Sciatic nerve injuries often cause partial or total loss of motor, sensory and autonomic functions due to the axon discontinuity, degeneration, and eventual death which finally result in substantial functional loss and decreased quality of life. Nerve growth factor (NGF) plays a critical role in peripheral nerve regeneration. However, the lack of efficient NGF delivery approach limits its clinical applications. We reported here by fusing with the N-terminal domain of agrin (NtA), NGF-β could target to nerve cells and improve nerve regeneration.in vitro were carried out. The bioactivity of LBD-NGF on laminin in vitro was also measured. Using the rat sciatic nerve crush injury model, the nerve repair and functional restoration by utilizing LBD-NGF were tested.Laminin-binding assay and sustained release assay of NGF-β fused with NtA (LBD-NGF) from laminin in vitro and in vivo. In the rat sciatic nerve crush injury model, we found that LBD-NGF could be retained and concentrated at the nerve injury sites to promote nerve repair and enhance functional restoration following nerve damages.LBD-NGF could specifically bind to laminin and maintain NGF activity both Fused with NtA, NGF-β could bind to laminin specifically. Since laminin is the major component of nerve extracellular matrix, laminin binding NGF could target to nerve cells and improve the repair of peripheral nerve injuries. Sciatic nerve injuries are often caused by injections, gunshot wounds, lacerations, contusions, compressions, and iatrogenic causes NGF promotes proliferation and differentiation of neurons, and also modulates the repair of injured nerves Laminin is the ubiquitous component of the tight network of glycoproteins, collagen IV and proteoglycans in basement membranes in vitro and in vivo. In the rat sciatic nerve crush injury model, LBD-NGF could target to the native nerve extracellular matrix component laminin, and could be retained and concentrated at the nerve injury sites to improve peripheral nerve regeneration.Agrin is a key organizer of postsynaptic differentiation at the neuromuscular junction. The binding of agrin to laminin is required for its localization to synaptic basal lamina and other basement membranes in vitro laminin-binding activities of NAT-NGF and LBD-NGF by ELISA. From the results, we found at each indicated point the OD405 of LBD-NGF was significantly higher than that of NAT-NGF, indicating that the retained LBD-NGF on laminin was more than that of NAT-NGF  (We measured the  P<0.01) .−4 M and 7.25×10−6 M respectively. The lower Kd value of LBD-NGF to laminin indicated that LBD-NGF bound to laminin specifically.Using the binding curve, we calculated the dissociation constant Kd values of NAT-NGF and LBD-NGF to laminin by Scatchard analysis. The slope of the resulting straight line equals −1/Kd . The Kd in vitro release experiment, sustained release of NAT-NGF and LBD-NGF was followed up to 8 days  (We then measured the bioactivity of NGF on laminin  P<0.05) . 3- . These rWe tested laminin content in the rat sciatic nerve. Immunohistochemistry showed the distribution of laminin in the extracellular matrix of rat sciatic nerves . Using wWe evaluated whether LBD-NGF could be retained at the injury sites of sciatic nerves. Three hours and twelve hours after injection of NAT-NGF or LBD-NGF, the sciatic nerve proteins were extracted and the retained NGF was assessed by western-blotting. At 3 h after injection, the level of exogenous LBD-NGF was significantly higher than that of NAT-NGF  . At 12 hAt day 0 after injury, there was no difference among the groups of the control, NAT-NGF, and LBD-NGF . Following several weeks after injury, the sciatic functional index (SFI) values were significantly increased in groups of NAT-NGF and LBD-NGF compared with the control . At day 35, 42, 49 and 56, there were also significant differences between groups of NAT-NGF and LBD-NGF  .The results showed after injury, the nerve conduction velocity (NCV) was significantly decreased  , and theThe distal compound muscle action potential (DCMAP) was also significantly decreased after injury  . At weekThe longitudinal sections at the injury sites of sciatic nerves were analyzed by hematoxylin and eosin (H.E.) staining. The results showed that at week 8 after injury, there was little nerve regeneration in the control group , while sAxonal regeneration was investigated by staining neurofilament-positive axons in transverse sections at the injury sites of sciatic nerves. Results showed that LBD-NGF promoted more axonal regeneration and nerve repair at week 8 after injury . The staImmunostaining with the glia/Schwann cell marker S100 also demonstrated that LBD-NGF increased S100 expression and improved Schwann cell regeneration in response to sciatic nerve crush injury at week 8 after injury compared with the control . The staTransverse semi-thin sections at the injury sites of sciatic nerves were analyzed by Toluidine blue staining. The results showed at week 4 after injury, a large number of well-myelinated axons with large diameter were observed in the group of LBD-NGF .Ultra-thin sections were observed under the transmission electron microscope. The results showed at week 12 after injury, the thickness of the myelin sheath around the axons was the greatest for native nerves, followed by LBD-NGF and then NAT-NGF treated nerves. The thickness of the myelin sheath in the control group was the thinnest .The statistical analysis revealed that at week 4 after injury, there were significant differences in the number of myelinated axons among the groups of the control, NAT-NGF and LBD-NGF  . HoweverIn the statistical analysis of the myelinated axon diameter, we found at week 4, myelinated axon diameter was significantly larger in groups of NAT-NGF  and LBD-NGF  compared with the control . But theWe also evaluated the thickness of myelin sheath by Image-Pro Plus software (Media Cybernetics). The statistical analysis revealed that at each time point, the myelin sheath thickness was significantly greater in the LBD-NGF group compared with the control  . At weekNGF plays a critical role in nerve injury repair. However in practice, NGF simply delivered in solution is difficult to be retained at the injury sites due to its rapid diffusion in extracellular fluids. In order to maintain NGF concentration, multiple injections are needed. However, they would increase cost and surgical risks. In addition, the excessive NGF at the injection sites and the diffusion of NGF may cause possible adverse effects. Thus, a delicate control of NGF both in dosage and in localization is critical to apply NGF safely and efficiently.One of the strategies is to incorporate NGF into polymeric materials and then implant directly into the targeted tissue sites Laminin is widely dispersed in the peripheral nervous system in vitro. As expected, LBD-NGF showed specific laminin-binding capacity compared with NAT-NGF which reflected by the lower Kd value. From the sustained release experiment, NAT-NGF was found to be quickly released at the first day, whereas LBD-NGF could gradually release from laminin during 6 days. These results demonstrated the LBD-NGF could be retained on laminin for a longer time compared with NAT-NGF.Agrin is a key organizer of acetylcholine receptor (AChR) clustering at the neuromuscular junction. Binding of agrin to laminin requires NtA, which comprises the first 135 amino acids. This region mediates high-affinity interaction with the coiled-coil domain of laminin in vitro also demonstrated that LBD-NGF maintained higher concentration and bioactivities on laminin. The results suggested LBD-NGF could target to laminin and could be helpful in nerve injury repair.We subsequently evaluated whether the bioactivity of NGF-β was influenced during the genetic engineering process. PC12 cells were used for examining the bioactivities of the refolded LBD-NGF by neurite outgrowth assay and MTT assay. The results showed that LBD-NGF retained the bioactivity of NGF. Bioactivity assay of NGF on laminin The rat sciatic nerve crush injury model was used to test the effect of nerve injury repair by LBD-NGF. In order to limit additional variations, such as the surgical technique, mismatch of proximal and distal axonal alignment and foreign body (suture) reaction, we chose a pure crush injury model in the study. Sciatic nerve crush injuries caused anatomical disruption of axons. Wallerian degeneration occured when there was a disruption of the axon in vivo work demonstrated that laminin binding NGF targeted specifically to the endogenous laminin of the sciatic nerves and maintained a higher concentration and stronger bioactivity of NGF at the injury sites. It was effective in enhancing histologically detectable nerve regeneration and improving functional recovery.LBD-NGF was retained at the injury sites, and the LBD-NGF group demonstrated the most favorable functional recovery as measured by SFI. The electrophysiological observation of LBD-NGF group showed significantly improved restoration of NCV and DCMAP than NAT-NGF and the control groups correlated well with the functional SFI findings. In addition, the histological findings indicated that the LBD-NGF group showed better regeneration in axonal regeneration, Schwann cell proliferation and remyelination of sciatic nerves. Thus the For nerve injuries such as crush and stretch, LBD-NGF could be directly utilized for repair by using the native nerve extracellular matrix component laminin as the target as well as the scaffold. However, if the nerve injury is extensive, forming an irreducible gap between the injured proximal and distal stumps, a nerve bridge technique would be preferred. Scaffolding biomaterials filled with laminin or laminin-rich biomaterials which can aid in the guidance of growing nerve fibers along appropriate paths could be used to enhance the precision of stump approximation. At the same time, the laminin binding NGF could be used to promote nerve regeneration and improve functional recovery. Laminin-rich nerve bridging scaffolds and the laminin binding NGF could be an effective strategy for the repair of severe nerve injuries.In conclusion, LBD-NGF promoted sciatic nerve regeneration. LBD-NGF maintained a higher concentration and stronger bioactivity on laminin. Using a rat sciatic nerve crush injury model, LBD-NGF was found to be retained and concentrated at the nerve injury sites to enhance the functional restoration following nerve damages. The laminin binding NGF targeted to the nerve extracellular matrix laminin and promoted the repair of peripheral nerve injuries.E. coli was transformed targeted vector, and then we induced the protein of NAT-NGF or LBD-NGF with 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) at 37°C for 5 h. we harvested the two recombinant proteins accumulating in the inclusion bodies from cell lysis by centrifugation and solubilized in 8 M urea containing 0.4% (vol/vol) β-Mercaptoethanol. Purification of the solubilized fusion proteins was then accomplished under denaturing conditions by nickel chelate chromatography (Amersham Biosciences). The expression of recombinant proteins was analyzed by electrophoresis on a 15% (wt/vol) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing condition and western-blotting with mouse antibody to polyhistidine  and sheep antibody to mouse Alkaline phosphatase . The purified NAT-NGF or LBD-NGF was refolded in the glutathione redox-refolding system by chromatographic method Gene of NAT-NGF or LBD-NGF was generated by PCR and inserted into vector pET-28a (Novagen), respectively. BL21 (DE3) strain of para-nitrophenylphosphate (pNPP) (Sigma) in AP buffer (pH 9.6) for 15 min at 37°C. The results were quantified at 405 nm using an ELISA reader (Molecular Devices). We calculated the dissociation constant Kd values for two proteins binding to laminin as described previouslyThe laminin binding assay was carried out by a modified ELISA assay In this study, we prepared the laminin containing membranes from pig amnion 3 cells/well and maintained under serum-free RPMI 1640 medium (HyClone) at 37°C for 1 h, then serially diluted refolded NAT-NGF or LBD-NGF was added to the plates. After 24 h incubation, the percentage of cells with neurites more than the average diameter of the cell in length was determined. The number of surviving cells was determined by MTT assay after 72 h culture with NAT-NGF or LBD-NGF. Cells cultured under identical conditions but without NGF served as a blank control. The assay was valued in duplicate by two independent assessors.We measured the bioactivity of NAT-NGF and LBD-NGF using PC12 cells  in cell neurite outgrowth assay 3 cells/well under the same condition as described above. After 24 h incubation, the percentage of cells with neurites more than the average diameter of the cell in length was determined. The number of surviving cells was also determined by MTT assay after 72 h culture. Cells cultured under identical conditions but without NGF served as a blank control. The assay was valued in duplicate by two independent assessors.In this study, laminin-coated 48-well plates (Costar) were incubated with NAT-NGF or LBD-NGF at 4°C for 1 h, and then the plates were extensively washed. PC12 cells were seeded at the density of 5×10Six rats were sacrificed by cervical dislocation. The sciatic nerves about 1 cm of 3 rats were rapidly excised and fixed in 4% (vol/vol) formaldehyde for 48 h. we embedded the samples in paraffin and 5-µm sections and examined the samples by immunohistochemistry using antibody to laminin . The sciatic nerves about 1 cm of the other 3 rats were also excised, frozen immediately in liquid nitrogen. Then we extracted the proteins for western-blotting analysis. In the analysis, we used the antibody to laminin  to test the laminin content in the rat sciatic nerve. At the same time, we used laminin (10 µg) purchased from Sigma-Aldrich.Inc as the positive control.in vivo, we used a rat sciatic nerve crush injury model. Seventy-eight male Sprague-Dawley rats weighting 200–220 g were anesthetized by an intraperitoneal injection of sodium pentobarbital (40 mg/kg body weight) and the hair on the right femur was removed. After sterilization, subcutaneously an incision was made at the thigh. Right sciatic nerve was carefully exposed from the intermuscular space, isolated, and subsequently crushed a 5 mm segment for 10 s above the bifurcation of the sciatic nerve with a hemostatic forceps. Then we used a non-degradable suture to mark the site of the lesion at the epineurium. 10 µl NAT-NGF (30 µM) or LBD-NGF (30 µM) was injected into the injury sites respectively. We injected PBS as negative control. Finally, we sutured the fascia and skin separately. During these animal experiments, we observed the Chinese Ministry of Public Health (CMPH) Guide and US National Institute of Health (NIH) Guide for the care and use of laboratory animals. All animals were kept under standardized laboratory conditions in an air-conditioned room with free access to food and water. At different time points, the rats were sacrificed by cervical dislocation.To test the laminin binding ability and biological function of NAT-NGF and LBD-NGF Three hours and twelve hours after injection of NAT-NGF or LBD-NGF, 24 rats were sacrificed by cervical dislocation. We excised the injured sciatic nerves about 1 cm, frozen them immediately in liquid nitrogen. Then we extracted proteins for western blot analysis. Antibody to polyhistidine  was used to distinguish exogenous proteins from endogenous proteins.We assessed the functional nerve recovery following sciatic nerve injury using rat walking track analysis by recording its footprints. At days 0, 7, 11, 14, 18, 21, 25, 28, 35, 42, 49, 56, 70 after nerve injury the animals were submitted to walking track analysis and measurement of the SFI using a method described by Bain et al Where EPL indicated the operated experimental paw length; NPL, normal paw length; ETS, operated experimental toe spread, i.e., the distance between the first and fifth toes; NTS, the normal toe spread; EIT, the operated experimental intermediary toe spread, i.e., the distance between the second and fourth toes; and NIT, normal intermediary toe spread. The SFI was scaled such that −100 represented the sciatic nerve was crushed completely and 0 represented normal function or completely recovery We performed the electrophysiological evaluation before, immediately after, and weeks 4, 8, 12 after the sciatic nerve injury. In this experiment we evaluated two parameters, NCV and DCMAP. Under anesthesia, the rats' sciatic nerves were exposed. Monopolar recording electrode and bipolar stimulating electrodes were used to induce and record electrical activity. We recorded NCV and DCMAP in responding to the stimuli using EMG recorder . The following settings were used: Low Filter, 3 Hz; High Filter, 10 KHz; Sensitivity, 0.1 mV; Sweep duration, 10 ms.We performed the morphological assessments at weeks 4, 8 and 12 after sciatic nerve injury. The sciatic nerves (about 1 cm) at the injury sites were rapidly excised and fixed in 4% (vol/vol) formaldehyde for 48 h. The segments were embedded in paraffin and 5-µm sections were cut from each segment examined by H.E. staining and immunohistochemistry using antibodies to neurofilament  and S100 . Using Image-Pro Plus software (Media Cybernetics), we performed the quantification of neurofilament-positive and S100-positive area from at least 6 randomly selected fields at 400× magnification.We stained the transverse semi-thin sections (1 µm thick) from each segment with Toluidine blue and observed under a light microscope . The number of myelinated axons was counted from at least 6 randomly selected fields under the magnification of ×400. Using Image-Pro Plus software (Media Cybernetics), we evaluated the myelinated axon diameter at 400× magnification. Ultra-thin sections (70 nm thick) were observed under transmission electron microscope . We also evaluated the thickness of myelin sheath by Image-Pro Plus software (Media Cybernetics).Data were presented as mean values±standard deviation. The statistical significance of differences in parameters was determined as *P<0.05 and **P<0.01, by two tailed student's t-test.


Rheumatoid arthritis (RA) is a common rheumatic disease in Caucasians and in other ethnic groups. Diagnosis is mainly based on clinical features. Before 1998, the only serological laboratory test that could contribute to the diagnosis was that for rheumatoid factor (RF). The disease activity markers for the evaluation of clinical symptoms or treatment outcome were the erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). As a matter of fact, the diagnosis of early RA is quite impossible, as the clinical criteria are insufficient at the beginning stage of the disease. In 1998, Schelleken reported that a high percentage of RA patients had a specific antibody that could interact with a synthetic peptide which contained the amino acid citrulline. The high specificity (98%) for RA of this new serological marker, anti-cyclic citrullinated antibody (anti-CCP antibody), can be detected early in RA, before the typical clinical features appear. The presence or absence of this antibody can easily distinguish other rheumatic diseases from RA. Additionally, the titer of anti-CCP can be used to predict the prognosis and treatment outcome after DMARDs or biological therapy. Therefore, with improvement of sensitivity, the anti-CCP antibody will be widely used as a routine laboratory test in the clinical practice for RA. Rheumatoid arthritis (RA) is a systemic autoimmune disease of unknown origin, characterized by chronic joint inflammation that may later develop into joint destruction, as well as functional limitation , 2. So fSince 1960, many investigators used indirect immunoflurorescence and enzyme-linked immunosorbent tests to detect serologic antibodies in RA patients –15. ThesSchelleken in 1988 reported that 76% of RA patients had a specific antibody which could interact with a synthetic peptide which contained the amino acid citrulline . The argIn the past 5–6 years, many studies have focused on the value of the clinical application of anti-CCP antibody in rheumatoid arthritis and other rheumatic diseases –25. The Many rheumatic or immune diseases can present the clinical symptoms of polyarticular, symmetrical arthritis and positive RF, which mimic RA or fulfill the diagnosis of RA. These consist of SLE, SS, psoriatic arthritis (PSA) with polyarticular involvement, HIV-related arthropathy, polymyalgia rheumatica (PMR), and even undifferentiated arthritis or palindromic rheumatism (PR) –37.Anti-CCP antibody can be detected in a small percentage of patients with either rheumatic disease or immune disease . One stuPalidromic rheumatism is a common rheumatic disease, characterized by recurrent self-limited arthritis. More than 50% of PR may evolve into RA years later. Salvador et al. demonstrated 18 of 32 (56.3%) patients with pure PR had serologically positive anti-CCP antibody. The clinical syndrome can be considered as an abortive form of RA . RussellRF can be detectable in cases with hepatitis C infection, with a prevalence ranging from 30% to 60% , 46–48. PMR is a rheumatic disease that mainly affects elderly people. The persistent shoulder pain with morning stiffness and remarkable elevation of ESR or acute phase reactant makes it difficult to distinguish PMR from elderly onset RA (EORA) with initial shoulder joint involvement . A recenThe extraarticular manifestations of RA consist of rheumatoid nodules, interstitial lung disease or pleuritis, vasculitis, amyloidosis, scleritis, mononeuritis, and others. The positive correlation between RF and entraarticular manifestations was reported by De Rycke . HoweverAlthough anti-CCP antibodies are associated with the severity of RA and erosion, both Rycke and Korkmaz could not demonstrate a positive correlation between anti-CCP antibodies and extra-articular manifestations , 51. ManRA has long been recognized as an autoimmune disease which is genetically determined. HLA-Class II alleles are the most important genetic marker that increases the risk of developing RA . The shaRecently, the North American RA Consortium family cohort and the Study of New Onset RA cohort further investigated 1723 Caucasian RA patients and reconfirmed that HLA-DRB1 SE was strongly associated with anti-CCP . In thisThe interaction between SE alleles and citrullinated peptide antigen may be the pathogenetic mechanism that increases the anti-CCP antibody production in RA . HoweverFor juvenile RA, HLA-DR4 positive patients with polyarticular onset were more likely to have anti-CCP antibodies than those without HLA-DR4  . InteresThe environmental factors in RA are multiple. Among them, smoking is a risk factors for RA, and this is evidenced by the fact that smokers have increased levels of RF –69 and aHowever, it is quite difficult to identify the possible pathogenic pathways through which smoking confers a high risk of developing RA in SE-positive individuals. Whether smoking can increase the proinflammatory cytokines or destroy the tolerance to citrinullated proteins remains unknown and needs further investigation .Many recent studies have shown that anti-CCP antibody can predict the severity of either the clinical or radiological outcome in RA patients , 74–80. We recently enrolled 155 ethnic Chinese RA patients and divided them into anti-CCP positive (110) and anti-CCP negative  patientsThe 2 most common laboratory tests that we use to evaluate RA disease activity and treatment efficacy are ESR and CRP. In certain RA patients, the clinical manifestations may not be correlated well with ESR and CRP. Searching for other disease activity markers in order to assess clinical improvement after a specific drug therapy is important. After biological therapy, a significant improvement in clinical features, including pain, stiffness and swelling has been noticed. Apart from that, both ESR and CRP can be reduced significantly within 3 months after treatment , 82. SevThe positive rate for anti-CCP antibody before infliximab or etanercept treatment ranged from 83% to 90%, and for RF, from 78% to 95%. The higher frequency of anti-CCP or RF was mainly due to the severe RA patients who were enrolled in the study , 89. AleIn conclusion, short-term biological therapy (<6 weeks) can effectively suppress the serum anti-CCP level in RA patients. The long-term (>1 year) efficacy of TNF-alpha inhibitor for the serum anti-CCP antibody level is not conclusively known now, and requires further study.Anti-CCP has become a “key” serologic marker in RA. It can be used (1) as a test for early diagnosis of RA; (2) for the differential diagnosis between RA and other rheumatic or immune diseases; (3) for prediction of prognosis; and (4) for evaluation of treatment outcome.


Smear-negative pulmonary tuberculosis (TB) accounts for a considerable proportion of TB transmission, which especially endangers health care workers (HCW). Novel Mycobacterium-tuberculosis-specific interferon-γ release assays (IGRAs) may offer the chance to define the burden of TB in HCW more accurately than the Mantoux tuberculin skin test (TST), but the data that is available regarding their performance in tracing smear-negative TB in the low-incidence, in-hospital setting, is limited. We conducted a large-scale, in-hospital contact investigation among HCW of a German university hospital after exposure to a single case of extensive smear-negative, culture-positive TB with pulmonary involvement. The objective of the present study was to evaluate an IGRA in comparison to the TST and to identify risk factors for test positivity.®-TB Gold in Tube (QFT-GIT) and the TST, and followed-up for two years. Active TB was ruled out by chest x-ray in QFT-GIT-positive subjects. Independent predictors of test positivity were established through the use of logistic regression analysis.Contacts were prospectively enrolled, evaluated using a standardized questionnaire, the IGRA QuantiFERONOut of the 143 subjects analyzed, 82 (57.3%) had close contact, but only four (2.8%) experienced cumulative exposure to the index case >40 hours. QFT-GIT results were positive in 13 subjects (9.1%), while TST results were positive in 40 subjects (28.0%) at an induration >5 mm. Overall agreement was poor between both tests (kappa = 0.15). Age was the only predictor of QFT-GIT-positivity , while TST-positivity was significantly related to Bacillus Calmette-Guérin vaccination and foreign origin. Logistic regression analysis showed no relation between test results and exposure. No secondary cases of active TB were detected over an observational period of two years.Our findings suggest a low contagiosity of the particular index case. The frequency of positive QFT-GIT results may in fact reflect the pre-existing prevalence of latent TB infection among the study population. TB transmission seems unlikely and contact tracing not generally warranted after cumulative exposure <40 hours. However, the substantially lower frequency of positive QFT-GIT results compared to the TST may contribute to enhanced TB control in health care. Tuberculosis (TB) is a major cause of illness and death worldwide . In contAlthough cross-reactivity following vaccination with Bacillus Calmette-Guérin (BCG) or exposure to non-tuberculous mycobacteria is common, the tuberculin skin test (TST) has been applied for the diagnosis of LTBI for about a century . In-vitrAcid-fast bacilli smear-negative, culture-positive pulmonary TB accounts for a considerable proportion of TB transmission. In 2007, 56.3% of all infectious pulmonary TB cases reported to the responsible German authority (Robert Koch Institute) were smear-negative ,11. The TB contact investigations in the in-hospital setting are often challenging due to patient movement and the changing work assignment of personnel . Particu®-TB Gold in Tube (QFT-GIT) with the Mantoux tuberculin skin test (TST) in a large-scale in-hospital contact investigation among German HCW after exposure to a single case of smear-negative, culture-positive pulmonary TB and to identify independent risk factors of test positivity.We conducted an in-hospital contact investigation of a single index patient with extensive smear-negative, culture-positive TB including non-cavitary pulmonary involvement, who had a complicated in-hospital course of about three months and numerous contacts in various medical departments and disciplines  until March 13th 2009. All HCW were informed of TB-related symptoms, instructed on self-monitoring and reporting and subject to routine follow-up screening according to German Occupational Safety and Health legislation. All QFT-GIT-positive subjects were radiologically followed up as recommended by national guidelines [We prospectively enrolled eligible HCW. The suspected time of in-hospital infectivity was 57 days from referral to our neurotraumatological department on January 10The TST was performed by the Mantoux method using 0.1 ml (two tuberculin units) of purified protein derivative (PPD) RT 23 . The test was administered strictly intradermally to the volar side of the forearm and was read 72 hours after application. The transverse diameter of induration was measured and documented as described previously . A diameAs an IGRA, the QFT-GIT  was used. ELISAs and the interpretation of QFT-GIT results were performed according to the manufacturer's instructions that consider a result positive if the IFN-γ response of TB antigen minus Nil was ≥ 0.35 IU/ml  . The cumData analysis was performed using SPSS, version 11.5 . Categorical data were compared by Pearson's chi-squared or Fisher's exact test, where appropriate. Normal distribution in continuous variables was determined with the Kolmogorov-Smirnov test and differences were subsequently determined either with the student's t-test or the Mann-Whitney-U test. Spearman correlation coefficients and kappa values were calculated for both tests. Relations were described as odds ratio (OR) and 95% confidence interval (CI). ORs for test results depending on different putative predictive variables were calculated using logistic regression. Model building was performed backwards using the chance criteria for variable selection . All p vBetween June and August 2007, mean 17 ± 2 weeks after last exposure to the source case, 202 HCW with suspected contact were evaluated. Of those, 44 had not had contact or had not been exposed during the time of infectivity. Of the 158 eligible contacts, 14 denied consent and 144 were recruited for the study. One subject (with a negative TST result) was excluded from data analysis due to an indeterminate QFT-GIT result .The median cumulative exposure time was 60 min and ranged from 3 to 4000 min (67 h). Eighty two subjects (57.3%) had had close contact to the index case. These included four individuals (2.8%), who had been exposed for >40 hours Table . The cumQFT-GIT results were positive in 13 of the 143 contacts (9.1%). The QFT-GIT-positive subjects were significantly older  and had been working in health care for a longer period of time than the QFT-GIT-negative subjects . However, there was no difference between median cumulative exposure times with regard to the QFT-GIT results . Remarkably, the only subject with a history of prior TB in 1976 had a negative QFT-GIT (IFN 0.046 IU/ml), but a positive TST result (15 mm induration). Figure The TST was read mean 72 ± 5 hours after application. Overall, 40 contacts (28.0%) and 28 contacts (19.6%) had a positive TST result when a cut-off >5 mm and >10 mm induration was applied, respectively. Mean age rather than mean duration of employment in health care, was significantly higher in TST-positive subjects when compared to TST-negative subjects . Whichever TST cutoff was applied, there was no difference between the median cumulative exposure times .More than one half of the contacts 51%) were BCG vaccinated (Table % were BCOne hundred and seventeen subjects 81.8%) had been tested with a prior TST median five years (range 3 mo to 38 yrs) ago. In most instances, prior TST had been administered by the multi-puncture method . Of those, 38.5% had had a positive prior TST result  administered preventive therapy with INH as recommended. None of the contacts developed active TB within a period of two years (106 ± 1.5 weeks) after the last exposure to the index case.The QFT-GIT proved to be feasible for contact tracing HCW in a low TB incidence in-hospital setting containing a high proportion of BCG vaccinated individuals even in a smear-negative index case. No secondary cases of active TB were detected within the observational period of two years, and the positive test results were not related to exposure. Altogether, relevant nosocomial TB transmission appears unlikely. The frequency of positive QFT-GIT results may in fact reflect the pre-existing prevalence of LTBI in the study population. Thus, IGRAs may offer the chance to increase the accuracy of diagnosing LTBI, enhance the implementation of preventive chemotherapy and further improve TB control in low-incidence countries and health care.®-TB Gold assay [We determined a low overall frequency of positive QFT-GIT results of 9.1%. This frequency was substantially lower compared with the recent Mantoux TST 28.0%) or to the prior TST (38.5%). These findings are in agreement with studies on comparable populations determining the prevalence of LTBI among HCW without recent TB exposure. Just about one decade ago, Kralj and colleagues proposed a LTBI prevalence among German HCW of 40% according to positive multi-puncture TST results . More reld assay . A recenld assay . In contld assay -28..0% or toAn informative comparison between the frequencies of positive IGRA results among the study population of HCW and the general German population is hampered by the lack of sufficient data on background IGRA positivity rates and the fact that the IGRA results depend to a great extent on the characteristics of exposure, the different settings and populations the test is applied to. Two recent contact studies that were conducted at an urban public health department among a population of non-HCW found QFT-GIT positivity rates of 10% and 11%, respectively, but included recent contacts of smear-positive index cases with extensive exposure >40 hours only ,30. MoreMoreover, we found a low level of overall agreement between TST and QFT-GIT results. This finding is consistent with previous studies in HCW and thus confirms that the BCG vaccination is a major confounder of TST results, while QFT-GIT results were not affected by BCG ,32,33. DLogistic regression analysis showed no obvious relation between exposure and positive results for either of the applied tests. Instead, we found age to be the only independent predictor of QFT-GIT positivity and demonstrated a further link between foreign origin, BCG vaccination and positive TST results. In previous contact tracing studies of profoundly contagious smear-positive pulmonary TB index cases, IGRA-positivity was well correlated with exposure ,17,30. IWe detected no secondary cases of active TB within a follow-up period of two years after the last exposure. Recently, first evidence for the relevance of positive QFT-GIT results was provided demonstrating a progression rate to active TB of 14.6% (6/41 subjects) over a two-year period in subjects who tested positive. However, this study only included subjects after recent exposure to smear-positive pulmonary TB >40 hours . AnotherThe present study is subject to limitations. Although only 82 HCW  had had close contact, all eligible subjects were included in this contact investigation contrary to current German and CDC guidelines and assigned to the medium to high priority category ,35. ThisMoreover, as there is no gold standard for the diagnosis of LTBI, both IGRAs and TST tend to indicate the lasting immune response after exposure to MTB rather than prove a genuine TB infection . DespiteThe finding of age-dependency of positive QFT-GIT results may be due to an age-cohort effect based on steadily decreasing TB-incidence rates in Germany over the past decades and, on the other hand, to a longer time at risk whilst being employed in health care. This suggests that a significant proportion of the QFT-GIT-positive results were caused by prior MTB infection and not by recent exposure. Hence, our findings suggest a low contagiosity of the particular index case. Consequently, the frequency of positive QFT-GIT results may in fact reflect the pre-existing prevalence of LTBI among the study population and makes any relevant nosocomial transmission unlikely. The observed link between foreign origin and TST positivity may be due to the proportion of subjects with unknown BCG vaccination status among the subgroup of foreign born subjects, and may indicate substantially different BCG vaccination policies among countries in the past, as documented for Europe .The QFT-GIT proved to be a feasible method in this large-scale, in-hospital contact investigation. Substantially lower prevalence rates of presumed LTBI resulted when different approaches of conducting contact investigations were employed, particularly compared with those that had been applied in the past . This indicates that IGRAs have the potential to profoundly change our clinical practice. The high frequency of discordant results observed in our study argues against a two-step screening procedure in a low-incidence country with a substantial proportion of BCG vaccinated subjects. Moreover, our results support a recent study by Diel and colleagues, who suggested the feasibility of IGRAs in contact investigations of smear-negative index cases and, in this context, an exposure-dependent performance with markedly increased positivity rates only after exposure >40 hours . FinallyWe did not detect any secondary case of active TB within the observational period of two years. Overall, the probability of relevant nosocomial transmission for the particular index case appears to be low. Our findings suggest that contact tracing is not generally warranted after cumulative exposure <40 hours if the index case is smear-negative. However, given the sensitivities of current IGRAs, they may not be used to sufficiently rule out the presence of LTBI. So far, no conclusive statement regarding the progression risk to active disease in our population and particular setting can be made. Both IGRAs and TST possess inherent limitations, and lack the ability to reliably discriminate between recently acquired or prior latent TB infection. Depending on the applied method, the prevalence of LTBI among the study population varied considerably. However, the substantially lower frequency of positive QFT-GIT results may provide the opportunity to target preventive therapy and thus contribute to enhanced TB control in health care.The authors declare that they have no competing interests.FCR conceived and designed the study, took care of adequate funding and equipment, performed the statistical analysis, took some blood samples, conducted and interpreted the ELISAs, interpreted the data, supervised the study and drafted the manuscript. SS participated in the study design, interviewed the HCW, applied and read the TST. AN participated in the study design, data interpretation, statistical analysis and revised the manuscript critically for important intellectual content. AS participated in the study design, data interpretation, statistical analysis and revised the manuscript critically for important intellectual content. GSW contributed to the study design and supervised the study. GR contributed to the study design, the analysis and interpretation of data, supervised the study and revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.th European Respiratory Society Annual Congress 2008 in Berlin, Germany [Part of the data was presented at the 18 Germany . The sitDefinition of the index case. The data provide radiological and microbiological details of the index patientClick here for fileAddendum methods section. The data provide details of the QFT-GIT processing and the questionnaire itemsClick here for fileDetailed description of the subject with indeterminate QFT-GIT result. The data provide clinical details of the subject with indeterminate IGRA resultClick here for file


Homeodomain proteins play critical roles in shaping the development of the embryonic central nervous system in mammals. After birth, neurogenic activities are relegated to stem cell niches, which include the subgranular layer of the dentate gyrus of the hippocampus. Here, we have analyzed the function of HOP (Homeodomain only protein) in this stem cell niche and in human glioblastomas.HOP is strongly expressed by radial astrocytes of the dentate gyrus in mice, which are stem cells that give rise to hippocampal granular neurons throughout adulthood. Deletion or down-regulation of HOP results in a decrease of apoptosis of these stem cells without changes in proliferation, and in an increase in the number of newly formed granule neurons. We also find that human glioblastomas largely lack HOP expression and that reintroduction of HOP function in glioma cells cultured as gliomaspheres leads to enhanced apoptosis in a subset of cases. In these cells, HOP function decreases clonogenicity.We find that These data suggest that HOP participates in the regulation of the adult mouse hippocampal stem cell niche by negatively affecting cell survival. In addition, HOP may work as a tumor suppressor in a subset of glioblastomas. HOP function thus appears to be critical in the adult brain in a region of continued plasticity, and its deregulation may contribute to disease. HOP  is a small 73 amino acid atypical homeodomain protein, composed simply of a homeodomain. HOP was first identified in the developing heart where it modulates cardiac growth . SurprisIn the initial reports describing HOP in the heart, its expression was also detected in the developing neural tube and in the adult brain ,2. This,HOP is expressed by radial astrocytic stem cells and increases neuronal production by promoting apoptosis of these cells.Cell lineages in these niches have been described -17. In tIt has been suggested that gliomas in general, and glioblastomas (GBMs) in particular, derive from the transformation of neural stem cells -22, whicHOP is down-regulated in GBMs. Its re-expression induces apoptosis in two of four GBM cultures tested and decreases GBM cancer stem cell clonogenicity in one of them. We conclude that HOP is a new regulator of stem cell survival in the adult brain and that its deregulation may participate in the tumorigenic process.We show that HOP expression in detail in stem cell niches of the adult mouse brain, we performed in situ hybridization and immunolocalization studies. HOP mRNA and protein were not detected in the SVZ of the lateral ventricle of the adult forebrain. In contrast, they were found in the DG and, in particular, in cells of the SGL , a general marker of astrocytes , which stains the cell bodies and processes of all astrocytes, including DG radial astrocytes [+ cells were 3PGDH+, thus confirming their astrocytic identity , which are nested along astrocytic processes  and exprP Figure . TogetheGiven that adult neurogenesis requires proliferation and survival of stem cells and their progeny, we tested the role of HOP in these two processes.+/GFAP+ in wild-type and HOP-/- mice (n = 5). No significant differences were detected  and epidermal growth factor (EGF) to stimulate cell proliferation  bromodeoxyuridine (BrdU) incorporation Figure , as descd Figure . MoreoveHOP in wild-type mice through RNA interference using cell-permeable small interfering RNAs (siRNAs). These siRNAs were rendered cell-permeable by reversibly coupling them to Penetratin, a small transducing peptide [HOP siRNA-Penetratin into the brain, at a location just above the DG with micro-osmotic pumps, led to a dramatic reduction in HOP immunoreactivity , as compared with control siRNA infusions. HOP-siRNA and control siRNA-treated mice infused for three days in the lateral ventricle showed similar levels of BrdU incorporation in SGL GFAP+ stem cells . The amount of TUNEL+ cells expressing HOP was 82% ± 4 SEM , indicating their SGL astrocytic and thus stem cell nature  or neurons, which are normally pruned [+ and that this number is reduced in the absence of HOP, we conclude that HOP is a pro-apoptotic factor in SGL radial astrocytic stem cells in the hippocampus.To evaluate a possible role of HOP in stem cell survival, we analyzed the number of TUNELe Figure . We foune Figure . TUNEL+/y pruned . Given tHOP-/- mice were pulsed with BrdU followed by a chase for three weeks before analyzing the number of new BrdU+/NeuN+ DG neurons .An effect in stem cell survival should be reflected on the rate of production of new neurons. To assess this possibility, wild-type and s Figure , as NeuNHOP-/- animals survive to adulthood [+/BrdU+ neurons in mice infused with HOP siRNA compared to mice infused with a control siRNA . The lower penetrance of the RNA interference phenotype (1.7-fold) versus that seen in knock out mice (2.5-fold) is consistent with the common incomplete effects of RNA interference compared with gene deletions. Notwithstanding this difference, the data support a direct role of HOP in the regulation of hippocampal stem cell apoptosis.This result could be due to a function of HOP in SGL adult cells or to indirect effects, perhaps through an earlier effect of HOP. Indeed, only 50% of dulthood ,2. Thus,HOP, which decreases apoptosis in the normal mouse hippocampal stem cell niche, marks human gliomas. Here we have focused on grade IV astrocytomas (or GBMs), which are the most aggressive and invasive type of gliomas and display poor prognosis.Faulty apoptotic control of stem cell lineages could underlie or favor the development of tumors. Gliomas are common adult human brain tumors that are thought to derive, at least in part, from stem cell lineages and harbor cancer stem cells -22,29,30HOP mRNA is down-regulated in tumor excisions compared to the normal cortex , but we could unfortunately not compare these levels to the matched biopsies. Interestingly, the HOP expression level was also extremely low in one oligodendroglioma and corresponding gliomasphere culture. There is thus a positive trend correlating low levels of HOP and glioma stem cell lineages in a subset of gliomas.Analysis by quantitative RT-PCR of four independent glioblastomas  . In contrast, HOP internalization triggered a 50% increase in apoptosis, as measured by the percentage of apoptotic cleaved-Caspase 3+ cells compared to Kn-HD . Restored HOP function in human GBM-6 stem cell lineages is thus able to induce apoptosis without affecting proliferation. Similarly, HOP-Penetratin increased apoptosis in GBM-11 cells, which express high levels of HOP, compared with Kn-HD , whereas no difference in their proliferation was measured by Ki67 staining (1.76% ± 0.05 SEM versus 1.7% ± 0.12 SEM). In contrast, still in comparison with Kn-HD, HOP-Penetratin did not modify apoptosis in GBM-8 cells , which express low levels of HOP, or in GBM-12 cells (3.75% ± 1.4 SEM versus 3.29% ± 0.47 SEM), which express higher levels. This also confirmed the lack of non-specific effects of HOP-Penetratin in GBM cells. HOP is thus able to induce apoptotic cell death only in a subset of gliomaspheres with varying levels of endogenous HOP. Analyses of the growth rates of these cultures revealed that HOP was effective only in those that grew poorly: GBM-12 gliomasphere cultures had three times more cells than GBM-6 or GBM-11 cultures after six days under identical conditions and starting with the same number of cells . The basis of this difference is not known.To assess HOP function in GBM cells, we increased HOP function by introducing cell-permeable HOP. Purified full-length HOP protein, which itself is not cell permeable, was coupled with Penetratin peptide, thus rendering it cell permeable and functional in GBM cells after internalization Figure . As contp = 0.01). HOP is thus able to induce apoptosis of glioma stem cells and decrease clonogenicity, used here as a measure of self-renewal.To test if HOP could affect stem cell self-renewal, we used GMB-6 cells that show a clear increase in apoptosis when treated with HOP-Penetratin. GMB-6 gliomaspheres were treated as above but in suspension to allow internalization of HOP-Penetratin or Kn-HD as control and to be able to plate one cell per well in 96-well plates. After two weeks of growth, gliomaspheres were counted in triplicates assays. It should be noted that clonal analyses of glioblastoma cells is stringent even in the presence of 50% conditioned media since these cells do not readily grow in isolation. HOP induced a ten-fold reduction in the number of clonogenic units (gliomaspheres) compared with Kn-HD , the only other radial astrocytes in the adult brain, confirming and extending previous mRNA localization data .Bax and Bak [We describe here a new mode of regulation of adult hippocampal neurogenesis through modulation of adult progenitor death. A recent study has shown that 85% of the variance in neurogenesis between strains could be explained by different cell-survival rates . However and Bak . Our wor+/BrdU+ cells (reflecting the number of progenitors) is the same in HOP null and wild-type siblings . The reduction of hippocampal progenitor death in mice lacking HOP, therefore, does not lead to an increase in their number. A reduction in progenitor death not leading to an increase in their abundance is puzzling, although it could be explained by a difference in the choice between symmetric and asymmetric divisions in wild-type versus HOP null hippocampal stem cells. Indeed, GBM cells that increase apoptosis in response to enhanced HOP levels also decrease their self-renewal (as measured by their clonogenicity). Therefore, there may be a mechanism that senses overall cell numbers and that regulates self-renewal and apoptosis in normal and tumorigenic stem cell lineages.As fewer hippocampal progenitors die in mice lacking HOP, one could predict that loss of HOP may lead to a change of the total number of progenitors. However, the number of GFAPs Figure . We haveBcl2 in embryonic stem cell and early embryogenesis [Bcl2 gene. We tried to test this hypothesis by comparing Bcl2 expression in wild-type and knock out mice by quantitative RT-PCR but since the vast majority of hippocampal cells express Bcl2, a potential difference in a minority of progenitors could not be quantified.HOP has been shown to interact with SRF in the heart ,2 where ogenesis . One couHOP in glioma excisions  compared to controls and even lower levels of HOP in gliomaspheres (which only contain tumor-derived cancer stem cell lineages). Experimentally, increased HOP expression enhanced apoptosis in a subset of gliomasphere cultures. Differences in responsiveness to HOP might depend, for instance, on the region of origin of the tumor or on the presence of co-factors necessary for HOP pro-apoptotic function. The apoptotic-inducing activity of HOP, albeit context-dependent, may be related to its normal function, as brain tumors are thought to derive from the transformation of stem cells and/or progenitors in adult neurogenic regions. Indeed, alterations in the cellular mechanisms that control adult neurogenesis might contribute to tumorigenesis [The deregulation of apoptosis in hippocampal stem cells could contribute to dysfunction and/or disease. We report a common down-regulation of igenesis -22. Thisigenesis , head anigenesis  and lungigenesis . HOP migHOP expression is low in gliomas and its re-expression in gliomaspheres induces apoptosis and a reduction of glioma cancer stem cell self-renewal in a subset of cases. We suggest that HOP acts as a tumor suppressor in the hippocampal stem cell niche, where its loss could favor the inappropriate survival of stem cells and their descendants, increasing cell numbers and contributing thus to tumor growth.We describe here a new function for the homeoprotein HOP as a negative regulator of hippocampal stem cell survival. Our work provides the first evidence that apoptosis of stem cells can regulate adult neurogenesis. In addition, HOP cDNA was cloned from hippocampal mRNA and subcloned into pGEM vectors for riboprobe synthesis and pGEX vectors for protein production. The recombinant HOP protein was purified and used to produce a rabbit polyclonal antibody .The mice used were either Swiss or the HOP knock out mice from the Epstein laboratory  analyzedin situ hybridization and TUNEL staining, adult mice (two months old) were perfused , their brains were removed, post-fixed overnight and sectioned with a vibratome .For immunostaining, In situ hybridizations were performed as described [Sections were blocked in phosphate-buffered saline, 10% fetal calf serum, 0.3% triton and incubated in primary antibodies in the same buffer for 36 h at 4°C under agitation. Antibodies were mouse anti-GFAP , rat anti-BrdU , mouse anti-NeuN , rabbit anti-Sox2 , guinea-pig anti-3PGDH , guinea-pig anti-Dcx  Fluorescent secondary antibodies were FITC, rhodamine or Cy3 anti-species . Immuno-labeled sections were observed and imaged with confocal Leica and Zeiss microscopes and a compound Axiophot microscope. escribed .Sections were dehydrated in successive baths of 50%, 70%, 90%, and 100% ethanol followed by rehydration along the inverse path. They were then incubated in the TUNEL  staining mix of the Roche kit  according to the manufacturer's instructions, thus leading to a FITC staining of apoptotic cellsHOP siRNA is: AAGGCTTGCCTTCGGAATGCA. It was produced by Qiagen  with a thiol modification on the 5' extremity of the sense strand. Annealed siRNA duplex was resuspended in the buffer provided by the manufaturer and heated at 90°C for 1 minute followed by a 1 h incubation at 37°C. It was then pretreated with an equimolar quantity of TCEP for 5 minutes at room temperature. An equimolar amount of activated penetratin was added and the reaction was allowed for 2 h at 37°C. The efficacy of the coupling was assessed on 22% polyacrylamide gel with and without dithiothreitol. The control siRNA was the standard control siRNA from Qiagen called Negative.The sequence of the sense strand of Coupled siRNAs (20 μM) or growth factors  were diluted in 0.09% NaCl. Mice were infused continuously for three days using micro-osmotic pumps  implanted streotaxically. The coordinates for intraventricular infusion of growth factors were: anterior/posterior, 0; lateral, 0.8 mm;depth, 2 mm (relative to bregma and surface of the brain). The coordinates for intraventricular infusion of siRNAs were: anterior/posterior, -0.82 mm; lateral, 1.5 mm; depth, 1.7 mm. The coordinates for intraventricular infusion of siRNA above the DG were: anterior/posterior, -2.46; lateral, 0.7 mm;depth, 2 mm.U test. After perfusion, the region of the brain containing the DG was cut frontally with a vibratome as four alternate series of nine 50 μm thick sections. For the proliferation assay, mice were injected intraperitoneally with BrdU (30 mg/kg) and perfused 2 h later. One of the four series of sections was immunohistochemically labeled with GFAP and BrdU. The sections were observed under the 20× objective of a Leica confocal microscope and all BrdU positive cells located in the SGL were zoomed upon (magnification of at least 5 times) and observed along the z-axis to assess their GFAP immunoreactivity. The DG length of each section was measured with NIH image and the total number of GFAP/BrdU positive cells of all sections was divided by this length to be expressed as GFAP/BrdU positive cells per DG length. For neuronal production, mice were injected intraperitoneally 3 times every 3 h with BrdU (30 mg/kg), perfused 3 weeks later and the quantification was performed as above after a NeuN/BrdU immunocytochemistry (ICC). For TUNEL staining, all 36 DG sections of each animal were processed and the total number of TUNEL positive SGL cells counted with the Leica SP2 confocal microscope.At least five mice per condition were used. Statistical significance was determined using Mann-Whitney To assess the reduction of immunoreactivity after siRNA infusions, the optical density of one series of sections per animal was measured with ImageJ as the mean gray value within the DG.et al. [et al. [Human glioma samples were obtained from the Hôpital Universitaire de Genève under approved protocols as in Clement et al. . Glioma  [et al. . GBM U87For internalization, gliomaspheres were cultured on poly-ornithine and poly-laminine coated wells and HOP coupled to Penetratin (Hop-Penetratin) or Knotted homeodomain were added to the medium at 1 μM . After 48 h, cells were fixed with 4% paraformaldehyde and stained with rabbit anti-activated Caspase-3  or mouse anti-Ki-67 . Stained cells were counted with a Zeiss Axiophot optical microscope.For cloning assays, gliomaspheres were dissociated manually with a 200 μl tip. Dissociated cells were then treated with HOP-Penetratin or controls for 48 h before plating in full media in 96-well plates at a density of one cell per well. Fourteen days after plating, gliomasphere growth was scored visually under the microscope.HOP-s, ACCACGCTGTGCCTCATC; HOP-a, TTGGTTAAGCGGAGGAGAGA; GAPDH-s, TGACATCAAGAAGGTGGTGAAGC; GAPDH-a, CCCTGTTGCTGTAGCCGTATTC.Quantitative real time RT-PCR was performed using the iQ™ SYBR Green supermix  at 60°C, according to the manufacturer's instructions. Primers were 5' to 3': The authors declare that they have no competing interests.ADT participated in the staining and counting procedures, MZ performed all the glioma/gliomasphere experiments, JE provided the HOP knock out mice, ARA conceived and supervised the glioma/gliomasphere experiments and conceptually revised the manuscript, IC and AP designed the study and collaborated in the writing of the manuscript, IC carried out the surgery experiments, staining and counting. All authors read and approved the final manuscript.(a-c) After a 2 h pulse of BrdU, mice were processed for 3PGDH/BrdU immunocytochemistry. Some 3PGDH immunoreactive cells incorporate BrdU. (c) Higher magnification of merged (a) and (b) with orthogonal projections. Scale bar: 10 μm. (d) After a 2 h pulse of BrdU, mice were processed for 3PGDH/BrdU ICC and the double-labeled SGL cells were counted under a confocal microscope. The data are presented as the number of 3PGDH/BrdU cells per mm of SGL. The number of 3PGDH/BrdU cells is unchanged in the HOP knock out (KO) mice compared to the wild type (WT).Proliferation of 3PGDH immunoreactive SGL cells. Click here for fileHOP interacts with SRF in the hippocampus. Western blot with HOP antibody shows that HOP is present in hippocampal extracts  and efficiently co-immunoprecipitated with SRF (IP SRF).Click here for file


In order to develop a framework for the analysis of sex-biased genes, we present a characterization of microarray data comparing male and female gene expression in 18 day chicken embryos for brain, gonad, and heart tissue.From the 15982 significantly expressed coding regions that have been assigned to either the autosomes or the Z chromosome , roughly 18% were significantly sex-biased in any one tissue, though only 4 gene targets were biased in all tissues. The gonad was the most sex-biased tissue, followed by the brain. Sex-biased autosomal genes tended to be expressed at lower levels and in fewer tissues than unbiased gene targets, and autosomal somatic sex-biased genes had more expression noise than similar unbiased genes. Sex-biased genes linked to the Z-chromosome showed reduced expression in females, but not in males, when compared to unbiased Z-linked genes, and sex-biased Z-linked genes were also expressed in fewer tissues than unbiased Z coding regions. Third position GC content, and codon usage bias showed some sex-biased effects, primarily for autosomal genes expressed in the gonad. Finally, there were several over-represented Gene Ontology terms in the sex-biased gene sets.On the whole, this analysis suggests that sex-biased genes have unique genomic and organismal properties that delineate them from genes that are expressed equally in males and females. Many genes are more actively transcribed in one sex than the other, and this sex-biased expression pattern is a mechanism by which heritable sexual dimorphisms can arise from a genome that is largely identical in males and females . Sex-biaAdditionally, sex-biased genes are by their very nature often linked to reproduction, and this makes many sex-biased genes subject not only to natural selection, but the powerful pressures of sexual selection as well ,16. The Drosophila melanogaster suggested that sex-biased genes have peculiar evolutionary properties. Specifically, male-sex-biased genes, particularly those associated with gonad, evolve at a faster functional rate [Microarray technologies have recently made it possible to study expression patterns for entire annotated transcriptomes, which has the potential to vastly increase our understanding of the underlying genomic mechanisms that give rise to recognizably dimorphic sexes. Initial evolutionary studies of transcriptome-wide sex-biased gene expression in nal rate ,21-23, hnal rate ,24, and nal rate .D. melanogaster may not extend to all metazoans. Work in other Drosophila species did not recover an elevated rate of evolution for male-biased genes [Drosophila lineages.Recent results have suggested that the syndrome of sex-biased genes observed in ed genes , and reced genes . Birds aed genes , which cElucidating underlying genomic and molecular differences between sex-biased and unbiased genes can give insight into the functionality and constraints for these expression classes, and ultimately help us understand the forces that produce their unique evolutionary properties. Global analysis of sex-biased gene expression in female heterogametic systems have so far been limited to birds ,28 and fHere we study several expression  and genomic  properties of gene expressed in somatic (brain and heart) and gonad tissue of embryonic chickens, and investigate how these properties relate to sex-biased genes.2 fold-change level >1, though this proportion decreased as the fold-change level increased. Z-linked targets were consistently more sex-biased across a range of fold-change cutoffs compared to autosomal targets. Specifically, male-biased genes more frequent on the Z, most likely due to the lack of dosage compensation recently suggested for birds [The genomic distribution of biased and unbiased genes is shown in Table or birds ,28. Femap < 0.00001 for both Z and autosomal genes), as determined by equality of proportions test for overlapping regions  and heart (p = 0.0114) although not in brain (p = 0.1848).Genes with similar breadth and level of expression have been shown to cluster in eukaryotic genomes -35, probp < 0.0001), heart (p = 0.0175) and brain (p = 0.0131). All these correlations remained significant at the same level upon removal of comparisons of neighboring paralogs. This suggests that tandem duplications are not responsible for the observed local similarities, and that genes with similar expression patterns for some tissues are more likely than chance to be proximately located.We also looked for clustering of sex-biased genes. Fold-change in expression, based only upon difference in expression between the sexes without a statistical cut-off p-value to define sex-bias, was significantly correlated in gonad . However, when brain or heart expression was considered, there were no such examples of neighboring sex-biased genes.We also examined the genome for neighboring genes that both had significant evidence for biased expression in the same sex using the absolute value of logD. melanogaster [p < 0.0001 in both cases). For the heart, female-biased genes had lower GC3 content than unbiased genes . GC3 for both male-and female-biased genes expressed in the gonad was significantly different than the unbiased gene class (p < 0.01 in both cases). There was no difference in GC3 among Z-linked expression classes  has been linked to codon bias , which hnogaster . We founses Fig. .p < 0.003 in both cases), though not as starkly as GC3 levels   also difels Fig. . Post hoFigure p < 0.01) in sex-biased categories within each tissue  in male-biased gonad-expressed genes, and the extracellular region (padj = 0.075) over-represented in female-biased gonad genes. Unbiased genes expressed in the gonad showed nineteen significant GO terms. This GO term dataset does not contain any gamete-specific terms, and this is likely due to the fact that the individual animals were collected at embryonic day 18, far before the onset of gametogenesis in chickens.Many Gene Ontology (GO) terms were significantly over-represented , and the brain, heart, and left gonad collected from four male and four female individuals. This data was originally collected for an analysis of dosage compensation, and further details about study design, tissue preparation, RNA isolation, and microarray hybridization and analysis have been previously published . We noteAnnotations and genomic locations for the microarray probe sets were extracted from Ensembl  using ve2  fold-change value was calculated. This treatment results in negative values for genes that are female-biased, positive values for genes that are male-biased, and values approaching 0 for genes with similar expression in both sexes. Genes were further divided into fold-change cutoff categories, based on the Wilcoxon test statistic of the log2 values, and adjusted for a 5% false discovery rate using the Benjamini Hochberg correction. Therefore, absolute value of log2 fold change > 1 corresponds to a 2× difference in expression in one sex compared to the other, absolute value of log2 fold change > 1.5 represents roughly a 3× increase, and absolute value of log2 fold change > 2 corresponds to a 4× difference. Relative expression was calculated across within-sex replicates for sex-biased  and unbiased gene classes for each tissue separately, and 95% confidence intervals were determined via bootstrapping (1000 repetitions).For each actively transcribed target, the log2 fold change > 1, padj > 0.05; female biased genes where log2 fold change < 1- and padj < 0.05; and unbiased probe sets), and computed 95% confidence intervals via bootstrapping (1000 replicates).In order to investigate the relationship between expression variance, an indicator of noise, and fold-change, we normalized the expression level for each gene target such that the averaged expression level was equal to one within each set of sex-specific replicates. This reduces the problem where genes with low expression levels will have associated small variance estimates, which could create a false signal. We then computed average within-sex variance for each sex-bias expression class . We examined the significance of these values by comparing them with datasets where gene order was randomized on each chromosome using 10000 replicates.For all genes that were significantly expressed in our study, we calculated the difference in average level of expression between it and its nearest neighbor for which gene expression had been measured in the same tissue. We used sex-averaged expression to correct for sex-bias effects. We also measured the difference in fold-change between neighboring gene pairs for which we had expression data, and counted the number of times gene pairs both had evidence for significant biased expression in the same sex . Within each tissue, we compared the GC3 and ENC among autosomal expression categories using one-way ANOVA (2 d.f.), and between Z-linked categories using a one-tailed t-test assuming unequal variance. As ENC is strongly governed by GC content in vertebrates, we also tested whether differences in ENC values between categories were significantly different from that predicted by the observed differences in GC3. To do this we fitted a smoothed cubic spline to the relationship between ENC and GC3, and used this to estimate predicted ENC values from GC3. Significance was tested performing a one-way ANOVA on the residuals of model fitting.Third codon GC (GC3) content has been linked to codon-bias ,47, whicosophila . Therefoosophila  from theGallus Gene Ontology (GO) [padj < 0.05 cutoffs to denote sex-bias). Significant over-representation was determined for each term with Fisher's exact test, and the Bonferroni correction was used for multiple comparisons. As the Bonferroni method can be unnecessarily over-conservative, we report over-represented terms that are significant before Bonferroni correction as well.We compared the functional role of biased and unbiased gene sets for each tissue using Ontologizer  and the ogy (GO)  databaseHE AND JEM conceived of the study. JEM, LH-R, and MW analyzed the data. All co-authors contributed to writing the manuscript.


OCT4 and NANOG genomic targets were identified in mouse embryonic stem cells by ChIP-chip and were compared with previously reported ChIP-PET results. Genome-wide approaches have begun to reveal the transcriptional networks responsible for pluripotency in embryonic stem (ES) cells. Chromatin Immunoprecipitation (ChIP) followed either by hybridization to a microarray platform (ChIP-chip) or by DNA sequencing (ChIP-PET), has identified binding targets of the ES cell transcription factors OCT4 and NANOG in humans and mice, respectively. These studies have provided an outline of the transcriptional framework involved in maintaining pluripotency. Recent evidence with comparing multiple technologies suggests that expanding these datasets using different platforms would be a useful resource for examining the mechanisms underlying pluripotency regulation.Oct4 or Nanog knockdown.We have now identified OCT4 and NANOG genomic targets in mouse ES cells by ChIP-chip and provided the means to compare these data with previously reported ChIP-PET results in mouse ES cells. We have mapped the sequences of OCT4 and NANOG binding events from each dataset to genomic coordinates, providing a valuable resource to facilitate a better understanding of the ES cell regulatory circuitry. Interestingly, although considerable differences are observed in OCT4 and NANOG occupancy as identified by each method, a substantial number of targets in both datasets are enriched for genes that have known roles in cell-fate specification and that are differentially expressed upon This study suggests that each dataset is a partial representation of the overall ES cell regulatory circuitry, and through integrating binding data obtained by ChIP-chip and ChIP-PET, the methods presented here provide a useful means for integrating datasets obtained by different techniques in the future. Oct4 knockout embryos and ES cells differentiate into trophectoderm, whereas over-expression of the gene leads to differentiation into primitive endoderm and mesoderm lineages  [Jarid2, Cdx2 and Sox2 have been identified previously as OCT4 or NANOG targets [Several lines of evidence indicated that this ChIP-chip dataset is of high quality . First, in accordance with previous findings in both mouse and human ES cells ,12, Gene targets ,12,15,16Given that it has been reported that a significant number of binding sites may be located outside of promoter regions , we nextIn order to compare genomic targets across different platforms, we re-analyzed previously reported ChIP-PET experimental data for OCT4 and NANOG . In the In order to analyze the ChIP-PET and ChIP-chip data together, all raw ChIP-PET sequence reads were re-mapped to the same version of the mouse genome (mm6) used in the ChIP-chip experiments. The sequence reads were between 34 and 36 bp, and only those that had at least 34 matched base pairs and a gap-length of 10 bp were considered to be uniquely mapped to the mouse mm6 genome. Out of 951,437 OCT4 reads, 198,802 (20.9%) could be uniquely mapped. Similarly, among 624,237 NANOG reads, 333,248 reads (53.4%) could be mapped uniquely to the genome. Importantly, the methods and criteria used to remap data to a different genome version will provide a useful resource for performing such analyses with other sequencing based platforms that use other genome versions.In ChIP-PET experiments, a minimum number of overlapping sequence reads was used as a criterion for identifying binding events. A region was considered occupied by OCT4 and NANOG if it had at least four or three overlapping sequence reads, respectively. In order to analyze our ChIP-chip findings in relation to these data, we examined only those ChIP-PET reads that had corresponding regions represented on the mouse promoter arrays (576 for OCT4 and 924 for NANOG). Additionally, for chromosome 19, 90 OCT4 targets and 224 NANOG targets could be remapped for the ChIP-PET data.To examine the binding events obtained by ChIP-chip and ChIP-PET, we used the 'Genomic Spatial Events' (GSE) Visualizer program  Figure  . We generated two types of recovery curves to analyze the ChIP-chip and ChIP-PET data. The ChIP-PET recovery curve examined the fraction of ChIP-PET regions overlapping with the ChIP-chip data at a wide range of p-value thresholds for the ChIP-chip experiments. In this instance, the threshold criteria were kept constant for the ChIP-PET experiments, and the ChIP-PET recovery (y-axis) was plotted against a range of ChIP-chip p-values (x-axis). Conversely, the other type of curve represented the ChIP-chip recovery at varying ChIP-PET 'overlapping read' threshold values. The ChIP-chip p-value threshold was kept constant at 0.001, and the ChIP-chip recovery (y-axis) was examined at different numbers of ChIP-PET sequence reads (x-axis). We examined each type of curve under a range of recovery distances, as binding events identified by both methods may not have exact overlaps due to differences in sample processing and technologies.In order to determine how the analysis methods and threshold criteria in ChIP-chip and Chip-PET experiments influenced the overall concordance between datasets, we examined the data by generating 'recovery curves'  within a distance of 1 kb. Conversely, using the same thresholds, 9.3% of the OCT4-bound peaks found by ChIP-chip were recovered in the ChIP-PET data . From the NANOG data we observed that 28.1% of the ChIP-PET peaks (>3 reads) were recovered in ChIP-chip bound regions  within a 1 kb area. Conversely, the ChIP-chip percent recovery from ChIP-PET bound regions (>3 reads) was 19.5% . Therefore, these recovery curves illustrate the importance of recovery distance and threshold calibration in examining data from different sources.Not surprisingly, we observed that the recoveries of OCT4 and NANOG targets obtained by one experimental method increased as the threshold value for the other method was relaxed. The recoveries also increased as the distance permitted between a ChIP-chip peak and corresponding ChIP-PET peak was increased  upon Oct4 knockdown. These results suggest that both NANOG and OCT4 can potentially activate or repress their binding targets. Therefore, these analyses have revealed a higher-value set of OCT4- and NANOG-regulated genes, by collectively examining the targets identified by ChIP-chip and ChIP-PET.Since protein-DNA binding alone is not indicative of a regulatory event, the expression of OCT4 and NANOG binding targets obtained through ChIP-chip and ChIP-PET was compared by comparing binding data with previously published OCT4 and NANOG RNA interference (RNAi) gene expression profiles in ES cells . The expOct4 RNAi-mediated knockdown, certain genes, including Sox2 and Rif1, which have important roles in development, were bound by OCT4 in both ChIP-chip and ChIP-PET experiments. However, other genes that play a part in cell-fate determination, such as Gdf3 and Notch4 [Yap1 and Foxd3, which have been shown to have developmentally important roles [Nanog RNAi knockdown data, which showed changes in expression of target genes identified both exclusively and collectively by the two technologies. Therefore, combining the binding data obtained by both techniques, along with gene expression data, has provided a more detailed overview of the factors involved in the ES cell transcriptional circuitry. Further genetic studies of these regions will lend deeper insight into the mechanisms governing ES cell biology.The functional relevance of the ChIP-chip and ChIP-PET data, as examined by GO analyses, had revealed that the OCT4 and NANOG bound regions were significantly enriched for transcriptional and developmentally important regulators of gene expression. Similar observations had been made earlier for these factors in human ES cells as well . For insd Notch4 ,19, werent roles ,21, wereChIP-based technologies are being used extensively in identifying protein-DNA interaction networks in a variety of cell types and a number of varying conditions. In particular, ChIP-PET and ChIP-chip have been used to identify the mouse and human ES cell transcriptional circuitries, which are largely regulated by the key pluripotency factors OCT4 and NANOG. Although each ChIP-based technology used in the identification of these networks has its distinct advantages, we find substantial differences in the data derived through these different experimental methods. Recent technological comparisons have shown differences in the results obtained by these methods, and illustrated the need to use these data in a complementary manner . We havep-value threshold was made more stringent. A similar trend was observed for the ChIP-chip recovery when the ChIP-PET read stringency was increased. Additionally, at the same thresholds, this overlap decreased when the recovery distance permitted between a ChIP-chip peak and ChIP-PET peak was narrowed. Therefore, these recovery curves revealed the necessity of recovery distance calibration in examining binding experiments from multiple sources. Interestingly, we also observed that the amount of recovery between ChIP-chip and ChIP-PET data increased when the whole chromosome arrays were used. Therefore, the criteria used to determine a binding event, as well as the extent of genome coverage, had an effect on the overlap between the data obtained by the two methods. The recovery curves illuminated the sensitivity of recovery to distance threshold, and provided a useful means to examine the datasets relative to each other.We analyzed our ChIP-chip results for OCT4 and NANOG in relation to existing ChIP-PET data. Since the criteria for identification of genomic targets is different between platforms, the datasets obtained by the two methods were examined against each other under an exhaustive range of significance values. Recovery curves were used to measure the recovery of targets obtained by keeping the binding threshold for one technique constant and varying the threshold values for the other method. As expected, for both OCT4 and NANOG targets, the ChIP-PET recovery decreased as the ChIP-chip Oct4 and Nanog RNAi expression profiling data in order to analyze the targets that are differentially regulated upon Oct4 or Nanog knockdown in ES cells. OCT4- and NANOG-bound regions uncovered by both technologies, as well as the ones obtained exclusively by each method, contained a number of differentially regulated genes. Many of these genes encode transcription factors and regulators of gene expression, which are important in development. For instance, the expanded OCT4 and NANOG regulatory network contained genes such as Hoxa1, Foxd3, Msx2 and Hexb, which showed changes in expression upon Oct4 or Nanog knockdown. These genes have been shown to be important in cell fate specification, and are involved in developmentally important signaling pathways. Such additional targets identified by each technique can be used to expand the ES cell transcriptional regulatory framework, and thereby provide more detailed groundwork to understand pluripotency mechanisms. Further genetic manipulations of each of these genes in ES cells would be necessary to independently validate their contributions to pluripotency.We combined the protein-DNA binding data with known Although both ChIP-chip and ChIP-PET technologies have been useful in studying protein-DNA interactions on a genome-wide scale, each method has its set of limitations. In ChIP-chip, our observations are restricted to regions tiled on the array platform, and the resolution is limited by the size of the probes, their spatial distribution, as well as the average fragment length of sonicated DNA hybridized to the arrays. In ChIP-PET experiments, the bacterial cloning and sequencing steps, as well as mapping issues, introduce scope for error. We feel that a combination of more stringent mapping criteria and the inherent noise in the sequencing procedure may be responsible for the number of sequence reads that did not match perfectly to the genome. Moreover, as indicated by our sequence-depth analysis, the number of sequences obtained from ChIP-PET experiments can be a limiting factor, since more binding targets can be recovered through greater depth in sequencing. Additionally, as in the case of ChIP-chip experiments, the resolution of binding is limited by the average DNA fragment size used in the ChIP experiment. We observed some of these limitations in this study since there was a significant number of OCT4 and NANOG targets that had been identified by ChIP-PET, and did not have corresponding probes tiled on the arrays used in the ChIP-chip experiments. Apart from these limitations, it is also important to consider that binding sites may be differentially occupied at different times in the cell cycle since the chromatin state changes at different times . HoweverApart from ChIP-chip and ChIP-PET, other ChIP based methodologies, such as ChIP-SACO   and STAGThe results of this study provide a useful way to integrate protein-DNA interaction data that are obtained by different techniques. We have used this to expand our current knowledge of the mouse ES cell regulatory network that is orchestrated by the transcription factors OCT4 and NANOG. Although both the ChIP-chip and ChIP-PET technology platforms identified different sets of binding events, a considerable number of these events represented genes that were regulated by OCT4 and NANOG. Since a number of these genes have known roles in important developmental pathways and in cell-fate specification, it will be interesting to explore their biological roles with respect to ES cell pluripotency. Therefore, this expanded network provides a stronger foundation to further examine biochemical and genetic interactions that regulate stem cell properties. Moreover, the methods described to compare datasets from different platforms would be very useful as data from newer technologies, such as Chip-Seq, become available. Since ES cells are a model system for studying developmental processes, and are thought to hold great promise in regenerative medicine, it will be important to gain a thorough understanding of the means by which a stem cell maintains its identity, and how it can be directed to form different cell types. Our work will allow for a more detailed examination of the components of this expanded stem cell circuitry and will lend better insight into the mechanisms of pluripotency.2 at 37°C on gelatinized tissue-culture plates. They were grown in DMEM  with 15% fetal bovine serum , Leukemia inhibitory factor, 1% penicillin/streptomycin , 1% L-glutamine , 1% non-essential amino acids  [V6.5 murine ES cells  were grown at 5% CO140-050)  were alsFor ChIP experiments, we used anti-NANOG  and anti-OCT4 , which have been previously characterized for immunoprecipitation.8 ES cells were grown at 70-80% confluency. The cells were cross-linked by adding fresh 11% formaldehyde solution to the ES media for 10 minutes at room temperature. The cells were washed twice with 1× phosphate-buffered saline and scraped off the plates, pelleted and stored at -80°C. They were then lysed and sonicated to solubilize chromatin and shear the cross-linked DNA. Sonications were performed with a Misonix Sonicator 3000 and sonicated at power 7 for 12 × 30 second pulses (60 second pause between pulses) at 4°C while the samples were immersed in a water bath. The whole cell extract was incubated overnight on a rotating platform at 4°C with 100 μl of Dyna1 Protein G magnetic beads, blocked with 0.05% bovine serum albumin/phosphate-buffered saline and pre-incubated for 6 hours to overnight with 10 μg of antibody of choice. The beads were washed five times with radio immunoprecipitation assay (RIPA) buffer and once with TE containing 50 mM NaCl. Bound protein-DNA complexes were eluted off the beads in elution buffer by occasional vortexing and heating at 65°C overnight. Whole cell extract (saved from the sonication step) was treated similarly for cross-link reversal. Following treatment with RNaseA and Proteinase K, the immunoprecipitated and whole cell extract DNA was purified by phenol:chloroform:isoamyl alcohol extraction. The DNA was blunted-end ligated to a universal linker and amplified using a two-stage expansion PCR protocol (three reactions per sample were done for the second expansion and combined). The amplified immunoprecipitated DNA and whole cell extract DNA were labeled with Cy5 and Cy3 fluorophores, respectively, using Invitrogen random primer labeling kits . DNA (1 μg) was used in a labeling reaction and three labeling reactions were done per sample and combined after purification. The labeled DNA was purified using Invitrogen BioPrime Array CGH module purification kit. Five micrograms each of immunoprecipitated and whole cell extract DNA was combined along with mouse Cot-1 DNA and hybridized to each of the arrays in Agilent hybridization chambers for 40 hours at 65°C using the Agilent hybridization protocol and reagents for 244 K arrays . Arrays were then washed and scanned as previously described. Three biological replicates were done for each transcription factor in order to determine statistical significance for binding targets.The ChIP protocol was similar to previously published studies ,28-30. Bp-value was below a pre-determined cutoff . For each bound probe, a region around the probe was marked 'bound' extending to the nearest unbound probe . A region was marked as a 'peak' if it was bound in all three replicates.Three biological replicates were examined for both OCT4 and NANOG using mouse promoter arrays. The same samples were also hybridized to mouse chromosome arrays. A probe was marked 'bound' in a particular replicate if its Rosetta The list of micro-RNAs to be analyzed was taken from version 8.0 of miRBase , which wTwo sets of previously published ChIP-PET experiments for OCT4 and NANOG were examined. These reads were mapped to the Mouse mm6 genome sequence in order to be consistent with the genome version used for the arrays in the ChIP-chip experiments. The mapping was done using the BLAT sequence alignment tool, with a step size of 5 and tile size of 11.We received two FASTA files of sequences, each between 34 and 36 bp in length. Only those sequence hits reported by BLAT that had at least 34 matched bases , and a gap-length of at least 10 bp, were retained. Any sequences with more than one retained hit to the mm6 genome were filtered out. The locations of the unique hits for the sequences that remained were stored in a database, and used for comparison with the ChIP-chip peaks.In order to examine if a bound region from ChIP-chip was 'matched' to another bound region from ChIP-PET, and vice versa, a simple genomic distance threshold was determined. If the two regions were on the same chromosome, and if the edge of one region was within the recovery distance from the edge of the other, then they were considered 'matched'. The typical recovery distance used was 1 kb, although this was varied systematically for the recovery curves. A recovery distance of '0' represents only strictly overlapping regions that were matched to each other.p-value) cutoff, and the other for the ChIP-PET (overlapping region) cutoff. In the ChIP-PET recovery curves, fixed cutoffs of four overlapping reads for Oct4 and three overlapping reads for Nanog were used to determine a 'background set' of ChIP-PET bound regions, as described above. At that ChIP-PET cut-off, the number of ChIP-chip bound regions was calculated for a range of p-value cut-offs. The percentage of background ChIP-PET bound regions that were matched by at least one ChIP-chip bound region, at each p-value threshold, was graphed. This was repeated for several different matching distances.Recovery curves were produced for promoter and chromosome array data for OCT4 and NANOG. One curve was for the ChIP-chip . This allowed us to divide the set of differentially regulated gene annotations into four categories: bound in both experimental types, bound in neither, and bound in one (but not the other).We wanted to determine the relevance to pluripotency for OCT4 and NANOG binding targets obtained by ChIP-chip or ChIP-PET. Previously published analysis . These ep-value < 0.001 for ChIP-chip, and a maximum distance of 4 kb from the gene start site for any annotated gene). The sets of RefSeq identifiers were converted into sets of 'known gene' gene symbols, using the kgXref table of the UCSC Genome database [p-value of the hypergeometric probability for the overlap of each test set with each GO category.A set of bound RefSeq gene identifiers was compiled for the OCT4 and NANOG bound genes according to a standard cutoff  the two-slide set promoter arrays and (b) chromosome 19 arrays.Click here for fileImmunoprecipitate versus whole cell extract enrichment in three biological replicate samples is shown for (a) the two-slide set promoter arrays and (b) chromosome 19 arrays.Click here for filePlots indicate the number of ChIP-chip targets recovered (y-axis) when different percentages of ChIP-PET sequences are randomly sampled. Error bars represent the standard deviation.Click here for fileThe binding of NANOG at the REST gene promoter is identified by both ChIP-chip and ChIP-PET. However, the binding event detected in the ChIP-PET experiment is not within the region used for combining the expression profiling information (± 4 kb around the transcription start site). Consequently, the changed expression of REST after NANOG knockdown is associated with NANOG binding detected by ChIP-chip only and not ChIP-PET.Click here for fileProtein coding gene promoter regions that were bound by OCT4 and NANOG in ChIP-chip and ChIP-PET experiments.Click here for fileMicro-RNA gene promoter regions that were bound by OCT4 and NANOG in ChIP-chip experiments.Click here for fileGO analyses for OCT4 and NANOG bound regions in ChIP-chip and ChIP-PET experiments.Click here for fileChromosome 19 regions that are bound by OCT4 and NANOG in ChIP-chip experiments.Click here for fileOCT4 ChIP-chip and ChIP-PET recovery curve values for promoter and chromosome array experiments.Click here for fileNANOG ChIP-chip and ChIP-PET recovery curve values for promoter and chromosome array experiments.Click here for fileOct4 knockdown.OCT4 bound genes that are differentially expressed upon Click here for fileNanog knockdown.NANOG bound genes that are differentially expressed upon Click here for fileGene-specific PCR binding data for OCT4 and NANOG.Click here for fileSequence depth analysis for OCT4 and NANOG bound regions on chromosome 19.Click here for fileSupplementary information on additional methods and materials and results.Click here for fileSummary of promoter array ChIP-chip binding data and corresponding ChIP-PET binding data for OCT4 and NANOG.Click here for fileSummary of chromosome array ChIP-chip binding data and corresponding ChIP-PET binding data for OCT4 and NANOG.Click here for file