Prion protein (PrP) level plays the central role in bovine spongiform encephalopathy (BSE) susceptibility. Increasing the level of PrP decreases incubation period for this disease. Therefore, studying the expression of the cellular PrP or at least the messenger RNA might be used in selection for preventing the propagation of BSE and other prion diseases. Two insertion/deletion (indel) variations have been tentatively associated with susceptibility/resistance of cattle to classical BSE.PRNP was significantly (p<0.0001) greater in the homozygous deletion genotype at the 23-bp locus in JB breed. Compared to the homozygous genotypes, the expression of PRNP was significantly greater in the heterozygous genotype at the 12-bp locus in JB (p<0.0001) and in JBr (p = 0.0394) breeds. In addition, there was a statistical significance in the PRNP levels between the insertion and the deletion alleles of the 23-bp locus in JB (p = 0.0003) as well as in JBr (p = 0.0032). There was no significance in relation to sex, age, geographical location or due to their interactions (p>0.05).We studied the expression of each genotype at the two indel sites in Japanese Black (JB) and Japanese Brown (JBr) cattle breeds by a standard curve method of real-time PCR. Five diplotypes subdivided into two categories were selected from each breed. The two cattle breeds were considered differently. Expression of PRNP at the 23-bp locus in the medulla oblongata of these cattle breeds.Our results suggest that the del/del genotype or at least its del allele may modulate the expression of  PRNP) are linked to the occurrence of transmissible spongiform encephalopathies (TSEs) also called prion diseases in humans, sheep and mice PRNP have been tentatively associated with classical BSE (cBSE) incidences in a few cattle populations PRNP and possibly the expression of PrP in species. Expression of the cellular PrP is necessary for the transmission and propagation of prion diseases Genetic variations in the prion protein gene  animals while three were reported in local Japanese Black (JB) cattle. So far a number of Japanese cattle populations have been analyzed for various polymorphisms in the PRNP. Both 23-bp and 12-bp loci were polymorphic in both breeds. At the 23-bp locus, frequency of the deletion homozygous (del/del) was 0.62, that of the heterozygous (ins/del) was 0.30 and that of the insertion homozygous (ins/ins) was 0.08 in JB breed and 0.31, 0.50 and 0.19 respectively in the JBr breed. At the 12-bp locus, the frequencies were 0.39 for the del/del (− −), 0.38 for the ins/del (+−) and 0.23 for the ins/ins (++) in JB breed whereas the frequencies were 0.30, 0.47 and 0.23 respectively in the JBr breed. Compared to the insertion (+) allele, the deletion (−) allele was higher in frequency at both loci in both breeds. JB breed had greater frequencies of the − allele at both loci as compared to the JBr breed. Genotypic and allelic data are presented in A total of 218 animals including 120 JB and 98 JBr were genotyped for polymorphisms in the 23-bp and 12-bp indel loci of the These genotypes were assembled into diplotypes. The left side of these diplotypes represents one of the three genotypes of the 23-bp indel loci whereas the right side constitutes genotypes of the 12-bp indel loci. As shown in PRNP among genotypes of the 23-bp promoter indel, three diplotypes were selected from each breed. Diplotypes ++/++ (n = 6), +−/++ (n = 8), and −−/++ (n = 8) were selected in the JB breed whereas diplotypes ++/+− (n = 3), +−/+− (n = 6) and −−/+− (n = 5) were selected in the JBr breed. The left side of these diplotypes constitute of genotypes ++, +− and −− of the 23-bp locus whereas the right which represent the 12-bp locus has a similar genotypes in all diplotypes. It was presumed that a similar genotype at the 12-bp locus would have similar effect in these diplotypes and therefore the gene expression was compared among genotypes of the 23-bp locus on the left side. The 12-bp locus was represented by the ++ genotype in the JB breed and +− in the JBr breed. The expression of PRNP relative to the reference ACTB gene were analysed. As presented in PRNP (mean±SE) were 0.2721±0.033 in the ++/++ diplotype, 0.3017±0.042 in the +−/++ diplotype and 0.3314±0.027 in the −−/++ diplotype in the JB breed. Therefore, the level of PRNP was higher in the −−/++ diplotype compared to ++/++ and +−/++ diplotypes suggesting the order of expression of PRNP in medulla oblongata of the JB breed as −−/++>+−/++>++/++. The levels of PRNP were 0.0801±0.007 in the ++/+− diplotype, 0.0838±0.007 in the +−/+− diplotype and 0.1076±0.022 in the −−/+− diplotype in the JBr breed. The PRNP expression was therefore greater in the −−/+− diplotype as compared to the other two diplotypes. The order of expression was −−/+−>+−/+−>++/+− in the JBr breed. Clearly, it can be shown that, diplotypes with the −− genotype had greater PRNP levels than those with the ++ or the +− at the 23-bp locus in both breeds (PRNP was significantly different (p = 0.0001) among genotypes of the 23-bp locus in JB breed but there was no statistical significance (p>0.05) among these genotypes in the JBr breed. Also, there was no significance in relation to sex, age, regions or farms of the animals and their interactions (p>0.05).To evaluate the expression of h breeds . The expPRNP expression among the genotypes of the 12-bp locus, we selected animals with diplotypes −−/++ (n = 8), −−/+− (n = 10) and −−/−− (10) from the JB breed. From the JBr breed, animals with diplotypes +−/++ (n = 6), +−/+− (n = 12) and +−/−− (n = 4) were selected. The 23-bp locus was represented by genotypes −− (JB breed) and +− (JBr breed) at the left side of these diplotypes. As summarized in PRNP levels were 0.3314±0.027 in the −−/++, 0.3971±0.043 in the −−/+− and 0.2552±0.011 in the −−/−− in the JB breed as well as 0.1212±0.004 in the +−/++, 0.1301±0.010 in the +−/+− and 0.0984±0.009 in the +−/−− in the JBr breed. As a result, animals with the −−/++ diplotype showed the greatest PRNP expression followed by those with the −−/+− and −−/−− in the JB breed whereas animals with the +−/++ diplotype showed the greatest PRNP expression than those with diplotypes +−/+− and +−/−− in the JBr breed. Our results indicate that, diplotypes with the +− genotype had greater PRNP levels compared to those with either ++ or −− at the 12-bp locus in both breeds  and JBr (p = 0.0394) breeds. There was no significant difference in relation to sex, age, regions or farms of the animals and their interactions (p>0.05).In order to evaluate h breeds . The PRNPRNP with respect to alleles + and − of the 23-bp locus were assayed using diplotypes +−/+− and −−/+− in the JB breed and diplotypes ++/++ and +−/++ in the JBr breed. Three of the four alleles in the diplotypes selected in the JB breed were the same (−/+−) so that it was possible to compare the expression of PRNP between two alleles. In the JBr breed, except for the alleles of interest the other three alleles were similar (+/++) in both diplotypes thus possible to detect the expression of PRNP between alleles + and −. The PRNP levels were 0.0492±0.003 and 0.0899±0.009 for the diplotypes +−/+− and −−/+− in the JB breed respectively and 0.1419±0.006 and 0.1787±0.015 respectively for the diplotypes ++/++ and +−/++ in the JBr breed (p = 0.0003) as well as in the JBr breed (p = 0.0032).Expression of Br breed . For botBr breed . The expPRNP loci have been tentatively shown to influence the cBSE incidence in some cattle populations Two PRNP expression assays and have not been submitted for publication. Our genotypic and allelic frequencies data agree with those published in earlier reports. Notably, the allelic frequencies at the 23-bp locus for the JBr breed resembled to those of the United States (US) HF We genotyped the 23-bp and 12-bp indel loci in healthy JB and JBr breeds and analysed the expression of its messenger RNA using ten genotype combinations (five diplotypes in JB and five in JBr). The genotypic and allelic data at these loci supplement the data available in Japan for these and other breeds PRNP expression. Firstly, we have shown that diplotypes with the −− genotype had higher expression than those with the +− and ++ genotypes at the 23-bp locus in both breeds. Secondly, we showed that diplotypes with the +− genotype had a greater expression than those with the ++ and −− genotypes at the 12-bp locus in both breeds. Considering the genotypes alone, our results suggest the order of PRNP expression as −−>+−>++ at the 23-bp locus and +−>++>−− at the 12-bp locus in the medulla oblongata of these breeds. Thirdly, we studied the difference of PRNP expression between diplotypes differing in one allele (the + and the −) at the 23-bp locus and showed that diplotypes with the + allele had lower PRNP levels compared to those with the − allele.We used the diplotypes to study the effect of the genotypes of the 23-bp and 12-bp loci in the PRNP expression compared to ++ and +− genotypes at the 23-bp locus. This genotype was reported to be significantly overrepresented in BSE-affected cattle presumably due to the homozygous deletion of a PRNP transcriptional RP58 binding site PRNP level compared to genotypes +− and ++. The +− genotype could show high PRNP than the other two genotypes due to a possible over-dominance whereby the − allele might be masked by the + allele PRNP but compared to the other two genotypes, the +− genotype has been reported to be high in frequency at the 12-bp locus in many cattle populations PRNP level compared to the −− of the 12-bp locus in animals with the −−/−− diplotype as we previously reported PRNPPRNP may elucidate the levels of PrP in the medulla oblongata of these animals. We further showed that a different between the + and − alleles at the same position in two diplotypes might significantly affect the level of PRNP. Our results showed that an increase of the − allele increased the level of PRNP in one of the two diplotypes examined for expression in alleles. If PRNP expression is an important factor for the PrP expression, animals with genotypes +− and −− at the 23-bp locus may show higher levels of PrP compared to those with the ++ genotype at this locus. It is worth to note that, the ++/++ diplotype had a low PRNP level compared to the other diplotypes in the JB breed an indication that the ++ of both loci may show a low PRNP expression. The + variants of either regulatory element have the potential to lower host PrP expression levels in vivo and also in the in vitroIn summary, the −− genotype was correlated with an increased PRNP expression in the 23/12 diplotypes based assays in the medulla oblongata of JB and JBr breeds. However, the deletion alleles are not entirely independent of one another as there is high linkage disequilibrium (LD) between the two polymorphic sites in Bos taurus cattle populations PRNP on incidence of cBSE may be better understood if PRNP haplotypes were considered in testing for association with disease incidence. Moreover, PRNP haplotypes, containing one or both of the two indel loci, may have a stronger association with either susceptibility or resistance to cBSE than if the indels are considering independently. Also, other functional variants may affect the expression of PRNP and therefore carrying out confirmatory studies in these loci or other polymorphisms using the same tissue or other tissues in other populations would be useful. We performed the assays in individual breeds and did not attempt to compare the breeds due to possible breed effect as we previously reported PRNP expression in these breeds because of their economic and socio-cultural importance in Japan. Our sampling places (Kagoshima and Kumamoto prefectures) were preferred because they are the number one producers of JB and JBr breeds respectively. However, there are only a few BSE cases in JB breed  but the disease has not been diagnosed in JBr breed.We report here that the 23-bp − allele and the −− genotype may contribute to a greater We used the medulla oblongata tissue sampled from healthy 120 JB and 98 JBr from public slaughterhouses in Kagoshima and Kumamoto, Japan. Samples were taken randomly, but later coded according to age and sex. JB cattle aged between 25–36 months in (average 27–30 months) while JBr cattle aged between 22–159 months in (average 24–27 months). Total RNA was extraction from 500 mg of the dry freezed medulla oblongata tissue samples using either TRIZOL® Reagent (invitrogen™) or TriPure isolation reagents  following the manufacturers' instructions. Cleanup of the isolated RNA was done using the RNeasy® Mini Kit with RNase-Free DNase Set  according to the manufacturer's instruction. Reverse transcription into cDNA was performed using 12.5-units of AMV Reverse Transcriptase , 2.5-µl of random hexamer primer  and 0.5 µg of the isolated RNA in a 50-µl reaction by a GeneAmp® PCR System 9700  at 30°C for 10 minutes, 37°C for 120 minutes and 99°C for 5 minutes. cDNA samples were stored at −20°C. Genomic DNA was isolated from 200 mg of the same tissue as that used for RNA isolation using PUREGENE™ Cell & Tissue kit  according to the manufacturer's instructions. Concentration and purity of RNA, mRNA and the DNA were calculated by a GeneQuant 100  according to the manufacturer's instructions.GTGCCAGCCATGTAAGTG and TGGACAGGCACAATGGG (23-bp indel) also CTCGGTTTTACCCTCCTGGT and CACTTCCCAGCATGTAGCCACCA (12-bp indel). PCR was carried out in a final volume of 20 µl containing 0.5 units of Ex Taq (TAKARA), 40 ng of genomic DNA, 200 mmol/l each of the four deoxynucleoside triphosphates (dNTPs), 10 pmoles of each primer and 2.0 µl of 10× Ex Taq buffer (TAKARA). The mixture was first treated at 94°C for 2 min, followed by 28–35 cycles of denaturation at 94°C for 30 s, annealing temperature 56°C (23-bp) ; 62°C (12-bp) for 30 s, and extension at 72°C for 30 s and a final elongation step at 72°C for 5 min. All PCRs were carried out in the GeneAmp PCR System 9700 (Applied Biosystems). PCR products were separated on 10% polyacrylamide gels and visualized with ethidium bromide staining under ultra-violet (UV) light. From the genotypes, diplotypes for the two loci were constructed. Animals with the selected diplotypes were used in the gene expression analysis. The diplotypes were selected based on the objectives of our studies while considering the sample sizes. To avoid analysis of animals of the same origin , sampling of the medulla oblongata tissue was restricted to 10–15 samples per a sampling day. Information of these animals was known through a Japanese traceability system at http://www.nlbc.go.jp.Genotyping with respect to the 23-bp indel within the promoter region and 12-bp indel within the first intron were amplified by polymerase chain reaction (PCR) using primer pairs PRNP and an endogenous control actin beta gene (ACTB) were constructed by PCR products including the target sequence and its flanking sequence as a template. 10-fold dilution series of each standard was used to generate the real-time PCR standard curve. The PCR used to amplify this standard was carried out in the GeneAmp® PCR System 9700 using primer pairs listed in PRNP and ACTB were quantitatively measured by the real-time PCR using primer pairs also listed in PRNP) and NM_173979 (ACTB) using Primer3 software PRNP 153-bp and ACTB 169-bp were sequenced and confirmed to be identical with those from which primer pairs were designed. The real-time PCR was carried out in a 15-µl reaction volume containing 7.5-µl of SYBR® Green Real-time PCR Master Mix , 0.03-µM of each primer and 3.0-µl of each cDNA in the 7300 Real-Time PCR System (Applied Biosystems). Thermal cycling conditions were 95°C for 10 minutes followed by 40 cycles of 95°C for 10 seconds and 60°C for one minute. All reactions  were performed in duplicates on the same 96-well plate. Results reported here are averages of the duplicates. The PRNP expression level was normalized by dividing it with the ACTB expression level. ACTB was favored against GAPDH because the latter performed poorly in our medulla oblongata samples.Real-time PCR standard curves for each of the PRNP expression of the normally distributed data. All decisions were made following the differences in dependent variables but also considered the values of coefficient of determination (R2) and the t grouping. R2 values ranging from 0.6725–0.9085 and P-values<0.05 were considered significant in our analyses.Raw data were developed by spreadsheets and later subjected to the GLM procedure of SAS® Proprietary Software, Release 8.2  for statistical analyses using a linear model including diplotypes, sex, age, region/farms and their interactions as shown below. We performed the independent t-tests to support the ijkl = ExpressionYμ = Overall meani = Random effect of the ith diplotypeDj = Random effect of the jth sexSk = Random effect of the kth ageAl = Random effect of the lth locationLijkl = Random residual effect of each observation (error effect)E


However, BVDV 1b, 1e and 1i were also identified and, for the first time in the UK, BVDV 1d. Through analysis of animal movement data alongside the phylogenetic analysis of these BVD isolates, it was possible to link animal movements to the viral isolates present on several premises and, for the first time, begin to elucidate the routes of viral transmission. With further work, this type of analysis would enable accurate determination and quantification of the true biosecurity risk factors associated with BVDV transmission.Bovine Viral Diarrhoea Virus (BVDV) is a pestivirus which infects cattle populations worldwide and is recognised as a significant source of economic loss through its impact on health and productivity. Studies investigating the molecular epidemiology of BVDV can give invaluable information about the diversity of viral strains present in a population and this, in turn, can inform control programs, drive vaccine development and determine likely infection sources. The current study investigated 104 viral isolates from forty farms across the UK. Through phylogenetic and nucleotide sequence analysis of the 5′UTR and N Flaviviridae family. It is a positive sense, single stranded RNA virus of approximately 12.5 kb in length[pro, C, Erns, E1, E2, P7, NS2/3, NS4A, NS4B, NS5A, NS4B). The genus is currently comprised of four recognised species; BVDV-1, BVDV-2, Border Disease Virus (BDV) and Classical Swine Fever Virus (CSFV) and four proposed “atypical” species Giraffe, “HoBi”, Pronghorn Antelope and Bungowannah[Bovine Viral Diarrhoea Virus (BVDV) infects cattle worldwide and is a cause of significant losses in both beef and dairy systems. The virus is a member of the pestivirus genus in the pro gene, has resulted in further sub-classification such that BVDV genotype 1 currently contains twelve sub-genotypes; BVDV 1a-k plus Deer[Pestiviruses are highly variable both antigenically and genetically, hence each species is further classified. BVDV is currently divided into two genotypes. More relus Deer,10. Six lus Deer,11-15. Blus Deer-18.Studies investigating the molecular epidemiology of BVDV can provide invaluable information about the diversity of viral strains present in a population and, in turn, inform control programs, drive vaccine development and determine likely infection sources. In a survey of bovine pestiviruses published in 1999, 62 BVD viral isolates across England and Wales were sequenced and were predominantly found to be BVDV 1a although BVDV 1b and 1i were also identified,19. A moSamples were collected from PI animals identified on farms that were screened across six regions of the UK between 2006 and 2011 . RNA was isolated from heparinised whole blood using a QIAamp RNA Blood Mini Kit  and from serum using a QIAGEN QIAamp Viral RNA Kit (QIAGEN Ltd.) according to the manufacturer’s instructions.6 Random Hexamers  and incubated at 70°C for 10 min. 8 μL of 5× Superscript II First Strand Buffer (Invitrogen), 4 μL of 0.1M DTT (Invitrogen), 2 μL of 10 mM dNTPs , 16 μL nuclease-free dH2O and 1 μL of 40 U/μL RNasin (Promega) was then added. After incubating the mixture at 42°C for 2 min, 1 μL of 200 U/μL Superscript II Reverse Transcriptase enzyme (Invitrogen) was then added to each sample and the incubation continued for a further 50 min followed by 10 min at 72°C. RNA samples were stored at −70°C and cDNA samples were stored at −20°C for future use.cDNA was synthesised from isolated RNA using Invitrogen Superscript II reverse transcription kit . For each cDNA reaction, 5 μL of RNA was mixed with 3 μL of pd(N)pro regions respectively were amplified. At least two PCR reactions for each region, using a high fidelity Pfu DNA polymerase enzyme , were performed on each sample. If consensus sequence could not be obtained from sequence analysis of two PCR reactions, further reactions and sequencing were performed in order to achieve this. Primers 324 and 326 were used for amplification of a 288bp region of the 5′ UTR as described by Vilcek et al.[pro under the same conditions described by Vilcek et al.[For each viral isolate, a 288bp and 428bp region of the 5′UTR and Nk et al.. Primersk et al. and a mok et al., named Bk et al. for the Pfu DNA polymerase reaction buffer (Stratagene), 1 μL 10mM dNTP, 0.5 μL Pfu DNA polymerase 2.5 U/μL (Stratagene) and 31.5 μL dH20. The PCR conditions were as follows: 94°C for 3 min initial denaturing followed by 30 cycles of 94°C for 1 min, 52°C for 1 min, 72°C for 1 min and then final elongation at 72°C for 10 min. Amplicons of the expected sizes (288bp for 5′UTR and 428bp for Npro) were gel purified or PCR purified  using QIAquick /PCRGel Extraction Kit (QIAGEN). Purified PCR products were sequenced by QIAGEN Genomic Services  using Dye Terminator cycle sequencing performed with the ABI PRISM BiG DYE V3.1 Terminator Cycle Sequencing. At least two independent PCR reactions were sequenced for each sample using forward and reverse primers 324/326 or BD1/BD3A for 5′UTR and Npro respectively.For PCR reactions, the following was setup; 2 μL template cDNA, 5 μL Forward primer (10 pmol/μL), 5 μL Reverse primer (10 pmol/μL), 5 μL 10 × MEGA version 5[pro sequences in FASTA format were imported into MEGA 5 and sequence alignments performed using ClustalW. Phylogenetic analysis was performed using the Neighbour Joining Method[The sequences received were initially analysed in Vector NTI Version 11 (Invitrogen) and consensus sequences were assembled in the Contig Express feature of Vector NTI where they were checked for discrepancies. Primer sequences were clipped from each consensus prior to phylogenetic analysis conducted using ersion 5. 5′UTR ag Method and evolg Method. Bootstrg Method.Sixty-two farms gave permission to obtain animal movement records relating to their premises. This network included thirty-one farms from which PI samples were genotyped, twenty-six farms where BVDV surveillance was conducted but no PI animals identified and five farms on which PI animals were identified but genotyping not yet performed. These are listed as Farms 1–62 in Table One hundred and four viral isolates were collected from PI cattle on forty farms across the UK. The geographical location of the farms and the way in which the samples were submitted is summarised in Table pro sequences were obtained for phylogenetic analysis and sequence comparison. Table pro and comparison to a set of reference strains detailed in Table Of the 104 viral isolates investigated, 101/104 5′UTR and 98/104 Npro phylogenetic trees in Figures pro, shown in Table pro analysis occurred for isolates 1–1 and 1–2, which both had the highest identity (95%) to the BVDV 1e 5′UTR reference strains, yet upon Npro analysis were most identical (83%) to the BVDV 1a reference strain SD1. However, removal of SD1 from the Npro analysis left isolates 1–1 and 1–2 with closest identity (82%) to BVDV 1e, 1i, 1j and 1m reference strains and only 81% identity to NADL (1a); hence, isolates from Farm 1 clustered phylogenetically with BVDV 1e in both Figures pro respectively).For the viral isolates investigated, the apparent sub-genotypes indicated by the 5′UTR and Nentical 8% to the Movement data was available for Farms 1–62 in Table pro sequences where more than one PI was identified on the same farm revealed that they shared 99%–100% nucleotide identity in both regions investigated.1. Same farm, same isolate: This situation is demonstrated in both Figures pro regions within each PI cluster. However, between clusters on the same farm, identity in the 5′UTR and Npro regions ranges from 95%–96% and 88%–90% respectively. For Farm 59, only two PIs were identified and these shared nucleotide sequence identities in the 5′UTR and Npro regions of only 95% and 91% respectively thus grouped separately from each other upon phylogenetic analysis , Farms 20 and 43 (isolates share 99% 5′UTR nucleotide identity), Farms 56 and 62 (isolates share 99%–100% 5′UTR nucleotide identity) and Farms 33 & 34 (isolates share 100% 5′UTR nucleotide identity) are seen to cluster as distinct groups in Figure pro phylogenetic tree in Figure pro phylogenetic analysis of Farms 20 & 43 and 56 & 62 with isolates from these farm groups returning 97% and 96% Npro nucleotide identity respectively. Whilst these identities are still high, the clustering of isolates from these farms is less convincing in Figure 3. Different farms, same isolate: This instance appears to occur on several farms if the 5′UTR in Figure Farms 33 and 34 have been linked by direct animal movements  and also through indirect animal movements through Farm 32 throughout the study period. Figure pro phylogenetic trees, they were not linked by direct animal movements between their premises. Despite this, the high identity noted in both the 5′UTR and Npro regions of viral isolates from these farms supports the theory that there is a common isolate in both units. It is known to the authors that the owners of Farms 40 and 41 exhibit animals at the same agricultural shows each year and it is quite feasible that animal contact may have occurred at one of these shows resulting in either concurrent infection of both farms with the same BVDV strain or one farm infecting the other at the show. As an adjunct to this, the authors are aware that Farm 40 has supplied colostrum to Farm 41 on several occasions; another potential route for transmission (this practice has now ceased consequent to our advice).Whilst the isolates from Farms 40 and 41 appear linked when analysing both the 5′UTR and Npro region. No direct animal movements were identified to link these farms. Upon initial investigation, there was no common link between Farms 56 and 52 since they were more than 400 miles apart. However, both use embryo transfer services which could explain a common source of infection although this would seem unlikely given the analysis of the Npro regions of BVDV isolates from both premises.Whilst Farms 56 & 52 and 20 & 43, initially appear to have a common BVDV isolate upon 5′UTR phylogenetic and nucleotide analysis, this is less well supported by analysis of the NThe predominant BVDV sub-genotype identified on the forty infected farms investigated in this study was BVDV 1a. This was isolated from the four main regions sampled in this study. BVDV 1b was isolated on two premises; one in the southwest and one in the southeast of England. Whilst these findings are largely in agreement with those of Vilcek et al.,19, the No type 2 BVDV isolates were identified during the investigations in this study and this is re-assuring since the vaccines currently available in the UK only confer good cross protection against type 1 strains. Isolated occurrences of BVDV 2 have been reported in the UK-40 but tCurrent literature suggests that there is a continuing increase in the diversity of the BVDV 1 sub-genotype,11-15. WThe phylogenetic analysis performed on isolates presented in this manuscript utilises the Neighbour Joining method which hapro region confirmed that where this confusion occurred, the isolates were actually BVDV 1a. Furthermore, one could argue that the BVDV 1i branch highlighted in Figure pro analysis was able to provide a more convincing definition of the isolates on this branch confirming them as likely BVDV 1i isolates. Becher et al.[pro region. It is however worth re-iterating this point again since many of the most recent publications sub-genotyping BVDV isolates still concentrate on the 5′UTR with little additional information from other genomic regions. As a result, ideally, at least two regions of the BVDV genome should be analysed and agreement sought between them in order to define the isolates investigated with some certainty. Several studies have explored the use of three or more regions of the BVDV genome for sub-genotyping[From the dataset investigated, it is evident from both Figure r et al. has prevnotyping,46. The notyping and Kadinotyping. Peterhanotyping has subsnotyping have alrIn addition to the more traditional use of phylogenetics to investigate and type viral isolates present in a region or country, the data presented in this manuscript links viral isolates to the farms investigated and explores the potential routes of viral transmission. The farm level data collected enabled a detailed analysis of animal movements to be performed between premises which could, in some cases, be related directly to the phylogeny of the viruses isolated. In effect we are less concerned with primary branching and definition of viral type but instead interested in the clade formation at the end of each branch. Observing clustering at branch ends on the phylogenetic trees enables rapid identification of isolates with a high identity and thus high chance of a common infection route. Three different situations were described in the results:pro regions. The phenomenon of clustering of strains on the same farm is not new and has been noted previously[Same farm, same isolate: Where more than one PI is isolated on the same farm, typing of those isolates commonly shows that the virus infecting each PI will share close to 100% nucleotide identity in both the 5′UTR and Neviously. As yet,eviously.Same farm, different isolate: Where the one farm demonstrates two (or more) markedly different groupings upon phylogenetic analysis, it is likely that more than one infection event has occurred (perhaps two PIs bought in from different locations) producing clusters of PIs with two unique viral isolates.Different farm, same isolate: Ridpath et al. used seqIn conclusion, the work in this study supports the findings of Strong et al. and has We have demonstrated that in depth investigations of animal movements and contacts between farms combined with phylogenetic analysis of BVDV can produce a “papertrail” of infection. This may have potential when utilised on a wider scale to quantify the risk factors associated with BVDV transmission between premises. These investigations may also have implications in legal cases where the origin of infection is under dispute.Finally, the analysis of the data presented in this paper has demonstrated areas of pestivirus classification that require clarification. Particular issues arising from this work have been highlighted which would benefit from set guidelines in order to standardise future BVDV phylogenetic analyses. This is particularly important where these processes are used to determine the existence of new pestivirus strains/subgroups. This research, as a consequence, may also develop discussion within the pestivirus community in order to develop agreed guidelines to attain consistent investigation and classification for future epidemiological studies and newly identified isolates.The authors declare that they have no competing interests.RB conducted the sample collection from herds 1–42, performed RT-PCR followed by sequence and phylogenetic analysis on samples from all farms involved. CT and LA gave extensive advice on the technical aspects of the phylogenetic and sequence analysis. CT, LA, GG and JB contributed to the drafting of the manuscript. All authors read and approved the final manuscript.


Aphis fabae alarm pheromone, i.e. (E)-β-farnesene (EβF), by ant scouts. The perception and behavioral impact of EβF on Lasius niger were investigated using electroantennography and two bio-assays measuring their attraction and orientation towards aphid semiochemicals. Pronounced electrical depolarizations were observed from L. niger scout antennae to stimulations of A. fabae alarm pheromone, while other sesquiterpenes elicited weak or no responses. L. niger scouts were significantly attracted toward EβF in a four-arm olfactometer, as well as in an two-choice bioassay. These laboratory results suggest for the first time that low amounts of aphid alarm pheromone can be used by L. niger scouts as a cue indicating the presence of aphid colonies and could therefore mediate the aphid-ant partnership in the field.The mutualistic relationships that occur between myrmecophilous aphids and ants are based on the rich food supply that honeydew represents for ants and on the protection they provide against aphid natural enemies. While aphid predators and parasitoids actively forage for oviposition sites by using aphid semiochemicals, scouts of aphid-tending ant species would also benefit from locating honeydew resources by orienting toward aphid pheromone sources. The present study aims to provide additional information on the use of  Aphids  and ants  are the protagonists of one of the most studied model of mutualistic relationships in the animal kingdom: the first ones produce a carbohydrate-rich excretion named honeydew, which is collected by some ant species who provide aphids in return with protection and hygiene Formica subsericea ability to react behaviorally to the main component of the alarm pheromone of most aphidinae species. When F. subsericea were attending aphids and suddenly exposed to huge amounts of (E)-β-farnesene (EβF), they extended their antennae and opened their mandibles being prepared for attacking potential aphid enemies. Besides, more recent studies have demonstrated that ants detect specific blends of cuticular hydrocarbons on aphids’ body what allows them to discriminate myrmecophilous aphids from potential prey The communication between both partners was thought to be essentially tactile, as ants palpate aphids’ abdomen using alternatively their two antennae to stimulate the ejection of honeydew droplets. But the interactions between ants and aphids are also chemically mediated. Nault and co-authors EβF and use it as a kairomonal substance to locate their host or prey EβF on Lasius niger were investigated using electroantennography and two bio-assays measuring their attraction and orientation towards aphid semiochemicals.Several laboratory studies have suggested that aphid natural enemies, including ladybeetles, hoverflies and chrysopids, may be able to detect Lasius niger L. colonies  were collected in Brussels in April 2007 and placed in plastic containers (35×25×8 cm) whose edges were covered with polytetrafluoroethylen to prevent them from escaping. Test tubes covered with a red transparent foil were disposed as laboratory rearing nests. Sucrose solutions (1 M), dead arthropods  and water filled test tubes were provided and renewed every two days. The colonies were kept in an environmentally controlled room . The black bean aphids, Aphis fabae Scopoli, were mass reared on broad beans (Vicia faba L.) grown in 10 cm3 plastic pots filled with a mix of perlite and vermiculite (1∶1) and placed in similar conditions as above.Queenless L. niger scout antenna was carefully excised from the head. Because of the important background noise registered from the ant antenna, the scape was removed to improve electrical contact and subsequently decrease background noise. The antenna was mounted and stimulated as described in our previous experiments on the perception of aphid alarm pheromone by beetle and fly antennae EβF solutions with concentrations ranging from 0.1 g/l to 100 g/l (by 10x increments). Stimulation with semiochemical-free paraffin oil was carried out as a negative control before and after the stimulations with the four EβF solutions cited above. Thirty seconds elapsed between successive stimulations. Preliminary results indicate that this length of time was adequate to allow the insect recover its full reactivity to stimuli. EβF was synthesized from farnesol with a chemical purity of 98% (determined by GC). In order to compare the scout antenna sensibility to EβF with other sesquiterpenes (but not associated to aphids), (E)-caryophyllene and α-humulene, both purchased from Sigma-Aldrich  were also tested following the same procedure as above. A total of 15 different antennae were tested: five per chemical. Each antenna was tested with the 5 concentrations of one single chemical .et al. L. niger colony. It was constructed entirely of Teflon® and was closed with a removable glass roof, both cleaned with n-hexane between each tested ant. Charcoal-filtered air was pushed in each of the four olfactometer arms through Teflon® tubing, and adjusted to 100 ml/min for each arm with a digital flowmeter. A pump ventilated the walking arena by removing air from the centre at 400 ml/min. A L. niger colony was placed under the olfactometer and a Teflon® tube allowed scouts to climb up to the walking arena. A “T” glass piece allowed the connection of the plastic tube to the olfactometer, and at the same time the aspiration of the outgoing air. This piece also allowed to close the access to the olfactometer and thus controlled the entrance of only one scout per replicate. A 0.5 l glass chamber was connected to one of the four olfactometer arms, and was used to introduce five unwinged A. fabae adults, that were rapidly crushed inside the glass chamber using a small glass pestle left inside the chamber . According to Pickett & Griffiths et al. A. fabae consist exclusively of EβF. Preliminary volatile analysis experiments allowed us to approximate the amount of EβF released by five crushed A. fabae. Five A. fabae individuals were quickly crushed in n-hexane and the supernatant was injected in a gas chromatograph. We found an average amount of 50.9 ng of EβF, which is similar to what a quiet non-preyed M. persicae colony made of about 75 individuals release The four-arm olfactometer was similar to that previously described by Verheggen EβF was placed alternatively (with similar number of replicates being conducted on both sides) on one of the two plants and switched after each observed ant scout. One ventilator was placed behind each plant to ensure an air flow of 0.6±0.1 m/s, going from the plant to the bioassay setup. The “T” setup was divided into different sections: the middle part of the “T” aluminium (8 cm length) was considered as an area of no-choice. The last 1.5 cm of the “T” foraging branches were considered as areas where the final choice was made by the ants which were removed after having reached one of these sections. The time spent by each ant scout in both “T” arms, the final choice and the number of U-turns were recorded. The walking speed has been calculated by dividing the time spent in one of the two sections of the bioassay by the length of the section. Ants that changed direction (i.e. side of the olfactometer) during the test were not taken into account for this calculation. The setup was surrounded by black plastic sheets to avoid visual bias and disturbances, and was placed under uniform light provided by three neon tubes. Three different ant colonies were tested and results were pooled, after having checked the absence of bias. A total of 43 ant scouts were observed.The setup  was madeEβF; (E)-caryophyllene; α-humulene) at the 5 different doses , a three-way ANOVA was conducted with factors being “chemicals” (systematic factor), “doses” (systematic factor) and “antennae” (random factor). Because every doses were tested on every antennae, and because only one chemical was tested per antenna, we used a partially hierarchized model: the factor “doses” is crossed with the factor “chemicals” and with the factor “antennae”, while the factor “antennae” is hierarchized with the factor “chemical”. Observed frequencies related to the final choice of L. niger scouts in olfactometer assays (four-arm and two-choice bioassays) were compared to corresponding theoretical frequencies by using a χ2 goodness-of-fit test. Paired t-test was conducted to compare, for each ant scout, the difference between the time spent in the branch leading to the EβF treated plant and the time spent in the branch leading to the control plant. ANOVA were conducted to compare the mean durations spent in the different branches of both bio-assays. Finally, Fisher’s exact tests were conducted to compare proportions of ants initiating specific types of behavior in the bioassays. All these tests were conducted with MINITAB v15 .To compare EAG responses for the three tested chemicals (EβF elicited EAG responses of −0.692±0.197 mV (mean ±SE). Two additional sesquiterpenes were also tested and both (E)-caryophyllene (−0.224±0.045 mV) and α-humulene (−0.036±0.031 mV) elicited weak electrical depolarizations from L. niger antennae -caryophyllene; α-humulene) at the 5 different doses . The electrical responses recorded differed statistically for all three semiochemicals tested . A positive dose–response relationship in EAG was also observed . The three-way ANOVA highlighted an interaction relationship between the two systematic factors, namely the chemicals and the doses . We have therefore conducted a two-way ANOVA for each tested chemical. A positive dose–response relationship in EAG was recorded to EβF  and to (E)-caryophyllene , but not to α-humulene . When conducting a two-way ANOVA separating each tested doses, we found that, at the highest tested dose, the recorded electrical responses differed statistically between the three semiochemicals tested , with EβF eliciting the highest electrical response.The highest dose of antennae .. A threEβF source. This visitation rate is significantly higher than expected from a random orientation (25% in the case of a random choice) . They also spent most of their time in the arena connected to the EβF source, as the tested scouts spent 42.7±6.2% of the observation time  in the EβF arm of the olfactometer .According to the first area visited, 53.3% of the scouts were first attracted to the EβF treated plant (67%) rather than to the branch leading to the control non treated plant (33%) . For each ant scout, the time spent on the branch leading to the EβF treated plant  was on average significantly higher than the time spent on the branch leading to the control plant  . Before ant scouts orient themselves toward one side or the other, we also recorded the time spent in the neutral area as an estimate of their difficulty to make a choice, this time being assumed to be shorter for ants being attracted by the EβF treated side. But, no difference was observed between the mean time spent in the neutral area by ant scouts choosing the EβF side (3.8±0.6 s) and that for ants choosing the untreated side (3.8±0.8 s) . On average, the walking speed were similar for ants orientating to the control (1.74±0.12 cm/s) and the EβF side (1.96±0.23 cm/s) of the bioassay . Finally, we noted the number of ant scouts “changing their mind” – i.e. first walking over one branch, then making U-turns and finally choosing the other side of the setup. Over the ants having finally chosen the EβF side, only 10% of scouts had initially strolled over the untreated zone. Indeed, a majority (90%) of scouts orienting themselves towards EβF treated side made this clear-cut choice from the start of the experiment. As regards ant scouts having finally chosen the untreated side, we found out a higher, but not statistically significant, percentage (29%) of “hesitating” individuals that first initiated a short movement towards the EβF treated plant before changing side .Ant scouts preferrentially orientated towards the branch leading to the L. niger have olfactory receptors perceiving A. fabae alarm pheromone, as shown by the positive dose–response relationship in EAG to EβF. The highest tested EβF dose elicited EAG responses of -0.692±0.197 mV (mean ±SE) statistically higher than the paraffin oil control , and three times lower with the Asian lady beetle, Harmonia axyridis . Moreover, other sesquiterpenes α-humulene and (E)-caryophyllene did not elicit pronounced electrical depolarizations. The olfactory system of foraging ant workers therefore seems to be sensitive and adapted for the perception of aphid alarm pheromone. We also showed that L. niger scouts detect (E)-caryophyllene, as low electrical responses were recorded from scouts antennae. As observed for aphid natural enemies EβF emitted by aphids and EβF from some plant species that is emitted along with other sesquiterpenes like (E)-caryophyllene.Like most aphid natural enemies that have evolved to adapt their olfactory system to the perception of aphid-related volatile chemicals and subsequently locate their prey, ants would have advantage to perceive aphid odorant cues, which would increase their chance to establish a mutualistic relationship. Our results demonstrate that  control .. While Formica subsericea ant species Acyrthosiphon pisum colonies, has reported an increase in the number of predating Lasius niger ants in the treated aphid colonies EβF also induced typical alarm and defensive behavior among Lasius niger ants (pers. obs.). That low EβF levels – i.e. the background level emitted by quiet and non-preyed aphid colonies – could be perceived by ant scouts, looking for food resources, and thus be used as a cue to locate their aphid partner has never been demonstrated earlier. Single ant scouts were clearly attracted by EβF in the four-arm olfactometer. This has been observed also in our two-choice bioassay. In both cases, none of the observed ant scouts exhibited agressive behaviour like that observed by Nault et al. L. niger scouts foraging for food (1.6 to 2.1 cm/s) EβF) have led to attraction rather than an alarm or defensive behaviour. The fact that EβF induces quite different behavioural responses among ant scouts depending on the perceived amounts might have strong ecological implications, and may explain the increase in ant predation behavior observed in pea aphid colonies where additional amounts of alarm pheromone were added M. persicae colonies EβF levels trigger aggressive behaviours among ants and thus speed up their chasing and killing of predators/parasitoids. Many ant species, including Lasius niger, are also known to switch continuously from a “breeder” to a “predator” behavior according to aphid colony size EβF could facilitate the shift from a “breeder” behavior of tending ants to a “predator” behavior, when the aphid colony gets crowded EβF is used as unique component of the alarm pheromone in most aphid species, including unattended ones Aphid alarm pheromone is known to elicit agonistic behaviour –i.e. raising of antennae and opening of mandibles – among EβF leads mostly to aphid colonies, and sounds like reliable semiochemical for aphid presence, one could consider its perception by ants as either an indicator of a mutualism opportunity, or a source of food. Regarding aphids, they would have strong advantage to emit low amounts of semiochemicals to attract ants at the first steps of this mutual relationship. Once this first contact established, ants will assess the profitability of this aphid colony such as the quality, the amount or the renewal rate of produced honeydew If EβF attract ant scouts. Because the aphid alarm pheromone is not the only semiochemical to be released by an aphid colony, one should also evaluate the biological activity of other aphid-related volatile chemicals, including those released by the aphid honeydew Within aphid-ant mutualism, aphid semiochemicals, including the aphid alarm pheromone, could act as synomones, being beneficial for the releasers (aphids), that will attract their bodyguards, and beneficial for the receivers (ant scouts), that will likely encounter a food source. This assumption should however be confirmed by performing field assays demonstrating that in natural conditions, emissions of


Peroxisome proliferator activated receptors (PPARs) are well studied for their role of peripheral metabolism, but they also may be involved in the pathogenesis of various disorders of the central nervous system (CNS) including multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's and, Parkinson's disease. The observation that PPARs are able to suppress the inflammatory response in peripheral macrophages and in several models of human autoimmune diseases, lead to the idea that PPARs might be beneficial for CNS disorders possessing an inflammatory component. The neuroinflammatory response during the course of Alzheimer's disease (AD) is triggered by the deposition of the β-amyloid peptide in extracellular plaques and ongoing neurodegeneration. Non-steroidal anti-inflammatory drugs (NSAIDs) have been considered to delay the onset and reduce the risk to develop Alzheimer’s disease, while they also directly activate PPARγ. This led to the hypothesis that NSAID protection in AD may be partly mediated by PPARγ. Several lines of evidence have supported this hypothesis, using AD related transgenic cellular and animal models. Stimulation of PPARγ by synthetic agonist (thiazolidinediones) inducing anti-inflammatory, anti-amyloidogenic and insulin sensitizing effects may account for the observed effects. Several clinical trials already revealed promising results using PPARγ agonists, therefore PPARγ represents an attractive therapeutic target for the treatment of AD. The peroxisome proliferator activated receptors (PPARs) belong to the family of nuclear hormone receptors (NHR) that comprise 48 human ligand-inducible transcription factors which activity is regulated by steroids and lipid metabolites [reviewed in PPARs act principally as lipid sensors and regulate whole body metabolism in response to dietary lipid intake and direct their subsequent metabolism and storage . The proPPARα acts primarily to regulate energy homoeostasis through its ability to stimulate the breakdown of fatty acids and cholesterol, driving gluconeogenesis and reduction in serum triglyceride levels. This receptor acts as a lipid sensor, binding fatty acids and initiating their subsequent metabolism. PPARγ binds a number of lipids including fatty acids, eicosanoids and other natural lipid ligands. Its dominant action is to stimulate adipocyte differentiation and to direct lipid metabolites to be deposited in this tissue. PPARγ operates at the critical metabolic intersection of lipid and carbohydrate metabolism. PPARγ activation is linked to reduction in serum glucose levels, likely as a secondary effect of its ability to regulate endocrine factors. It is this latter activity that has led to the development of specific PPARγ agonists for the treatment of type II diabetes . PPARγ/δPPARs regulate gene expression by forming heterodimers with retinoid-X-receptors (RXRs). Stimulation of target gene expression is controlled by specific PPAR-response elements in the promoter region (PPREs). Under unstimulated conditions these heterodimers are associated with corepressors, like N-CoR and SMRT, which suppress gene transcription . Upon livia the ubiquitination degradation pathway [There are several mechanisms involved in PPARγ inactivation. Thus, phosphorylation can negatively or positively affect PPARγ activity depending on which specific protein residue is modified. It has been shown that S82 (for PPARγ1) /S112 (PPARγ2) phosphorylation, by ERK and JNK pathways result in PPARγ inhibition . Serine  pathway . Alterna pathway ,15. In a pathway . One S82 pathway . On the  pathway .transcriptional transrepression, which is not mediated by their binding to PPREs. PPARγ is able to suppress expression of proinflammatory genes in myeloid lineage cells, such as microglia and macrophages, and in the vasculature, by suppressing the action of other transcription factors like NFκB, AP-1 and STAT1 [Like other NHR, PPARs also inhibit proinflammatory gene expression by a controversial mechanism of nd STAT1 . One mecnd STAT1 ,21.Binding of PPARs to their specific ligands leads to conformational changes which allow co-repressor release and co-activator recruitment. Even though all PPARs can be attributed to a common ancestral nuclear receptor, each PPAR isotype has its own properties with regard to ligand binding. Synthetic thiazolidinediones (TZDs), which are commonly prescribed for the treatment of type II diabetes, are selective PPARγ ligands. Naturally occurring PPARγ ligands include eicosanoids and the prostaglandin 15d-PGJ2. The best characterized PPARγ agonists are pioglitazone and rosiglitazone which are Food and Drug Administration (FDA) approved for treatment of type II diabetes and troglitazone, which has been withdrawn in 2000. PPARα agonistic ligands include fibrates that are commonly used for the treatment of hypertriglyceridemia and WY14,643 and GW7647. PPARβ/δ agonists include the prostacyclin PGI2, and synthetic compounds GW0742, GW501516, and GW7842. In addition, all PPARs can be activated by polyunsaturated fatty acids with different affinities . An overPPARα and γ transcripts appear late during fetal development of rat and mouse (day 13.5 of gestation), with similar expression pattern to their adult distribution. PPARα is found in the liver, the kidney, the intestine, the heart, the skeletal muscle, the adrenal gland and the pancreas. PPARγ expression is restricted to the brown adipose tissue (day 18.5 of gestation), and to the CNS (day 13.5 to 15.5 of gestation). Compared to the two other isotypes, PPARβ/δ is expressed ubiquitously and earlier during fetal development . In adulNot much is known about the expression of the PPARs during human development -27. PPARAll three PPAR isotypes are co-expressed in the nervous system during late rat embryogenesis, and PPARβ/δ is the prevalent isotype. The expression of the three PPAR isotypes peaks in the rat CNS between day 13.5. and 18.5 of gestation. Whereas PPARβ/δ remains highly expressed in this tissue, the expression of PPARα and .The number of individuals with the Alzheimer’s disease (AD) is dramatically increasing as a consequence of a longer life expectancy in our societies. The large number of affected individuals and the increasing prevalence of the AD presents a substantial challenge to health care systems and does so in the face of substantial economic costs. The pathological hallmarks of AD are the formation of extracellular plaques consisting of amyloid-β peptides and intracellular neurofibrillary tangles made up from hyperphosphorylated tau protein, causing neuronal death that is responsible for progressive memory loss and inexorable decline of cognitive functions ,46. AnalPPARγ is expressed in the brain at low levels under physiological conditions. Recently, a detailed gene expression analysis has demonstrated that mRNA levels are elevated in AD patients . This suin vitro and in vivo [in vitro and prevented cortical or hippocampal neuronal cell death [The initial studies exploring the actions of PPARγ in AD were based on the ability of non-steroidal anti-inflammatory drugs (NSAID) to activate this receptor. A number of epidemiological studies demonstrated that NSAID treatment reduces AD risk by as much as 80% and it was suggested that these effects arise from the ability of these drugs to stimulate PPARγ and to inhibit inflammatory responses in the AD brain -59. This in vivo ,60. In a in vivo . These l in vivo . PPARγ all death -63. In all death . The effll death . 12 montll death .The modest effects of pioglitazone in this study were thought to be due to poor drug penetration into the brain. In a subsequent study treatment with larger doses of pioglitazone in aged APPV717I transgenic mice significantly decreased microglial and astroglial activation as well as Aβ plaque burden . The finAdditionally, modulation of the Wnt/β-catenin signalling pathway may also account for some PPARγ mediated beneficial effects in AD since recent findings show that PPARγ mediated protection of hippocampal neurons against Aβ-induced toxicity directly correlates with β-catenin levels, inhibition of GSK 3β activity and increased levels of Wnt-target genes ,75. FurtIn a further animal study, Pedersen and colleagues have demonstrated that rosiglitazone treatment of Tg2576 mice resulted in behavioural improvement in these animals as well as in reduction of Aβ1-42 in the brain. Treatment with rosiglitazone for 7 months enhanced spatial working and reference memory . SignifiPro12Ala polymorphism, albeit a change in PPARγ activity by this mutation will most likely affect the adipose tissue. However, a recent study revealed a significant overrepresentation of the Ala12 allele in octogenarian AD patients [The influence of genetic mutations on the course or overall risk of Alzheimer`s disease have almost only be addressed for the PPARγ2patients , suggestpatients . Two furpatients ,87. Howepatients . Importapatients . In diabpatients ,90. Excepatients . TherefoClinical investigations of insulin-sensitizing TZDs that are in clinical use for type II diabetes are currently ongoing. A small study of 30 patients with mild AD or MCI found that 6 months of treatment with rosiglitazone resulted in improved memory and selective attention. A larger trial of rosiglitazone in AD patients has recently been reported . More th1). The compelling results from animal models of Alzheimer’s disease underline the beneficial effects of PPARγ agonists for future therapies. The importance of these activities for the disease altering actions of PPARγ agonist as well as the underlying molecular mechanisms have to be elucidated in future research.PPARs exhibit a wide range of activities to positively influence the pathology of Alzheimer’s disease. Beside the ameliorating effect of PPARγ agonists on the inflammatory status of the AD brain by repressing the secretion of proinflammatory molecules and the enhancement of mitochondrial function, a direct involvement in the processing of the Aβ peptide has been demonstrated Fig. (


Here we report that expression of CREB regulated genes was altered in the lungs of CREBαΔ mice. CREBαΔ mice had greater pulmonary vascular resistance than wild types, both basally in normoxia and following exposure to hypoxic conditions for three weeks. There was no difference in rho kinase mediated vasoconstriction between CREBαΔ and wild type mice. Stereological analysis of pulmonary vascular structure showed characteristic wall thickening and lumen reduction in hypoxic wild-type mice, with similar changes observed in CREBαΔ. CREBαΔ mice had larger lungs with reduced epithelial surface density suggesting increased pulmonary compliance. These findings show that α and Δ isoforms of CREB1 regulate homeostatic gene expression in the lung and that normal activity of these isoforms is essential to maintain low pulmonary vascular resistance in both normoxic and hypoxic conditions and to maintain the normal alveolar structure. Interventions that enhance the actions of α and Δ isoforms of CREB1 warrant further investigation in hypoxic lung diseases.Chronic hypoxia causes pulmonary hypertension associated with structural alterations in pulmonary vessels and sustained vasoconstriction. The transcriptional mechanisms responsible for these distinctive changes are unclear. We have previously reported that CREB1 is activated in the lung in response to alveolar hypoxia but not in other organs. To directly investigate the role of α and Δ isoforms of CREB1 in the regulation of pulmonary vascular resistance we examined the responses of mice in which these isoforms of CREB1 had been inactivated by gene mutation, leaving only the β isoform intact (CREB Chronic alveolar hypoxia leads to the development of pulmonary hypertension (PH) which is characterized by a sustained elevation of pulmonary arterial pressure and pulmonary vascular resistance leading to the development of right ventricular hypertrophy. This direct effect of hypoxia on vascular resistance is unique to the pulmonary circulation. On moving to high altitude previously normal but susceptible lowlanders can develop progressive pulmonary hypertension leading to right ventricular failure which is fatal if not corrected by return to low altitude Two important mechanisms that cause elevation in pulmonary vascular resistance in hypoxic pulmonary hypertension are vasoconstriction and structural changes in the vascular bed. First, sustained rho kinase dependent vasoconstriction contributes a major part of the total increase, 50–90% depending on the species in vivo that we had previously reported acted to worsen or to ameliorate hypoxic pulmonary hypertension Previous work had shown that adenoviral mediated expression of a dominant-negative form of CREB (Ad-CREB-M1) attenuated systemic vascular smooth muscle cell hypertrophy induced by angiotensin-II The CREB1 gene encodes three functional isoforms of CREB α , Δ (isoform A) and β (isoform C) produced by alternative splicing. CREB Δ is ubiquitously expressed in adult tissues and is the most abundant isoform in normal adult tissues, comprising approximately 60–70% of the total CREB in a tissue in vivo, we investigated the pulmonary vascular responses of these hypomorphic, CREBαΔ−/− mice in normoxia and following sustained hypoxia and revealed a central role for these two isoforms of CREB1 in maintaining the normally low pulmonary vascular resistance.Deletion of the three functional splice variants of CREB1, α, β and Δ, is not compatible with postnatal survival Detailed methods are available in All procedures involving using mice were approved by the University College Dublin Animal Research Ethics Committee and carried out under license (B100/3430) issued by the Department of Health & Children in accordance with European Communities Regulations 2002  and EU Directive 2010/63/EU. All surgery was performed under sodium pentobarbital anesthesia that induced abolition of reflex withdrawal responses.αΔ mutant mice were generated as described previously CREBNote that the exon structure and numbers used throughout the manuscript are those of the most recent update (NCBI Reference Sequence: NC_000067.6). However, the original CREB α, Δ and β nomenclature of the three functional isoforms has been used to facilitate reference to the previous literature; these correspond to isoforms B, A and C respectively (see 2 = 0.10) for 3 weeks, and age- and weight-matched controls were maintained in normoxic conditions (FiO2 = 0.21) Adult male and female mice  were exposed to hypoxic conditions in an environmental chamber (FiOPulmonary vascular resistance was assessed with an isolated ventilated lung preparation perfused at constant flow 2O) and fixed with intratracheal glutaraldehyde (25 cm H2O). Left lung volumes were measured, and lungs were then processed to obtain isotropic, uniform, random resin-embedded sections (1 μm) for stereological quantification of the pulmonary vasculature by a blinded reviewer After anesthesia, anticoagulation, and exsanguination, mouse lungs were perfused with horse blood at standard pressure  to cDNA according to the manufacturer's protocol. TaqMan real-time PCR was performed using 18S rRNA as the endogenous loading control gene. Reactions were carried out on the ABI PRISM 7900 Sequence Detection System. Relative quantification of mRNA expression levels was determined using the standard curve method and normalized to 18S.Whole lung lysates were prepared in radioimmuno-precipitation assay (RIPA) buffer and homogenized by mechanical disruption. Equal amounts of protein extract from each lung (15 μg per sample) were separated by SDS-PAGE, transferred onto PVDF membranes and detected using appropriate antibodies and secondary antibodies labeled with horseradish peroxidase. An identical amount of protein from a standard sample formed by pooling homogenate from a panel of normal lungs was loaded onto each gel to act as a loading and transfer control. Optical intensity of each sample was recorded by digital imaging, quantified using ImageJ software and normalized to the pooled standard sample.P values were computed using the exact (permutation) method. For four group designs in which the data were non-normally distributed, correction for multiple post hoc comparisons were made using the Holms-Sidak step-down procedure P<0.05 were accepted as statistically significant.Normally distributed data are reported as means ± SEM, while non-normally distributed data are presented as medians ± interquartile range (IQR). For normally distributed data, statistical significance of differences between two group means was determined using t-tests. For four group designs, normally distributed data were analyzed using 2–factor analysis of variance to seek statistically significant effects of oxygen concentration and genotype, and interactions between these two. For non-normally distributed data, statistical significance was determined using the Mann-Whitney rank sum (unpaired); αΔ−/− mice (subsequently referred to as CREBαΔ) appeared healthy and showed no developmental abnormalities, the yield of CREBαΔ mice (approximately 3%) following crossing of pairs of mice heterozygous for the deletion was substantially below the expected Mendelian frequency had the mutation been without effect, in agreement with previous reports Although CREBαΔ and wild type mice showed a similar loss of weight in response to sustained hypoxia  interaction between genotype and hypoxic exposure was observed.CREB hypoxia . Both wiildtypes  and a stαΔ although expression of these CREB family members was not further altered by hypoxia in either genotype (Expression of CREB β and cAMP responsive element modulator (CREM) mRNA was markedly increased in the mice with mutation of CREBgenotype .αΔ mice showed no expression of α and Δ isoforms due to the disruption of exon 2 , we showed that activated CREB protein remained at similar levels in normoxia and hypoxia in wildtype mice, while the phosphorylated CREB protein was not detectable by western blot in CREBαΔ mice , endothelin1 (EDN1), follistatin (FST) and tissue plasminogen activator (PLAT) αΔ mice when compared to normoxic wild type mice  in homozygous CREBαΔ mice. We observed that pulmonary vascular resistance was significantly greater in both normoxic and hypoxic conditions in homozygous CREBαΔ mice than in wildtypes  and found that chronic hypoxia caused a significantly enhanced vasodilator response  was significantly elevated in the CREBildtypes . In wildobserved .αΔ mice under normoxic conditions but increased significantly in both groups following sustained hypoxic exposure , in keeping with the well known increase in lung volume caused by sustained hypoxia αΔ mutation also caused significant lung enlargement when compared to wild type mice (Hypoxia caused significant increases in lung volume  when infype mice .αΔ mouse lungs appeared to have larger airspaces (αΔ mutation (αΔ mice were more compliant (reduced elastic recoil) than those of wild type animals.Microscopic examination of the lung demonstrated that the alveoli of CREBirspaces . Quantitmutation  althoughmutation . Taken tin vitroWe have previously reported that alveolar hypoxia, similar to that found in lung diseases and at high altitude, leads to selective phosphorylation and activation of CREB1 in the absence of similar activation in other organs, suggesting that this transcription factor has an important role to play in the pulmonary response to hypoxia αΔ mice. Of note, the right ventricular hypertrophy was significantly greater in hypoxic CREBαΔ mice. In addition, a greater increase in lung volume in response to hypoxia was observed in CREBαΔ mice associated with a reduction in the alveolar epithelial surface density suggestive of an emphysematous phenotype.In the present study, we further explored the role of CREB1 by examining the pulmonary response to hypoxia following partial loss of CREB1 due to homozygous, ubiquitous deletion of two of its three isoforms, α and Δ, and found an increase in pulmonary vascular resistance in these mice under both normoxic and hypoxic conditions. Right ventricular hypertrophy was observed in both hypoxic wildtype and CREBαΔ mouse used in these studies, only the CREBβ isoform is expressed; no mRNA for CREB α or Δ can be detected αΔ mice, it has been shown that the CREBβ isoform protein is markedly upregulated in the brain Mice in which all three functional isoforms of CREB, α, β and Δ, have been knocked out, die shortly after birth due to respiratory distress syndrome demonstrating that CREB activity is required for normal lung development αΔ mice using an antibody that detects these two isoforms , often antagonizing NFAT actions αΔ mice. Nonetheless, it is clear that CREB α and Δ are required for normal pulmonary vascular function and a normal response to hypoxia.Other mechanisms could also contribute to the changes in gene expression observed in the CREBαΔ deletion on pulmonary arterial pressure mediated indirectly by changes in cardiac output, right or left heart function, endocrine and reflex influences acting on the pulmonary circulation. Thus, the increased pulmonary vascular resistance that we observed in the CREBαΔ mice . None of the ratios differs significantly from 1.00.(DOC)Click here for additional data file.


Delichon urbicum) are sensitive to factors influencing the availability of flying insects, and have declined in recent decades. The strict aerial behaviour of martins severely limits ring recoveries on wintering grounds and so there is a dearth of information on where European breeding populations over-winter in Africa, and the relative effects of population regulation on breeding vs. wintering grounds. We used a newly developed multi-isotope  feather isoscape for Africa together with inferences from summarized ring return data based on longitude, to assign winter origins to birds captured at a breeding colony in The Netherlands.The ability to connect breeding, stopover and wintering locations of populations of migratory birds greatly enhances our understanding of the phenomenon of migration and improves our chances of effectively conserving these species. Among Palearctic-Afrotropical migratory species, aerial insectivores like the house martin  in Africa and population patterns in Italy and the United Kingdom. These confirmatory data-sets underscore the importance of suitable habitats in West Africa to the conservation of migratory aerial insectivores and other species. Delichon urbicum) are among the most common migratory aerial insectivores in the Old World Hirundo rustica), house martins rarely yield recoveries away from their European breeding grounds Migrationes Avium) that swallows and martins hibernate at the bottom of lakes was officially rejected by the Swedish Academy of Sciences in 1854, the wintering distribution of house martins has remained enigmatic House martins  of martin feathers between juvenile and adult birds captured at a single breeding site in The Netherlands over six years. We then used the triple isotopic  composition of primary feathers of adult martins returning to their breeding grounds in The Netherlands to provide a first approximation of the most likely regions of Africa in which these feathers were grown by assigning them to isotopic “clusters” in Africa δ2H alone.In the absence of wing molt in martins on the breeding grounds, and with molt unlikely to happen during migration, feather replacement takes place at the overwintering sites This study was conducted under permits issued by the Dutch bird ringing centre Vogeltrekstation-Arnhem.House martins were captured at a colony in the village of Gaast , The Netherlands, from June-August 2005–2010. We captured birds using mist-nets in late afternoon and early evening before nightfall, with small nets at the entrance of individual nests during daytime, by open bags in the case of birds emerging at dawn by paper-plugging of nest entrances. To minimize disturbance, we did not attempt to capture birds until their breeding season was well underway. All birds were processed  flash pyrolysis of 350±10 ug feather subsamples and keratin standards using continuous-flow isotope-ratio mass spectrometry. Measurement of the two keratin laboratory reference materials  corrected for linear instrumental drift were both accurate and precise with typical mean δ2H ± SD values of –197±0.79 ‰ (n = 5) and −54.1±0.33 ‰ (n = 5), respectively. All results are reported for non-exchangeable H expressed in the typical delta notation, in units of per mil (‰), and normalized on the Vienna Standard Mean Ocean Water – Standard Light Antarctic Precipitation (VSMOW-SLAP) standard scale.All feathers were cleaned of surface oils in 2∶1 chloroform:methanol solvent rinse and prepared for δ13C and δ15N analyses, between 0.5 and 1.0 mg of feather material was combusted online using a Eurovector 3000  elemental analyzer. The resulting CO2 and N2 was separated by Gas Chromatograph (GC) and introduced into a Nu Horizon  triple-collector isotope-ratio mass-spectrometer via an open split and compared to a pure CO2 or N2 reference gas. Stable nitrogen (15N/14N) and carbon (13C/12C) isotope ratios were expressed in delta (δ) notation, as parts per thousand (‰) deviation from the primary standards: atmospheric nitrogen and VPDB (Vienna Pee Dee Belemnite carbonate) standards, respectively. Using previously calibrated internal laboratory C and N standards (powdered keratin and gelatin), within runs, precisions for δ15N and δ13C were better than ±0.15 ‰.For δ2H, δ13C, and δ15N in feathers of adult versus juvenile house martins using a two sample t-test. Data from adult house martins were subsequently analyzed using linear mixed effects models (LME) We compared δ2H, δ13C, and δ15N) for samples collected from 2007–2010. However, the 2009–2010 overwintering period appeared climatically anomalous within Africa based on examination of climate indices ; thus we expected that samples collected in 2010 would not fit the long-term isoscapes (particularly δ2H) well. As a result, we omitted data from 42 birds for which we had data on all three isotopes from 2010. Data for δ13C and δ15N were not available for samples collected in 2005 and 2006. Thus, we used data from 171 house martins sampled during 2007–2009 in assignment of individuals to isotopic clusters’ representing isotopically distinct regions of Africa δ13C δ15N δ2H δ13C and δ15N isoscapes were converted to feather isoscapes by assuming isotopic discrimination of +2 ‰ and +5 ‰ respectively from diet to tissue δ2H isoscape of δ2Hf = −6.77+1.42 δ2H),  assigned to a given isotopic cluster across simulations. In order to further interpret our geographic assignments based upon stable isotope assays we also examined ring-recovery data for trans-Saharan house martin recoveries reported in 13C and 2H and enriched in 15N . The high HY δ15N values reflect the strong influence of the agricultural landscape of the Netherlands.As expected, all feathers grown by hatching-year birds in The Netherlands differed isotopically from adults that grew their feathers in Africa, being comparatively depleted in δ2H values  and most enriched (2005) years, respectively. Similarly, feather δ13C suggested an isotopic enrichment of approximately 1 ‰ in 2008 and 2010 relative to 2007 and 2009  feather basemap for Africa which portrayed four isotopically distinct clusters and ring recovery data that pointed toward a longitudinal band centred on Cameroon, we assigned the wintering origins of our adult house martins from the Netherlands primarily to regions in West Africa, especially central Cameroon and the southern Congo. While our approach relied on weak ring recovery data to provide constraints on longitude of wintering grounds, our identification of these areas of West Africa agreed remarkably well with non-isotopic work by http://www.ace-eco.org/vol6/iss1/art3/figure2.html). This, together with the analysis by Using a triple isotope , 2006 (n = 27), 2007 (n = 71), 2008 (n = 66), 2009 (n = 84), and 2010 (n = 51). Red ellipses represent the approximate location of putative origins for house martins breeding in Italy based on correlation between winter Normalized Difference Vegetation Index and breeding ground population indices as reported by Likelihood based assignment of house martins sampled at Gaast, The Netherlands to the (TIF)Click here for additional data file.Table S1δ2Hf, δ13C, and δ15N isoscapes falling within regions defined by cluster analysis of those isoscapes. δ2Hf represents a calibration of the predicted amount weighted growing season average isotopic composition of rainfall.Summary of the isotopic composition of cells within (DOC)Click here for additional data file.Table S2Stable isotope composition of feathers and frequency of times the individuals was assigned to the stated isotopic province of feather growth out of 1000 simulations see  for hous(DOC)Click here for additional data file.Text S12H, δ13C, δ15N)(δ African feather isoscape.Derivation of a 3-isotope (DOC)Click here for additional data file.


P<0.05), in males aged 50–55 during the second (P = 0.031) and third exercises (P = 0.028) and in males aged 45–49 during the final exercise bout (P = 0.046). Although not significantly different, 40–44 years old males also had a lower rate of heat loss compared to younger males. Over the sum of two hours, the change in body heat content was greater in males 40–70 years compared to young males . Our findings suggest that middle-aged and older adults have impairments in heat dissipation when doing physical activity in the heat, thus possibly increasing their risk of heat-related illness under such conditions.Older adults typically experience greater levels of thermal strain during physical efforts in the heat compared to young individuals. While this may be related to an age-dependent reduction in whole-body sweating, no study has clearly delineated at what age this occurs. In the present study, we report direct measurements of human heat dissipation during physical activity in the heat in males ranging in age from 20–70 years. Eighty-five males performed four 15-min bouts of cycling separated by 15-min rest periods, in a calorimeter regulated to 35°C and 20% relative humidity. Direct calorimetry was used to measure total heat loss (whole-body evaporative heat loss and dry heat exchange). We also used indirect calorimetry as a continuous measure of metabolic heat production. Body heat storage was calculated as the temporal summation of heat production and total heat loss over the experimental session. Whole-body sweat rate (WBSR) was calculated from measurements of evaporative heat loss. Males were divided into five age categories for the analysis of WBSR and body heat storage: 20–31 years (n = 18), 40–44 years (n = 15), 45–49 years (n = 15), 50–55 years (n = 21) and 56–70 years (n = 16). Relative to young males, WBSR was reduced in males aged 56–70 during each exercise (all  It is generally understood that older adults (≥60 years) experience greater thermal and/or cardiovascular strain during exposure to hot environments Engaging in exercise or physical activity is strongly advocated to reduce the risk of many adverse health outcomes that have a tendency to occur as people age The experimental procedures were approved by the University of Ottawa Health Sciences and Science Research Ethics Board. Written informed consent was obtained from all volunteers prior to their participation in the study.2peak) were determined prior to the experimental session. Measurements of body density were obtained using the hydrostatic weighing technique and were used to calculate body fat percentage 2peak. Continuous electrocardiographic monitoring was used during the maximal exercise test in males 50 years of age or above.Eighty-five males volunteered to participate in this study. All participants were healthy and physically active with no history of cardiovascular, metabolic or respiratory disease. Our participants were also not taking medication related to these conditions. Measurements of height, body mass, body density and peak oxygen uptake  separated by 15-min rest periods, for a total duration of two hours.EH) and dry heat exchange (DH):−1), 2,426 is the latent heat of vaporization of sweat (J·g sweat−1) and 1,005 is the specific heat of air [J·(kg air·°C)−1]. A full peer-reviewed technical description of the performance and calibration characteristics of the whole-body calorimeter is available M) from measurements of oxygen and carbon dioxide concentrations in a known volume of expired gas . We subsequently calculated the change in body heat content as the temporal summation of heat production and total heat loss during each exercise and recovery period, as well as over the two hour experimental session: heat storage = M−EH±DH. Evaporative heat loss was used to measure whole-body sweat rate in g·min−1 which was calculated as: evaporative heat loss (in W) multiplied by 60 s and divided by the latent heat of vaporization of sweat . Rectal temperature was measured by inserting a thermocouple probe  to a minimum of 12 cm past the anal sphincter.We used the world's only direct calorimeter (a device for making extremely accurate measurements of the heat emitted by the human body) to determine total heat loss from measures of evaporative heat loss and dry heat exchange . The rates of evaporative heat loss and dry heat exchange were measured as the difference in humidity and temperature, respectively, between the incoming and outgoing air. The following equations were used to calculate evaporative heat loss . Given that previous work showed no differences in whole-body heat loss and heat storage between young and middle-aged males (40–45 years) 2peak, body surface area and body fat percentage) using Pearson product-moment correlation coefficients. An alpha level ≤0.05 was considered statistically significant. The statistical software package SPSS 20  was used for all analyses.The values used for the analyses of whole-body sweat rate, body heat content and rectal temperature were obtained by averaging the last minute of each exercise  and recovery bout . A mixed-design analysis of variance was used to test for differences in whole-body sweat rate, body heat content and rectal temperature between age groups (five levels) and across the exercise or recovery bouts (four levels). Measurements obtained during the exercise and recovery periods were analysed separately. We also compared the net change in body heat content at the end of the two hour experimental session as well as participant characteristics using one-way analysis of variance. In a secondary analysis, the data for whole-body sweat rate  as well as change in body heat content were pooled (separately) to examine whether these two variables correlated with relevant participant characteristics  were not different between age groups. Body fat percentage was significantly greater in males in the age categories 45–49 (P = 0.028) and 56–70 years (P = 0.001) compared to young males. The values for whole-body sweat rate, body heat content and rectal temperature during the exercise and recovery periods are provided in Participant characteristics are presented in P = 0.004). In comparison to the youngest age group, whole-body sweat rate was significantly reduced in males aged 56–70 during each exercise , in males 50–55 during the second (P = 0.031) and third exercises (P = 0.028) and in males 45–49 during the final exercise bout (P = 0.046) (Whole-body sweat rate was significantly different between groups (= 0.046) . Whole-bP = 0.001). Heat storage was greater in males 56–70 years of age compared to young males during Ex2 (P = 0.012), Ex3 (P = 0.008) and Ex4 (P = 0.014). Males between the ages of 50–55 and 45–49 stored significantly more heat during Ex2 (P = 0.006 and P = 0.024 respectively) and Ex4 (P = 0.032 and P = 0.035 respectively) compared to younger males. There was a significant group difference in the change in body heat content during the recovery periods (P = 0.002). Males in the age group 40–44 had a lower negative change in body heat content  than young males during R4 (P = 0.049).As a result of reduced whole-body sweat rate, changes in body heat content also differed between groups during exercise (P = 0.001), 45–49 (P<0.001), 50–55 (P = 0.005) and 56–70 years of age (P = 0.003) relative to young males , whereby advancing age was associated with a reduction in whole-body sweating. Age was also correlated with body heat content , where advancing age was associated with a greater change in body heat content. VO2peak did not significantly correlate with whole-body sweat rate  or body heat content . Body surface area and body fat percentage both correlated with body heat content  but not with whole-body sweat rate .Age was significantly correlated with whole-body sweat rate  of repeated physical activity in the heat. Interestingly, as depicted in 2peak and/or body composition had a greater influence on heat loss capacity than “age” per se2peak (range 34.1 to 68.9 mLO2·kg LBM·min−1), body surface area (range 1.67 to 2.55 m2) or body fat percentage (range 7 to 40%). On the other hand, age was the only variable to significantly correlate with whole-body sweat rate. This would suggest that when the stimulus for sweating is equal for all participants, which we achieved by using a fixed heat load (i.e. 400 W) under fixed environmental conditions, aging may have a larger influence on whole-body heat loss capacity than the fitness level or specific physical characteristics of the individual.The present study examined thermoregulatory responses across an age continuum and our results suggest that deteriorations in heat loss capacity occur progressively from middle adulthood through to older adulthood. Previous studies have either compared thermoregulatory function during exercise in the heat between young and middle-aged adults Our laboratory is equipped with the world's only direct calorimeter, the gold standard for making extremely accurate measurements of the heat emitted by the human body. Therefore the major strength of the present study was that we were able to precisely measure whole-body heat dissipation, and consequently whole-body heat storage, in a large sample of males between the ages of 20 and 70 years. Alternatively, studies in the field of thermoregulatory physiology typically measure local sweat rate as an estimate of whole-body heat dissipation and/or core temperature as an indication of whole-body heat storage. It is important to consider that conclusions based solely on these local measurements could be misleading. For example, our results indicate that the change in rectal temperature after the two hour experimental session was very similar across age groups. Core temperature measurements however, only reflect how much heat is stored within a specific region of the body and have been shown to underestimate how much heat is stored throughout the entire body The intensity of the physical activity performed by our participants was very moderate and was done in a dry environment inside a laboratory. Thus, it is unclear if impairments in heat dissipation as a function of age would be more pronounced at higher workloads or on the other hand, if differences would occur at even lower workloads. Our participants were habitually active by nature of their occupation , but none reported being highly trained in endurance sports. Nevertheless, it is possible that some of our middle-aged and older participants were more fit than the average population, and that age-related decrements in whole-body heat loss capacity may be greater in sedentary individuals. Finally, it remains to be determined whether thermal strain would be exacerbated for middle-aged and older adults under conditions which restrict evaporative cooling, for example in more humid environmental conditions or with protective clothing.Older adults are encouraged to engage in regular physical activity as this has been shown to elicit important health benefits that contribute to healthy aging. However, with the predicted increase in the Earth's average temperature combined with the rapidly aging global population and workforce in coming years, a greater proportion of older individuals will be required to work and play in the heat. While adults over the age of 60 are generally regarded as being especially vulnerable in the heat, we showed that people as young as 40 years of age stored a greater amount of heat as a result of reduced heat dissipation in comparison to younger adults. Since middle-aged and older adults appeared to experience similar levels of thermal strain during physical activity in the heat, the present findings might alter the age at which it is recommended that adults take precautionary measures on hot days .Decrements in whole-body heat loss capacity were apparent as early as the age of 40 and declined with advancing age. We conclude that not only should older adults be cautious of the risks associated with performing physical activity when ambient air temperature rises, but middle-aged adults should also be aware that they could be more prone to heat-related illness compared to young individuals.