND specimen examination, because SLN can be thoroughly evaluated by serial sectioning and immunohistochemical methods. This has led to the increased detection of nodal micrometastases in patients with invasive breast cancer, and has raised the expectation that the rate of positive nodal findings in patients with DCIS could also be increased. Currently, the indications for SLN biopsy in patients with a diagnosis of pure DCIS are controversial. The aim of the present study was to determine the prevalence of SLN metastasis in a multi-institutional series of patients with a diagnosis of pure DCIS in order to determine the usefulness of routine SLN biopsy in such cases. Methods Patients Our series consisted of 102 patients with a final histopathological diagnosis of pure DCIS who underwent SLN biopsy in five Institutions between January 1999 and January 2004. Patients with DCIS with microinvasion were excluded from the study. All patients underwent SLN biopsy after they had given their written informed consent, and had decided whether they would undergo a completion ALND if SLN were found to be positive. Lymphoscintigraphy On the day before surgery, all the patients received an injection of 30–50 MBq of 99 m-Tc-nanocolloidal albumin (Nanocoll, Nicomed-Italia, Saluggia, Italy) in 0.2 cc of saline. In patients with palpable tumours, the radiocolloid was injected subdermally into the cutaneous projection of the tumour. In those with non-palpable tumours, the lesion was localized by stereotactic or ultrasonographic placement of a self-retaining wire and the cutaneous projection was marked by an ink spot on the skin, which served as a guide for subdermal radiocolloid injection. Patients with extensive microcalcifications or multifocal tumours were given subareolar radiotracer injection. Finally, patients who underwent delayed SLN biopsy after excision of the primary lesion were given subdermal radiotracer injection in two fractions, at the sides of the surgical scar. Twenty minutes and 2 hours after injection, scintigraphic images were obtained using a large-field-of-view gamma camera (Orbiter, Siemens, IL, USA) equipped with a parallel hole, low energy and a high resolution collimator. Surgical procedure Primary tumour resection with simultaneous SLN biopsy was performed in patients with a preoperative diagnosis of DCIS made by stereotactically-guided directional vacuum-assisted biopsy and in those with mammographical finding pathognomonic of DCIS, with or without a preoperative cytologic diagnosis of malignancy. A diagnostic excisional biopsy was performed in patients with a doubtful pre-operative diagnosis, and SLN biopsy was performed as a second operation in those with a final histologic diagnosis of DCIS. As a rule, in patients undergoing conservative surgery for non-palpable lesions, an intraoperative x-ray examination of the resected specimen was performed, in order to confirm that complete tumour excision had been achieved. SLN biopsy was performed 16 to 24 hours after radiocolloid injection. When conservative surgery was performed, the SLN was excised through the incision used for resecting the primary tumour, if located in the upper outer quadrant, whereas a separate axillary incision was performed if the tumour was located in another breast site. A gamma-ray-detecting probe was used for intraoperative SLN identification. All lymph nodes with a probe-detected radioactive count >10% of that of the hottest node were excised. Histopathology The excised breast lesions were sampled with serial cuts and the margins were identified by ink. The histopathologic diagnosis and classification of DCIS was made according to the Holland grading system [ 4 ]. The Van Nuys prognostic index [ 5 ] was used only in some of the cases. The search for microinvasive foci was performed in selected cases both with haematoxylin-eosin serial sections and immunostains to smooth muscle actin and CD10 for the detection of myoepithelial cells. In all cases, the tumour size and margin status were specified in the histopathological report. The SLN examination was performed as described elsewhere [ 6 ]. Briefly, for frozen-section examination, nodes with diameters of ≤ 0.5 cm were bisected, while nodes measuring >0.5 cm were sectioned each 2 to 3 mm. For each sample, two frozen sections made at 40 μm intervals were examined. The frozen tissue was then thawed, fixed and embedded to obtain permanent sections. For definitive histological examination, two consecutive 5 μm thick tissue sections were cut from a paraffin block at two levels, 40 μm apart from each other. The sections were then stained with haematoxylin-eosin and immunostained with monoclonal antibody to cytokeratin. Results The 102 patients with pure DCIS had a median age of 59.4 years (range 37 to 85). The histologic characteristics of the tumours are reported in Table 1 . Of the 102 patients, 20 (19.6%) had palpable, and 82 (80.4%) non-palpable breast tumours. Seventy-four patients (72.5%) were treated with conservative surgery (quadrantectomy or lumpectomy) and 28 (27.5%) with mastectomy. Ninety-one patients underwent primary tumour resection with simultaneous SLN biopsy: 16 on the basis of a preoperative histologic diagnosis of DCIS obtained by a vacuum-assisted large core biopsy, 63 with a preoperative cytology suggestive of malignancy and 12 only on the basis of the mammographical findings alone. Patients with a doubtful preoperative diagnosis underwent excisional biopsy of the breast lesion, and the 11 patients with pure DCIS at definitive histology, underwent SLN biopsy and, if required, definitive treatment of the primary tumour as a second operation. A total of 147 SLNs were identified and excised from the 102 patients: a single SLN was found in 61 cases (59.8%), two SLNs in 37 (36.3%), and three SLNs in 4 (3.9%). A positive SLN was found in one (0.98%) patient, who had one micrometastases (0.6 mm) detected by haematoxylin-eosin staining. The pathologic review of the surgical specimen confirmed that the tumour had been completely excised, and no microinfiltration was detected. In this SLN positive case, the primary tumour was a micropapillary DCIS, G2, with a diameter of 16 mm. The patient did not undergone previous microbiopsy or FNAC, and SLN biopsy was performed at the time of the primary tumour excision. Completion ALND was not performed. Discussion Indications for routine SLN biopsy in patients with a diagnosis of pure DCIS are still controversial. The first study proposing SLN biopsy for DCIS patients came from the H. Lee Moffitt Cancer Center in the year 2000 [ 7 ]: in a study of 87 DCIS patients, 5 (5.7%) presented SLN metastases, and the authors concluded that SLN biopsy should be routinely used in DCIS patients in order to identify and correctly stage patients with undetected invasive disease. In a later report on 195 DCIS patients from the same Institution, 26 (13%) patients were SLN positive and this finding strengthened the recommendation that SLN biopsy should become a routine part of surgical treatment for all DCIS patients [ 8 ]. The Memorial Sloan-Kettering Cancer Center group reviewed patients with "high risk" DCIS and found that 9/76 (12%) patients had SLNs positive for metastases. The authors suggested that SLN biopsy should be performed in all DCIS patients with one or more of the following characteristics: palpable or mammographic mass, suspicion of microinvasion, multicentric disease, high nuclear grade or necrosis [ 9 ]. On the other hand, in subsequent studies negligible rates of nodal involvement were found in patients with pure DCIS: Kelly et al. [ 10 ] found nodal metastases in only 3/134 (2.2%) patients, Intra et al. reported SLN metastases in 7/223 (3.1%) patients [ 11 ], Farkas et al. found no cases of SLN metastasis among 44 patients [ 12 ]. All these investigators concluded that SLN biopsy should not be routinely performed in patients with DCIS, with the exception of those undergoing mastectomy, because this operation precludes the possibility of performing a subsequent SLN biopsy if invasive foci are found at histology of the mastectomy specimen. Our results are consistent with the latter series of reports, since we found only one case of SLN micrometastasis among 102 patients with a diagnosis of pure DCIS (0.98%). The variability in the reported rates of nodal metastases probably reflects differences in the accuracy of pathological evaluation of the primary breast tumour: extensive sampling and a thorough histological examination of the DCIS are of crucial importance in ruling out microinvasive foci. Microinvasive DCIS is a different pathological entity, with a well-defined metastatic potential, and should be excluded from studies evaluating the role of axillary staging in non-invasive cancer. Our results support the view that the presence of axillary nodal metastases in patients with a final histopathological diagnosis of pure DCIS is a very unusual phenomenon, if the primary breast tumour has been completely excised and thoroughly examined by the pathologist. We, therefore, believe that routine SLN biopsy in all DCIS patients represents an over-treatment and should be avoided. Enthusiasm for SLN biopsy in DCIS patients is partly due to the low morbidity of the procedure. However, complications such as lymphedema, seroma, infection and sensory neuropathy have been reported after SLN biopsy [ 13 - 15 ]. Moreover, performing SLN biopsy in patients with a small DCIS treated with a conservative operation precludes the possibility of using this procedure in patients with a subsequent homolateral invasive tumour, which is not infrequent in this setting [ 1 ]. Finally, the policy of performing primary tumour excision with simultaneous SLN biopsy in all patients with a preoperative diagnosis of DCIS may incur a risk of performing axillary biopsy in patients with benign breast lesions, since intraoperative frozen section histology is usually unreliable in patients with small areas of microcalcifications. We therefore believe that this procedure should be used cautiously, being reserved for cases in which a real advantage can be expected. Another factor that has prompted interest in SLN biopsy for DCIS patients is the widespread use of image-guided core needle biopsy for the diagnosis of mammographically-detected abnormalities, a diagnostic technique that often fails to identify invasive foci: 14 to 29% of patients with a preoperative core needle biopsy of DCIS are found to have invasive cancer at surgical excision [ 16 , 17 ], and require a second operation for axillary staging. With vacuum-assisted large core biopsy the under-diagnosis rate is lower, but it is still not negligible. Supporters of routine SLN biopsy claim that this procedure is not reliable after primary tumour excision. They therefore believe that all patients with a preoperative diagnosis of DCIS who undergo definitive surgery and are upstaged because the pathologist finds foci of invasion on the specimen, must undergo an ALND if a SLN biopsy was not performed at the time of the first operation [ 18 ]. We disagree with this view, because it has been clearly demonstrated that SLN biopsy can be safely performed as a second procedure after primary tumour excision [ 19 , 20 ]. The only exceptions to this are patients who undergo mastectomy and those who require a wide quadrantectomy of the upper outer quadrant, which can disrupt lymphatic pathways toward the axilla. We therefore believe that only patients with a preoperative diagnosis of DCIS who need mastectomy or wide excision close to the axilla should undergo a concomitant SLN biopsy. In all other cases, only the primary tumour excision should be performed and SLN biopsy should be reserved, as a second procedure, for patients found to have infiltration at the histologic examination of the primary tumour. It has been claimed that some features of DCIS (large dimensions, high grade, comedo-type, mass forming lesions) are associated with a higher risk of invasive disease and nodal metastases. Therefore, some authors suggest that SLN biopsy should be reserved for all patients with these "high- risk" DCIS [ 9 , 21 , 22 ]. However, several investigators fail to correlate any histopathologic parameter with lymph node metastases [ 8 , 10 , 11 ]. The majority of patients in our series presented with low risk DCIS: most of the tumours were small (<1 cm) and only 20/102 patients had a palpable breast mass. Therefore, we cannot rule out that a higher incidence of microinvasion and nodal metastases might be found in a patient population with more aggressive forms of DCIS. However, in our series, the one DCIS patient with SLN metastasis had a small micropapillary, G2, non-palpable tumour. In our opinion, clinical judgement should be used in patients with large solid tumours or diffuse comedo-type DCIS, bearing in mind that most of these patients require a mastectomy and are therefore candidates for SLN biopsy. Conclusion Our results confirm that the finding of SLN metastasis in pure breast DCIS is a very rare occurence, if the primary tumour has been completely excised and microinvasion has been ruled out by a thorough histologic examination. Therefore, our current policy is to avoid routine SLN biopsy at the time of primary tumour resection in the presence of a preoperative diagnosis of DCIS. We reserve this procedure, as a second operation, for cases in which an invasive component is identified at the histologic examination of the surgical specimen. Only patients with a diagnosis of DCIS requiring a mastectomy or a wide resection close to the axilla should undergo concomitant SLN biopsy. However, particularly if a large high-grade tumour is found, patients should be informed of the risk that invasive disease may be found, and that a second procedure for SLN biopsy may be required. In these cases, the patient can decide whether to undergo a potentially unnecessary SLN biopsy at the time of the first operation or whether to run the risk of requiring a second operation. Competing interests The author(s) declare that they have no competing interests Authors' contributions GZ planned the study and drafted the manuscript PC, RM, ZF, GS, PB, PP, AB contributed with their own cases to the final multi-institutional series RM and GC reviewed the pathologic specimens of selected cases and drafted the histopatology section of the manuscript MEP and GM were in charge of data collection ML and DN co-ordinated the study Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC555738.xml
503387	A protease activated receptor-2 (PAR-2) activating peptide, tc-LIGRLO-NH2, induces protease release from mast cells: role in TNF degradation	Background Mast cell (MC)-derived serine proteases have been implicated in a variety of inflammatory processes. We have previously shown that rat peritoneal MC (PMC) express mRNA for protease activated receptor 2 (PAR-2), a G-coupled receptor activated by trypsin-like proteases. Recent evidence also suggests that MC-induced inflammation can be mediated through PAR. Therefore, we hypothesized that specific PAR-2 agonist peptides (PAR-2ap) induce protease release from PMC. Results Western blot analysis of PMC supernatants revealed that a PAR-2ap, tc-LIGRLO (10 μM), stimulated the release of rat MC protease (RMCP)-1, RMCP-5 and carboxypeptidase-A. The release was evident by 20 min but further increased up to 8 h. To study the biological effects of protease release we tested supernatants from tc-LIGRLO, tc-OLRGIL (inactive control peptide) and antigen-activated PMC for proteolytic activity by seeding with TNF (150 pg/ml), incubating for 8 h at 37°C, and measuring TNF remaining in the supernatants. Supernatants from tc-LIGRLO-stimulated PMC degraded 44 % of seeded TNF (n = 5). Moreover, this TNF proteolysis was dependent on the concentration of tc-LIGRLO used to stimulate PMC, and was significantly inhibited (94 %) by soybean trypsin inhibitor. Antigen and tc-OLRGIL induced no significant release of such proteolytic activity. Conclusions These data indicate that a PAR-2ap induces the release of proteases from mast cells, which may degrade extracellular cytokines and other substrates thus modulating the inflammatory response.	Background Protease activated receptor-2 (PAR-2) has been identified on a variety of cell types including eosinophils [ 1 ], neutrophils [ 2 ], neurons and smooth muscle cells [ 3 ]. It can be activated by a variety of serine proteases including MC tryptase [ 4 ], pancreatic trypsin [ 5 ], and coagulation factors [ 6 ] to induce inflammatory, mitogenic and chemotactic functions. Serine proteases cleave PAR-2 at a specific site in the extracellular NH 2 -terminus unmasking a new NH 2 -terminus (tethered ligand) and changing the conformation of the receptor to allow the tethered ligand to interact with the activation site on the 2 nd extracellular loop of the receptor. Peptides that are similar in sequence to the tethered ligand domains of PAR-2, such as SLIGRL-NH 2 (SLI) or tc-LIGRLO-NH 2 (tc-LIG) are able to interact directly with the activation site and act as potent agonists [ 7 ]. A growing number of studies have identified a role for PAR-2 in inflammation. There is a delayed onset of inflammation in PAR-2 knock out mice [ 8 ], and PAR-2-activating peptides (PAR-2ap) stimulate leukocyte rolling, adherence, and recruitment in rat mesenteric postcapillary venules [ 9 ]. Furthermore, PAR-2 activation of human airway epithelial cells mediates the release of the eosinophil survival-promoting factor GM-CSF and matrix metalloproteases [ 10 , 11 ]. The ability of serine proteases to activate MC and the observation that MC express PAR-2 [ 12 , 13 ], suggest that PAR-2-induced proinflammatory functions in vivo could be MC-mediated. The administration of PAR-2ap or trypsin into the rat hind paw enhanced vascular permeability and caused edema formation, which can be abolished by repeated pre-treatment with compound 48/80, known to deplete the MC of its granular content [ 14 ]. By contrast, Vergnolle et al ., 1999 [ 15 ] showed that edema induced by injection of PAR-2ap was only slightly reduced in rats pre-treated with compound 48/80, and the pre-treatment of rats with cromolyn, a MC stabilizer, had no effects on PAR-2ap induced inflammation of the paw. These studies showed that the administration of PAR-2ap induces an acute inflammatory response characterized by persistent edema and granulocyte infiltration, but the involvement of MC in these responses requires further investigation. Activation of PAR-2 on the surface of mast cells could act as part of an autocrine and paracrine positive feedback loop through the release of serine proteases that could activate further PAR-2 on mast cells or other neighboring cells. Therefore, we investigated the direct effects of PAR-2ap on the release of serine proteases from purified PMC and the effects of these released proteases on extracellular protein degradation. In particular we studied the release of rat mast cell protease-1 (RMCP-1), RMCP-5 and carboxypeptidase A (CPA). Results The PAR-2ap, tc-LIG, induces release of RMCP-1, RMCP-5 and CPA from PMC To identify proteases released by mast cells following PAR-2ap stimulation we activated PMC with tc-LIG (10 μM), and analyzed the supernatants for various mast cell proteases by western blotting, using antisera against the amino-terminal sequences of RMCP-5 and MC-CPA and an antiserum against RMCP-1 protein. In supernatants from tc-LIG-treated PMC one band for RMCP-1 (30 kDa), two bands for RMCP-5 (34 and 35 kDa), and three bands for CPA (40, 41 and 42 kDa) were detected (Fig. 1A ). The PAR-2ap tc-LIG induced most of the protease release in the first 20 min. However, proteases accumulated in the conditioned media up to 8 hr (Fig 1B ). The release of all three proteases was dose-dependent and was detectable in supernatants of PMC stimulated with tc-LIG at concentrations 0.1 μM and higher (Fig 1C ). PMC activation with 48/80 (0.5 mg/ml) induced the release of all three proteases in similar levels to 0.5 μM of tc-LIG (Fig. 1C ). Figure 1 Release of RMCP-1, -5 and CPA from PMC following activation with tc-LIG (PAR-2ap), compound 48/80 and Ag. (A) Supernatants from tc-LIG (10 μM), Ag (10 We/mL) and sham-treated (spon) mast cells were concentrated (10 ×) and Western blot analysis preformed for CPA, RMCP-1 and RMCP-5. Left panel shows Coomassie blue staining of the same gel and right panel Western blot with normal rabbit serum as a negative control. (B) Release of RMCP-1, RMCP-5 and CPA following 20 min and 8 h activation of PMC with tc-LIG (10 μM). (C) Dose response for the release of RMCP-1, -5 and CPA by tc-LIG-stimulated or compound 48/80-stimulated PMC. In all cases representative blots from three experiments with similar results are shown. Nippostrongylus brasiliensis antigen ( Nippo Ag) (10 We/ml) induced no detectable release of any of the three proteases studied (Fig 1A ). Nippo Ag-activated cells under these conditions released 10 ± 2% of β-hex; level similar with that released by 0.1 μM of tc-LIG (12 ± 2%), which however, was associated with protease release (Fig 1C ). This amount of β-hex release was the highest we could obtain following PMC activation with Nippo Ag, under the conditions used in our experiments. PAR-2ap-induced proteolytic activity released from mast cells degrades TNF On further comparing Fcε RI with tc-LIG-induced PMC activation we noted that Fcε RI-mediated activation induced TNF release while tc-LIG-mediated activation did not induce significant TNF release following PMC activation for up to 8 hr (Fig 2 ). One possible hypothesis to explain this effect was that TNF released by tc-LIG activated PMC was degraded by some of the proteases we showed to be released from mast cells at the same time. Therefore, we examined the ability of supernatants of PAR-2ap-activated PMC to degrade extracellular proteins. We used a bioassay for released protease activity, employing TNF as the cytokine to be degraded. PMC were incubated with no activators (sham treatment), tc-LIG (10 μM), tc-OLR (10 μM) or compound 48/80 (0.5 μg/ml) for 20 min or 8 hr at 37°C and the supernatants were collected. These supernatants or media were then seeded with 150 pg/ml of rat recombinant TNF and incubated for an additional 8 hr. At the end of the incubation TNF was measured by ELISA and the proteolytic activity was calculated as % degraded TNF (as discussed in the methods section). Proteolytic activity in the supernatants of sham-treated cells was subtracted from that in the supernatants of activated PMC. Supernatants from sham-treated PMC showed significant loss of seeded TNF (17 ± 7 % at 20 min and 22 ± 5 % at 8 hr) as compared to media. At both 20 min and 8 hr of treatment supernatants from tc-LIG-treated MC (10-0.1 μM) showed a greater loss of seeded TNF compared to supernatants of sham-treated MC (p < 0.05), suggesting tc-LIG-mediated activation induced the release of proteolytic activity. Proteolytic activity, following subtraction of spontaneous proteolytic activity released, was 44 ± 5 % at 8 hr and 30 ± 4 % at 20 min following PMC activation with 10 μM of tc-LIG (Fig. 3A and 3B respectively). Supernatants from tc-OLR- or Nippo Ag-treated cells showed no significant loss of TNF over that which occurred in sham-treated cells (Fig. 3A and 3B ). Figure 2 TNF release from PMC (1 × 10 6 cells) after 8 hr incubation with PAR-2ap (tc-LIG, 10 μM), PAR-2cp (tc-OLR, 10 μM) and Ag (10 We/ml). (Mean ± SEM, n = 4). Star indicates statistically significant difference from spontaneous (p < 0.05, n = 4). Figure 3 PAR-2ap, PAR-2cp compound 48/80 and Ag-mediated release of proteolytic activity from mast cells. (A) Supernatants from PMC treated with tc-LIG (10 μM), tc-OLR (10 μM), or Ag (10 We/mL) for 8 hr were incubated with 150 pg/mL of TNF. Proteolytic activity was calculated as % degraded TNF according to the formula given in Methods section. Values in the graph indicate proteolytic activity after the subtraction of activity released by sham-treated cells (17 ± 7 %). (B) TNF-degrading proteolytic activity released from PMC by various doses of tc-LIG (20 min). Values in the graph indicate proteolytic activity after the subtraction of activity released by sham-treated cells (22 ± 5 %). (C) TNF-degrading proteolytic activity released by SLI (PAR 2 -ap, 40 μM), LSI (PAR 2 -cp, 40 μM) and tc-LIG (10 μM) treated PMC (20 min). Values in the graph indicate proteolytic activity after the subtraction of activity released by sham-treated cells (23 ± 7 %). Values are shown as "mean ± SEM" (n = 3–5). Star indicates statistically significant difference from spontaneous (p < 0.05). We also examined the ability of another PAR-2ap, SLI and its PAR-2cp, LSI, to release proteolytic activity from PMC (Fig. 3C ). A small but significant increase in proteolytic activity over spontaneous release was induced by SLI (40 μM, 7 ± 1 %, p < 0.05). However, net SLI-mediated proteolytic activity released was not significantly different than that released by the inactive control peptide, LSI (40 μM, 7 ± 5 %). To study whether tc-LIG-mediated TNF proteolytic activity was a result of serine protease activity, the supernatants were mixed with the broad spectrum serine protease inhibitor, SBTI (1 mg/ml) before seeding with TNF. SBTI inhibited TNF loss from the supernatants of tc-LIG (10 μM) stimulated PMC by 82% (Fig. 4 ), confirming that tc-LIG-induced loss of TNF was by the proteolytic activity of serine proteases. Figure 4 Effect of SBTI on the proteolytic activity in supernatants of tc-LIG-stimulated mast cells. Supernatants from PMC stimulated by tc-LIG, tc-OLR, and Ag for 8 hr were incubated with or without SBTI (1 mg/mL) before 150 pg/mL of TNF was added and % degradation calculated. Values indicate proteolytic activity after the subtraction of spontaneous release (17 % ± 7). Star indicates statistically significant difference from spontaneous (p < 0.05, n = 4–5). (Mean ± SEM). Discussion We have previously shown that PMC express PAR-2 mRNA, that can be regulated by cytokines and PAR-2ap [ 13 ]. We have also shown that RMCP-1, RMCP-5 and CPA are stored in PMC and are the most prominent proteases produced by PMC [ 16 ]. In the present study we demonstrated that tc-LIG, a PAR-2ap, induces the release of RMCP-1, RMCP-5 and CPA from mast cells. Compound 48/80 induced comparable release of proteases form PMC, but FcεRI-mediated activation did not. We also showed that these, and possibly other proteases released at the same time from PMC, are capable of degrading TNF. In this study, we provided the first direct evidence for serine protease release from PMC measured by Western blot analysis of the supernatants, in addition to proteolytic activity assays. The sizes of released RMCP-1 (~30 kDa), RMCP-5 (2 close bands, ~34 kDa) and CPA (3 close bands, ~41 kDa) are similar to the sizes of the stored forms of these proteases that we published previously [ 16 ]. The different bands for RMCP-5 and CPA are likely due to differential glycosylation, as has been shown before [ 16 ]. PAR-2 ap have been shown to release proteases from gastric pepsinogen secreting cells [ 17 ] and from epithelial cells [ 10 ]. A recent report showed release of tryptase from human colon mast cells following PAR-2ap-mediated activation [ 18 ]. In that case the concentration of PAR-2ap needed was higher than in our experiments and the effect was similar with FcεRI-mediated activation, while in our experiments proteases were released by PAR-2ap but not with Nippo Ag. Previous studies have shown that activation of MC to release protease activity may be induced by a variety of agents both in vivo and in vitro . The release of RMCP-2 by rat mucosal mast cells has been reported to be induced by antigen challenge in parasitic infections, and during anaphylaxis [ 19 - 21 ]. The release of RMCP-2 mouse counterpart, MMCP-1, can be increased during parasitic infections [ 22 ]. Furthermore, rat CPA can be released by 48/80, Ca 2+ ionophore and antigen activation of PMC [ 23 ]. In our experiments FcεRI-mediated PMC stimulation did not release detectable levels of RMCP-1, RMCP-5 or CPA, or any proteolytic activity with the ability to degrade TNF. It is possible that FcεRI-mediated activation induces low levels of protease release which is undetectable by Western blotting. Furthermore, the lack of demonstrable proteolytic activity in the supernatants does not necessarily indicate that proteases are not released since it may be due to an FcεRI-mediated simultaneous release of protease inhibitors stored in the MC. Indeed, MC produce and release secretory leukocyte protease inhibitor (a chymase inhibitor) and latexin (a CPA inhibitor) [ 23 , 24 ]. Finally, FcεRI-mediated activation may selectively release proteases different from the ones released by 48/80 or PAR-2ap. Given that RMCP-1 and RMCP-5 are present in the supernatants of tc-LIG-stimulated MC, it is likely that these proteases are involved in the TNF degradation. However, antibodies to RMCP-1 inhibited TNF degradation by supernatants of sham treated cells but did not affect the additional degradation of supernatants from tc-LIG activated MC (unpublished observation). We cannot rule out that PAR-2ap activated PMC release other proteases, including the tryptases RMCP-6 and RMCP-7 [ 25 ], which may contribute to TNF-degradation. It is also interesting that chymases and CPA, which are released in parallel, have synergistic effects [ 26 ]. Other proteases from leukocytes are known to be able to degrade TNF. These include cathepsin G [ 27 ], neutrophil elastase [ 28 , 29 ] and by proteases released from bacteria [ 39 ]. The same proteases can also degrade other cytokines, such as endothelin [ 31 , 32 ], lymphotoxin [ 27 ] and IFNγ [ 30 ]. Our data further suggest that MC may regulate TNF function by releasing proteases that can directly degrade this cytokine. Given that both TNF and serine proteases are stored and released from MC, our present findings suggest an important mechanism by which MC may regulate TNF function in vivo . It may be that such proteolytic activity directed against TNF, and possibly other cytokines, is an important anti-inflammatory function for mast cell serine proteases. The expression of PAR-2 by mast cells and the involvement of MC in PAR-2-mediated inflammation has been controversial. In vitro , MC tryptase can stimulate histamine release by human tonsillar [ 33 ] and guinea pig [ 34 ] MC, but not from foreskin mast cells. The tryptase inhibitor APC366 inhibits IgE-dependent MC activation, and also inhibits calcium ionophore-induced histamine release [ 33 ]. Tryptase-mediated bronchoconstriction in sheep is histamine mediated [ 35 ], indicating that tryptase induces lung MC activation. PAR-2 has been identified on human [ 12 ] and rat [ 13 ] mast cells. MC have been implicated in rat paw oedema caused by PAR-2ap or trypsin administration [ 14 ], but other reports failed to confirm this observation [ 15 ]. Taken together these reports strongly suggest a role for tryptase and possibly PAR-2 in MC activation. In a previous study we have shown that only one of two PAR-2ap (tc-LIG) activates β-hex release from PMC [ 13 ]. The other peptide, SLI, although it is a potent and selective PAR-2 agonist, was unable to induce release of β-hex as had also been shown before [ 36 ], although others showed that higher concentrations of SLI can induce the release of β-hex and to a greater extend histamine from rat PMC [ 37 ]. However, our study was the only one to use tc-LIG. In this study again only tc-LIG induced the release of proteases from PMC. SLI induced slightly increased release of proteolytic activity compared to sham treated cells but this release was not significantly different than the release induced by the control peptide LSI. We have previously shown that SLI is sensitive to proteases and its effects on MC increases in the presence of amastatin, an aminopeptidase inhibitor [ 13 ]. In contrast tc-LIG possesses a trans-cinnamoyl group, which acts to stabilize the peptide and prevent its degradation by aminopeptidases. It is unlikely that the different sensitivity to proteases can explain fully the difference between the effects of the two PAR-2ap peptides. It is also unlikely that the trans-cinnamoyl modification on tc-LIG is solely responsible for tc-LIG-mediated activation of MC, because it is also present on the reverse sequence peptide tc-OLR, but does not have the same effects with tc-LIG on protease release, as shown in this study, or in the release of β-hex, as we showed before. Compound 48/80 along with other cationic compounds can activate MC by directly interacting with a pertussis toxin sensitive component [ 38 ]. Our previous work suggested that tc-LIG may activate MC through a 48/80-like mechanism, but appears to also possess a second mechanism of signalling that is distinct from that of 48/80 [ 13 ]. Thus, we cannot rule out the possibility that tc-LIG-mediated release of proteolytic activity may be mediated in part through a 48/80-like mechanism. Indeed, 48/80 induced similar levels of proteolytic activity and protease release to tc-LIG. Recently, a new receptor activated by the PAR-2 activating peptide tc-LIGRLO-NH 2 has been identified pharmacologically in murine vascular smooth muscle [ 39 ]. In that case, tc-LIG induced vasoconstriction, while the other PAR-2 activating peptide, SLI, did not have similar effects. The structure or the exact function of this receptor is not known. In our case also tc-LIG had a significant effect on protease release from mast cells while SLI had a very small effect. These data suggest that mast cells may express the same receptor as the one identified pharmacologically in smooth muscle cells. Conclusions Our study provides evidence that a PAR-2ap, tc-LIG, activates MC to release proteases and proteolytic activity that could potentially have both pro- and anti-inflammatory functions. We further showed that these proteases may degrade extracellular proteins and affect the inflammatory environment in areas of mast cell activation. Although the presence and function of PAR-2 on MC is still controversial, our findings indicate that PAR-2 may be part of an autocrine loop. PAR-2 activation leads to the release of serine proteases which in turn may further activate more PAR-2 receptors on mast cells and also on other cells. Methods Reagents Compound 48/80, 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (β-hexosaminidase (β-hex) substrate) and soybean trypsin inhibitor (SBTI) were purchased from Sigma Chemical Co. (St. Louis, MO). PAR-2ap and PAR-2 control peptides (PAR-2cp) were synthesized by the Peptide Synthesis Facility, Faculty of Medicine, University of Calgary. These peptides were determined to be ≥ 95 % pure by mass spectrometry and HPLC. Polyclonal RMCP-5 and CPA antibodies were produced and characterized as described previously [ 16 ]. Briefly, RMCP-5 (15 amino acids) and CPA (12 amino acids) NH 2 -terminal sequences were synthesized at Zymogenetics Inc, Seattle, WA, and used to immunize rabbits to develop specific polyclonal anti-protease antibodies. Professor H. Miller, Edinburgh, Scotland, kindly provided rabbit antibody to RMCP-1. Animal sensitization Outbred male Sprague-Dawley rats (weight 250–500 g) were purchased from Charles River Canada Inc., (St. Constant, Quebec). Rats were maintained in an isolation room with filter-topped cages to minimize unwanted infections. For the experiments where MC were activated through their IgE receptor, rats were sensitized to Nippostrongylus brasiliensis , by a single subcutaneous injection of 3000 third-stage larvae in 0.5 mL of saline as described previously [ 40 ]. The experimental protocol was approved by the University of Alberta Animal Care Committee in accordance with the guidelines of the Canadian Council on Animal Care. Harvesting and enrichment of peritoneal mast cells Fifteen mL of ice-cold Hepes-buffered (10 mM, pH 7.3) Tyrodes buffer supplemented with 0.1% BSA was injected into the peritoneal cavity of each rat for the isolation of PMC. MC in peritoneal lavage fluids were enriched by centrifugation through a discontinuous density gradient of Percoll, as described previously [ 41 ]. Recovered MC were >95 % pure. Cell viability was >97 %. Mast cell activation After isolation and enrichment, PMC were rested in RPMI (Invitrogen, Burlington, Ontario) supplemented with 5% FBS for 2 hr at 37°C. After incubation, the cells were washed twice by centrifugation (150 g) and resuspended in RPMI at 1 × 10 6 cells/mL. Cells were placed in 1.5 mL Eppendorf tubes or in 48 well plates, incubated at 37°C for 10 min, and then the same volume of pre-warmed (37°C) PAR-2ap or controls in complete RPMI were added, to give a final cell concentration of 0.5 × 10 6 cells/mL. The cells were incubated for different times (20 min to 8 hr) depending on the experiment. To measure spontaneous release of mediators by PMC, cells were mixed with media alone. As positive controls, either compound 48/80 (0.5 μg/mL) or Nippostrongylus brasiliensis Antigen (1–100 worm equivalents (WE)/mL [ 40 ]) were mixed with cells under the same conditions. After incubation, tubes were placed on ice for 10 min and then centrifuged (150 g) to separate supernatant from cells. The supernatants were collected in tubes and the same volume of fresh media was added to the pellets, which were then resuspended. Cell viability was assessed at different times. Cell pellets and supernatants were stored at -70°C until assayed for their content of cytokines or proteolytic activity. SDS-PAGE and western blot analysis Supernatants were concentrated (10×) using Centricon (YM-10) centrifugal filter devices (Millipore, Bedford, MA). For Western blot analysis, proteins were transferred electrophoretically (25 V, 35 min) to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Mississauga, ON) using the Semi-Dry Trans Blot System. The membranes were blocked in Tris-buffered saline containing 0.02% Tween, 5% w/vmilk (Bio-Rad Laboratories) and 5 % v/vgoat serum (Invitrogen) for 1 hr. The membranes were probed with 1/1000 dilution of anti-RMCP-1, 1/600 anti-CPA, 1/5000 anti-RMCP-5 and then incubated with donkey anti-rabbit IgG HRP-conjugated antibody (1:5000). Protein bands were detected by enhanced chemiluminescence using ECL Western blotting detection system (Amersham Pharmacia Biotech, Quebec, Canada). β-hexosaminidase (β-hex) assay β-hex was measured in the supernatants and cell pellets, as described [ 42 ]. Results are expressed as β-hex released as a percent of total β-hex (pellet + supernatant). Values shown have been corrected for the spontaneous β-hex release. Proteolytic activity assay and protease inhibition assay To measure release of proteolytic activity, supernatants from stimulated PMC (containing secreted TNF) were transferred to a 96-well plate. After 2 min incubation, exogenous TNF or medium was added to the supernatants and mixed to give a final concentration of 150 pg/ml. Plates were incubated at 37°C for 8 hr and then TNF content was measured by ELISA. Percent TNF proteolysis was calculated by the following formula: % TNF degraded = 1 - (TNF recovered / (rat recombinant TNF seeded + measured TNF release) × 100) For protease inhibition experiments SBTI (1 mg/ml) was added to the supernatants before seeding with TNF. The supernatants were then processed as above and used to measure TNF degradation. TNF measurements Supernatants from activated PMC were analysed for TNF using a rat TNF ELISA kit (Endogen, Woburn, MA), according to manufacturer's instruction. The sensitivity of the TNF assay was < 10 pg/ml. To exclude the possibility that proteases contained in PMC supernatants interfere with ELISA determination of TNF we incubated the TNF antibody coated wells with PMC supernatants washed them and then added specified amounts of TNF for determination. Pre-incubation with PMC supernatants did not affect the ability to measure TNF, indicating that the proteases in PMC supernatants do not degrade the antibodies of the assay. Statistics All values are given as mean ± standard error of mean (SEM) for the numbers of experiments noted and statistical analyses were performed using the Student's t -test and ANOVA. Abbreviations β-hex β-hexosaminidase CPA carboxypeptidase-A LSI LSIGRL-NH 2 (PAR2-cp) MC mast cell PAR protease-activated receptor PAR-ap protease-activated receptor-agonist peptide PAR-cp protease-activated receptor-control peptide PMC peritoneal mast cells RMCP-1,5 rat mast cell protease-1, 5 SBTI soybean trypsin inhibitor SLI SLIGRL-NH 2 (PAR2-ap) tc-LIG trans-cinnamoyl -LIGRLO-NH 2 tc-OLR trans-cinnamoyl -OLRGIL-NH 2 Nippo Ag Nippostrongylus brasiliensis Antigen WE worm equivalent . Authors' contributions HNA carried out the majority of the experiments presented and drafted the manuscript. This work was part of his MSc thesis. GRS assisted with mast cell isolation and some of the activation experiments and participated in the experimental design. JLW participated in the design of the study. MDH participated in the design of the study. ADB participated in the design of the study, supervised the work shown and made substantial contributions in manuscript preparation. HV participated in the design of the study and contributed in the preparation of the final manuscript. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC503387.xml

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