When transplanted into infarcted mouse hearts, CPCs engrafted long-term in the infarct zone and surrounding myocardium without causing teratomas or arrhythmias. The grafted cells differentiated into cross-striated cardiomyocytes forming gap junctions with the host cells, while also contributing to neovascularization. Serial echocardiography and pressure-volume catheterization demonstrated attenuated ventricular dilatation and preserved left ventricular fractional shortening, systolic and diastolic function. Our results demonstrate that CPCs can engraft, differentiate, and preserve the functional output of the infarcted heart.Stem cell transplantation holds great promise for the treatment of myocardial infarction injury. We recently described the embryonic stem cell-derived cardiac progenitor cells (CPCs) capable of differentiating into cardiomyocytes, vascular endothelium, and smooth muscle. In this study, we hypothesized that transplanted CPCs will preserve function of the infarcted heart by participating in both muscle replacement and neovascularization. Differentiated CPCs formed functional electromechanical junctions with cardiomyocytes  Cardiomyocyte loss as a result of myocardial infarction (MI) injury is considered irreversible with the heart lacking sufficient capacity for self-regeneration. Cell-based cardiac therapies are proposed as an attractive therapeutic alternative to reverse cardiomyocyte loss, repair the injured myocardium and ultimately prevent heart failure. To date, a variety of cell sources, both of adult and embryonic origin, have been investigated for use in heart repair with mixed outcomes Brachyury/Flk1Isl1/Flk1/Nkx2-5cKit/Nkx2-5Nkx2-5We and others have recently described the identification, isolation, and characterization of the novel mouse ESC-derived cardiac progenitor cells (CPCs) on the basis of D3Rosa26Nkx2-5 expression on differentiation day 5  indicated the formation of nascent mesoderm. By day 7, the CPCs underwent differentiation-commitment into cardiac cell lineages including cardiomyocytes as indicated by the initiation of Mhc6 expression along with the detection of spontaneously contracting cell clusters.Temporal quantitative RT-PCR analysis performed on differentiating ESCs indicated a time period (days 5–6) during which the CPCs were present but not yet committed into specific cell lineages Fig. 1g.(+) CPCs decreased temporally between days 5 and 10  progenitor population, the cells were reaggregated and the cell clusters were induced to differentiate for 7–30 days. Several foci of GFP(+) spontaneously contracting cardiomyocytes (α-Actinin expression) were detected in culture . Gap junctions indicated by punctuate Connexin 43 localization at the site of cell-cell contacts were detected in the differentiated cardiomyocytes .Optical mapping in all co-cultures revealed spontaneous electrical activity at a relatively low rate (1.2±0.3 Hz) that originated in the CPC islands, suggesting the existence of pacemaking cells amongst the CPC-differentiated cardiomyocytes. Consistent with positive staining for Connexin 43, both spontaneous and pacing-induced action potentials (2 Hz rate) within the CPC island propagated outwards into the surrounding NRVM area Fig. 2e.in vivo gap junctions, and ultimately improve the functional output of the injured hearts. To determine the extent of injury and scar tissue formation in the infarcted hearts, we performed Mason's trichrome staining on thin heart sections that allowed us to distinguish healthy muscle tissue (red) from scar tissue (blue)  Fig. 3. β-Galactosidase, which is constitutively expressed in the Rosa26-derived CPCs  cells were detected within the infarcted region in an area comprising a thin muscle band underneath the scar tissue  cells was detected and summed in multiple sections both within and in the surrounding area of the myocardial infarction region. The survival rate was calculated by extrapolating the number of βGal(+) cells over the entire estimated area of the affected myocardium (≈4 mm) and determined to be 12.7±2.1% of the initial total number of cells injected.Anti β-Galactosidase and α-Actinin  CPCs readily differentiated into vascular smooth muscle and incorporated in large and small vessels with βGal(-) host cells  uncommitted progenitors were also detected. Taken together, ESC-derived CPCs committed predominantly to the cardiac muscle cell fate both when transplanted in the infarcted heart and when densely seeded in co-culture with rat cardiomyocytes in addition to inducing vascularization.In the infarcted region of animals which received CPC injections several blood vessels comprised of both donor and host cells were detected  cardiomyocytes containing a single nucleus. In our analysis approximately 96% of the identified cells contained a single nucleus indicating that at least that number of cells were the result of direct differentiation versus cell fusion.Since cardiomyocytes in the adult mouse myocardium are binucleated whereas embryonic stem cell-derived cardiomyocytes are immature and primarily uninucleated in vivo with the host myocardium was examined by immunohistochemical analysis  cardiomyocytes which also exhibited Connexin 43 gap-junction staining were detected at the site of the infarct (The capacity of the differentiated CPCs to electrocouple in vitro ((+) CPCs were co-cultured with NRVMs and subsequently assayed for GFP and N-Cadherin expression which mediates adhesion in the intercalated discs at the termini of cardiomyocytes thereby serving as a mechanical anchor for myofibrils at cell-cell contacts (+) cardiomyocytes, which were readily distinguishable from the NRVMs, formed N-Cadherin(+) intercalated disc cell contracts with the NRVMs.The capacity of the differentiated CPCs to form mechanical inter-connections with neonatal rat ventricular cardiomyocytes was assessed Myocardial infarction injury produced a time-dependent ventricular dilatation and left ventricular dysfunction Fig. 6. Over the span of 4 weeks the ejection fraction EF, Fig. 6c Fig. S2). Forty mice finished the 4 weeks of the study, of which 26 underwent successful hemodynamic analysis with a pressure-volume catheter.A total of 55 surgical procedures were performed with a surgical mortality of 10.9%  is a measure of the fastest pressure rise within the left ventricle during the isovolumetric phase of myocardial contraction and it can be correlated with the peak acceleration of blood in the descending aorta through the ventricle. The left ventricular peak pressure rise ((dP/dt)max) for the control group  was lower than that of the CPC-treated infracted animals (8427.3±1850.8 mmHg/sec), which in turn was comparable to the ((dP/dt)max) values measured for the saline-treated sham animals  but lower than those measured for the cell-treated sham animals .Hemodynamic catheterization also allowed the measurement of the indices of myocardial contractility Fig. 7b.The measurement of the relaxation time constant Fig. 7c,preload recruitable stroke work (PRSW) relationship, which describes the dependence of the stroke work on the end diastolic volume, demonstrated a preservation of the cardiac functional capacity in the cell-treated infarcted group Nkx2-5 CPCs and the relative ease to obtain large cell numbers represent major advantages for their potential use as a single therapeutic cell source. Importantly, since these CPCs are capable of generating both cardiomyocytes and vascular cells, they may obviate a need for use of additional vascular cell types A significant aspect of the study is the utilization of a novel cell progenitor: ESC-derived CPCs selected based on the activity of a cardiac specific Nanog or Pou5f1 was detected following (+)Nkx2-5 CPC derivation and extended culture in vitro. In contrast, colonies of undifferentiated ESCs and high expression levels of Nanog and Pou5f1 were found in cultures of the sorted GFP(−) cells One of the major concerns in using ESCs or their differentiated progeny for cell-based interventions is the risk of neoplastic tumor or teratoma A major drawback of any allogeneic transplantation therapy is the immune response which leads to the rejection of transplanted tissue. In our study we successfully utilized animals with common genetic background to the donor cells in vitro. This low conduction velocity was consistent with observed sparse Connexin 43 expression and possibly contributed by immature  electrophysiological phenotype of CPC-derived cardiomyocytes in vivo integration. In support, although no systematic studies were done to assess the arrhythmogenic risk of implanted cells, we observed no difference in the incidence of sudden cardiac death between cell-treated and untreated mice. Moreover, we demonstrate in vivo electrical coupling through gap junction formation of the differentiated CPCs with the host myocardium.The success of any cell-based therapy for myocardial infarction injury is ultimately judged by the improvement of the heart function, i.e., decreased heart dilatation and increased cardiac contractility and output. Based on the optical mapping of electrical propagation in co-cultures of CPCs and primary ventricular cardiomyocytes, the observed improvement in cardiac mechanical indices in treated hearts could be attributed in part to the functional integration of CPC-derived cardiomyocytes with the host cells. Of note, however, is the relatively low velocity of impulse propagation measured within the CPC islands in vivo and in vitro microenvironments may all significantly affect their lineage commitment. The systematic studies to understand the factors that control lineage commitment are currently underway.It is interesting that CPCs plated at high density within neonatal rat ventricular myocytes primarily differentiated into the cardiomyocyte lineage, while abundant smooth muscle cells were found within the sparsely plated islands βGal(+) cardiomyocytes (α-Actinin(+) or MYH6(+)) in the host myocardium suggesting the direct differentiation of the delivered CPCs towards the cardiac muscle lineage. Admittedly there is a possibility that some of the detected differentiation events are the result of cell fusion between the host cardiomyocytes and the CPCs. Recently Kolossov et al. examined the capacity of both bone marrow derived cells and embryonic stem cell derived cardiomyocytes to engraft into the infracted mouse myocardium (+) cells. Although these cells expressed cardiac markers as determined by immunocytochemistry they were small in size and resembled immature cardiomyocytes. Most importantly, one month, post injection 96% of βGal(+) cells contained a single nucleus suggesting their direct differentiation into cardiomyocytes versus their fusion with the host cells.We detected a number of Importantly, through the systematic assessment of cardiac contractile function, we demonstrated a superior systolic and diastolic performance of the CPC-treated relative to untreated hearts. In particular, while the function of untreated hearts was significantly compromised as early as one week after the infarction (based on echocardiographic parameters), the functional indices in CPC-treated hearts remained relatively constant. The preserved cardiac function was independently confirmed at 4 weeks post-infarction using pressure-volume analysis. Mechanistically, the cardiac function was maintained not only due to attenuated ventricular remodeling including a decrease of scar size and relatively preserved ventricular volume but more importantly due to independently preserved contractile function as evidenced from a higher PRSW slope .in vivo cardiac improvement following myocardial infarction Vegf, Igf1, and Notch signaling pathways are active in the cells both of which have been shown to have pro-vascular and cardioprotective effects in vivo functional integration of the donor cells with the host myocardium we plan to utilize genetic calcium indicators and two-photon laser scanning microscopy These findings are consistent with previous reports in demonstrating that transplantation of ESC-derived cells leads to the An additional important determinant of a successful long-term cell-based cardiac therapy involves the level of cell survival following direct intramyocardial delivery. Previous studies have demonstrated a clear correlation between graft size and the level of functional improvement The recent identification and characterization of adult File S1.Mouse ESCs D3  lines weThe previously described (+) CPCs were re-aggregated for 24 hours in suspension and re-plated on fibronectin coated tissue culture dishes and further maintained for seven days in ESC differentiation medium.To assay their differentiation capacity, sorted GFPEmbryoid bodies cultured in hanging droplets or in suspension were harvested every 24 hours in order to isolate total RNA. For the quantitative RT-PCR analysis total RNA (2 µg) was reverse transcribed using random decamers as primers , and the MMLV-RT enzyme . Relative RNA expression levels were measured using quantitative RTPCR. Detection of the described markers was achieved with FAM/MGB probes (Applied Biosystems): Nkx2.5 (Mm00657783_m1), Myh6 (Mm00440354_m1), Brachyury (Mm00436877_m1). The instrument used was the Applied Biosystems ABI Prism 7900HT sequence detection system and the software for data collection and analysis was the SDS2.1. An 18S RNA probe (Hs99999901_s1) was used in order to normalize the data.4 cells, high density: 2.5×105 cells). Following CPC attachment floating cells were washed off and the PDMS ring was removed. Isolated NRVMs were subsequently plated around the CPCs. Washing of unattached NRVMs within 6–8 hours following plating ensured minimum NRVM attachment on top of the CPCs.Fibronectin-coated coverslips and polydimethylsiloxane (PDMS) rings were used to selectively plate isolated CPCs only in the central region (5 mm diameter) of the coverslips  providing a spatial resolution of 750 µm in a 19.5 mm field of view, as previously described Following optical mapping analysis the samples were fixed in 4% parafolmaldehyde and stained with antibodies against the GFP protein , α-Actinin , Acta2 , and Connexin 43 . Secondary antibodies were purchased from Molecular probes and were conjugates to the fluorochromes Alexa 488, and Alexa 546. Nuclei were stained with 4′,6-diamidino-2-phenylindole (Molecular probes). The samples were imaged with a Nikon TE2000 and a confocal Zeiss 510 microscope.6 cells total) in the boundaries and the center of the infarcted region. The cells used in these experiments were GFP+ CPCs isolated on differentiation day 6. The animals were sacrificed four weeks post-transplantation and the hearts prepared for immunohistological analysis.All research involving experimentation on vertebrate animals have been specifically approved by Johns Hopkins Medical Institutions . Using a left anterior thoracotomy, the heart of SVE129 female mice (8–10 weeks of age) was exposed and LAD was ligated permanently. For sham surgeries, the needle was passed under the LAD without ligation. The mice were monitored twice a day for four weeks. Four experimental groups were established consisting of animals that underwent LAD ligation and received phosphate buffered saline injections with (n = 15) or without cells (n = 10) and animals that underwent sham surgeries and also received saline injections with (n = 7) or without cells (n = 11). Immediately following infarction injury, each animal received three injections (10 µl/injection) of saline or saline with CPCs (0.5–1×105 cells in three doses (10 µl/injection) directly into myocardium with a 30-gauge needle. Injections were done immediately following LAD ligation, into the mid infarct zone and border-zones. The infarct zone was defined by blanching of the heart after ligation.Using a tubing system attached to a standing syringe, we injected 4–5×10The analysis was done using a HP/PHILIPS/AGILENT SONOS 5500. The animals were analyzed prior to surgery (same day) and at 1, 2, and 4 weeks after it. The procedures were performed under general anesthesia using isoflurane 1–2%.4 weeks after the surgery the mice were anesthetized using a combination of etomidate, morphine and urethane, which were injected intraperitoneally. Using the right carotid approach a Millar SPS 839 catheter progressed to the left ventricle. During the procedure, a diluted 6.25% albumin solution was infused into jugular vein at a rate of 5 mcl/min. Pressure volume data were obtained at baseline with closed chest and after opening the thorax with IVC occlusion. The volume calibration was done using thoracic aortic flow and hypertonic saline infusion.Graph-pad prism 4.03).We obtained average and standard error of mean for all values and used one way and two way ANOVAs for statistical analysis Click here for additional data file.Figure S1In vitro co-culture of ESC-derived CPCs with NRVMs. The CPCs were selectively plated at low density in the center region of the coverslip and subsequently differentiated into cardiomyocytes and smooth muscle cells. A confluent layer of NRVMs surrounded the CPCs. Dashed lines denote the border between the two cell types. Mouse ESC-derived cardiomyocytes as well as NRVMs stain positive for α-Actinin (red), whereas only mouse ESC-derived CPCs and mouse ESC-derived cardiomyocytes express GFP (green). (c–d) High magnification immunostaining analysis illustrates the interface of the two cell types demonstrating formation of Connexin 43 gap junctions. (e) The majority of CPCs plated at low density differentiated into smooth muscle cells  with some cardiomyocyte differentiation . (f) Sequential isopotential maps reveal electrical propagation from the central island of differentiated CPCs to the outside regions containing primary cardiomyocytes . Color bar shows relative transmembrane potential normalized from resting (blue) to peak (red) value. The monolayer was electrically stimulated by a point electrode in the center, indicated by the yellow pulse sign in 0 ms frame. The timing index has been set relative to the stimulus pulse . Membrane potential traces are from three channels . Note that electrical propagation was very slow inside the central island due to predominance of CPC-derived smooth muscle cells and significantly accelerated after exiting to the outside cardiomyocyte region.Cardiomyocyte and smooth muscle lineage committed mouse ESC-derived CPCs functionally electrocouple with neonatal rat ventricular cardiomyocytes. (a–b) (3.21 MB TIF)Click here for additional data file.Figure S2Kaplan-Meyer survival graph showing the four study arms during the four weeks after myocardial infarction. Logrank analysis for survival demonstrated no significance in the number of animal deaths in the four experimental groups.(1.25 MB TIF)Click here for additional data file.


Using a double-blind randomized crossover design, each subject performed two isokinetic tests separated by a period of at least 6 weeks: once with placebo, and once with piroxicam (Feldene®). They were given one capsule containing either placebo or piroxicam (20 mg) per day for 6 days with initial doses given starting 3 days prior to isokinetic testing. Exercise consisted of eight stages of five maximal contractions of the knee extensor and flexor muscle groups of both legs separated by 1 min rest phases, on a Kin Trex device at 60°/s angular velocity. The subjective presence and intensity of DOMS were evaluated using a visual analogue scale immediately after, and 24 and 48 h after each test. The mean plasma concentration of PGE2 measured at rest and after exercise was significantly lower in the group treated with piroxicam (p < 0.05). However, statistical analysis (two-way ANOVA test) revealed that exercise did not cause any significant change of mean plasma PGE2 over time in either of the two groups. Eccentric work was followed by severe muscle pain in extensor and flexor muscle groups. Maximal soreness was noted 48 h postexercise. Serum creatine kinase activity and the serum concentration of myoglobin increased significantly, and reached peak values 48 h after exercise in both experimental conditions (p < 0.001). By paired t-test, it appeared that there were no significant differences in the serum levels of these two markers of muscle damage between the two groups at any time point. We conclude that: (1) oral administration of piroxicam fails to reduce muscle damage and DOMS caused by strenuous eccentric exercise; and (2) the hypothetical role of increased PGE2 production in eccentric exercise-induced muscle damage, DOMS, and reduced isokinetic performance is not substantiated by the present results.To test the hypothesis that delayed onset muscular soreness (DOMS) following intense eccentric muscle contraction could be due to increased production of prostaglandin E


A three-dimensional (3D) weighted helical cone beam filteredbackprojection (CB-FBP) algorithm  has already been proposed to reconstructimages from the projection data acquired along a helicaltrajectory in angular ranges up to . However, anoverscan is usually employed in the clinic to reconstructtomographic images with superior noise characteristics at the mostchallenging anatomic structures, such as head and spine,extremity imaging, and CT angiography as well. To obtain the mostachievable noise characteristics or dose efficiency in a helicaloverscan, we extended the 3D weighted helical CB-FBP algorithm tohandle helical pitches that are smaller than 1: 1 . By decomposing ahelical over scan with an angular range of  into a union of full scans corresponding to an angularrange of , the extended 3D weighted function is asummation of all 3D weighting functions corresponding to each fullscan. An experimental evaluation shows that the extended 3Dweighted helical CB-FBP algorithm can improve noisecharacteristics or dose efficiency of the 3D weighted helicalCB-FBP algorithm at a helical pitch smaller than 1: 1, while itsreconstruction accuracy and computational efficiency aremaintained. It is believed that, such an efficient CBreconstruction algorithm that can provide superior noisecharacteristics or dose efficiency at low helical pitches may findits extensive applications in CT medical imaging.


B. melitensis infection, a protein microarray was fabricated containing 1,406 predicted B. melitensis proteins. The array was probed with sera from experimentally infected goats and naturally infected humans from an endemic region in Peru. The assay identified 18 antigens differentially recognized by infected and non-infected goats, and 13 serodiagnostic antigens that differentiate human patients proven to have acute brucellosis from syndromically similar patients. There were 31 cross-reactive antigens in healthy goats and 20 cross-reactive antigens in healthy humans. Only two of the serodiagnostic antigens and eight of the cross-reactive antigens overlap between humans and goats. Based on these results, a nitrocellulose line blot containing the human serodiagnostic antigens was fabricated and applied in a simple assay that validated the accuracy of the protein microarray results in the diagnosis of humans. These data demonstrate that an experimentally infected natural reservoir host produces a fundamentally different immune response than a naturally infected accidental human host.Brucellosis is a widespread zoonotic disease that is also a potential agent of bioterrorism. Current serological assays to diagnose human brucellosis in clinical settings are based on detection of agglutinating anti-LPS antibodies. To better understand the universe of antibody responses that develop after  Brucella melitensis proteins was used to study the antibody response of experimentally infected goats and naturally infected humans in B. melitensis infection. Goats recognized 18 proteins and humans recognized 13 proteins as serodiagnostic antigens; antibody detection of only two of these antigens was shared by goats and humans, suggesting either fundamentally different immune responses or different responses in relation to mode or setting of infection. The human serodiagnostic antigens were evaluated in a simple nitrocellulose line blot assay, which validated the protein microarray results. The approach described here will lead to the development of new diagnostics for brucellosis and other infectious diseases, and aid in understanding the human and animal host immune response to pathogenic organisms.Brucellosis is a bacterial disease transmitted from infected animals to humans. This disease often presents as a prolonged but non-specific illness primarily characterized as fever without specific organ localization. Because infections can result after ingestion  or inhalation  of small numbers of organisms, the bacteria that cause brucellosis are potential biological warfare agents. Here, a protein microarray containing 1406  Brucella melitensis, the most virulent species affecting humans, include goats and sheep Brucellosis is a zoonotic infectious disease endemic in regions around the world where agricultural, animal husbandry and vaccination practices have not controlled infection among livestock reservoirs Brucella abortus proteins recognized by cattle, mice and sheep and limited studies on human and goat recognition of Brucella melitensis antigens B. melitensis infection of natural reservoir host (goat) and accidental host (humans) is similar despite potentially different routes of infection. For this we constructed a protein microarray consisting of 1406 B. melitensis proteins and probed with a collection of sera from naturally infected and control human sera from Lima Peru, and goats experimentally infected with virulent B. melitensis 16M.The current knowledge of protein antigens recognized by humans and reservoir animals is limited to a relatively small number of immunogenic Human sera were obtained from patients enrolled in a prospective clinical study of brucellosis in Lima, Peru. The human subjects part of the study was approved by the Humans Research Protections Committee of the University of California San Diego, the Comite de Ética of Universidad Peruana Cayetano Heredia, Lima, Peru and the Comite de Ética of Asociación Benéfica PRISMA, Lima, Peru, all of whom have maintained federal wide assurances with the United States Department of Health and Human Services. All patients provided written informed consent prior to enrollment in the study, and signed consent forms have been stored in locked files in study offices at UPCH and AB PRISMA, Lima, Peru.Goat sera were obtained from previously stored samples from experimentally infected goats under Institutional Animal Care and Use protocols approved by Texas A&M University, College Station, Texas, USA. Animals were housed in an outdoor, restricted access, large-animal isolation facility operated under guidelines approved by the United States Department of Agriculture/Animal and Plant Health Inspection Service (USDA/APHIS). At the termination of the experiments, adult animals were euthanized by captive bolt. All animals were disposed of by University approved protocols.Brucella melitensis 16M genomic DNA were identified using GenBank NC_003317 and NC_003318, amplified using gene specific primers containing 33bp nucleotide extension complementary to ends of linearized pXT7 vector. Homologous recombination takes place between the PCR product and pXT7 vector in competent DH5a cells. The recombinant plasmids were isolated from this culture using QIAprep 96 Turbo kit (Qiagen). Around one quarter of the cloned genes were sequenced and verified that the correct sequence was inserted. The resulting fusion proteins also harbor a hemagglutinin epitope at 3′ end and polyhistidine at the 5′ end.Genes were amplified and cloned using high-throughput PCR and recombination method as described previously in vitro transcription/translation E. coli system (RTS 100 kits from Roche), according to the manufacturer's instructions. For microarrays, 15 µl of reaction was mixed with 5 µl 0.2% Tween 20 to give a final concentration of 0.05% Tween 20, and 15-µl volumes were transferred to 384-well plates and printed onto nitrocellulose coated glass FAST slides (Whatman) using Omni Grid 100 microarray printer (Genomic Solutions). Protein expression and printing was monitored by immunoprobing with anti-polyhistidine  and anti-hemagglutinin . For all array staining, sera samples were diluted to 1∶200 in Protein Array Blocking Buffer (Schleicher & Schuell). Slides were first blocked for 30 min in protein array-blocking buffer before incubation with primary antibody at 4°C overnight with agitation. The slides are then washed extensively and incubated in biotin-conjugated secondary antibody (Jackson Immuno Research) diluted 1/200 in blocking buffer. After washing, bound antibodies are detected by incubation with streptavidin-conjugated SureLight® P-3 (Columbia Biosciences). The slides are washed and air dried after brief centrifugation. Slides were scanned and analyzed using a Perkin Elmer ScanArray Express HT microarray scanner. Intensities are quantified using QuantArray software. All signal intensities are corrected for spot-specific background.Plasmids were expressed at 30°C in 5 hour- from B. melitenis culture-positive patients all of whom were positive by the Rose Bengal screening test and had tube agglutination tests > = 1/160; and 18 samples from culture negative, Rose Bengal serology-positive patients. These latter 18 samples were from culture negative individuals diagnosed with brucellosis and treated according to standard antibiotic therapy within 2 days of serum sampling. Additional control patient samples included 13 sera from Rose Bengal-negative patients, 44 samples from ambulatory healthy controls from north Lima where brucellosis occasionally affects patients, and sera from humans in the U.S. where brucellosis is not found.Human sera tested in this study were obtained from the following patient groups: patients confirmed (by positive blood culture) to have acute brucellosis in Lima, Peru; from culture-negative, Rose Bengal-positive patients presenting with brucellosis-compatible syndromes; Rose Bengal-negative patients referred by their physicians for possible brucellosis; and ambulatory, apparently healthy control patients from the north Lima neighborhood of Puente Piedra where brucellosis is known to occur with risk factors similar to those in the rest of Lima. No patients in this study were known to be directly exposed to goats; risk factors for all were reported to be ingestion of unpasteurized goat's milk products, the typical risk factor in Lima for acquisition of brucellosis. All patients included in this study had their first known episode of brucellosis, with clinical presentation within 1–3 weeks of onset of symptoms. The patient samples were as follows: 42 serum samples B. melitensis 16M-infected) and negative (uninfected) controls from a previously conducted vaccine safety study B. melitensis. Goats were experimentally infected with 1×107 CFU of Brucella melitensis strain 16M by bilateral conjunctival instillation at 110 days' gestation, and sera were collected 8 weeks after infection. As an additional negative control, 15 serum samples from a specific pathogen-free goat flock were obtained .Goat sera tested in this study were positive (in vitro transcription-translation reactions (RTS 100 E. coli HY Kit from Roche) according to the manufacturer's instructions. VIG was obtained from ADi as a gift, and the concentration of VIG was diluted to 0.05 mg/ml. Proteins were printed on Optitran BA-S 85 0.45 µm Nitrocellulose membrane (Whatman) using BioJet dispenser (BioDot) at 1 µl/cm, and cut into 3 mm strips. Individual strips were then blocked in 10% non fat dry milk dissolved in 10 mM Tris (pH 8.0) and 150 mM NaCl containing 0.05% (v/v) Tween 20 buffer for 30 minutes. Prior to immunostrip probing, forty two culture positive and forty four Peruvian naive sera were diluted to 1/250 in 10% non fat dry milk solution containing 20% E. coli lysate (McLab) and incubated for 30 minutes with constant mixing at room temperature. Each strip was then incubated with pretreated sera overnight at 4°C with gentle mixing. Strips were then washed five times in Tris buffer containing 0.05% (v/v) tween 20, and then incubated for 1 hour at room temperature in alkaline phosphatase conjugated donkey anti-human immunoglobulin , diluted to 1/5000 in tris buffer containing 0.05% (v/v) tween 20. Strips were then washed extensively and reactive bands were visualized by incubating with 1-step Nitro-Blue Tetrazolium Chloride/5-Bromo-4-Chloro-3′-Indolyphosphate p-Toluidine Salt (NBT/BCIP) developing buffer (Thermo Fisher Scientific) for 1 minute at room temperature. Strips were scanned with Hewlett-Packard scanner, and were quantified using Image J software.Thirteen plasmids of interest were expressed in five hour http://www.r-project.org). It has been noted in the literature that data derived from microarray platforms is heteroskedatic www.bioconductor.org) is applied to the quantified array intensities. In addition to removing heteroskedacity, this procedure corrects for non-specific noise effects by finding maximum likelihood shifting and scaling parameters for each array such that control probe variance is minimized. This calibration has been shown to be effective on a number of platforms All analysis was performed using the R statistical environment  genomic DNA and cloned using the high throughput recombination method previously described Bm ORFs cloned in pXT7 vector were expressed under T7 promoter in the E. coli in vitro transcription/translation system, and printed in duplicates on microarrays as described in A set of 1406 ORFs from Bm protein arrays were probed with sera from experimentally infected goats, naïve goats from the same pasture, and specific pathogen free (SPF) goats from a different location. Reactivity of sera from the individual goats is shown as a heat map with samples grouped according to their description  receiver operating characteristic (ROC) curves were generated for individual serodiagnostic antigens to assess the ability to separate the control and disease cases Fig. 4. B. melitensis positive sera.To test the feasibility of using the serodiagnostic antigens in an alternative analytical assay, thirteen serodiagnostic proteins were printed onto Nitrocellulose membranes using a BioDot jet dispenser. The paper was then cut into 3 mm strips Fig. 5a.The sensitivity and specificity of the top 5 serodiagnostic antigens discovered using the protein microarrays had sensitivity and specificity of 95% and 96%, better than that of the 13 antigens on the immunostrips (94% and 89%).Bm. In the present study, goats were infected by B. melitensis strain 16M which would be expected to be virtually identical to the strains infected by patients in Lima given the limited diversity of the strains found there Bm infection between humans and goats, we compared serodominant antigens for both humans and goats. In the current study, two antigens are found to be serodiagnostic for both humans and goats , identification of diagnostic protein antigens may facilitate the development of more specific serodiagnostic assays Serological diagnosis of both human and animal brucellosis can suffer from the inability to distinguish new from previous infection , have been found to be upregulated during intracellular infection by B. suis or B. abortusB. melitensis infection may be related to species differences between humans and goats but further comment on this possibility is limited by the lack of available data. Third, while humans in this study were thought to have been infected by ingestion of infected goat's milk products containing a small inoculum, the goats were infected with a high dose (107 CFU) of B. melitensis strain 16M via the conjunctiva. Both dose and route of inoculum may have contributed to the differential antigen recognition between goats and humans, and, in fact, among humans as well. Finally, while it seems unlikely that the different patterns of immune response variation between goats and humans were due to protein expression differences by different strains of B. melitensis, this possibility must be considered as well.The differences in the patterns of goat and human seroreactivity to B. melitensis 16M genome. Of these 1406 proteins, we only observed less than two fold enrichment of serodiagnostic antigens in the 1009 selected versus randomly selected antigens (not shown). Completion of the proteome array is currently underway, which will allow a more complete genome-level analysis of all immunogenic B. melitensis proteins. The subset of diagnostic antigens identified here provided an initial estimated accuracy rate of 95% for diagnosis of human cases and it is likely that this set of antigens will form the basis of a new and accurate serodiagnostic assay for human brucellosis. The clinical and veterinary utility of the protein antigens discovered in this study for diagnosis of acute and chronic brucellosis awaits validation in prospective studies in endemic regions.The results presented here represent an analysis of 1406 proteins of 3198 predicted proteins in the


Sir,The presence of CD4+CD25+ regulatory T cells (Treg) in cancer patients might be, in part, responsible for downregulation of antitumour immune responses. There are various inhibitory mechanisms in T cells to prevent pathological autoimmunity. Blocking CTLA4 on T cells or depleting CD25+ Treg is sufficient to break self tolerance, causing nonspecific autoimmune pathologies (As interferon alfa-2b has the ability to induce an immune response against autoantigens with serologic and clinical manifestations of autoimmunity, it is possible that it also induces an immune response against tumours. The appearance of autoantibodies or clinical manifestations of autoimmunity during treatment with high-dose adjuvant interferon alfa-2b was associated with statistically significant improvements in relapse-free survival and overall survival (By contrast, the early presence of autoantibodies in the serum has been described to correlate with an unfavourable prognosis (While it could be possible to use autoimmune responses as surrogate markers to evaluate new treatments (


GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively. Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date. The discovery of over 50 novel DNA methylation-based biomarkers of breast cancer may provide new routes for development of DNA methylation-based diagnostics and prognostics, as well as reveal epigenetically regulated mechanism involved in breast tumorigenesis.Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis. The inherent thermodynamic stability of cytosine methylation and the apparent high specificity of the alterations for disease may accelerate the development of powerful molecular diagnostics for cancer. We report a genome-wide analysis of DNA methylation alterations in breast cancer. The approach efficiently identified a large collection of novel differentially DNA methylated loci (∼200), a subset of which was independently validated across a panel of over 230 clinical samples. The differential cytosine methylation events were independent of patient age, tumor stage, estrogen receptor status or family history of breast cancer. The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the  Recent declines in breast cancer-associated mortality are partly attributed to the use of screening mammography, however, the benefit is significant only for women over 40 years of age BRCA1 and BRCA2BRIP1CHEK2ATMTP53Genetic mutations in In addition to genetic alterations, epigenetic abnormalities such as changes in genomic DNA cytosine methylation patterns are associated with all cancer types. The spectrum of alterations includes both gain and loss of DNA methylation involving multi-copy elements as well as single-copy genes  of the cytosine methylation dependent restriction enzyme McrBC and, therefore, is capable of determining the regional DNA methylation density associated with the plurality of molecules present. In the present study, the approach revealed numerous novel epigenetic biomarkers capable of distinguishing infiltrating ductal breast carcinomas from normal and benign breast tissues. A subset were extensively validated by screening a panel of over 230 clinical samples, revealing biomarkers that display clinical sensitivity and specificity up to 90% and 96%, respectively. Bisulphite sequencing analyses confirmed the DNA methylation changes and validated the qPCR-based assay adapted for high-throughput DNA methylation screening. In addition to identifying exceptionally promising biomarkers for improved disease detection, the functions of the associated genes suggest that the approach may also provide critical insights into molecular mechanisms of breast tumorigenesis.Genome-wide DNA methylation analysis at more than 21,000 loci was performed in nine infiltrating ductal breast carcinoma (IDC) and nine patient-matched adjacent histology normal samples. The IDC panel included six stage IIA and three stage IIB tumors from women ranging from 32 to 57 years of age (median age 47). Tumor and adjacent normal samples contained ≥65% and 0% neoplastic cellularity by H&E histology, respectively. Tumor samples included three IDCs positive for both estrogen and progesterone receptors and six IDCs negative for both receptors. Demographic information for these patients is provided in Following statistical analyses, 220 loci were identified that provided optimal distinction between tumor and adjacent normal DNA samples. As expected, unsupervised hierarchical clustering of the data derived from these microarray features divided samples into two major clusters . All nin2 ratios of DNA methylation measurements for 16 genes reported to be differentially methylated in breast cancer are depicted in A powerful novel discovery approach should recapitulate previous findings, as well as identify novel molecular abnormalities that offer advantages relative to what is known. The logAccuracy of the microarray-based DNA methylation measurements was assessed by a quantitative PCR (qPCR) i.e. <40% sensitivity). This observation has been made previously by Bestor and colleagues ). As shown in i.e. the percentage of tumors displaying intermediate to dense DNA methylation) and clinical specificity . Among loci displaying 100% specificity relative to normal breast tissue, sensitivities ranged from 6%  to 81%   and 25 normal or benign breast tissues. We focused on hypermethylation events because hypomethylation events were considerably less frequent than hypermethylation events  . Becaused; GHSR) . TherefoThe 16 differentially methylated regions that displayed greater than 95% specificity relative to normal and benign breast tissue and normal blood were analyzed in a larger validation panel, including a total of 103 IDC samples , 104 normal or benign breast samples and 25 peripheral blood samples from cancer-free women . Demograi.e. the percentage of Stage I tumors scoring as intermediately to densely methylated) versus the Stage II and III tumors. The directly proportional relationship between the two sensitivity calculations  indicates that the frequency of hypermethylation of these loci is similar regardless of tumor stage. Therefore, for the majority of loci, the differential methylation events are just as likely to be present in a Stage I tumor as they are in later stage tumors. The proportion of methylated molecules in tumors at each stage was then analyzed for three selected loci  analysis was performed for each of the 16 loci to determine optimal thresholds for calculation of sensitivity and specificity of the differential DNA methylation event. Examples of the primary qPCR data for four selected loci are shown in GHSR qPCR measurements for the 16 samples near the 0.64 dCt threshold (0.5–1.5). Three replicates were preformed using a different lot of McrBC. All 11 tumor samples again scored above the dCt threshold in all three replicates  involves an inherent level of technical variability. This variability could potentially have an impact on calculated sensitivity and specificity when ROC-based thresholds are close to the 0.5 cycle variability range of the qPCR platform. We addressed this issue first by performing technical replicates of all measurements, and excluding measurements with standard deviations ≥1.0. To further investigate the impact of this variability, we repeated the plicates . Three oGHSR alone. Models including one to four biomarkers were constructed and tested by determining the error rate of classifying tumor and normal samples in a leave-one-out cross-validation paradigm. As expected, methylation of GHSR was the optimal single biomarker, resulting in an overall average error rate of 8.3% . No two- or three-biomarker panel reduced this error rate. Six four-biomarker panels resulted in less than 1% reduction in error rate. Therefore, biomarker combinations did not result in a biologically significant increase in sensitivity and specificity relative to that of differential DNA methylation of the GHSR locus alone. Future experiments aimed at direct identification of epigenetic abnormalities associated with the minor class of tumors that were not hypermethylated at GHSR  may identify low frequency differential methylation events that, when combined with GHSR hypermethylation, lead to sensitivity approaching 100%.Other biomarkers displayed similar specificity, with decreasing sensitivity. Discriminant analysis was performed  to identify potential biomarker panels with greater sensitivity and specificity than methylation of GHSR, MGA, NFIX and the uncharacterized region corresponding to chr7-8256880 (UCSC hg.18(NCBI36)) for extensive bisulphite sequencing analysis . While tumor samples displayed higher DNA methylation load at MGA than normal breast and adjacent histology normal breast samples, the non-tumor tissues displayed higher baseline DNA methylation densities than at the other loci  of infiltrating ductal breast carcinomas, independently of patient age or tumor stage. Conversely, 4 of 103 cancer-free breast tissues reported a DNA methylation density measurement slightly above the ROC curve-established threshold (96% specificity). To our knowledge, this locus represents the highest combined sensitivity and specificity for a DNA-based (genetic or epigenetic) biomarker of breast cancer reported to date.Strikingly, the approach identified a single locus within the promoter region of the in situ to malignancy. Notably, we have confirmed the ability to detect tumor-associated hypermethylation of several loci in fine needle aspirate specimens collected from breast cancer patients. The frequency of detection of hypermethylation of these loci is similar regardless of whether the tested DNA is derived from primary tumors or from fine needle aspirates  for the circulating peptide hormone, ghrelin. A second transcript encodes a truncated form of the receptor (GHSR 1b), presumably via alternative splicing and polyadenylation within the single intron GHSR 1a mRNA expression in 3 of 4 cell lines tested (consistent with the frequency of GHSR hypermethylation reported here). Loss of GHSR 1a expression and overexpression of GHSR 1b has also been reported in adrenocortical carcinomas GHSR locus is involved in the switch between GHSR 1a and GHSR 1b isoform expression, potentially by directing a switch to alternative upstream transcription start sites, is a focus of future research. Furthermore, since GHSR 1b is reported to be an inactive isoform of the receptor In addition to revealing novel and powerful epigenetic biomarkers of breast cancer, our results provide insights into potential epigenetic mechanisms of breast tumorigenesis. As described above, hypermethylation of the promoter region of MGA gene, encoding a transcriptional repressor that intersects with the Myc pathway. Myc gene amplification occurs in approximately 15–20% of breast cancer patients myc occurs in up to 50% of tumors Max Gene Associated) also forms hetero-dimers with Max to form a transcriptional repressor co-complex that antagonizes the activity of Myc. MGA-Max complexes have been identified as part of the E2F6 repression complex that occupies and represses E2F- and Myc-responsive promoters at G0in vivo. The frequency of MGA hypermethylation in breast tumors suggests that it may serve to accentuate the activity of Myc, even in the absence of Myc amplification or overexpression. Future studies of the association between MGA hypermethyaltion, transcriptional repression and Myc amplification/overexpression may reveal directly cooperative mechanisms involving oncogene activation and epigenetic inactivation of negative regulators of oncogene activity.Another high-frequency hypermethylation event is associated with the GHSR . This region is located on chromosome 7, from approximate nucleotide 8449665 to 8450724 ), and includes an annotated CpG island. The region is devoid of confirmed genes within approximately 0.2 Mb upstream or downstream of this sequence, however the sequence falls within the 3′ regions of two juxtaposed predicted transcripts  or provided through North Glasgow NHS Trust. Whole blood samples were obtained from Research Blood Components. All samples were collected with appropriate institute ethics approval and written consent was provided by each patient. Demographic and source information for patients included in this study is provided in DNA samples were fractionated based on DNA methylation density as previously described The OGHAv1.0 microarray has been previously described TH2B gene, which is densely methylated in all tissues except the testes TH2B delta Ct≥4.0 (>90% depletion of the amplified region in the McrBC-digested portion relative to the mock-treated portion). The percent TH2B depletion for tumor, normal or benign breast tissue and blood samples did not differ significantly . For each McrBC-digested/mock-treated reaction pair, a delta Ct (Ct McrBC–Ct Mock) was calculated. Duplicate delta Ct values were averaged, and the standard deviations between delta Ct values were determined. All reported average delta Ct values had standard deviations less than 1 cycle.PCR primers were designed to amplify approximately 400–600 bp amplicons within a 1 Kb sequence window spanning the sequence represented by the associated microarray feature. Primer selection was guided by uniqueness of the oligonucleotide sequence across the human genome, as well as the CpG distribution within the 1 Kb sequence window. All primer pairs were confirmed to amplify a single product of appropriate size. Oligonucleotide sequences are provided in Bisulphite sequencing primers were designed to amplify DNA corresponding to the amplicons used for quantitative PCR analyses. Primer pairs  flanked,GHSR were as described GAPDH were 1 cycle of 95°C for 5 min., 30 cycles of 95°C for 30 sec., 65°C for 15 sec., 72°C for 15 sec., and 1 cycle of 72°C for 10 min. Amplification products (1 µL) were visualized and quantified using an Agilent Bioanalyzer 3100 and DNA 1000 LabChips (Agilent). All reactions were performed at least three times using two independently synthesized cDNA preparations.For IDC samples, 5 µg total RNA was used as template for cDNA synthesis by the Superscript III First Strand Synthesis System (Invitrogen) using the manufacturer's protocol. Prior to cDNA synthesis, RNA samples were treated with recombinant DNase I (Ambion) by the manufacturer's protocol. Normal breast cDNA was obtained from Invitrogen and was prepared using the same protocol. cDNA concentrations were normalized between samples and serial dilutions were used as template for PCR amplification. PCR primers  were desFigure S1Repeated GHSR qPCR analyses for samples near the 0.64 threshold for sensitivity and specificity calculations. 16 samples were analyzed three times . Digestions were performed using a different lot of McrBC enzyme than that used for the experiments summarized in (0.01 MB PDF)Click here for additional data file.Figure S2Detection of tumor-specific DNA hypermethylation in fine needle aspirate specimens. Eight biomarker loci were screened in seven FNA samples obtained from confirmed breast cancer cases. For each locus, the percentage of FNA samples that reported hypermethylation was plotted against the percentage of independent tumor samples that reported hypermethylation. If the biomarkers are detecting breast cancer at the same frequency as in tissue samples the expectation is that the two results should be directly proportional (i.e. exhibit a sensitivity slope of 1.0). This theoretical maxim is indicated by the dashed y = x line. The actual slope (solid line) and its R2 are indicated. The theoretical and experimental results are not significantly different (n = 8 data points).(0.01 MB PDF)Click here for additional data file.Figure S3Ensembl contig view of the uncharacterized locus corresponding to chr7-8256680(NCBI35). The position of the microarray feature that reported differential DNA methylation and Ensembl annotated CpG islands are indicated by arrows (NCBI36(hg18)).(0.16 MB PDF)Click here for additional data file.Table S1Patient demographics for samples used in microarray analyses(0.04 MB DOC)Click here for additional data file.Table S2Microarray data.(1.06 MB XLS)Click here for additional data file.Table S3Sensitivity/Specificity of Breast Cancer Biomarkers in Initial Validation Panel.(0.03 MB XLS)Click here for additional data file.Table S4Patient demographics for samples used in validation analyses.(0.08 MB DOC)Click here for additional data file.Table S5Oligo sequences(0.03 MB XLS)Click here for additional data file.


DNA barcoding of life using a standardized COI sequence was proposed as a species identification system, and as a method for detecting putative new species. Previous tests in birds showed that individuals can be correctly assigned to species in ~94% of the cases and suggested a threshold of 10× mean intraspecific difference to detect potential new species. However, these tests were criticized because they were based on a single maternally inherited gene rather than multiple nuclear genes, did not compare phylogenetically identified sister species, and thus likely overestimated the efficacy of DNA barcodes in identifying species.COI in 60 sister-species pairs identified in multigene phylogenies from 10 orders of birds. In all pairs, individuals of each species were monophyletic in a neighbor-joining (NJ) tree, and each species possessed fixed mutational differences distinguishing them from their sister species. Consequently, individuals were correctly assigned to species using a statistical coalescent framework. A coalescent test of taxonomic distinctiveness based on chance occurrence of reciprocal monophyly in two lineages was verified in known sister species, and used to identify recently separated lineages that represent putative species. This approach avoids the use of a universal distance cutoff which is invalidated by variation in times to common ancestry of sister species and in rates of evolution.To test the efficacy of DNA barcodes we compared ~650 bp of COI sequences, verifying coalescent-based statistical tests of reciprocal monophyly for taxonomic distinctiveness. Contrary to recent criticisms, a single DNA barcode is a rapid way to discover monophyletic lineages within a metapopulation that might represent undiscovered cryptic species, as envisaged in the unified species concept. This identifies a smaller set of lineages that can also be tested independently for species status with multiple nuclear gene approaches and other phenotypic characters.Closely related sister species of birds can be identified reliably by barcodes of fixed diagnostic substitutions in  COI, known as DNA barcoding, has two main goals: 1) to develop a species identification system that also allows unknown individuals to be assigned to species; 2) and to enhance the discovery of new species [Large scale sequencing of a predefined region of approximately 650 (base pairs) bp of the mitochondrial gene  species . Althoug species -11, the  species -16.COI clusters [COI barcodes of cowries and found extensive overlap of overall intraspecific distances with interspecific distances, resulting in minimum error rates of ~17% to screen for putative new species. Additionally, a simulation study using the neutral coalescent and the Bateson-Dobzhansky-Muller (BDM) model of speciation suggested that mtDNA barcodes will have error rates lower than 10% in assigning individuals to species only when populations have been isolated for more than 4 million generations [A major test performed on 643 previously recognized species of birds of North America demonstrated the effectiveness of DNA barcoding because 94% possessed unique monophyletic clusters ,11. The clusters ,11. Critclusters ,15,17, wclusters . The sugclusters  reportedclusters ,11, althclusters  tested derations .Additionally, Hickerson et al.  argued tSome of the advantages of using a single mtDNA barcode to identify species are that it has a higher rate of evolution (and thus more mutations), and because matrilineal lineages sort into reciprocally monophyletic clades much faster than nuclear genes . This reIn response to the above criticisms we initiated a more comprehensive study of 60 sister-species pairs of birds defined rigorously with multigene phylogenies to determine whether mtDNA barcodes can reliably distinguish closely related sister species. Instead of the much criticized 10× rule, which may not apply in recently diverged sister-species pairs, we use coalescent-based statistical tests for species distinctiveness under reciprocal monophyly . AdditioSturnella magna), with the majority of the values (93.1%) above 85% , Black-chinned Hummingbird , Gunnison Sage-Grouse (Centrocercus minimus), Dusky Grouse (Dendragapus obscurus), Nuttall's Woodpecker , Jackass Penguin (Spheniscus demersus), and Magellanic Penguin (Spheniscus magellanicus). These species were distinguished by <10 fixed nucleotide substitutional differences or had multiple intraspecific clusters. Probabilities of chance occurrence of reciprocal monophyly arising from random-branching within a single taxon were smaller than the level of significance (α) of 5%  tree under the Kimura 2-parameter (K2P) model in all the sister-species pairs compared Table , 2. Multalues 93.% above 8alues 93.% above 8Apteryx australis, Figure Gelochelidon nilotica) and Gentoo Penguin (Pygoscelis papua), the query individual was assigned correctly to the each intraspecific cluster  which are thought to be two species [Mean among sister-species distances of mtDNA barcodes varied from 0.78% to 11.77%, with 20 out of 60 (28.6%) distances smaller than the 2.7% threshold used to flag potential new species of birds. Among-species distances overlapped maximum within-species distances in 39 of 60 (65%) sister-species pairs. Excluding cases that are likely to represent overlooked species based on other attributes, the overlap was observed in 21 of 60 sister-species pairs , Gentoo Penguin (Pygoscelis papua), Gull-billed Tern (Gelochelidon nilotica), Eastern Meadowlark (Sturnella magna), Common Redshank (Tringa totanus), and Little Penguin  and S. n. vanrossemini (Russia), but they were poorly sampled (2 samples each) [Six species had distinctive intraspecific clusters with probabilities of chance reciprocal monophyly below a conservative level of α = 1%: Kittlitz's Murrelet  .Eudyptula minor, are probably two species (Table Anthus novaeseelandiae) from New Zealand and Australia differ by 4.1% in their barcodes, and Little Terns  from England and Australia differ by about 1%. However, increased sampling of these species is required to properly test whether they represent separate taxonomic entities.Using the test for chance reciprocal monophyly, the Little Penguins of Australia and New Zealand, respectively, currently lumped into es Table . This coes Table . Other sCOI evolution in different clades and short species divergence times. However, all sister-species pairs were shown to possess unique DNA barcodes by which they could be identified. In particular, the COI sequences of even very closely related sister species were found to have diagnostic combinations of 5–64 fixed substitutional differences that better fit the analogy of a short DNA barcode. Individuals were correctly assigned to each sister species for which we had moderate sample sizes (N ≥ 4) using different lines of evidence: NJ clustering, diagnostic fixed substitutions, and a decision-theoretic framework based on coalescent theory implemented in Assigner [Our study of 60 pairs of sister species from a broad range of bird clades showed that closely related pairs could not be distinguished using the 10× rule of among to within species divergence, as predicted by critics of this criterion ,15. SimiAssigner . The conCOI sequences representing within-species variation can potentially be confounded with recently diverged sister species, so to objectively discriminate between these two possibilities we applied a statistical test of the null hypothesis that reciprocal monophyly has arisen by random branching of lineages within a single species. The null hypothesis could be rejected in all closely related sister species (P < 0.05), verifying the power of the test. In addition, putative new species were strongly supported by the distinctive signatures of >12 fixed substitutional differences and low probabilities of chance reciprocal monophyly within a single species. For example, the barcodes of Little Penguins from Australia and New Zealand, and of Gentoo Penguins from Macquarie Island and the Falklands, provide strong inferences of separate lineages that may warrant species status for these groups. The existence of separately evolving metapopulation lineages is the species delimitation criterion for a recently proposed unified species concept [Phylogroups of  concept , though  concept , but rat concept . However concept ).COI haplotypes were detected would seem to be fertile ground for further investigation with independent multiple nuclear gene trees in a coalescent framework. For example, the split between Australian and New Zealand populations of Little Penguins was dated at approximately 1.3 Mya using the neutral coalescent method in IM [COI evolution of 0.01354 substitutions/site/Myr [COI sequences might be an advantage in identifying possible recent speciation events, and they can be combined with nuclear gene sequences in IM to estimate whether the divergence is due to isolation or if gene flow has been ongoing. Thus we view DNA barcodes as useful complements in multigene data sets that might include more than one mtDNA gene [One of the most cogent criticisms of single locus mtDNA barcodes is that a pattern of reciprocal monophyly in maternally inherited genes can also arise when female dispersal is very restricted, often contrasting with widespread apparent panmixia of autosomal and paternally inherited genes . Howeverod in IM , and a psite/Myr . Given asite/Myr  this equDNA gene , contrarCOI sequences. All pairs were characterized by reciprocally monophyletic lineages, and tests of the null hypothesis of random branching within a single species were rejected. Thus in well studied groups like birds, mtDNA barcodes are extremely effective in identifying sister species. In species that are shown by COI barcodes to be comprised of several divergent monophyletic lineages that might flag unrecognized species, it is important to test these splits with multiple independent gene trees in a coalescent framework to guard against the alternative inference of population subdivision via restricted female dispersal. Combination of multiple genes including mtDNA barcodes should counter any biases in species detection and the high variance in associated genetic processes [We show that in a broad range of birds even closely related sister species delimited with independent evidence could be identified with mtDNA barcodes and diagnostic substitutions using standard rocesses .COI barcoding in detecting species boundaries of birds we analyzed sister-species pairs defined rigorously by previous phylogenetic studies . COI sequences obtained from previous work are available in the Completed Projects selection of the BOLD, in the "Birds of North America" project [DQ432694–DQ433261, DQ433274–DQ433846, DQ434243–DQ434805).To evaluate the performance of es Table . We exclem  [Taq polymerase (Invitrogen), and 20–25 ng of DNA. Cycle conditions were 36 cycles of 94°C for 40 s, 50°C for 40s, and 72°C for 1 m, with an initial denaturation of 94°C for 5 m and a final extension at 72°C for 7 m. Bird universal primers used were as follows: LTyr – TGTAAAAAGGWCTACAGCCTAACGC,  and COI907aH2 – GTRGCNGAYGTRAARTATGCTCG,  Amplified segments were purified by excising bands from agarose gels and centrifuging each through a filter tip. Sequences were obtained on an ABI3100 (Applied Biosystems) according to the manufacturers' suggested protocols using the internal primers COIaRt  and COI748Ht (reverse-TGGGARATAATTCCRAAGCCTGG), or alternatively LTyr (primer used in amplification) and COI745h2 . Sequences were checked for ambiguities in Sequencher 4.1.2  and the multiple alignments was performed in MacClade 4 [DNA was extracted from blood, muscle or liver by phenol, chelex or a membrane purification procedure with glass fiber filtration plates . PCR amin (BSA) , 0.4 mM To check for reciprocal monophyly in sister-species with DNA barcodes, a Neighbor-Joining (NJ) tree was constructed in PAUP 4.10b  with theBecause compound diagnostic characters are a valuable source of information to diagnose species  we filteThe test for chance occurrence of reciprocal monophyly  was applCOI sequence of one randomly selected individual was excluded from the matrix and used as the query sequence. For each of the sister species of the pair (target groups), the evolutionary parameter θ  with corresponding maximum likelihood was estimated from the data in FLUCTUATE [The correct assignment of individuals to species was performed in a decision-theoretic framework based on coalescent theory in Assigner . The speLUCTUATE . These vLUCTUATE .COI in different lineages of birds [COI distances between sister species of Terns, Shanks and Penguins were mapped on the corresponding chronograms.Distances under the K2P model were calculated among sister-species and within-species in MEGA 3.1 . Completof birds  affect dof birds , shanks of birds , alcids of birds , penguinof birds , and kiwof birds ). K2P diAJB and EST designed the scope of the research. EST carried out the lab work, data assembly, and analysis except for coalescent simulations in IM which were done by AJB. Both authors wrote and approved the final manuscript.COI barcode sequencesSister-species differences in . Sister-species pairs and sampling, fixed substitutions (fixed subst.), bootstrap support, mean interspecific K2P distances (Dinter), and maximum intraspecific K2P distance within each species (Dintra).Click here for fileNeighbor-joining tree topology constructed from DNA barcodes of sister species of birds. Neighbor-joining tree topology of ~650 bp of mitochondrial gene COI, under the K2P model and pairwise deletion. File in nexus format, opens in TreeView.Click here for file


Drosophila and is required for H3K9 trimethylation in the female germ line. Here we show that Windei (Wde), the Drosophila homolog of mouse mAM and human MCAF1, is an essential cofactor of Egg required for its nuclear localization and function in female germ line cells. By deletion analysis combined with coimmunoprecipitation, we have identified the protein regions in Wde and Egg that are necessary and sufficient for the interaction between the two proteins. We furthermore identified a region of Egg that gets covalently modified by SUMOylation, which may facilitate the formation of higher order chromatin-modifying complexes. Together with Egg, Wde localizes to euchromatin, is enriched on chromosome 4, and binds to the Painting of fourth (POF) protein. Our data provide the first genetic and phenotypic analysis of a mAM/MCAF1 homolog in a model organism and demonstrate its essential function in the survival of germ line cells.The epigenetic regulation of gene expression by the covalent modification of histones is a fundamental mechanism required for the proper differentiation of germ line cells during development. Trimethylation of histone 3 lysine 9 (H3K9me3) leads to chromatin silencing and the formation of heterochromatin by recruitment of heterochromatin protein 1 (HP1). dSETDB1/Eggless (Egg), the ortholog of the human methyltransferase SETDB1, is the only essential H3K9 methyltransferase in  Drosophila is controlled by three different histone methyl transferases, but only one of these, dSETDB1/Eggless, is essential for viability and fertility of the fly. Here we describe an essential cofactor for dSETDB1/Eggless that is specifically required in germ line cells for their survival. This cofactor, that we called Windei, binds to dSETDB1/Eggless and recruits it to the nucleus. Null mutations in windei show strongly reduced trimethylation of H3K9 in germ line cells, demonstrating that Windei is one of the factors required for controlling chromatin organization in the germ line.Germ line cells are the only cells in an organism that are able to transmit their genetic material to the next generation by forming eggs or sperm. They do not participate in the formation or function of tissues and organs and therefore show a unique pattern of transcription, with many genes being silenced that are only required for somatic functions. The covalent modification of histones by methylation, acetylation, and other mechanisms is crucial for these global alterations in the transcriptional program. Among the modifications involved in silencing of chromatin regions, methylation of histone 3 lysine 9 (H3K9) is among the most important ones. Methylation of this residue in  The epigenetic regulation of gene expression by the modification of histone proteins is a very important mechanism to control the differentiation of many cell types during development. The N-terminal, outward protruding histone tails are targets of posttranslational modifications, such as acetylation, ubiquitination, phosphorylation and methylation. These histone modifications are supposed to act sequentially or in combination to form a histone code that can be deciphered by different chromatin-associated proteins to mediate changes in chromatin structure and transcriptional activity Drosophila as a suppressor of position effect variegation Su(var)2–5 One of the best-studied histone modifications is the methylation of the histone 3 lysine residue 9 (H3K9), which generally correlates with transcriptional repression DrosophilaSeveral histone methyltransferases (HMTs) have been identified that specifically methylate H3K9, the first being Su(var)3–9 of A third class of H3K9 specific HMTs is represented by SETDB1/ESET Knockout mice lacking the function of SETDB1 Many proteins involved in transcriptional repression are either covalently modified by conjugation to the small ubiquitin-related modifier (SUMO) or they contain SUMO binding domains Drosophila, only the SETDB1 homolog dSETDB1/Eggless (Egg) is essential for viability and fertility egg mutants loose most of the H3K9 methylation marks as well as binding of HP1 on chromosome 4, which is consistent with global changes in the transcription level of genes located on chromosome 4 that were observed in egg mutants egg mutant females possess only rudimentary ovaries, due to massive apoptosis at early stages of oogenesis in somatic and germ cells egg mutant germ line cells, particularly at the earliest stages of oogenesis in the germarium In Drosophila ortholog of mAM/MCAF1 precisely colocalizes with Egg in ovaries and binds to Egg in vivo. We furthermore show that Egg gets covalently modified by SUMOylation, which is a hallmark of many chromatin-associated proteins involved in transcriptional repression. Wde localizes to euchromatic regions of salivary gland polytene chromosomes, in particular to chromosome 4, and associates with POF in a protein complex. We have generated null mutations in wde, which are homozygous lethal and can be fully rescued by a transgene encoding a GFP-Wde fusion protein. Surviving homozygous wde mutant females are sterile and possess only rudimentary ovaries. Loss of wde function in germ line clones eliminates nuclear localization of Egg, leads to the arrest of oogenesis before stage 10 and to subsequent degeneration of mutant egg chambers by apoptosis. Like egg mutant cells, germ line cells mutant for wde show strongly reduced H3K9 trimethylation. According to the indistinguishable subcellular localization and mutant phenotypes of the two interactors, we propose that Wde is an essential binding partner of Egg required for the conversion of H3K9me2 to H3K9me3.So far it was not known whether Egg requires a binding partner homologous to mammalian mAM/MCAF1 for its function. Here we show that Windei (Wde), the Drosophila homolog of SETDB1/ESET called dSETDB1/Eggless (Egg) is essential for oogenesis http://blast.ncbi.nlm.nih.gov/Blast.cgi) revealed the existence of a single Drosophila homolog of mAM encoded by the CG12340 transcription unit located at position 47C1 on the right arm of the second chromosome  and will use this name throughout the manuscript. wde encodes a strongly acidic protein  of 1420 amino acids and a calculated molecular weight of 157.776 Dalton. Although the overall sequence identity between mAM and Wde is only 14,8%, the domain structure with an internal coiled-coil region and a C-terminal fibronectin type III repeat is identical  romosome . Due to dentical . Within dentical .wde mutant and Wde overexpressing embryos . In wild type embryos, the antibody detected several bands with a molecular weight around 250 kD that were absent in extracts of homozygous mutant embryos (http://smart.embl-heidelberg.de/).The precise colocalization of Wde with Egg and the fact that the mammalian homologs of Wde and Egg bind to each other  embryos . We then embryos . The sam embryos  and test embryos . The sma embryos , a regioMany proteins involved in transcriptional repression can either bind to SUMO and SUMOylated proteins or are covalently modified by SUMOylation. We noticed that full length Egg ran at a higher molecular weight in SDS-PAGE than predicted from its sequence . This waDrosophila melanogaster  by FLP/FRT mediated recombination in trans of two P-elements flanking the wde locus on both sides (00884wde and 06198wde) caused by insertion of the P-elements P{Epgy2}CG12340EY00884 and P{XP}CG12340d06198, respectively, into the coding region of wde , proving that the observed defects were due to mutation of wde and not to a second site mutation elsewhere in the genome.To analyze the function of th sides . Two addn of wde  are predn of wde , which eermarium . The letwde during germ line development without affecting the function of wde in follicle cells, we generated germ line clones of the 00884wde and TD63wde alleles using the autosomal FLP-DFS technique 00884wde and TD63wde germ line clones did not produce any eggs, whereas control clones using the same FRT chromosome without a wde mutation produced eggs at the expected frequency (data not shown). To analyze the oogenesis defect of wde mutants in more detail, we generated germ line clones marked by the absence of GFP expression using the FLP/FRT technique. While control clones showed robust nuclear Wde staining (00884wde completely lacked nuclear Wde staining (06198wde and TD63wde (data not shown). We noted that egg chambers with wde mutant germ line clones did not develop beyond stage 8 of oogenesis. Closer inspection of late stage egg chambers with wde germ line clones revealed that the nuclei of germ line cells were highly condensed and degenerated subsequently , demonstrating that Wde is indispensable for H3K9 trimethylation by Egg. In general, the germ line clone phenotypes of wde and 1473egg mutants were indistinguishable with respect to apoptosis and the timing and severity of egg chamber degeneration (data not shown). Moreover, germ line clones doubly mutant for 06198wde and 1473egg showed the same phenotype as either single mutant (data not shown), indicating that both proteins function in the same process and are nonfunctional in the absence of their binding partner.The oogenesis phenotype of 1473egg 3–9 and G9a mutants egg mutants, the second study egg mutants, measured in a microarray experiment. Nonetheless, the involvement of both Egg and Wde in the transcriptional regulation of genes on chromosome 4 appears very likely, since both Egg On polytene chromosomes Wde binds strongly to the fourth chromosome and to multiple euchromatic bands on all other chromosomes. Strong binding to the fourth chromosome has also been reported for Egg Drosophila melanogasterpof mutants, the transcription level of genes on the fourth chromosome is reduced, indicating that POF promotes transcription of genes on chromosome 4 POF is a unique example of a protein that specifically associates with a single autosome, the fourth chromosome of Drosophila. Ovaries of homozygous egg mutant females are rudimentary and degenerate by apoptosis before egg chambers bud off the germarium wde mutant females show exactly the same phenotype. From these observations it was not clear whether the function of Egg and Wde is required in the germ line cells, the somatic follicle cells, or both. To address this question, we eliminated the function of egg and wde in germ line cells by FLP/FRT mediated mitotic recombination. Egg chambers with egg or wde germ line clones did develop up to stage 8 of oogenesis, but subsequently degenerated due to apoptosis. Because the ovary phenotype of homozygous mutant egg and wde females was more severe than the germ line clone phenotype of mutants in both genes, we conclude that wde and egg may also be required for proper development of somatic follicle cells.Two recent studies showed that Egg is required for the development of ovaries in wde and egg lead to apoptosis of germ line cells, which obviously cannot be rescued by the presence of Su(var)3–9 which is already expressed in the germ line at the time when apoptosis starts.It has been speculated that Egg may be dispensable for trimethylation of H3K9 at later stages of oogenesis because this function could be taken over by Su(var)3–9 Modification by SUMOylation and binding to SUMO is a common hallmark of many chromatin regulators involved in transcriptional repression Drosophila Stock Center), P{XP}CG12340d06198, P{XP}d03942, P{XP}d01917 (Exelixis collection at Harvard), P{EPgy2}CG12340EY00884 (#15045), Df(2R)27 (#8109), daughterless-GAL4 (#5460), engrailed GAL4 (#6356), tubulin-GAL4 (#5138), mat67-GAL4 (#7062), P{w+FRTG13}GFP (#5826), P{neoFRT40A} P{w+FRTG13} (#8217), y w hsFlp; Sco/CyO (#1929) , egg147306198wde and 1473egg was generated by meiotic recombination. The TD63wde null allele was generated by FLP/FRT mediated recombination in trans of the P{XP}d03942 and P{XP}d01917 P element insertions wde and egg were generated as described using a heat shock promoter driven flippase on the X-chromosome wde mutant adults, living embryos lacking GFP fluorescence derived from the CyO[twi::GFP] balancer chromosome were separated from their GFP positive siblings under a fluorescence stereo microscope and raised in separate vials.The following stocks were used in this study: P{EP}EP2024 (Szeged Antibodies against Wde were generated by immunizing two rabbits with the following peptides: DKPKKISDRERNPGS (aa 70–84) and RSENTPPPASRLRYSH (aa 1286–1301). Final bleeds of both rabbits were pooled and affinity purified against the peptide corresponding to aa 70–84 . For immunohistochemical stainings of embryos, ovaries and salivary gland polytene chromosomes the following antibodies were used: rabbit anti Wde, affinity purified (see above), 1∶5000 (1∶500 for polytene chromosomes); rabbit anti POF, 1∶1000 (1∶400 for polytene chromosomes) Lysates of S2 cells were prepared in TNT buffer  supplemented with protease inhibitors (Roche). Western blots and coimmunoprecipitations were done as described Figure S1TD63wde homozygous mutant embryo at the same stage. Note that residual maternal Wde can be detected in the primordial germ cells ((D), arrows). Homozygous mutant TD63wde embryos were identified by absence of lacZ staining derived from the Cyo[ftz::lacZ] balancer chromosome (lac Z staining not shown). (E–G) Overexpressed full length GFP-Wde is detected by the anti Wde antibody. pUASP-GFP-Wde was overexpressed under control of the engrailed-GAL4 driver line, which is expressed in segmentally repeated stripes in the epidermis. The GFP fluorescence (E) matches precisely the staining with the anti Wde antibody .  Endogenous Wde was overexpressed under control of engrailed GAL4 using the P{EP}EP2024 insertion line (I) in which the EP element is inserted 40 bp upstream of the transcription start site of the wde locus. The overexpressed Wde was detected by the anti Wde antibody (H). (J–M) In the embryonic ectoderm, Wde ) is nuclear in interphase cells and shows partial colocalization with the DNA dye YoYo-1 ). In mitotic neuroblasts (asterisks), Wde is cytoplasmic and does not colocalize with DNA (K–M). Neuroblasts were marked by expression of Miranda ). Scale bars in (A) and (E) = 100 µm, Scale bar in (J) = 10 µm. Anterior is to the left in all panels.The anti Wde antibody specifically detects endogenous Wde and overexpressed GFP-Wde.  A wild type embryo at stage 16 was stained for DNA ) and Wde ). Note the intense staining of Wde in primordial germ cells (arrows).  Wde staining is strongly reduced in a (7.66 MB TIF)Click here for additional data file.Figure S2wde and egg homozygous mutant females possess only rudimentary ovaries. Whole ovaries of 2 day old wild type , TD63wde , and 1473egg homozygous mutant females  were stained with DAPI. Whereas wild type ovaries contain approximately 16 ovarioles each with egg chambers at different developmental stages , both wde and egg mutant ovaries are tiny and do not contain any egg chambers that have separated from the germarium (C–F). Boxes indicate regions shown at higher magnification in the right panels. Scale bars = 200 µm.(1.89 MB TIF)Click here for additional data file.Figure S300884wde mutant germ line clones (F–J) were induced using the FLP/FRT technique. Ovaries were stained for DNA with DAPI and for Wde  and Orb  using specific antibodies. Germ line clones are marked by the absence of GFP fluorescence , dotted circles). While control germ line clones show nuclear Wde staining in nurse cells and in the oocyte (C), wde mutant germ line cells lack nuclear Wde staining (H). Both in control clones and in wde mutant clones, oocyte determination appears normal, because staining for Orb is restricted to a single cell at the posterior pole of each egg chamber . Scale bars = 50 µm.Wde is dispensable for oocyte determination. (A–J) GFP marked control germ line clones (A–E) and (2.46 MB TIF)Click here for additional data file.Text S1Supplemental methods.(0.03 MB DOC)Click here for additional data file.


P< 0.001). p53 mutations were observed in 8 carcinomas, but no K-ras mutation was detected. Interestingly, although none of the overexpressions of p53 protein was detected immunohistochemically in the BGP positive foci, the p53 gene frequently (41.2% of the BGP foci tested) mutated in spite of no K-ras mutation. The present study demonstrates potentially premalignant foci in the colorectal transitional mucosa with frequent p53 gene mutation. It is suggested that BGP foci are promising candidates for the further investigation of ‘de novo’ colorectal carcinogenesis. © 2001Cancer Research Campaign http://www.bjcancer.com‘de novo’ carcinogenesis has been advocated besides ‘adenoma carcinoma sequence’ as another dominant pathway leading to colorectal carcinoma. Our recent study has demonstrated that the distribution of brain -type glycogen phosphorylase (BGP) positive foci (BGP foci) has a close relationship with the location of ‘de novo’ carcinoma. The aims of the present study are to investigate genetic alteration in the BGP foci and to characterize them in the ‘de novo’ carcinogenesis. 17 colorectal carcinomas without any adenoma component expressing both immunoreactive p53 and BGP protein were selected from 96 resected specimens from our previous study. Further investigations to examine the proliferating cell nuclear antigen (PCNA)-labelling index, and the p53 and the codon 12 of K-ras mutation using the polymerase chain reaction-single strand conformation polymorphism were performed in the BGP foci, BGP negative mucosa and carcinoma. The BGP foci were observed sporadically in the transitional mucosa adjacent to the carcinoma in all cases. The PCNA labelling index in the BGP foci was significantly higher than that in the BGP negative mucosa (