Vimentin is one of the cytoplasmic intermediate filament proteins which are the major component of the cytoskeleton. In our study we checked the usefulness of vimentin expression in identifying cases of breast cancer with poorer prognosis, by adding vimentin to the immunopanel consisting of basal type cytokeratins, estrogen, progesterone, and HER2 receptors.179 tissue specimens of invasive operable ductal breast cancer were assessed by the use of immunohistochemistry. The median follow-up period for censored cases was 90 months.38 cases (21.2%) were identified as being vimentin-positive. Vimentin-positive tumours affected younger women (p = 0.024), usually lacked estrogen and progesterone receptor (p < 0.001), more often expressed basal cytokeratins (<0.001), and were high-grade cancers (p < 0.001). Survival analysis showed that vimentin did not help to delineate basal type phenotype in a triple negative  group. For patients with 'vimentin or CK5/6, 14, 17-positive' tumours, 5-year estimated survival rate was 78.6%, whereas for patients with 'vimentin, or CK5/6, 14, 17-negative' tumours it was 58.3% (log-rank p = 0.227).We were not able to better delineate an immunohistochemical definition of basal type of breast cancer by adding vimentin to the immunopanel consisted of ER, PgR, HER2, CK5/6, 14 and 17 markers, when overall survival was a primary end-point. Vimentin is a 57 kDa intermediate filament (IF) protein, which forms a part of the cytoskeleton. Six major classes of IFs are believed to be relatively specific for certain cell types, for example keratin in epithelial cells, neurofilaments in neurons, glial fibrillary acid protein in glial cells, desmin in muscule cells and vimentin in mesenchymal cells. Obviously, they are variably expressed in different cell types and in corresponding tumours. Expression of vimentin and cytokeratins has also been described in breast carcinomas ,2.Moreover, vimentin is selectively expressed in aggressive breast cancer cell lines . ElevateVimentin reactive cells in benign and malignant breast tissue have been described by many authors ,7. The sFew reports are in opposite, as they showed that vimentin expression did not inversely predict patient survival .ERBB2 oncogene) and basal-like phenotypes  could reliably be defined as basal-like tumours, making these two subgroups synonymous . Others Recent studies have compared the prognostic significance of three- and five-biomarker  surrogate panels to define intrinsic breast cancer subtypes and have suggested that the extended immunopanel provided more specific definition of basal like breast cancer, which can better predict survival of breast cancer patients .The aim of our study was to assess if the immunopanel consisted of triple negative phenotype  with the addition of basal cytokeratins  or vimentin could better delineate a basal type tumour group and better predict patient survival when compared to only pure ER, PgR, HER2 negative phenotype.A series of 179 formalin fixed, paraffin-embedded invasive ductal carcinomas not otherwise specified were acquired from the archives of the Pathology Department of Copernicus Memorial Hospital, Lodz, Poland. Patients had undergone surgery  between 1997 and 2001. The median patient age at surgery was 56 years .The primary pathologic diagnosis was confirmed in H&E staining. All operative and pathologic reports were reviewed to confirm disease stage. Follow-up period was defined as a time from surgery to the last observation for censored cases or death for complete observations.Sections of 2 μm thickness were cut and mounted onto polylysine-coated slides, which were stained for vimentin, estrogen receptor (ER), progesterone receptor (PgR), HER2, cytokeratin 5/6, 14 and 17, Ki-67, cyclin E and p-cadherin.Antibodies against:- vimentin (Dako), dilution 1:50, antigen retrieval: autoclave, high pH;- CK5/6 (Dako), 1:100, autoclave, high pH;- CK 14 (Novocastra), 1:20, microwave oven, citrate buffer, pH 6;- CK17 (Novocastra), 1:40, microwave oven, citrate buffer, pH 6;- ER (Dako),1:35, microwave oven, citrate buffer, pH 6;- PgR (Dako),1:75, microwave oven, citrate buffer, pH 6;- HER2  and Ki-67 (Dako), 1:200, microwave oven, citrate buffer, pH 6;- cyclin E (Dako), 1:40, microwave oven, citrate buffer, pH 6;- p-cadherin (Dako), 1:200, microwave oven, citrate buffer, pH 6.Any distinct positive staining of tumour parenchyma with vimentin antibody was regarded as vimentin expression. Positive staining in fibroblasts, endothelial cells, lymphocytes and macrophages served as 'built-in' positive control, furthermore, negative staining of epithelial cells in non-neoplastic tubules served as negative control.For CK5/6, CK14 and CK17, membranous staining results were classified as follows: negative – no staining seen in invasive tumour cells, positive – weak or strong staining seen in invasive cancer cells.ER and PgR nuclear staining scoring was done using the method described by McCarty et al. . TumoursHER2 staining was scored according to Herceptest kit manufacturer's instructions and score 3+ denoted HER2-positive tumours.Ki-67 and cyclin E labeling indices were defined as the percentages of tumour cells displaying nuclear immunoreactivity and were calculated by counting nuclear stained tumour cells in 1000 tumour cells. For cyclin E, samples were classified as being negative (<2%) or positive (≥ 2%).For p-cadherin, a semiquantitative scoring system was used, taking into account both the intensity of staining and the proportion of tumour cells showing the positive reaction. The scores of staining intensity were recorded from 0 (no staining) to 3 (strong staining). The scores of staining area were recorded as 1 (<10%), 2 (10–50%) or 3 (>50%). A staining index (SI) was obtained by multiplying the score of staining intensity by the score of staining area, negative cases had SI = 0–1, positive ones had SI = 2–9.Pearson's chi-square test or Fisher exact test were used to test for contingency between dichotomized values of vimentin expression (negative and positive) and values of other histopathological and clinical parameters. Patient survival was calculated from the date of primary surgery to the date of death or the last follow-up according to the Kaplan-Meier method. Data for patients who died from other causes than breast cancer were censored at the time of death. Differences in survival distributions were evaluated by a log-rank test. Univariate survival analyses were performed with the use of Cox proportional hazards method. All results were considered statistically significant when two-sided p was less than 0.05. The analyses were performed using the StatsDirect software .The median follow-up period for all patients was 71 months , and for 113 censored (living) patients it was 90 months .Vimentin expression was observed in 38 cases 21.2%)   and significant in triple negative tumours (p = 0.035). In the non-triple negative group only not significant tendency towards such relationship was observed (p = 0.065).There was also a statistically insignificant but quite obvious tendency towards a relationship between vimentin and cyclin E. Vimentin-positive tumours more frequently expressed cyclin E (p = 0.058) Table . Relatio54 patients had triple negative tumours 30.2%)  Table .In 54 30.2%) triple negative patients vimentin as a single marker did not predict clinical outcome  -positive and triple negative ' markers was formulated and its prognostic value has been checked out by the comparison with 'basal cytokeratin -positive and triple negative ' panel, in which vimentin is omitted.These two basal phenotype immunopanels were adversely associated with survival in patients with non-triple negative cancer Table . This efThe present study also supports our previous analysis which showed that basal cytokeratins (CK5/6 and CK17) expression had not any impact on survival in patients with breast cancer .The possible association of vimentin with clinically aggressive behaviour of tumours described by others -9,11 mayHowever, we cannot offer a better indicator of basal type breast cancers by adding vimentin to the diagnostic panel when overall survival is a primary end-point. Also, an immunopanel defined as CK5/6 or 14 or 17-positivity did not show any significant prognostic value in survival analysis in a triple negative group. Five marker method proposed by Cheang et al.  showed sIn summary, may we conclude that adding vimentin to an immunopanel consisted of basal cytokeratins  appears to be inefficient at predicting survival of triple negative breast cancer patients.The authors declare that they have no competing interests.RUK – conceived and coordinated the study, performed experiments, analyses, interpreted data and wrote the manuscript. RK – acquisition of funding, general supervision of the research group. EP, AKB, JHP – acquisition of data, edition of the draft manuscript. PP – participated in analysis and interpretation of data, performed the statistical analysis, was involved in drafting the manuscript and revised it critically. All authors read and approved the final manuscript.


Human adenovirus type 37 (HAdV-37) is a major etiologic agent of epidemic keratoconjunctivitis, a common and severe eye infection associated with long-term visual morbidity due to persistent corneal inflammation. While HAdV-37 has been known for over 20 years as an important cause, the complete genome sequence of this serotype has yet to be reported. A detailed bioinformatics analysis of the genome sequence of HAdV-37 is extremely important to understanding its unique pathogenicity in the eye.We sequenced and annotated the complete genome of HAdV-37, and performed genomic and bioinformatics comparisons with other HAdVs to identify differences that might underlie the unique corneal tropism of HAdV-37. Global pairwise genome alignment with HAdV-9, a human species D adenovirus not associated with corneal infection, revealed areas of non-conserved sequence principally in genes for the virus fiber (site of host cell binding), penton , hexon , and E3 . Phylogenetic analysis revealed close similarities between predicted proteins from HAdV-37 of species D and HAdVs from species B and E. However, virtual 2D gel analyses of predicted viral proteins uncovered unexpected differences in pI and/or size of specific proteins thought to be highly similar by phylogenetics.This genomic and bioinformatics analysis of the HAdV-37 genome provides a valuable tool for understanding the corneal tropism of this clinically important virus. Although disparities between HAdV-37 and other HAdV within species D in genes encoding structural and host receptor-binding proteins were to some extent expected, differences in the E3 region suggest as yet unknown roles for this area of the genome. The whole genome comparisons and virtual 2D gel analyses reported herein suggest potent areas for future studies. Mastadenovirus, Aviadenovirus, Atadenovirus, and Siadenovirus , HAdV-4 [AY487947], HAdV-7 [AC_000018], HAdV-9 [AJ854486], HAdV-12 [AC_000005], HAdV-17 [AC_000006], HAdV-40 [L19443]. Previously sequenced HAdV-37 penton base protein, hexon protein, fiber protein, and VA RNA gene accession numbers are AAG00906, ABA00016, AAB71734, and U10679, respectively. The GenBank accession number for HAdV-37 is DQ900900.The nucleotide sequence for the following HAdVs can be found in GenBank: HAdV-2 [in silico 2D gel program JVirGel 2.2.3b [Percent identities and similarities between proteins of HAdV-37 and other HAdVs were determined using Fasta3 ,79 and Bl 2.2.3b . Phylogel 2.2.3b . BootstrCMR designed primers, annotated the virus, performed the bioinformatics analysis, and drafted the manuscript. FS performed the PCR, and assisted with compilation of the sequence. AFG and DWD participated in primer design, sequence compilation and analysis, and manuscript writing. JC conceived the project design, and participated in the data analysis writing of the manuscript. All authors read and approved the final manuscript.2D Gel Analysis. Virtual 2D gel analysis. Protein migration patterns for select HAdV proteins by virtual 2D gel. Each spot represents a given serotype's homologue based on its predicted amino acid sequence. A. DNA binding protein, B. Viral polymerase, C. Penton base, D. Hexon, E. Protease, and F. pVIII. One protein from HAdV-37 and a homologue from a representative serotype of all 6 HAdV species are represented in each gel.Click here for file


The skeletal neuromuscular junction is a useful model for elucidating mechanisms that regulate synaptogenesis. Developmentally important intercellular interactions at the neuromuscular junction are mediated by the synaptic portion of a basal lamina that completely ensheaths each muscle fiber. Basal laminas in general are composed of four main types of glycosylated proteins: laminins, collagens IV, heparan sulfate proteoglycans and nidogens (entactins). The portion of the muscle fiber basal lamina that passes between the motor nerve terminal and postsynaptic membrane has been shown to bear distinct isoforms of the first three of these. For laminins and collagens IV, the proteins are deposited by the muscle; a synaptic proteoglycan, z-agrin, is deposited by the nerve. In each case, the synaptic isoform plays key roles in organizing the neuromuscular junction. Here, we analyze the fourth family, composed of nidogen-1 and -2.In adult muscle, nidogen-1 is present throughout muscle fiber basal lamina, while nidogen-2 is concentrated at synapses. Nidogen-2 is initially present throughout muscle basal lamina, but is lost from extrasynaptic regions during the first three postnatal weeks. Neuromuscular junctions in mutant mice lacking nidogen-2 appear normal at birth, but become topologically abnormal as they mature. Synaptic laminins, collagens IV and heparan sulfate proteoglycans persist in the absence of nidogen-2, suggesting the phenotype is not secondary to a general defect in the integrity of synaptic basal lamina. Further genetic studies suggest that synaptic localization of each of the four families of synaptic basal lamina components is independent of the other three.All four core components of the basal lamina have synaptically enriched isoforms. Together, they form a highly specialized synaptic cleft material. Individually, they play distinct roles in the formation, maturation and maintenance of the neuromuscular junction. The formation, maturation and maintenance of chemical synapses require multiple interactions between pre- and postsynaptic elements. Many of these interactions are mediated by membrane- or matrix-associated proteins that occupy the narrow cleft separating the pre- and postsynaptic membranes -3. At th2[α2(IV)]1 trimer, whereas the α3–6 chains are selectively associated with synaptic BL, presumably in [α3(IV)][α4(IV)][α5(IV)] and [α5(IV)]2[α6(IV)]1 trimers [in vivo. Laminin β2 promotes the maturation of motor nerve terminals [Members of four families of proteins are present in BLs throughout the body: laminins, collagens IV, heparan sulfate proteoglycans (HSPGs), and nidogens/entactins (referred to here as nidogens) . Synapti trimers ,11,12. T trimers -15. Impoerminals ,17, lamierminals , and togerminals . Synaptierminals -25.Caenorhabditis elegans, the lone nidogen gene is necessary for the formation of the NMJ, although the protein is not synaptically enriched [In contrast to this wealth of knowledge about laminins, collagens IV and HSPGs, little is known about localization or roles of nidogens at the neuromuscular synapse. In enriched . Unlike enriched -30. In menriched -33, and enriched -36. Moreenriched , supportNidogens-1 and -2 are similar in domain structure but differ in sequence . As shown previously , nidogenen face-sectioned NMJs, allowing us to distinguish three distinct domains in BL near synaptic sites: synaptic BL, which occupies the synaptic cleft; extrasynaptic BL, in directly adjoining stretches of the muscle fiber surface; and Schwann cell BL, atop Schwann cell processes that themselves cap nerve terminals  receptor tyrosine phosphatase ,37,39,47Another possible explanation for the defects in nidogen-2-deficient NMJs is that nidogen-2 might be required to recruit or retain other synaptic BL components that are, in turn, required for synaptic integrity. To test this possibility, we immunostained nidogen-2 mutant muscle with a panel of antibodies to synaptic BL components. As expected, no nidogen-2 was present in mutants Figure . AlthougC. elegans and zebrafish [nid2-/- mice. Collagen XIII was concentrated at NMJs in control muscle, as reported previously [nid2-/- muscles, collagen XVIII was undetectable in synaptic BL, although it was associated with the BL of terminal Schwann cells (data not shown). Thus, the mechanisms by which nidogen-2 contributes to synaptic maturation and maintenance remain to be determined.A third possibility is that nidogen-2 selectively binds and presents a matrix-associated synaptic organizing molecule to nerve or muscle. Indeed, several bioactive matrix molecules have been reported to bind with much higher affinity to nidogen-2 than nidogen-1, including tropoelastin, collagen XIII and collagen XVIII/endostatin ,51-53. Iebrafish ,55,56. Weviously ,57 and tRecently, we and others have observed striking intermuscular differences in neuromuscular phenotypes in mutants lacking agrin or collagen IV chains α3–6 ,58,59. Tet al. [nid2-/- mice, tibialis anterior and extensor digitorum longus, are 'fast synapsing' muscles [Muscles, and the NMJs within them, differ from each other in many respects  and it iet al. . Called et al. ,61,62. I muscles .Biochemical studies have shown that interactions among three main structural components, laminins, collagens IV, and nidogens, are involved in BL assembly ,64. MoreFragmentation and immaturity similar to that observed in nidogen-2 mutants has been reported in collagen α5(IV) and laminin α4 mutants and in laminin α4/α5 double mutants, respectively ,18,19. HFinally, we broadened our inquiry to ask whether any synaptic BL components are necessary for the recruitment and restriction of other BL components to the synaptic cleft. As noted above, nidogen-2 is not required for synaptic localization of laminins, collagens IV or agrin, nor does it require synaptic laminins or collagens IV to become synaptically localized. On the other hand, previous results show that loss of a single laminin subunit or collagen IV chain can lead indirectly to absence of other components of the trimer from the synaptic cleft ,12,65. HC. elegans it has been proposed that nidogen exerts it synaptic organizing activity through a receptor protein tyrosine phosphatase present in presynaptic terminals [We report here that nidogen-2 is selectively associated with synaptic BL at the NMJ and required for maturation or maintenance of this synapse. Combined with previous studies on laminins, collagens IV and HSPGs, these findings establish that each of the four major families of BL components has isoforms that are enriched in synaptic BL and required for synaptogenesis: laminins α4, α5, and β2, collagens α3–6(IV), agrin ,11,12,15erminals . We are Our studies also provide the foundation for analyzing assembly of the synaptic cleft. Biochemical evidence has suggested that laminins, collagens IV, and nidogens are all necessary for BL assembly: laminins for initial BL formation; collagens IV for BL stabilization and maintenance; and nidogens for cross-linking laminin and collagen IV networks ,66-68. Anid2-/-) [lamb2-/-) [lama4-/-) [col4a5Y-/) [lama5flox/flox) [lama5flox/flox and HAS-Cre mice; we refer to the lama5flox/flox;HSA-Cre mice as lama5M/M. Mice lacking both laminin α4 and α5  were generated by crossing lama4-/-;lama5flox/+; HSA-Cre males and females. All mutants and transgenics were maintained on a C57/B6 background. In most cases, littermates of mutants were used as controls. CD1 mice were obtained from Charles River Laboratories, Inc. . All analyses conformed to NIH guidelines and were carried out under an animal protocol approved by the Harvard University Standing Committee on the Use of Animals in Research and Teaching.Targeted mutant and transgenic mice used in this study have been described previously. They are: nidogen-2 mutants (nid2-/-) , lamininamb2-/-) , lamininama4-/-) , collagel4a5-/Y) , obtaineox/flox) , and micox/flox) . MutantsPrimary antibodies used in this study are listed in Table Full-length human nidogen-1 and -2 fusion proteins were purchased from R&D Systems . Recombinant proteins (250 ng) were denatured by boiling in Laemmli buffer and separated by SDS-PAGE. Electrophoretically separated proteins were transferred to Immuno-Blot PVDF membrane  in Tris-Glycine buffer /20% methanol at 300 mA for 2 hours. Immunoblotted proteins were detected as previously described .Mice were perfused with phosphate-buffered saline (PBS) and tibialis muscles were dissected. Tissue was immediately frozen in OCT on dry ice and 4 μm sections were cut on a cryostat. Sections were collected on gelatin-coated slides and allowed to air-dry for 15 minutes before tissue was fixed by incubating in ice-cold acetone for 10 minutes. For collagen IV antibodies, tissue was treated for 10 minutes in a 1:1 mix of 1 M KCl and 1 M HCl following fixation . Sectionet al. [C2C12 cells  were cultured as previously described by Kummer et al. . MyotubeAchR: acetylcholine receptor; BL: basal lamina; HSPG: heparan sulfate proteoglycan; NMJ: neuromuscular junction; P: postnatal day; PBS: phosphate-buffered saline.The authors declare that they have no competing interests.JRS and MAF conceived the study, constructed the experimental design, and drafted the final manuscript together. MAF performed the experiments. MSPH and NRS provided nidogen mutant mice. All authors read and approved the final manuscript.


Stephania, possesses dopamine (DA) D1 partial agonistic and D2 antagonistic properties in the nigrostriatal and mesocorticolimbic DAergic pathways. These unique dual effects have suggested that SPD can effectively restore previously imbalanced functional linkage between D1 and D2 receptors under schizophrenic conditions, in which, SPD improves both the negative and positive symptoms of schizophrenia. SPD also relieves the motor symptoms of Parkinson’s disease (PD) when co-administered with Levodopa. Furthermore, SPD exhibits neuroprotective effects through an antioxidative mechanism and slows down the progression of neuronal degeneration in the substantia nigra (SN) of PD patients and/or animal models. Therefore, SPD is a novel, natural compound with potentially therapeutic roles in the treatment of schizophrenia and/or PD.(-)-Stepholidine (SPD), a natural product isolated from the Chinese herb  Initially, SPD was reported to decrease blood pressure without exerting any adverse effect on the heart [(-)-Stepholidine (SPD), extracted from the Chinese medicinal plant he heart , 84, andhe heart , 75. Thehe heart , 83, 87.2.via specific DAergic receptors, which are particularly expressed in the nigrostriatal and mesocorticolimbic pathways.The DAergic system in the CNS plays important roles in the control of cognitive, motivational, motor and endocrine functions. DA exerts its pharmacological effects The nigrostriatal pathway, which is comprised of DA-synthesizing neurons of the substantia nigra pars compacta (SNc) that project to the striatum, contains nearly 80% of all the DA in the CNS. This pathway is crucial for motor coordination, and the degeneration or dysfunction of this pathway results in the characteristics of Parkinson’s disease (PD). The mesocorticolimbic pathway, which arises in the VTA and projects to the PFC and limbic areas, such as the NAc and the olfactory tubercle, is involved in regulating motivational behavior, learning, memory and cognition. The degeneration or dysfunction of this DAergic pathway is implicated in the development of schizophrenia. Therefore, this pathway may be the target of antipsychotics.s proteins to positively activate adenylyl cyclase, while D2 receptors act through Gi and Go proteins to inhibit cAMP production. Thus, D1 and D2 receptors exert opposing effects and balance the two signaling pathways through the activation and inhibition of adenylyl cyclase activity. Although D1 and D2 receptors belong to distinct subfamilies of DA receptors, several lines of evidence indicate there is an important functional linkage between these two subclasses of receptors.Recently, five subtypes of DAergic receptors, which belong to the family of seven-transmembrane heterotrimeric G-protein coupled receptors, have been cloned and identified. Two different categories of DAergic receptors, termed D1-like (D1 and D5) and D2-like (D2-D4), have been identified on the basis of DNA sequence similarities . D1 receet al. demonstrated that the abolished long-term depression (LTD) of synaptic transmission in the striatum after DA depletion could be restored by exogenous DA or co-administration of specific D1 and D2 agonists, but not by application of either selective agonist alone [et al. observed that virtually all striatal neurons contained both D1 and D2 receptors  [et al. demonstrated that D1 and D2 receptors are co-expressed and co-localized within striatal neurons and that these two receptors are part of the same heteromeric protein complex and a phospholipase C-mediated calcium pathway that is only activated by both D1 and D2 receptor co-activation [Behavioral, biochemical and electrophysiological evidence has revealed synergism between D1 and D2 receptors , 44, 65.st alone . Therefoceptors  . Using btivation . These rTherefore, functional synergism between D1 and D2 receptors is necessary for maintaining some normal physiological functions, and once the functional linkage between the two receptors is broken, disorders, such as schizophrenia, may develop.3.The DAergic system in the SNc, substantia nigra pars reticulata (SNr), neostriatum, globus pallidus, and subthalamic nucleus is known to play a pivotal role in the control and coordination of movement. In 1968, Ungerstedt developed a new technique for lesioning the nigrostriatal DA system by injecting 6-hydroxy-DA (6-OHDA) into the SN, which produced unilateral anterograde degeneration of the nigro-neostriatal DA system and constituted the first rat hemiparkinsonian model . Using tin vitro radioligand binding assay demonstrated that SPD was able to bind to the D1 receptor at two sites, a high-affinity site (Ki = 3.9 nM) and a low-affinity site (Ki = 126 nM), using calf striatal membrane preparations [In the 1980s, Jin and colleagues started examining the pharmacological effects of SPD and its analogs using the 6-OHDA-induced PD rat model . They fiarations . These ret al. demonstrated that SPD displays D1 agonistic effects at a low dose (0.5 or 1 mg/kg) but D1 antagonistic effects at a high dose (4 or 10 mg/kg) when co-administered with APO during behavioral experiments [i = 13 nM, [In contrast, in control and reserpinized rats (rodents treated with reserpine at 1 mg/kg for 6 days to deplete endogenous DA), SPD exerts an antagonist effect on the D1 receptor. SPD reversed and/or significantly attenuated SNc DA cell firing induced by SKF38393 or by the mixed DA receptor agonist apomorphine (APO) in reserpinized rats. This effect was similar to that produced by SCH23390 , 64. It = 13 nM, ), but ex= 13 nM, , 84.What is the mechanism by which SPD exhibits this controversial effect on the D1 receptor? Although the exact reasons for this difference are still uncertain, it is clear that SPD shows D1 agonistic action in the presence of SCH23390 and exhibits antagonistic effects in the presence of SKF38393, meaning that as a partial agonist, SPD displays a large range of intrinsic activities at the same D1 receptors depending on different conditions. Another possible reason explaining this controversial effect is that the D1 receptor may exhibit different sensitivities under these two conditions. In the 6-OHDA-induced lesioned model, a relatively large amount of endogenous DA is depleted, which consequently enhances D1 receptor sensitivity. However, in normal rats, D1 receptors are not sensitive enough to observe the agonistic effects of SPD , 90.In conclusion, SPD modulates D1 receptors depending on the receptor’s status. Under normal conditions, SPD works as a D1 receptor antagonist, while in the 6-OHDA-induced PD model, SPD serves as a partial agonist.i = 85 nM) is 4-7 times lower compared to the D1 receptor (Ki = 13 nM) [i proteins on D2 receptor-coupled AC [In addition to modulating D1 receptor function, SPD also show the ability to bind to the D2 receptor. Although its affinity for the D2 receptor (K= 13 nM) , SPD sho= 13 nM) , 84. It = 13 nM) , increas= 13 nM) , and inc= 13 nM) . Moreoveupled AC . In reseupled AC . In 6-OHupled AC , 37, 86.4.in vivo electrophysiological study in rats demonstrated that at a low dose (1-64 μg/kg), SPD increased VTA DA cell firing and attenuated APO-induced inhibition of DA cell spontaneous firing, suggesting that SPD blocks D2 receptors located on VTA DA cells, thereby feed-back regulating spontaneous activity [SPD-induced dual D1 receptor agonistic and D2 receptor antagonistic effects have also been demonstrated in mesocorticolimbic structures, including the mPFC, NAc, and VTA DAergic systems. An activity . Howeveractivity , 63.in vivo experiments using rats have demonstrated that intravenous (i.v.) administration of SPD  produced two opposing effects on the spontaneous firing of NAc neurons. Since D2 receptors on VTA DA neurons are 3-10-fold more sensitive to SPD than those in the NAc , a low dose (0.02-0.08 mg/kg) of SPD blocked D2 receptors on VTA DA neurons [via blockade of VTA D2 receptors. With pretreatment of spiperone (a D2 antagonist), only an excitatory effect is observed, but the excitatory effect is reversed by SCH23390, which suggests that SPD works as a D1 receptor agonist and induces excitation [via the mPFC D1 receptor [Additional  neurons  and redu neurons . Therefo neurons , meaningcitation . The D1 citation , suggestreceptor , 88, 89.receptor . Thereforeceptor .5.. In particular, DA receptors are present on vascular smooth muscle cells (vascular DA receptor) [It is well-known that specific DA receptors are expressed in both the CNS and extracerebral structureseceptor)  and on peceptor) . It has eceptor) . Functioeceptor) , 45, 48.eceptor)  and renaeceptor) .+-K+-ATPase activity, and rat renal protein kinase C isoform expression between normotensive and hypertensive rats suggest that renal D1-like receptor dysfunction may be the mechanism responsible for generation of hypertensive rats [et al. discovered that aberrant DAergic regulation of aldosterone secretion mediated via DA D2-like receptors may also contribute to hypertension [Evidence suggests that hypertension results from overactivity of central DA neurons in spontaneously hypertensive rats (SHRs). The prevailing opinion is that D2-like receptor function is upregulated in SHRs , 51, 71.ive rats . Gordon rtension . The absrtension . In addirtension .As mentioned above, SPD is a D1 receptor partial agonist and D2 receptor antagonist in the CNS, and studies have demonstrated that SPD also exerts dual effects on peripheral DA receptors. SPD (0.1-10 μM) shifts dose-response curves to the right and decreases the maximal response of both fenoldopam (a selective D1 agonist)-induced and propyl-butyl-DA (a selective D2 agonist)-induced vasorelaxation, showing a non-competitive antagonistic action on both D1 and D2 receptors. In addition, SPD (0.1-100 μM) induces slight but dose-dependent vasorelaxation of renal and pulmonary arteries, thereby displaying D1 agonistic activity .It is possible that SPD may be a promising antihypertensive agent due to its interaction with both central and peripheral DA receptors. Furthermore, the inhibitory effect of SPD on α1-adrenoceptor-mediated contractile responses evoked by nerve-released and exogenous noradrenaline in peripheral small- resistance arteries might contribute to its antihypertensive effect  .2+ release and block Ca2+ channels. Using aortic strip preparations, a low dose (0.3-100 μM) of SPD inhibited norepinephrine (NE)-induced Ca2+ release from intracellular stores [2+ channels [2+ induced by high extracellular Ca2+ and NE [2+ channels by SPD was weaker than that of verapamil  [In addition, SPD has been reported to inhibit Car stores  whereas channels , 52, 58.+ and NE . Howeverblocker) .Collectively, SPD reverses the contraction of peripheral arteries in cerebral, mesenteric, renal and coronary regions, and results in a decrease of total peripheral resistance, thus leading to its antihypertensive effects .6.· [et al. found that SPD increased levels of phospho-AKt, thus keeping neurons viable following exposure to H2O2 neurotoxicity. These results suggest that SPD can maintain cell survival following exposure to certain neurotoxins or serve as an antiapoptotic factor [Morphological and biochemical experiments have demonstrated that SPD possesses neuroprotective effects . The pos· . Recentlc factor . Finallyc factor .Considering all of its effects, SPD may be utilized as a cytoprotective drug to attenuate ischemia-induced neuronal injury, thereby maintaining neuronal survival, and may even be used to promote neuronal regeneration. These properties of SPD suggest potential therapeutic applications for the treatment of neurodegeneration.7.7.1.Schizophrenia is a chronic, devastating mental disorder. It is characterized by the presence of both negative and positive symptoms. Schizophrenia is the most serious of all psychiatric illnesses and affects about 1% of the world's population, but the etiology of schizophrenia remains obscure. According to recent clinical and experimental studies, the cause of schizophrenia may involve an imbalance of cortical/subcortical DA systems. Specifically, hyperactivity of D2 receptors in subcortical mesolimbic regions, such as the NAc, may produce the positive symptoms of schizophrenia, while hypofunction of D1 receptors in the mPFC is considered to be responsible for the negative symptoms and cognitive impairment , 14, 55.D1 and D2 receptors have important functional interactions (see section 2). However, these interactions were abnormal in over half of the postmortem striata from schizophrenic patients, suggesting an impaired functional linkage between D1 and D2 receptors in schizophrenic patients . TherefoBefore atypical neuroleptics were discovered, clinical doses of the available antipsychotic drugs primarily blocked D2 receptors , 56, butClinical trials are currently testing the potential use of SPD as an antipsychotic drug. Co-administration of SPD and typical antipsychotic drugs  could significantly enhance the effectiveness of drug therapy for mental disorders. In addition, SPD alone has been shown to exhibit a significant therapeutic effect. Furthermore, a double-blind comparative study demonstrated that SPD not only improved the positive symptoms of schizophrenia, but also significantly alleviated the negative symptoms compared to perphenazine, a common drug prescribed for schizophrenic patients , 37, 74.7.2.PD, after Alzheimer’s disease, is the second most common progressive neurodegenerative disorder in the United States, affecting about one million people, with more than 50,000 new cases being diagnosed each year. Its prevalence in the U.S. population aged over 50-years old is above one percent, and likely exceeds five percent in individuals aged over 80-years old . As a re·, may play a key role [The pathogenesis of PD typically is slow-paced but relentlessly progressive. Understanding of the etiopathogenesis of PD is still limited. According to current knowledge, PD develops as a consequence of DAergic neuronal degeneration in the SNc. It appears that several factors  contribute to this degenerative process , 42, butkey role , 42, 53.The most accepted therapeutic strategy for the treatment of PD is essentially DA replacement. L-dopa, the direct precursor of DA, in combination with carbidopa, remains the most effective drug combination. The majority of patients taking these drugs experience benefits initially, but drug effectiveness declines after 2-5 years, evidenced by the emergence of significant adverse effects associated with L-dopa. Long-term L-dopa therapy increases oxidative burden , thus wo2O2 neurotoxicity [SPD may be potentially used as a novel therapeutic strategy for PD. As previously described, SPD not only protects striatal cells against transient cerebral ischemic injury , but alstoxicity , 80. Mortoxicity . Also, Stoxicity . In additoxicity . Furthertoxicity . Thus, SAnimal experiments have demonstrated that SPD is well-absorbed in the digestive tract, can be widely distributed in body tissues, and can easily penetrate the blood-brain barrier . SPD may8.SPD has been shown to possess dual D1 agonistic and D2 antagonistic effects in both the nigrostriatal and mesocorticolimbic DAergic pathways. Thus, SPD potentially reverses hyperactivity of subcortical D2 receptors and restores PFC dysfunctional D1 receptors in patients with schizophrenia, which would result in the recovery of functional linkage between D1 and D2 receptors. SPD not only treats the positive symptoms of schizophrenia, but also alleviates the negative symptoms. In addition, SPD also potentially relieves the motor symptoms of PD due to its D1 agonistic and D3 antagonistic effects. Furthermore, SPD exhibits neuroprotective effects by scavenging hydroxyl free radicals, and consequently, results in the slowing down of neuronal degeneration in the SN of patients with PD. Considering its features, such as being well-absorbed in the digestive tract, can be widely distributed in body tissues, and can easily penetrate the blood-brain barrie  , as well


Oligoryzomys species. This is the first report of molecular identification of a human hantavirus associated with Bermejo virus.Two suspected hantavirus pulmonary syndrome (HPS) cases from Bolivia were confirmed by enzyme-linked immunosorbent assay. (ELISA)-ANDES was performed using N-Andes recombinant antigen serology in May and July 2000. Clot RNAs from the two patients were subjected to reverse transcription–polymerase chain reaction (PCR) amplification and sequencing. We describe two characterized cases of HPS. One was caused by infection with Bermejo virus and the other with Andes Nort viral lineage, both previously obtained from  Andes virus (ANDV) has been responsible for most of the >400 cases recorded in Argentina, Chile, and Uruguay; Oligoryzomys is the predominant rodent carrier. A different genus, Calomys laucha, was responsible for most cases in Paraguay associated with Laguna Negra virus (LNV) Rio Mamore virus (RIOM) from Oligoryzomys microtis. RIOMV has not been associated with human cases yet Hantaviruses cause hemorragic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome (HPS) in the Americas. Infection occurs primarily by the respiratory route via aerosolized virus in rodent excreta. Field investigations have identified sigmodontine rodents as the host of the respiratory hantaviruses. HPS cases have been reported in several American countries during the last 8 years including Argentina, Bolivia, Brazil, Canada, Chile, Panama, Paraguay, United States, and Uruguay  were serologically confirmed to have HPS; five died. Five out of the six cases occurred between April and July; the sixth case occurred in November. All the patients lived and worked in rural areas in a 70-km ratio around Bermejo, although it was not clear whether one of them (Patient 2) was infected in Argentina or in Bolivia. In addition, two HPS cases were previously reported in Bolivia, one in June 1998 and the other in October 1999.2 pressure (PaO2) 50mm Hg, arterial CO2 pressure (PaCO2) 32mm Hg, leukocytes 6,700 /mm3, urea 0.36 g/L, bilirubin 0.52. mg/100 mL. Laboratory results 4 days after he was hospitalized were as follows: hematocrit 46%, 46.8%, 48%, 43%; platelets/mm3 50,000, 32,000, 30,000, and 128,000; and white blood cells/mm3 6,700, 15,400, 20,700, and 18,300.On May 18, 2000, a 49-year-old man from Bermejo, Bolivia, who worked as a woodcutter in San Telmo, had a onset of HPS symptoms. Seven days later he was hospitalized at the San Vicente de Paul Hospital and his case was confirmed by enzyme-linked immunosorbent assay (ELISA)-ANDES specific immunoglobulin(Ig) M and immunoglobulin(Ig) G test 2 73, PaCO2 35, and leukocytes 64,300 /μL. The patient died the same day he was hospitalized. HPS was confirmed by ELISA-ANDES-specific IgM and IgG test. Although the patient lived in Bolivia, he worked as a muleteer in Rio Blanco River in Salta Province, Argentina for 2 months and came back to Bolivia 3 weeks before his illness.In July 2000, a 20-year-old man from Bermejo was admitted to the San Vicente de Paul Hospital 4 days after symptoms began; a preliminary diagnosis of HPS was made. The patient’s condition deteriorated rapidly, showing respiratory compromise with bilateral interstitial infiltrates and renal compromise with oliguria. Laboratory results were as follows: serum creatinine 2.3 mg/dL, lactic dehydrogenase (LDH) 572 IU/L, platelets 26,000 /μL, hematocrit 71%, PaOO. chacoensis captured in Orán City, Salta Clot samples from the two patients were used to prepare RNA with an RNA matrix . The RNA was subjected to reverse transcription-polymerase chain reaction (PCR) amplification by nested or heminested reactions. Synthesized DNA products were separated on agarose gels, gel-purified, and directly sequenced with an ABI 377 sequencer. Three cDNA products were produced, allowing generation of nucleotide (nt) sequences of 289 nt in length for an S segment region encoding the most conserved region of the nucleoprotein (N), and 243 nt and 226 nt in length for two M segment regions of G1 and G2, respectively. The nucleotide sequences were determined and compared with known hantaviruses . SequencO. chacoensis was tentatively implicated as the predominant rodent reservoir for Bermejo virus; however, the sample size was too small since Bermejo virus was identified in only 1 rodent O. flavescens), two rodents (O. chacoensis)O. longicaudatus)Oligoryzomys species is now being investigated.In previous studies, Molecular and epidemiologic data showed the presence of Andes Nort lineage circulating in Bolivia. Considering that the incubation period was estimated to be 19.5 days


Seven patients had biliary tumors: 3 cholangiocarcinomas, 2 gall bladder cancers, cystadenocarcinoma, and dysplasia of intrahepaticducts. Nineteen patients received bilioenteric anastomosis. Retained stones and recurrentstones developed in 3 and 4 patients, respectively. Twenty-six patients had no remainingsymptoms; 2 died of operative complication or cholangiocarcinoma; 6 presented symptomscaused by retained stones (n=2), recurrent stones (n=2), bile stasis (n=1), or neuralgia (n=1). In4 of the 6 patients, unrelieved posterior duct strictures caused the symptoms. With a meanfollow-up period of 4.5 years, 30 patients are symptoms free, and 27 are stone free. In patientswith right lobar or bilobar type, intra- and extrahepatic type, and confluence strictures,bilioenteric anastomosis is required. Hepatic resection is a rational treatment for hepatolithiasis,however, meticulous management of biliary tract abnormalities, particularly theposterior duct stricture, is mandatory.Long-term results of hepatic resection for hepatolithiasis in 34 patients having intrahepaticbiliary strictures were studied. The left lateral and the right posterior segmental ducts werecommonly and often simultaneously involved. Fourteen patients had multiple segmentalinvolvement. Hepatic resection included left sided resection (


Cytoplasmic phospholipase A2 (PLA2) is known to be phosphorylated and activated by MAP kinase , an important downstream component of signal transduction, whereas paclitaxel has been shown to inhibit isoprenylation of ras proteins . Given that quinacrine (Q), a PLA2 inhibitor, and paclitaxel (P) might act at different sites in the cell signalling pathway, our aim was to test whether they were synergistic in combination against prostate cancer cells. Cell viability of PC-3, PC-3M and DU145 cells in 96 - well plates was assessed 96 h after drugs were added concurrently. Using Chou analysis, we demonstrated synergy for the combination against all three cell lines. Further, synergy was present under both conservative  and non-conservative  models. Studies in the nude mouse xenograft model support the finding of synergy in vitro. In DU145-bearing mice, Q (50 mg kg(-1)) and P (0.5 mg kg(-1)) given daily for 12 consecutive days, either concurrently or sequentially, was more effective than either drug alone, at twice the dose intensity. In an enzyme-linked immunosorbent (ELISA) apoptosis assay, arachidonic acid was able to partially reverse Q- and P-induced apoptosis, suggesting PLA2 pathway involvement. Finally, the combination of lovastatin, another inhibitor of ras isoprenylation, and quinacrine had synergistic inhibitory effects on the growth of PC-3 cells in vitro, suggesting that the combination of these two classes of compounds might serve as an attractive therapeutic approach for prostate cancer.


We describe an outbreak of vomiting, wasting, and encephalomyelitis syndrome in piglets in Argentina, caused by porcine hemagglutinating encephalomyelitis coronavirus (PHE-CoV) infection. Diagnosis was made by epidemiologic factors, pathologic features, immunohistochemistry, reverse transcription–PCR, and genomic sequencing. This study documents PHE-CoV infection in South America. Of 19 farrowing barns, 10 (52.6%) were affected. Total proportion of deaths in pigs that had not been weaned was 16.9% ; an estimated 12.6% of pigs that died had suspected PHE-CoV infection .>4 days of age and consisted of vomiting, listlessness, pallor, and dehydration. Neurologic signs such as abnormal gait, dullness, inability to eat, tremors, and nystagmus were observed in some animals. Vomiting and wasting occurred in 27.6% of pigs <1 week of age and gradually declined to 1.6% in pigs 3 weeks of age (mean 13.6%). Twenty-nine percent of weaned pigs housed in nursery barns that received affected animals from site 1 showed wasting (panel A), and the proportion of deaths was 15%–40%. In total (sites 1 and 2), 3,683 piglets died or were euthanized.Clinical signs were observed in pigs  wasting , panel APostmortem examinations were performed on 16 affected piglets, 2–11 days of age. Samples submitted for histopathologic examination included brainstem, trigeminal ganglia, tonsils, pyloric gland area of the stomach, jejunum, ileum, lymph nodes, heart, spleen, liver, kidneys, and lung.-mouse immunoglobulin G (IgG) goat antiserum at room temperature for 2 h. The color reaction was detected by alkaline phosphatase and 5-bromo-4-chloro-3-indoyl phosphate by using nitroblue-tetrazolium and true red as cromogens.Selected paraffin sections of brainstem from 5 piglets that had characteristic microscopic lesions were examined for PHE-CoV antigen by IHC tests with an anti PHE- CoV-67N strain mouse antibody; the samples were diluted 1:1,000 and incubated overnight at 4°C. Samples were then labeled with biotinylated conjugated antiRNA was isolated with a commercial kit , from brain samples of 7 symptomatic piglets (6–11 days of age) that had nonsuppurative encephalomyelitis, from 1 asymptomatic piglet, and from a PK-15 cell culture suspension inoculated with a pool of tissues from 1 symptomatic piglet. Ribonuclease-A (RNase)–free water was used as negative control. The RT-PCR reaction was performed immediately after RNA isolation by using the specific primer pair for CoV, Cor-FW 5′→3′ (DNA) ACTCAAATGAATTTGAAATATGC, and Cor-RV 5′→3′ (DNA) TCACACTTTGGATAA TCCCA that amplifies a 251-bp fragment of the polymerase gene (www.ncbi.hlm.hig.gov).The amplicons were purified by using the QIAquick PCR purification kit (QIAGEN) and sequenced on a MegaBase 1000 DNA sequencer . The obtained sequence was analyzed by using NCBI BLAST , ganglioneuritis in the trigeminal ganglion, and nonsuppurative encephalomyelitis (panel C). PHE-CoV–positive neurons were found in the brainstem (panel D) and trigeminal ganglion.Microscopic changes were observed in samples taken from 5- to 11-day-old affected pigs. The most remarkable changes were perivascular cuffing around Meissner and Auerbach ganglia in the muscle layer of stomach , panel Bmyelitis , panel Crainstem , panel DRT-PCR analysis showed a product of the expected size for CoV (≈250 bp) in all analyzed brain samples. No amplification was observed in inoculated PK 15 cells . The relFrom an epidemiologic standpoint, the clinical course of the disease (3 weeks), age of affected pigs (<3 weeks), and clinical signs were in agreement with those of VWD caused by PHE-CoV (Pensaert (


To the Editor: Picornaviruses (family Picornaviridae) are small, nonenveloped viruses with single-stranded, positive-sense genomic RNA. They are divided into 12 genera: Enterovirus, Aphthovirus, Cardiovirus, Hepatovirus, Parechovirus, Erbovirus, Teschovirus, Sapelovirus, Senecavirus, Tremovirus, Avihepatovirus, and Kobuvirus. The genus Kobuvirus consists of 2 officially recognized species, Aichi virus  <3 weeks of age in a herd of 400 animals in central Hungary. At this farm, merino ewes from Hungary were mated with blackhead meat rams from Germany. At the time of sampling, no clinical signs of diarrhea were reported. Reverse transcription–PCR was performed by using generic kobuvirus screening primers (UNIV-kobu-R/F) reported previously (www.megasoftware.net). The sequence for kobuvirus/sheep/TB3-HUN/2009/Hungary was submitted to GenBank under accession no. GU245693.On March 17, 2009, a total of 8 fecal samples were collected from young, healthy, domestic sheep  were positive for kobuvirus. The partial 3D region (216 nt) was genetically identical for all 5 strains. The 3′ continuous nucleotide sequence of the partial 3D (688 nt) and 3′ UTR (174 nt) regions of strain kobuvirus/sheep/TB3-HUN/2009/Hungary  was determined. TB3-HUN had 59%–66% (862) nt and 77%–84% aa identities to Aichi and porcine kobuviruses, respectively. Strain TB3-HUN had 89/97% nt/aa and 86% nt identities to bovine kobuvirus in the 3D/3′ UTR (862 nt) and 3′ UTR (174 nt) regions, respectively. Phylogenetic analysis of the overlapping partial 3D/3′ UTR nucleotide sequence of TB3-HUN from sheep and of reference bovine, porcine, and human kobuviruses confirmed that ovine kobuvirus strain TB3-HUN is related to bovine kobuviruses .The nucleotide sequence of the partial 3D/3′ UTR region of kobuvirus in sheep has high nucleotide identity to bovine kobuviruses and forms the same lineage (but a different sublineage) with the kobuvirus strains in cattle. This result raised the following questions: can a highly similar kobuvirus be present in (and pathogenic for) 2 animal species (cattle and sheep), or is this result a consequence of natural contamination? The concept of sheep as host is supported by the high prevalence of kobuvirus in young healthy sheep; by the sublineage position of the sheep strain on the phylogenetic tree according to the most conserved genetic region; and by the genetic relation between the 2 potential ruminant hosts, cattle and sheep. The existence of 1 pathogen in 2 host species (cattle and sheep) is well known, e.g., for bluetongue virus, adenoviruses, ovine herpesvirus type 2, and foot-and-mouth disease picornaviruses (


Sus scrofa) chromosome 10 (SSC10) using the IMNpRH212,000-rad porcine WG-RH panel, integrated with the IMpRH7000-rad WG-RH, genetic and BAC fingerprinted contig (FPC) maps.Whole genome radiation hybrid (WG-RH) maps serve as "scaffolds" to significantly improve the orientation of small bacterial artificial chromosome (BAC) contigs, order genes within the contigs and assist assembly of a sequence-ready map for virtually any species. Here, we report the construction of a porcine: human comparative map for pig  maps, within which FW markers common to both panels have an identical order. This strategy reduced map discrepancies between the two panels and significantly improved map accuracy. A total of 216 markers, including 50 microsatellites (MSs), 97 genes and ESTs, and 69 BAC end sequences (BESs), were ordered within two linkage groups at two point (2 pt) LOD score of 8. One linkage group covers SSC10p with accumulated map distances of 738.2 cR7,000 and 1814.5 cR12,000, respectively. The second group covers SSC10q at map distances of 1336.9 cR7,000 and 3353.6 cR12,000, yielding an overall average map resolution of 16.4 kb/cR12,000 or 393.5 kb per marker on SSC10. This represents a ~2.5-fold increase in map resolution over the IMpRH7,000-rad panel. Based on 127 porcine markers that have homologous sequences in the human genome, a detailed comparative map between SSC10 and human (Homo sapiens) chromosome (HSA) 1, 9 and 10 was built.Map vectors from the IMNpRH212,000-rad and IMpRH7,000-rad, genetic and BAC FPC maps and provides a scaffold to close potential gaps between contigs prior to genome sequencing and assembly. This map is also useful in fine mapping of QTLs on SSC10.This initial comparative RH map of SSC10 refines the syntenic regions between SSC10 and HSA1, 9 and 10. It integrates the IMNpRH2 Therefo is based on the BAC fingerprinted contig (FPC) map  and the BES RH map [7,000-rad panel. An initial draft assembly of SSC10 sequence has recently been released in Pre-Ensembl , but only contains ~30 Mb, roughly one-third of the entire chromosome. Here we report the generation of an initial high-resolution RH map of SSC10 that integrates the IMpRH7,000-rad, IMNpRH212,000-rad RH, porcine genetic and BAC FPC maps, and allowed us to construct a comparative map of SSC10 and HSA1, 9 and 10.SSC10 is a small metacentric chromosome, comprising ~3.1% (85/2700 Mb) of the pig haploid genome. Sequencing of SSC10 by the International Swine Genome Sequencing Consortium (SGSC) and Pig Genome Sequence Project S RH map ,26, whic12,000-rad and the IMpRH7,000-rad panels were used to construct the RH FW map of SSC10. The vectors were merged into one data-set and analyzed using a maximum multipoint likelihood linkage strategy with CarthaGene [7,000-rad  at 1.2  with the average -16. Ther average -34. SSC1 average . The rat, and 22 . Unfortu12,000-rad panel. As shown in Fig. 12,000-rad SSC10 map [It is also worth noting that a pair of markers for five genes  was typed separately on the IMNpRH2SC10 map .  of SSC10  MSs ordered on the IMNpRH2 panel were mapped on the latest SSC10 genetic map  Fig. , of whic Fig. . The ord Fig. . In addiC10 Fig.   were almap Fig.  and 1d, 7,000-rad maps for SSC10 reported by Aldenhoven et al. [However, two small regions of SSC10 did not completely align with the genetic map. The first is the NOR-centromere region. The four singleton MSs  Fig.  were alln et al.  and Rink except for the two small regions also encountered in the genetic map. We estimate the 4 singleton MSs  in the genetic map, the NOR and centromere region of SSC10 cover ~5.5 cM (from 50 cM to 55.5 cM)  Fig.  with therad Fig.  where serad Fig. . One wasThe order of three non-FW BESs  at the end of BAC contig 10006 was also inverted Fig. . Four FW and BLAST  searched against the human genome sequence (Build 36.3) to identify regions of synteny between the human and porcine genomes  that SSC10 is in synteny with regions of HSA1, 9 and 10 [12,000) between FBP1 and CTSL2 (cathepsin L2)  remains a difficult target for physical as well as linkage mapping, the current map provides a platform to integrate additional STS from the wealth of data submitted to the IMNpRH212,000-rad and IMpRH7,000-rad, genetic and BAC FPC maps. This should improve our ability to identify breakpoints and micro-rearrangements and close potential gaps between BAC contigs prior to sequencing and assembly, as well as provide a useful starting point for higher resolution mapping of QTLs including those for meat quality, growth, feed intake and reproductive traits [We merged mapping vectors from the IMNpRH2e traits ,39-41 onPrimer pair sequences were acquired from normalized, essentially full-length cDNA libraries  or from 12,000-rad panel, where they served as internal controls to evaluate mapping accuracy and resolution. Additional file As discussed by Liu et al. , we avoi12,000-rad and the IMpRH7,000-rad panels were used to construct the RH FW map of SSC10 RH as previously described [12,000-rad panel  map was constructed for each linkage group with a likelihood difference threshold of 1000:1. Additional (non-FW) markers were then mapped on the IMNpRH212,000-rad panel with CarthaGene [.Two sets of mapping vectors from the IMNpRH2escribed ,16. The l Tables . The secnal file  and mergrthaGene . Final m or BLAST  searched against human genome sequence (Build 36.3). Once a sequence match (95% and greater similarity of length 40 bp or more) [. Cartesian coordinates of 127 genes/ESTs and BESs based on their map position in HSA 1, 9, and 10 in SSC10 were also developed   was idenJGM participated in data analysis, developed the SSC10 7000-rad and 12,000-rad RH maps, and the pig-human comparative map; the main author for drafting and revising the manuscript. HY participated in porcine gene isolation, primers design, RH typing on the 7000-rad panel, and data analysis. KE carried out RH typing on both 12,000-rad and 7000-rad panels and the consensus mapping (vector); participated in porcine EST sequencing, EST primers design, and data collection and analysis. HH participated in porcine gene isolation, primers design, RH typing on the 7000-rad panel, and data analysis. TS participated in porcine gene isolation, primers design, data collection and analysis. SNM participated in porcine BAC end sequencing, BES primers design, RH typing on the 7000-rad panel, and data analysis. JEB participated in porcine BAC end sequencing, BES primers design, RH typing on the 7000-rad panel, and data analysis. LBS participated in porcine BAC end sequencing, BES primers design, RH typing on the 7000-rad panel, and data analysis. CWB participated in the research design, coordination, porcine EST sequencing, EST primers design and mapping, and helped to revise this manuscript. WSL, principal author, coordinated the research project, carried out consensus mapping (vector), CarthaGene analysis, and map construction; involved in drafting the manuscript and revising it critically for important intellectual content; and edited the final version for publication.SSC10 marker information. This file provides detailed information regarding all markers mapped in this work.Click here for fileIMpRH7,000-rad mapping vectors for the markers on SSC10. This file provides mapping vector and retention frequency of all SSC10 markers in the IMpRH panel.Click here for fileIMNpRH2-12,000-rad mapping vectors for the markers on SSC10. This file provides mapping vector and retention frequency of all SSC10 markers in the IMNpRH2 panel.Click here for file


The objectives of this study were to investigate the molecular mechanisms by which curcumin sensitized TRAIL-resistant LNCaP xenografts in vivo.We have recently shown that curcumin  enhances apoptosis-inducing potential of TRAIL in prostate cancer PC-3 cells, and sensitizes TRAIL-resistant LNCaP cells Prostate cancer TRAIL-resistant LNCaP cells were implanted in Balb c nude mice to examine the effects of curcumin and/or TRAIL on tumor growth and genes related to apoptosis, metastasis and angiogenesis./WAF1, and p27/KIP1, and inhibited the activation of NFκB and its gene products such as cyclin D1, VEGF, uPA, MMP-2, MMP-9, Bcl-2 and Bcl-XL. The regulation of death receptors and members of Bcl-2 family, and inactivation of NFκB may sensitize TRAIL-resistant LNCaP xenografts. Curcumin also inhibited number of blood vessels in tumors, and circulating endothelial growth factor receptor 2-positive endothelial cells in mice.Curcumin inhibited growth of LNCaP xenografts in nude mice by inducing apoptosis (TUNEL staining) and inhibiting proliferation (PCNA and Ki67 staining), and sensitized these tumors to undergo apoptosis by TRAIL. In xenogrfated tumors, curcumin upregulated the expression of TRAIL-R1/DR4, TRAIL-R2/DR5, Bax, Bak, p21The ability of curcumin to inhibit tumor growth, metastasis and angiogenesis, and enhance the therapeutic potential of TRAIL suggests that curcumin alone or in combination with TRAIL can be used for prostate cancer prevention and/or therapy. The process of malignant transformation involves the sequential acquisition of a number of genetic and epigenetic alterations as a result of increasing genomic instability caused by defects in checkpoint controls ,2. TheseCurcuma spp. , a major constituent of the yellow spice turmeric, is derived from the rhizomes of uma spp. . In addiuma spp. -12. We hroblasts , and curroblasts . These dNuclear factor (NFκB) is a dimeric DNA binding protein consisting of members of the NFκB/Rel family . Its expin vitro [in vivo has not yet been demonstrated.TNF-related apoptosis-inducing ligand (TRAIL) binds to TRAIL-R1/DR4 and TRAIL-R2/DR5. TRAIL induces apoptosis in cancer cells of various origins -30. Datain vitro -35. Howein vivo. Our results indicated that curcumin inhibited growth, metastasis, and angiogenesis of TRAIL-resistant LNCaP xenografts in nude mice through regulation of NFκB and its gene products, and sensitized these xenografts to TRAIL treatment. Thus, curcumin can be used alone or combined with TRAIL for prostate cancer prevention and/or therapy.The purpose of our studies was to investigate the molecular mechanisms by which curcumin sensitized TRAIL-resistant prostate cancer cells in vitro [in vivo. LNCaP cells were xenografted in Balb c nude mice. After tumor formation, these mice were treated with curcumin, TRAIL, or curcumin plus TRAIL for 6 weeks to examine their effects on tumor growth and markers of proliferation, apoptosis, metastasis, invasion and angiogenesis. While TRAIL was ineffective in inhibiting the tumor growth, curcumin inhibited the growth of LNCaP xenografts in nude mice  and death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5) by immunohistochemistry in tumor tissues derived from in vivo experiment  and death receptors (DR4 and DR5). By comparison, curcumin induced the expression of Bak, Bax, DR4 and DR5, and inhibited the expression of Bcl-2 and Bcl-XL. These data are in agreement with immunohistochemistry data where the expression of proapoptotic Bak, Bax, DR4 and DR5 proteins were induced and the expression of antiapoptotic Bcl-2 and Bcl-XL proteins were inhibited.We confirmed the immunohistochemistry data by examining the expression of these proteins by the Western blot analysis Fig. . TRAIL h/WAF1/CIP1 and p27/KIP1 in vitro, we sought to validate this phenomenon in vivo in xenografted tumors. Treatment of mice with curcumin resulted in the induction of cell cycle inhibitors p21/WAF1/CIP1 and p27/KIP1 and inhibition of cyclin D1 -positive endothelial cells correlate directly with increase in tumor angiogenesis and can serve as ogenesis ,37,38. AWe next confirmed the immunohistochemistry data of VEGF expression by examining the protein levels by the Western blot analysis Fig. . TRAIL hElevated expression of matrix metalloproteinases (MMPs) and uPA are associated with increased metastatic potential in many tumor cells -42. We tin vitro through activation of multiple signaling pathways [L) and IAPs (XIAP and survivin) [We have recently shown that curcumin induces apoptosis in TRAIL-sensitive PC-3 cells, and sensitizes TRAIL-resistant LNCaP cells pathways . Curcumipathways . Curcumiurvivin) . Since tIn vitro curcumin downregulated the expression of Bcl-2, and Bcl-XL and upregulated the expression of p53, Bax, Bak, PUMA, Noxa, and Bim at mRNA and protein levels in prostate cancer cells [L, and induced the expression of Bax and Bak. The combinatioin of curcumin and TRAIL was more effective in regulating Bcl-2 family members than single agent alone. Our in vitro and in vivo studies demonstrate that curcumin can engage cell-intrinsic pathway of apoptosis by regulating the expression of Bcl-2 family of proteins.er cells . We haveer cells . The abier cells . Furtherer cells . Furtherer cells . In the /WAF1, p27/KIP1, and p16, and inhibition of cyclin D1, cyclin E, Cdk4 and cdk 6 [/CIP1 and p27/KIP1 and inhibit cyclin D1 expression was also confirmed in our xenograft experiment. In a recent study, we have demonstrated that inhibition of p21/CIP1 inhibited curcumin-induced cell cycle arrest and apoptosis [We and others have recently shown that curcumin caused a growth arrest at G1/S stage in several cancers including prostate -45. The poptosis . We and poptosis ,45. Intepoptosis , malignapoptosis , tumor ppoptosis , and patpoptosis . Overallin vivo, tumor cells induce angiogenesis in both primary solid tumors and metastatic foci. Our results showed that curcumin significantly inhibited the growth of TRAIL-resistant LNCaP xenografts and sensitized these xenografts to undergo apoptosis by TRAIL. Tumor tissues derived from curcumin treated mice showed that curcumin inhibibited proliferation (PCNA and Ki67 staining), induced apoptosis (TUNEL staining), metastasis , and angiogenesis (CD31 and VEGF staining). Curcumin also inhibited VEGFR2-positive circulating endothelial cells. Treatment of LNCaP xenografted mice with TRAIL alone had no effect on tumor growth, apoptosis, metastasis and angiogenesis. Our recent in vitro studies demonstrated that curcumin inhibits capillary tube formation and endothelial cell migration, and the inhibitory effects of curcumin were enhanced in the presence of ERK MAP kinase inhibitor [Entry of malignant cells into the vasculature (i.e. intravasation) requires proteolytic remodeling of the extracellular matrix so that tumor cells may pass through the local stroma and penetrate the vessel wall. The circulatory system then provides a means of transporting tumor cells to distant sites where they extravasate and establish metastatic lesions. Matrix metalloproteinase (MMP) is up-regulated in many tumor types and has been implicated in tumor progression and metastasis. MMP is critical for pericellular degradation of the extracellular matrix, thereby promoting tumor cell invasion and dissemination. To grow efficiently nhibitor . These din vitro and in vivo through upregulation of death receptors DR4 and/or DR5 [in vitro study has demonstrated the upregulation of DR4 and DR5 in PC-3 and LNCaP cells by curcumin [in vivo promoted bioluminescent colon tumor xenograft growth and confers resistance to chemotherapeutic agent 5-fluorouracil [TRAIL induces apoptosis in cancer cells which express TRAIL-R1/DR4 and TRAIL-R2/DR5. We have shown that the upregulation of death receptors by chemotherapeutic drugs, irradiation and chemopreventive agents enhance or sensitize cancer cells to TRAIL treatment ,35,51-58d/or DR5 ,28. Simicurcumin -64. Indurouracil . These fL. NFκB-mediated expression of genes, involved in angiogenesis , invasion and metastasis , may further contribute to the progression of prostate cancer. Constitutive NFκB activity has also been demonstrated in primary prostate cancer tissue samples and suggested to have prognostic importance for a subset of primary tumors. In the present study, curcumin inhibited the activation of NFκB and its gene products such as VEGF, Bcl-2, Bcl-XL, uPA, cyclin D1, MMP-2, MMP-9, COX-2 and IL-8 in LNCaP xenografted tumors. These findings suggest that NFκB may play a role in human prostate cancer development, and/or progression, and curcumin can inhibit these processes through regulation of NFκB-regulated gene products.The NFκB family of transcription factors has been shown to be constitutively activated in various human malignancies, including a number of solid tumors, leukemias, and lymphomas . NFκB isin vivo experiments have demonstrated that curcumin sensitizes TRAIL-resistant LNCaP cells through multiple mechanisms. It induces death receptors, upregulates proapoptotic members of Bcl-2 family (Bax and Bak), inhibits antiapoptotic Bcl-2 proteins (Bcl-2 and Bcl-XL) and markers of cell proliferation (PCNA and Ki67), and induces expression of cell cycle inhibitors p21/CIP1, and p27/KIP1. Furthermore, curcumin can also inhibit the activation of NFκB and its gene products  in vitro [in vivo (current study), which play significant roles in invasion, metastasis and angiogenesis. All these events will significantly contribute to the antiproliferative and antitumor activities of curcumin. Clinical trials on curcumin have demonstrated that (1) curcuma extract can be administered safely to patients at doses of up to 2.2 g daily, equivalent to 180 mg of curcumin; and (2) curcumin has low oral bioavailability in humans and may undergo intestinal metabolism [Our in vitro , and in tabolism . Our stuL, Bax, Bak, TRAIL-R1/DR4, TRAIL-R2/DR5 and β-actin were purchased from Santa Cruz Biotechnology Inc. . Antibodies against p21, p27, phospho-p65-NFκB, Cox-2, IL-8, cyclin D1, uPA, MMP-2, and MMP-9 were purchased from Cell Signaling Technology, Inc. . Enhanced chemiluminescence (ECL) Western blot detection reagents were from Amersham Life Sciences Inc. . Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) assay kit was purchased from EMD Biosciences/Calbiochem . TRAIL was purified as described elsewhere [Antibodies against CD31, VEGF, VEGFR2, Bcl-2, Bcl-Xlsewhere . CurcumiWestern blot analysis was performed as we described earlier . Protein6cells as a 50% suspension in matrigel, Becton Dickinson, Bedford, MA) in a final volume of 0.1 ml were injected subcutaneously at right flank of Balb c nude mice. When the average tumor volume reached about 100 mm3, mice were randomized into four groups of 10 mice/group, and the following treatment protocol was implemented: Group 1, vehicle control  administered by oral injection, three times/week  beginning the day of tumor cell implantation through out the duration of experiment; Group 2, TRAIL (15 mg/kg) administered i.v. on day 1, 7, 14, and 21; Group 3, curcumin  administered by oral injection, three times/week  beginning the day of tumor cell implantation through out the duration of experiment; Group 4, curcumin and TRAIL, curcumin administered through oral injection, and TRAIL administered i.v. Mice were housed under pathogen-free conditions and maintained on a 12 h light/12 h dark cycle, with food and water supplied ad libitum. Tumor volume was calculated using the equation: (volume = length × width × depth × 0.5236 mm3). The in vivo experiment was performed under IACUC's approved protocol.Athymic nude mice  were purchased from the National Cancer Institute . LNCaP cells . After washing with PBS, Vectashield (Vector Laboratories) mounting medium was applied and sections were coverslipped and imaged.Immunohistochemistry was performed as described earlier . In brief, tumor tissues were collected on week 6, excised and fixed with 10% formalin, embedded in paraffin and sectioned. Tissue sections were stained with primary antibodies against Bax, Bak, Bcl-2, Bcl-XEMSA was performed as we described elsewhere .The mean and SD were calculated for each experimental group. Differences between groups were analyzed by one or two way ANOVA. The non-parametric Mann-Whitney U test was performed to assess the difference of tumor volume between control and treatment group. To assess the difference between two groups under multiple conditions, one-way ANOWA followed by Bonferoni's multiple comparison tests were performed using PRISM statistical analysis software . Significant differences among groups were calculated at P < 0.05.The author(s) declare that they have no competing interests.SS, SG, and QC have performed the experiments and drafted the manuscript. RS has directed the project and edited the manuscript. All authors read and approved the final manuscript.


Epitheloid hemangioendothelioma (EHE) is a vascular tumor of intermediate malignancy that arises in soft tissues, liver, lung, bone, and spleen. The hepaA 39-year-old woman presented with right upper quadrant abdominal pain of six months duration. She denied any fever, chills, or malaise. On physical examination, there was painless hepatomegaly. Laboratory values, including serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), bilirubin, and alpha-fetoprotein (AFP) levels, were all normal; there was increased alkaline phosphatase (620 U/l) and gamma-glutamyl-transpeptidase (300 U/l) and mild hypoalbuminemia (3 g/dl). She underwent abdominal CT, which revealed conglomerate mass lesions suggestive of metastases in the liver. MRI of the abdomen was then performed on a 1.5-T unit  using T1W, T2W, and dynamic gadolinium-enhanced sequences. Innumerable small, rounded, predominantly peripheral hepatic lesions, measuring between 1 and 3 cm in size, were identified throughout segments 2 and 3 as well as in the subcapsular right lobe of the liver. These were centrally isointense on T2W and hypointense on T1W images, with an unusual peripheral rim of T2 hyperintensity . There wHepatic EHE is a rare clinical entity that was first described by Weiss and Enzinger in 1982. Its etioAmong all radiological modalities, MRI provides the best tissue contrast and helps in the diagnosis of EHE. It depicts the internal architecture of hepatic EHE better than CT scan. The MRI 7The differential diagnosis includes cholangiocarcinoma, fibrolamellar hepatocellular carcinoma, and metastatic carcinoma.7 These c6Two histological types, nodular and diffuse, have been described. Infiltration into sinusoids and into intrahepatic veins, resulting in luminal narrowing and obliteration with polypoidal projections, are characteristic features of hepatic EHE.36 Demons36Multifocal hepatic EHE has a better prognosis and better long-term survival than any other hepatic malignancy. The typical MRI features should raise a suspicion in the correct clinical setting and an appropriately guided biopsy will usually give the answer, thus avoiding more complicated diagnostic or therapeutic interventions.


Enterovirus 71 (EV71) is a common cause of hand, foot, and mouth disease and sometimes causes severe neurologic complications, mainly in children. We report a case of adult-onset encephalitis caused by intrafamilial transmission of a subgenogroup C4 strain of EV71. This case elucidates the risk for EV71 encephalitis even in adults. In children, enterovirus 71 (EV71) is a common cause of hand, foot, and mouth disease (HFMD), and most patients recover within 4–6 days. However, severe neurologic complications, such as acute encephalitis and poliolike paralysis, develop in some patients with EV71 infection. In the largest and most severe EV71-associated HFMD outbreak occurring in Taiwan in 1998, 405 children had severe neurologic complications, pulmonary edema, or both; 78 children died (3 (82.5% polymorphonuclear leukocytes and 17.5% lymphocytes) and total protein concentration of 62 mg/dL with normal glucose levels. Empirical therapy with acyclovir and cefotaxime was initiated. On day 4, the patient reported diplopia and slurred speech. Ocular movements were not obviously restricted, and the extremities showed ataxia without weakness. She could not sit on the bed without support because of severe unsteadiness. Deep tendon reflexes were absent, and the patient had no pathologic reflexes. Brain MRI showed hyperintense lesions in the tegmentum of the medulla oblongata, pons, and midbrain in T2-weighted and fluid attenuated inversion recovery images (3 (100% lymphocytes) and a total protein concentration of 50 mg/dL. On day 22, MRI showed that the brain had normalized. Three months after the onset of disease, she had completely recovered.In November 2006, a 37-year-old woman without serious past illness sought treatment at our hospital with hand tremor, unsteadiness, and a 2-day history of headache (day 1). Examination showed high fever (39.3°C), neck stiffness, intentional tremor of bilateral upper extremities, and truncal ataxia. Brain magnetic resonance images (MRI) and results of laboratory blood tests were normal. A cerebrospinal fluid (CSF) tap showed 305 leukocytes/mmy images . No abnoDuring the illness, CSF was negative for bacteria and viruses. Enterovirus-specific RNA was detected from a stool sample on day 16 by a seminested reverse transcription–PCR (RT-PCR) with consensus-degenerative primers from Nix et al.  of the PCR products directly amplified from stool samples of all 4 cases were 100% identical. The entire VP1 sequences (891 bp) of the EV71 isolates from 2 of her sons with HFMD (07-Ishikawa and 08-Ishikawa) were also 100% identical. Phylogenetically, by using VP1-based genetic classification, the isolates were classified as subgenogroup C4  (6). TheSeveral EV71 outbreaks have been documented throughout the world, and clinical manifestations of EV71 infections can be diverse, including HFMD, herpangina, central nervous system (CNS) complications, and pulmonary edema. Recently, EV71-associated HFMD outbreaks with severe CNS complications have frequently been reported, especially in the Asian-Pacific region (We diagnosed the mother’s illness as EV71 encephalitis because the clinical features were similar to those of EV71 rhombencephalitis in children (Several previous studies have demonstrated a rather low virus isolation rate in CNS specimens compared with that in other clinical samples, such as throat swab, rectal swab, and stool samples from EV71-associated cases with HFMD, encephalitis, or both (Genetic analysis among 4 different EV71 isolates from the patients indicated probable intrafamilial transmission of EV71. In a recent study, EV71 transmission rate to household contacts was 52%, and the transmission rate from children to parents was 41% (


Three hundred and twenty Chinese hamsters (Cricetulus griseus) (CH) were treated (s.c.) with 1/5, 1/10 or 1/20 LD50 of dimethylnitrosamine (DMN) or N-diethylnitrosamine (DEN). These substances, in several respects, showed a different organotropy in this species than in both the Syrian golden (SGH) and the European hamster (EH). In CH, DEN produced up to 100% squamous cell papillomata and occasionally also carcinomata of the cheek pouch, tongue, pharynix, oesophagus and forestomach. With DEN a high rate of hepatomata was simultaneously realized. DMN induced a considerable quantity of liver tumours, the highest incidence being demonstrated in the lowest dosage group.


SIX6, EGR2, PKNOX2, HOXD4, HOXD10, DLX5 and RAXL1, known to regulate developmental processes, are retained in AFiPSCs and FiPSCs. Surprisingly, expression of the self-renewal-associated gene PRDM14 or the developmental processes-regulating genes WNT3A and GSC are restricted to ESCs. Implications of this, with respect to the stability of the undifferentiated state and long-term differentiation potential of iPSCs, warrant further studies.Human amniotic fluid cells (AFCs) are routinely obtained for prenatal diagnostics procedures. Recently, it has been illustrated that these cells may also serve as a valuable model system to study developmental processes and for application in regenerative therapies. Cellular reprogramming is a means of assigning greater value to primary AFCs by inducing self-renewal and pluripotency and, thus, bypassing senescence. Here, we report the generation and characterization of human amniotic fluid-derived induced pluripotent stem cells (AFiPSCs) and demonstrate their ability to differentiate into the trophoblast lineage after stimulation with BMP2/BMP4. We further carried out comparative transcriptome analyses of primary human AFCs, AFiPSCs, fibroblast-derived iPSCs (FiPSCs) and embryonic stem cells (ESCs). This revealed that the expression of key senescence-associated genes are down-regulated upon the induction of pluripotency in primary AFCs (AFiPSCs). By defining distinct and overlapping gene expression patterns and deriving the LARGE  Principle of Cellular Reprogramming, we could further highlight that AFiPSCs, FiPSCs and ESCs share a core self-renewal gene regulatory network driven by OCT4, SOX2 and NANOG. Nevertheless, these cell types are marked by distinct gene expression signatures. For example, expression of the transcription factors,  Besides, the fact that AFCs do not form teratomas in vivobona fide pluripotent cells. Hence, their ability to form complex, mature differentiated cell types may be restricted. Indeed, the capacity of AFCs to form specialized cell types and to contribute to the formation of certain tissues or organs in vitro and in xenotransplantation experiments in vivo is a subject of debate in vitro model system to investigate developmental processes, to conduct disease modeling, toxicological studies, drug research and exploitation in regenerative medicine could be achieved by cellular reprogramming of these cells to an undifferentiated ground state. As a result, AFCs acquire the ability to self-renew and become pluripotent.Human amniotic fluid cells (AFCs) represent a heterogeneous mixture of cells originating from different fetal tissues. They have been used for prenatal diagnosis of various congenital fetal abnormalities for more than fifty years OCT4, SOX2, KLF4, and c-MYC or OCT4, SOX2, NANOG, and LIN28, respectively The induction of pluripotency was first achieved in human somatic cells employing combinations of retroviral or lentiviral vectors encoding either During the course of this study, the generation of iPSCs from human AFCs were described Auxiliary samples of human AFCs obtained during routine amniocentesis were kindly donated by the clinical laboratory of Prof. Dr. Wegner/PD Dr. Stumm  after written informed consent. Utilization of these cells was approved by the ethics commission of the Charité Universitätsmedizin Berlin.www.invitrogen.com). Cells were subsequently cultured in alpha-MEM supplemented with 15% embryonic stem cell-qualified fetal bovine serum, 1% L-glutamine, 1% penicillin/streptomycin , 18% Chang B and 2% Chang C  at 37°C in a humidified 5% CO2 atmosphere. AFCs were routinely passaged using 0.05% Trypsin/EDTA (Invitrogen) at varying ratios of 1∶3 or even 1∶6 when cells were sub-confluent.For the initial culture period (up to passage 5) AFCs were grown in AmnioMAX-C100 , AFiPSCs (derived from human AFCs) and FiPSCs ) were cultured under feeder-dependent and feeder-free conditions as described by Prigione et al. www.stemcell.com).Human ESC lines H1 and H9  for 16 h. The cells in one flask were then transfected with 12 µg of one of the retroviral DNA vectors encoding either OCT4, SOX2, KLF4 or c-MYC using the FuGENE HD transfection reagent  according to the manufacturer's instructions. The retrovirus-containing medium was harvested 48 and 72 h post-transfection. For the generation of AFiPSCs, 180,000 AFCs were transduced with a cocktail of the respective retrovirus-containing media, supplemented with 4 µg/ml Polybrene  at a rate of 1.25 or 2.5 MOI on days 1 and 2 after plating. Each time, directly after the addition of retroviruses, the plates were centrifuged at 800×g, at 37°C for 99 min before replacement of the infectious medium by fresh medium (DMEM/10% FBS). The next day, the infected cells were plated onto irradiated MEFs on Matrigel-coated dishes in DMEM/10% FBS. Another 24 h later, the medium was switched to ESC medium OCT4, SOX2, KLF4 and c-MYC retroviruses were generated using pMX vectors as described previously The generation of FiPSCs used for the comparative transcriptome analysis has been described in vitro differentiation, embryoid body (EB) formation of AFiPSC lines 4, 5 and 41 was induced in ESC medium without bFGF supplementation using the hanging-drop method In vivo differentiation experiments were performed by EPO-Berlin GmbH . Basically, approximately 2*10(6) cells of the AFiPSC lines 4 and 41 were collected by type IV collagenase-treatment or 0.05% Trypsin/EDTA-treatment, washed, pooled and injected s.c. into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice, commonly known as NOD scid gamma (NSG). Teratomas were collected approximately 63 d after injection and processed according to standard procedures for paraffin embedding and hematoxylin and eosin staining. Histological analysis was performed by a pathologist.For www.peprotech.com) until they attained about 30 to 50% confluency. At this point, medium was changed to defined N2B27 medium (Invitrogen) lacking bFGF but including either 100 ng/ml BMP2 (PeproTech) or BMP4  for a period of five days or a combination of 10 ng/ml BMP4 and 10 µM SB431542  for 7 days. Undifferentiated controls were grown in N2B27 including 20 ng/ml bFGF only. After a period of 5 d or 7 d, including daily replacement of media, the cells were harvested for RNA isolation for qRT-PCR and global gene expression profiling analyses or fixed for immunofluorescence microscopy analysis.To induce differentiation into the trophoblast lineage, AFiPSC lines 5 and 41 were transferred onto Matrigel-coated cell culture dishes and grown in MEF-CM including 8 ng/ml bFGF . In each case, 500 ng RNA were used as input for the bead chip hybridization . Processing of samples and the conversion of raw data was previously described http://www.geneontology.org, 28th of March 2010) http://david.abcc.ncifcrf.gov/home.jsp) th of April 2010).Total RNA was isolated using the RNeasy Mini Kit  and Microsoft Excel as described elsewhere www.clontech.com) was used as a positive control. Data are presented as mean LOG2 ratios with respect to biological controls and standard deviation.Quantitative real-time PCR (qRT-PCR) was performed and analyzed using ABI PRISM SDS 2.1 software .For the identification of ESC markers in undifferentiated AFiPSCs  and detection of lineage markers in AFiPSCs differentiated www.millipore.com).Alkaline phosphatase (AP) staining of all manually picked AFiPSC lines was performed following the manufacturer's instructions  was used according to the manufacturer's protocol. All stainings were visualized and images were acquired using the confocal microscope LSM 510 Meta . Processing of images was done with the help of AxioVision V4.6.3.0 (Zeiss) and Adobe Photoshop CS version 8.0  software.For staining of senescent cells, the Senescence beta-Galactosidase Staining Kit . In contrast, the AFiPSC transcriptomes are distinct from those of primary AFCs at passage 6 (average linear R2≈0.67) and passage 17 (average linear R2≈0.50) , self-renewal and pluripotency , a donor cell memory , ESC-specificity , donor cell (AFCs)-specificity  and an iPSC (AFiPSC)-specific gene expression signature   as well as linear correlation coefficient analysis based on expression signals of detected genes revealed similar, though not identical, transcriptomes of AFiPSCs and ESCs (average linear RR2≈0.50) . The samR, SIX6) .MAD2L2, PARP1, RPA3, DKC1, MSH6, CHEK1, PLK1, POU2F1, CDC2, BLM, WRN, DNMT1, DNMT3B, LMNB1, and CDT1, were down-regulated in primary AFCs compared to AFiPSCs and ESCs. In contrast, PIN1, LMNA, GADD45A, CBX6, NOX4, ENG, HIST2H2BE, CDKN2A, CDKN1A, GDF15 and SERPINE1, among others, were up-regulated in primary AFCs compared to AFiPSCs and ESCs.To investigate the effect of reprogramming on bypassing senescence observed in primary AFC cultures , we analPOU5F1, SOX2, NANOG, DPPA4, LEFTY2 and CDH1 , and those acquired gene expressions, which are rather iPSC type-dependent  or PKNOX2, HOXD4, HOXD10 (FiPSCs); DLX5 (AFiPSCs & FiPSCs)). The retained gene expression sets included genes like PKNOX2 (AFiPSCs); HMBOX1, MGA (FiPSCs) or RAXL1 (AFiPSCs & FiPSCs).What can be gleaned from the global gene expression analyses presented here but also from other iPSC-based transcriptome analyses We have shown that cellular reprogramming of primary AFCs results in a fully pluripotent iPSC type, which is in line with recent publications MAD2L2, PARP1, RPA3, DKC1, MSH6, CHEK1, PLK1, POU2F1, CDC2, LMNB1 and CDT1, as well as TERT itself in AFiPSCs in contrast to primary AFCs. The p53/p21 pathway plays a pivotal role in inducing and maintaining senescence CDKN1A (p21), GDF15, and SERPINE1, were strikingly elevated in primary AFCs compared to AFiPSCs and ESCs. In contrast, low level gene expression of DNMT1 and DNMT3B were detected in bulk primary AFCs, whereas these genes are significantly up-regulated in AFiPSCs. Besides their function in establishing and maintaining CpG methylation patterns during embryonal development, they are also known to repress CDKN1A transcription in opposition to and potentially independent of p53 DNMTs may repress CDKN1A and, thus, senescence in AFiPSCs. Taken together, there is evidence that senescence is bypassed upon the activation of a self-renewal and pluripotency program in reprogrammed AFCs, which is in line with our previous findings One of the great advantages of AFiPSCs over their bulk primary counterparts for any desirable application is their acquisition of the ability to propagate indefinitely. The data presented herein suggest, that this phenotypically rejuvenated appearance of AFiPSCs is based on a gene regulatory network, which averts or at least markedly delays the onset of senescence. This is based on the fact that primary AFCs and AFiPSCs and ESCs exhibit opposing expression patterns related to a large number of senescence-associated genes. In particular, we could detect high expression levels of various cell cycle and telomere elongation-associated genes, such as Mechanistic and functional aspects of cellular reprogramming in general, and of AFCs in particular, can be highlighted on the basis of the presented comparative transcriptome analyses of AFiPSCs, ESCs  and FiPSCs and the corresponding parental cell lines. For the different iPSC types we have identified genes, the expression of which are either lost (L), acquired (A) or retained (R) upon the induction of pluripotency. We refer to this as the LARGE  Principle of Cellular Reprogramming. Some of these gene expression patterns, including several signature genes, are discussed.CD59, TNFSF10, and NT5E (CD73) MYC (AFiPSCs & FiPSCs) and KLF4 (FiPSCs). This supports the idea that the main function of KLF4 and c-MYC in the process of reprogramming is to accelerate or enhance the efficiency by increasing a balanced cellular proliferation, while in pluripotent cells they seem to be dispensable The donor cell (AFC)-specific gene signature contains putative immune-suppressive factors, such as POU5F1, SOX2 and NANOG. These establish a core gene regulatory network essential for maintaining self-renewal and pluripotency EOMES and HAND1, are acquired OCT4 knockdown, and, therefore, negatively regulated by OCT4 http://amazonia.transcriptome.eu/temp/histo_7ebe096c97857d933b94cd30a6a120cf.png) SIX6, EGR2, HOXD4, HOXD10, PKNOX2 and DLX5RAXL1, which is also involved in the regulation of developmental processes Among the expressed genes, which are universally acquired during reprogramming processes, independent of the cell source, are key pluripotency-regulating factors, such as It is tempting to speculate, that the expression of some of the distinct signature genes are due to viral integrations into the target cell's genome. However, since these signature gene expressions were identified as a result of a group-wise analysis of several AFiPSCs/FiPSCs lines versus the parental cell lines and ESCs, they are unlikely to be attributable to clone-specific viral integrations in most cases. A probable explanation could be that these gene expression patterns are due to an incomplete erasure of epigenetic imprints in iPSCs depending on the nature of chromatin modifications of the original cell type, or in other words, a kind of cell type-specific epigenetic memory PRDM14, WNT3A and GSC. PRDM14 has been implicated in maintaining the undifferentiated ESC state WNT3A and GSC are primitive streak/mesendoderm markers known to regulate developmental processes In addition to the above-mentioned results of our LARGE analysis, we identified ESC-specific genes in the Venn diagrams, including, for example, Both, primary AFCs, in particular stem cell-like subpopulations of primary AFCs, as well as AFiPSCs are considered to be valuable for the application in basic and applied research. Taken together, our results propose that, for these purposes, cellular reprogramming of AFCs is beneficial as it represses senescence and leads to a phenotype very similar, though not identical, to ESCs. These findings are even more significant, considering that due to the presence of fetal stem cells within bulk primary AFCs, amniotic fluid seems to be a very suitable source of cells for the realization of non-integrating reprogramming strategies. Yet, as a main result of this study, we identified gene expression signatures and LARGE patterns for different types of iPSCs, corresponding parental cells and ESCs. Two conclusions can be drawn from this. First, this kind of comparative transcriptome analysis should be extended integrating iPSC lines derived from several distinct cell sources and generated using various reprogramming techniques, as it would aid in enhancing our meagre understanding of mechanisms underlying cellular reprogramming. Secondly, the functional relevance of such distinct expression patterns, especially of AFCs, AFiPSCs and ESCs, will have to be investigated profoundly in order to estimate limitations and to exploit the full potential associated with putative future utilization of amniotic fluid-derived cells.Table S1List of primers used for qRT-PCR and DNA fingerprinting analyses.(0.03 MB XLS)Click here for additional data file.Table S2List of antibodies used for immunofluorescence stainings.(0.02 MB XLS)Click here for additional data file.Table S3Complete gene lists reported in the ESC/AFiPSC/AFC-Venn diagram . Spreads(5.87 MB XLS)Click here for additional data file.Table S4Senescence-associated genes. List of 116 senescence-associated genes derived from the Gene Ontology database (0.04 MB XLS)Click here for additional data file.Table S5Complete gene lists reported in the ESC/FiPSC/Fibs-Venn diagram . Spreads(6.57 MB XLS)Click here for additional data file.Table S6Complete list of 525 core self-renewal genes as reported in (0.67 MB XLS)Click here for additional data file.