The purpose of this study was to use high resolution synchrotron radiation-based Fourier Transform Infrared (FTIR) micro-spectroscopy coupled with multivariate analysis to investigate the characteristics of adult stem cell (SC) and transit amplifying (TA) cell populations of the human corneal epithelium.Spectra of individual SC and TA cells in situ from cryosections of human cornea were collected using a synchrotron micro-spectroscopy facility at Daresbury laboratory, UK. Multivariate analysis and Mann Whitney U tests were used to analyse the spectral data from the SC and TA cell populations.There were marked differences between the median spectra of the two cell populations. This correlated with their level of differentiation and functional specialization. Multivariate  analysis revealed that the cell populations could be segregated into distinct clusters, with only slight overlap between the two cell types. Significant (p<0.05) spectral differences were found in the spectral regions associated with nucleic acid, protein and lipids.Synchrotron FTIR micro-spectroscopy together with principal component analysis is able to discriminate between SC and TA cell populations. Our results also suggest a small sub-population of corneal epithelial cells in the SC niche have TA cell-like characteristics. Many of the spectral differences between the SC and TA cell populations relate to differences in nucleic acid conformation. Adult stem cells (SC) are responsible for the regeneration of cell populations in tissues that undergo continuous turnover. These cells are slow-cycling cells with a high capacity for unlimited or prolonged self-renewal extending throughout adult life . One of It is known that corneal SCs can divide either symmetrically to produce two daughter SCs or asymmetrically to produce one daughter SC and one progenitor or transit amplifying (TA) cell. These TA cells, which have only limited proliferative capacity, have been observed to migrate centripetally from the periphery of the cornea forming a basal cell layer. It is also thought that TA cells in turn form terminally-differentiated (TD) cells, which are highly specialized and have no proliferative capacity . The apiLimbal basal cells are sometimes described as putative SCs, because as yet there is no definitive SC marker. There are, however, a number of molecules which are differentially expressed in comparison with other cells . These sStudies of the corneal epithelium have contributed enormously to our basic understanding of the way adult SCs function. This is because the SCs here are spatially well-defined and easily accessible for both surgical intervention and observation. It has also facilitated the rapid development of ex vivo SC expansion and transplantation techniques for ocular surface disorders -11. AlthAdvances in high resolution Fourier transform infrared (FTIR) micro-spectroscopy may provide valuable new information on SCs. This powerful technique has been around for almost 40 years but only now is becoming used in cell biology. It has been used to detect subtle intracellular changes associated with diseases such as Alzheimer's disease and osteoporosis, and to discriminate between malignant and non-malignant cells in several different tissues -15. It hFTIR micro-spectroscopy uses the mid-IR (λ=2-20 μm) absorbance of cellular biomolecules. Each individual chemical bond within the cells gives rise to characteristic spectra. This can provide unique information about structural and conformational changes of the molecules inside the cell -21. ConvIn contrast, synchrotrons provide a highly collimated beam of light that is orders of magnitudes brighter. Using the IR portion of synchrotron radiation, at a spatial resolution of 10 μm, the S/N is about 1,000 times greater . AlthougFTIR micro-spectroscopy using a bright IR synchrotron radiation source has tremendous potential for the interrogation of intracellular dynamics and molecular changes, e.g., the transition of SCs to TA cells.FTIR generates massive amounts of complex data, with each individual spectra containing more than 1,000 wavebands. With each experiment requiring the acquisition of tens of spectra, it is often difficult to identify important and usually subtle, differences from the information that is collected. However, recent developments in the use of multivariate analysis and in particular principal component analysis (PCA) now allow more efficient analysis of spectra.In PCA, each spectrum is assigned a point in n-dimensional space when selected principal components (PCs) are used as coordinates. Cluster analysis of the data is achieved when data plot is viewed in a particular direction. Each of the PCs are eigenvectors of the correlation coefficient matrix of squared deviations. They comprise of a set of new variables, retaining almost all the variation present in the original spectra, with the first PC presenting the most variance, the second PC presenting the maximum amount of the remaining variance and so forth . The infIn this study, we have employed synchrotron radiation-based FTIR (SR-FTIR) micro-spectroscopy to acquire unique in situ information about the putative SC and TA cell populations in human corneal epithelium.2 slides . The sections were stored in desiccators until required. Parallel sections were stained with hematoxylin and eosin for histological comparison.Corneal specimens were obtained from the Northwest Lions Eye Bank, Seattle, WA. The corneas were all from male Caucasians aged 23, 64, and 66 years. The eyes were harvested within 12 h of death, the corneas placed in Optisol corneal preservation medium  and stored at 4 °C for up to 5 days before use. Corneal samples were dissected and frozen. Cryosections (10 μm thick) were collected onto BaF-1 spectral resolution and co-added for 1,024 scans. Spectra were baseline corrected and normalized to the amide II  absorbance band using Opus software . SC spectra were collected from the basal cell layer in the limbal region which consisted of small primitive cells that were poorly differentiated. The location and appearance of these was consistent with that of limbal SCs. TA cell spectra were collected from the basal cell layer in the cornea approximately 3 mm away from the limbus.Spectral data was collected from cryosections of human cornea at the Daresbury Synchrotron Radiation Source on beamline 11.1  . SpectraPrincipal Component Analysis was conducted on spectra using the Pirouette software package . After baseline correction and normalization, the spectra were processed as first-derivative (15 points) spectra using Pirouette. Nine PCs were selected for analysis, and loading curves for each PC were plotted for each sample. These loading curves allowed the influence of specific spectral features on each PC to be identified. Score plots  of each PC pair were then plotted for each sample and by combining the clustering evident in these figures with the analysis of the loading curves, the most appropriate 3 PCs were selected for the 3-D cluster analysis. The Mann-Whitney test  was employed to compare absorption spectra.-1, which is associated with C=O stretching vibrations of lipids [-1 is associated with C=O stretching vibrations of nucleic acids [-1 is associated with amide I [-1 is related to C=N stretching in guanine [-1 are related to vibrational bonds within sugar rings [-1 is due to CH2 scissoring in lipids [-1 and 1,260 cm-1 have been linked to changes in glycoproteins and amide III, respectively [f lipids . The regic acids . The reg amide I . The reg guanine . The wavar rings . The regn lipids . The peaectively ,18. Finaectively .Previous workers have used FTIR micro-spectroscopy with some success in the identification and characterization of many cell types e.g., distinguishing both malignant and pre-malignant changes in several different tissues -14 and iThe results of our investigation shows that synchrotron-FTIR micro-spectroscopy coupled with multivariate analysis segregates spectra derived from SC and TA cell populations into discrete clusters . This teExamination of the difference spectrum shown in Our finding that SR-FTIR is able to discriminate between SC and TA cells is in agreement with observations made on bovine tissue , which aTo date, FTIR micro-spectroscopy is an under-exploited technique in cell biology. Recent developments mean that it is now possible to obtain spectra from individual cells or even intracellular compartments, and that such spectra can be very conveniently collected from cryosections of tissues. It is even possible now to collect spectra from certain types of live cells . Until rIn conclusion, this study was able to show that synchrotron-FTIR micro-spectroscopy coupled with PCA is capable of discriminating and segregating populations of SC and TA cells in human cornea. Analysis of the different populations suggests that a small subpopulation of cells within the corneal epithelial SC niche appear to possess TA cell-like characteristics, suggesting that these are newly generated TA cells prior to their migration. Analysis also shows that some of the spectral differences are due to changes in nucleic acid conformation. This finding fits in with recent work showing that the control of SC differentiation is related to major changes in chromatin structure .


Two-dimensional echocardiography is a useful tool in diagnosing cardiac masses. However, the three-dimensional offline reconstruction technique of transesophageal echocardiography might be superior to two-dimensional transesophageal echocardiography in providing additional information of structural details.We report the case of a 76-year-old Caucasian man with a permanent dual-chamber pacemaker and a worm-like right-heart thrombus in transit. Two-dimensional transthoracic echocardiography and two-dimensional transesophageal echocardiography showed that it was debatable as to whether "the worm" was originating from the leads. Offline three-dimensional transesophageal echocardiography reconstruction technique proved superior in identifying the cardiac mass as a thrombus trapped between the leads of the pacemaker. The thrombus was successfully dissolved by systemic heparin therapy.The three-dimensional transesophageal echocardiography is useful and effective in patients with implanted pacemakers or defibrillators when other closely competing imaging modalities are contraindicated, such as magnetic resonance imaging. In patients with pacemakers and trapped thrombus in transit for whom surgical therapy might be a high risk, medical therapy seems to offer a safer and convincing alternative. Whether the management of right-heart thrombi has to be modified due to the presence of pacemaker leads is controversial. In the context of different imaging modalities, two-dimensional (2D) transesophageal echocardiography (TEE) is a useful tool in diagnosing cardiac masses. It is superior to transthoracic technique in defining the morphology of intracardial structures . By cont®) was admitted to our centre with a two-day history of progressive dyspnea. Our exam findings were consistent with right and left heart failure. 2D transthoracic echocardiography  with a 2.5 to 3.5 MHz ultrasound transducer revealed an enlarged right ventricle (RV), a systolic pulmonary artery pressure of 46 mmHg calculated by tricuspid regurgitation, and a reduced left ventricular ejection fraction of 20 percent.A 76-year-old Caucasian man with a 24-year history of myocardial infarction, coronary artery bypass graft and a permanent dual-chamber pacemaker  was confirmed by venous limb sonography. Pulmonary embolism (PE) was immediately ruled out by computed tomography (CT). The electrocardiogram revealed an atrial flutter with a ventricular rate of 150 per minute; a possible pathomechanism for his symptoms. For the preparation of an external cardioversion, another 2D TTE was performed, demonstrating an echogenic worm-like mass with a length of 10 cm in the right atrium (RA)  to be aThe development of RH thrombotic complications in the presence of permanent PM leads has been described in the literature . SeriousWe conclude that 3D TEE seems to be very helpful for the assessment of RH cavities and intracardiac masses in patients with implanted PM or defibrillators. Medical therapy might offer a safe and convincing alternative to surgical therapy in high-risk patients with PM and trapped thrombus in transit. Even though the number of patients with both PM leads and RH thrombus is very small, it is possible that PE is prevented by PM in some patients.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.PM analyzed and interpreted patient data regarding the cardiac disease and was a major contributor in writing the manuscript. PM performed the transthoracic echocardiography. PM, TB and MP performed the transthoracal and transesophageal echocardiography. PM, TB, MP and AZ analyzed the images of the offline reconstruction. AM and HT have been involved in drafting the manuscript and revising it critically for important intellectual content. All authors read and approved the final manuscript."The worm" presented in two-dimensional transthoracic echocardiography. Two-dimensional transthoracic echocardiography demonstrated an echogenic worm-like mass with a length of 10 cm in the right atrium trapped between the permanent pacemaker leads and prolapsing through the tricuspid orifice in the right ventricle.Click here for file"The worm" presented in two-dimensional transesophageal echocardiography. Two-dimensional transesophageal echocardiography from "a worm in the heart".Click here for file"The worm" presented in three-dimensional offline reconstruction of the two-dimensional transesophageal data. Three-dimensional offline reconstruction of the two-dimensional transesophageal data helped us to identify the origin of the thrombus. 3D visualization of the thrombus demonstrated that the mass was not attached to the leads in the RA and RV but trapped between them.Click here for file


In addition to shotgun sequencing, next generation sequencing has been shown to be suitable for deep sequencing of many specific PCR-amplified target genes in parallel. However, unspecific product formation is a common problem in amplicon sequencing since these fragments are difficult to fully remove by gel purification, and their presence inevitably reduces the number of mappable sequence reads that can be obtained in each sequencing run.We have used a novel flow cytometric sorting approach to specifically enrich Roche/454 DNA Capture beads carrying target DNA sequences on their surface, and reject beads carrying unspecific sequences. This procedure gives a nearly three-fold increase in the fraction of informative sequences obtained. Presented results also show that there are no significant differences in the distribution or presence of different genotypes between a FACS-enriched sample and a standard-enriched control sample.Target-specific FACS enrichment prior to Roche/454 sequencing provides a quick, inexpensive way of increasing the amount of high quality data obtained in a single sequencing run, without introducing any sequence bias. Next-generation sequencing approaches have proved to be highly valuable for the analysis of genomes -3, transHowever, we have observed that the introduction of Genome Sequencer primer handle sequences  can result in the formation of short unspecific products; fragments that amplify more readily than target sequences and thus threaten to severely reduce the frequencies of target sequence reads in a sample. Unspecific products can be removed, to some extent, by gel purification if there is sufficient size difference between them and the target sequences, or alternatively the handle sequences can be omitted in the PCR and the A, B handle sequences can be added through more laborious linker ligation.We have previously demonstrated that Fluorescence Activated Cell Sorting (FACS) can be used for the enrichment of DNA-covered beads in sample preparation for Roche/454 sequencing. This is a quick, inexpensive way of ensuring that all of the analyzed beads carry DNA, without affecting the sequence quality . Here, wIn efforts to perform ultra-deep amplicon sequencing of the hypervariable Dog Leukocyte Antigen (DLA) target using the Roche/454 Genome Sequencer we have developed a strategy based on dual-labelling of emulsion PCR (emPCR) beads and fluorescence activated cell sorting (FACS) to isolate beads carrying the target sequence on their surface as described below and illustrated in Figure The aim was to eliminate the large amounts of unspecific products present in many amplicon libraries, which are difficult to fully remove by gel or SPRI-based (eg AMPure) purification and take up precious sequencing capacity. We have achieved this by using SYBR Green to label all types of DNA-carrying beads and a target-specific probe facilitating Alexa647 detection and collection of beads carrying the target sequence.The analyzed sample consisted of an amplicon library prepared from 34 individuals. Amplification of the library resulted in significant formation of unspecific products, as shown in Figure Flow cytometric analysis of the labelled sample showed a major peak representing naked beads that had failed to capture and amplify a DNA sequence while the DNA-covered, SYBR Green positive beads formed two minor populations with strong and weak positive SYBR Green fluorescence, respectively, as indicated by green arrows in Figure The average sequence length was greatly improved by FACS enrichment  in the two FACS-enriched samples as compared to the standard-enriched control sample Figure . Only miWe further analyzed the genotypes and found that all genotypes were represented in both FACS-enriched and control samples. Up to five mismatches between FACS probe and target sequences was observed. An example is shown in Figure To investigate whether similarity to FACS probe sequence had an effect on the enrichment efficiency of particular genotypes, the frequency of each probe region sequence present for every individual in the FACS sorted sample and the control sample, was calculated  can be effectively used to identify DNA-carrying beads to simplify the titration step in Roche/454 sequencing and (more importantly) facilitate enrichment of pure samples with DNA-carrying beads produced by a shotgun sample preparation procedure . In our In the technique presented here we exploit an additional feature of FACS (its ability to detect more than one fluorescent label) to increase the resolution of the procedure and remove the high numbers of non-target amplicons. This approach could be used for most cases of amplicon deep sequencing, such as analysis of 16S rRNA, mtDNA, cancer genes etc. The probe-mediated FACS enrichment protocol, using a probe tolerating at least five mismatches, provides sufficient flexibility to enrich beads carrying DLA target sequence (with significant between-amplicon variation). The results also show that the relative frequencies of sequences from different individuals in the amplicon library were not affected by FACS enrichment. Further we show that genotypes with a probe region sequence very similar to the FACS probe were not favoured by the FACS enrichment protocol over genotypes with up to five mismatches against the FACS probe.The beads carrying the target sequence could be detected using solely the target-specific probe. However, the dual labelling system confirms the efficiency of the target-specific labelling. Further, some stickiness of the probe to naked and unspecific product- carrying beads was observed (data not shown), probably due to the length of the target-specific probe, and use of two labels greatly increases the resolution and scope for optimising the FACS sort gate positions.Further, this report illustrates the possibility of fishing out specific sequences carried on beads using FACS, a feature that could be used for colour-coded MID -specific FACS enrichment, in order to load and sequence optimal numbers of beads carrying each MID-tag. The value of this would be the ability to compensate for differences in amplification efficiency between members of a MID library.Moreover, the results show that FACS-based quantification of DNA contents, and thus active selection of beads carrying long DNA fragments, could be used to increase average sequence read lengths in Roche/454 sequencing runs.Another future possibility would be to enhance sort speeds to further increase throughput. In order to maximise yield we used a modest sorting speed, of 1000 beads/s, but with a maximal sorting speed of 70,000 beads/s FACS enrichment of beads for large-scale sequencing would not be a rate-limiting step in the sample preparation procedure for Roche/454 Sequencing.Our results demonstrate that the number of informative sequence reads can be substantially increased by using gene-specific FACS sorting to remove most beads carrying amplified unspecific fragments on their surface, prior to sequencing using a Roche/454 Genome Sequencer.Using a biotinylated 50 mer FACS probe and streptavidin-conjugated Alexa647 florophore, beads with an amplified target gene product on their surface could be collected by FACS sorting for subsequent sequencing using the Roche/454 Genome Sequencer. This procedure gives a nearly three-fold increase in the fraction of reads with the target amplicon sequence. The method also allows for several mismatches between the sequence and the FACS probe and does not significantly affect the distribution of sequence reads over different individuals or genotypes obtained.An amplicon library of 34 individuals was prepared by PCR of the Dog Leukocyte Antigen gene DLA-DRB1 exon 2 (target size: 303 bp) , introduFor detecting beads carrying the amplified gene fragment by FACS, a biotinylated 50 mer was designed to match the central part of the amplicon product and still allow for some mismatches in the hypervariable DLA target sequence. To investigate whether the amount of DNA on the beads could be visualised by FACS, beads were also labelled with random hexamers and SYBR Green.Dual labelling was carried out by adding 20 pmol random hexamers and 20 pmol biotinylated gene-specific 50 mer probe, both from Eurofins MWG Operon  in a total volume of 100 μl 1× Annealing Buffer  and applying the following conditions: 1 min at 95°C, then decreasing to 25°C over 10 min. 1 μl 50 × SYBR Green I Nucleic Acid Gel Stain  and 4 μl streptavidin-conjugated Alexa647 dye  were then added to each sample, and the resulting mixture was incubated for 10 min at room temperature. Samples were then transferred to 5 ml Polystyrene Round-Bottom FACS Tubes  and diluted by adding 500 μl phosphate-buffered saline (PBS) containing 0.1% Pluronic F108 NF surfactant (PBSP) . As negative FACS control, unreacted beads were labelled in the same manner.®-MC Centrifugal Filter Units  and washed twice in 140 μl 0.1 M NaOH for 40 s and twice in 250 μl 95°C 1×AB for 40 s, followed by re-suspension in 1×AB.FACS enrichment of beads carrying an amplified gene fragment on their surface was achieved by sorting out beads that were Alexa647 positive using a FACSAria instrument . Beads were transferred to UltrafreeFACS-enriched and standard-enriched control samples were sequenced in separate 1/16-lanes of a picotiter plate according to the instructions of the sequencing system.Sequences were sorted by individual using an in-house developed Perl script, aligned using Muscle , JalviewThe authors declare that they have no competing interests.JS participated in the design of the study, collection of data, data analysis and interpretation and manuscript writing; MN participated in the design of the study, collection of data, data analysis and interpretation; AA participated in the design of the study, data analysis and interpretation; and JL participated in the design of the study and writing of the manuscript. All authors read and approved the final manuscript.Genotype frequency in standard-enriched sample and FACS-enriched sample. The data provided represent the frequency of genotypes of the different individuals in the standard-enriched sample and the FACS-enriched sample. Frequencies of individuals with less than ten sequence reads (N/A) and individuals with two different genotypes but with identical consensus sequences in the probe region were omitted.Click here for file


Using these cell lines, we observed that EpoR stimulation by Epo down-regulated expression of the SCF receptor Kit and induced expression of the Src kinase Lyn. Furthermore, enforced expression of Lyn in the cell lines increased cell proliferation in response to Epo, but reduced cell growth in response to SCF in accordance with Lyn ability to down-regulate Kit expression. Together, the data suggest that Epo-R/Lyn signaling pathway is essential for extinction of SCF signaling leading the proerythroblast to strict Epo dependency. These results highlight a new role for Lyn as an effector of EpoR in controlling Kit expression. They suggest that Lyn may play a central role in during erythroid differentiation at the switch between proliferation and maturation.Overexpression of the transcription factor Spi-1/PU.1 by transgenesis in mice induces a maturation arrest at the proerythroblastic stage of differentiation. We have previously isolated a panel of  Likewise, mice with null mutations in the genes encoding either Epo or EpoR die at midgestation with a severe anemia. Fetal livers from these mice contain BFU-E and CFU-E progenitors, although in reduced number, indicating that the Epo/EpoR pathway is crucial in regulating survival, proliferation and terminal differentiation of CFU-E Erythropoiesis is critically regulated by a number of growth factors acting through specific receptors, among which erythropoietin (Epo) and stem cell factor (SCF) are essential factors Signaling induced by Epo/EpoR and SCF/Kit is determined by the temporal and spatial expression of their cognate receptors at the surface of responsive cells. Kit is expressed from the earliest committed erythroid progenitor up to the basophilic erythroblastic stage of differentiation spi-1 transgenic mice spi-1 gene encodes the ETS transcription factor Spi-1/PU.1, a main player regulating the commitment of multipotent hematopoietic progenitors and the development of the B lymphoid and monocytic lineages spi-1 transgene induces a differentiation arrest in the erythroid lineage at the CFU-E/proerythroblast transition leading to severe anemia spi-1 transgenic proerythroblasts express both Epo and SCF receptors and can be expanded in vitro in the presence of Epo or SCF.In an attempt to dissect the signaling determinants controlling the expression of EpoR and Kit, we used proerythroblastic cell lines isolated during the preleukemic step of erythroleukemia developing in spi-transgenic proerythroblasts, Lyn favored cell proliferation in response to Epo, but not to SCF. These biological effects are consistent with the ability of Lyn to induce a down-regulation of Kit expression. Our findings reveal a novel aspect of signaling crosstalk between Kit and EpoR and highlight a central role for Lyn in SCF signaling extinction at the CFU-E/proerythroblast stage.Using cell lines established from the spleen of various diseased mice, we observed that each of these cell lines exhibited a particular growth rate in response to either Epo alone or SCF alone, and expressed EpoR and Kit in a ratio modulated by the cytokine used to sustain their proliferation. Starting from this observation, we investigated the molecular mechanisms controlling the expression of Kit and EpoR. We show that Epo down-regulated Kit expression and induced expression of the Lyn kinase. When ectopically expressed in spi-1-transgenic proerythroblastic cell lines  have been previously described in details 5 cells/mL and the number of living cells was monitored at 48 hours by Trypan blue exclusion using a Vi-Cell analyzer . For cytokine switching experiments, exponentially growing cells were washed 3 times in MEM and then plated at 2×105 cells/mL in culture medium with the cytokines indicated. AG490  and JAK inhibitor1  were used at a concentration of 10 µM and 20 nM, respectively.The 2b control monoclonal antibodies . After washing, cells were analyzed on a FACsort® with the Cellquest software package .Untreated cells were incubated for 30 min at 4°C with Phycoerythrin (PE)-conjugated anti-CD117 (c-Kit) or PE-conjugated IgGLyn cDNA was amplified by RT-PCR from mRNAs prepared from 663 cells. The Myc epitope (MT) was added at the cDNA C-terminus by PCR and the WTMT-Lyn construct was cloned into the pEF-BOS expression vector by standard cloning procedures. The mutant Y397FLyn cDNA was generated by mutagenesis of the WTMT-Lyn construct using the quickchange site-directed mutagenesis system  according to the manufacturer's recommendations. Cells were nucleofected with 5 µg of plasmid using an Amaxa nucleofector . Stable transfectants were selected in growth medium containing 800 µg/mL G418  and Epo (1 U/mL).The wild-type (WT) Whole cell extracts were fractionated by SDS-PAGE, blotted and visualized as previously described Kit, EpoR and Lyn: Kit-fw5′-TCCTCgCCTCCAAgAATTg-3′ and Kit-rev 5′-ggAAgCCTTCCTTgATCATC-3′, EpoR-fw 5′- ggCTCCgAAgAAcTTCTgTg-3′ and EpoR-rev 5′- CCAggAgCACTACTTCATTg-3′, Lyn-fw 5′-GATCCAGAGGAACAAGGTGA-3′ and Lyn-rev 5′-TGACATCACCATGCATAGGG-3′.RNAs were prepared as previously descibed spi-1 transgenic mice in vitro in the presence of either Epo (1 U/mL) or SCF (100 ng/mL) The three proerythroblastic cell lines  used in this study were derived from erythroleukemic spleens of three individual Next, we compared the EpoR and Kit expression levels in the cell lines cultured in a combination of Epo+SCF (SCF was added in Epo-cultured cells for 5 days) to cells cultured with either Epo alone or SCF alone. On Western blotting, Kit expression levels were high with SCF alone, but significantly reduced when Epo was present . SimilarWe next assessed whether the modulation of Kit expression by Epo affected the proliferation of cells cultured in the presence of a combination of Epo+SCF . When EpKit transcription. The expression level of kit transcripts was analyzed by semi-quantitative RT-PCR using RNAs extracted from cells cultured in the presence of either Epo or SCF  were extensively washed and then switched to SCF (100 ng/mL). 48 hrs later, cell extracts were analyzed by immunobloting with an anti-Kit antibody. Switching from Epo to SCF induced a marked increase in Kit level . Reciprospi-1 transgenic proerythroblasts.The next issue was to identify the effectors downstream of EpoR that may participate in the regulation of Kit expression. According to the known function of Src kinases in signaling from growth factors receptors Lyn transcripts, RT-PCR analyses of RNAs prepared from cells continuously cultured with either Epo or SCF were performed. Lyn transcripts were only detected in Epo-cultured cells (Lyn expression.To determine whether the variations in Lyn protein expression were associated to variations in the expression level of ed cells  suggestiWT) and the neomycine resistance gene (NeoR) was stably transfected in Epo-cultured 663 cells. Control cells were transfected with an empty vector encoding NeoR. G418-selected clones were selected in the presence of Epo and then amplified in the presence of Epo or SCF. Among those, two MT-LynWT and two control clones were studied in details and gave similar results. Only data with one clone are shown herein. Expression of exogenous MT-LynWT was detected by Western blotting with an anti-MT antibody and Lyn global expression was measured with an anti-Lyn antibody  to inhibit Lyn function. Stable G418-resistant transfectants were selected and amplified in the presence of Epo. As illustrated in Y397F detected by immunoblotting with the MT antibody was associated with an increase in Kit level in the Epo-cultured cells. Thus, Lyn inactivation by a dominant-negative mutant allowed an up-regulation of Kit expression.To explore whether Lyn was a link between EpoR activation and Kit down-regulation, we used an enforced expression strategy. An expression vector encoding Lyn tagged with a Myc epitope (MT) at the C-terminus  and that this modulation was rapidly reversible by a change in cytokine. Modifications in Kit levels were detected both in whole cell extracts and at the cell surface indicating that they in fine may lead to a modified response to SCF. The variations in surface expression of Kit analyzed by flow cytometry were significantly lower than that of total amount of cellular Kit analyzed by SDS-PAGE. These differences might be a consequence of the occurrence of Kit receptor internalization following SCF-induced activation We took advantage of this heterogeneity to investigate the molecular processes involved in the control of Kit and EpoR expression in response to their cognate ligands. Each cell line (six were studied and three are shown) had a proliferation rate in the presence of SCF that was correlated with the level of Kit expression. However, we observed that Kit levels were modulated by the cytokine used to expand the cells  in combination with SCF, Epo was the only player in controlling cell proliferation. It is reasonable to propose that the down-regulation of Kit expression leads to the loss of SCF responsiveness. In this regard, SCF and Epo cooperation would be restricted to early stages of erythropoiesis when Kit level is high. This agrees with data localizing the physical interaction of Kit and EpoR before or at the CFU-E stage in vivo erythropoiesis Several laboratories have described synergistic features between Kit and EpoR co-signaling to maintain the growth and survival of erythroid progenitors Our findings add to our knowledge of Lyn action in erythropoiesis. Analyses of Lyn-deficient mice revealed that CFU-E exhibited a reduced proliferative capacity associated with attenuated responses to Epo and SCF and that erythroblasts presented defects in survival and maturation spi-1 transgenic proerythroblasts. In contrast, Stat5 was activated by Epo. Because phosphorylated Stat5 levels in cells cultured under Epo paralleled the expression level of Lyn, a prediction could be that Stat5 was an effector downstream of Lyn in Epo-dependent proliferation of cells. Though Stat5 has been characterized as a direct substrat for Lyn during Epo-induced differentiation of the J2E cell line Stat5 phosphorylation is induced by Epo in erythroid cells In conclusion, our findings on the down-regulation of Kit expression by the EpoR/Lyn pathway in the context of a late erythroid progenitor cell (CFU-E/proerythroblast transition) provide some insights into the mechanisms leading to Kit extinction in maturing erythroblasts. We postulate that the control of Kit expression during erythroid development results from interference between Epo and SCF signaling where activation of the EpoR/Lyn pathway would lead the proerythroblast to the strict Epo dependency through mediating extinction of Kit expression. In this light, it will be interesting to determine if such a down-regulation of the activity of a cytokine active on early multipotent progenitors by a cytokine active later on the maturation of a lineage specific progenitor illustrates a general process in the hematopoietic development.


The Expanded Program on Immunization (EPI) is the most cost-effective measures to control vaccine-preventable diseases. Currently, the EPI schedule is similar for HIV-infected children; the introduction of antiretroviral therapy (ART) should considerably prolong their life expectancy.+ T cells <25%).To evaluate the persistence of antibodies to the EPI vaccines in HIV-infected and HIV-exposed uninfected children who previously received these vaccines in routine clinical practice, we conducted a cross-sectional study of children, aged 18 to 36 months, born to HIV-infected mothers and living in Central Africa. We tested blood samples for antibodies to the combined diphtheria, tetanus, and whole-cell pertussis (DTwP), the measles and the oral polio (OPV) vaccines. We enrolled 51 HIV-infected children of whom 33 were receiving ART, and 78 HIV-uninfected children born to HIV-infected women. A lower proportion of HIV-infected children than uninfected children had antibodies to the tested antigens with the exception of the OPV types 1 and 2. This difference was substantial for the measles vaccine . We observed a high risk of low antibody levels for all EPI vaccines, except OPV types 1 and 2, in HIV-infected children with severe immunodeficiency (CD4+ T cell counts. This study supports the need for a second dose of the measles vaccine and for a booster dose of the DTwP and OPV vaccines to maintain the antibody concentrations in HIV-infected and HIV-exposed uninfected children.Children were examined at a time when their antibody concentrations to EPI vaccines would have still not undergone significant decay. However, we showed that the antibody concentrations were lowered in HIV-infected children. Moreover, antibody concentration after a single dose of the measles vaccine was substantially lower than expected, particularly low in HIV-infected children with low CD4 Pediatric HIV infection is a major public health threat. Two thirds of the 700,000  children less than 15 years old newly infected with HIV in 2005 were living in sub-Saharan Africa The World Health Organization (WHO) recommendations for immunization of HIV-infected children differ slightly from the general guidelines for HIV-uninfected children In this study, we evaluated the persistence of antibody levels in HIV-infected and HIV-exposed uninfected children born to HIV-infected mothers, living in Central Africa and who previously received EPI vaccines in routine clinical practice. In addition, we assessed the influence of host and viral related factors  on the association between HIV infection and the absence of antibody to EPI vaccines.i.e. ≥ 37 weeks) to an HIV-infected mother who was diagnosed in the context of the mother to child transmission prevention program; and (iii) they had received at least a primary vaccine series of three doses of the combined diphtheria, tetanus, and whole-cell pertussis (DTwP) vaccine, the total oral polio (TOPV) before one year of age, and one dose of the measles vaccine (documented on an immunization card). The HIV-1 status has been already confirmed by serology in all included children between 15 and 18 months of age. Children were not included in the study if (i) their clinical status was severely impaired (including advanced or severe HIV-associated symptoms) or (ii) they were breastfed six months before the enrolment period to avoid recent HIV infection through breastfeeding.We conducted a cross-sectional study in 4 pediatric care centers ). Children were recruited from November 2004 to June 2005. Children were eligible if: (i) they were between 18 and 36 months of age; (ii) they were born  given at birth; a primary vaccination with three doses of the DTwP combined vaccine and TOPV given at 6, 10, and 14 weeks; and recommended booster doses of the DTwP combined vaccine and TOPV given at 18 months of age. The measles vaccine was scheduled to be given at nine months of age and a second dose was recommended at 12 to 15 months of age.All parents or legal guardians gave written informed consent. The study was approved by National Ethics Committees in Cameroon and in CAR. The study was conducted in accordance with the Declaration of Helsinki.+ T cells were counted with a fluorescence activated cell sorter (FacScan) flow cytometer .Blood samples were collected and processed at the Centre Pasteur in Yaoundé (Cameroon) and at the Institut Pasteur in Bangui (CAR). Complete blood count, proteinemia and tests for HIV-1-infected children including assessment of HIV-1 viral load and lymphocyte subpopulation counts were done. Thick blood smears were also performed for malaria diagnostics. In Cameroon, the plasma HIV-1 RNA viral load (VL) was quantified by a commercial assay  according to the manufacturer's instructions. The threshold for quantification was 50 HIV-1 RNA copies/ml. In Bangui, values for the plasma HIV-1 RNA were determined by real-time TaqMan RT-PCR with the protocol established by the Working Group for Viral Quantification of the ANRS Bordetella. All the samples were tested in duplicates, and results were expressed as the mean values for the two determinations. The antibody level was considered adequate if the antibody concentration or titer was above the cut-off value defined by the manufacturers or by WHO recommendations.The antibody concentrations or titers to measles, diphtheria, tetanus, and polio types 1, 2 and 3 were analyzed at the laboratory sites. Serological testing for pertussis was performed by the French National Reference Center for Antibodies to measles were initially measured with a commercial ELISA kit  in which plates were coated with inactivated measles virus Antibodies to diphtheria and tetanus toxoids were measured using commercial ELISA kits . Sera with antibody concentrations of, at least, 100 mIU/ml were considered positive. For the wP vaccine, agglutinins (AGG), i.e. antibodies that agglutinate bacteria mostly directed against fimbrial antigens, were measured. These AGG were detected by the classical micro-agglutination test 50 doses of the corresponding Sabin-type poliovirus. HEp-2 cells were added and plates were scored after five days. The neutralizing antibody titer was the end point dilution of sera that protected 50% of the cell culture. Detectable antibody at a dilution of 1∶8 was considered positive for that poliovirus serotype . The geometric mean concentration (GMC) for each poliovirus serotype was calculated as IU for all samples.Neutralizing antibody titers against each of the three poliovirus types were measured using a micro-neutralizing assay, based on the WHO recommended procedure The chi-square or Fisher's exact test was used as appropriate to compare categorical variables of HIV-infected and uninfected children. The student's t-test was used to compare continuous variables if the data adhered to normality assumptions; otherwise the Mann-Whitney test was used. The results for antibodies to the DTwP vaccine, the measles vaccine and the TOPV were reported as the GMC or GM titers (GMT) with the 95% confidence intervals (CI) for each group.+ T cells  Univariate and multivariate logistic regression models were used to assess the effect of covariates on the odds ratios (OR) of a low antibody concentration to vaccine (only the serotype 3 for the OPV). Composite categorical variables were created to evaluate the effect of HIV infection: i) HIV status combined with the percentage of CD4The first two criteria for eligibility were met by 177 children (54 HIV-infected and 123 HIV-exposed uninfected), but the third criteria (EPI schedule) was not fulfilled in three HIV-infected and 45 HIV-exposed uninfected children (44 from CAR). Thus, 129 children were included in the study: 51 perinatally HIV-infected (16 in the CAR and 35 in Cameroon) and 78 HIV-exposed uninfected (44 in the CAR and 34 in Cameroon). The mean age of children enrolled was 24.6 months . Sex ratio, duration of breastfeeding, weight, MUAC + T cells was as follows: <15% (n = 15), 15–24% (n = 16) and ≥ 25% (n = 20).According to the WHO pediatric clinical staging system + T-cell counts lower than 25% had significantly low levels of antibodies, particularly to the live attenuated measles vaccine. A short duration of ART or no ART was significantly associated with a low measles antibody concentration (tested by EIA) as shown in the + T cells, HIV VL ≥10,000 copies) was also associated with a low antibody concentration. Only the covariates linked to HIV status  were statistically significant. More children with low measles antibody concentration than those with adequate antibody concentrations were breastfed; however, the difference was weakly significant.We observed that the proportion of HIV-infected children with adequate antibody levels to all the tested vaccine antigens was lower than that of HIV-exposed uninfected children . The dif+ T-cell percentage was considered in these analysis, due to the high agreement between the composite categorical variables . For each EPI vaccine, HIV-infected children with severe immunodeficiency (CD4+ T-cell percentage <25%) had a significantly higher risk than HIV-exposed uninfected children of having low antibody levels  in CAR could not be included because they did not meet the criteria about the EPI vaccine schedule showing how much a successful EPI could be difficult to implement and to maintain at a national level. Logistics related to supply, storage and administration of vaccines can lead to difficulty in applying the EPI recommended schedule. A recent study in Zambia showed a lower rate of EPI vaccine coverage in HIV-infected children et al. linked low antibody response to the measles vaccine to nutritional status (stunting) rather than to HIV infection + T-cell deficiency in HIV-infected children likely impairs mounting an effective memory against vaccines, and secondary vaccine failure due to a decline in vaccine-induced antibody over time has been described Low responsiveness to the measles vaccine in HIV-infected children has been reported previously + memory T cells, whereas the regenerative capacity of resting central memory and naïve T cells remains largely intact + memory T cells in the mucosa, affecting pathogen-specific adaptive responsiveness. These mechanisms have not been studied in HIV-infected children born to HIV-infected mothers. Children were examined at a time when their antibody concentrations would have peaked and still not undergone significant decay, suggesting either a lack of inducing immediate vaccine response or, at least, a lack of immune capacity to maintain an adequate antibody level.The poor antibody levels to EPI vaccines observed in perinatally HIV-infected children is probably related to major immunological dysfunction involving both cellular and humoral responses. The peripartum transmission of HIV correlates with rapid disease progression and a high mortality rate in HIV-infected infants We included, as a reference group, perinatally HIV-exposed uninfected children; this was initially for practical reason because the HIV status of the mothers and the children were already determined at the time of the cross-sectional study. We expected similar antibody concentrations in this reference group and in the HIV unexposed and uninfected children We also observed that the persistence of antibody to the TOPV, particularly to types 1 and 2 was less affected in HIV-infected children than to the other EPI vaccines, as reported previously H. influenzae type b conjugate, hepatitis B, pneumococcus and yellow fever vaccines also deserve further investigation in this population. Our findings also highlight the urgent need to delineate the mechanisms of cellular and humoral responses to EPI vaccines in HIV-infected children.We unambiguously observed that the proportion of children with low antibodies levels to the EPI vaccines was associated with the severity of HIV-induced immunosuppression. The benefits of using ART for HIV-infected children


Down-regulation of AR protein expression occurred already after 8 h of bFGF treatment and a maximal inhibition was observed 24 h after addition of bFGF to culture media. As AR expression can be reduced by an increase in intracellular calcium levels, we investigated whether the bFGF effect on AR protein is mediated by this mechanism. Calcium release from intracellular stores and store-operated calcium influx after treatment with either bFGF or calcium ionophore A 23187 were measured by single cell fluorescence technique. The ionophore A 23187 was able to induce calcium influx and an increase in cytoplasmic calcium concentration in LNCaP cells. In contrast, bFGF was incapable of eliciting a similar effect. In contrast to AR protein, AR mRNA levels were not affected by bFGF as shown by semiquantitative reverse transcription polymerase chain reaction. In summary, these studies show that bFGF is a potent negative regulator of AR protein expression in the human prostate cancer cell line LNCaP. © 2000 Cancer Research CampaignInteractions between polypeptide growth factors and the androgen receptor (AR) are important for regulation of cellular events in carcinoma of the prostate. Basic fibroblast growth factor (bFGF), the prototype of heparin-binding growth factors, and the AR are commonly expressed in prostate cancer. bFGF diminished prostate-specific antigen protein in the supernatants of androgen-stimulated human prostate cancer cells LNCaP by 80%. In the present study, we asked whether the bFGF effect on prostate-specific antigen is preceded by action on AR expression. LNCaP cells were treated with bFGF and AR protein expression was determined by immunoblotting and ligand binding assay. bFGF down-regulated AR protein in a dose-dependent manner showing a maximal effect at 50 ng ml


In this study we have determined the effects of the n-6 essential fatty acid gamma-linolenic acid (GLA) on the motility and invasive/metastatic nature of the human colon cancer cell lines HT115, HT29 and HRT18. Cell motility was induced by hepatocyte growth factor/scatter factor (HGF/SF) and measured by both colony scattering and dissociation from carrier beads. Invasiveness was measured in vitro by cellular invasion into extracellular matrix. At concentrations up to 100 microM (which had no effect on cell growth over the duration of the experiments) both cell motility and invasion induced by HGF/SF were markedly reduced by GLA and its lithium salt. The attachment of these cells to the extracellular matrix components (Matrigel and fibronectin) was also inhibited. There were also changes in the cell-surface E-cadherin, but not fibronectin receptor at similar concentrations. It is concluded that n-6 essential fatty acids have the ability to inhibit both motility and invasiveness of human colon cancer cells, perhaps by modifying cell-surface adhesion molecules.


Monocyte-mediated antibody-dependent cellular cytotoxicity (ADCC) was tested in 23 patients with histologically proven Hodgkin's disease and 29 healthy normal controls. Seven patients presented with active and 16 with inactive disease. The lytic capacity of the individual monocytes was significantly (P < 0.02) higher in patients with Hodgkin's disease than in normals. However, no significant difference was found between the numbers of monocytes in both groups of individuals, as determined by non-specific esterase staining. No correlation was found between the lytic capacity of monocytes and the activity of the disease.


Multiple homologs and splice variants of V-ATPase subunits are thought to explain its varied spatial and temporal expression pattern in different cell types. Recently subunit nomenclature was standardized with a total of 22 subunit variants identified. However this standardization did not accommodate the existence of splice variants and is therefore incomplete. Thus, we propose here an extension of subunit nomenclature along with a literature and sequence database scan for additional V-ATPase subunits. An additional 17 variants were pulled from a literature search while 4 uncharacterized potential subunit variants were found in sequence databases. These findings have been integrated with the current V-ATPase knowledge base to create a new V-ATPase subunit catalogue. It is envisioned this catalogue will form a new platform on which future studies into tissue- and organelle-specific V-ATPase expression, localization and function can be based.The vacuolar-type H Partially embedded within the membrane, its function is to transfer two hydrogen ions out of the cytoplasm at the expense of 1 ATP molecule The vacuolar-type HThe V-ATPase is also functionally important at the plasma membrane of specialized cell types in certain tissues. There, it is responsible for critical homeostatic functions such as body acid-base regulation , bone remodeling (osteoclasts), and sperm storage and maturation (clear cells in the epididymis) Loss of V-ATPase activity due to subunit mutations in specific cell types has been implicated in diverse pathophysiological states such as kidney and bone disease, sensorineural deafness and wrinkly skin syndrome 0 and V10 domain is composed of several transmembrane subunits that are involved in hydrogen ion translocation across the bilayer, while the V1 domain is a peripheral to the membrane and is the site of ATP hydrolysis. V0 is composed of 5 subunits labeled a to e while V1 consists of 8 subunits denoted A to H. All 13 different subunits are encoded by separate genes located throughout the genome.The V-ATPase is a large (800 kDa) and complex molecular motor. It is made up of at least 13 individual components/protein subunits organized into two functional domains: VMany of the 13 V-ATPase subunits exist as homologs, thereby adding another level of complexity to the motor. The diverse functions and locations of V-ATPases are believed to be encoded within the various homologs. For example the d1 subunit is ubiquitously expressed, while the d2 homolog is seen only in the kidney, osteoclast and lung An additional level of V-ATPase subunit variation is encoded through splice variants. To date, splice variants have been identified for the a, d, e, C, G and H subunits To date, discovery of V-ATPase homologs and splice variants has largely been subunit focused and experimentally based. This method has proven successful as demonstrated by the large number of identified variants. However, this fragmented discovery process led to a fragmented naming system which was recently standardized At the time of the first V-ATPase nomenclature standardization, the incorporation of splice-variants was not implemented The V-ATPase proton pump is composed of 13 subunits. According to the data presented in Smith et al (2003) these 13 are encoded through 22 homologs. These are denoted with a “K” for known in Experimentally identifying and characterizing the associated functional proteins was outside the scope of this study but should be the subject of future work by investigators interested in V-ATPase function, including our own groups. The results of this study suggest that the V-ATPase is regulated in a much more complex manner than is currently assumed. At what level this regulation is exerted remains to be determined experimentally, and having a complete systems overview of all V-ATPase components will help expedite this process. This almost two-fold increase in the number of V-ATPase subunit forms demonstrates how the output of broad genomic scale projects can be utilized for a specialized pursuit. It also highlights the importance of computational methods for sifting and sorting through vast amounts of data deposited in sequence databases worldwide. Identification and standardization of transcript variation offers a powerful approach to guide the future assignation of functional significance among protein variants.Sequences corresponding to the 13 human V-ATPase subunits described previously


Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. The molecular mechanisms underlying hepatocarcinogenesis are still poorly understood. Genetically modified mice are powerful tools to further investigate the mechanisms of HCC development. However, this approach is limited due to the lack of non-invasive detection methods in small rodents. The aim of this study was to establish a protocol for the non-invasive analysis of hepatocarcinogenesis in transgenic mice using a clinical 1.5 Tesla Magnetic Resonance Imaging scanner.As a model system we used hepatocyte-specific c-myc transgenic mice developing hepatocellular carcinoma at the age of 12-15 months. The scans of the murine livers included axial T2-weighted turbo-spin echo (TSE) images, axial T1-weighted and contrast enhanced T1-weighted gradient echo  and sagittal true Fast Imaging with Steady state Precession (true-FISP) images. Application of contrast agent was performed via tail vein-catheter and confirmed by evaluation of the altered longitudinal relaxation T1 time before and after application. Through technical adaptation and optimization we could detect murine liver lesions with a minimum diameter of approximately 2 mm and provided histopathological evidence that these MR findings correspond to hepatocellular carcinoma. Tumor growth was repeatedly measured using sequential MRI with intervals of 5 weeks and subsequent volumetric analysis facilitating direct comparison of tumor progression between individual animals. We finally demonstrated that our protocol is also applicable in the widely- used chemical model of N-nitrosodiethylamine-induced hepatocarcinogenesis.Our protocol allows the non-invasive, early detection of HCC and the subsequent continuous monitoring of liver tumorgenesis in transgenic mice thereby facilitating future investigations of transgenic tumor mouse models of the liver. Hepatocellular carcinomas (HCC) represent one of the most common cancers with growing incidence in several regions around the world. The major cause for HCC development is chronic liver disease. Although the exact molecular mechanism leading to HCC progression is still poorly understood, it is well accepted that chronic liver damage is associated with continuous cycles of apoptosis and/or necrosis followed by compensatory proliferation of hepatocytes. This triggers liver fibrosis and cirrhosis but also results in the accumulation of mutations and transformed hepatocytes due to excessive DNA replication and increased mitosis during liver regeneration. Accordingly, a number of genes involved in cell cycle progression and apoptosis including E2F ,2, c-mycDuring the last decades, the treatment options for patients with advanced HCC have been very limited due to a lack of effective conventional systemic chemotherapy. Promising progress was reported recently by the use of the multi-kinase inhibitor Sorafenib in patients with advanced hepatocellular carcinoma leading to significantly increased overall survival . Consequin vivo. Thus, current reports still rely on a huge amount of animals per group which have to be sacrificed at distinct time points for the measurement of tumor size. Subsequently, methods for the continuous monitoring of liver tumor growth in individual transgenic mice need to be developed.In order to define new and better therapeutical targets, strong efforts have been made during the last years to better understand the molecular mechanisms of hepatocarcinogenesis. One promising approach is the analysis of transgenic - or knockout mice specifically targeting tumor promoters or tumor suppressors such as c-myc, p53, p21 and others -11. IndeMagnetic Resonance Imaging (MRI) has been used in many clinical, biomedical, chemical and engineering applications. In clinical practice, MRI is used to visualize the internal structure of organs and tissue of the body with a special focus on neurological (brain), cardiovascular and oncological imaging including HCC in particular .In comparison to ultrasound and Computed tomography (CT), MRI has proven to be more reliable when focal liver lesions have to be detected and characterized ,16. ThisCT can also be used for the detection and characterisation of focal liver lesions. However, this modality exposes small rodents to relatively high doses of radiation therefore potentially influencing hepatocarcinogenesis. Ultrasound on the other hand may not allow objective imaging of all anatomical regions in mice. Based on these considerations, MRI was preferred for longitudinal follow up imaging in rodents. MRI is not associated with radiation exposure and can be repeatedly used for imaging of small animals without any long term effects or alteration of the course of the disease.Therefore the aim of this study was to establish a protocol for the non-invasive and reliable detection of murine HCC - induction and progression using MRI techniques. As specialized MR scanner for small rodents with higher magnetic field strength are expensive and not available in most clinical research centres, an additional requirement for this protocol was the applicability for regular clinical 1.5 Tesla MR-scanning systems.After correct positioning of the mouse in the MRI scanner in the supine position, a short localizer sequence  mice Figure  by modifIn T2-weighted spin echo sequences water- and fluid-containing tissues (e.g. vessels) are strongly demarcated from the surrounding tissue and have a bright appearance. T2-weighted imaging of a healthy murine liver clearly demonstrated the dark healthy liver parenchyma crossed by a hyperintense mesh-work of connective blood vessels Figure .In summary, these initial experiments demonstrated that the optimized parameters shown in Tab. tg) are a well established animal model for hepatocarcinogenesis. These animals develop slow growing liver tumors similar to a group of human HCCs with better prognosis and survival [tg mice showing less than 40% of HCCs in 45 weeks old animals, whereas 80% of the 65 weeks old mice had a minimum of one liver tumor  were scanned by MRI and checked for hypointense signals in contrast-enhanced T1-weighted images. Immediately after MRI the animals were sacrificed and the respective livers were macroscopically and histologically analyzed for HCCs. The size and location of the macroscopic tumor nodes was measured and correlated to the MRI findings. Overall, 22 out of 33 animals (66%) with evidence of hypointense signals in contrast-enhanced T1-weighted images displayed also tumor nodes with a size between 2 and 10 mm of diameter.The data for one representative animal is shown in Fig Of note, out of all animals between 45 and 65 weeks of age 100% of tumors with ≥ 4 mm of diameter were verified by MRI, whereas only 70% of tumors with a diameter ≤ 4 mm could be detected with the same settings.So far, our experiments were exclusively performed in c-myc transgenic mice and wildtype controls. In order to prove that our MR protocol also allows detection of murine HCC in tumor models with different pathogenesis, we decided to use the widely-used, well accepted model of chemically induced HCC in mice using N-nitrosodiethylamine (DEN).The carcinogenic effect of DEN is mediated via its capability of alkylating DNA structures. In most mouse strains, a single injection of DEN in juvenile mice at the age of 14 days leads to multinodular hepatocarcinogenesis within 40-50 weeks in 80-100% of the animals . GeneticThe histopathological analysis Figure  confirmeIn summary, these experiments demonstrated that our MRI protocol for HCC imaging in mice is not restricted to c-myc transgenic animals, but also applicable to chemical HCC models with completely different pathogenesis.Imaging of small animals on a clinical scanner in this study was challenging, given the small body volume of the rodents. The application of a dedicated small coil allowed for acquisition of images with a high signal/noise ratio, so that spatial resolution was adapted to small sized animals. Another important issue was the development of a stable logistic protocol, including optimized anaesthesia and positioning of the animals for imaging. Sub-optimal anaesthesia allowed the movement of the animal during image acquisition leading to imaging artefacts. This was especially problematic for T1-weighted images acquired shortly after injection of the contrast medium, since this sequence could not be repeated in the same imaging session, due to the already injected dose. Overall, the standardization and streamlining of the logistics was an important prerequisite for acquisition of high quality images in this study.Although the major aim of this study was the development of a non-invasive imaging technique for murine HCCs which can be used for further basic research in mice, our results could also potentially contribute to improvement of MR imaging of human hepatocellular carcinoma. In the clinical situation the analysis of MR images with respect to HCC detection mostly depends on the experience of the radiologist as matching of MR results with liver histology is restricted to patients with subsequent liver resection or biopsy. In contrast, MR imaging in mice allows the immediate comparison with liver histology in large cohorts. Moreover, multiple genetic and chemical mouse models are available reflecting the pathogenesis of human hepatocarcinogenesis including steatosis-, fibrosis- and cirrhosis models ,22 thus Although several studies have demonstrated that MRI represents one of the highest imaging accuracy of all imaging methods available to date , the diaThe majority of experiments performed in this study were carried out in c-myc transgenic animals as this is an established and well characterized model of hepatocarcinogenesis. Extensive DNA-profiling analysis revealed that this model corresponds to slow growing human HCC associated with better survival ,25. HoweOur own analysis revealed, that during the early phase of DEN-mediated tumor initiation (24 weeks after DEN-induction), MRI was not efficient in detecting small tumor nodules in the liver (data not shown). This might be due to the nature of these tumors with small, multinodular appearance with the challenge to distinguish between single lesions. These problems are enhanced by the small size of the analyzed object (mice versus humans) and a relatively low signal/noise ratio due to the magnetic field strength of 1.5T of the used MR scanner thus defining some limitations of clinical MR scanners in monitoring murine HCC nodules. However, such difficulties might also have been observed in larger subjects, since the multiple small tumours have a low contrast compared to the surrounding tissue. Nevertheless, we clearly could detect advanced, DEN-induced liver tumors in mice 45 weeks after tumor induction, which were retrospectively characterized as well- or moderately differentiated HCC, further demonstrating that clinical 1.5T MR scanners are appropriate tools for liver cancer research in mice.It was shown before that it is basically possible to detect hepatocellular carcinoma in rodents with a clinical 1.5T MR scanner e.g. by using an orthotopic tumor cell implantation model in rats . NeverthOnly a few studies have described the monitoring of murine HCC by MRI so far and these studies mainly took advantage of MR scanners specialized for small animals with a magnetic field strength of 4.7 T or higher . HoweverIn summary, we demonstrated that detection of murine HCC initiation and progression in transgenic mouse models is feasible with clinical 1.5 T MR scanners thus allowing further analysis of potential oncogenes and tumor suppressors in the liver by comparison of respective tumor-susceptible transgenic mouse models. Nevertheless, limitations in reliable detection and characterization of small HCC remain an area of further investigation. This will help to gain better insight and to understand the underlying molecular mechanisms of HCC initiation and development.tg) of male gender as previously described [All animals used for experiments were maintained in a temperature-controlled room with 12-hour light/dark cycle. Animal experiments were approved by the authority for environment conservation and consumer protection of the state North Rhine-Westfalia  and the University Hospital Aachen Animal Care Facility's guidelines. For our study we used transgenic mice carrying a c-myc transgene under the control of the hepatocyte-specific albumin promoter  intraperitoneally as described earlier ,31. Usuai.p.). For contrast enhancement 0.025 mmol/kg body weight gadoxetic acid disodium  was administered by tail vein injection using a catheter with injection port .All animal interventions including MRI analysis in mice were carried out under anaesthesia using 100 mg/kg Ketamin and 10 mg/kg Rompun . A 47 mm diameter coil  was used for signal reception. For volumetric analysis we used an off-line workstation .For preparation of whole livers, mice were anesthetized and livers were immediately perfused through the inferior vena cava with 4% formaldehyde/PBS for 20 minutes. Subsequently the complete liver was extracted and fixed in 4% formaldehyde/PBS for 24-48 hours and then embedded in paraffin  according to its original three-dimensional topography. In order to perform tissue sections from formalin-fixed and paraffin-embedded liver which exactly parallels MR images, the surface of the formalin-fixed liver was marked with a color code corresponding to topographical hallmarks . Afterwards the whole organ was embedded in paraffin and orientated as found in the MR images. Serial 4 μm sections were performed and subsequently stained with hematoxylin/eosin following routine protocols.PCNA immunhistochemistry was performed on 5 μm paraffin embedded sections with fixation in 4% formaldehyde/PBS for 5 min using an anti-PCNA antibody  at a dilution of 1:200 and a secondary goat anti-mouse-HRP antibody  at a dilution of 1:100. Final staining was performed with DAB solution  and counterstaining with hematoxylin.HCC: hepatocellular carcinoma; MRI: magnetic resonance imaging; TSE: turbo spin echo; FFE: fast field echo; true-FISP: true fast imaging in steady state precession; TR: repetition time; TE: time of echo; FOV: field of view; CT: computed tomography; WT: wildtype; PBS: phosphate-buffered saline; PCNA: proliferation cell nuclear antigen.The authors declare that they have no competing interests.in vivo experiments, analyzed data, carried out immunohistochemical staining of tissue slides and contributed to manuscript draft. NG performed histological and pathological analysis of liver samples and contributed to manuscript draft. NM performed the chemical induced hepatocarcinogenesis in wildtype mice and analyzed tumor samples. RWG provided MRI equipment and supervised imaging experiments. CT coordinated the study and contributed to manuscript preparation. CL designed the study and wrote the manuscript. GAK coordinated the experiments, analyzed the MRI data and wrote the manuscript.JF performed most of the All authors read and approved the final manuscript.


Cell responses to environmental stimuli are usually organized as relatively separate responsive gene modules at the molecular level. Identification of responsive gene modules rather than individual differentially expressed (DE) genes will provide important information about the underlying molecular mechanisms. Most of current methods formulate module identification as an optimization problem: find the active sub-networks in the genome-wide gene network by maximizing the objective function considering the gene differential expression and/or the gene-gene co-expression information. Here we presented a new formulation of this task: a group of closely-connected and co-expressed DE genes in the gene network are regarded as the signatures of the underlying responsive gene modules; the modules can be identified by finding the signatures and then recovering the "missing parts" by adding the intermediate genes that connect the DE genes in the gene network.ClustEx, a two-step method based on the new formulation, was developed and applied to identify the responsive gene modules of human umbilical vein endothelial cells (HUVECs) in inflammation and angiogenesis models by integrating the time-course microarray data and genome-wide PPI data. It shows better performance than several available module identification tools by testing on the reference responsive gene sets. Gene set analysis of KEGG pathways, GO terms and microRNAs (miRNAs) target gene sets further supports the ClustEx predictions.Taking the closely-connected and co-expressed DE genes in the condition-specific gene network as the signatures of the underlying responsive gene modules provides a new strategy to solve the module identification problem. The identified responsive gene modules of HUVECs and the corresponding enriched pathways/miRNAs provide useful resources for understanding the inflammatory and angiogenic responses of vascular systems. Understanding of cell responses to environmental stimuli is one of the central tasks of molecular biology. Genome-wide gene expression profiling techniques, such as microarray and deep sequencing, are widely used to identify the responsive genes whose expressions are significantly changed after the stimulus. But identifying the responsive genes by differential expressions does not consider the complex gene-gene interactions or regulation information. Increasing evidences suggest that cell responses are usually organized as pathways or responsive gene modules consisting of a group of interacted genes at the molecular level -4. IdentHere we presented a new formulation of the module identification task: a group of closely connected and co-expressed differentially expressed (DE) genes in genome-wide gene networks are regarded as the signatures of the underlying responsive gene modules at the RNA expression level. Our method named ClustEx was designed to find those signatures in the first step. Many studies show that the genes which are co-expressed in RNA level and/or interacted in protein level tend to involve in the same biological process, and promising new discoveries have been found by using the co-expression ,17 and/oin vitro models to study the vascular systems in inflammation and angiogenesis. We collected two time-course microarray datasets: one is for tumor necrosis factor alpha (TNF) stimulated HUVECs, an inflammation model [Human umbilical vein endothelial cells (HUVECs) are widely used as on model -24, and on model -28. Thenon model -31. Resuon model , microRNon model ,34 and Gon model  identifiClustEx is a two-step method for identifying the responsive gene modules by combining gene expression and interaction information. In the clustering step, average linkage hierarchical clustering was used to cluster and partition the DE genes into different gene groups according to their distances in gene networks, based on the assumption that a group of closely-connected and co-expressed DE genes are the signatures of the underlying responsive gene modules. In the extending step, the intermediate genes on the k-shortest paths between the DE genes were added to form the final responsive gene modules Figure . The detz-score = 50.279 for the biggest module; z-score = 9.72 for the second module).ClustEx was applied to identified the responsive gene modules of HUVECs in inflammation model using the 0~8 h time-course microarray expression profiling data  and To validate our predictions, three different TNF reference responsive gene sets were collected from 1) NetPath "TNF/NF-kB signaling pathway", 2) PID/BioCarta/Reactome annotated TNF signaling pathways, and 3) PubMed abstracts. We compared our predictions with several available module identification tools. The original node-based approach using simulated annealing (CytoScape jActiveModules plug-in ) and theApoptosis, Notch signaling pathway, Jak-STAT signaling pathway, Toll-like receptor signaling pathway and Cell cycle  target genes were conducted to find additional supporting evidence. Sixteen pathways were enriched in the biggest responsive gene module identified by ClustEx, including many known pathways affected by TNF, such as le Table . Years ad by TNF -42. Our  process . jActiveFor the enriched miRNA target gene sets (the target gene sets are downloaded from the TargetScan website ): comparapoptosis, protein kinase cascade and I-kB kinase/NF-kB cascade. Apoptosis and I-kB kinase/NF-kB cascade are two main programs activated by TNF. These two GO terms are consistent with the enriched KEGG pathways. The detail information of the enriched GO terms is documented in Additional file We also analyzed the enriched GO terms of the biggest responsive gene module. The enriched terms for TNF are mainly divided into three classes: z-score of the biggest module is 39.81. On the literature reference gene set (VEGFLitRef), FoldChange_2.0 achieves highest sensitivity and ClustEx show competitive performance with jActiveModules, while on the reference gene set collected from pathway databases (VEGFPathDBRef), ClustEx achieves highest specificity and competitive sensitivity to FoldChange_2.0 , a canoTGF-beta signaling pathway, Cell cycle and Wnt signaling pathway are frequently reported to be related to VEGF stimulus [(anti-)apoptosis and RNA/nucleic acid transport related terms , which are significantly up-regulated by TNF stimulus and covered in the biggest TNF responsive gene module . TheTaking the closely-connected and co-expressed differentially expressed (DE) genes in condition-specific gene networks as the signatures of the underlying responsive gene modules provides a new strategy to solve the module identification problem. The responsive gene modules can be identified by finding the extended sub-networks from groups of clustered DE genes. Following this strategy, a two-step method named ClustEx was proposed and applied to identify the responsive gene modules of HUVECs within inflammation and angiogenesis. ClustEx shows better performances than several available module identification tools on reference responsive gene sets. The following gene set analysis of pathways and miRNA target genes also support ClustEx predictions.Two time-course datasets were downloaded from NCBI GEO database ,61: GSE9PPI data were downloaded from HPRD (Release 7) -31. OnlyFirst, the maximum fold change  respect to the 0 h00 m signal was calculated for each gene. Then the genes with minimum 2-fold changes (either up-regulated or down-regulated) were selected as the DE genes. We found 1421 DE genes (15.7%) in the TNF dataset and 709 DE genes (9.36%) in the VEGF dataset.Cell responses to environmental stimuli are usually organized as relatively separate responsive gene modules. We clustered and partitioned the DE genes into different groups based on their interactions and their dynamic expression correlations. Each edge of the gene network derived from HPRD PPIs was weighted asAnd the distance between two direct-interacting genes was defined asThe gene-gene distance was defined as the length of the shortest path between the two genes in the gene network. The shortest path length between any pair of DE genes was calculated using Dijkstra's algorithm. Then average linkage hierarchical clustering was used to cluster the DE genes according to the gene-gene distances. Distance cutoff was set to partition the DE gene into separate gene groups.Hierarchical model analysis (HMA), a basic density-based clustering algorithm, is also used to cluster the DE genes. The detail description of this algorithm and the corresponding results are presented in  [To reduce the false positives on the long paths and the huge computational cost for finding the k-shortest paths between all pairs of nodes in the whole gene network, the extending step was implemented as follows: first, the genes on the shortest paths between the DE genes were added to form a connected sub-network; then the sub-network was extended by one step in the whole gene network ; finally, the responsive gene modules were identified by extracting all the genes and edges on the 10-shortest paths between all the pairs of the DE genes in the extended sub-network. The k-shortest paths were calculated using an implementation of Yen's algorithm (k-shortest paths mean the shortest k [1network) . NecessaSimilar to find the cutoff of the hierarchical clustering, we traced the size expansion of the biggest responsive gene module by increasing "k" from 1 to 20. No obvious cutoff was observed as in the curve of the size of the biggest DE gene cluster in the previous section. We empirically selected "k" as 10: the increased module size from 0 to 10 is more than 5 times as the increased size from 10 to 20   and sd(y) are the standard derivations of the expressions of gene x and y in microarray datasets, respectively. |cor| is the Pearson correlation of gene x and y . The module score (mscore) was calculated by summing the escores of all edges in the moduleThen we randomly sampled the same number of edges in the whole network and calculated the shuffled module scorez-score:The random sampling processes were repeated 10,000 times and the statistical significance was evaluated by http://bioinfo.au.tsinghua.edu.cn/member/~gujin/clustex/ or via email: jgu@tsinghua.edu.cn. Current release requires huge computational cost, especially long waiting time. We will develop future version to solve this problem. We will also include the scripts to help determine the parameters of ClustEx  in the future version.To facilitate the usage of ClustEx, we prepared the ClustEx package including two network distance calculation programs (modified Yen source codes are included in the package), several Perl scripts and the installation script. Users can download the package via our website: We prepared several reference responsive gene sets to evaluate the performances of the computational approaches:TNFLitRef (TNF literature reference gene set), 376 genes. The gene symbols were analyzed and extracted from the 998 PubMed abstracts (before 2009/11/10) using keyword (TNF AND HUVEC*) by Agilent Literature Search (v2.71), a CytoScape plug-in. Then gene symbols were converted to Entrez Gene IDs by IDConverter [TNFNetPathRef (TNF NetPath pathway reference gene set), 184 genes. All Entrez Gene IDs were derived from "TNF signaling pathway" curated in NetPath database [TNFPathDBRef (TNF pathway database reference gene set), 63 genes. Entrez Gene IDs of the reference genes were derived from following TNF related signaling pathways: BioCarta "TNF/stress related signaling", "TNFR1 signaling pathway and TNFR2 signaling pathway" [onverter  , 342 genes. The gene symbols were analyzed and extracted from the 871 PubMed abstracts (before 2009/11/10) using keyword (VEGF AND HUVEC*) by Agilent Literature Search (v2.71). Then gene symbols were converted to Entrez Gene IDs by IDConverter. The genes not covered by HPRD or Affy U133A array were removed. VEGFPathDBRef (VEGF pathway database reference gene set), 109 genes. Entrez Gene IDs of the reference genes were derived from BioCarta "VEGF, Hypoxia, and Angiogenesis", PID "Signaling events mediated by VEGFR1 and VEGFR2" and KEGG "VEGF signaling pathway" [pathway" . The genWe compared the gene lists between the identified responsive gene modules and the reference gene sets. The sensitivity is defined as the percentage of genes in the reference gene set covered by the identified responsive gene module:S/N is calculated as the following definition:The signal-to-noise ratio (S/N) was used to evaluate the significance of overlapping. The signal is defined as the number of overlapped genes between the identified responsive gene module and the reference gene set; the noise is defined as the mean of the numbers of the overlapped genes between control modules and the reference gene set: 10,000 control gene sets each with the same size as the studied module were randomly sampling from the complete gene list and then jActiveModules with simulated annealing searching  and edgea) The edge-based scoring method. The Matlab and Java codes are obtained by email. The package was run as the following parameters: simulated annealing start temperature 1 (default), end temperature 0.01 (default)/0.001 and iteration 30000 (default)/10000. The package was run multiple times with different random seeds. The produced biggest gene modules are no larger than 20 genes for the TNF dataset. Similar results are observed for the VEGF dataset.b) jActiveModules with simulated annealing. This Cytoscape plug-in was run with the default parameter except changing the iteration to 100,000  and switching the Hubfinding On/Off. We ran multiple times with different random seeds. No significant modules were produced by the plug-in.c) jActiveModules with greedy search. The program was run with its default parameter ("search depth" = 1 and "max depth from start node" = 2). The produced modules with the highest scores were used in the comparisons.d) GXNA. The program was run with "-depth 300" for the TNF dataset  and "-depth 250" for the VEGF dataset .Meet/Min values, commonly used to evaluate the overlapping of the two gene sets [Meet/Min values mean higher enrichments:ene sets , were adz-scores of Meet/Min were calculated as:Degree preserving permutation methods were used to generate 1,000 random pathways and the z-score > 3.0 were reported as enriched in the corresponding responsive gene modules.The pathways with gscores) were proposed to evaluate the importance of gene x in the responsive gene module:Based on the assumption that the genes with higher expression changes, higher correlation with their neighbors and higher connection degrees would be more important, the network-based gene importance scores . TargetScan (v5.1) [Degree preserving permutation methods were used to generate 1,000 random miRNA target gene sets and the n (v5.1) ,34 miRNAGJ did most of the computational analyses and manuscript writings. CY focused on the biological background and the interpretation of the computational results. LS and LY lead the project and gave extensive instructions for this work. All authors read and approved the final manuscript.The biggest responsive gene modules. The list of the Entrez IDs of the genes in the biggest responsive gene modules and the differentially expressed genes of TNF/VEGF stimulated HUVECs.Click here for fileThe enriched KEGG pathways. the detail results of the gene set analysis of KEGG pathways in the biggest responsive gene modules for TNF/VEGF stimuli.Click here for fileThe enriched miRNA target gene sets. the detail results of the gene set analysis of miRNA target gene sets in the biggest responsive gene modules for TNF/VEGF stimuli.Click here for fileThe enriched GO terms. The detail results of the gene set analysis of GO biological process terms in the biggest responsive gene modules for TNF/VEGF stimuli.Click here for fileThe hierarchical mode analysis. the description of the hierarchical mode analysis (HMA) algorithm and the corresponding results.Click here for fileThe performance comparison among different module identification methods. the detail results of the performance comparison among different methods, including ClustEx, ClustEx_HMA, jActiveModules and GXNA.Click here for file