Cerebral palsy (CP) is an upper motor neuron disease that results in a spectrumof movement disorders. Secondary to the neurological lesion, muscles frompatients with CP are often spastic and form debilitating contractures that limitrange of motion and joint function. With no genetic component, the pathology ofskeletal muscle in CP is a response to aberrant complex neurological input inways that are not fully understood. This study was designed to gain furtherunderstanding of the skeletal muscle response in CP using transcriptionalprofiling correlated with functional measures to broadly investigate muscleadaptations leading to mechanical deficits.Biospsies were obtained from both the gracilis and semitendinosus muscles from acohort of patients with CP (n = 10) and typicallydeveloping patients (n = 10) undergoing surgery. Biopsieswere obtained to define the unique expression profile of the contractures andpassive mechanical testing was conducted to determine stiffness values inpreviously published work. Affymetrix HG-U133A 2.0 chips(n = 40) generated expression data, which was validated forselected transcripts using quantitative real-time PCR. Chips were clusteredbased on their expression and those from patients with CP clustered separately.Significant genes were determined conservatively based on the overlap of threesummarization algorithms . Significantly alteredgenes were analyzed for over-representation among gene ontologies and musclespecific networks.The majority of altered transcripts were related to increased extracellularmatrix expression in CP and a decrease in metabolism and ubiquitin ligaseactivity. The increase in extracellular matrix products was correlated withmechanical measures demonstrating the importance in disability. These data lay aframework for further studies and development of novel therapies. CP (CP) is a movement disorder caused by an upper motor neuron (UMN) lesion in thedeveloping brain There are a variety of treatments designed to inhibit muscle activity in CP andprevent contracture formation. Physical therapy techniques, oral muscle relaxants,intrathecal placement of medication (baclofen), chemical neurectomies with phenol oralcohol, chemodenervation using neurotoxins, and surgical neurectomies have all beenemployed to decrease spasticity in children with CP There are no known genetic defects in patients with CP and their muscles, as it is adirect consequence of the UMN lesion Previous research demonstrated that muscle stiffness in contracture is independent ofactive muscle contraction As a purely adaptive muscle disorder, contractures are believed to have an alteredtranscriptional profile. The current study has taken advantage of a large surgicalpopulation of both children with CP and typically developing children to conduct arobust microarray analysis correlated to mechanical parameters. Our previous studywas limited by a very small control subject population which was not age matched,(N = 2) For this transcriptional study, a cohort of 20 subjects were recruited. 10 patientswith cerebral palsy were undergoing hamstring lengthening surgery, making biopsiesof gracilis and semitendinosus accessible. The disease parameters of these subjectsare listed in MAB21L1, SIM1, and EN1)with unknown roles in muscle as significantly different between muscles,demonstrating that both muscles have similar expression profiles. There werealso no genes that produced a significant interaction between muscle type anddisease state, indicating that both muscles undergo similar changes in CP.Among the 22,283 probesets on the microarray, 13,787 were considered present forfurther analysis. Of those 1,398 genes were identified as significantlydifferent in CP , 3,954 for RobustMultiarray Analysis (RMA), and 4,009 for GC-RMA; The condition tree was conducted based on all present genes on the microarray andresulted in a clustering of CP subject biopsies distinct from typicallydeveloping subject biopsies, with the exception of one mild CP subject . The conTRIM63 and (0.79)FBXO32. TRIM63 expression had asignificant main effect of CP (p<0.01)  in all 6 genes examined . qRT-PCR(p<0.01) .Secondary analysis was performed using predefined gene sets for Gene Ontologyallowing the categorization of significantly altered genes in CP. There were 87gene ontologies over-represented from the list of significantly genesup-regulated in CP , Table 2We recently created networks of genes related to muscle function to enabledetailed investigation of muscle-specific gene expression. A heatmap, usingexpression ratios, is created with genes listed in their 9 respective functionswith genes in those determined as significantly different in CP according toprevious analysis . Some geMost of the networks had a selection of genes that were both up and downregulated in CP. These include: the neuromuscular junction, excitationcontraction coupling, cytoskeleton elements, and muscle signaling. Othernetworks had transcripts that were altered in a more uniform manner. Moststrikingly, the extracellular matrix had 14 out of 20 transcripts with increasedtranscription. On the other hand muscle metabolic factors had 11 out of 37transcripts down regulated and only one up regulated. Unfortunately only 7 ofthe inflammatory markers were present and available for analysis on themicroarray. Some proteins function with a different isoform in fast or slowmuscle that is transcribed from a separate gene. There was no significant changein expression among the slow isoforms, however 5 out of the 11 transcripts forfast isoforms were significantly down regulated. This indicates a relative shiftto slow fibers that corresponds to the myosin heavy chain protein content of themuscles A unique aspect of this study was the corresponding mechanical measurements fromthe same muscle biopsies in 17 of the 20 subjects. Individual fibers wereisolated and mechanically tested to determine stiffness The objective of this study was to describe the transcriptional adaptations thatoccur in skeletal muscle of patients with CP and incorporate functional data todetermine the cellular mechanisms that drive the muscle pathology secondary to theupper motor neuron lesion. This research has seen little prior work, as there is nocommonly accepted animal model necessitating the challenges of direct human researchThe condition tree clustering demonstrates the difference between the transcriptionalprofile of muscles from patients with CP or typically developing . This seTo determine the various functions that are altered in CP we first established alist of genes with altered transcription. We sought to narrow our results byusing a more stringent analysis that uses the congruence of three summarizationalgorithms as well as a restrictive false discovery rate of 1%. Thisstill produced a large gene list due to the large sample size, but we chose notto implement a fold change cutoff as this assumes an arbitrary level of changeis required to be functionally important PARV was drastically altered in CP muscle PARV was unchanged. The impact of growth factor binding isalso crucial in skeletal muscle, with many of the transcripts being IGF bindingproteins known to be important in skeletal muscle.Among the up-regulated transcripts there were a total of 87 gene ontologiesover-represented, but they generally fell into only a few categories. Theincrease in extracellular matrix transcripts has been well documented in musclefrom CP Contrastingly, there were 62 ontologies that were over-represented indown-regulated genes. The most extensive of these were related to metabolicprocesses and ubiquitin ligases. Skeletal muscle is a very metabolically activetissue and these results suggest that, despite spasticity, the muscle has lessmetabolic machinery. This decrease was also seen in the previous transcriptionalstudy in CP, however this was accompanied by a shift to faster fiber types,which was not the case here CHRN) wasdown-regulated. CHRN is dramatically up-regulated when muscleis denervated COL4 was down-regulated as wasUTRN an important link to the extracellular matrix of theneuromuscular junction. It is unclear how these adaptations change the functionof the neuromuscular junction, but it does support evidence for disorganizationCategorical analysis provides an avenue to analyze vast gene lists intomanageable pieces. However, we also sought to investigate more deeply intonetworks of genes critical to muscle function using a recently established genenetworks . As CP is neurological in origin, theneuromuscular junction could be altered and has indeed been shown to have adisorganized nature in CP ASPH andDHPR which have a role in activating the -ryanodinereceptor are down-regulated. As are mechanisms for pumping calcium back in tothe sarcoplasmic reticulum with ATP2A and its regulatorSLN. Further, a decrease in transcripts of calcium bindingproteins of CALM1 and downstream CAMK2 suggestthat there is less calcium cycling in CP muscle. The only up-regulatedtranscript was RYR3, which is a ryanodine receptor expressed inimmature muscle, and reinforces the theme that there are a number of immaturetranscripts in these muscles.Excitation-contraction coupling has been largely unexplored in CP, but has beenshown to be altered and is also the target of therapeutic intervention MYH3) and embryonic myosin light chain(MYL4). Many of the contractile elements have well definedisoforms that have genes expressed in either fast or slow muscle CAPZ and titin filament Z-disc cappingprotein TCAP are down-regulated in CP. This could lead toZ-disc disorganization and TCAP itself is associated withdestabilization in limb girdle muscular dystrophy 2G That theme is also observed in the contractile transcripts of skeletal muscle.The only up-regulated genes are immature isoforms embryonic myosin heavy chain(SGC) and snytrophins (SNT) as well asUTRN are down-regulated. This is while many crosslinkingtranscripts of the cytoskeleton are up-regulated (ankyrin(ANK), sprectrin (SPT), filamin(FLNC)) along with an important connector to the Z-disc(MYOZ2). What role these increased cytoskeletal filamentconnections may play is unknown, but they did not lead to increased fiberstiffness EMD and LMNA, which both lead toDreifuss-Emery muscular dystrophy when absent are not co-regulated withEMD decreased and LMNA increased VIM is up-regulated, which isthe primary immature muscle intermediate filament that is replaced with desmin(DES) during development.The force generated in the sarcomere is transmitted through the cytoskeleton tothe cell periphery, but the effects in CP have been largely unexplored. Ourresults are somewhat difficult to interpret with many genes both up anddown-regulated. Of those associated with the dystroglycan complex, sarcoglycans and aheat-shock transcription factor (HSF2). The role of muscledamage in pathologic CP muscle is not discernable from these data.While the state of damage within a static muscle contracture is relativelyunknown, it is known that inflammation is present within damaged muscle MSTN) a critical muscle growth inhibitor, itsreceptor (ACVR2B), and up-regulation of natural inhibitor(FST). FGF2 also plays a role instimulating muscle growth and differentiation TRIM63) and itstranscription factor FOXOMSTN signal is in contrast to the previous study in CPmuscle and is a candidate for being responsible for blocking the growth signalin these muscles. The question of what limits muscle growth in CP is notapparent from these results.The critical aspect of muscle growth is a complicated system of many genes andviewed the lack of muscle growth is considered the primary cause of contractureThe role of fiber type has been discussed in regard to multiple networks. Theresults here show clearly that while slow isoform levels remain unchanged manyfast isoforms have significant decreases in transcription. The role of fibertype in upper motor neuron lesions has been inconsistent TTN) in being the major contributor to passive tension ofindividual fibers Having mechanical stiffness measurements for both the muscle fibers and themuscle fiber bundles with their extracellular matrix was a unique aspect of thisstudy. There is literature to support the role of the giant protein titinwere recruited into the study based on undergoing distal hamstring lengtheningsurgery involving both the gracilis and semitendinosus muscles such that 2muscle biopsies could be acquired per subject. “Control” subjects(n = 10) were pediatric patients undergoing ACL (anteriorcruciate ligament) reconstructive surgery of the knee with hamstring autograftusing gracilis and semitendinosus tendons that were excised along with a distalportion of the muscle that was obtained prior to trimming of the tendon. Controlpatients did not have any neuromuscular disorders and were ambulatory prior tosurgery suggesting no damage to the hamstring muscle from the injury. Theirindex injury occurred at least 6 weeks before their surgery. However, becausethey were having surgery to repair a torn ligament we acknowledge that these arenot truly normal muscles. However, given the ethical constraints associated withtaking muscle biopsies, we believe that this is the best possible comparisongroup that can be envisioned. Patients with CP had developed a fixed contracturerequiring surgery and were classified based on the clinical measures of GrossMotor Function Classification System RNA was extracted using a combination of standard Trizol  and RNeasy  protocols. Briefly, approximately 30 mg offrozen muscle tissue was homogenized using approximately 50 mg of RNase free 0.5mm zirconium oxide beads  in 0.5 ml Trizol usinga Bullet Blender . 0.1 ml of chloroform wasadded to the solution, then vigorously vortexed for 15 seconds, then centrifugedat 4°C for 15 min. The upper aqueous layer was removed and mixed with anequal volume of 70% ethanol before being added to the RNeasy spin column.The column was washed and then incubated with RNAse-free DNAse (Qiagen) for 15minutes and then washed again three times prior being eluted as described in themanufacturer's protocol. RNA concentration was determined by absorbance at260 nm, and the 260 nm-to-280 nm absorbance ratios were calculated to define RNApurity.Affymetrix microarrays  were used foreach individual muscle biopsy (n = 40 chips). RNAprocessing including cDNA synthesis, cDNA labeling, microarray hybridization,microarray scanning, and stringent quality control measures were performed bythe Gene Chip Core at the Department of Veterans Affairs San Diego Health CareSystem, . The raw data are available (.cel files) at the GeneExpression Omnibus (GEO) under accession number GSE31243.DMD, COL1A2,MSTN, IGF1, COL4A2, andMYH1) and also to provide expression values for twotranscripts of interest not present on the microarray (FBXO32and TRIM63). The same RNA extracted for microarray analysis wasused for qRT-PCR. Isolated RNA was diluted 1∶5 with DNase/RNase free water(Invitrogen) and 1 µl of each sample was reverse transcribed usingstandard protocols . cDNA was amplified with theCepheid SmartCycle  with primers designed specific to each geneof interest (2(Invitrogen), 0.2 mM sense and antisense primers, 0.2 mM dNTP, 0.2×SYBRgreen, and 1 U of platinum Taq polymerase (Invitrogen). Amplification conditionsincluded an initial hold at 95°C for 2 minutes with 40 cycles of denaturingat 95°C for 15 seconds, followed by annealing and extension phases adjustedfor each transcript. Success of each the reaction was determined based onobservation of a single reaction product on an agarose gel and a single peak onthe DNA melting temperature curve determined after the 40 cycles. The results ofqRT-PCR were expressed using a standard curve method with the “cycles tothreshold” values represented the number of PCR cycles at which the SYBRgreen signal was increased above threshold. The triplicate measures werenormalized to the housekeeping gene GAPDH and then averaged.qRT-PCR data were normalized to the median value of the gene to facilitatecomparisons to microarray data.Quantitative real-time PCR (qRT-PCR) was conducted to validate the expressionlevels of select genes  for determination of significantly altered genes andclustering analysis. Present genes were determined from the MAS5 (Affymetrix)probe set algorithm based on a 12.5% (5/40) present call. Each sample wasclustered based on MAS5 for present genes based on Pearson Correlationsimilarity score and average linkage clustering algorithm.To provide a conservative choice of significantly altered genes in CP, threeindependent probe set algorithms were used: MAS5, RMA, and GCRMA. Requiringconcordance among different probe set algorithms has recently been used as anapproach to reduce false positives in data sets specific to any individualalgorithm. Each probe set was normalized to the median of the microarray andthen to the median of that probe set on all chips. The probe set data were thencondensed into gene level data in GeneSpring by calculating the median value ofall probe sets belonging to a single gene. Gene values were analyzed by2×2 Welch ANOVA based on pathology (CP vs. Control) and muscle (gracilisvs. semitendinosus) with a Benjamini and Hochberg False Discovery Rate formultiple testing correction setting the required statistical significance to(p<0.01). For each algorithm significant genes were separated into lists forincreased or decreased expression with CP, so that concordances of significantgenes were all in the same direction.Accordingly, 1% of the genes deemedsignificant for an individual probeset algorithm are suspected to be falsepositives and genes that passed in all three algorithms were designated assignificantly different for further analysis.http://bioinfo.vanderbilt.edu/webgestalt/) on Gene Ontology,KEGG Pathways, transcription factor targets, and microRNA targets After the significantly altered genes were defined, categorical analysis was usedto provide information on over represented subsets of genes. Enrichment analysiswas performed on up-regulated and down-regulated genes independently withWebGausalt . As these networks are specific to skeletalmuscle function, each gene in the network was also investigated in order toavoid arbitrary cutoffs. The networks were visualized using Cytoscape  The same biopsies used for microarray analysis also underwent mechanicalexperiments, published separately, that allowed matching of microarray data tomechanical function Correlations analysis was performed using MATLAB software . Each gene that was considered significantly altered in children with CPfrom the analysis above was correlated with each of the physical parametersmeasured. Correlations were considered significant with p<0.05. No multipletesting correction was used in determining genes significantly correlated withmechanical data.Table S1A list of each gene that was significantly altered in CP.Gene_ID is affymetrix microarray spot ID. Ratio is expression ratio of CP totypically developing controls. P-values are listed for all 3 summarizationalgorithms used.(XLSX)Click here for additional data file.Table S2Significantly over-represented ontologies from genes significantlyup-regulated (A) or down regulated (B) in CP. Category (C),Observed (O), Expected (E), Ratio of observed over expected (R), raw p-value(rawP), and multiple testing adjusted p-value (adjP).(XLSX)Click here for additional data file.Table S3List of gene families and complexes used for . Complexes are separated by the functionalmuscle network.(XLSX)Click here for additional data file.Table S4Genes significantly correlated with passive stiffness measurements onfibers and fiber bundles.(XLSX)Click here for additional data file.Table S5Primer pairs used for quantitative real-time PCR analysis.(XLSX)Click here for additional data file.Table S6Significantly over-represented ontologies from genes with positively ornegatively significantly correlated with fiber or bundle passivestiffness. Category, Observed, Expected, and multiple testingadjusted p-value (adjP).(XLSX)Click here for additional data file.Table S7Similarity between upper and lower extremity transcriptional studies ofskeletal muscle in ceregbral palsy. The list of genes that aresignificantly regulated in the same direction with cerebral palsy betweenthe current study in hamstring muscle and a previous study in wrist muscle.Only 13 genes were up-regulated in both and only 6 down regulated in both.The 13 up-regulated genes were significantly over-represented in the 4extracellular matrix gene ontologies listed.(XLSX)Click here for additional data file.


Sc immunohistochemistry (IHC), paraffin embedded tissue blots (PET-blot) and Western blotting approaches were used to determine the neuroanatomical distribution and molecular profile of PrPSc associated with each strain, in conjunction with traditional methodologies. Results revealed three strains isolated through each mouse line, including a previously unidentified strain. Moreover IHC and PET-blot methodologies were effective in characterising the strain-associated types and neuroanatomical locations of PrPSc. The use of Western blotting as a parameter to define classical scrapie strains was limited. These data provide a comprehensive description of classical scrapie strain phenotypes on isolation through the mouse bioassay that can provide a reference for further scrapie strain identification.Mouse bioassay can be readily employed for strain typing of naturally occurring transmissible spongiform encephalopathy cases. Classical scrapie strains have been characterised historically based on the established methodology of assessing incubation period of disease and the distribution of disease-specific vacuolation across the brain following strain stabilisation in a given mouse line. More recent research has shown that additional methods could be used to characterise strains and thereby expand the definition of strain “phenotype”. Here we present the phenotypic characteristics of classical scrapie strains isolated from 24 UK ovine field cases through the wild-type mouse bioassay. PrP C) to a pathogenic isoform termed PrPSc[Sc is detectable by immunochemical methods, can precede clinical and histopathological signs and is widespread in the CNS of classical scrapie infected animals at end stage disease [Classical scrapie, which affects sheep and goats, is the prototypic transmissible spongiform encephalopathy (TSE), a family of fatal neurodegenerative diseases that also affect humans and other mammals. Histopathologically, classical scrapie manifests primarily as spongiform change and gliosis in the central nervous system (CNS) of affected animals . The primed PrPSc. PrPSc i disease .Prnp amino acid sequence (Prnpa) whilst VM mice (Prnpb) differ at codons 108 and 189 [A fundamental trait of prions is their ability to manifest as distinct strains that demonstrate characteristic and reproducible phenotypes when passaged in a given species . Classic and 189 . This di and 189 . Indeed Traditional strain typing methodology dictates that an isolate is independently passaged through the C57BL/6 and VM line until the strain is stabilised, as measured by a non significant difference in the IP between consecutive serial passages  and thenSc as the probable disease-causing agent has augmented the development and application of alternative methods for the diagnosis and characterisation of TSE strains. These methods can also be applied to murine models and as such represent additional parameters to characterise strain phenotype. They include immunochemical methods that detect PrPSc in tissue sections, i.e. immunohistochemistry (IHC) [Sc following proteinase K digestion and ratio of PrPSc glycoforms [Sc sensitivity to proteinase K, and its conformational properties and stability (conformation-dependent immunoassay) [The identification of PrPry (IHC) -12, parary (IHC) ,14 and hry (IHC) , those wycoforms  and assanoassay) .Sc deposition patterns indicative of specific strains were identified following first passage of ovine classical scrapie isolates to wild-type mice [Ultimately the use of alternative methodologies in conjunction with the traditional bioassay approach can expand the definition of strain phenotype and crucially, if defined phenotypic parameters are identifiable at earlier passages, would reduce the time and number of mice required to perform strain characterisation. In accordance with this hypothesis we have previously reported that PrPype mice ,18,19.Sc deposition patterns revealed by IHC and PET-blot analysis and by Western blot profiling in addition to standard strain typing methodology. Using the approach suggested here we identify and characterise several scrapie strains and suggest how the strain of TSE sources can be determined without the requirement for a full standard bioassay protocol.The purpose of this study was to devise a methodological approach by which a large number of classical scrapie sources could be characterised following mouse bioassay. The phenotypic properties arising from 24 classical scrapie field sources that have undergone full strain typing through either the C57BL/6, VM, or both mouse lines are reported, according to PrPPrimary isolates were generated within two projects where wild-type mouse bioassays were initiated for cases of natural, ovine classical scrapie representing several breeds and PrP genotypes Table . These tnd and 3rd passage) would be adequate to reach stabilisation following which the 4th “characterisation” passage into 10 C57BL/6, 10 VM and 10 C57BL/6 × VM mice would ensue. Where IP did not stabilise and/or if the transmission rate did not reach 100% for each panel of mice, additional serial passages were carried out before final characterisation of the agent. All analyses were carried out on mice at final passage which was the passage used for strain characterisation.Mice were monitored and euthanised, and tissue collected and stored as described above for generation of primary isolates. During serial passage of each scrapie source, the portion of frozen brain of the first C57BL/6 and VM mouse whose fixed tissue was identified histopathologically as TSE positive was homogenised with normal saline (1% w/v) and inoculated via the intra-cerebral route into a panel of 10 mice of the same line (20 μL per mouse). Twenty four classical scrapie sources underwent full bioassay. Twenty nine transmissions are reported here because five sources were characterised through both the C57BL/6 and VM mouse lines. It was anticipated that two serial passages  and the relative band intensity (%) of each PrPSc glycosylation state calculated.Tissues were extracted and analysed by Bio-Rad TeSeE™ Western blot . Briefly, 20% (w/v) tissue homogenates were treated with proteinase K (PK) before alcohol precipitation. After centrifugation pellets were solubilised in Laemmli buffer and proteins separated on 12% Bis/Tris gels, electrotransferred to membrane, blocked with 5% (w/v) bovine serum albumin and labelled with biotinylated Sha31 anti-prion antibody . FollowiParaffin-embedded tissue (PET) blots were performed as described previously  with minOf 87 transmissions  attack rates for 74 transmissions were 90-100%. For 10 transmissions attack rates were 70% or greater. These are high attack rates as expected at this stage of passaging with very few intercurrent deaths. The remaining three transmissions had lower attack rates as follows: inoculum 53; 40% in C57BL/6 × VM mice (following stabilisation through the VM line), inoculum 59; no transmissions in C57BL/6 or C57BL/6 × VM mice (following stabilisation through the VM line).Figure Corresponding LP are shown in Figure Of the two sources that were grouped as VLA-C1 based on IP data, the LP from source 55 following characterisation in all three mouse lines closely resembled the ME7 LP , for Western blot analysis following proteinase-K digestion with Sha31 anti-PrP antibody  compared to VM mice challenged with other scrapie strains , the midbrain (more extensively in 87A than 87V), all nuclei of the hypothalamus and thalamus and specific areas of the frontal level including the vertical limb of the diagonal band, ventral pallidum including medial forebrain bundle and islands of Calleja, the caudate putamen and lateral accumbens shell. Within the cerebellum the granular layer was most prominently affected with granular deposition (more so for 87A than 87V) and relatively large diffuse aggregates of PrPSc (87A), or smaller more defined aggregates (87V). Similar deposition was noted less extensively in the molecular layer. Intraneuronal labelling in the medulla was more prominent with 87V and extended to the cerebellar nuclei. Aggregates and plaques were a common and extensive feature of both 87A and 87V at all coronal levels. In 87A the plaques were of fibrillar type, exhibiting a dense core and radiating fibrils  in conjunction with the whole brain mapping approach, thus increasing the discriminatory power of this method to distinguish different strains. A distinct advantage of this method is that it is less affected by titre and patterns can be identified on an individual mouse basis, in contrast with traditional IP and LP approaches which are based on mean data of all mice inoculated with a given source. Thus with the IHC method it is possible to compare the PrPSc pattern in mice associated with a given TSE strain both within and between inocula derived from different sources.It has been previously reported that the deposition pattern of PrPt strain -12, alth protein -36. In t sources  and seco sources , althougSc deposition patterns: ME7 through both C57BL/6 and VM mouse lines gave a very similar pattern of predominantly widespread granular deposition within the neuropil and the presence of many small aggregates. The characteristic perpendicular linear streaks observed were in agreement with those reported in previous IHC [Sc pattern for VLA-V1 was very similar but had not been previously observed in association with any other strain in this mouse line.Each of the strains identified in the current study gave distinct PrPious IHC  and PET-ious IHC  studies ious IHC . 87A thrSc patterns following transmission of classical field scrapie sources to wild-type mice were reported [Sc-like pattern was reported in RIII and C57BL/6 mice, which suggests that this parameter stabilises before IP and LP. If used on primary passage IHC may therefore better reflect the repertoire of strains in the host and reduce the number of murine passages required for strain typing. IHC can also help to resolve strain identification when IP and LP are uninformative, even after full characterisation as was shown for VLA-C1 in the current study. Conversely this method may help to establish identification of novel strains, i.e. VLA-V1.In an earlier study the PrPreported . NotablySc detection. It has been reported that in C57BL/6 mice inoculated with ME7, PrPSc was detected in the brain by PET-blot 30 days after inoculation and 145 days before clinical signs [Sc across the brain associated with different prion types [Sc associated with different classical scrapie strains and in this respect correlated well with IHC staining. In agreement with previous studies, IHC showed comparably better microscopic resolution at the cellular and subcellular levels, enabling different PrPSc types to be more clearly defined, although certain strain specific markers, i.e. perpendicular streaks of PrPSc through the molecular layer of the cerebellum, associated with ME7, were also identifiable by PET-blot as were denser structures including aggregates and plaques.The PET-blot method has previously been proven to be a highly sensitive method for PrPal signs , with gron types  and as aon types . For strAccording to our data, Western blotting cannot be utilised to strain type classical scrapie following passage in wild-type mice: it was not possible to distinguish the scrapie strains identified in C57BL/6 mice by Western blotting whilst in VM mice only 87V could be distinguished from ME7 and VLA-V1. This was in agreement with a previous study  where 87In conclusion we have characterised 29 transmissions of classical scrapie field sources through the wild-type mouse bioassay and attained a comprehensive phenotypic description of each identified strain based on standard and alternative methodologies. Numerous scrapie strains have been reported using the wild-type mouse bioassay  however,Sc types as revealed by IHC on individual mice which may negate the requirement for serial passages currently required for strain typing in mice and in addition, may reveal more than one phenotype emerging from a single source.The current study will serve as a standard reference which will be used to further analyse the dynamics of strain evolution and adaptation of classical scrapie through serial passage in wild-type mice, including the identification of strain specific parameters which can be used to identify strains during primary passage. Upon identification of the strains from at least 100 field cases of classical scrapie using the methodology presented here, we will attempt to analyse the strain demographics of classical scrapie in the UK and identify host parameters that may influence them. These data suggest that analysis of these sources should include whole brain mapping based on the distribution of different PrPThe authors declare that they have no competing interests.KB participated in lesion profiling, analysed IHC data and drafted the manuscript. CMV participated in lesion profiling and IHC analysis. RL participated in experimental design. TH, LT and LAT performed and analysed Western blotting data. MD and PW performed IHC and PET-blots. MMS conceived and designed experiments. JS conceived and designed experiments, participated in all data analysis and drafted the manuscript. All authors reviewed and approved the final manuscript.


Aspergillus nidulans using a combination of molecular genetics, biochemistry, cell biology and genomic approaches. Here we describe dual localization-affinity purification (DLAP) tags enabling endogenous N or C-terminal protein tagging for localization and biochemical studies in A. nidulans. To establish DLAP tag utility we endogenously tagged 17 protein kinases for analysis by live cell imaging and affinity purification. Proteomic analysis of purifications by mass spectrometry confirmed association of the CotA and NimXCdk1 kinases with known binding partners and verified a predicted interaction of the SldABub1/R1 spindle assembly checkpoint kinase with SldBBub3. We demonstrate that the single TOR kinase of A. nidulans locates to vacuoles and vesicles, suggesting that the function of endomembranes as major TOR cellular hubs is conserved in filamentous fungi. Comparative analysis revealed 7 kinases with mitotic specific locations including An-Cdc7 which unexpectedly located to mitotic spindle pole bodies (SPBs), the first such localization described for this family of DNA replication kinases. We show that the SepH septation kinase locates to SPBs specifically in the basal region of apical cells in a biphasic manner during mitosis and again during septation. This results in gradients of SepH between G1 SPBs which shift along hyphae as each septum forms. We propose that SepH regulates the septation initiation network (SIN) specifically at SPBs in the basal region of G1 cells and that localized gradients of SIN activity promote asymmetric septation.Filamentous fungi occupy critical environmental niches and have numerous beneficial industrial applications but devastating effects as pathogens and agents of food spoilage. As regulators of essentially all biological processes protein kinases have been intensively studied but how they regulate the often unique biology of filamentous fungi is not completely understood. Significant understanding of filamentous fungal biology has come from the study of the model organism  Aspergillus fumigatus, are opportunistic pathogens having the ability to cause systemic Aspergillosis in immuno-compromised patients Aspergillus nidulans is a powerful model genetic system and one of an impressive and growing number of sequenced filamentous fungal genomes including 19 species of Aspergilli (http://www.aspgd.org/) A. nidulans genome based on RNA-seq data together with advances in transcriptome analysis, endogenous gene targeting and the availability of gene deletion constructs for over 93% of genes have set the stage for further functional genomics Filamentous fungi have enormous ecological, medical, agricultural and industrial impact and understanding their often unique cell biology is of great importance A. nidulans hyphae arise from the growth of uninucleate conidiospores because septation is initially suppressed during the first few cell cycles Differential regulation of asymmetric septation in between nuclei along filamentous fungal hyphae contributes greatly to their often unique cell biology. Multinucleate A. nidulans has been analyzed using functional genomics to generate and phenotypically analyze deletion mutants of all 128 protein kinases A. nidulans has provided insights into both universally conserved kinase functions as well as fungal specific functions. For example the multifunctional NIMA kinase is the founding member of the NIMA related kinase (NEK) family and is essential for mitotic entry in A. nidulans and Magnaporthe griseaProtein kinases are involved in the regulation of virtually all eukaryotic biological processes through the reversible phosphorylation of their substrates and have thus been the subject of intense study Protein localization is often indicative of function and this is perhaps most clearly illustrated by the location of mitotic kinases. For example during mitotic progression mammalian Aurora B kinases sequentially locate to, and have functions at, chromosomal arms, centromeres, the spindle midzone, the equatorial cortex and the midbody A. nidulans offers many advantages for proteomic studies defining protein interactions and subcellular localizations. Aiding biochemical analysis is the ability to produce an enormous number of genetically identical conidia  for the rapid generation of biomass which can easily be harvested by filtration A. nidulans is also compatible with live cell imaging of proteins endogenously tagged with fluorescent markers and attaches to glass coverslips helping facilitate imaging over long time periods of polarized growth A. nidulans we describe here dual localization-affinity purification (DLAP) tags comprised of GFP in tandem with the S-tag for endogenous C-terminal or N-terminal protein tagging. To establish the utility of DLAP tags and advance the understanding of fungal kinase biology, we have endogenously tagged, localized and affinity purified 17 A. nidulans protein kinases and completed mass spectrometry analysis for 10 affinity purified kinases. Analysis of this data demonstrated that DLAP tagged versions of the CotA, SldABub1/R1 and NimXCdk1 kinases each specifically co-purify associated proteins. Comparative analysis of interphase and mitotic localizations indicated that 13 kinases changed location from interphase to mitosis and 7 kinases located to mitotic structures. Our data suggest functions for the An-Cdc7 DNA replication kinase at mitotic SPBs, the TorA kinase at endomembranes and provide novel insights into how SepH kinase location to SPBs differentially regulates septation generating asymmetric cell divisions along the length of hyphae.A. nidulans proteins endogenously tagged with the S-tag pyrGAf) for endogenous C-terminal tagging using standard A. nidulans primers AfpyrG tagging construct generated by standard 3-way fusion PCR was transformed into a ΔKu70nkuA recipient strain in order to facilitate a high frequency of homologous gene replacement  protein which localizes to the nuclear periphery and forms a complex with Nup159 Cdk1, UvsBATR, CmkA, SepH, SldABub1/R1, and SudDRio1, were tagged at experimentally verified C-terminals http://www.aspgd.org/) Hrr25 C-terminal with orthologous Aspergillus kinases was ambiguous, we performed 3′-RACE PCR which defined the C-terminus as GLGRQWYYEA*. For each of these kinases specific C-terminal DLAP constructs were generated, transformed into the same recipient strain and site specific transformants identified as above. In total 15 of the 17 C-terminally DLAP tagged kinases were functional as they complemented the phenotypes caused by deletion of the respective kinase genes . We note that localization studies of the UvsBATR orthologue Mec1p in S. cerevisiae were also unsuccessful ATR compromises its fluorescence.Apart from An-Cak1, which is predicted to activate cyclin dependent kinases, and the UvsBd levels  even thod levels . Given tRio1 and BckA kinases each localized throughout the cytoplasm but were excluded from regions evenly spaced along the germtubes, as was CotA, in a manner suggesting such shadows represent nuclei  before complete Nup49 dispersal suggesting that its kinetochore recruitment is a very early mitotic event. Given this, we determined if SldABub1/R1 kinetochore recruitment preceded that of the Mad1 SAC protein. This revealed that SldABub1/R1 initially accumulated at kinetochores while Mad1-mCherry was still present at its interphase NPC residence and before Mad1 transition to kinetochores and the surrounding spindle matrix  SAC protein. Although SldABub1/R1 and SldBBub3 were both identified in a genetic screen for mutants synthetically lethal without dynein function Bub1/R1 is a low abundance kinase and only a weakly silver staining band corresponding to the size of SldBBub3 was present in the SldABub1/R1 purification  Cdk1 partially dispersed from nuclei although a pool remained concentrated around the SPBs, as occurs during normal mitotic entry  with 9 peptides representing 21% sequence coverage as well as 71% sequence coverage for NimXCdk1  indicated that it was enriched in interphase nuclei, consistent with the well established function of the Cdc7/Dbf4 complex, called DDK for Dbf4/Drf1-dependent kinase, during DNA replication  after NPC reassembly which reestablishes nuclear transport . HoweverWe next determined if An-Cdc7 mitotic location was dependent upon microtubules by treating cells with benomyl. Microtubule depolymerization did not affect An-Cdc7 location to interphase nuclei and when these cells entered mitosis a pool of An-Cdc7 persisted at the SPBs . ImportaAn-cdc7 is essential we have recently utilized heterokaryon rescue to study the phenotype of the null allele Dbf4 in A. nidulansAn-cdc7 mutants do not undergo DNA replication but still attempt mitosis due to a failure to activate the checkpoint which prevents mitotic entry when DNA replication is incomplete A. nidulans cells still undergo multiple cell cycles, each consisting of a round of DNA replication and a delayed failed mitosis An-cdc7 mutants multiple cell cycles could occur in the absence of both DNA replication and successful mitosis. To determine this we utilized a heterokaryon containing both wild type and ΔAn-cdc7 nuclei as well as endogenously tagged GFP-tubulin and histone-H1-mCherry ΔAn-cdc7 spores generated from this heterokaryon were identified by their ability to form germtubes on selective media whereas An-cdc7 wild type cells from the same heterokaryon did not form germtubes. During interphase histone H1-mCherry fluorescence levels increased in wild type cells but not in ΔAn-cdc7 cells, consistent with the expected failure in DNA replication in cells lacking An-cdc7 function  which helps establish centromeric cohesion S. pombe Swi6/HP1 locates to SPBs A. nidulans HepAHP1 (AN1905) also locates to SPBs  HP1 at SPBs to help maintain centromeric cohesion until anaphase.The location of An-Cdc7 to SPBs might also be related to the function of the DDK complex in regulating centromeric cohesion Polo kinase, also locates to mitotic SPBs in A. nidulansS. cerevisiae DDK binds to the Cdc5p Polo kinase inhibiting the ability of Polo to promote mitotic exit Polo at SPBs during mitosis in A. nidulans.Another potential substrate of An-Cdc7, the PlkAHrr25-DLAP casein kinase 1 (CK1) located to multiple cell structures consistent with the diverse functions of this kinase family including roles in trafficking amino acid transporters to the plasma membrane in A. nidulansHrr25 was weakly enriched in interphase nuclei Hrr25 concentrated to a single nuclear focus which subsequently segregated into two foci  of septa . However, as indicated by the location of the type II myosin MyoB, septal precursors can begin to form at one site and then disassemble and reassemble at another site along hyphae Hrr25 negatively regulates septation, one possibility is that cytoplasmic CkiAHrr25 foci mark sites of septation which are subsequently aborted, perhaps due to the activity of CkiAHrr25.Intriguingly, in addition to nuclei, SPBs and septa, CkiAterphase . These fterphase . In addiThe SepH-DLAP septation kinase was difficult to detect in short cells but periodically became prominent at foci as cells grew longer. These SepH foci were unevenly distributed along germtubes typically concentrating in regions near the spore bulb of unseptated cells and/or near mature septa . This reS. pombe Cdc7 septation kinase (distinct from S. cerevisiae Cdc7p or An-Cdc7) which is the most upstream of the three septation initiation network (SIN) kinases, orthologues of which are required for septation in A. nidulansS. pombe Cdc7 locates to mitotic SPBs SepH is orthologous to the S. pombe Cdc7 initially associates with both SPBs during metaphase, during late anaphase and telophase it concentrates to only one SPB and this asymmetric distribution is important for SIN regulation Although A. nidulans germlings. To examine this we followed SepH location during entry into a prolonged mitotic SAC arrest in the absence of microtubules and followed mitotic entry and nuclear position using an NLS-DsRed (nuclear location sequence) reporter construct which disperses from nuclei when cells enter mitosis The preferential location of SepH to SPBs distal from the cell tip could be due to the affinity of these SPBs for SepH or potentially because SepH does not have time to spread from tip distal SPBs and foci to tip proximal SPBs during the rapid parasynchronous mitoses of cdk1, An-Cdc7 and CkiAHrr25, SepH is not maintained at mitotic SPBs during a mitotic SAC arrest. Notably however, during SIME after the SAC is inactivated, SepH again appeared at SPBs as cells returned to interphase . In 71% of cells (n = 14) septa formed between daughter nuclei originating from the same parental nucleus  as occurS. pombe in which SIN activation can be visualized by Cdc7 association with SPBs A. nidulans. During late mitosis S. pombe Cdc7 locates to only the new SPB indicating differential SIN activity between the two SPBs A. nidulans SepH locates to SPBs distal from the cell tip and suggest that the resulting gradient of SIN activity along the germ tube is important for septal positioning. Supporting this, when cells formed multiple septa the gradient of SIN activity (indicated by the SPB association of SepH) shifted along the germ tube as each septum forms. Therefore although still manifested at SPBs the asymmetric nature of SIN activity in A. nidulans occurs along the length of germ tubes and not necessarily between the SPBs of daughter nuclei. Thus the location of SepH during septation potentially explains how septation is restricted to the basal region of apical cells, as well as how such cells can form multiple septa in one cell cycle and how septa can form in between G1 nuclei originating from different parental nuclei.Septation is best understood in S. pombe Cdc7 and SepH at other stages in the cell cycle. Unlike S. pombe Cdc7, SepH locates to multiple non-SPB foci which are most prominent during early G2 in regions distal from the cell tip. Although the nature of these foci remains to be determined one possibility is that they help concentrate SepH in basal regions of apical cells. It is also notable that SepH prominently associates with a subset of SPBs distal from the cell tip during in early mitosis. It is likely that high NimXCdk1/Cyclin B activity at mitotic SPBs inhibits septation during mitosis in a manner similar to what occurs in S. pombe in which Cdc7 first associates with SPBs in metaphase S. pombeThere are also interesting differences between the locations of A. nidulans kinome by completing proteomic analysis of 17 A. nidulans kinases. The basis for this analysis is the DLAP tag comprised of GFP in tandem with the S-tag affinity purification peptide and developed here for endogenous protein tagging using standard primers and methods. Using this tag we confirmed 3 known or predicted kinase complexes by LC-MS/MS analysis of DLAP tagged kinases purified using a single step affinity purification procedure. Live cell imaging confirmed localizations for 4 previously studied A. nidulans kinases and experimentally verified the predicted location of 13 kinases for the first time in filamentous fungi. In addition the An-Cdc7 DNA replication kinase was unexpectedly located to mitotic SPBs suggesting an unknown mitotic function for this kinase. Finally the SepH kinase displayed a biphasic location to SPBs distal from the cell tip during mitosis and then again during septation providing novel insights into how septation is potentially regulated along the length of filamentous fungal hyphae. Collectively the data demonstrate the utility of the DLAP tag for proteomic and localization analysis in A. nidulans.We have extended and complemented previous functional analysis of the pyrGAf) cassette was amplified from plasmid pCDS65  was used for fragment amplification, fusion PCR and diagnostic PCR Both the N and C-terminal DLAP tags contained 10 amino acid spacers which separated the GFP and S-tag as well as the GFP and the tagged protein. The C-terminal DLAP  in which the deletion of the ku70nkuA gene facilitates a high frequency of homologous recombination pyrGAf marker selection and containing 1 M sucrose to maintain osmotic stability. Transformants were streaked to single colony and tested for site specific integration by diagnostic PCR using primers situated external to the targeting sequence for each tagging construct. External primers for C-terminally tagged kinases were the same as those used to confirm correct integration of kinase deletion constructs Tagging constructs were transformed into strain SO451 . To quantify histone H1 fluorescence levels the average pixel intensity less the background was determined for an identical sized area for each cell. Each time series was aligned for mitotic entry and values normalized relative to the last frame in G2 before mitotic entry which was set to 100%. Data points represent the mean +/− standard deviation.Conidiospores were inoculated in 35 mm glass-bottom microwell dishes (MatTech) containing minimal media with glucose as the carbon source and urea as the nitrogen source. Imaging was carried out at room temperature using an Ultraview ERS spinning disk confocal system (Perkin-Elmer) fitted with an Orca-AG camera (Hamamatsu) on a TE2000-U inverted microscope (Nikon) with a 60× 1.40 NA Plan Apochromatic objective (Nikon). For some experiments, imaging was with an Ultraview Vox spinning disk confocal system (Perkin-Elmer) fitted with dual C9100-13 cameras (Hamamatsu) run by Volocity software (Perkin-Elmer). Data are displayed as maximal intensity profiles. Benomyl (Sigma) was used at a concentration of 2.4 µg/ml which is sufficient to depolymerize all microtubules 8 conidiospores and grown until asexual spore production was apparent, typically 28–32 hr. 10 dishes grown in this manner typically generated ∼0.6 mg lyophilized dry weight and ∼80–100 mg of total protein extract. A control DLAP construct expressed from the pyrG promoter in place of pyrG served as a control. For lowly abundant kinases, as estimated by kinase-DLAP fluorescence levels, the volume of S-protein agarose beads (Novagen) was decreased two to four fold from the standard 150 µl packed bead volume/100 mg total protein. To minimize protein degradation, extracts were incubated with S-protein agarose for 15 min at 4° in the presence of protease inhibitors Endogenously DLAP tagged kinases were purified from lysates generated from lyophilized mycelia with the following modifications to methodologies previously described File S1Supplemental Figures S1–S4, Supplemental table S1.(PDF)Click here for additional data file.File S2Plasmid pCDS65 for C-terminal DLAP tagging.(PDF)Click here for additional data file.File S3Plasmid pCDS67 for N-terminal DLAP tagging.(PDF)Click here for additional data file.File S4Primer List.(PDF)Click here for additional data file.File S5Strain List.(PDF)Click here for additional data file.Video S1An-Cdc7 locates to mitotic SPBs. The cell in (AVI)Click here for additional data file.Video S2Cells lacking An-Cdc7 undergo multiple cell cycles. The movie used to generate An-Cdc7 spore containing GFP-Tubulin and Histone H1-mCherry. Although DNA replication does not occur due to the lack of An-Cdc7 function, this cell attempts mitosis twice separated by an intervening period of interphase. Time is in min.(AVI)Click here for additional data file.Video S3Hrr25 locates to SPBs and a cytoplasmic focus during SAC arrest.CkiA The movie used to generate Hrr25 -DLAP together with Ndc80-mCherry in a cell transiting the mitotic state without microtubules. Time is in min.(AVI)Click here for additional data file.Video S4SepH displays a biphasic location to SPBs distal from the cell tip during mitosis and septation. The movie used to generate (AVI)Click here for additional data file.


Corticosteroid hormones regulate appraisal and consolidation of information via mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) respectively. How activation of these receptors modulates retrieval of fearful information and the subsequent expression of fear is largely unknown. We tested here whether blockade of MRs or GRs during retrieval also affects subsequent expression of fear memory.Mice were trained in contextual or tone cue fear conditioning paradigms, by pairing mild foot shocks with a particular context or tone respectively. Twenty-four hours after training, context-conditioned animals were re-exposed to the context for 3 or 30 minutes (day 2); tone-conditioned animals were placed in a different context and re-exposed to one or six tones. Twenty-four hours (day 3) and one month later, freezing behavior to the aversive context/tone was scored again. MR or GR blockade was achieved by giving spironolactone or RU486 subcutaneously one hour before retrieval on day 2. Spironolactone administered prior to brief context re-exposure reduced freezing behavior during retrieval and 24 hours later, but not one month later. Administration of spironolactone without retrieval of the context or immediately after retrieval on day 2 did not reduce freezing on day 3. Re-exposure to the context for 30 minutes on day 2 significantly reduced freezing on day 3 and one month later, but freezing was not further reduced by spironolactone. Administration of spironolactone prior to tone-cue re-exposure on day 2 did not affect freezing behavior. Treatment with RU486 prior to re-exposure did not affect context or tone-cue fear memories at any time point.We conclude that MR blockade prior to retrieval strongly reduces the expression of contextual fear, implying that MRs, rather than GRs, play an important role in retrieval of emotional information and subsequent fear expression. Memories for emotionally arousing and stressful events are generally well retained Surprisingly little is known about how corticosteroid hormones and their receptors regulate the retrieval of fearful information. While exposure to stressful experiences and elevated corticosteroid hormones has been reported to suppress the retrieval of spatial information 3, 99 = 29.91, P<0.01). Importantly, freezing behavior during training was comparable for the groups that were later treated with vehicle or the MR-antagonist spironolactone . Twenty three hours later, animals received either spironolactone or vehicle injection. One hour after drug administration, animals were re-exposed to the same context as used during training and freezing behavior was measured at that time (day 2) as well as 24 hours (day 3) and one month later. In the first series of experiments we tested the effect of spironolactone given prior to brief (3 minutes) context re-exposure  and spironolactone treated animals . Multiple mean comparisons with post hoc Bonfferoni test revealed that both vehicle and spironolactone treated mice showed significantly less freezing behavior on day 3 (P<0.01) and one month later (P<0.05) respectively, when compared to day 2 . Post hoc test showed that treatment with spironolactone reduced freezing behavior during retrieval  as well as 24 hours after the retrieval trial  when compared to vehicle treatment. No significant treatment effect was found one month after retrieval (P>0.05). These results indicate that spironolactone, when applied before a brief retrieval of contextual information reduces the expression of fear for at least one day.During training animals displayed a progressive increase in freezing behavior  was observed. This indicates that spironolactone is only effective in reducing the expression of fear when the drug is present during retrieval of information. This was confirmed by another set of experiments where spironolactone was administered immediately after retrieval  and spironolactone treated mice . Post hoc test showed (as expected) that compared to day 2, both vehicle and spironolactone treated animals displayed significantly less freezing on day 3 (P<0.01) though one month later only vehicle treated animals showed a significant reduction in freezing scores (P<0.01). A between group effect of spironolactone was also found . On day 2, spironolactone treated animals displayed less freezing during the first 3 minutes of the retrieval period when compared to vehicle treatment  and spironolactone treated mice . When compared to day 2, both groups showed significant less freezing behavior on day 3  though one month later only spironolactone treated group showed less freezing (P<0.01), No between group-effect of spironolactone treatment was found .We next tested if spironolactone modifies the expression of fear when the drug is administered prior to retrieval of tone-cue memories. During training in context A, freezing behavior during and immediately after the tone was comparable between the groups that were later treated with vehicle or spironolactone (data not shown). One day after training, both vehicle and spironolactone treated mice displayed high levels of freezing in response to one tone in context B . Data an2,24 = 66.32, P<0.01). Freezing behavior was also scored one day (day 3) and one month later  and spironolactone  treated mice; when compared to freezing behavior during the first tone on day 2, less freezing was scored both on day 3 .Repeated exposure to six tones on day 2 significantly decreased the freezing behavior over time during extinction training . No difference was found between the groups that were later treated with vehicle or the GR-antagonist RU486 . One hour after drug treatment on day 2, freezing behavior to the training context was measured for 3 minutes and then compared to that measured one day (day 3) or one month later. Repeated measures ANOVA showed a main time effect in both vehicle  and RU486  treated animals. Post hoc test revealed that, when compared to day 2, only vehicle treated animals displayed significantly less freezing on day 3 (P<0.01). No significant differences were found in spironolactone treated animals or one month later (P>0.05). Also no between-group effect of RU486 was found . These results suggest that activation of GRs is not critically involved in retrieval of contextual fearful information and subsequent expression of fear.During training animals displayed a progressive increase in freezing behavior  and RU486  treated mice. Post hoc test showed that compared to the first 3 min re-exposure on day 2, both groups showed a significant reduction in freezing behavior on day 3 (P<0.01) bu not (P>0.05). No between group effects of RU486 were found . These results suggest that GR activation is not critically involved in the effect of 30-minute retrieval process of contextual fear information and subsequent expression of fear behavior.In a separate experiment animals were injected with RU486 or vehicle 23 hours after training and subsequently placed animals into the training context for 30 minutes; freezing behavior was scored on day 2, day 3 and one month later. Repeated measures ANOVA indicated a significant time effect in both vehicle  and RU486  treated mice. Post hoc test revealed that vehicle treated group displayed a significant decrease in freezing behavior on day 3 compared to day 2 (P<0.05), while RU486 treated mice showed reduced freezing behavior one month later (P<0.05). No between group effect of RU486 treatment was found on tone-cue freezing .We next tested if RU486 affects the expression of tone-cue fear memory when administered prior to retrieval. During training, freezing behavior during and immediately after the tone was comparable between the mice that were later treated with either vehicle or RU486 (data not shown). Twenty-three hours later, mice were treated with either vehicle or RU486, and one hour later exposed to one tone. Freezing behavior was scored on day 2, day 3 and one month later respectively . Repeate2, 20 = 23.93, P<0.01). Freezing behavior was further scored one day (day 3) and one month later  and RU486 treated groups . Compared to day 2, vehicle group displayed less freezing behavior both on day 3 .Repeated exposures to six tones on day 2 resulted in a significant decrease in freezing behavior  and glucocorticoid receptors (GRs). We tested here whether activation of MRs and GRs is also critical for retrieval and subsequent expression of fear. This is particularly interesting since retrieval of fearful events supposedly turns these memories into a labile state The protocol that we used to modulate the expression of fear–i.e. re-exposure on day 2 to the training context for 3 or 30 minutes-was previously described to examine reconsolidation and extinction of fearful memories respectively, which are two opposing processes that may be triggered by a similar memory retrieval procedure Our results demonstrate that MRs are critical during the retrieval of contextual emotional information for the subsequent expression of fear behavior, lasting at least one day but less than one month. Mineralocorticoid receptors (MRs) have been implicated in appraisal of novel and stressful events and response selection By blocking MRs, the relative contribution of GRs in behavioral effects might be increased as blocking MRs could have impaired the fast negative feedback of endogenous corticosterone on HPA-axis activity, leading to increased secretion of corticosterone While administration of spironolactone prior to retrieval reduced the expression of contextual fear memory, tone-cue memory remained unaffected. This also strongly argues against a non-specific role of the drug in reducing fear expression. The differential effectiveness of spironolactone in context versus tone-cued memories could be explained by the MR distribution in areas playing a major role in these two types of memories, that is, the hippocampus and basolateral amygdala respectively. MRs are highly expressed in all hippocampal fields, whereas MR expression is much lower in the basolateral amygdala Several studies have shown that post-training application of GR agonists promote (fearful) memory consolidation In conclusion, our results demonstrate that blocking MRs, but not GRs, prior to retrieval of contextual fearful information reduces the expression of contextual fear later on, lasting for at least one day.Male C57/BL6 mice  were housed (2 mice per cage) for at least one week after arrival. All animals were kept on a light/dark cycle of 12 h  and food and water were given without restriction. The experiments  were performed between 8:30–11:30 a.m. and approved by the local Animal Ethics Committee of the University of Amsterdam (DED212). All efforts were made to minimize suffering of the animals.The mineralocorticoid receptor antagonist spironolactone , glucocorticoid receptor antagonist RU486  or vehicle  was injected subcutaneously one hour before the retrieval of contextual or tone-cued information (i.e. twenty three hours after training) or immediately after retrieval. The dosage and drug delivery route were chosen based on previous studies in which MR mediated effects were effectively blocked by the administration of MR antagonist Animals were trained in a fear conditioning chamber  that contained a grid floor with 37 stainless steel rods and was connected to a shock generator and sound generator (Med-Farm LION-ELD) developed in-house. During training, one animal at one time was placed into context A. After three minutes of free exploration, three foot shocks  were delivered with an interval of 90 seconds. Sixty seconds after the end of the last foot shock, the mouse was placed back into its home cage. Freezing behavior, defined as no body movements except those related to respiration, was determined every 2 seconds throughout the experiment In separate control experiments, animals received the injection with spironolactone or vehicle one day after training without or immediately after the retrieval of contextual information. Freezing behavior was monitored on day 2, day 3 and one month later.All animals were handled for three days before the start of the experiments and placed for 20 minutes/day in context B which has the same size as context A, but different contextual background . During training, one mouse at a time was placed into context A. After three minutes of free exploration, the mouse was exposed to a tone  that lasted for 30 seconds and co-terminated with a mild foot shock  post hoc test. P values smaller than 0.05 were considered to be significantly different.Freezing behavior is expressed as percentage of freezing time versus total testing time. All results are presented as mean ± SEM. Freezing behavior was scored at 24 hours (Day 2), 48 hours (Day 3) and also one month after training. Freezing behavior was therefore analyzed with repeated measures ANOVA (in SPSS 9.0), with time as a repeated measure and drug treatment as between-subject factor. Significant within-subject (time) effects and between-subject (treatment) effects were followed up using multiple mean comparions with Bonfferoni


Escherichia coli, LTB) were studied in sheep following oral immunisation with plant-made and delivered vaccines. Delivery from a root-based vehicle resulted in antigen-specific immune responses in mucosal secretions of the abomasum and small intestine and mesenteric lymph nodes. Immune responses from the corresponding leaf-based vaccine were more robust and included stimulation of antigen-specific antibodies in mucosal secretions of the abomasum. These findings suggest that oral delivery of a plant bioencapsulated antigen can survive passage through the rumen to elicit mucosal and systemic immune responses in sheep. Moreover, the plant tissue used as the vaccine delivery vehicle affects the magnitude of these responses.Antigen-specific antibody responses against a model antigen (the B subunit of the heat labile toxin of enterotoxigenic  Vaccines administered via mucosal routes are sought-after because they can induce both mucosal and systemic immune responses to protect against infections caused by pathogens entering and colonising mucosal surfaces such as the gastrointestinal tract (GIT). Mucosal, humoral responses are characterised by secretory antibodies of which the IgA isotype is the most prominent and IgG less abundant Escherichia coli is a well characterised, mucosal antigen often used as an adjuvant The heat labile toxin (LT) of enterotoxigenic In an earlier study we examined different plant tissues as potential vehicles for oral delivery of recombinant LTB (rLTB) in the mouse GIT Petunia parodii (petunia) plants producing rLTB were generated and maintained as described previously 2 then freeze-dried using a Dynavac freeze drier (Model FD12) for 48 h with a maximum shelf temperature of 20°C. Nicotiana benthamiana leaves transiently expressing apoplast-targeted LTB or GFP were produced as described previously 2 then freeze-dried using a Dynavac freeze drier (Model FD12) for 48 h with a maximum shelf temperature of 20°C. Freeze-dried plant materials were powdered using a commercial coffee grinder and sieved to standardise particle size to 0.5–1 mm2. Accumulation of rLTB pentamer, the functional form required for binding to GM1-gangliosides on the mucosal surface of the gut epithelium, was confirmed in N. bethamiana leaves and petunia hairy roots as per Hairy root cultures of transgenic 2HPO4, 2 mM KH2PO4) supplemented with 0.05% Tween 20] with two 3 mm tungsten carbide beads for 1 min at a frequency of 28/s in a Qiagen Mixer Mill. The homogenate was cleared by centrifugation at 13,000 rpm at 4°C for 5 min.Crude protein was extracted from freeze-dried plant material by homogenising in 1∶60 (w/v) PBST [PBS  coated with 50 µl/well of chicken anti-cholera enterotoxin subunit B (CTB) antibody (Sigma-Aldrich) diluted 1∶5,000 in PBS. Plates were sealed and incubated at 4°C overnight. Unless stated otherwise, all subsequent incubations were performed at 37°C for 1 h and antibodies diluted in 1% dry skim milk powder (DM) in PBST. Following all incubations, plates were washed three times with PBST.Pichia pastoris-made rLTB (Sigma-Aldrich) standard. Accumulation of the functional pentameric form of rLTB was confirmed by western blot Plates were blocked with 5% DM in PBST before a 2 h room temperature (22–25°C) incubation with serially diluted crude plant extract starting with 1∶100 in PBS. Plates were then incubated with 1∶2,000 rabbit anti-LTB (Benchmark Biolabs), then 1∶15,000 goat anti-rabbit IgG HRP conjugate (Sigma-Aldrich). Bound LTB-specific antibodies were visualised using TMB-peroxidase substrate (Bio-Rad Laboratories) according to manufacturer's instructions. The amount of rLTB in the freeze-dried plant materials was calculated against a Ovis aries, Merion/Merino) aged between 4.5 to 12 months were obtained from the Commercial Registered Pfizer Animal Health Woodend Farm and housed at the Monash University Werribee Animal Facility under conditions approved by the Monash University Animal Ethics Committee (AEC SOBSA/P/2009/98). Sheep were provided with water and standard feed ad lib and fasted 16 h before oral immunisation. Sheep were randomly assigned into four groups of 2–5 animals each . When receiving the transgenic rLTB plant-based vaccines (LTB-HR or LTB-Leaf), each dose was sufficient to deliver 5 mg rLTB. Sheep receiving the CtHR or CtLeaf vaccines were immunised with the equivalent volume of formulated control plant materials. The formulated vaccines were administered orally to sheep by gavage directly into the rumen to simulate drenching, a common delivery system used routinely to worm domestic sheep flocks. At trial termination (day 42), sheep were humanely killed by intravenous injection with a lethal dose of lethobabarb (100 mg/kg bodyweight).Sheep were immunised on days 0, 14 and 28 followed by a boost dose on day 38, four days before sacrifice. Vaccine materials were formulated immediately before delivery by mixing 19 g freeze-dried plant material with 200 ml of an oil based emulsion (125 ml peanut oil:75 ml dHg for 10 min and stored at −20°C until required for LTB-specific antibody detection by ELISA.Blood samples were taken from the jugular vein using an 18 G needle immediately before the first immunisation (pre-immune), 14 days after each of the first three doses and four days  after the boost. The blood was clotted at room temperature (20–22°C) overnight and serum separated by centrifugation at 400 2 for 24 h. Culture supernatants (ASC supernatants) were collected and stored at −20°C and the presence of LTB-specific antibodies determined by ELISA.At post-mortem, four lymph nodes were taken from the mesentery, the first at the abomasum/duodenum junction (MLN 1) and the next three along the first 0.5 m of the small intestine (MLN 2–4). MLNs were subjected to an antigen-specific antibody secreting cell (ASC) assay for detection of LTB-specific antibody responses using a protocol modified from those previously described g for 15 min at 4°C and stored at −20°C until required.The mucosal lining of the abomasum was sampled by scraping the inside surface with a glass slide. Mucus scrapings were prepared for ELISA as described by Four sections of the small intestine were excised, each section measured 0.5 m in length and was taken 3 m apart, beginning at the abomasum/duodenum junction . Sections 2–4 were sampled at 3.5–4 m, 7–7.5 m and 10.5–11 m respectively. Each segment was flushed with 20 ml saline then incubated for 30 min with 10 ml saline and gentle rocking. Each end of the intestinal segments was clamped during washes to prevent leakage. Washes containing intestinal secretions were collected and stored at −20°C until required.Faecal samples were collected before vaccination on day 0 and again at day 16 and 36 h after immunisation with the second oral dose allowing administered vaccine material to complete transit through the sheep GIT Pichia-made rLTB (Sigma-Aldrich). Serial dilutions of the various biological samples were made on the plate with starting dilutions in PBST as follows – 1∶1000 or undiluted serum for IgG or IgA determination respectively, 1∶2 ASC supernatant, 1∶4 small intestine wash and undiluted abomasum mucus. Plates were incubated overnight at 4°C before incubation with 1∶2,000 mouse anti-sheep/goat IgG HRP conjugate (Enzo Life Sciences), or rabbit anti-sheep IgA HRP conjugate  at 37°C for 2 h. Bound LTB-specific antibodies were visualised using TMB-peroxidase substrate (Bio-Rad Laboratories) according to manufacturer's instructions. Endpoint antibody tire was reported as the highest dilution with an absorbance of four standard deviation units above background . All measurements were performed in triplicate, the geometric mean titre determined and data subjected to statistical analysis using the non-parametic one way analysis of variance (ANOVA) and student's t-test. Data from sheep receiving control vaccine treatments (CtHR and CtLeaf) were combined for analysis. An antigen-specific antibody response exceeding the geometric mean titre of the control group (background) by at least three standard deviations was considered a positive response.LTB-specific ELISA was used to assess IgG and IgA antibody responses in immunised sheep. Costar 9018 96-well microtitre plates (Corning Life Sciences) were coated with 50 µl/well chicken CTB antibody (Sigma-Aldrich) diluted 1∶5,000 in PBS. Plates were sealed and incubated at 4°C overnight. Three washes with PBST were performed following each incubation. Plates were blocked with 5% DM in PBST at 37°C for 1 h before a further incubation for 1 h at 37°C with 0.25 µg/ml N. benthamiana and Petunia parodii (petunia) were chosen due to their lack of use in the animal or human food chain to reduce the chance of contamination of the food chain and due to their ease of transformation. Although this resulted in more than one variable in the study our previous study demonstrated their optimal nature for oral delivery to mice Two different plant species E. coli colonisation in animals, which were not housed in germ-free conditions.Immunisation of sheep with the LTB-Leaf vaccine resulted in a higher number of sheep with positive antigen-specific IgG-serum responses than those receiving the LTB-HR vaccine . The meaDetection of antibody production in serum following oral immunisation may not be indicative of immune responses at mucosal sites All five sheep immunised with the LTB-Leaf vaccine assayed positive for an LTB-specific ASC-IgG response at one or more of the MLN sites sampled . One sheInduction of LTB-specific antibody responses in the mucosa of the abomasum was identified only after immunisation with the LTB-Leaf vaccine . At thisLTB-specific IgG antibody was detected in intestinal washes of two of the five sheep immunised with the LTB-Leaf vaccine . In one Faecal samples were assayed for LTB to determine whether the vaccine plant materials had resisted breakdown during passage through the sheep GIT. LTB was not detected in faecal samples taken from pre- and post-immune sheep from control, LTB-HR or LTB-Leaf groups (data not shown).The pharmaceutical industry is constantly assessing methods for improved delivery for vaccines, pharmaceuticals and nutraceuticals. The oral route increases ease of delivery, is less expensive, and encourages increased compliance by eliminating the need for needles. Moreover, oral delivery is particularly desired for immunising free-ranging domestic animals that are typically ruminants. Numerous studies have reported immunogenicity of orally delivered plant-made vaccines in humans and small animal models, but few have demonstrated their efficacy in ruminants LTB was chosen as our model antigen because it can be produced in a wide variety of plant systems 2 to better protect the antigen from degradation by minimising the time spent in the rumen. In sheep, particles with diameters larger than 1.18 mm transit through the rumen slower than smaller particles Vaccine materials were formulated in oil and administered directly into the rumen of the sheep via a tube inserted down the oesophagus. The delivered plant materials were sieved to achieve a uniform particle size of 0.5–1 mmFrom the rumen, the vaccine transits through the reticulum and omasum before reaching the abomasum (true stomach) where enzymatic digestion of protein, carbohydrates and lipids is initiated. It is anticipated that breakdown of the plant cells encapsulating the rLTB antigen begins in the rumen and continues in the reticulum, the principal sites for cellulose digestion in ruminant species. It was in the abomasum mucus that antibody responses were first observed following administration of the LTB-Leaf vaccine. This suggests that as the leaf material begins to degrade the antigen remains sufficiently protected during rumination, presumably by the lipid coating provided by the oil formulation matrix. In contrast, the lack of antibody response in abomasum mucus from the LTB-HR vaccine suggests that root tissue may be comparatively more resistant to rumination and enzymatic digestion resulting in delayed antigen release.Although GALT is absent in the abomasum, immune responses can be induced when the mucosal epithelium is penetrated From the abomasum, the vaccine materials enter the small intestine. By this stage breakdown of the plant cells and formulation matrix should be completed, releasing the remainder of its antigenic cargo. It was in the small intestine that the most robust mucosal immune responses were detected from both the LTB-Leaf and LTB-HR vaccines, the leaf material producing elevated IgA titres compared to other treatments in all five sheep receiving this vaccine. It was of interest that section 4, the section further through the GIT, was the site where the most robust antigen-specific IgG responses were found while IgA responses expanded to earlier sites (sections 2 to 4). The consistency in the immune response observed at the small intestine, particularly for the LTB-Leaf group, is noteworthy given the potential for variable responses when using an outbred sample of sheep.LTB-specific IgA antibodies were absent in all sera, irrespective of vaccine treatment or number of doses administered. This is not unexpected as detection of antibody production in serum following mucosal immunisation can be typically difficult particularly when responses are low Whilst most of the immune inductive sites of the GIT are located in the GALT of the small intestine, the potency of the LTB-Leaf vaccine benefitted from an early release in the abomasum perhaps due to the stability of LTB and the resulting prolonged antigen exposure at mucosal surfaces and priming distal sites in the small intestine. Antibody responses at the tonsils or other lymphoid tissues of the oral and nasopharyngeal cavities were not sampled in this study but should not be discounted as additional sites within the mucosal epithelium that could be exploited for induction of immune responses from plant-made vaccines. Plant material in its nature is fibrous and as such is often regurgitated from the rumen during fermentation for further mechanical breakdown by chewing and can result in repeated and sustained exposure of the plant-delivered antigen to the tonsils priming more distal sites of the GIT or respiratory system It is apparent that both the leaf- and root-based vaccine preparations protected the antigenic load sufficiently during rumination and enzymatic digestion to enable its delivery to relevant immune responsive sites. Furthermore, the type of plant tissue used can manipulate timing of antigen release. In our experience, antigen release from both leaf- and root-based vaccines has been consistent across sheep (present study) and mouse N. benthamiana leaf material provided the optimal oral delivery vehicle for induction of mucosal immune responses to LTB in both monogastric (mouse) and ruminant (sheep) models, it is anticipated that plant choice will need to be assessed on a case by case basis, taking into account antigen stability.The plant material used to deliver LTB orally to sheep affected immunogenicity. This finding suggests that a delicate balance between protecting the vaccine antigen against digestive degradation and enabling release for presentation of the antigen at immune responsive sites needs to be struck to maximise vaccine efficacy. Although Optimising oral delivery of plant-made, valuable proteins will have broad ramifications to animal as well as human health. Oral delivery will facilitate treatment of free-ranging domesticated and native animal populations that may otherwise go untreated, broaden opportunities for existing pharmaceuticals and create opportunities for new compounds and target populations.


We found that multiple measures of metabolism in cells from tropical birds were significantly lower than their temperate counterparts. Basal and uncoupled cellular metabolism were 29% and 35% lower in cells from tropical birds, respectively, a decrease closely aligned with differences in whole-animal metabolism between tropical and temperate birds. Proton leak was significantly lower in cells from tropical birds compared with cells from temperate birds. Our results offer compelling evidence that whole-animal metabolism is linked to cellular respiration as a function of an animal’s life-history evolution. These findings are consistent with the idea that natural selection has uniquely fashioned cells of long-lived tropical bird species to have lower rates of metabolism than cells from shorter-lived temperate species.In general, tropical birds have a “slow pace of life,” lower rates of whole-animal metabolism and higher survival rates, than temperate species. A fundamental challenge facing physiological ecologists is the understanding of how variation in life-history at the whole-organism level might be linked to cellular function. Because tropical birds have lower rates of whole-animal metabolism, we hypothesized that cells from tropical species would also have lower rates of cellular metabolism than cells from temperate species of similar body size and common phylogenetic history. We cultured primary dermal fibroblasts from 17 tropical and 17 temperate phylogenetically-paired species of birds in a common nutritive and thermal environment and then examined basal, uncoupled, and non-mitochondrial cellular O Life history theory is based on the idea that the schedule and duration of key events in an organism's lifetime are shaped by natural selection to ensure the largest number of surviving offspring in the next generation. These events, juvenile development, age of sexual maturity, age of first reproduction, number of offspring, level of parental investment, and the rate of senescence, are influenced by the abiotic and biotic environment of the organism Although it is thought that physiological processes underlie many life-history trade-offs, the physiological mechanisms underlying the diversification of life-histories remain elusive in situ scaling characteristics when they are grown in common nutrient environment, and thus, cultured cells from animals with different rates of mass-specific metabolism will assume a uniform rate of cellular O2 consumption The connection between whole-animal metabolic rate and the metabolic rate of cells cultured from animals remains controversial Tropical birds were collected by mist-net in the lowland forest around Gamboa, Panama (Latitude: 9.12° and Longitude: −79.72°), and temperate birds were collected in and around Ohio, USA (Latitude: 41.28° and Longitude: 83.1°). All collected birds were adults of unknown age. Temperate birds were collected under an Ohio Division of Wildlife permit number 15–29. All procedures were approved by the Institutional Animal Care and Use Committee of The Ohio State University (protocol IACUC2004A0093) and capture of birds in Panama was accomplished under permit from Panamanian Autoridad Nacional del Ambiente (No. SEX/A-22-12) and Autoridad del Canal de Panamá. All birds were sacrificed by cervical dislocation, prior to collection of skin. Tissues were exported from Panamá the same day as collected under USDA permit 118465 and sent overnight to our lab in Ohio, where they were immediately cultured. Our study was designed to minimize differences in cell physiology as a result of acclimatization of adults because we collected temperate birds during May–July when the average air temperature was at or near ∼23°C, which is only slight below the average air temperature of Panama ∼27°C.2 consumption capacity and glycolytic capacity. For all species, primary cell cultures were established from the skin of free-living adult birds. Immediately after birds were sacrificed, their feathers were plucked and the exposed skin was washed with anti-microbial soap. We excised a 5×5 mm2 piece of skin and placed it into cold complete bird cell culture media . Skin from Panamanian birds was sent overnight to our lab at the Ohio State University, where skin was treated exactly as the skin from temperate birds.We evaluated primary dermal fibroblast cell lines from 17 temperate and 17 tropical bird species for their cellular O22 flasks at day 7–10. After cells grew for another 7 to 10 days, they were harvested and cryopreserved at 106 cells/mL in DMEM supplemented with 40% fetal bovine serum and dimethylsulfoxide (DMSO) at a final concentration of 10%. We stored cells in liquid N2 for up to 12 months prior to assessment of their metabolic profile. Previous work from our lab comparing primary dermal fibroblasts across passages and between bird species has found that large scale changes among passages, such as cell size, ratio of non-phospholipids to phospholipids, and the concentrations of the two most common phospholipid classes, tend to be similar among species. Some changes do differ between species, but they occur in such a way that species are becoming more similar to each other 2 consumption measurements were conducted using cells at passage 2 (P2). All cell lines were thawed, resuspended and allowed 5 days to recover from freezing before Oxygen consumption rate (OCR) and Extracellular acidification rate (ECAR) experiments.We established primary fibroblast cell cultures after the skin was exposed to 0.5% Collagenase B solution overnight. Cells were grown in culture flasks at 37°C in an atmosphere of 5% O2 uptake and CO2 efflux in isolated primary dermal fibroblasts from adult birds. Optimization assays were performed prior to experiments to determine the optimal cell seeding density, and optimal concentrations of each compound used for the assay. We seeded 50,000 cells per well per species into XF24 cell culture plates 48 h before experiments. O2 consumption rate (OCR) and Extracellular acidification rate (ECAR) was determined following o portion of the ATP synthase. This can be used to distinguish the percentage of O2 consumption devoted to ATP synthesis and the oxygen consumption required to overcome the natural proton leak across the inner mitochondrial membrane plus any non-mitochondrial O2 consumption. Next, we injected 300 nm FCCP, an ionophore which disrupts the proton gradient, providing a maximal respiratory rate. Finally, we injected 2 µM Antimycin A, a Complex III inhibitor and 100 nm rotenone, a Complex I inhibitor. This combination of chemicals stops mitochondrial respiration and the remaining O2 consumption is non-mitochondrial in origin A Seahorse XF-24 Extracellular flux analyzer was used to measure the rate of O2 consumption is subtracted from OCR obtained after the addition of oligomycin, a measurement of proton leak can be estimated. Proton leak is driven by the magnitude of the proton motive force across the inner mitochondrial membrane (Δp) and the rate of proton leak is determined by the inner membrane conductivity to protons at a given Δp When non-mitochondrial O2 consumption assays were performed, we counted the final concentration of cells in each well and normalized all rates to a total of 50,000 cells as a standard. We additionally measured cell diameters at P2 for each species to determine if fibroblast cells from different species were of different size. To measure cell diameter, we took images of rounded cells after trypsinization for all species using a CCD digital camera through a 10x power objective, and measured cell size using Labkit Image J. We measured 30 cells per individual to obtain mean values. For OCR measurements, we used the third basal measurement made by the XF24, for maximal metabolic rate, we took the OCR value after injection of FCCP, and non-mitochondrial respiration was the lowest measurement after the injection with Antimycin A and rotenone. The spare respiratory capacity was calculated as the maximal metabolic rate divided by the basal respiratory rate with the minimum non-mitochondrial respiration subtracted from both. Spare respiratory capacity provides an idea of a cell’s maximum ATP production, therefore, cells with higher capacity have greater ability to respond to stress After OWe used a Shapiro-Wilk test to check for normality in our data. To determine statistical difference between tropical and temperate pairs, we used mean values for each parameter as a function of environment and applied a Wilcoxon signed ranks test In comparative biology, most investigators attempt to avoid statistical problems associated with the lack of independence of data from closely related species by using one or more methods that take into account phylogenetic relatedness We measured the diameter of cells for each species at passage 2 and found no significant differences between cell size across species.   , Fig. 1A2 consumption of cells from species of tropical birds was significantly lower than cells from temperate species . Anaerobic breakdown of glucose produces an excess of protons, resulting in acidification of the medium surrounding the cells. Basal ECARs were not statistically different between cells from tropical and temperate birds  rather than the lower O2 tensions found within a living cell (3–5%), conclusions should be regarded as tentative Mitochondria typically show a leak of protons back across inner mitochondrial membrane, which results in oxidative phosphorylation not being completely coupled to electron flow Because differences in cellular metabolism are directly related to mitochondrial function, differences in mitochondrial density or morphology may account for the differences in cellular metabolic rates that we have found. Fibroblasts from tropical birds had 26% less mitochondrial lipid than did cells from tropical species, and cells from tropical birds had 26% less cardiolipin 2 consumption than did tissue slices from small mammals 2 consumption Some colleagues might suggest that it is intuitive that animals with lower whole-organism basal metabolism, like tropical birds, would also have cells with a lower intrinsic rate of metabolism when those cells are grown in culture. However, the connection between whole-organism physiology and intrinsic physiological function of isolated cells in a culture environment has been controversial, especially in the medical community. The idea that intrinsic rates of cell metabolism should mirror whole-organism metabolism has its roots in experiments which related metabolism of slices of liver, or a heterogeneous collection of hepatocytes, to animals of different body size: tissue slices or liver cells from large mammals, with their lower mass-adjusted whole-organism metabolism, had lower OFibroblast cells are widely used as a tool to study a range of physiological, medical, and pathological processes Life-history theory suggests that lower extrinsic mortality results in low reproductive effort and more energy allocation to self-maintenance in the tropics In summary, we have isolated primary dermal fibroblasts from temperate and tropical birds with differing life-history traits and have cultured these cells under the same conditions through two passages. We found that intrinsic rate of metabolism of cells from tropical birds was significantly lower than was cells from temperate birds. Tropical birds had lower basal and peak whole-animal metabolic rates, and these rates appear to be reflected by metabolic rates of individual cells. Physiological differences at the cellular level such as reduction in mitochondrial density and/or efficiency, has apparently allowed tropical and temperate birds to have different cellular metabolic rates. Our study links aspects of cellular function to the life-history evolution of tropical and temperate bird species.Figure S1Example of a typical metabolic profile illustrating OCR responses for avian dermal fibroblasts when exposed to metabolic inhibitors.(TIF)Click here for additional data file.Figure S2Phylogenetic tree of 34 tropical and temperate bird species. Phylogenetic relationships are based on information assembled by . Branch lengths were derived from Sibley and Alquist (1990) and shown in units of difference in melting temperatures of bonded DNA strands of different species. Tropical species are designated in red lettering, and temperate species in black lettering.(TIFF)Click here for additional data file.Figure S3Cell size comparison across species. Images were taken using an Olympus light microscope at 10 x after trypsinazation at passage 2 (P2). Cell diameters were measured using Image J. Values represent mean ± SEM. (p>0.2).(TIF)Click here for additional data file.