C). In genetic forms of these diseases, mutations in the globular C-terminal domain are hypothesized to favor the spontaneous generation of misfolded PrP conformers (including the transmissible PrPSc form) that trigger downstream pathways leading to neuronal death. A mechanistic understanding of these diseases therefore requires knowledge of the quality control pathways that recognize and degrade aberrant PrPs. Here, we present comparative analyses of the biosynthesis, trafficking, and metabolism of a panel of genetic disease-causing prion protein mutants in the C-terminal domain. Using quantitative imaging and biochemistry, we identify a misfolded subpopulation of each mutant PrP characterized by relative detergent insolubility, inaccessibility to the cell surface, and incomplete glycan modifications. The misfolded populations of mutant PrPs were neither recognized by ER quality control pathways nor routed to ER-associated degradation despite demonstrable misfolding in the ER. Instead, mutant PrPs trafficked to the Golgi, from where the misfolded subpopulation was selectively trafficked for degradation in acidic compartments. Surprisingly, selective re-routing was dependent not only on a mutant globular domain, but on an additional lysine-based motif in the highly conserved unstructured N-terminus. These results define a specific trafficking and degradation pathway shared by many disease-causing PrP mutants. As the acidic lysosomal environment has been implicated in facilitating the conversion of PrPC to PrPSc, our identification of a mutant-selective trafficking pathway to this compartment may provide a cell biological basis for spontaneous generation of PrPSc in familial prion disease.Prion diseases are fatal neurodegenerative disorders caused by aberrant metabolism of the cellular prion protein (PrP C). The transmissible agent is PrPSc, a misfolded version (conformer) of PrP capable of converting PrPC into PrPSc. PrPSc can be generated de novo in inherited prion diseases due to synthesis of aberrant PrP forms from a mutated PrP gene. Such mutant PrP forms, analogous to other aberrant proteins, should typically be destroyed by various cellular ‘quality control’ (QC) pathways; however, several human diseases result from an eventual breakdown in these QC systems, often due to prolonged bombardment by mutant proteins. We have therefore sought to identify the specific pathways that normally cope with disease-causing misfolded PrPs. By carefully following the generation and turnover of these mutant PrPs in cells, we have discovered an intracellular QC pathway that selectively routes biochemically aberrant PrP species to lysosomes. As the lysosomal system has been implicated as a site for conversion of PrPC to PrPSc, our identification of a mutant-selective trafficking pathway to this compartment may provide a cell biological basis for spontaneous generation of PrPSc in familial prion disease. Importantly, these findings suggest that eventual changes or breakdown of this QC pathway may contribute to disease progression.Prion diseases are transmissible fatal neurodegenerative diseases caused by aberrant metabolism of the cellular prion protein (PrP Several diseases are caused by mutations that generate aberrant proteins with adverse cytotoxic consequences For proteins transiting the secretory pathway, the major QC pathways reside in the ER Sc) directs the templated conversion of normal cellular PrP into additional PrPSc. How or why PrPSc accumulation leads to neurodegeneration remains largely unknown. A primary obstacle to such studies is the difficulty in classifying cellular alterations as causes, direct consequences or secondary adaptations to PrPSc accumulation. One way to circumvent this challenge may be to investigate inherited mutations, where the root cause of the disease, a specific mutation in PrP, is established Prion diseases are one example of disorders in which the generation of aberrant misfolded proteins has dire consequences for the cell. These neurodegenerative diseases can be acquired by a transmissible route, sporadically, or through an inherited mutation. In each case, the central event involves aberrant metabolism of the cell surface prion protein (PrP) CtmPrP CtmPrP levels can lead to neurodegeneration in both mouse models and humans This type of approach has already shed light on the mechanism of pathogenesis of a subset of inherited PrP mutations that occur within the central hydrophobic domain of the protein. These mutations cause increased generation of a transmembrane form of PrP termed Interestingly, however, most inherited mutations do not occur within the hydrophobic region, but in the globular C-terminal domain To address this problem, we have initiated a systematic and quantitative comparison of multiple inherited mutations within the C-terminal globular domain of PrP. Our focus was on identifying the ways that cells distinguish and differentially handle wild type versus mutant PrPs. We reasoned that deviations shared by many or all of the mutants may represent the cellular quality control response to aberrant PrP species. A clear delineation of these pathways is almost certainly of direct relevance to the associated diseases. Not only would such analyses identify the routes of mutant PrP trafficking, but also provide strong candidates for pathways that, when perturbed, would lead to accumulation of aberrant PrP. Indeed, age-dependent decline in QC and degradation pathways is emerging as a common theme in many neurodegenerative diseases, underscoring the importance of defining their role in PrP metabolism. In this study, our analysis has led to the identification of an intracellular trafficking pathway that is shared by several mutant PrPs to selectively route misfolded species for degradation in lysosomes. Interestingly, this misfolded mutant PrP-specific trafficking pathway is dependent not only on the C-terminal mutation but on a highly conserved lysine-based motif in the N-terminus of PrP. These data highlight an unappreciated role for post-ER quality control in PrP metabolism and further suggest important hypotheses for the mechanisms underlying prion disease pathogenesis.Wild type human PrP (wtPrP) and several human disease-causing PrP mutants were expressed in mouse N2a cells and selectively visualized by indirect immunofluorescence with the human-specific 3F4 PrP antibody . As expeAmong the mutants analyzed, PrP(A117V) was most similar to wtPrP not only in its general pattern of localization, but also in the relative amounts seen in the different cellular compartments. This is consistent with the fact that this mutation is not located within the structured globular C-terminal domain and therefore does not influence the folding of PrP, but is instead known to influence the topology of a small subset (<5%) of total PrP in vivo Further support for a different relative distribution was provided by analysis of cells co-expressing fluorescently tagged wtPrP and PrP(H187R). Consistent with the analyses of non-tagged proteins, wtPrP-CFP and PrP(H187R)-YFP displayed largely overlapping patterns of localization . HoweverCtmPrP , as cells blocked in ER-to-Golgi transport with Brefeldin A showed identical, exclusively ER localization patterns for both wtPrP and PrP(H187R) . ConsistThe heterogeneity of localization patterns seen with both wtPrP and the mutants, combined with their largely overlapping distributions, precluded clear and direct qualitative comparisons. The co-expression analyses , while iWhen analyzed in this manner, wtPrP showed a wide distribution (ranging from ∼5% to 60%) with an average of ∼28% intracellular PrP . SurprisIt is noteworthy that the absolute proportion of intracellular PrP changed among different experiments and appeared to be influenced by culture conditions . Despite this variability, the relative differences among the mutants remained reproducibly consistent when sufficient numbers of cells were analyzed. Shown in The differential steady-state distribution of globular domain mutants suggested the presence of a mutant-selective subpopulation that was either trafficked or metabolized differently than wtPrP, resulting in its intracellular localization. To identify and biochemically characterize this population, we analyzed PrP in detergent lysates of N2a cells expressing either wtPrP or the different mutants. In a typical experiment, wtPrP and PrP(H187R) recovered in the detergent soluble fraction (using 0.5% Triton X-100 and 0.5% deoxycholate) showed little or no differences in the glycosylation pattern or isoforms . This inBy contrast, the detergent insoluble fraction , but comSimilar analyses of several other globular domain mutants revealed the existence of an increased population (to varying extents in different experiments) of detergent-insoluble, trypsin inaccessible (intracellular), incompletely glycosylated, EndoH-resistant isoforms . This miC to PrPSc, the folding, trafficking, and metabolism of PrP mutants appears to be more generally conserved. This is consistent with intracellular protein trafficking and metabolism being mediated by very generic and highly conserved machinery.Several of the mutants were also analyzed in HeLa cells, a completely unrelated human cell line that expresses very low (but detectable) levels of endogenous PrP. As in N2a cells, PrP(H187R) and PrP(E200K) expressed in HeLa cells showed increased amounts of detergent-insoluble, incompletely glycosylated, intracellular forms . This inFurthermore, the finding of similar results in two cell lines that express different amounts and species (human versus mouse) of endogenous PrP suggests that the behavior of PrP mutants may not be significantly influenced by the co-expressed wild type counterpart. Indeed, in the short term of our experiments, the presence or absence of endogenous PrP in co-expression experiments , singly-glycosylated (∼25%), and doubly-glycosylated (∼50%) PrP  PrP . Upon chPrP(H187R) looked qualitatively similar to wtPrP at early time points, as no differences were noted in its core glycosylation and initial trimming up to 30 minutes of chase . Mature Thus, a normally transient, glycan-trimmed minor population of immature PrP seen during the normal maturation of wtPrP persists for a prolonged period during the biogenesis of several globular domain mutants, but not a hydrophobic domain mutant. The migration of this immature population corresponds to the migration of the insoluble, intracellular, EndoH-resistant, mutant-specific form characterized at steady state . It is iWe next addressed the location and nature of this minor subpopulation of PrP over time by combining pulse-chase analysis with assays of subcellular localization. Extracellular trypsin digestion at each time point during the pulse-chase showed that the mutant-specific immature species, but not the mature species, was largely shielded from the cell surface . DensitoThe rapid acquisition of EndoH resistance of the mutant-specific immature species indicates that it passes the major chaperone-based quality control systems in the ER without significant impediment. This could be explained in two ways. One possibility is that this species folds correctly when in the ER environment , but misfolds upon delivery to the different environment of the Golgi. Alternatively, it might misfold in a manner that is invisible to the ER QC systems, allowing its exit. In order to distinguish between these two models, we combined pulse-chase analyses with assays of detergent insolubility.Upon pulse labeling, when PrP is quantitatively in the ER , we could detect a difference in relative solubility between wtPrP and PrP(H187R) . As expeAdditional evidence to support the notion that this mutant-specific species was not recognized by ER-based QC was provided by the failure of this subpopulation to be degraded even upon prolonged retention in the ER with Brefeldin A . Further+- ATPase) led to the steady-state enhancement of the mutant-specific detergent-insoluble form of PrP(H187R)  , suggestP(H187R) . Mature To investigate how mutant PrP was being metabolized, we analyzed both total protein and exosomes from the conditioned media of PrP-expressing cells . A smallInterestingly, although the immature species of mutant PrPs are trafficked to and degraded in lysosomes, this subpopulation neither accumulates in this compartment nor colocalizes significantly with lysosomal markers (such as Lgp-120) or lysotracker (data not shown). This indicates that upon reaching the lysosome, its degradation is probably relatively rapid (evidenced by its dramatic stabilization upon Bafilomycin 1A treatment). Our data further suggests that this mutant subpopulation traffics from the ER to Golgi in under 30 minutes . Yet itsTo gain insight into the misfolding-selective sorting operating on mutant PrP, we sought to identify potential sorting determinant(s) by analyzing PrP deletion mutants. We found that deletion of residues 23–48 (the ΔN constructs) markedly reduced the amount of detergent-insoluble, immaturely glycosylated species of several PrP mutants relative to their full length (FL) counterparts . In mostAnalysis of PrP(H187R) and PrP(H187R)ΔN by pulse-chase showed that the ΔN construct generated mature PrP species in amounts and kinetics very similar to wild type PrP (Enhanced maturation of PrP(H187R)ΔN could transpire because the N-terminal deletion either prevents the initial misfolding of the C-terminal globular domain or prevents its selective retention within the secretory pathway. At present, we cannot fully discriminate between these possibilities. However, we did observe that the detergent insolubility of pulse-labeled PrP(H187R) was only partially improved by the N-terminal deletion , even thIn order to narrow the motif within this N-terminal domain that influences the generation and/or metabolism of misfolded PrP, we engineered a version of PrP(H187R) in which 3 highly conserved lysine residues  were changed to arginines [PrP(H187R)-KR3]. This construct was then quantitatively assessed for localization using our single cell analyses . RemarkaWe have quantitatively analyzed the trafficking and metabolism of disease-causing PrP mutants. Our aims were two-fold. First, we hoped to gain insight into the QC pathways that discriminate wtPrP from mutants that are ostensibly prone to misfolding. Second, we wanted to determine whether many or all of the PrP mutants would share certain features in their metabolism that could represent potential pathways of direct importance to disease pathogenesis. By determining how cells metabolize PrP mutants relative to the wild type counterpart, we discovered a prominent role for post-ER pathways of differential sorting and trafficking. Interestingly, we found that despite the multitude and diversity of ER quality control pathways, all of the PrP mutants we analyzed were refractory to recognition by these components. They are instead recognized in a more distal compartment of the secretory pathway and sorted for degradation in lysosomes. The implications of these findings for inherited and transmissible prion disease as well as cellular quality control in general are discussed in turn below.Our primary conclusion that disease-causing PrP mutations use a QC pathway that culminates in lysosomal degradation has several potential implications for inherited prion disease pathogenesis. The most important is that reduced lysosomal function would lead to accumulation of misfolded PrP species. This could represent a mechanism of neuronal cytotoxicity in inherited prion disease. Indeed, age-dependent changes in the lysosomal system such as decreased activity of lysosomal hydrolases and decreased regulation of lysosomal pH have been documented In addition, increased production of misfolded PrP species may lead to saturation of the trafficking pathway from the trans-Golgi network (TGN) to lysosomes. It is unlikely that this pathway is used solely by PrP as most cellular trafficking and QC systems have a wide range of substrates. This means that increased traffic of misfolded PrPs (as would occur with these mutants) could lead to compromised function of the secretory pathway, saturation of the Golgi QC machinery, and/or inappropriate secretion of immature or misfolded cellular proteins. In addition, sorting of lysosomal enzymes that are critical for the degradative capacity of this compartment could be compromised. This would lead to reduced lysosomal activity, perhaps in an age-dependent manner Sc isoform. While not all inherited PrP mutants generate infectivity , a few mutants  are unambiguously converted to PrPSc. Lysosomes (or related acidic compartments) have long been implicated as the site of conversion of PrPC to PrPSc during transmissible prion disease Sc.Lysosomal trafficking could also play a role in the spontaneous conversion of some mutant PrPs into the PrPOur data indicate that wild type and mutant PrPs are distinguished and trafficked differentially within the cell after they have left the ER, the predominant site of QC in the secretory pathway. This conclusion is consistent with several previous observations providing evidence for altered mutant PrP species in post-ER intracellular compartments on the basis of imaging, glycosylation analysis, or inhibitor studies The second line of evidence for ER-based QC is the accumulation of unglycosylated PrP upon prolonged proteasome inhibition The existence of Golgi-mediated QC was first suggested by studies in yeast showing that certain mutants of plasma membrane proteins were retained in the Golgi and degraded in the vacuole Results from our current study are consistent with a similar model, suggesting that PrP may use a more general QC pathway that remains to be studied in detail. Of crucial importance is the machinery and recognition motifs on mutant PrPs used to discriminate folded from misfolded molecules. While additional studies are required to answer this question, the currently available information suggests an intriguing hypothesis. Many PrP mutations may act by favoring dimerization or multimerization of PrP molecules without grossly affecting the overall protein fold. This could explain why ER QC pathways fail to recognize misfolded mutant PrPs. However, this multimerization could create novel motifs  that are recognized by sorting factors in the ER, ERGIC or Golgi, as has been suggested for Furin All PrP constructs are based on human PrP. This is recognized by the 3F4 monoclonal antibody, which does not recognize mouse PrP expressed endogenously by N2a cells. All mutant PrPs were generated by site-directed mutagenesis and the mutation verified by sequencing. The data shown are from human PrPs containing a Valine at position 129, although very similar results were observed for the Methionine variant. Human wtPrP, all mutant PrPs, and fluorescent protein (FP) tagged PrPs were subcloned into and expressed from pCDNA3.1-based vectors  by standard procedures. FPs were the monomeric variants of CFP, GFP, and YFP. FP tagged PrPs were created by insertion of PCR amplified FPs  into the Bsu36I site in PrP (at residue 50). The monomeric Cerulean and Venus variants of CFP and YFP were used. Co-localization with ER was carried out using GFP-KDEL 2). Trypsin Inhibitor was added to 250 µg/ml, and after 2 min, the cells sedimented by centrifugation, and lysed in detergent as above for further analysis. As a control, detergent (0.2% Triton X-100) was included during the trypsin digestion of a parallel sample to rule out any intrinsic protease resistance of PrP under these conditions. Limited trypsin digestion to assess PrP folding status was performed on total detergent lysates prepared in ice-cold DB. Trypsin was added to the indicated final concentrations from 10 to 100 µg/ml, and incubated for 60 min on ice. Trypsin inhibitor was added to 500 µg/ml, incubated for 5 min on ice, and the samples separated into soluble and insoluble fractions by centrifugation for 30 min in a microcentrifuge at 4°C. 5× SDS-PAGE sample buffer was added to the supernatants and pellets for analysis by immunoblotting. For analysis of total PrP excretion, transfected cells were placed into Opti-MEM media containing or lacking 250 nM Bafilomycin A1 16 h after transfection. After culturing for another 12 h, the conditioned media was collected, debris removed by centrifugation for 2 min in a microcentrifuge at full speed, and the supernatant subjected to TCA precipitation to collect all of the proteins. The precipitated proteins were washed in acetone, and dissolved in SDS-PAGE sample buffer. To isolate exosomes, conditioned media (prepared as above) was centrifuged for 2 min at full speed in a microfuge to remove debris. The supernatant was then subjected to ultracentrifugation at 70,000 rpms in a TLA100.3 rotor with microtest tube adaptors. The pellet containing exosomes was dissolved in SDS-PAGE sample buffer. SDS-PAGE resolution of proteins was performed on 12% Tris-Tricine gels. Quantification was by either phosphorimaging or densitometry. Where densitometry was used, multiple exposures of the gels/blots were obtained and those in the linear range of the film were used. Pulse-chase data was quantified using a Typhoon Phosphorimager and company software  and autoradiographs were digitized using Adobe software .Detergent solubility assays, glycosidase digestions, pulse-chase analysis, immunoblotting, and immunoprecipitations have been described Indirect immunofluorescence and confocal microscopy was as before Figure S1Mutant and wtPrPs occupy heterogeneous but similar cellular locales. (A) Indirect immunofluorescent detection of wtPrP reveals considerable heterogeneity in localization patterns, three of which are shown: predominantly cell surface (left), significant perinuclear in addition to cell surface (middle), and surface, perinuclear, and ER/nuclear envelope. (B) PrP(H187R) analyzed as in panel A also show comparable heterogeneity. The percent intracellular PrP for each cell (quantified in (1.22 MB TIF)Click here for additional data file.Figure S2Image analysis for quantitation of total and intracellular PrP. To quantify the surface and intracellular populations of PrP on a per-cell basis, random fields (chosen blindly using a co-transfected RFP marker) of PrP-expressing cells were first imaged under multiple detector conditions  that allow imaging of cells with expression levels spanning ∼25 fold (see example in panel A). Each image is a 1 µm thick confocal section focused at roughly the mid-nuclear level. Each cell was quantified from the respective image in which the fluorescence intensity of that cell falls entirely within the linear range of the detector setting . Regions of interest (see example in panel B) were drawn around the cell periphery , and just within the plasma membrane (to quantify intracellular fluorescence). After subtracting background, raw values were obtained for the total, intracellular, and surface  fluorescence. These values were normalized for the detector setting used so that values obtained from cells quantified from different exposures could be directly compared. The normalized values were than used to calculate the % intracellular and surface-to-intracellular parameters that are plotted in (1.68 MB TIF)Click here for additional data file.Figure S3Heterogeneity in wtPrP and mutant PrP behavior. (A) Cells were transiently transfected with wtPrP or PrP(E200K) using either Lipofectamine (2 µg DNA) or Effectene (0.4 µg DNA) transfection methods. The soluble (S) and insoluble (P) fraction of the detergent lysates were immunoblotted for PrP. The two blots are taken from the same exposure and processed in parallel, illustrating the markedly different expression levels. The migration of different PrP species are indicated on the left as in (0.88 MB TIF)Click here for additional data file.Figure S4Pulse-chase analysis of PrP(E200K) and PrP(A117V). The metabolism of E200K (panel A) and A117V (panel B) were compared to wtPrP by pulse-chase analysis exactly as in (0.36 MB TIF)Click here for additional data file.


Xenopus laevis tadpoles may avoid potentially destructive antiselfresponses by largely discarding the larval immune system at metamorphosis andacquiring a new one. By implanting triploid (3N) thymuses into diploid (2N) hosts, weexamined the influx and expansion of host T-cell precursors in the donor thymus ofnormally metamorphosing and metamorphosis-inhibited frogs. We observed that donorthymocytes are replaced by host-derived cells during metamorphosis, but inhibition ofmetamorphosis does not prevent this exchange of cells. The implanted thymuses exportT cells to the spleen. This donor-derived pool of cells declines after metamorphosis innormally developing frogs but is retained to a greater extent if metamorphosis isinhibited. These studies confirm previous observations of a metamorphosis-associatedwave of expansion of T cells and demonstrate that it is not dependent on the relativelyhigh concentrations of thyroid hormones required for metamorphosis. Although somelarval T cells persist through metamorphosis, others may be destroyed or the larvalpopulation is significantly diluted by the expanding adult population.Metamorphosis in amphibians presents a unique problem for the developing immunesystem. Because tadpoles are free-living, they need an immune system to protect againstpotential pathogens. However, at metamorphosis, they acquire a variety of new adultspecificmolecules to which the tadpole immune system must become tolerant. Wehypothesized that


Aphelocoma ultramarina). We predicted that populations on different sky islands would share common, ancestral alleles that existed during the last glaciation, but that populations on each sky island, owing to their isolation, would contain unique variants of postglacial origin. We also predicted that divergence times estimated from corrected genetic distance and a coalescence model would post-date the last glacial maximum.Genetic tests of paleoecological hypotheses have been rare, partly because recent genetic divergence is difficult to detect and time. According to fossil plant data, continuous woodland in the southwestern USA and northern Mexico became fragmented during the last 10,000 years, as warming caused cool-adapted species to retreat to high elevations. Most genetic studies of resulting 'sky islands' have either failed to detect recent divergence or have found discordant evidence for ancient divergence. We test this paleoecological hypothesis for the region with intraspecific mitochondrial DNA and microsatellite data from sky-island populations of a sedentary bird, the Mexican jay (Our results provide multiple independent lines of support for postglacial divergence, with the predicted pattern of shared and unique mitochondrial DNA haplotypes appearing in two independent sky-island archipelagos, and most estimates of divergence time based on corrected genetic distance post-dating the last glacial maximum. Likewise, an isolation model based on multilocus gene coalescence indicated postglacial divergence of five pairs of sky islands. In contrast to their similar recent histories, the two archipelagos had dissimilar historical patterns in that sky islands in Arizona showed evidence for older divergence, suggesting different responses to the last glaciation.This study is one of the first to provide explicit support from genetic data for a postglacial divergence scenario predicted by one of the best paleoecological records in the world. Our results demonstrate that sky islands act as generators of genetic diversity at both recent and historical timescales and underscore the importance of thorough sampling and the use of loci with fast mutation rates to studies that test hypotheses concerning recent genetic divergence. Current patterns of genetic diversity in North American species were shaped to a large degree by quaternary glacial cycles -4, but eNeotoma spp.) middens [In the southwestern USA and northern Mexico, a detailed history of the last 40,000 years of habitat change has been reconstructed from fossilized plant material preserved in packrat , a sedentary bird species found in pine-oak woodlands in the sky islands. Replication is rare in large-scale biogeographic studies, leading to the widespread use of comparative phylogeography of multiple codistributed species [With this study, we attempt to reconcile paleoecological and genetic estimates of the timing of postglacial habitat fragmentation for this region using as our model the Mexican jay . To test this prediction, we assessed genetic diversity in mitochondrial DNA (mtDNA) and nuclear (microsatellite) DNA. We constructed minimum-spanning networks of mtDNA haplotypes to help clarify recent genealogies where ancestral and derived haplotypes often coexist, making inferences based on traditional bifurcating trees difficult . Using oOur results validate the idea that the two archipelagos can be considered independent replicates for the purpose of investigating postglacial divergence. Sequences from 525 base pairs (bp) of the mtDNA control region of 265 Mexican jays from 10 different sky islands contained no missing data  and revealed 32 variable sites, identifying 21 unique haplotypes in the Arizona archipelago and 18 in the Trans-Pecos archipelago Figure ; yet, thin situ postglacial mutation within sky islands and limited gene flow among sky islands, in agreement with the natural history of the Mexican jay [Results from within each archipelago provide multiple lines of evidence to support the paleoecological hypothesis of postglacial sky-island fragmentation set forth by the fossil midden data. We first discuss patterns found in mtDNA haplotype networks and then divergence times estimated from corrected genetic distance and a coalescence model. The predicted mtDNA pattern for postglacial divergence, a 'star-shaped' haplotype network with rare, peripheral haplotypes unique to individual sky islands Figure , was eviican jay . Alternaican jay . Furtherican jay . Stabiliican jay -28. It sn = 155; Figure Private haplotypes could also represent haplotypes that went undetected in other sky islands due to limited sampling, but we believe this is unlikely because in most cases sample sizes were high relative to the number of haplotypes. For example, one sky island with a large sample size (site 7 where ND2) and nuclear microsatellites. The large-scale genetic structure seen in our data is largely corroborated by a smaller data set from ND2 , Masta [Daktulosphaira vitifoliae) [Melanoplus oregonensis) show evidence for recent divergence and in situ generation of unique haplotypes [Until now, evidence for postglacial fragmentation of sky islands has eluded intraspecific studies of poorly dispersing sky-island organisms. In a study of sky-island jumping spiders , Masta  found evifoliae) . Of the plotypes . Quite rplotypes . Our resThere are various reasons why previous studies might have failed to detect genetic divergence among sky islands. Reconstructing genetic histories for recently diverged lineages is hindered by stochasticity in the coalescent process and the confounding influences of migration and incomplete lineage sorting . ThorougND2, especially over short timescales where saturation is not an important factor [ND2 haplotype networks for the Trans-Pecos archipelago , which replace Mexican jays at high elevation throughout most of their distribution, provided a barrier to east-west dispersal of Mexican jays (and possibly other organisms) in Arizona during the last glaciation. Hypothetical maps of habitat at the LGM inferred from the midden data support this idea and suggest two glacial refugia, one in southwestern Arizona and another in southwestern New Mexico and northern Chihuahua, with connections between the two interrupted by large expanses of montane conifer forest  and sky islands in southwestern Texas and northern Coahuila, Mexico ('Trans-Pecos archipelago') are composed of mountain ranges of roughly similar size and distance Figure . For the® DNeasy™ Tissue Kit . We amplified a 525-base-pair portion of the mtDNA control region using primers JCR03 (5'-CCCCCCCATGTTTTTACR-3') [We extracted genomic DNA from blood and tissue using a QIAGENTACR-3')  and H124TACR-3') . ReactioSEQUENCHER 4.1 (GeneCodes) and checked variable sites visually for accuracy. All sequences used in this study have been deposited in GenBank under accession numbers EU121284 to EU121375. We encountered no problems suggesting nuclear inserts ; nevertheless, a subset of the data, including all unique haplotypes from blood extractions, was re-extracted from feather tissue and resequenced with identical results.Polymerase chain reaction (PCR) products were purified with an UltraClean™ GelSpin kit . We obtained sequences from PCR products using a 10-μl total volume, dydeoxy-terminator cycle-sequencing reaction using a CEQ DTCS kit . We purified products from this reaction with an ethanol precipitation and sequenced them in a Beckman-Coulter CEQ 2000 automated sequencer. We aligned sequences automatically using ND2) gene for subset of our samples using primers L-5216 (5'-GGCCCATACCCCGRAAATG-3') and H-6313 (5'-CTCTTATTTAAGGCTTTGAAGGC-3') [Ap) and calculated a percentage compared with total alleles.As differences in substitution patterns and stochasticity in the lineage-sorting process can result in disparity between gene history and population history , particuAGGC-3') , using tAGGC-3') , ApCo2, AGGC-3') . For theARLEQUIN 2.0 [Dxy), taking into account intra-population polymorphism [Dxy = dixy - 0.5(dix + diy), where x and y are the groups being compared and di is the uncorrected average genetic distance [ND2.We constructed minimum-spanning networks of absolute distances between mtDNA haplotypes using the molecular variance parsimony algorithm  implemenQUIN 2.0 . We usedmorphism , so thatdistance . We calcdistance ,52 and hdistance . The SieIM, a program that implements the isolation-with-migration coalescence model. IM uses a Markov chain Monte Carlo method to estimate jointly several demographic parameters of two diverging populations [t). Two types of data sets were used, one with only mtDNA sequence data under an Hasegawa-Kishino-Yano model of sequence evolution [IM, particularly for data sets like ours that use microsatellite data involving many individuals and loci is slow convergence on the true posterior density of the parameters [t to certain intervals and to alter the heating scheme to achieve sufficient mixing among chains, as advised in IM documentation. Mixing was monitored by observing effective sample size (ESS) values and inspecting parameter plots for trends.We also estimated divergence times using ulations . For thivolution , and onevolution . Microsarameters . Runningg1 = 0.9, g2 = 0.8), particularly for the joint data sets. MtDNA-only data sets generally achieved convergence quickly and the five different pairwise runs took between 3 and 7 days on a personal laptop with a dual-core Intel 1.83-GHz processor. Runs with the joint data sets took considerably longer, up to 30 days to exceed 10,000,000 steps and approach convergence. ESS values for these data sets were never particularly high , and here we relied primarily on the lack of trends in the parameter plot for t to determine if convergence had been reached. Importantly, results for the joint data sets were consistent among independent runs and are very similar to results for mtDNA data alone, in which convergence was confidently achieved. We present results from the longest run for any particular data set and conditions.Although conditions varied among runs depending on the needs of the data, we ran each pairwise analysis for at least 10,000,000 steps after a long burn-in of 3,000,000 steps using 10 to 30 chains for the mtDNA data set and 40 to 70 chains for the joint data set. We used a geometric heating scheme, generally using high values for the heating parameters . However, population parameters, particularly migration rates, proved difficult to estimate in IM under this model. Similar difficulty in estimating parameters with high confidence in IM compared with MDIV has been reported elsewhere [IM. When migration was unconstrained in IM, likelihood curves for this parameter were relatively flat and included zero as well as values that seemed unrealistically high (more than 20 migrants per generation). When migration was capped at more realistic values (m1 = m2 ≤ 4 ≈ 2 migrants per generation), divergence times were often multimodal, but had peak likelihood post-dating the LGM. However, estimates of migration rate under this model never peaked, with likelihood steadily increasing across the range of values. This could indicate insufficient information in the data to estimate migration and it is advisable in this situation to reduce the number of parameters . Importantly, postglacial divergence was not ruled out under realistic isolation-with-migration scenarios (data not shown). However, because for this study we were concerned with obtaining precise estimates of divergence time, and as our haplotype networks and knowledge of the natural history of the Mexican jay suggested little or no current gene flow among sky islands, we present results from an isolation model where migration rate was set to zero (m1 = m2 = 0). We acknowledge that some migration among sky islands likely occurred during the initial stages of habitat fragmentation, and this has probably contributed to the difficulty of finding a clear signature of migration in the results.Initially, we estimated divergence times under an isolation-with-migration model that allowed the possibility of migration between sky islands. Previous results using MDIV, the prelsewhere , which cbp: base-pair; ESS: effective sample size; LGM: last glacial maximum; Ma: million years ago; mtDNA: mitochondrial DNA; PCR: polymerase chain reaction.JEM collected samples, carried out the molecular genetic studies and analyses and drafted the manuscript. BSB collected samples and helped to draft the manuscript. TBS participated in the design of the project and helped to draft the manuscript. All authors read and approved the final manuscript.


Histone deacetylase inhibitors have progressed rapidly from the laboratory to clinical testing. This review highlights the promising data for their combination with a wide range of established and novel anticancer agents and discusses the mechanisms that underpin these interactions. Histone deacetylase inhibitors (HDIs) have progressed rapidly from the laboratory to clinical testing as novel anticancer agents, culminating in the approval of SAHA  for the treatment of recurrent cutaneous T-cell lymphoma. However, despite their promising activity in pre-clinical models, HDIs have demonstrated only modest antitumour activity in initial clinical trials in solid malignancies. In this review, we will discuss current findings to support the hypothesis that in most scenarios, combination with other therapeutic modalities will be required to optimise efficacy and current evidence for the molecular mechanisms that underpin potential combinations.ε-amino acetyl moiety on specific acetylated lysine residues within core histones  enzymes counter the activity of histone acetyltransferases by inducing hydrolysis of the histones . HistoneAlthough the modulation of transcription through histone modification serves as a useful paradigm, the mechanisms that mediate the anticancer effects of HDIs are more complex, with many non-histone targets identified .There are multiple HDIs in clinical development and key representatives are shown in In vitro, synergistic induction of apoptosis is seen when HDIs are combined with epirubicin in breast cancer cells and with etoposide or cisplatin in melanoma cells .In an attempt to find a niche for HDIs, they have been tested in combination with a variety of conventional cytotoxic chemotherapeutic agents. Pre-clinical data in multiple cancer cell lines  have shown the potentiation by HDIs of the effects of topoisomerase I inhibitors  topoisomerase II inhibitors  and other DNA-damaging agents . A number of mechanisms may account for the potentiation of DNA-damaging agents by HDIs, reflecting their pleiotropic actions. For topoisomerase inhibitors, HDAC1 and -2 have been shown to bind and interact with topoisomerase II, and form an integral part of the NuRD complex , and this was confirmed in mouse xenograft studies . With reThese data show a clear rationale for combining HDIs with a range of chemotherapeutic agents, but that clinical trials must be underpinned by a clear mechanistic rationale specific to both experimental agents and tumour types.In vitro studies indicate that HDIs have single-agent activity in HER2-overexpressing breast cancer cell lines including attenuation of HER2 expression, its tyrosine kinase activity, its cell membrane localisation and dimerisation with HER3 , synergy has been shown between HDIs and the HER1 (EGFR) tyrosine kinase inhibitors erlotinib and gefitinib , and HDIs manipulate gene expression patterns resulting from NF-κB activation both directly through NF-κB subunit acetylation and indirectly through chromatin remodelling  gene most commonly with the PML gene. The resulting fusion protein transcription factor has enhanced co-repressor-binding properties, increasing HDAC and DNA methyltransferase recruitment. This aberrant retinoid signalling results in potent transcriptional silencing of target genes. All trans-retinoic acid (ATRA), alone or combined with chemotherapy, is effective in reversing this silencing but resistance may occur. Addition of an HDI to this combination is logical in view of the enhanced co-repressor binding and HDAC recruitment by RARA fusion proteins. Histone deacetylase inhibitor therapy alone does not induce differentiation in APL but can induce this in retinoic acid-resistant cell lines when the two agents are combined  modulators tamoxifen and raloxifene, the pure antioestrogen fulvestrant and the aromatase inhibitor letrozole in breast cancer cell lines. Interestingly, the partial agonistic effect of endometrial adenocarcinoma cell proliferation induced by tamoxifen was blocked by HDI co-administration  for which the BCR/ABL tyrosine kinase inhibitor imatinib is now a standard therapy but resistance can develop. WAF1/CIP1, perhaps the most consistently observed HDI downstream molecular effect. Forced expression of p21WAF1/CIP1 depleted the combined HDI/sorafenib effect, implying that this may be required for synergism. Potential mechanisms include disruption of p21WAF1/CIP1-mediated G1 arrest, interference with its direct antiapoptotic actions such as inhibition of caspase-3 or c-Jun NH2-terminal kinase activation or disruption of the upstream Raf/MEK/ERK axis  produced synergistic growth inhibition and induction of apoptosis in leukaemia cell lines  promoters reversed methylation during the first cycle of therapy whereas none of the six non-responders showed any demethylation  where the unique tumour microenvironment may account for their unpredicted efficacy, their ability to synergise with, and potentially overcome resistance to, many other agents represents a promising strategy for clinical development. Current evidence to support this assertion is predominantly pre-clinical, with only a small number of non-randomised early-phase clinical trials reported . We therefore wait for clear proof that the multiple promising combinations tested in pre-clinical studies can in fact translate to added clinical value for patients above use of single or other agents. If this can be achieved, then the next few years should herald clinical data from phase II and III studies to define the exact value of combination approaches and improved understanding of mechanisms that underpin activity.


Attamyces fungi for food, which are thought to be grown by the ants in single-strain, clonal monoculture throughout the hundreds to thousands of gardens within a leafcutter nest. Monoculture eliminates cultivar-cultivar competition that would select for competitive fungal traits that are detrimental to the ants, whereas polyculture of several fungi could increase nutritional diversity and disease resistance of genetically variable gardens.Leafcutter ants depend on the cultivation of symbiotic Atta leafcutter ants, which are most likely among all fungus-growing ants to cultivate distinct cultivar genotypes per nest because of the nests' enormous sizes (up to 5000 gardens) and extended lifespans (10–20 years). In Atta texana and in A. cephalotes, we resampled nests over a 5-year period to test for persistence of resident cultivar genotypes within each nest, and we tested for genetic differences between fungi from different nest sectors accessed through excavation. In A. texana, we also determined the number of Attamyces cells carried as a starter inoculum by a dispersing queens , and we tested for genetic differences between Attamyces carried by sister queens dispersing from the same nest. Except for mutational variation arising during clonal Attamyces propagation, DNA fingerprinting revealed no evidence for fungal polyculture and no genotype turnover during the 5-year surveys.Using three experimental approaches, we assessed cultivar diversity within nests of Atta leafcutter ants can achieve stable, fungal monoculture over many years. Mutational variation emerging within an Attamyces monoculture could provide genetic diversity for symbiont choice , an analog of artificial selection. Cooperation and conflict within host-symbiont associations evolve under the constraints of stabilizing and destabilizing mechanisms Atta and Acromyrmex) are dependent on symbiotic fungi for food, which are thought to be grown by the ants in single-strain, clonal monoculture throughout the multiple gardens within a leafcutter nest. This traditional assumption of fungal monoculture derives primarily from natural-history observations collected 100 years ago Leafcutter ants  may have been lost during the transfer into the laboratory. However, coexistence of two fungal genotypes in a chimaeric garden is thought to be unlikely in these two Acromyrmex species because the fungi appear to secrete incompatibility compounds that are distributed by the ants throughout gardens Acromyrmex and Atta are closely related and can even be shared through lateral transfer of fungal clones between nests of these two ant genera Atta texana, which is able to co-cultivate several genotypically diverged fungi in experimentally created, chimaeric laboratory gardens Only a single study has so far tested for monoculture of leafcutter gardens with molecular methods Atta leafcutters are thought to be most likely to cultivate several distinct cultivar genotypes per nest because of the enormous sizes of the nests. Mature Atta nests have hundreds to thousands of gardens . An estimate of the number of cultivar cells carried by a foundress is critical to assessing whether the ants passage their cultivar through an extreme bottleneck to help reduce evolutionary conflicts among genetically different cultivars, as predicted by theory Atta nests and no genotype turnover during the 5-year surveys, indicating that nests of these Atta species can achieve stable, fungal monoculture over many years.We assessed within-nest cultivar diversity with highly polymorphic microsatellite DNA markers Attamyces bromatificus, teleomorph Leucocoprinus gongylophorus, Agaricales, Basidiomycota; Attamyces strains genotyped so far were all polyploid because of the multinucleate cells of AttamycesAttamyces fungi appear to be obligate symbionts, as they have not been found so far to grow independently of the ants Attamyces fungi are fruiting-competent and can produce spore-bearing mushrooms in laboratory colonies, or, so far known only from Acromyrmex leafcutter ants that thatch gardens at ground level, on mounds of field nests  and extreme worker polymorphism, but the two species differ in many other respects. A. cephalotes has one of the largest distributions of any Atta species, ranging throughout lowland tropical rainforest from the Amazon Basin and the Atlantic Coast forests in Brazil (about latitude 15°S), to the Sierra de Los Tuxtlas in southern Mexico , nest-founding in A. cephalotes appears to be strictly monogynous . A. cephalotes constructs mounds with both perennial shallow and deep gardens  to evade summer droughts and winter temperatures in the seasonally variable subtropical habitat.Attamyces genotypes differed between Atta nests in each population studied, we found no evidence of Attamyces polyculture within each of the surveyed Atta nests, with the exception of minor mutational variation that emerges during the continuous clonal propagation of Attamyces within nests of fungus-growing ants.Although A. texana nests in 2006, all Attamyces pellets carried by females from each particular nest were genetically identical (44–50 pellets genotyped per nest), with the exception of two possible mutations , all Attamyces pellets carried by females from the same nest were genetically identical , this result is less conclusive than the corresponding results for A. cephalotes .We found no evidence of within-nest genetic diversity of Attamyces diversity within each of the six A. cephalotes nests , except for five putative mutant Attamyces strains in five different gardens  per pellet was 543.5  that are expected for the kind of di- and tri-nucleotide microsatellite loci used for DNA fingerprinting of AttamycesA. cephalotes and A. texana.DNA fingerprinting reveals no evidence for the coexistence of diverged cultivar genotypes in single nests of Atta species is consistent with the reported monoculture in small laboratory gardens of two Acromyrmex species Acromyrmex gardens screened in Acromyrmex may culture different fungi in different gardens of a nest. Our study on Atta tested for differences between different gardens in field nests, and establishes monoculture by sampling across the hundreds to thousands of gardens of single Atta nests.The finding of monoculture in the two A. texana pellets estimates a population of several thousand Attamyces cells in the starter culture at nest founding); (b) possible weeding of secondary Attamyces strains if they were to enter an established garden  the transgenerational passage of the cultivar through a small bottleneck , because of the clonal transfer of cultivars between ant generations, and because of the persistence of the same cultivar genotype across many years within a nest documented here for the first time, partner fidelity feedback A. texana, but a second mechanism, ant-mediated symbiont choice that biases cultivar propagation against inferior cultivar mutants, may also operate in the Atta ant-fungus mutualism A. texana suggest that choice may be a comparatively weak mechanism, as workers do not show a strong and consistent cultivation-bias between closely-related Attamyces strains presented to the ants in laboratory experiments .(a) Because of the longevity of Atta nests is expected to facilitate build-up of specialized diseases, which is the bane of clonally propagated crops in human agriculture  that may help suppress diseases, as first suggested by Atta ants appear to manage, in addition to the primary cultivars, an array of “auxiliary” microbes providing disease suppression and other services (b) Monoculture of fungi in the long-lived, sessile Atta colonies, it would seem sufficient to sample only a single chamber in population-genetic studies of Attamyces, or sample from infrabuccal pellets carried by extranidal workers. However, detailed studies of within-nest cultivar diversity remain necessary for the massive nests of Atta laevigata and A. capiguara , such as the desert leafcutter Acromyrmex versicolorAttamyces genotypes may co-found a common garden. Preliminary investigation of gardens from polygynously-founded Acro. versicolor colonies did not indicate co-cultivation of several Attamyces strains or recombination between Attamyces strains in such polygynous colonies (R. Clark & U.G. Mueller unpublished).(c) Because of monoculture in A. texana carries a population of minimally several thousand cultivar cells per infrabuccal pellet as a starter culture for her incipient garden. This could suggest that Atta ants do not passage their cultivar through an extreme bottleneck to help reduce evolutionary conflicts among co-cultivated and genetically distinct cultivars, as is typical for many symbioses between macro- and microorganisms Attamyces cells derives from a drastically bottlenecked founder population of a few cultivar cells gathered by a female for her pellet. Females incorporate multiple fragments of substrate into their pellet , suggesting that foundress queens sample from both mature and from young garden for their pellets, rather than from a single source. Future studies should elucidate how foundress queens amass the mycelium in their pellet and specifically determine (i) whether females gather mycelium from a single garden fragment or from multiple fragments; (ii) whether females gather only a few cells initially then permit growth to thousands of cells within a pellet; and (iii) whether females stress the mycelium to depress population size  and at the same time test for fitness and viability of the chosen mycelium. Evolutionary theory of cooperation predicts that attine females should exhibit careful partner choice when picking mycelium for the pellet (d) A foundress queen of Atta nest-founding first suggested the hypothesis that Atta cultivate monocultures in their enormous nests Atta leafcutter ants can indeed achieve stable, fungal monoculture over many years and that mutational variation can arise within a nest's Attamyces monoculture. Additional variation may be introduced if novel Attamyces strains enter the nest and recombine with the resident strain; recombination appears to occur in rare cases in experimental laboratory colonies of A. texana , but recombination has so far not been observed directly in natural Atta nests in the field. Any mutational and recombinational variation within a single Atta nest could provide the raw material for Attamyces evolution driven by symbiont choice (cultivation biases of the ants favoring or disfavoring Attamyces variants coexisting in the same nest), an analog of artificial selection operating in human agriculture.A century after natural-history observations on A. texana nests at Brackenridge Field Laboratory (BFL), University of Texas at Austin  and two A. texana nests at Hornsby Bend Environmental Research Center . The five Atta colonies were chosen because of easy access to the mounds during the mating flights. The five nests were mature with large mounds (>7 m mound diameter), and therefore at least 5–10 years old. Based on field records collected at BFL, nest UGM050509-01 was probably founded between 1994–1996, but the founding dates of the other nests are unknown. Fungal pellets were expelled sterile from the females as described in A. texana was conducted in May 2006, when three nests were sampled intensively, nest UGM050509-01 , nest UGM060121-01 , and nest UGM060121-02  . For nest UGM050509-01, additional samples were available for the years 2004, 2005, 2007–2010 , yielding for this nest a continuous seven-year record . For nest UGM050509-02 (not part of the 2006 survey), three pellets were genotyped for each of 2004, 2007–2010 . For nest UGM050509-07 , two, three, and one pellet were available for 2004, 2009, and 2010 . A. texana was assessed primarily by genotyping the fungal pellets carried by females emerging from nests for a mating flight (see above), but a few nests were also repeat-sampled by excavation as part of a larger population-genetic survey of A. texana cultivars [U.G. Mueller unpublished] and as part of a phenological survey of the non-cultivar fungi growing in gardens of A. texanaFungal diversity within single nests of Atta cephalotes were excavated in December 2003 along Pipeline Road, Parque Soberanía, Republic of Panamá. Nest mounds had a diameter of at least 14 meters . In sum, to evaluate genetic differences between fungal samples within A. cephalotes nests, we screened an average number of 730 loci per nest in an average of 73 garden fragments per nest (10 loci screened per fragment).The average number of samples per nest was 73.0 garden fragments (range 68–76), for a total of 438 garden fragments between all six nests. Each sample was genotyped at ten microsatellite loci . Two fragments of a single garden were collected for each of these four nests and preserved in 100% ethanol. These 2008 samples, and corresponding samples for each nest collected in 2003, were genotyped at 15 loci using the multiplexed microsatellite screen  supplemented with the antibacterial chloramphenicol. Plates were sealed with parafilm and incubated at room temperature (about 20–23°C). The number of colony-forming units  of the Attamyces cultivar growing on each PDA plate was counted two weeks after plating. Twenty pellets were screened from females from Nest 1 (UGM050509-01) at Brackenridge Field Lab, and 8 pellets from females from Nest A (UGM060121-01) at Hornsby Bend Environmental Research Center  the particular plant substrate incorporated in the pellet (different substrates may anchor or protect Attamyces cells differently), (b) viability differences between Attamyces genotypes on the growth medium, and (c) pellet health or age . Absolute counts of CFUs per pellet and any differences between samples , but these genotyping differences do not affect any of the conclusions. See Results S1Genotyping Methods and Results.(0.06 MB DOC)Click here for additional data file.Table S1Attamyces pellet genotyping 2004-2010 for Atta texana.(0.18 MB PDF)Click here for additional data file.Table S2Attamyces diversity for Atta texana.Within-nest (0.07 MB PDF)Click here for additional data file.Table S3Attamyces diversity for Atta cephalotes.Within-nest (0.30 MB PDF)Click here for additional data file.Table S4Attamyces garden genotyping 2004&2008 for Atta cephalotes.(0.06 MB PDF)Click here for additional data file.Table S5Attamyces colony-forming units per pellet for Atta texana.(0.06 MB PDF)Click here for additional data file.


Hair follicles                        undergo continuous cycles of growth, involution and rest. This process, referred                        to as the hair growth cycle, has a periodicity of weeks to months.  At the                        same time, skin and hair follicles harbor a functional circadian clock that                        regulates gene expression with a periodicity of approximately twenty four                        hours.  In our recent study we found that circadian clock genes play a role                        in regulation of the hair growth cycle during synchronized hair follicle                        cycling, uncovering an unexpected connection between these two timing                        systems within skin. This work, therefore, indicates a role for circadian                        clock genes in a cyclical process of much longer periodicity than twenty                        four hours.                 Beginning after completion of hair morphogenesis                            , hair growth cycles commence with catagen, an                            involution process of the hair follicle during which the majority of its                            epithelial compartments undergo apoptosis .  In micIn order to systematically discover                            transcriptional activity associated with the hair growth cycle, we                            profiled mRNA expression at a genome-wide level over multiple time points                            corresponding to morphogenesis and two synchronized hair growth cycles.                             Interestingly, a large fraction of the genome, more than six thousand genes,                            exhibits changes in expression that correlates with the progression of the hair                            growth cycle, thus underscoring the complexity of this process ,5.    OnOn a molecular level, the circadian clock consists of                            positive and negative feedback loops.  At its core are the bHLH-PAS                            transcriptional activators CLOCK and BMAL1 (ARNTL), which form a heterodimer                            and activate target genes containing E-boxes in their enhancer regions,                            including Periods  and Cryptochromes (Cry1 and 2).  PERs and                            CRYs form heterodimeric complexes that translocate into the nucleus where they                            inhibit BMAL1-CLOCK transcriptional activity, thus constituting the negative                            feedback loop . In otheSince our mRNA expression studies                            were performed using whole skin, we asked which compartments of the skin and                            hair follicles contribute to the robust rhythmic circadian gene expression in telogen.  The hair                            follicle contains several functionally and                            structurally distinct compartments, including the bulge region, which harbors                            slow-cycling hair follicle stem cells ; the secThe robust circadian clock gene                            expression within the secondary hair germ led us to test the possibility that                            circadian clock genes might play a role in the hair growth cycle.  For these                            studies, we turned to Clock and Bmal1 mutant mouse models. We found a                            significant delay in anagen progression in both mutants and this delay was more                            pronounced in Bmal1 deficient mice, possibly due to partial functional                            redundancy between Clock and its homologue Npas2. Clock and Bmal1 mutant mice                            have no visible defects in hair follicle morphogenesis and enter the first                            stage of anagen, characterized by the expansion of the secondary hair germ, at                            approximately the same time .  Yet by day 28, when the                            majority of hair follicles in control  littermates                            have  developed hair matrix  and hair shaft with the hair bulb growing into the                            subcutis, the Bmal1 mutant mice remained in the first anagen phase .  AfterFurther analysis revealed absence of mitotic cells in                            the early anagen secondary hair germ in Bmal1 mutant hair follicles, while                            wild-type secondary hair germs at the same stage contained mitotic cells.                             Importantly, epidermis and dermis of Bmal1 mutant mice contained mitotic cells,                            indicating that the proliferation defect was hair follicle specific.                             Phosphorylated Retinoblastoma Protein (Rb), a marker of cell cycle progression                            through the G1-S cell cycle checkpoint , was absTo gain insights into the                            molecular mechanisms underlying the G1 arrest of progenitor cells in the                            secondary hair germ, we profiled gene expression in the skin of Bmal1 deficient                            mice during telogen.  As expected, the expression of multiple known CLOCK-BMAL1                            target genes was affected, including that of Rev-Erbα, which was                            downregulated approximately fifteen fold.  Studies in hepatocytes have                            demonstrated that REV-ERBα directly represses expression of the gene                            encoding the G1 cell cycle inhibitor p21WAF1/CIP , and conIn the mouse, the first two hair growth cycles are                            synchronized.  After completion of the second telogen, which can last up to                            thirty days, the coat begins to grow asynchronously in complex domains created                            by waves of anagen moving through the domain until a wave reaches "refractory"                            telogen, an area of skin unresponsive to the propagating anagen stimulus.  As                            the mouse ages, this process creates increasingly complex patterns of hair                            growth with each domain consisting of a telogen competent to be activated, a                            propagating anagen wave, a catagen, and a refractive telogen ,25.  InOne plausible role for circadian                            mechanisms in the hair growth cycle is in animals with seasonal hair growth, commonly found in mammals living in the wild -30.    Hair loss and hair graying are commonly recognized                            symptoms of aging in mammals. In addition to the visible location of hair, the                            highly regenerative nature of hair follicles may explain why hair loss is a                            prominent feature of aging syndromes. Several mouse models with premature aging                            phenotypes show progressive hair loss or graying -38, and Age-associated hair graying has been linked to                            ultraviolet light and reactive oxygen species (ROS)-induced cell damage.  The                            hair follicle bulge harbors melanocyte stem cells that give rise to mature                            melanocytes which synthesize and secrete hair pigments during anagen.  Graying                            human hair follicles have been shown to contain melanocytes with accumulated                            oxidative stress , and conThe mechanisms underlying senescent alopecia have not                            been extensively studied. However, a plausible hypothesis is that analogous to                            hair graying, alopecia is related to loss of hair follicle epithelial stem                            cells either through decreased renewal, premature differentiation, apoptosis or                            cellular senescence. While the link between genotoxic stress and hair graying                            has been established using mouse models, only correlative data from human                            patients are available in regards to life-long UV exposure and hair loss ,45.  SkiRecent studies indicate that circadian clock proteins                            may be involved in DNA repair and in regulating accumulation of cellular ROS,                            thus making them plausible actors in the aging processes .  Fu et.Direct evidence for clock involvement in                            the aging process comes from the study of Kontdravov et. al.  , showingIn conclusion, our study demonstrated that                            circadian clock genes can regulate the non-circadian cyclical hair growth                            cycle, presumably via an effect on the progression of the cell cycle in a                            progenitor cell compartment of the hair follicle, the secondary hair germ .


Microarray technologies are widely used to quantify the abundance of transcripts corresponding to thousands of genes. To maximise the robustness of transcriptome results, we have tested the performance and reproducibility of rat and mouse gene expression data obtained with Affymetrix, Illumina and Operon platforms.We present a thorough analysis of the degree of reproducibility provided by analysing the transcriptomic profile of the same animals of several experimental groups under different popular microarray technologies in different tissues. Concordant results from inter- and intra-platform comparisons were maximised by testing many popular computational methods for generating fold changes and significances and by only considering oligonucleotides giving high expression levels. The choice of Affymetrix signal extraction technique was shown to have the greatest effect on the concordance across platforms. In both species, when choosing optimal methods, the agreement between data generated on the Affymetrix and Illumina was excellent; this was verified using qRT-PCR on a selection of genes present on all platforms.This study provides an extensive assessment of analytical methods best suited for processing data from different microarray technologies and can assist integration of technologically different gene expression datasets in biological systems. SeveraleneChip®  and Illup arrays , which b® have an entirely unique layout of 50 base pairs long oligonucleotides synthesised in a separate procedure and attached covalently to silica beads, which are randomly dispersed over the array so that they each lie in an unique well [On Affymetrix arrays, genes are represented by one or more probe sets, which are short oligonucleotides (25 base pairs) covering distinct sections of the gene synthesised in place through photolithography. Illumina BeadChipsque well . This raThe evaluation of the reproducibility of biological results across microarray platforms is essential for interpreting data independently generated with different technologies and reducing the need to duplicate experiments. Transcriptomic platforms produce both random and systematic errors in predicting actual biological changes. When data from two platforms are compared, the two platform-specific systematic errors are combined with the random error, hence the superior platform which minimises its own systematic errors should have higher agreement with the other platforms.Growing interest in testing consistency of gene transcription results from different sources has paralleled the increasing number of genome-wide gene expression datasets and the emergence of various microarray platforms and technologies . Some stStudies on multiple platforms have found generally poor reproducibility across gene expression platforms ,17, althAlthough numerous methods have been developed for transcriptomic data analysis, the vast majority of these studies used only one method of data normalisation for each platform, and a single agreement measure (for example fold change), hence many methodologically biases may not be recognised. Here we report the first comparative analyses of multiple gene transcription data sets obtained in parallel with different microarray technologies over several popular normalisation methods using multiple cross-platform concordance criteria. We use biologically-relevant transcriptomic data for the mouse and rat genomes in the context of insulin resistance in inbred strains. Probe alignment was performed both by sequence identity and assignment to the Ensembl Gene database. After applying multiple background, intra- and inter-array effect corrections and signal extraction and probe summarisation techniques, we demonstrate the factors affecting the concordance of both magnitude and statistical significance of the transcriptomic effects between platforms.To test the performance of widely used microarray technologies in generating consistent genome-wide gene expression data and to assess the effects of various calibration methods on the degree of cross-platform data reproducibility in terms of magnitude and statistical significance of gene expression changes, we used transcriptomic profiles of various mouse and rat organs derived with different microarray systems.To determine the degree of consistency in gene content between platforms, we initially tested both individual oligonucleotide mapping and gene level agreements. In the mouse arrays, we aligned 8,901 Illumina oligonucleotides to Affymetrix probe sets using "target sequence" identity and a total of 14,242 Ensembl genes were overlapping between the two platforms. In the rat arrays, analysis of gene level matches via the Ensembl gene database allowed the identification of a core of 1,804 individual genes represented on all three platforms, as opposed to 149 using "target" sequence identity, thus enabling a more reliable three-way comparison to be made.r) between the measured intensities for gene matches between any two platforms for all experiments was greater than 0.7, and the majority greater than 0.8. In the rat datasets, the minimal Illumina-Operon pairwise correlations were all greater than 0.8 for all strain comparisons , but had a large impact when correlating fold changes for the Illumina and Affymetrix mouse datasets  analysis of the expression of a selection of genes in the rat kidney . The Affymetrix signal extractions showed a much larger heterogeneity than the background corrections and normalisations used on the other platforms. This is largely due to the more complex design of the Affymetrix microarrays, including the presence of distinct oligonucleotides in a probe set and mismatch probes, which are handled differently. The difference was greatest at low fluorescence. In the mouse dataset, the Li-Wong method and MAS 5.0 had high variance Tables  and 3 anWhen qRT-PCR data derived in rats for a small election of genes were used to assess the effects of normalisation methods on microarray-based gene expression data, we found that for Affymetrix, MAS 5.0, RMA, GC-RMA and the Li-Wong method all had very high correlations with the logarithm of the qRT-PCR fold change, and the highest was comparison-dependent  , which represent significant challenges due to the existence of synonyms and evolving or inconsistent annotations. Our inter-platform correlations in log fold changes and agreements in the most affected genes obtained using "target" sequence identity were often surpassed by aligning the oligonucleotides to Ensembl gene sequences. This is surprising, as the precise matching of oligonucleotide sequences within a gene provided by "target matching" is expected to be most reproducible, as the effects of complicating factors, such as alternative splicing, are reduced. However, the removal of poorly annotated probes in a probe set and combining information across entire genes create more reproducible results, and demonstrate the power of this novel alignment tool.The Affymetrix signal extractions showed a much larger heterogeneity than the background corrections and normalisations used on the other platforms. This is largely due to the way the methods treat the more complicated design of the Affymetrix platform (distinct probe sequences in a probe set and MM probes).RMA was the method which performed consistently well in all comparisons. It produced high correlation in log fold change of gene expression between platforms, regardless of filtering, annotation or biological comparison and high agreement in gene lists for both statistical significance and fold change magnitude. However, the fold change can be underestimated by RMA . This waThe Affymetrix own method MAS 5.0 and the popular Li-Wong method showed very mixed results, especially extreme for MAS 5.0. These methods showed poor agreement with other platforms when all genes were used, but occasionally produced excellent results when intensity-based filtering was utilised. These methods often showed extremely high agreement in top fold change and  p value lists. These two methods also produced very high correlations with the qRT-PCR data, and MAS 5.0 fold change was almost identical. These findings suggest that these methods may be useful when searching for large effect sizes in highly-expressed known genes, but are poor for whole genome studies or detection of effects of small magnitude. Other Affymetrix normalisations, which used the PM values without correction often showed intermediate results. This implies that using either the mismatch values or a statistical framework to calculate and remove background effects is likely to improve the accuracy, sensitivity and reproducibility of transcriptomic data generated using the Affymetrix platform.Testing multiple analytical methods in microarray experiments is essential to maximise the robustness of results of gene transcription profiling in terms of metabolic and hormonal regulations of systems and interpretations of biological processes. This study provides an exhaustive and stringent assessment of analytical methods best suited for processing data from different microarray-based gene expression profiling technologies. These results are important for comparing transcriptomic data generated with different platforms and explaining inconsistencies between results that can be reanalysed using the most appropriate method. Our analyses can assist the integration of gene expression data obtained with different technologies in a single biological system.Based on results from our analyses, the following recommendations can be made:1. The choice of platform depends on the design of the experiment. Illumina microarrays can be more appropriate for models with large transcriptomic differences, whereas Affymetrix have a larger dynamic range.2. For Affymetrix studies, the choice of signal extraction significantly affects reproducibility. RMA performs well in all tests, whereas MAS 5.0 and the Li-Wong method have high accuracy and sensitivity in detecting highly differentially expressed genes.3. Filtering results on intensity level, through platform-specific present calls, is very important for obtaining reliable data.t-test statistic on the unstable estimate of sample error, and the comparison of observed fold changes is recommended.4. In agreement with published findings , for mod5. Comparing platforms on a gene-level summary basis rather than individual nucleotide or accession number improves data reproducibility for the arrays studied.ad libitum as previously described [Male mice of the inbred strain C57BL/6JOxjr were bred locally using stocks from the Jackson laboratory. They are referred as C57BL/6 throughout the text. Mice were fed a normal carbohydrate (CHD) diet  or a 40% high fat diet (HFD)  escribed . For theescribed , the norOrgans used for gene expression profiling were chosen for their role in glucose homeostasis (liver) and diabetic complications (kidney). Total RNA was prepared from liver (mice and rats) and kidney (rat) biopsies as previously described .® GeneChip® Mouse Genome 430 2.0 arrays, which contain 45,101 probe sets, and Illumina® Sentrix® BeadChip Mouse-6 Expression arrays (Beta Version 1), which contain 46,120 distinct oligonucleotide sequences.Targets prepared from C57/Bl6 mice (n = 5 per diet group) were individually hybridised to both AffymetrixThree separate types of microarray were used to carry out rat gene expression profiling:- Operon Rat v1.0 OpArray™ is a two-colour gene expression system, containing 5,717 oligonucleotides, almost entirely representative for unique well-documented genes. A full factorial dye-swap design was implemented, where a target from each animal's mRNA was hybridised on a separate array with all animals from different strains, and each array was repeated with the corresponding dyes switched, to correct for dye biases. A total of 108 slides were used for the liver experiment and 54 for the kidney, as three animals per group were used. RNA from WKY-STZ kidneys were not used on this array type.® GeneChip® Rat Expression Set 230A GeneChip® arrays contain 15,923 probe sets, corresponding to over 10,000 distinct annotated genes. A total of twelve microarrays were hybridised using samples from three animals for each of the four groups.- Affymetrix® Sentrix® BeadChip RatRef-12_V1_Eval Expression microarrays carry twelve arrays for every slide, and each array contained 22,612 distinct oligonucleotides. Technical replicates were used for all samples.- Beta test version of the Illumina under the accession E-MEXP-1195 (Rat kidney transcriptome on Affymetrix), E-MEXP-889 (Rat liver transcriptome on Affymetrix), E-TABM-500 (Rat kidney transcriptome on Operon), E-TABM-502 (Rat kidney transcriptome on Illumina), E-MEXP-1755 (C57BL/6J mouse liver transcriptome on Affymetrix).All experiments are MIAME compliant. Protocols and data are available through ArrayExpress et al. 2004) and is analysed, by default, with no background removal technique.These techniques are very platform-dependent because of the various background measurement techniques and scanning technologies. Different signal extraction techniques, especially for the Affymetrix platform, are expected to be less comparable than different between-array normalisations applied to the same signal extraction, as the extraction models glean distinct information from the raw foreground (and background) intensities, whereas the normalisation methods seek to minimise distribution differences through global corrections. For Operon arrays we used background subtraction, Normal and Exponential Convolution model (normexp) , and met® BeadStudio® software. All the R normalisations were implemented in the "LIMMA" [The variance stabilisation (vsn) method  was used "LIMMA" , "affy"  "LIMMA"  or "vsn" "LIMMA"  R packagQuantitative Real-Time Polymerase Chain Reaction (qRT-PCR) was performed for a total of 17 genes with kidney samples from all four rat models. Genes were selected primarily due to differential expression in either the Affymetrix or Operon-generated rat kidney microarray data sets. Experiments were conducted using samples from the same animals as profiled with microarrays, and technical triplicates were used for all genes. Actin was used as the control "house-keeping" gene.We labelled as cross-platform "target matches", sets where the oligonucleotide sequence of a probe aligned identically to the sequence spanned by all probes of an Affymetrix probe set. Dai et al. observed that Affymetrix probe set design for each microarray could not evolve as the genome and transcriptome information improved over time, and that, in some cases, probes in the same probe set derived from distinct genes . They cr. For Illumina and Operon, no such annotations existed in the literature. However, as on both these platforms oligonucleotides in a set are identical, we aligned all probes against Ensembl genes , with the version used for the Affymetrix probe sets, to ensure the same genome builds were consistently used (Mouse NCBI m34 and Rat RGSC 3.4).Publicly available gene annotations were used Prior to analysis, the data was filtered by removing a user-specified proportion of oligonucleotides with the lowest mean log2 intensity in the comparison of interest. Only matches where at least one oligonucleotide from each platform under investigation remained after filtering were used.As different signal extractions and normalisations produce outputs in absolute or logarithmic values (in varying bases), all expression intensities and fold changes from all platforms were converted into logarithmic (base 2) values after normalisation. This transformation converts multiplicative effects (such as fold changes in gene expression) into additive effects, which increases ease of both analysis and interpretation . PearsonWe also investigated whether the platforms produce the same genes as most worthy of investigation, if one only considers the "top" genes ranked by either most significant p value or highest absolute fold change. Although any overlap is likely to be very highly pointwise significant when compared to the null hypothesis of no relation between the platforms, whether the agreement is sufficient to be biologically useful is difficult to assess.When comparing all three platforms, three pairwise tables would have to be created. In order to illustrate all intra-platform concordances simultaneously, a new plot which was labelled a "dartboard plot" was specifically designed Figures  and 4. TIt must be emphasised that when compiling these concordance lists, only oligonucleotides or genes which match to the other platform or platforms are included, implying there are no missing values, so that the above statistics can be applied and the lists directly compared for their technical performance. However, this is an important concern for experimental reasons; if two platforms show high concordance for genes assayed on both, but one measures the expression of many more genes, the two will not be of equivalent biological value. For the mouse experiment, the Affymetrix and Illumina microarrays had very similar oligonucleotide numbers, while in the rat the Operon platform had fewer oligonucleotides and the Affymetrix GeneChip utilised was designed in a prior generation of feature size to the Illumina BeadChip, so fewer probe sets were present. Hence for a valid comparison, only matching oligonucleotides were used. In general, similar generation Affymetrix and Illumina microarrays contain similar oligonucleotide numbers.BN: Brown-Norway; GK: Goto-Kakizaki; LIMMA: Linear Models for Microarray Analysis; MAS5.0: MicroArray Suite version 5.0; RMA: Robust Multi-array Analysis; STZ: Streptozotocin; WKY: Wistar-Kyoto; MM: mismatch; PM: perfect match; qRT-PCR: quantitative real time PCR; vsn: variance stabilisation.AS and JR produced the Operon arrays. AS, KA, MTB and PJK performed gene transcription profiling experiments and data interpretation. SPW carried out statistical analyses, bioinformatic studies and gene annotations of microarray data. SPW and DG drafted the manuscript. DG, PJK and MTB conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.y) against Affymetrix (x) for most normalisations for the 25% most intense oligonucleotides.Scatterplots of mouse target log2 fold changes for Illumina , Illumina  and Operon  for all tissues and strain comparisons. Analysis of rat gene expression changes for genes represented on the Illumina, Affymetrix and Operon arrays.Click here for fileScatterplots of Rat Ensembl Gene match log2 fold changes between Affymetrix , Illumina  and Operon  for all tissues and strain comparisons, restricting analysis to the 25% most intense genes on each platform (approximately 320 genes). Analysis of the magnitude of rat gene transcription changes for genes represented on the Illumina, Affymetrix and Operon arrays and showing strong level of expression.Click here for filey) against Affymetrix (x) mouse "Target matches" for all normalisations, restricting analysis to the 25% most intense oligonucleotides .Gradient of log2 fold change scatterplots of Illumina . Comparative analysis of statistical significance of mouse gene expression data derived by Illumina and Affymetrix arrays.Click here for fileConcordance of all mouse Affymetrix and quantile normalised Illumina unique "Target match" fold change data between different Affymetrix normalisations . Comparative analysis of the magnitude of mouse gene expression changes derived by Illumina and Affymetrix arrays.Click here for fileX fold change lists between Affymetrix , Illumina  and Operon  for both tissues and all comparisons, using unfiltered  and top 25% intensity-based filtering (approximately 280 genes).Total concordance in top  Comparative analysis of the magnitude of rat gene expression changes derived by Illumina, Affymetrix and Operon arrays.Click here for fileX p value lists between Affymetrix , Illumina (loess) and Operon  for both tissues and all comparisons, using unfiltered  and top 25% intensity-based filtering (approximately 280 genes).Total concordance in top  Comparative analysis of the statistical significance of rat gene expression changes derived by Illumina, Affymetrix and Operon arrays.Click here for fileCorrelation in log2 fold change between qRT-PCR and all Affymetrix normalisations by strain comparison for all seventeen genes in rat kidney. Comparative analysis of gene expression changes given by quantitative RT-PCR and Affymetrix array data normalised using several methods.Click here for fileCorrelation in log2 fold change between qRT-PCR and all Illumina normalisations by strain comparison for all seventeen genes in rat kidney. Comparative analysis of gene expression changes given by quantitative RT-PCR and Illumina array data normalised using several methods.Click here for fileCorrelation in log2 fold change between qRT-PCR and all Operon normalisations by strain comparison for all seventeen genes in rat kidney. Comparative analysis of gene expression changes given by quantitative RT-PCR and Operon array data normalised using several methods.Click here for fileDescriptions and log2 fold changes for all seventeen genes selected for qRT-PCR in all strain comparisons for the rat kidney experiment. Gene expression ratios derived by quantitative RT-PCR of renal samples from diabetic and control rats.Click here for filey) against microarray (x) for seventeen genes for selected normalisations for all microarray platforms for all rat kidney comparisons.Gradient of log2 fold change fit of qRT-PCR ( Comparative analysis of renal gene expression ratios in rat models of diabetes and controls given by quantitative RT-PCR and normalised data from the corresponding genes on Illumina, Affymetrix and Operon arrays.Click here for file


Background. Spindle cell lipoma (SCL) is a benign lipomatous tumour, typically occurring in the posterior neck, shoulder or upper back of elderly males. They compose of fat, CD34 positive spindle cells, and ropey collagen on a myxoid matrix. This case highlights a rare presentation of SCL and the need for pre-operative diagnosis. Case Report. A 63-year-old gentleman presented with a pre-existing left supraclavicular mass that had recently increased in size. FNA and CT Scans were performed and results discussed in the mutidisciplinary team meeting. Excisional biopsy was recommended. Radiology. CT neck showed a left supraclavicular mass of fatty density with fine internal septations. A low-grade liposarcoma could not be excluded. Histopathology. FNA was indeterminate. Histology of specimen showed bland spindle cells with no evidence of malignancy. Immuno-histochemistry showed SCL with CD34 positivity and negative staining on CDK4 and p16. Management. Excision biopsy of the mass was performed which was technically difficult as the mass invaginated around the brachial plexus. The patient recovered well post-operatively with no neurological deficits. Conclusion. Spindle cell lipoma is a rare benign tumour and a pre-operative diagnosis based on the clinical context, imaging and immuno-histochemistry is crucial to management. Lipomasare slow growing fatty tumours of mesenchymal origin and can occur in any organ of the body. Spindlecell lipomas (SCLs) are lipoma variants which have the potential of neuromuscular invasion despite their benign histopathology.  This case report highlights a spindle cell lipoma variant in the supraclavicular area found intraoperatively to invade both the paravertebral muscles and the brachial plexus. Computerised tomography (CT) and Magnetic Resonance Imaging (MRI) findings were suggestive of possible malignancy but the biopsy and immune-histochemical stains confirmed the diagnosis as SCL.  This report highlights the need for making a preoperative diagnosis of spindle cell lipoma. This ensures accurate patient consent and appropriate preoperative surgical planning including surgical approach and extent of surgery.A-sixty-three-year-old gentleman presented with a two-year history of a left supraclavicular mass which had increased in size over the previous three months prior to presentation. The swelling caused discomfort with movement but there were no neurological deficits. He was a smoker with a past medical history of hypertension and type 2 diabetes mellitus.  On examination, there was a large tennis ball sized swelling in the left neck level V area with no palpable cervical lymphadenopathy. This swelling was smooth and nontender on palpation. ENT examination was normal.  Fine Needle Aspiration (FNA) of the mass was performed but it was nondiagnostic. CT neck showed a 46 × 69 × 91 mm left supraclavicular mass posterior to the sternocleidomastoid muscle and superficial to the paravertebral muscles, of fatty density with fine internal septations Figures and 2. Th The case was discussed at the multidisciplinary meeting and due to the suspicion of malignancy, and a nondiagnostic FNA, excision biopsy was recommended. The operation was technically difficult because the mass invaginated around the brachial plexus and extended posteriorly through the prevertebral fascia. Complete excision of the mass was undertaken with mobilisation and preservation of the brachial plexus. The patient made a full recovery with no neurological deficits.The specimen weighed 132  grams and consisted of well-circumscribed fatty tissue. Histological examination showed mature adipocytes interspersed with fibrous septa containing bland spindle cells with focal pseudo-angiomatous arrangement. There was the presence of ropey collagen, a very characteristic finding in spindle cell lipoma. No lipoblasts were seen and the spindle cells showed strong uniform immuno-histochemical positivity for CD34.  Subsequent review also showed negative staining for CDK4 and p16, supporting the diagnosis of spindle cell lipoma, and excluding atypical lipomatous neoplasm.Spindle cell lipoma (SCL) is a benign tumour which typically occurs on the posterior neck, shoulder, or upper back in predominantly elderly males. There is a marked male predilection especially in the sixth decade which may be partly explained by androgen receptors reactivity . Other r Histologically it consists of a proliferation of mature adipocytes, collagen fibres, and spindle cells within a myxoid stroma , and 7 aDifferential diagnoses include atypical lipomatous tumour/well-differentiated liposarcoma, pleomorphic lipoma, neurofibroma, nuchal fibroma, lipoblastoma, hibernoma, cellular angiofibroma , extrama It is important to note that reactivity to CD34 immuno-histochemical stain is not specific to only spindle cell lipoma as other entities can demonstrate immunoreactivity to CD34 such as atypical lipomatous neoplasm (ALN)/well-differentiated liposarcoma (WDL).  ALN/WDL is the most common form of liposarcoma seen in late adult life with equal gender affectation generally. They contain a significant component of mature fat and tend to have less well-defined borders than lipomas . The fav SCL shows cytogenetic aberrations—mostly loss of 16q material and less frequently material from 13q. This coupled with the usual absence of giant marker and ring chromosomes that are typically seen in atypical lipomatous tumour/well-differentiated liposarcoma supports their distinction.  Immunostaining for MDM2 and CDK4 has been shown to be a relatively sensitive and specific means of identifying and separating atypical lipomatous neoplasm/well-differentiated liposarcoma from various benign lipomatous lesions , 16.  In this case report, histologic findings did not demonstrate sufficient nuclear atypia to suggest a diagnosis of atypical lipomatous tumour and further immuno-histochemical markers that would make such a diagnosis possible  were found to be negative.  SCL shares overlapping clinical, morphologic, and immuno-histochemical findings with pleomorphic lipoma as well as the same cytogenetic aberrations. The differential diagnosis of SCL or pleomorphic lipoma depends on which elements predominate .  Dermatofibrosarcoma protuberans (DFSP) is also a differential of SCL. It however typically arises in the dermis, often in younger patients and lacks the characteristic ropey collagen of SCL .  Schwannoma and neurofibroma have a similar striking nuclear palisading and conspicuous mast cell infiltrate present in some SCL. These however invariably express S-100 protein which is negative in SCL .  SCL may also be mistaken for several sarcomas, including myxoid liposarcoma or spindle cell liposarcoma. SCL is however more circumscribed, lacks lipoblasts, with characteristic ropey collagen bundles. The presence of multinucleated floret-like giant cells is characteristic of pleomorphic lipoma and is occasionally seen in atypical lipomatous tumour/well-differentiated liposarcoma.  In this case report, the spindle cell lipoma presented a diagnostic and management dilemma as it could not be clinically differentiated from a liposarcoma which would require a more radical approach to treatment. The radiologic findings of a complex internal architecture with fine septations allowed differentiation of the preoperative differential diagnosis from a benign lipoma or possible malignancy but could not differentiate between a liposarcoma or a spindle cell lipoma.  The authors suggest that when radiologic findings suggest a complex internal architecture suspicious for a diagnosis of a possible lipomatous tumour, it is imperative to obtain a biopsy prior to definitive surgery. Histologic interpretation is crucial and this can be supported by the use of immune-histochemical stains. Immuno-histochemical stains demonstrating positivity for CD34 can be helpful in diagnosis of CD34 positive tumours such as spindle cell lipoma, and the differential diagnoses discussed such as atypical lipomatous tumour and dermatofibrosarcoma protuberans (DFSP). As discussed, further differentiation can be made based on clinical presentation, immunoreactivity for actin or desmin, S-100 protein staining of mature lipocytes, the presence/absence of ropey collagen bundles, multinucleated floret-like giant cells and cytogenetic aberrations. In difficult cases, fluorescence in situ hybridization (FISH) analysis can be helpful. A preoperative diagnosis of spindle cell lipoma is crucial to surgical management as it impacts on the consent process, surgical approach, extent of surgery, and management.  In making a preoperative diagnosis, tissue sampling is required with either FNA or core biopsy. However, it is important to note that FNA may be nondiagnostic as it was in this case, because the specimen consisted largely of fatty cells. Core biopsy is preferable and can be obtained by an initial procedure under anaesthesia prior to definitive surgery based on a histological diagnosis. A preoperative diagnosis will guide the consent process as such patients can be warned of the risks of neurovascular injury as well as determining the extent of surgery.