Insulin receptor substrate (IRS) proteins are key moderators of insulin action. Their specific regulation determines downstream protein-protein interactions and confers specificity on growth factor signalling. Regulatory mechanisms that have been identified include phosphorylation of IRS proteins on tyrosine and serine residues and ubiquitination of lysine residues. This study investigated other potential molecular mechanisms of IRS-1 regulation.in vitro of recombinant IRS-1 and HDAC2. HDAC2 bound to IRS-1 in mammalian cells treated with phorbol ester or after prolonged treatment with insulin/IGF-1 and also in the livers of ob/ob mice but not PTP1B knockout mice. Thus, the association occurs under conditions of compromised insulin signalling. We found that IRS-1 is an acetylated protein, of which the acetylation is increased by treatment of cells with Trichostatin A (TSA), an inhibitor of HDAC activity. TSA-induced increases in acetylation of IRS-1 were concomitant with increases in tyrosine phosphorylation in response to insulin. These effects were confirmed using RNA interference against HDAC2, indicating that HDAC2 specifically prevents phosphorylation of IRS-1 by the insulin receptor.Using the sos recruitment yeast two-hybrid system we found that IRS-1 and histone deacetylase 2 (HDAC2) interact in the cytoplasmic compartment of yeast cells. The interaction mapped to the C-terminus of IRS-1 and was confirmed through co-immunoprecipitation Our results show that IRS-1 is an acetylated protein, a post-translational modification that has not been previously described. Acetylation of IRS-1 is permissive for tyrosine phosphorylation and facilitates insulin-stimulated signal transduction. Specific inhibition of HDAC2 may increase insulin sensitivity in otherwise insulin resistant conditions. This leads to activation of different intracellular cascades [The insulin receptor substrate (IRS) proteins represent key elements in insulin and insulin-like growth factor (IGF) actions, transducing pleiotropic effects on cellular function and regulating processes such as metabolism, growth, cell differentiation and survival . At leascascades .The best-substantiated post-translational modification of IRS proteins, in addition to tyrosine phosphorylation, is phosphorylation of specific serine residues. Phosphorylation on these residues is associated both with inhibition of insulin-induced tyrosine phosphorylation of IRS proteins and with facilitation of the effects of insulin . PhosphoIn addition to phosphorylation of different amino acid residues, insulin signalling through IRS proteins has been shown to be regulated by at least two other mechanisms. Prolonged signal transduction via phosphoinositide 3-kinase (PI3K), which generates the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate, has been shown to induce a state of insulin resistance in cells , in partMultiple histone acetyltransferases (HATs) and histone deacetylases (HDACs) control the state of histone acetylation and hence play a regulatory role in modulating the structure and function of chromatin . About 2We now demonstrate that HDAC2 interacts with IRS-1 under conditions when the ability of cells to respond to insulin is compromised. As such, this interaction may constitute a new component of the negative regulation of IRS protein function. We also show that IRS-1 is acetylated, and that augmenting the acetylation level by treating cells with Trichostatin A (TSA, a non-specific inhibitor of HDACs) or with short inhibitory RNA oligonucleotides against HDAC2 partially restores normal responsiveness to insulin.5 cDNAs encoded the N-terminal portion of a 488 amino acid protein identified as histone deacetylase 2 , the PH-PTB domains (residues 1–578) and the PH-PTB-pre-C-terminal domains (residues 1–895). Using growth of yeast cells on selective medium as a readout for interaction between HDAC2 and IRS-1 showed that the interaction requires the C-terminal portion of the IRS-1 protein Figure . In ordes Figure  and theis Figure  lane 2 os Figure  lane 3, To validate the interaction between IRS-1 and HDAC2 further and to ascertain whether IRS-1 and HDAC2 are associated in mammalian systems, we chose to work with MCF-7 cells (a human breast adenocarcinoma cell line), with a high endogenous expression of IRS-1 . The celin vitro as described above occurs in vivo, we prepared lysates of liver tissues prepared from different mouse lines. The ob/ob mouse, which lacks functional leptin, was chosen as an insulin resistant animal model, and C57/bl6 was used as its genotype control. A PTP1B knockout mouse [in vitro data, these results showed that IRS-1 and HDAC2 are able to interact with each other in the cytoplasmic compartment of cells and that the interaction occurs under conditions of reduced insulin sensitivity, both in mammalian cells and in animals. The cytoplasmic location of the interaction is interesting in view of the fact that HDAC2 is considered to be largely a nuclear protein. In our work with cells and tissues, we have utilised lysis methods that are designed to retain nuclei intact and thereby minimise cross-contamination of compartments [To assess whether the interaction measured between IRS-1 and HDAC2 ut mouse  was usedartments ,40. Whilartments .The finding that HDAC2 binds to IRS-1 indicated that IRS-1 might be an acetylated protein in which acetylation might be a regulated post-translational modification of the protein. Indeed, the acetyl transferase Tip60 has been reported to bind to the PH domain of IRS-1 , suggestThe regulation and function of proteins such as sterol regulatory element binding protein 1c (SREBP1c)  and p53 To assess the relative roles of altered intracellular protein acetylation and binding of HDAC2 to IRS-1 on insulin signalling, we treated cells with the general HAT inhibitor, desulfo coenzyme A (DesCoA, ) and exaTo ascertain if more distal insulin signalling was also enhanced, we examined the activation of protein kinase B (PKB) by western blotting, using an antibody against PKB phosphorylated on serine 474; this phosphorylation is induced in a PI3K-sensitive manner resulting in enhanced protein kinase activity. The data showed that PMA treatment reduced the activation of PKB by 50% Figure  lane 3, A major functional response downstream of the PI3K arm of insulin signal transduction is increased glucose transport mediated by the GLUT4 transporter. We sought to examine the effects of TSA on glucose transport in rat L6 myotubes to see if the enhanced insulin signalling mediated by TSA treatment of cells translated into increased glucose uptake. We found that treatment of L6 myotubes with PMA resulted in increased basal glucose transport and had no effect on insulin-stimulated glucose transport (Kaiser & James, unpublished). Such effects are in line with data presented for rat epitrochlearis muscle  and indiTSA is an efficacious inhibitor of all class I and class II HDAC enzymes, with a potency in the low nanomolar range. To ascertain whether specific inhibition of HDAC2 activity is able to enhance insulin signalling in otherwise non-permissive conditions (PMA treatment), we used RNA interference to reduce HDAC2 activity specifically. MCF-7 cells were transiently transfected with a 21 base RNA duplex oligonucleotide against HDAC2 which reduced the HDAC2 protein content of the cells by approximately 70%  and TSA (0.1 mg/kg) and treated subcutaneously for three days. At the same time as drug injection, all food was withdrawn from the animals and 4 hours later, blood was collected from the tail vein for blood glucose and plasma insulin analysis. On the third day, an insulin tolerance test (ITT) was performed 4 hours after administration of the drug. After 24 hours, fasting blood glucose tended to be lower in treated animals than vehicle controls, but after three days no difference was evident. Furthermore, we were unable to detect a change in insulin sensitivity after drug treatment during the ITT on day 3 (Kaiser, Warpman & James, unpublished). In addition, no changes in lysine acetylation of IRS-1 were observed, indicating that the lack of effect on insulin sensitivity could be due to the inability of TSA to work through the molecular mechanism of increasing IRS-1 acetylation. TSA is rapidly metabolised by liver cells in culture in two stages, initially by reduction to the imide followed by demethylation, leading to inactive metabolites [The data we present here imply that treatment of insulin-resistant or diabetic animals with inhibitors of HDAC2 should increase insulin responsiveness. We attempted to assess the effects of TSA on insulin sensitivity in abolites . It is tabolites  has led in vitro and constitutively associates in cells to the PH-domain of IRS-1 [The mechanism whereby lysine acetylation of IRS-1 leads to increased tyrosine phosphorylation by the insulin receptor is not known. Time course experiments, in which cells were stimulated with insulin for one to ten minutes, showed that the kinetics of IRS-1 phosphorylation were the same, irrespective of pre-treatment of cells with TSA (Kaiser & James, unpublished). However, the IRS-1 tyrosine phosphorylation signal was greater at all times in cells treated with TSA, suggesting that lysine acetylation of IRS-1 simply increases the amount of phosphorylated IRS-1. It has recently been shown that lysine acetylation protects the transcription factor SREBP1C from ubiquitination and degradation via the proteasomal pathway by competing for the same lysine residues. IRS-1 has also been shown to be degraded via ubiquitination and subsequent proteasomal degradation . We inveof IRS-1 . PHIP isof IRS-1 . Consideof IRS-1 , the modin vitro and in vivo during conditions of compromised insulin signalling, as seen by reductions in insulin-stimulated IRS-1 tyrosine phosphorylation and PKB activation and increased phosphorylation of the negative regulatory phosphorylation site, serine 312. Our data indicate that it is the interaction with HDAC2 itself rather than its catalytic activity that is integral to the insulin unresponsiveness that ensues. Furthermore, our data show that IRS-1 is a lysine-acetylated protein, a previously unidentified post-translational modification of IRS-1, and that increases in the level lysine acetylation of IRS-1 result in improved insulin signal transduction. Increases in IRS-1 acetylation can be achieved pharmacologically (with TSA) or by ablation of HDAC2 specifically by use of RNAi. Out data therefore indicate that a new dimension to the physiology and pathophysiology of insulin sensitivity and insulin resistance involves changes in the degree of lysine acetylation of IRS-1 and that specific small molecule inhibitors of HDAC2 activity could represent novel therapeutics for the treatment of diseases that centre around insulin resistance, such as type 2 diabetes and obesity.In this study, we have identified a previously undescribed interaction between IRS-1 and HDAC2 in the cytosolic compartment of cells. The interaction is observed both The CytoTrap™ (Stratagene) yeast two-hybrid system was used to discover protein-protein interactions in the cytoplasm of yeast cells. Interactions were detected by recruitment to the cell membrane of the human Sos (hSos) gene product, which activates the Ras pathway. The yeast strain used (cdc25H) harbours a temperature sensitive mutation in the cdc25 gene, the yeast homologue for hSos, which means that the cells can grow at 25°C but not at 37°C unless rescued with a protein-protein interaction. A human foetal brain plasmid cDNA library (Stratagene), harboured in the pMyr vector , was used as "prey" and the sub-cloned full length IRS-1 gene in the pSos vector was used as "bait". When prey and bait proteins interact the hSos is brought into close proximity to Ras and subsequently the yeast survive and are selected by growth at 37°C. The IRS-1/HDAC2 interaction rescued growth at 37°C in this way. The corresponding pMyr yeast plasmid was isolated and co-transformed with the pSos bait construct to perform false positive tests.HDAC2 was full length cloned using RACE cDNA obtained from human heart tissue together with gene specific primers and the Advantage 2 polymerase mix (Clontech). With the purpose of mapping the interaction site of HDAC2 on IRS-1 we used the Matchmaker 3 yeast two-hybrid system (Clontech). This is a GAL4-based two-hybrid system that provides a transcriptional assay for detecting specific protein-protein interactions in yeast. Two nutritional markers and one enzymatic reporter gene were used to detect interactions. Different domains of IRS-1  were sub-cloned into a "bait" vector (pGBKT7), fused to the DNA-binding domain of GAL4. Full length HDAC2 was sub-cloned into the "prey" vector (pGADT7), fused to the activation domain of GAL4. Cell growth on medium lacking the two nutritional markers was used as a readout of the interaction between the predator and prey.in vitro, we used a coupled transcription/translation system (Promega) comprising a rabbit reticulocyte lysate solution with RNA polymerase, nucleotides, salts, a ribonucleoside inhibitor, and [35S]-methionine (Amersham Biosciences) to allow detection of translated proteins. Since the prey vector pMyr already contains a T7 promoter, this was used directly in the system. However, the bait vector pSos lacks a T7 promoter and thus the IRS-1 gene was subcloned into a T7-containing vector  to permit transcription. The individually transcribed and translated proteins were mixed and co-immunoprecipitated with anti-IRS-1 antibodies (Upstate Biotechnologies) and subsequently analysed by polyacrylamide gel electrophoresis (4–12%). The gel was dried analysed by phosphorimagery.In order to confirm the IRS-1/HDAC2 interaction 2, 0.2 mM EDTA, 0.1% NP40, 25 mM NaF, 25 mM β-glycerophosphate, 1 mM DTT, 1 mM Na-orthovanadate and protease inhibitors.The human breast adenocarcinoma cell line MCF-7 was cultured in a mixed medium of Dulbecco's Modified Eagle Medium with nutrient mixture F12 (Invitrogen) lacking phenol red with 10% Foetal Bovine Serum (Gibco). At near confluency, cells were starved of serum for 16 h and subsequently treated with IGF-1, insulin, PMA  or TSA , or combinations thereof, for different lengths of time as indicated in individual figures. Cells were harvested in hypotonic cell lysis buffer comprising 20 mM Hepes, pH 7.6, 20% glycerol, 10 mM NaCl, 1.5 mM MgClCell lysates were cleared by centrifugation at 16000 g for 10 min at 4°C, and protein content was determined using the Bradford method (BioRad). For immunoprecipitations, matched amounts of protein were incubated with primary antibody  for 2 h at 4°C followed by addition of 20μl of protein A/G agarose suspension (Santa Cruz) for 1 h at 4°C with rotating tube. After washing  and twice with isotonic buffer), beads were heated with SDS-PAGE sample buffer for 10 minutes at 70°C and proteins were resolved by 4–12% gradient SDS-PAGE. After blotting, membranes were blocked in 5% non-fat dried milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h prior to addition of the primary antibody. After incubation with secondary horseradish peroxidase-conjugated antibody, protein bands were visualised using enhanced chemiluminescence (ECL-plus detection kit, Amersham Biosciences).Antibodies used were anti-IRS-1 (Upstate, cat. no 06–248); anti-HDAC2 (Santa Cruz, cat. no. sc-9959 and sc-6296); anti-phosphotyrosine (Santa Cruz, cat. no. sc-7020); anti-acetyl lysine ; anti-ubiquitin (Santa Cruz, cat. no. sc-6085 and sc-9133); anti-phospho-serine 307 IRS-1 (Upstate, cat. no. 07–247), HRP-conjugated anti-mouse IgG (Amersham Biosciences, cat. no. NA931V); HRP-conjugated anti-goat IgG (Dako cat. no. PO449) and HRP-conjugated anti-rabbit IgG (Upstate, cat. no. 12–348).Double stranded RNA duplexes corresponding to amino acids from the C-terminal part of human HDAC2 (5'CAGCUCAGCAACCCCUGAAtt3') were annealed and transfected into human MCF-7 cells (Lipofectamine 2000 from Invitrogen was used as transfection agent): The effect of RNAi on HDAC2 expression and on insulin dependent IRS-1 tyrosine phosphorylation was measured after 48 hours. A second oligonucleotide (5'GGAGCAAAGAAAGCUAGAAtt3') was found to be non-silencing at a dose of 80 pmol, in contrast to the silencing oligonucleotide above, and was used in control experiments showing that no effect on IRS-1 phosphorylation or acetylation was observed (data not shown).ob/ob mice were obtained from Bomhultsgard, Denmark and housed according to standard procedures. C57/bl6 genotype control mice were obtained from Scanbur BK AB (Sollentuna, Sweden). PTP1B knockout animals on a balb/cJJ background were purchased from McGill University, Montreal, Canada. Balb/cJJ genotype controls were obtained from Scanbur BK AB. In our hands, balb/cJJ mice are generally a healthy mouse strain that breeds well. In side-by-side experiments, the mice are more insulin sensitive than C57/bl6 mice whilst being less insulin sensitive than the PTP1B knockout animals on the same genetic background. The animals are somewhat smaller than C57/bl6 mice and have a relatively high body fat content.Male 8-week old For compound treatment experiments and insulin tolerance tests, animals were divided into two groups: vehicle (1% DMSO sub-cutaneous injection (s.c.), n = 15 and TSA 0.1 mg/kg s.c., n = 15) and subsequently treated s.c. at 09.00 h for three days. At the same time as injection, all food was withdrawn from the animals. Four hours later, blood was collected from the tail vein for blood glucose and plasma insulin analysis. On the third day, an insulin tolerance test (ITT) was performed 4 h after administration of the drug. ITT: insulin (actrapid 0.5 U/kg) was administered i.p. Following insulin administration, blood samples were collected after 15, 30, 60, 120 and 180 min from the tail vein for glucose analysis. Animals were then sacrificed and livers were dissected and immediately frozen in liquid nitrogen and stored at -70°C. All experiments were performed in accordance with permission from the local Swedish ethics committee and the company Pharmacology ethics review team.For western blotting of liver proteins, frozen liver was powdered finely under liquid nitrogen using a pestle and mortar pre-cooled to -70°C. Powdered liver (1 g) was homogenized at 4°C using a Polytron in 3 ml of homogenisation buffer (4 mM EDTA, 50 mM NaF pH 8.0, 1 mM Na-orthovanadate, 1μM okadaic acid, 0.1% (v/v) 2-mercaptoethanol, with protease inhibitor cocktail). The homogenates were centrifuged at 13000 × g for 10 minutes at 4°C and the supernatant removed and used immediately for Western blot analysis or snap frozen in aliquots at -70°C until needed.DesCoA: desulfo coenzyme AHAT: histone acetyl transferaseHDAC: histone deacetylaseIGF: insulin-like growth factorIRS: insulin receptor substratePH: pleckstrin homologyPKB: protein kinase BPI3K: phosphoinositide 3-kinasePMA: Phorbol myristic acidPTB: phosphotyrosine binding domainTSA: Trichostatin ACK carried out all of the experimental procedures reported in this study and drafted the manuscript. SRJ conceived of the study, participated in the design of all the experiments and coordinated the study.


Cumulative trauma disorders (CTDs) are common in musicians and their prevalence has been the subject of a number of studies in most western countries. Such studies are scarce in developing countries despite the possibility that CTDs may have a different prevalence in these countries, especially when considering traditional musical instruments and different methods of playing. Although not formally studied before, according to our experience the prevalence of CTDs seemed to be high among Iranian instrumentalists.We proposed this study to determine the prevalence of CTDs in amateur music students playing one of the two traditional Iranian instruments: Daf and Setar.In a prospective cross sectional study, we interviewed and examined the students of three music training centers in Iran. Seventy eight instrumentalists, who were playing Daf or Setar and twelve students who had not started playing yet were regarded as case and control groups respectively. Some of them also underwent electrodiagnostic studies.Forty-seven percent (17 of 36) of the Setar players and 57% (24 of 42) of the Daf players and fifty-three percent (41 of 78) of the instrumentalists as a whole had CTDs. None of them had carpal tunnel syndrome.Our study revealed that the prevalence of CTDs in Iranian instrumentalists was unusually high. In addition to age, other variables may be contributory. This needs to be further studied. Cumulative trauma disorders, also called repetitive stress injuries, overuse syndromes or repetitive motion injuries -3 are coTo date no study has been performed about the prevalence of cumulative trauma disorders (CTDs) in players of Iranian instruments. According to our experience, it seemed to be unusually high when compared with related prevalence in nonprofessional players of classical instruments as reported by Fry .This study was performed to determine the prevalence of cumulative trauma disorders in amateur music students playing two traditional Iranian instruments: Daf and Setar.Daf is a percussion musical instrument that has a circular wooden frame covered with goat skin with or without metal discs around its edge. To play Daf the player shakes it and hits it with both hands fig. .Setar is a string musical instrument that has 4 strings. It is played by the index of right hand fig. .In comparison to classic musical instruments, Setar resembles the Guitar, but Daf doesn't have any similar equivalent.In a prospective cross sectional study, we interviewed and examined the students of three music training centers, numbering 94. Twelve students who were at their first sessions and hadn't begun to play were selected as control group.Age, sex and duration of playing (date of starting and daily playing time) as well as vocational and avocational risk factors for developing CTDs were recorded after a direct interview. Then the students were referred to a physician who did not know whether the student belonged to the case or control group. He then evaluated their upper limbs and necks. Specific attention was paid to pain, paresthesia, sensory changes, tenderness, range of motion, muscle power and muscle stretch reflexes. In addition, Phalen, Tinel and carpal compression tests  were perSince the standard diagnostic test for CTS is electrodiagnostic study , all of The data were analyzed by SPSS software using Chi square and Fisher's exact tests.Ninety four students were included in this study. Four students were excluded from the study, two because of a history of musculoskeletal pain before attending the music center, one because of playing two instruments and one, serving as a typist.Twelve of the students who had not started to play were assigned to the control group and the remaining 78 students; 42 in Daf and 36 in Setar groups; were considered as case group table .Mean age of students in the case group was 21.2 years (SD: 3.8) including 47 females and 31 males. Mean duration of instrument playing in this group was 7.9 months (SD: 5.4).Mean age of students in control group was 25.2 years (SD: 9.2). This group consisted of 9 females and 3 males.Mean Duration of daily playing in Setar students was more than Daf students (1.6 Vs 1.5 hours) which was not statistically significant .Mean duration of daily playing in male and female students was 1.8 hours and 1.4 hours respectively. Which was not statistically significant .Forty-one students in case group  had musculoskeletal pain and there was a significant correlation between playing Daf and Setar and development of musculoskeletal symptoms.The prevalence of pain among females was twice as much as males but the difference was not statistically significant  ; again, this difference was not significant table .Regarding the location of pain, hand was the most common site; it was painful in 65% of cases table .Twenty six students, all from the case group attended our electrodiagnostic center, none of them had carpal tunnel syndrome.None of the students in control group had problems in their exams and none of them attended for electrodiagnostic studies.A large number of amateur Daf and Setar players with a history of playing of less than 1 year (7.9 months) and almost 1.5 hours a day had musculoskeletal pain (that is considered a form of CTDs). The prevalence of pain in this group was much greater than students in tertiary music schools who train for some years for 6 hours a day (53% vs. 9.3–21%, respectively) and almost equals professional orchestra players (73–75%) [In a group of instrumentalists (Guitarists, Harpists, Pianists etc.) Bejjani et al found a 77.5% prevalence of upper extremity disorders serious enough to impair the performance or to cause the musician to stop playing at least temporarily . similarHow can we explain this high prevalence?The most important cause of CTDs is repetitive motions and in fact multiplication of duration and intensity of exercise . Since bAccording to Fry , other iThe particular instrument has been shown to be a risk factor for developing CTDs ,14. On tAnother risk factor is age . It has This hypothesis can explain, at least in part, the wide range (9–49%) -12 of thAs mentioned, we did not find any case of symptomatic carpal tunnel syndrome or nerve conduction abnormalities suggesting subclinical median neuropathy at the wrist, implying that CTS is a more advanced form of cumulative trauma disorders when compared to musculotendinous unit CTDs.Hand was the most common painful site in this study (65%); a finding which is in concert with other published studies 41–54 %)[1–54 %4,.There are two other findings in the current study left to be explained:First: Daf is played being held using both hands but Setar is being held like Guitar. So it can be postulated that playing Daf is more harmful than Setar and we expected more CTDs in Daf players. Although, the prevalence of CTDs was higher in Daf players, the difference was not significant . A significant difference may be found with a large scale study.Second: it has been known that CTDs are more common in females ,12,13. IOur study revealed that the prevalence of CTDs in Iranian instrumentalists was abnormally high. This is an unusual finding that can't be fully explained by the difference in the instruments , playing method or intensity of the exercise. Other susceptibility factors such as age at the starting of playing or genetic predisposition may be contributory. Larger studies focusing on individual characteristics and genetic analyses are needed to delineate other important factors.The authors declare that they have no competing interests.CTD: cumulative trauma disordersCTS: carpal tunnel syndromeSS: suggesting the proposal, examining the volunteers, writing the paper.BK & SMJS: examining the volunteersAB: great help in writing the paper and statistical analysisPJ: statistical analysisThe pre-publication history for this paper can be accessed here:


We use ancient DNA to compare two independent estimates of population size  and corroborate our results with gene diversity and serial coalescent simulations. Our data and analyses indicate that, with population size decreasing at times of climatic change, some species will exhibit declining gene diversity as expected from simple population genetic models, whereas others will not. While our results could be consistent with selection, independent lines of evidence implicate differences in gene flow, which depends on the life history strategy of species.Understanding how climatic change impacts biological diversity is critical to conservation. Yet despite demonstrated effects of climatic perturbation on geographic ranges and population persistence, surprisingly little is known of the genetic response of species. Even less is known over ecologically long time scales pertinent to understanding the interplay between microevolution and environmental change. Here, we present a study of population variation by directly tracking genetic change and population size in two geographically widespread mammal species  A novel analysis of paleoclimatic data, fossil abundance and contemporary and ancient DNA from different time periods introduces "phylochronology" and reveals how species respond genetically to climate change Phylogeography has advanced our understanding of the spatial distribution of genetic diversity within and between species . HoweverOur study takes advantage of a continuous, well-sampled mammalian fossil sequence spanning the last 3,000 years . Lamar Cave has an extraordinarily complete representation of the local species in the vicinity, with over 10,000 identified mammalian specimens representing over 80% of the mammal species in the local habitat . Late-HoMicrotus montanus (montane vole) and Thomomys talpoides (northern pocket gopher), species that presently are widespread in mountain habitats of western North America. Due to their preferences for wetter habitats, both responded demographically by increasing in relative abundance during wetter climates and declining during warmer climates. M. montanus showed an increase in abundance relative to other common rodents during periods of wet, cool climate in Yellowstone  techniques allows us to investigate directly the impacts of these environmental perturbations on neutral genetic diversity concurrent with these species population responses. We obtained ancient and modern mitochondrial DNA sequences from M. montanus and used previously published data for T. talpoides. Although these two species are broadly similar in body size  and are principally herbivorous, they differ in their natural history. T. talpoides is characterized by low population densities (1–62 gophers/hectare [ha]), a fossorial mode of life, maximum dispersal distances of a few hundred meters, and fiercely territorial behavior  despite the absence of obvious migration barriers.Although the population responses of behavior . Populatcalities . This spM. montanus achieves higher average population densities (60–186 voles/ha)  have found little evidence for population subdivision over the scale of hundreds of kilometers   than T. lometers , as expelometers . Such deplotypes .The primary advantage of a phylochronologic approach as opposed to a single time slice for understanding mammalian response is the ability to reveal changes in genetic variation through time. This is in contrast to modern genetic studies that seek to reconstruct demographic history based on inferences from past climate or geologic records e.g., . Our stuM. montanus and T. talpoides fossil specimens and modern population densities. (2) We calculated gene diversity over this 3,000-year period to determine the impact of environmental perturbations on genetic effective population size and gene diversity in M. montanus and T. talpoides. Unlike previous aDNA work  might influence the record of gene diversity over time. Thus, we combined four methods of estimating population size, determining statistical significance, and assessing gene diversity through time. (1) We derived independent ecological estimates of population size through time from abundances of DNA work , we usedDNA work . Our appT. talpoides, we predicted that changes in genetic variation through time would be dominated by drift, as suggested by the modern life history characteristics of small effective population size, low dispersal, and high amounts of population substructure. Therefore, as the ecological effective population size of T. talpoides declined with warmer climates, we expected genetic variation to decline. For M. montanus, we predicted that changes in genetic variation through time may be influenced more by migration, as suggested by large effective population sizes, high rates of dispersal, and low amounts of population substructure. As a result, past declines in ecological estimates of population size of M. montanus would not necessarily have resulted in a decrease in genetic variation.Despite similar population-level responses to climatic change of the late Holocene, we expected differences in gene diversity change for the two species. For e_ecol(N >> e_gen)N  . Howeveralpoides A and M. montanus B. For M.M. montanus suggests that the sequences we obtained for this study are target mtDNA. Of the 312 bp we sequenced for M. montanus, 96.5% of all the mutations were third-position codon changes, with first- and second-position mutations accounting for 3.5% and 0%, respectively. These ratios of variation are concordant with expectations for within-species variation and small overall sequence divergences . Most of the mutations are synonymous (97.7%); the transition-to-transversion ratio of the entire data set was 4.1 to 1, which is consistent with expectations for mammalian cytochrome b and evolution in other Microtus species  and translated successfully. Together with the frequency distribution of our pairwise differences, the prevalence of silent and third-position codon changes, and the standard of obtaining both forward and reverse fragments of overlapping sequence regions, these data permit us to conclude that the genetic diversity we have sampled represents authentic mitochondrial population variation and is unlikely to be from nuclear copies or pseudogenes.The genetic evidence we have assembled from ergences . Nucleot species , with an species . Fossil R2 = 0.004, not significant). All but one (out of 121) of the extraction controls were negative. When sequenced, this extraction blank BLASTed similar to Montanus townsendii, a taxon we had never worked on in the facility; this sequence has not since been amplified in the lab, and that extraction was not used further. Out of 87 successfully amplified samples and one sequence obtained from GenBank (AF119280), we identified 17 haplotypes within four haplogroups (A–D) of M. montanus  fall within haplogroups A and D, with 84% of the samples within one substitution of the locally ancestral haplotype A  and a deficit of guanine (11.4%). All mutations were synonymous; the transition-to-transversion ratio of the entire data set was 4.0 to 1.Protocols for M. montanus and T. talpoides T. talpoides (N exhibit the same trend as the genetic estimates e-tt_gen),(N are lower than those derived from genetic data e-mm_gen) for all time points except for 2,525 ybp, where observed gene diversity was significantly lower than could have been calculated given our sample size (n = 4), suggesting that we do not have sufficient power to detect processes at this time period.For ple size . Since tT. talpoides is not different given expectations from a closed population, we attempted to determine the statistical limitations of these data. Investigation of the effects of sampling for T. talpoides revealed that given the smaller number of base pairs (64 bp), we do not have enough statistical power to reject the null hypothesis. For every sampling time point, the Ewens distribution predicted that only one haplotype would be present in the genetic samples (unpublished data). As a result, the predicted gene diversity was zero for all time points. Because the observed gene diversity for T. talpoides was higher than predicted we also simulated five combinations of mutation rate and abundance, which could result in a higher predicted diversity  for 166 ybp (n = 34). Results for all five combinations predicted presence of a single haplotype. Since we do not have adequate statistical power given the genetic data for T. talpoides, we did not conduct significance tests for this species. However, the observed values of gene diversity are not unexpected from dynamics within a closed population.Although the gene diversity for M. montanus gene diversity throughout the entire time series (2,525 to 166 ybp)  based on the observed change in 166 ybp) . The expM. montanus prior to 2,525 ybp. We modeled population reduction to 104,577 (0.75 × e-mm_ecol2525ybpN), 69,718 (0.5 × e-mm_ecol2525ybpN), and 34,859 (0.25 × e-mm_ecol2525ybpN) prior to 2,525 ybp. For a 75% bottleneck prior to 2,525 ybp, we were no longer able to reject the null hypothesis of a closed population. These results indicate that an extreme bottleneck where population size was reduced to 75% or more might result in the observed change in gene diversity. Our simulations reveal that unless an extreme bottleneck happened prior to 2,525 ybp, we can be confident that the observed change in gene diversity is not likely to be from events that occurred immediately prior to our historic data, and thus is due to migration or selection.Our results may depend on our assumptions of equilibrium population size prior to 2,525 ybp. We investigated sensitivity to this assumption by modeling a population bottleneck in M. montanus. Historic genetic data allow us to discriminate between population processes over millennia much better than do modern data alone.Coalescent simulations used to investigate the significance of the observed gene diversity value for the modern samples demonstrated that the null hypothesis of past population size change could be rejected at only two of the nine mutation rate and density combinations. These results reveal that given data from only the modern samples, it was not possible to reject the null hypothesis of past population size change in M. montanus populations. Estimates of β-diversity (used here to measure haplotypic turnover) from haplotypic data reveal that turnover was highest between 2,525 and 845 ybp . Closer examination of the haplotypic distributions demonstrates that five novel haplotypes appeared by 845 ybp, three of which are ≥3.2% different from the most common haplogroup (A)  A at 2,52T. talpoides and M. montanus between 1,438 and 470 ybp, a period spanning the Medieval Warm Period  results in an increase in gene diversity. We attempted to statistically validate our results by the use of serial coalescent simulations to demonstrate that the change in gene diversity of M. montanus between 2,525 and 845 ybp is significantly different from that expected based on the decrease in ecological estimates of population size. Taken together, these results indicate a departure from conditions of equilibrium (closed population without selection) for M. montanus. Our results have the following possible explanations: (1) the sampling area for fossils changed, (2) the local population size expanded, (3) selection occurred, and/or (4) gene flow occurred.Our results demonstrate different genetic responses by two species of small mammals to changes in population size driven by climatic change. Fossil abundance data reveal population decline for both m Period . For T. Microtus oeconomus, indicate that migration occurs most frequently in and between low-density patches  and between populations (gene flow). Discrepancy between the ecological and genetic estimates of population size and significant changes in haplotypic diversity prior to the Medieval Warm Period implicate increased gene flow into the Lamar Cave M. montanus population. Additionally, the observed haplotypic turnover in the Yellowstone population during this period suggests that as abundance of M. montanus declined through the last 845 years, relatively more individuals carried newly introduced haplotypes.Here we demonstrate, using a phylochronologic approach, that it is possible to distinguish the dynamic processes that govern gene diversity over relatively short time scales (hundreds to thousands of years). We have documented environmental change, population response, genetic diversity change, and the correlations between the three. Without serial data, we would capture just a single record of these historic processes: modern genetic diversity. Although it is possible to hypothesize about historic events using modern data, phylochronology affords a unique look into the past and the potential ability to separate cause from effect. In particular, we show that M. montanus was maintained. In contrast, gene flow has not played a significant role in the recent genetic history of T. talpoides. This species, instead, responded more as a closed population over this time. The disparate nature of population response to climatic change of these two species is likely due to differences in demographic dispersal patterns between their populations. Such differences in species demography have resulted in differential genetic response to climatic change, even when ecological response is similar. Thus, genetic response to environmental change can be viewed as “individualistic,” similar to unique adjustments of species ranges  declined while others expanded. Our data show that even with a prolonged ecological population size decline, the genetic diversity of s ranges . Life hin = 8,589]) and are concordant with expectations of taxonomic diversity in montane mammal communities of western North America .Lamar Cave contains well-stratified, thoroughly radiocarbon-dated deposits, which display high fidelity to the local mammalian community , 1999. T America . The cumMicrotus samples encompass 15 of 16 radiocarbon-dated stratigraphic levels from Lamar Cave N and e-mm_ecol (M. montanus).NIn order to compare ecological estimates of effective population size to genetic estimates, we estimated mitochondrial effective population size assuming that Microtus species are cryptic with respect to these teeth. Genetic diagnosis indicated that these 78 fossil samples are derived from multiple arvicoline species, with a total of 47 M. montanus specimens (see Microtus molariform teeth) ranged from 3.5 to 15.3 mg (average, 8.8 mg). We used two previously described extraction methods on the fossil teeth (b from ancient samples (n = 47) using the following primers: forward primers (5′–3′), CLETH 37 TAY AAY ATA ATY GAA ACH TGA A , CLETH 37L AYG GMT CTT AYA ACA TAA TCG AAA CAT G (cyt b 311, M. musculus 14450), MMONT 1 CAG TAA TTA CAA AYC TWC TAT CA (cyt b 452, M. musculus 14591), and MMONT 3 AGT GAA TCT GAG GGG GCT TCT CAG TAG A (cyt b 485, M. musculus 14621); reverse primers (5′–3′), ARVIC 08 CAG ATY CAY TCY ACT AGT GTT G (cyt b 473, M. musculus 14612), ARVIC 08L CTC AGA TTC ACT CTA CTA GTG TTG TG (cyt b 471, M. musculus 14610), MMONT 4 TTR TTT GAT CCT GTT TCG TGT AGG AAT A (cyt b 595, M. musculus 14631), and MMONT 2L TTG ACT GTG TAG TAA GGG TGA AAT GGG A (cyt b 653, M. musculus 14792). Attempts were made to amplify the region in two overlapping fragments using CLETH37/ARVIC 08, CLETH37L/ARVIC 08L, and MMONT 1/MMONT 2L. However, the low rate of success for MMONT 1/MMONT 2L (40%) in the fossil samples necessitated the breaking of the second fragment into two overlapping fragments using MMONT 1/MMONT 4 and MMONT 3/MMONT 2L.From the fossil material, we used the upper first molar (from only one side of the jaw per level) to avoid sampling the same individual multiple times. Some mens see . Fossil il teeth . We obtaAncient DNA samples were run on an ABI PRISM 310 Genetic Analyzer in the post-PCR lab, and modern DNA samples were run on an ABI PRISM 377 Sequencer in a separate sequencing facility . Fragments were sequenced in both directions, primer regions were overlapped, and sequences with any ambiguous sites were rerun until completely resolved in order to provide additional corroboration and eliminate ambiguity.We adhered to strict extraction and amplification protocols . The pro and 12 teeth  from the vicinity (within 10 km) of the fossil site. In addition, DNA was successfully extracted from 51 modern specimens (liver tissue and museum skins) collected within a 400-km radius of Lamar Cave. Of these 51 samples, 22 were derived from museum skins  and 29 from liver tissue  of specimens trapped in the field. We followed the animal tissue protocol using the Qiagen Dneasy Tissue Kit  on 6.5 mg of liver samples and 7.5 mg of tooth samples (n = 3). For museum samples, DNA was extracted from the ventral skin incision . We amplified the entire cytochrome b gene (1,143 bp) for some modern skins (n = 13) and liver tissue (n = 29) with the following primers: forward primers (5′–3′), MVZ 05 CGA AGC TTG ATA TGA AAA ACC ATC GTT (cyt b −51, M. musculus 14088) and ARVIC 07 AAA GCC ACC CTC ACA CGA TT (cyt b 514, M. musculus 14653); reverse primers (5′–3′) MICRO 06 GGA TTA TTT GAT CCT GTT TCG T (cyt b 602, M. musculus 14741) and VOLE 14 TTT CAT TAC TGG TTT ACA AGA C (cyt b 1170, M. musculus 15309).Modern  genetic sampling was obtained from a variety of sources including modern skins, modern liver tissue, museum skins, and teeth derived from modern raptor pellets. DNA was successfully extracted from 16 modern skins Microtus samples, and 79 of those yielded successful amplification. Of these 79 samples, 48 were positively identified as M. montanus. Of the M. montanus samples, 41 specimens were successfully haplotyped. Sequences have been deposited in GenBank  , which is not found elsewhere in a 400-km radius around Lamar Cave. This constancy in the genetic lineage of T. talpoides within a single locality persists in spite of climatic changes and concurrent significant population and body size changes (We have built upon the previously published data set  with add= 3) see . T. talp changes .e) was used to estimate e. θSN was preferred over other estimators of θ since Hθ is biased for single locus estimates, kθ does not incorporate sequence information, and πθ has higher variance. These estimates are determined entirely from genetic data; thus we label them e-tt_gen N and e-mm_gen (M. montanus).N We assumed a range of μ (2% [low], 4% [moderate], or 10% [high] per million years per bp)  using Cody's index (β-diversity each haplotype was treated as a “species.”Arlequin v. 2.000  was used per bp) . Additio's index . For β-dN, a gene with mutation rate μ, and an infinite alleles model given a sample size n. Here we use the Ewens distribution to ascertain the ability of our data to detect variation in values of gene diversity. In addition, this distribution allows us to (1) predict the distribution of expected gene diversity at each point in time (independently) given estimates of e_ecol,N a moderate mutation rate of 4% per million years, typed sequence length , and sample size  and (2) predict change in the expected gene diversity distribution for a large sample size of 100. A modified version of MONTE CARLO ((θ, K, and n), and gene diversity was calculated for each configuration. Simulations were repeated 1,000 times for each sampling time point, resulting in the 95% confidence intervals for the distribution of predicted gene diversities given a closed, selectively neutral population through time.Given the general limitations of obtaining aDNA sequence data, sample sizes will always present challenges to ancient population genetic studies. To investigate whether our samples are adequate for addressing temporal gene diversity in both species, we used a neutral population model to evaluate expected diversity. TE CARLO , 1996 waM. montanus evolution during the past 2,525 years. Simulations were repeated 1,000 times to generate genetic data and changes in gene diversity for the null hypothesis. The significance of the observed changes was then inferred by comparing it to the generated null values (we thus used a Monte Carlo significance test). Estimates of e_ecolN were used to set up a null hypothesis specifying population size change through time in a closed population. We assumed that past changes in population size between intervals were due to exponential growth or decline. The effective population size at two time points and the time between the points was used to calculate a growth rate. The estimated growth rates were 1438–2525ybpr = −0.000178, 845–1438ybpr = 0.000732, 470–845ybpr = 0.0004385, and 166–470ybpr = 0.0003212. The null hypothesis thus corresponds to effective population sizes at particular points in the past and exponential growth or decline between those intervals, and represents an ecologically realistic description of the past 2,525 years. We assumed a constant population size prior to 2,525 ybp, as we have no data before this point in time. The coalescent program SIMCOAL , we investigated the significance of observed changes in gene diversity at all nine parameter combinations over the entire time span (2,525 to 166 ybp) and between 2,525 and 845 ybp .We assessed the significance of the observed changes in gene diversity between time points using simulations. The serial coalescent was used as a framework to model  SIMCOAL  was modialescent  has beenAdditionally, we also investigated the relevance of temporal data to our ability to reject the null hypothesis. Significance tests were repeated assuming the observed data were from only the most recent genetic samples. Again, simulations were repeated for all nine combinations of mutation rate and density estimates.M. montanus specimens described in Sequences for the successfully haplotyped


CLN3 mutation. The resulting loss of battenin activity leads to deposition of mitochondrial ATP synthase, subunit c and a specific loss of CNS neurons. We previously generated Cln3Δex7/8 knock-in mice, which replicate the common JNCL mutation, express mutant battenin and display JNCL-like pathology.JNCL is a recessively inherited, childhood-onset neurodegenerative disease most-commonly caused by a ~1 kb Cln3 wild-type, heterozygous, and homozygous neuronal precursor cell lines, which can be differentiated into MAP-2 and NeuN-positive, neuron-like cells. Homozygous CbCln3Δex7/8 precursor cells express low levels of mutant battenin and, when aged at confluency, accumulate ATPase subunit c. Recessive phenotypes are also observed at sub-confluent growth; cathepsin D transport and processing are altered, although enzyme activity is not significantly affected, lysosomal size and distribution are altered, and endocytosis is reduced. In addition, mitochondria are abnormally elongated, cellular ATP levels are decreased, and survival following oxidative stress is reduced.To elucidate the consequences of the common JNCL mutation in neuronal cells, we used P4 knock-in mouse cerebella to establish conditionally immortalized CbThese findings reveal that battenin is required for intracellular membrane trafficking and mitochondrial function. Moreover, these deficiencies are likely to be early events in the JNCL disease process and may particularly impact neuronal survival. CLN3 gene, which eliminates exons 7 and 8 and surrounding intronic DNA, predicting a non-functional protein product [Juvenile neuronal ceroid lipofuscinosis (JNCL), or Batten disease, is a recessively inherited childhood-onset neurodegenerative disorder characterized by progressive blindness, seizures, motor and cognitive decline, and early death . The pri product .The pathological hallmark of JNCL is autofluorescent ceroid lipofuscin deposits within autolysosomes that are enriched in subunit c of the mitochondrial ATP synthase complex -5. RemarCLN3-encoded protein  is a highly conserved, ubiquitously expressed, multi-pass membrane protein [btn1, the yeast CLN3 ortholog, have implicated battenin in lysosomal pH homeostasis and amino acid transport [The  protein  that loc protein -9. Batteransport ,11.Cln3Δex7/8 knock-in mice harbor the ~1 kb common JNCL mutation and express a non-truncated mutant battenin isoform that is detectable with antibodies recognizing C-terminal epitopes. Homozygous Cln3Δex7/8 knock-in mice exhibit a progressive JNCL-like disease, with perinatal onset of subunit c deposition in many cell types and later onset of neuronal dysfunction and behavioral deficits [To explore JNCL pathogenesis and battenin function, we previously generated a genetically precise JNCL mouse model. deficits . These fCln3Δex7/8 knock-in mice. Homozygous CbCln3Δex7/8 cells exhibit pathological hallmarks of the disease, and a survey of membrane organelles revealed membrane trafficking defects and mitochondrial dysfunction in homozygous mutant CbCln3Δex7/8 cells.Currently, there is no suitable neuronal cell system to investigate the impact of the common JNCL mutation on biological processes. Therefore, we have established cerebellar neuronal precursor cell lines from Cln3Δex7/8 knock-in mice, and wild-type littermates. Primary cell cultures enriched for granule neurons were transduced with retroviral vector bearing a selection cassette and temperature-sensitive tsA58 SV40 large T antigen. Growth in G418 containing medium at the permissive temperature (33°C) allowed for selection and isolation of multiple clonal nestin-positive  cerebella of homozygous and heterozygous ive Fig. , and GFAive Fig. , cell liive Fig. , but notive Fig. .Cln3Δex7/8 cells were first examined for JNCL-like characteristics. Homozygous Cln3Δex7/8 knock-in mice express multiple Cln3 mRNA splice variants and mutant battenin protein that is detectable by batp1 antibody recognizing C-terminal epitopes [Cln3Δex7/8 cells, RT-PCR and anti-battenin (batp1) immunostaining were performed. As shown in Figure Cln3 mRNA isoforms in wild-type and homozygous cells were similar to those observed in total RNA isolated from wild-type or homozygous Cln3Δex7/8 knock-in brain, respectively  and the late endosomal marker, Rab7 .Homozygous Cb epitopes . To asseely Fig. . In addiCln3Δex7/8 cells displayed autofluorescence or subunit c inclusion formation (data not shown). However, when cells were aged at confluency (3+ days post-confluency), homozygous CbCln3Δex7/8 cellular subunit c levels were elevated beyond normal wild-type levels by immunostaining .We next investigated the basis for subunit c accumulation, testing the hypothesis that cathepsin D is abnormal since it is required for ATP synthase subunit c degradation in the lysosome . We firsome Fig. . Howeverrms Fig. . Cathepsin vitro assay, cathepsin D activity in total cellular extracts was not significantly altered in homozygous CbCln3Δex7/8 cells , versus wild-type cells , although a consistent trend towards increased enzymatic activity in mutant cells was observed. Thus, cathepsin D transport and processing are disrupted in homozygous CbCln3Δex7/8 cells in a manner such that enzymatic activity appears to be relatively unaffected.The impact of the altered cathepsin D processing on enzymatic activity was next tested to determine if altered enzymatic activity accounts for inefficient subunit c turnover. In a fluorogenic Cln3Δex7/8 cells; therefore, we surveyed the subcellular distribution and morphology of membrane organelles. Components of the secretory pathway, including the ER, cis-Golgi, and trans-Golgi, did not appear altered from wild-type appearance when labeled with the respective markers, protein disulfide isomerase (PDI), GM130, and VVL (data not shown). By contrast, the lysosomal markers, Lysotracker and Lamp 2 had significantly altered signal in homozygous CbCln3Δex7/8 cells, versus wild-type cells. Wild-type cells exhibited brightly stained lysosomes that were large and clustered in the perinuclear region whereas homozygous CbCln3Δex7/8 lysosomes were lightly stained, smaller vesicles that were more diffusely scattered in the cytoplasm of the cell . It is noteworthy that Lysotracker dye, which selectively accumulates in acidic compartments, exhibited the most marked reduction in lysosomal labeling. This observation may reflect altered lysosomal pH, an established finding in JNCL [The abnormal transport and processing of cathepsin D suggested membrane trafficking disruptions in homozygous Cbell Fig. . Lamp 1  in JNCL ,15.Cln3Δex7/8 cells, as measured by dextran-FITC uptake  signal observed by immunostaining Fig. , fluid-pCln3Δex7/8 cell mitochondrial morphology and function. Mitochondrial distribution in homozygous CbCln3Δex7/8 cells was indistinguishable from wild-type and heterozygous cells; however, homozygous CbCln3Δex7/8 mitochondria appeared more elongated by grp75 marker immunostaining and TEM analysis . Moreover, compared to wild-type or heterozygous cells, homozygous CbCln3Δex7/8 cells had significantly reduced cellular ATP levels , Lamp 1 (clone 1D4B, 6 μg/ml), and Lamp 2  monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank, maintained by The University of Iowa, Department of Biological Sciences. Batp1 (1 μg/ml) was previously described . Anti-suCln3Δex7/8 knock-in mice have been previously described [2, in granule neuron growth media . Infection was performed the following day with defective retrovirus transducing the temperature-sensitive tsA58/U19 large T antigen and a selection neomycin-resistance cassette [Cln3 cell lines. CbCln3 cell lines were maintained on 0.01% poly-ornithine coated dishes at 30–90% confluency, in 33°C and 5% CO2 atmosphere. Passage number was recorded (up to ~20 passages), but had no apparent impact on phenotype. Neuronal differentiation was as previously described [escribed . Littermescribed . Postnatcassette , as prevcassette . Followiescribed  with theCln3Δex7/8 knock-in allele PCR genotyping was with wild-type primers, WtF (5'-CAGCATCTCCTCAGGGCTA-3') and WtR (5'-CCAACATAGAAAGTAGGGTGTGC-3') to yield a ~250 bp band and knock-in primers, 552F (5'-GAGCTTTGTTCTGGTTGCCTTC-3') and Ex9RA (5'-GCAGTCTCTGCCTCGTTTTCT-3') to yield a ~500 bp band. PCR cycling conditions were 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 35 seconds, repeated for 34 cycles. Total RNA isolation and Cln3 RT-PCR primers and procedures have been previously described [Genomic DNA was extracted from tail biopsies and cell pellets as described . escribed .4 cells per well for microscopy studies, or into 100 mm dishes  at a density of 5 × 105 cells per dish for protein extraction. Cells were typically >95% confluent one day post-plating, and the following day was considered 1-day post-confluency. At the indicated times, cells were either fixed with 4% formaldehyde in phosphate buffered saline (PBS), pH 7.4, for 20 minutes and processed for autofluorescence/subunit c immunostaining, or cell pellets were collected for total protein extraction.Cells were seeded into 4-well chamber-slides  at a density of 5 × 10Alternatively, cells were fixed with 2.5% glutaraldehyde/2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4 for 1 hour and subsequently post-fixed and processed for TEM analysis as described . In conf4 cells per well in 4-well chamber-slides and grown overnight at 33°C, unless indicated otherwise. Fixation was with ice-cold 4% formaldehyde in PBS, pH 7.4, for 20 minutes, or with ice-cold methanol/acetone (1:1) for 10 minutes at -20°C followed by air-drying (antibody-dependent). Cells were washed with PBS at least 2 times, 5 minutes per wash, between each of the following steps of the staining procedure: 0.1 M glycine in PBS for 5 minutes, 0.05% or 0.1% (antibody-dependent) Triton X-100 (Fisher Scientific) in PBS for 5 minutes, 2% bovine serum albumin (BSA) in PBS for 30 minutes, primary antibody diluted in 2% BSA/PBS for 90 minutes, secondary antibody diluted in 2% BSA/PBS for 60 minutes. All incubations were carried out at room temperature. Following staining procedures, slides were coverslipped with Vectashield mounting medium (Vector Laboratories) and analyzed on a BioRad Radiance 2100 confocal microscope (Biorad), with identical exposure settings for wild-type and mutant like images. All comparisons of wild-type and mutant staining were performed in Adobe Photoshop with identical brightness and contrast adjustments.For immunostaining, cells were seeded at a density of 3–5 × 10c Protein Assay) was separated by SDS-PAGE, for subsequent immunoblotting, as described [Total proteins were isolated from cell pellets by extraction with ice-cold 20 mM Tris, pH 7.4, 1% Triton X-100 (membrane-research grade, Roche), plus protease inhibitors (Complete mini tablet, 0.7 μg/ml pepstatin A, 2 μg/ml aprotinin, 5 μg/ml leupeptin [Roche]). Following homogenization through a 25-gauge needle (~10 passes), extracts were centrifuged at 1000 × g for 10 minutes, at 4°C, to remove debris. Typically, 20–40 μg of protein  according to the manufacturer's recommendations. Relative fluorescence was measured using an Analyst AD plate reader (Molecular Devices) with the following filters and settings: excitation filter, 360-35; emission filter, 400-20, Flash lamp with 100 readings/well, 100 ms between readings, and 100,000 μs integration time.100 mm tissue culture dishes, which were approximately 80–90% confluent, were washed briefly with ice-cold PBS, and total protein extracts were isolated by scraping cells into 10 mM Tris, pH 7.4, 0.1% Triton X-100 followed by incubation on ice, for 20 minutes. The insoluble material was centrifuged at 14,000 g, the supernatant was isolated, and protein concentration was determined using the Bio-rad D4 cells per well in 4-well chamber-slides and grown overnight at 33°C. Growth media was exchanged for fresh, pre-warmed growth media containing 500 nM Lysotracker or 1 mg/ml dextran-FITC, and cells were incubated at 33°C for 45 minutes or 15 minutes, respectively. Following labeling, cells were immediately placed on ice and washed for 10 minutes in ice-cold dye-free media, and fixed with 4% formaldehyde in PBS, for 20 minutes on ice. Chambers were removed and slides were coverslipped with Vectashield mounting media for confocal microscopy analysis, as described above.Cells were seeded at a density of 3–5 × 10TEM photomicrographs (10,000 × – 40,000 × magnification) were taken from random grid fields. For length measurements, the longest side of each mitochondria was measured in centimeters, and along the length of the mitochondria, width measurements were taken every 2.5–4 mm (dependent on the magnification of the micrograph image). Following measurement, all numbers were normalized to reflect one magnification and data was analyzed using Microsoft Excel software. To ascertain unmagnified mitochondrial size, final measurement data, in centimeters, was converted to nanometers according to scale bar representation.® Luminescent Cell Viability kit (Promega), according to the manufacturer's recommendations. Briefly, cells were plated in a black opaque-walled 96 well plate (Packard Bioscience) at a density of 20000/well and incubated at 33°C overnight. The following day, CellTiter-GLO® Reagent was added to each well and cell lysis was induced by mixing 2 minutes. An ATP standard curve was prepared in the same plate. Before recording luminescence with a microplate luminometer (MicroLumat Plus LB 96V, Berthold Techonologies), the plate was dark adapted for 10 minutes at room temperature to stabilize the luminescence signal.ATP was measured by using the CellTiter-GLO® AQueous Non-Radioactive Cell Proliferation Assay (Promega), according to the manufacturer's specifications.Cells were plated at a density of 10,000 cells/well in 96-well plates and incubated at 33°C overnight. The following day, fresh media containing varying concentrations of hydrogen peroxide was dispersed to each well. Cells were incubated in the presence of hydrogen peroxide for 24 hours, at 33°C, and viability was measured using the CellTiter-96EF participated in establishment and characterization of cell lines and performed ATP determinations. PW participated in mitochondrial analysis and immunocytochemistry. JE, TL-N, AMT, and HG participated in genotypic and additional phenotypic analysis of cell lines. DR and EC generated virus-conditioned medium for conditional immortalization of cells. MEM co-conceived of the study and assisted on drafting of the manuscript. SLC co-conceived of the study, participated in establishment and phenotypic analysis of cell lines, and drafted the manuscript. All authors read and approved the final manuscript.


Ki-1 (CD30) antigen expression is not found on peripheral blood cells but its expression can be induced in vitro on T and B lymphocytes by viruses and lectins. Expression of CD30 in normal tissues is very limited, being restricted mainly to a subpopulation of large lymphoid cells; in particular, cells of the recently described anaplastic large cell lymphoma (ALCL), the Reed-Sternberg (RS) cells of Hodgkin's lymphoma and scattered large parafollicular cells in normal lymphoid tissues. More recent reports have described CD30 expression in non-hematopoietic and malignant cells such as cultured human macrophages, human decidual cells, histiocytic neoplastic cells, mesothelioma cells, embryonal carcinoma and seminoma cells.We investigated the immunohistochemical expression of CD30 antigen in 15 paraffin-embedded tissue samples representing small intestines from fetuses after spontaneous abortion in the 8th, 10th and 12th weeks using the monoclonal antibody Ki-1. Hormones had been administered to all our pregnant women to support gestation. In addition, a panel of monoclonal antibodies was used to identify leukocytes (CD45/LCA), B-lymphocytes (CD20/L-26) and T-lymphocytes (CD3). Our findings were correlated with those obtained simultaneously from intestinal tissue samples obtained from 15 fetuses after therapeutic or voluntary abortions.The results showed that: (1) epithelial cells in the developing intestinal crypts express the CD30 (Ki-1) antigen; (2) CD30 expression in these epithelial cells is higher in cases of hormonal administration than in normal gestation. In the former cases  a mild mononuclear intraepithelial infiltrate composed of CD3 (T-marker)-positive cells accompanies the CD30-positive cells. CD30 antigen, a member of the tumor necrosis factor (TNF) receptor superfamily -3, was oCD30 appears to have an important immunoregulatory role in normal T cell development. Within the thymus, CD30L is highly expressed on medullary thymic epithelial cells and on Hassal's corpuscles .Pallesen and Hamilton-Dutoir  were theth and 12th weeks of embryo development. Goblet cells are present in small numbers after 8 weeks, Paneth cells differentiate at the base of the crypts in weeks 11 and 12, and enteroendocrine cells appear between weeks 9 and 11.Primitive crypts , appear in the postpharyngeal foregut between the 9The fact that the CD30 molecule can mediate signals for cell proliferation or apoptosis  prompted2O2 in methanol. The sections were preincubated in 20% serum of the species from which the secondary antibody was raised, and the primary antibody was applied. After overnight incubation at room temperature, the secondary biotinylated antibody was applied for 30 min. Staining was visualized with a Vector Elite System (Vector Laboratories, Burlingame, CA) using diaminobenzidine as the chromogen. The sections were counterstained with dilute hematoxylin. The primary antibodies used were as follows: (CD30/Ki-1) activated lymphoid cells, mouse monoclonal antibody (Novocastra); (CD45/LCA) leukocyte common antigen, mouse monoclonal antibody (Dako); (CD20/L-26) B-lymphocytes, mouse monoclonal antibody (Dako); and (CD3) T-lymphocytes, mouse monoclonal antibody (Dako). We used the high temperature antigen unmasking technique for immunohistochemical demonstration of CD30/Ki-1 on paraffin sections (Novocastra). Control slides were incubated with nonimmunized rabbit serum. An anaplastic lymphoma case-slide (positive control) was run in parallel with the assay.Samples representing 15 small intestines from fetuses after spontaneous (involuntary) abortion occurring in pregnant women treated with progesterone , and 15 small intestines from fetuses after therapeutic or voluntary abortion, were obtained in the 8th, 10th and 12th weeks of gestation. The Regional Ethics Committees approved the study. Written informed consent was obtained from all individuals and the procedures followed accorded with institutional guidelines. Small intestines were cut in 3 mm slices and fixed in 10% neutral buffered formaldehyde at 4°C for 24 h, then processed for routine paraffin embedding. Paraffin blocks were available in all cases, and 3 μm thick tissue sections were stained routinely with hematoxylin-eosin, PAS and Giemsa, and subsequently by immunohistochemistry. Immunoperoxidase labeling was performed as follows: sections were deparaffinized in 70% alcohol and endogenous peroxidase was blocked with 3% H2 of tissue section. The counted areas were selected from random tissue sections, taking into account that the ratio of the area of the intestinal stroma to the area of surface epithelium covering the crypts was representative of the entire field. Areas with obvious necrosis or haemorrhages were excluded. Statistical analysis was performed using the ANOVA test.For each sample, the CD30/Ki-1 positive population was assessed by enumeration of labeled cells in each tissue compartment for a minimum of five random fields per section viewed at 40-fold magnification through a grid. Cell numbers were calculated per mmFive microscopic fields of the small intestines were evaluated in each case without knowledge of the clinical data TABLE . Two obsIn cases of spontaneous (involuntary) abortion, immunohistochemistry revealed small clusters or scattered, large-sized CD30/Ki-1 positive cryptae cells within the intestine in all settings examined Fig. , with peIn cases of spontaneous abortion, immunohistochemistry showed a higher number of positive CD30/Ki-1 cryptae cells than at the 8th week of gestation . The number in cases of voluntary or therapeutic abortions was more or less the same as at 8th and 10th weeks, with percentages varying from 3.2 to 3.7 (mean ± sd = 3.41 ± 0.17). No differences in immune reaction were noted in the neighbouring intestinal stroma in cases of either spontaneous or voluntary/therapeutic abortion in comparison to the 8th and 10th gestational weeks.The differences among the numbers of CD30/Ki-1 positive cells at the 8th, 10th and 12th gestational week after spontaneous abortion were statistically significant (p < 0.0001). No significant differences were observed in the numbers of these cells after voluntary or therapeutic abortions (p = 0.95).The value of the CD30 antigen as a diagnostic marker for Hodgkin's lymphoma and anaplastic large cell lymphoma is well documented ,5,31. HoCD30 appears to have an important immunoregulatory role in normal T cell development. In normal cells, this transmembrane glycoprotein can be induced on B and T lymphocytes by mitogen stimulation or viral transformation -34. cDNARecent in vitro data indicate that the CD30 receptor-ligand complex can mediate signals for cell proliferation, apoptosis and cytotoxicity in lymphoid cells ,36,37. OSecond, these findings indicate that outside the lymphatic system, CD30 antigen expression in the epithelial cells of developing intestinal crypts can mediate signals for cell proliferation and differentiation in a region where other cell types (stem, goblet, Paneth and enteroendocrine) grow throughout life.Third, CD30 expression in the epithelial cells of the developing intestinal crypts is induced by progesterone. This is a novel mechanism of CD30 induction, distinct from neoplastic transformation and viral infection of lymphocytes. The demonstration of large R-S like cells in the developing crypts within a lymphoplasmacytic infiltrate, in the same way that similar R-S like cells are observed in reactive lymph nodes, especially within the parafollicular areas, is evidence that such cells might represent the physiological counterparts of R-S cells.The possibility that CD30 is an oncofetal antigen is supported by our positive findings in fetal intestinal cryptae cells. We have so far been able to investigate only a single tissue from a small number of fetuses of early gestational age. Pallesen and Hamilton-Dutoit  examined


Pain is highly prevalent among older adults, but little is known about how patient involvement in medical decision-making may play a role in limiting its occurrence or severity. The purpose of this study was to evaluate whether physician-driven and patient-driven participation in decision-making were associated with the odds of frequent and severe pain.A cross-sectional population-based survey of 3,135 persons age 65 and older was conducted in the 108-county region comprising West Texas. The survey included self-reports of frequent pain and, among those with frequent pain, the severity of pain.Findings from multivariate logistic regression analyses showed that higher patient-driven participation in decision-making was associated with lower odds  of frequent pain, but was not significantly associated with severe pain. Physician-driven participation was not significantly associated with frequent or severe pain.The findings suggest that patients may need to initiate involvement in medical decision-making to reduce their chances of experiencing frequent pain. Changes to other modifiable health care characteristics, including access to a personal doctor and health insurance coverage, may be more conducive to limiting the risk of severe pain. Persons age 65 years and older commonly endure a multitude of chronic and debilitating conditions which contribute to persistent pain . EstimatIncreasing patients' involvement in the medical decision-making process is one potentially fruitful means of improving pain management. Several studies suggest that patients, especially those with chronic conditions, who have opportunities to participate in care have more positive health outcomes than those who do not ,7. WhileAlthough patients' participation may improve their health outcomes, the effect can be diminished among elderly patients. Elderly patients, as compared to younger patients, have been shown to be less participatory in medical-decision making -11. UsinThe primary objective of the present study was to examine how participation in decision-making was associated with the occurrence of pain among a cohort of community-dwelling elders. In contrast to previous studies, we differentiated two types of participation in decision-making. The first type is physician-driven in which the physician takes the initiative to ask questions and offer choices to patients. The second type is patient-driven, in which the patient takes the initiative to ask questions and express preferences. We hypothesized that stronger physician and patient-driven participation in decision-making would be associated with lower odds of frequent pain and, among those with frequent pain, lower odds of severe pain. We also tested for the effects of other health care factors which might be conducive to pain management, such as tenure with a personal doctor. The findings have implications for how older patients interact with their physicians as well as how physicians and clinic managers organize health services.Data were obtained through a longitudinal, population-based study of community-dwelling elders, the Texas Tech 5000 Survey. The Texas Tech 5000 Survey was conducted in a 108-county region of West Texas, a geographically and ethnically diverse area encompassing approximately half of the state's land mass. The survey has been described in detail elsewhere -21. BrieThe occurrence and severity of pain were measured in Wave 3 using items developed for and included in two nationally representative surveys of older persons, the Health and Retirement Study (HRS) and Assets and Health Dynamics among the Oldest Old (AHEAD) . First, A number of sociodemographic, health care, and health status measures were included. Sociodemographic factors were gender, age (continuous), marital status, educational status (high school graduate vs. less than high school education), and place of residence . An urban area is a metropolitan county, or a county with a total population of at least 50,000, whereas a rural area is a county with fewer than 50,000 persons. Rural counties were further classified according to whether they were frontier areas, or counties with fewer than 7 persons per square mile .Health care variables were health insurance coverage, the number of physician visits in the last 6 months, tenure with a personal doctor, an index measuring physician-driven participation in decision-making, and an index measuring patient-driven participation in decision-making. Health insurance coverage was coded as Medicaid, Medicare, Medicare plus other private or government coverage, other private or government coverage, and no insurance. Tenure with a personal doctor was measured using a single question asking if the individual had a personal doctor and, if so, the duration of tenure with the physician (less than 1 year, 1 to 2 years, 3 to 4 years, and 5 or more years). An index of physician-driven participation in decision-making was created using three items taken from the Medical Outcomes Study . The phyThree questions adopted from a study of older patients' communication during medical visits  were useOverall health status (categorized as excellent, very good, good, and fair or poor) was measured using a general health item from a brief health-related quality of life instrument (the SF-12) . Mental Chi-square tests were first conducted to determine whether there was an association between each categorical sociodemographic, health care, and health status factor and frequent and severe pain. T-tests were conducted to determine if there was a difference in each continuous sociodemographic, health care, and health status factor between individuals with and without frequent pain and individuals with and without severe pain. Next, multivariate logistic regression analyses were conducted to determine if physician or patient-driven participation in decision-making were associated with the odds of frequent pain and, among those with frequent pain, the odds of severe pain. The potential for multicollinearity between the covariates was assessed by calculating their variance inflation factors; no problems with multicollinearity were found.A total of 1,333 42.5%) of the 3,135 survey participants had frequent pain. Table .5% of thAmong the 1,333 individuals with frequent pain, a total of 287 (21.5%) had severe pain. As shown in Table Findings from multivariate logistics analyses are shown in Table Physician-driven participation was not significantly associated with the odds of frequent pain. However, elders with higher patient-driven participation had lower odds  of frequent pain, confirming our hypothesis that persons who take a more active role in their medical treatment are less likely to experience pain. Individuals who had been diagnosed with arthritis at some point in their lives had a higher odds of frequent pain  than those without arthritis. Those who rated their general health as excellent , very good , good , and fair (OR, 0.69, CI 0.52, 0.92) had lower odds of frequent pain than those who rated their health as poor.Among those with frequent pain, there were no gender difference in the odds of severe pain. Persons of other race/ethnicity (primarily Black/African Americans) had a higher odds  of severe pain than non-Hispanic whites. Income, marital status, rural residence, and frontier residence were not significantly associated with the odds of severe pain.The number of physician visits was not significantly associated with severe pain. However, having insurance had a significant impact on the odds of severe pain. Compared to those without health insurance coverage, those with Medicare , Medicaid , Medicare plus other private or government coverage , and those with other private or government coverage  had a significantly lower odds of severe pain. Although physician and patient-driven participation were not significantly related to the odds of severe pain, tenure with one's personal doctor was a significant factor. Elders who had been seeing their doctor for less than 1 year , 3–4 years , or 5 or more years  had lower odds of severe pain than elders who had no personal doctor.As expected, several health status measures were also significant. Those who had arthritis had higher odds of severe pain  than those without arthritis. Finally, individuals who rated their general health as very good , good , and fair  had lower odds of severe pain than those who rated their health as poor.We found that patient-driven participation in decision-making was associated with lower odds of frequent pain, which is supported by previous research indicating that adult patients who are more actively engaged in their treatment have greater reductions in symptoms and improvement in health status , better While neither physician nor patient-driven participation in decision making were significantly associated with pain severity, another factor related to the strength of the doctor-patient relationship was significant. In the present study, having a personal doctor, no matter how long the tenure of the relationship was, reduced the odds of severe pain. The finding of a beneficial effect of having a personal doctor, at least in terms of the severity of pain, is consistent with prior studies which have shown that having a usual source of care is positively correlated with an individual's access to the health care system -33, satiIn addition to having a personal doctor, access to any type of health insurance coverage was associated with the odds of severe pain. Approximately 12 percent of persons in the study sample reported that they had no health insurance coverage at all, including Medicare, and 21.5% of those without insurance had severe pain. The percentage of patients in this group with severe pain was nearly 2 times higher than the percentage of patients in the other insurance categories. Many older persons may not be eligible for public health insurance because they have not contributed to the social security system for a minimal amount of time. This may be particularly common in the southwestern United States where there are larger numbers of Hispanic immigrants. Expansion of health insurance coverage to this group could improve their ability to visit physicians and other health providers when they experience pain and, ultimately, lead to better pain management. Further research is warranted to more clearly elucidate how characteristics of different health insurance plans, such as gatekeeping and cost sharing, affect access to physician services for pain treatment.Several demographic indicators were also significantly associated with frequent and severe pain. The gender differences beckon the question of whether medical care providers treat older women's pain less effectively or appropriately than men's. No differences were found between Hispanics and non-Hispanic whites, but other races (the majority of whom were Black/African American) had higher odds of severe pain than non-Hispanic whites. However, because of the heterogeneity of the other racial category, it is difficult to discern which particular racial groups experience severe pain. Research which includes greater numbers of other racial categories is thus warranted.In regard to health status, the results support that individuals with three or more comorbid diseases have a relatively higher odds of frequent pain and severe pain than those with no comorbid diseases. One disease, arthritis, was of particular interest and therefore was treated as a separate variable. Almost two-thirds of the subjects had arthritis, which is not unexpected for persons age 65 and older. Persons with arthritis had a much higher odds (over 3 times the odds) odds of frequent pain than individuals without arthritis. Moreover, those with arthritis had approximately 1.5 times the odds of severe pain. The magnitudes of these associations imply that efforts to more effectively treat arthritis could lead to improvements in pain management.While the present study has contributed to our understanding of the relationship between doctor-patient interactions and persistent pain, it is not without several limitations. Because the study was cross-sectional in design, it is impossible to infer any causal relationships. Although the pain measures were adapted from a nationally representative cohort study of older persons , they maDespite these potential limitations, the present study suggests that several strategies could be implemented to limit the incidence and severity of pain among community-dwelling elders. Health policy makers and insurance companies might implement new reimbursement schemes to encourage visits to a personal physicians in order to improve pain and other health outcomes. Managers of physician clinics should consider organizing practices to ensure that older patients are able to make timely appointments with a personal provider. Finally, patients themselves could help reduce their chances of having frequent pain by becoming more involved in their care. These are just a few examples of how changes to the organization and delivery of care might affect pain-related health outcomes. Future research should evaluate how a range of physician characteristics , physician clinic characteristics (e.g. solo or group practice), insurance characteristics (e.g. HMO, PPO, or FFS coverage), and patient characteristics (e.g. trust in physician) influence pain and pain treatment.The author(s) declare that they have no competing interests.TFB conceived the overall study, performed the statistical analyses, and led the drafting of the manuscript. KTX assisted with the study design, interpretation of statistical findings, and drafting of the manuscript. JH assisted with interpretation of the findings and drafting of the clinical implications. GK contributed to data management, statistical analyses, and drafting of the methods section. All authors read and approved the manuscript.The pre-publication history for this paper can be accessed here:


Patients with upper limb pain often have a slumped sitting position and poor shoulder posture. Pain could be due to poor posture causing mechanical changes  that in turn affect the function of major limb nerves (e.g. median nerve). This study examines (1) whether the individual components of slumped sitting (forward head position, trunk flexion and shoulder protraction) cause median nerve stretch and (2) whether shoulder protraction restricts normal nerve movements.Longitudinal nerve movement was measured using frame-by-frame cross-correlation analysis from high frequency ultrasound images during individual components of slumped sitting. The effects of protraction on nerve movement through the shoulder region were investigated by examining nerve movement in the arm in response to contralateral neck side flexion.Neither moving the head forward or trunk flexion caused significant movement of the median nerve. In contrast, 4.3 mm of movement, adding 0.7% strain, occurred in the forearm during shoulder protraction. A delay in movement at the start of protraction and straightening of the nerve trunk provided evidence of unloading with the shoulder flexed and elbow extended and the scapulothoracic joint in neutral. There was a 60% reduction in nerve movement in the arm during contralateral neck side flexion when the shoulder was protracted compared to scapulothoracic neutral.Slumped sitting is unlikely to increase nerve strain sufficient to cause changes to nerve function. However, shoulder protraction may place the median nerve at risk of injury, since nerve movement is reduced through the shoulder region when the shoulder is protracted and other joints are moved. Both altered nerve dynamics in response to moving other joints and local changes to blood supply may adversely affect nerve function and increase the risk of developing upper quadrant pain. Non-specific arm pain (NSAP), often called repetitive strain injury, describes the common problem of upper limb pain and functional impairment without objective physical findings. The contributing factors to the development of NSAP are not fully understood but ergonomic guidelines commonly suggest that good upper body posture protects against NSAP (e.g. ). In a sTogether, shoulder protraction, forward head position and flexion of the trunk form the main components of slumped sitting. The present study examines the effects of each of these components on longitudinal sliding of the median nerve using high frequency ultrasound imaging in asymptomatic normal subjects. In addition, the effects of sustained protraction on nerve sliding through the shoulder region are examined.Longitudinal nerve movement was measured using high frequency ultrasound imaging, as previously described by Dilley et al. ,6. A DiaFourteen healthy subjects, 5 male and 9 female, aged 25–38 years (mean = 32 years) were screened to exclude upper limb or cervical spine pathologies, rheumatological or neurological conditions. In each subject the nerve bed length was estimated, from the C6 spinous process to the tip of the index finger (mean = 97.0 cm (SD, 5.9)), and used to normalise the ultrasound transducer position between individuals. All measurements were taken from the right upper limb only.The median nerve was imaged in longitudinal section in the forearm during forward head position, trunk flexion and protraction and in the forearm and upper arm during contralateral neck side flexion (CNSF). Each movement was repeated three times, including some reverse trials.Each subject (n = 8) was imaged in the proximal forearm whilst positioned upright on a chair fitted with a back and head support, hips and knees at 90° flexion, and the trunk fixed with Velcro strapping. The right upper limb was strapped to a Perspex plate in 90° flexion and 20° abduction at the glenohumeral joint, with the elbow fully extended, 45° forearm supination, and the wrist, hand and fingers in neutral. An active forward head position movement was performed, which included lower cervical spine flexion and upper cervical spine extension.Each subject (n = 8) was imaged in the proximal forearm whilst positioned upright on a chair, with hips and knees at 90 degrees flexion. The right upper limb was positioned as for the forward head position trials. The subject was taught to actively flex their trunk whilst posteriorly tilting their pelvis.Each subject (n = 13) was imaged at two locations in the forearm and positioned as for the forward head position. The distal upper arm was imaged in three of the 13 subjects. For each trial the shoulder girdle was passively protracted from neutral  by sliding the Perspex plate supporting the arm on an adjustable table. In three of these subjects, additional data was also obtained during ultrasound imaging. A potentiometer attached using strong thread to the acromion process allowed measurement of the amount of protraction. Protraction data was captured on to a PC and synchronised offline to the recorded ultrasound sequence.In four subjects, good quality images of the median nerve within the upper arm could be obtained. In the majority of subjects, it was difficult to acquire good quality images because of dense tissue overlying the nerve that reduced the image quality. From these images, nerve trunk bowing was measured in the distal upper arm with the shoulder girdle in the neutral and protracted positions. The maximum deviation of the nerve from a straight line across single ultrasound frames was measured offline in both positions and the difference used as a measure of additional bowing. Repeat trials were averaged.Each subject (n = 11) was imaged in the distal forearm and distal upper arm, whilst lying supine with the right upper limb abducted to 90° at the glenohumeral joint. Ninety degree abduction at the glenohumeral joint rather than 90° glenohumeral flexion was used, so that the present data could be related to previous work  which haThe head was supported on a movable plate, with the centre of rotation positioned at the C7 spinous process. In each subject, the neck was passively moved to 35° CNSF with (a) the scapulothoracic joint in neutral (i.e. relaxed lying in supine) and (b) in full protraction. This movement was repeated several times. In four subjects, a potentiometer attached to the plate allowed continuous measurement of the angle of CNSF. Joint angle data was captured on to a PC and synchronised offline to the recorded ultrasound sequence.For each procedure the range of movement was determined from pictures obtained using a digital camera. Changes in joint angle and distance for each movement were determined from skin surface markers and measured using either CorelDraw  or "tpsDig" (F. James Rohlf, Department of Ecology and Evolution, State University of New York). Measurements for the individual components of slumped sitting are summarised in figure Strain is defined by the difference in the amount of elongation that occurs at two points along a nerve divided by the distance between these two points. In practice strain was determined by using regression lines fitted to plots of nerve movement against the distance along the arm. Note that the strain estimates represent the additional strain produced by the movement rather than the total nerve strain.Comparisons of nerve movement and strain in scapulothoracic neutral and protraction during CNSF were performed using paired t-tests.Moving the head forward while maintaining the shoulder and trunk position was tested in 8 subjects. This movement produced no detectable median nerve excursion in the forearm, the average trend being a movement of 0.1 mm (SEM, 0.02) occurring in a proximal direction. The repeat measure variability within subjects was very low, with a standard deviation ranging from 0–0.2 mm (mean = 0.1 mm). The mean change in the angle of lower cervical spine flexion and upper cervical spine extension was 23.6° (SD, 2.8) and 2.9° (SD, 1.9) respectively.Trunk flexion also produced minimal median nerve excursion with a mean over 8 subjects of 0.1 mm (SEM, 0.1) proximal movement. The mean change in the angle of trunk flexion was 19.7° (SD, 4.7).In 13 subjects the median nerve moved in a proximal direction during shoulder protraction with more movement at proximal locations (mean in forearm = 3.5 mm (SEM, 0.3), mean in upper arm = 5.9 mm (SEM, 0.6)) figure .Median nerve excursion was measured in 3 subjects with simultaneous measurement of protraction. The results revealed an initial delay of 6.5–33.0 mm  of the total protraction) before significant nerve movement occurred . The nerve straightened during protraction figure .In 11 subjects the median nerve moved in a proximal direction during 35° CNSF when the scapulothoracic joint was in neutral, as reported previously . The movMedian nerve excursion was measured in 4 subjects with simultaneous measurement of CNSF. The results revealed no obvious delay in the onset of nerve movement in protraction compared to scapulothoracic neutral. Despite less movement in protraction, the pattern of nerve movement mimicked that observed in scapulothoracic neutral figure .Nine of 11 subjects reported paraesthesia in the distribution of the median nerve dermatone once the shoulder girdle was sustained in protraction. The onset of symptoms ranged from 1 to 4 minutes. Symptoms disappeared when the shoulder was repositioned in scapulothoracic neutral.Nerves are designed to slide and stretch to accommodate joint movement. Using the method of Dilley et al. ,6, mediaAs the shoulder girdle is protracted there is a delay in nerve movement, which is followed by a steady increase in nerve excursion. During this initial toe region the median nerve appears bowed in the upper arm. The nerve trunk appears to straighten as the range of protraction progresses. It therefore seems that with the upper limb in scapulothoracic neutral and the glenohumeral joint in 90° flexion, the median nerve is unloaded. If this is the case, the strain value of 0.7% will represent the total strain.The results for CNSF provide evidence for a possible restriction within the shoulder region during shoulder protraction. With the shoulder protracted there was a 60% reduction in the transmission of nerve movement through the upper limb. Consistent with a reduction in movement, there was also significantly less strain in the forearm. The possibility that the nerve becomes unloaded when the shoulder is protracted is unlikely since it had been found that protraction itself causes some median nerve stretch. In addition, there was no obvious delay in nerve movement in response to CNSF  might reduce the space between the clavicle and the first rib, restricting nerve sliding.The components of slumped sitting (i.e. forward head position, trunk flexion and protraction) are associated with poor posture ,9-11, anProblems are more likely to result from local effects of shoulder protraction on the chords of the brachial plexus. The present study shows that protraction restricts nerve sliding through the shoulder region. Most subjects also experienced paraesthesia when maintaining shoulder protraction plus elbow extension and shoulder abduction. Therefore, sustained shoulder protraction may place the median nerve at enhanced risk of injury and possibly cause a vascular compromise. This may in turn explain for the trend that a high number of NSAP patients have poor shoulder posture. (e.g. ).The direct effects of slumped sitting on median nerve strain are not sufficient to alter nerve function. However, shoulder protraction does appear to restrict nerve sliding, and prolonged protraction leads to pareasthesias.None declared.AJ and RL participated in the study design, data collection, analysis and manuscript preparation. AD participated in the study design, analysis and manuscript preparation. BL participated in the study design and manuscript preparation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Median nerve sliding in the forearm during protraction. Ultrasound sequence of median nerve sliding in the distal forearm during protraction. The subject was imaged with the limb in 90° flexion and 20° abduction at the glenohumeral joint and elbow neutral. The median nerve can be seen to slide in a proximal direction as the shoulder is protracted. In a repeat of the sequence the nerve movement is tracked using cross-correlation analysis (yellow plus sign). The total nerve movement was 4.70 mm.Click here for file