DICER1 indicates that mis-expression of miRNAs in cancer may result from defects in their processing. As part of a recent study of DICER1 RNase III domains in 96 testicular germ cell tumors, a single RNase IIIb domain mutation was identified in a seminoma. To further explore the importance of DICER1 mutations in the etiology of testicular germ cell tumors (TGCT), we studied germ-line DNA samples from 43 probands diagnosed with familial TGCT.The RNase III enzyme DICER1 plays a central role in maturation of microRNAs. Identification of neoplasia-associated germ-line and somatic mutations in DICER1 exons 2–12, 14–19, 21 and 24–27. All questionable melt curves were subjected to confirmatory Sanger sequencing.We carried out High Resolution Melting Curve Analysis of Sanger sequencing was used for exons 13, 20, 22 and 23. Intron-exon boundaries were included in all analyses. We identified 12 previously reported single nucleotide polymorphisms and two novel single nucleotide variants. No likely deleterious variants were identified; notably no mutations that were predicted to truncate the protein were identified.DICER1 mutations in the etiology of TGCT.Taken together with previous studies, the findings reported here suggest a very limited role for either germ-line or somatic  DICER1 that are associated with a range of mainly childhood-onset cancers and dysontogenic or hyperplastic conditions, notably “blastoma”-type tumors such as pleuropulmonary blastoma (PPB), ovarian Sertoli- Leydig cell tumor (SLCT), embryonal rhabdomyosarcoma and Wilms tumor, as well as benign tumors such as cystic nephroma  exons 2–12, 14–19, 21, the 3’ half of 23 and 24–27 using lymphocyte DNA from one proband from each family as described previously [DICER1 by (HRM) using the LightScanner instrument . The PCR reactions were done in 96 well plates from Bio Rad  using the mastermix and the LCGreen Plus from Transition Technologies . The plates were then transferred to the LightScanner instrument and the melted curves were analyzed by the software provided by Idaho Technologies. This technique was used as a presequencing selection for amplicons harboring variants. The PCR primers used are shown in Table DICER1 analysis for exons 13, 20, 22 and the 5’ half of exon 23. Intron-exon boundaries were included in all analyses.We conducted High Resolution Melting Curve (HRM) analysis of Among the 43 probands, we identified 14 different single nucleotide variants or polymorphisms, 12 of which have been previously reported, but no likely deleterious variants; notably no mutations that were predicted to truncate the protein were identified .Multiple epidemiological studies point toward a strong genetic basis for TGCT susceptibility. A large Swedish study estimates the genetic contribution of TGCT susceptibility to be about 25%, the third highest among cancers. Although familial aggregation of TGCT is rare with only 1.4% of families having two or more first degree relatives with the disease, multiple studies in different populations have shown that sons of an affected father are at 4–6 fold increased risk of developing TGCT while brothers of an affected male are at 8–10 fold increased risk, a familial relative risk that is much higher than most other cancers. Ethnic variability is also observed with an incidence five times higher in Caucasian males than in African-Americans (as reviewed by Rapley and Nathanson) .KITLG, SPRY4, BAK1, TERT, ATF7IP and DMRT1 in disease pathogenesis. Nevertheless, these six loci together with the “gr/gr” deletion account for less than 15% of the excess familial risk, suggesting that many more risk alleles remain unaccounted for (reviewed in [de novo germline copy number variants (CNVs); such variants were seen in 7% of 43 TGCT trios, greater than the expected background rate of CNVs [A genome-wide linkage search for susceptibility loci initially identified a region on Xp27 as a possible candidate, however this finding was not replicated in an independent data set, the results of which suggested that no single highly penetrant allele is responsible for a substantial proportion of familial TGCT . Candidaiewed in ,22). A r of CNVs . With thDICER1 mutations are commonly associated with TGCT. These results strongly suggest that TGCTs do not fall within the spectrum of diseases associated with germ-line DICER1 mutations and thus clinical screening for such cancers is not warranted in DICER1 mutation carriers.The findings reported herein, when combined with the previously reported studies discussed above, suggest that neither germ-line nor somatic CNVs: Copy number variants; PPB: Pleuropulmonary blastoma; SLCT: Sertoli-Leydig cell tumor; TGCT: Testicular germ cell tumor.The authors declare that they have no competing interests.AB and NS did the HRM analysis and sequencing, AYS helped with interpretation of the results and writing of the paper, PAK and KLN ascertained the patients and maintained the database, MDT and WDF oversaw the project. WDF wrote the paper, which was edited by all authors, who commented on and approved the final version. All authors read and approved the final manuscript.


Consistent with previous studies, corneal epithelial stripe patterns coarsened with age in WT mosaics. Mosaic patterns were also coarser in +/−Pax6 mosaics than WT at 15 weeks but not at 3 weeks, which excludes a developmental explanation and strengthens the prediction that +/−Pax6 mice have a LESC-deficiency. To investigate how Pax6 genotype and age affected corneal homeostasis, we compared corneal epithelial cell turnover and label-retaining cells  in +/−Pax6 and WT mice at 15 and 30 weeks. Limbal BrdU-LRC numbers were not reduced in the older WT mice, so this analysis failed to support the predicted age-related decline in slow-cycling LESC numbers in WT corneas. Similarly, limbal BrdU-LRC numbers were not reduced in +/−Pax6 heterozygotes but BrdU-LRCs were also present in +/−Pax6 corneas. It seems likely that +/−Pax6 LRCs are not exclusively stem cells and some may be terminally differentiated CD31-positive blood vessel cells, which invade the +/−Pax6 cornea. It was not, therefore, possible to use this approach to test the prediction that +/−Pax6 corneas had fewer LESCs than WT. However, short-term BrdU labelling showed that basal to suprabasal movement (leading to cell loss) occurred more rapidly in +/−Pax6 than WT mice. This implies that epithelial cell loss is higher in +/−Pax6 mice. If increased corneal epithelial cell loss exceeds the cell production capacity it could cause corneal homeostasis to become unstable, resulting in progressive corneal deterioration. Although it remains unclear whether +/−Pax6 mice have LESC-deficiency, we suggest that features of corneal deterioration, that are often taken as evidence of LESC-deficiency, might occur in the absence of stem cell deficiency if corneal homeostasis is destabilised by excessive cell loss.We aimed to test previous predictions that limbal epithelial stem cells (LESCs) are quantitatively deficient or qualitatively defective in  The adult corneal epithelium is a constantly renewing tissue and it is widely accepted that, during normal homeostasis, it is maintained by a stem cell population in the basal limbal region that proliferates slowly unless stimulated by injury The absence of reliable markers, able to distinguish adult stem cell populations from early TACs in the corneal epithelium, means that various indirect methods have been used to deduce that the basal limbal epithelium is the niche for corneal epithelial stem cells. Two threads of information from mouse studies have been important: the demonstration of centripetal migration of corneal keratinocytes from the limbus towards the central cornea Early studies revealed that a characteristic feature of epithelial stem cells is that they divide relatively infrequently PAX6 gene. The phenotype involves developmental eye abnormalities, including a reduced or absent iris, Sey-NeuPax6 mutant allele is considered to be a −Pax6 null allele and heterozygous +/Sey-NeuPax6 (here abbreviated to +/−Pax6) mice have small eyes, hypoplastic irides and a range of other ocular abnormalities, including corneal deterioration, so provide a good model for human +/−PAX6 aniridia and ARK +/−Pax6 corneal abnormalities arise during development  Human aniridia is an inherited eye disease caused by heterozygosity for a defective +/−PAX6 humans and +/−Pax6 mice can be considered to represent abnormal tissue homeostasis. From a purely quantitative perspective, tissue homeostasis can be defined as the maintenance of an approximately constant cell number in a renewing tissue and so involves a balance of cell production and cell loss. However, to conserve full tissue functionality it is critical that lost cells are replaced by cells of the appropriate phenotype. Thus, tissue homeostasis can be considered to have both quantitative and qualitative aspects. Normal corneal epithelial homeostasis requires maintenance of an adequate number of cells of the phenotype required to ensure the cornea is able to maintain full transparency and an adequate barrier function. The accumulation of goblet cells that occurs during the deterioration of the corneal epithelia in +/−PAX6 humans and +/−Pax6 mice implies that homeostasis is qualitatively abnormal. This could arise either because corneal epithelial cell differentiation is abnormal or because conjunctival or other cells encroach onto the corneal surface to compensate for a numerical deficiency in corneal epithelial cells caused by reduced cell production and/or excessive cell loss.The corneal epithelial deterioration seen in +/−Pax6 mice +/−Pax6 X-inactivation mosaic corneas had fewer radial stripes than WT mosaic corneas at 15 weeks +/−Pax6 mosaic corneas have fewer, wider stripes. Hypothesis 1 , so there is a stem cell deficiency. Hypothesis 2  and clonally related LESCs are more likely to be adjacent to each other. It is important to evaluate whether cell mixing is reduced during development (hypothesis 2) before drawing the conclusion that the mosaic analysis predicts that LESC function is impaired in +/−Pax6 mice (hypothesis 1).It has been proposed that LESC deficiency is the principal cause of deterioration of the ocular surface in aniridia patients thesis 1  proposesthesis 2  proposes+/−Pax6 heterozygotes. Two observations suggest that cell turnover is more rapid in the corneal epithelium. First, the frequency of BrdU labelled cells has been reported to be higher in +/−Pax6 mice than WT mice, either for the whole corneal epithelium ex vivo evidence suggests that the +/−Pax6 corneal epithelium is more fragile than normal +/−Pax6 corneal epithelia suggests that differentiation of the +/−Pax6 corneal epithelium is abnormal +/−Pax6 epithelial fragility, as reported for K12 knockout mice +/−Pax6 cornea is in a chronic wound-healing state +/−Pax6 corneal epithelial cell loss would be greater than normal but this has not been investigated directly.Other factors also probably contribute to abnormal corneal homeostasis in +/−Pax6 and WT mice, as this is the irreversible first step of cell loss; (2) to re-evaluate the prediction from studies of mosaic mice that +/−Pax6 mice have fewer LESCs by testing whether the coarser mosaic patterns could be accounted for by reduced cell mixing during development of the +/−Pax6 ocular surface, so producing larger coherent clones (hypothesis 2 shown in +/−Pax6 and WT mice at two ages (15 and 30 weeks), as a means of comparing relative LESC numbers, to test whether +/−Pax6 mice have fewer LESCs than WT (hypothesis 1 shown in The aims of the present study were: (1) to compare the rate of movement of cells from the basal corneal epithelium to the suprabasal layers in All the animal work in this study was approved by the University of Edinburgh Ethical Review Committee (application PL21-06) and performed in accordance with UK Home Office regulations under project license number PPL 60/3635. All surgery was performed under general anaesthesia and all efforts were made to minimise suffering.+/Sey-NeuPax6 (+/−Pax6) and +/+Pax6, wild-type (WT) littermates were maintained on a congenic CBA/Ca genetic background. They were distinguished by eye size and their genotypes confirmed by PCR Tg(Hmgcr-LacZ)H253Sest, nLacZ transgene (abbreviated to XLacZ), were maintained on a mixed genetic background (predominantly C57BL/6 and CBA/Ca). Hemizygous males and females are designated respectively Tg/YXLacZ and Tg/−XLacZ. Both +/−, XLacZTg/−Pax6 and WT, Tg/−XLacZ X-inactivation mosaics were produced from +/−Pax6 female × Tg/YXLacZ male crosses. Groups of mice were compared at 15 and 30 weeks because previous comparisons of WT X-inactivation mosaics showed that an age-related decline in corrected corneal epithelial stripe numbers could be detected between these ages +/−Pax6 and WT X-inactivation mosaics at 15 and 30 weeks, as described later. Differences between these ages are considered to be age-related differences but not effects of “old age” as laboratory mice can live to about 2 years Heterozygous For acute BrdU labelling of the adult cornea, 12-week old mice were given single intraperitoneal (i.p.) injections of BrdU  at 10.00 am. Mice were killed after 4 hours or 1, 3, 7 or 14 days and samples collected. For BrdU pulse-chase identification of label-retaining cells (LRCs), 0.1 ml Alzet mini-osmotic pumps , containing BrdU solution  were surgically implanted, sub-cutaneously, under Isoflurane anaesthesia, into 15- or 30-week old mice to provide 0.5 µl BrdU solution/h. The pumps were removed after 7 days and mice were killed 10 weeks later and samples collected.Eyes were fixed in 4% paraformaldehyde for 24 h. and embedded in paraffin wax after dehydration in a graded ethanol series. Longitudinal sections were cut at 7 µm thickness, mounted on Polysine slides (VWR laboratory supplies), deparaffinised in Histoclear and processed for BrdU immunoshistochemistry with a 3-3′diaminobenzidine (DAB) endpoint and lightly counterstained with haematoxylin, as described previously +/−Pax6 corneal epithelium has fewer cell layers than WT and this could exaggerate or mask differences in suprabasal LI. To take account of this, an adjusted % BrdU-positive cells in the suprabasal layers  was calculated by expressing the number of BrdU-positive cells in the suprabasal layers as a percentage of the total cell number in the underlying basal layer rather than the total cell number in the suprabasal layers.After BrdU immunohistochemistry, calibrated digital images of mid-sections of the corneal epithelium were captured with a Nikon Coolpix 995 digital camera on a Zeiss Axioplan 2 compound microscope. Every basal corneal epithelial cell was scored as positive or negative, across the corneal diameter in a section from the middle of the eye. These data were divided into six equal sized groups according to corneal region, so the % BrdU-labelled nuclei in the basal layer  could be determined separately for two peripheral (P), intermediate (I) and central (C) regions which were then combined to provide one mean value per eye for the P, I and C regions. The % BrdU-positive cells in all the suprabasal layers  were scored in an equivalent way. The Whole-mount corneal immunofluorescence was adapted from a published method http://rsb.info.nih.gov/ij/). Composite images were constructed using the MosaicJ plug-in (http://bigwww.epfl.ch/thevenaz/mosaicj). BrdU immunofluorescence analysis is described below.The primary antibodies used were rat anti-BrdU (Abcam), rabbit anti-K19 (LifeSpan Biosciences) and rat anti-CD31 (BD Pharmingen). The secondary antibodies were goat Alexa 488 Anti-Rat IgG (Invitrogen), goat Alexa 488 Anti-Rabbit IgG (Invitrogen) and goat Alexa 568 Anti-Rat IgG (Invitrogen). For double immunofluorescent staining, antibodies were applied sequentially and incubated overnight at 4°C. To image K19 and CD31 immunofluorescence, overlapping Z-stack images were acquired with a Leica TCS NT confocal microscope and exported to ImageJ , in a clockwise direction with a Zeiss LSM510 CLSM confocal microscope using a ×20 lens. Images (with the limbus in the middle) were acquired at 1024×1024 pixel resolution. Individual photographs were compiled using Zeiss LSM Image Browser software (version 4.2.0.121) and projected to a single image based on maximum intensities. ImageJ was used for thresholding, to include particle sizes of 150–5000 voxels (3–15 µm diameter nuclei). A calibrated 340×200 µm sampling box was superimposed on the image across the width of the limbal region in each sector and BrdU-positive nuclei were counted. Fluorescent spots outside the size range for BrdU-positive nuclei were not included in the analysis.Counts of BrdU-positive nuclei were expressed as the mean number of LRCs per sampling box (one sampling box per sector). To correct for differences in corneal diameters, the mean number of LRCs per sampling box was multiplied by the corneal circumference (estimated from 3 diameter measurements of the flattened cornea) and divided by the length of the sampling box (340 µm) to provide an estimate of the number of LRCs in a 200 µm wide ring around the circumference.To reduce the effect of variability among individual immunofluorescence experiments, the mean number of LRCs per sampling box was also expressed as a normalised ‘LRC index per sampling box’. This was calculated separately for each immunofluorescence experiment that yielded results for at least one eye from each of the four groups compared. First, to accommodate experiments with unequal number of eyes in each group, a normalised total number of LRCs was calculated for each experiment as the sum of the mean number of LRCs per sampling box for each of the four groups. The ‘LRC index per sampling box’ was then calculated separately for each of the four groups as the number of LRCs per sampling box, expressed as the percentage of the normalised total number of LRCs per sampling box  for each experiment. The sum of the LRC indices for the four groups is, therefore, equal to 100% in each experiment. The estimated number of LRCs per circumference was similarly expressed as the ‘LRC index per circumference’.The confocal corneal images were also used to compare cell-packing densities in the four groups, using 3 regions from each of 3 corneas per group. Nuclei were counted manually in a 150×150 µm sampling box that was superimposed on the image by an independent person using Adobe Photoshop 7.0.Tg/−XLacZ, X-inactivation mosaic mice were stained with X-gal as described elsewhere Whole eyes from Whole adult eyes were stained with X-gal and the radial stripe patterns in the corneal epithelia were analysed semi-automatically from digital photographs showing the entire cornea, using the ImageJ plugin ‘Clonal Tools’ in batch mode as described previously to provide a ‘corrected stripe number’ GraphPad Prism software was used for statistical tests as indicated in the text and figure legends. The choice of parametric or non-parametric tests was guided, in part, by normality tests. In some cases, data  were log-transformed for analysis with parametric statistical tests. The error bars in the figures are 95% confidence intervals (CI).+/−Pax6 corneas were smaller in diameter  and had fewer suprabasal layers . As expected, BrdU-positive cells were almost entirely confined to the basal layer four hours after labelling  and 669±21.9 broader and flatter suprabasal cells. abelling . The perdeclined . There wnjection  as repornjection  or indivnjection .+/−Pax6 corneal epithelia had fewer suprabasal layers than WT, an ‘adjusted suprabasal LI’ was calculated as well as the suprabasal LI, to compare the percentage of BrdU-positive cells in the suprabasal layers . The frequencies of BrdU-positive cells increased significantly in both WT and +/−Pax6 suprabasal layers during the first three days and then declined  but not WT corneas. At this time the suprabasal LI in the central cornea was significantly higher for +/−Pax6 than WT  and the uncorrected median patch length calculated for the minority cell population  lengths . Thus, b+/−XLacZ corneas whereas the mean patch number decreases, implying that the patches increase in length over this period. This is consistent with observations on whole mount preparations showing that stripes emerge from the periphery at or before 5 weeks as a consequence of LESC activation and replace the randomly orientated patches formed during development +/−Pax6, +/−XLacZ corneas , which is present in the limbal and conjunctival epithelia but not the corneal epithelium +/−Pax6 eyes, both K19 positive cells and, as previously reported +/−Pax6 mice (+/−Pax6 eyes (data not shown). This infolding has been described previously for WT mouse eyes Before testing whether limbal-LRCs were reduced in +/− mice . Using T+/−Pax6 ocular surface  around the whole limbal circumference. Again, there were no significant differences among the four groups by 2-way ANOVA  per 150×150 µm sized sampling box in 3 fields from 3 eyes per group were estimated as 279±41 cells for 15-week WT, 207±45 for 15-week 30 weeks  was a co+/−Pax6 limbus it was noted that there were also some LRCs in the +/−Pax6 cornea itself . The images used for counting limbal LRCs were also used to count corneal LRCs so this analysis was restricted to the peripheral areas of the cornea. A few corneal LRCs were identified and counted in WT eyes but, if the limbal sampling box was imprecisely positioned over the limbus, these could have been limbal LRCs. Significantly more corneal LRCs were identified in +/−Pax6 corneas than in WT corneas at both ages  and eyes were immunostained for BrdU and CD31 blood vessels. The distribution of LRCs appeared very similar to the blood vessels  to the suprabasal layers, was followed by a decline in labelled suprabasal cells, after 3 days. This showed that most cells labelled in the basal layer were lost from the corneal epithelium by day 14 and implies that the corneal epithelium ‘turnover time’ is ≤14 days. However, most BrdU-positive cells will survive for less than this, once they leave the basal layer, because some will remain in the basal layer for several cell generations. The decline of labelled suprabasal cell numbers between 3 and 7 days suggests that some basal cells are shed from the surface within 7 days of labelling. This is similar to previous turnover time estimates of 6–7 days in mice The turnover time is shorter than the time required for LESCs to replace the corneal epithelium. This has been termed the ‘renewal time’ For WT corneas, there was little evidence of regional differences in cell proliferation or movement to the suprabasal layers. Results of previous studies are inconsistent +/−Pax6 corneal epithelium compared to WT +/−Pax6 and WT corneas. This demonstrated that relatively more labelled cells move from the basal to the suprabasal layers within the first 24 hours of labelling in +/−Pax6 than WT corneas and the difference is greatest in the central cornea. As this vertical cell movement is irreversible and is the beginning of cell loss, we conclude that greater corneal epithelial cell loss occurs in +/−Pax6 than WT corneas. Overall, this shows that +/−Pax6 corneal epithelial cell turnover is faster because basal cells both proliferate more frequently +/−Pax6 central corneal epithelium may be because the +/−Pax6 cornea is more fragile and the central region is most vulnerable to injury.A previous BrdU study showed significantly higher labelling in the basal We used LRCs to identify putative LESCs but this is not specific for stem cells. If both actively proliferating and relatively quiescent stem cell populations exist, as they do in some other tissues, only the quiescent ones will be identified as LRCs +/−Pax6 mice had fewer LRCs than WT mice at 15 weeks. The mosaic analysis compares corrected stripe numbers, which relates to the numbers of active coherent clones of stem cells (comprising 1 or more stem cells) rather than the numbers of individual stem cells. Thus, stripe numbers depend both on numbers of stem cell clones (≤ number of stem cells) and the ability of the stem cells to function . Different results may be obtained for the mosaic and LRC analyses for two reasons. First, LESC function may change in a way that affects stripe numbers and LRC numbers differently. Second, LESC function may not change with age but stripe numbers could decline for an unrelated reason.The presence of LRCs in the limbus but not the central cornea in WT mice is consistent with the conventional view that the adult corneal epithelium is maintained by slow-cycling LESCs, some of which are identified as label-retaining cells (LRCs). However, the quantitative LRC results failed to support two predictions prompted by analysis of radial mosaic corneal epithelial stripe patterns. There was no evidence that LRC numbers declined between 15 and 30 weeks in WT mice and no evidence that In the first case, several age-related changes could affect LESCs and cause stripe numbers to change independently of LRC numbers. (a) LESCs in older mice may continue to divide, and be detectable as LRCs, but more may be defective and unable to establish long-lived clones of corneal epithelial cells, identifiable as stripes in mosaic eyes. (b) Age-related changes in LESC cell cycle kinetics may cause longer or more frequent periods of quiescence, which reduce stripe production, but this might not reduce LRC numbers if, for example, reduced BrdU labelling was balanced by increased BrdU retention. (c) If the limbus harbours separate populations of actively proliferating stem cells (which produce most of the stripes) and more quiescent stem cells (detectable as LRCs) the age-related change could be restricted to stripe numbers if only the more active stem cell population decreased with age. (d) Both stripe numbers and LRCs produced by LESCs might decline with age but if the cell cycle slowed in some other cell types these might also be identified as LRCs and the overall LRC number would not decline in parallel with the stripe numbers.clones without any reduction in the numbers of active stem cells or their function if stochastic neutral drift in LESC populations eliminated some clones and expanded others. This has been suggested as an explanation of the coarsening of mosaic patterns derived from stem cell clones in the mouse testis In the second case, an age-related decline in corrected stripe number might reflect a decline in numbers of active LESC +/−Pax6 mice had fewer LRCs than WT mice at 15 weeks. Moreover, the limbal LRC index per unit area was significantly higher (not lower) for +/−Pax6 than WT eyes at 30 weeks  so +/−Pax6 LESC numbers might actually increase with age. However, the increase in LRCs between 15 and 30 weeks was not statistically significant and there is no other evidence for an age-related increase in +/−Pax6 LESCs.If the  vessels  is consi30 weeks  also rai+/−Pax6 limbus, and perhaps all of those in the cornea, are other types of cells that divided during the BrdU exposure period but then dropped out of the cell cycle. The close association between corneal LRCs and blood vessels in +/−Pax6 corneas suggests that some LRCs may actually be vascular endothelial cells or their associated pericytes, immediately underlying the epithelium, which proliferated during the period of BrdU exposure and then terminally differentiated. Moreover, the LRC index per unit area was only significantly greater in +/−Pax6 than WT eyes in the older age group +XTAChigh = ZLhigh if corneal homeostasis is in equilibrium or YSC(?)+XTAChigh<ZLhigh if corneal epithelial cell numbers decline (excluding other encroaching cell types). Corneal epithelial TACs might be stimulated to be more proliferative in response to greater cell loss from a fragile corneal epithelium and/or to compensate for deficient or defective LESCs. This could involve decreasing cell cycle times, increasing the number of TAC divisions beyond the normal maximum or changing the balance of cell divisions so more TACs fulfil their maximum proliferative potential, as may occur in response to wounding To ensure the corneal epithelium is maintained at a uniform thickness, cell production, movement and loss must be co-ordinated and balanced. For WT corneas this balance was originally described in terms of the X, Y, Z hypothesis +/−Pax6 mice is already thin by E18.5, just before birth +/−Pax6 mice, it seems likely that increased corneal epithelial cell loss exceeds the cell production capacity and causes corneal homeostasis to become unstable, resulting in progressive corneal deterioration. This is supported by morphological evidence of cells sloughing off an irregular, vesiculated superficial +/−Pax6 epithelial layer +/Sey-NeuPax6 heterozygotes and a similar difference was seen in the present investigation (compare WT in +/−Pax6+/Sey-NeuPax6 and +/Sey-DeyPax6 heterozygotes on a different genetic background The corneal epithelium of +/−Pax6 corneal epithelium exceeds the renewal capacity of even a normal complement of LESCs. We suggest that hallmarks of corneal deterioration, including ‘conjunctivilisation’ and the appearance of goblet cells within the corneal epithelium, that are often taken as evidence of LESC-deficiency, might also occur in the absence of LESC deficiency if corneal homeostasis is destabilised by excessive cell loss.Qualitative corneal epithelial deterioration is likely, even if additional encroaching cells maintain total cell numbers adequately, because these will not be phenotypically identical to corneal epithelial stem cells and so corneal function will be compromised. A shortfall in corneal epithelial cell production may arise simply because the increased cell loss from the +/−PAX6 corneal epithelium is accompanied by a down-regulation of K12 +/−Pax6 mice and indicates that corneal epithelial differentiation is abnormal. Reduced K12 in +/−PAX6 aniridia also probably increases epithelial fragility, so contributing to the recurrent corneal erosions that are characteristic of human aniridia-related keratopathy +/−PAX6 aniridia as well as heterozygous +/−Pax6 mice. These observations indicate that the effect of Pax6 depletion on the human corneal epithelium is not restricted to the LESCs so the abnormal phenotype associated with ARK is probably not mediated entirely via LESC-deficiency.There is also evidence that reduced Pax6 levels in the human +/−Pax6 mice have a LESC-deficiency. Several possible explanations are discussed that require further investigation. Although it remains unclear whether +/−Pax6 mice have LESC-deficiency, our BrdU-labelling analysis implied that epithelial cell turnover is faster than normal and this is probably driven by increased cell loss. Excessive cell loss might destabilise corneal homeostasis and cause +/−Pax6 corneal epithelial deterioration, even in the absence of LESC-deficiency. This also raises the possibility that excessive cell loss could also play a causal role in human aniridia-related keratopathy.Quantitative LRC experiments did not support the prediction that LESCs decline with age in WT mice and failed to test adequately whether


Carpodacus mexicanus). First, we confirmed the potential for pathogen-mediated balancing selection by experimentally demonstrating that house finches with intermediate to high multi-locus MHC diversity are more resistant to challenge with Mycoplasma gallisepticum. Second, we documented sequence and diversity-based signatures of pathogen-mediated balancing selection at class II MHC in exposed host populations that were absent in unexposed, control populations across an equivalent time period. Multi-locus MHC diversity significantly increased in exposed host populations following the epidemic despite initial compromised diversity levels from a recent introduction bottleneck in the exposed host range. We did not observe equivalent changes in allelic diversity or heterozygosity across eight neutral microsatellite loci, suggesting that the observations reflect selection rather than neutral demographic processes. Our results indicate that a virulent pathogen can exert sufficient balancing selection on class II MHC to rescue compromised levels of genetic variation for host resistance in a recently bottlenecked population. These results provide evidence for Haldane's long-standing hypothesis that pathogens directly contribute to the maintenance of the tremendous levels of genetic variation detected in natural populations of vertebrates.The extent to which pathogens maintain the extraordinary polymorphism at vertebrate Major Histocompatibility Complex (MHC) genes via balancing selection has intrigued evolutionary biologists for over half a century, but direct tests remain challenging. Here we examine whether a well-characterized epidemic of Mycoplasmal conjunctivitis resulted in balancing selection on class II MHC in a wild songbird host, the house finch ( A potential role for pathogens in maintaining the extraordinary polymorphism at the vertebrate Major Histocompatibility Complex (MHC) was suspected long before the molecular role of MHC genes was understood Geothlypis trichasAvian systems are particularly promising yet challenging candidates for the study of the evolutionary ecology of MHC genes Carpodacus mexicanus), to examine whether a well-characterized pathogen epidemic caused by Mycoplasma gallisepticum (hereafter “MG”) altered levels of individual variant diversity or molecular signatures of selection at the region of class II MHC containing the peptide-binding region. We chose to examine class II MHC due to the documented importance of this MHC subtype in mediating responses to bacterial infections by initiating T-cell responses to extracellular antigens The large extent of gene duplication which characterizes passerine MHC structure necessitates a multi-locus approach to characterize functionally similar loci of unknown homology The host jump of MG from commercial poultry into free-ranging house finches in eastern North America occurred in the early 1990s, and has been unusually well documented due to the involvement of “citizen scientist” birdwatchers who can readily distinguish the conjunctival signs associated with MG infection in house finches We used paired temporal comparisons to examine changes in MHC diversity across equivalent time periods (∼10 years) for exposed, eastern North American house finches versus unexposed, western populations . We characterized individual-level MHC diversity as the number of segregating variants with unknown locus homology, and we amplified and sequenced a representative subset of variants to obtain information on sequence identity and variant frequency. Our diversity and sequence measures were based on a portion of exon 2 of the class II MHC in house finches, a region well characterized in several passerine species Guide for the Care and Use of Laboratory Animals.All procedures involving animals were approved by Cornell University's Institutional Animal Care and Use Committee (Protocol 00-90) and were carried out in strict accordance with the standards published in the National Institute of Health's pre-epidemic eastern sample only: the average SSCP variants obtained per individual from toe-pad samples (2.81 bands) and tissue samples (2.75 bands) were statistically identical, indicating that allelic drop-out from toe-pad samples did not influence our results.We used a suite of house finch tissues, museum specimens, and blood samples in order to test for signatures of balancing selection at house finch class II MHC. Tissue and toe pad samples of house finches collected across an 18 year period (1971 to 1993) were obtained from the Louisiana Museum of Natural History, the Smithsonian Museum of Natural History, and the Cornell University Museum of Vertebrates . In ordeDNA from blood and tissue samples was isolated using Qiagen Tissue Kits . Since a large proportion of our pre-epidemic DNA samples were obtained from museum toe-pads collected prior to MG emergence, all pre-PCR laboratory work was conducted using stringent ancient DNA methodologies in an isolated and dedicated facility as described previously PBR 1.3 and PBR 1.5HofiEx2F (5′ AGT GTT ACT ACA CCA ACG GCA) and HofiExR4 (5′ GTT GTG CCG GCA GTA CCT) and used these to amplify 209-bp fragments, including primers, for the current study. Cycling conditions for the initial amplification began with 10 min at 94°C, followed by 40 cycles of 94°C for 50 sec, 51°C for 50 sec, and 72°C for 50 sec, and ended with a 10 min 72°C extension. PCR products re-amplified from extracted SSCP bands (see below) were subjected to similar cycling conditions but the annealing temperature was increased to 52°C and the number of cycles reduced to 30 in order to minimize potential contamination from nearby gel bands.We used published primers We used two multi-locus approaches —cloning and SSCP— to characterize functionally similar class II MHC sequences of unknown homology E. coli (Invitrogen C8620-03). Transformed cells were grown on ampicillin-coated plates overnight, and 16 colonies were randomly selected per individual, amplified by PCR directly following colony growth using M13 primers and sequenced. Due to the high potential for mosaic, or artifactual sequences, as well as cloning errors, we only included sequences obtained from multiple colonies.We directly cloned PCR products from a total of 16 individuals, including the two individuals for which we had both DNA and cDNA (see below). We ligated fresh PCR product into pCR® 4-TOPO® using a TOPO TA Cloning Kit (Invitrogen K4530-20) and transformed plasmids into One Shot® Mach1-T1 Chemically Competent We quantified individual-level MHC diversity as the number of segregating bands produced from a single multi-locus PCR reaction. To separate variants in each PCR that differed by conformation rather than size, we used a 0.5× MDE® gel  with 0.6× TBE, 20 uL Temed and 200 uL 10% ammonium persulphate. We denatured 10 uL unpurified PCR product and 10 uL loading buffer  for 10 min at 95°C and then snap froze the samples before electrophoresing them on an OWL Vertical System (Model ADJ-2) at 4°C and 200–300 W for 18–24 hours in 0.6× TBE running buffer. SSCP gels were stained using Gelstar and visualized on UV light. We extracted visualized bands from each individual using wide-bore 10 uL pipet tips, placing the gel core immediately into PCR master mix for amplification, purification (Qiaquick Purification Kits), and sequencing on an ABI 3100 (Applied Biosystems).Due to the potential for ambiguity when scoring gels, all gels were scored blind to individual identity a total of three times. The median value was used for all further analyses. We confirmed the validity of our SSCP methods by running single variants isolated via cloning side-by-side our multi-locus amplifications of individuals known to harbor the cloned variant. In every case, banding patterns confirmed that the isolated cloned variant was present in the multi-locus amplification of the individual from which we had previously amplified that variant via SSCP. Furthermore, SSCP band segregation patterns and the sequence amplified from each band were repeatable within individuals on multiple gel runs. Because lighter sub-bands universally did not sequence cleanly following band extraction and reamplification, we did not include these potential PCR artifacts in our analysis of variant number.later (Qiagen) using Trizol® (Invitrogen). We synthesized cDNA from our extracted RNA using the AccuScript High Fidelity 1st-strand cDNA synthesis Kit (Strategene 200820). We amplified MHC from the cDNA using our primer set, cloned PCR product from cDNA and corresponding DNA for both individuals, and performed side-by-side SSCP separation. Variants obtained from both cloning and SSCP were identical for both house finch individuals, indicating that the amplified loci are expressed in house finch spleens.We extracted mRNA from two house finch spleens stored at a 1∶10 ratio in RNAIn total, we obtained 210 unique sequences from 120 house finches comprising 55 DNA variants [HQ202977-HQ203031]. The number of segregating bands per individual as determined via SCCP, which served as our measure of individual MHC diversity, varied from 1 to 9, with a mean number of 3.51 variants per individual. Because we were unable to obtain sequence for all variants detected via SSCP, we use SSCP diversity (the number of segregating bands) as our measure of individual-level MHC diversity. We use the sequenced variants as representative subsets from each population for molecular analyses. Due to the large number of variants detected at very low frequency , we wereAll microsatellite markers used had a null allele frequency less than 0.10 in our population. Two out of 32 population-locus combinations significantly deviated from the assumptions of Hardy-Weinberg equilibrium . Two of twenty-eight locus pairs showed significant linkage disequilibrium . However, population-level comparisons revealed that LD between these locus pairs was only detected in one of four populations (pre-epidemic eastern), suggesting that the loci are unlikely to be tightly linked. Microsatellite diversity indices were analyzed using GENEPOP v. 3.4 5 CFUs) of a 1994 MG isolate from a VA house finch (7994-1 6P). Eye lesions were scored biweekly for 10 weeks following infection on a 0 to 3 scale: 1 for minor swelling, 2 for moderate swelling and eversion of conjunctival tissue, and 3 if the eye was nearly hidden by swelling and crusted exudate. Mortality following experimental infection was low (2.5%) relative to that experienced in the wild (up to 60%) during the early stages of the epidemic ad libitum food and the absence of predators in captivity. We quantified disease severity for each individual as the sum of weekly conjunctival symptoms, and square-root transformed values in order to meet the assumptions of normality In order to link MHC diversity with resistance to MG, we used 39 birds that have been subject to experimental disease challenges in previous studies We used JMP 9.0  for all non-molecular statistical analyses. For the experimental challenge data, we used mixed linear models to examine associations between individual-level variant diversity and disease severity across individuals in our experimental population. We included bird identity as a random effect and microsatellite heterozygosity, which has been shown to influence disease severity in house finches To examine temporal changes in individual-level MHC diversity and microsatellite diversity  prior to and following the epidemic, we used general linear models, including population (western versus eastern), time (pre- versus post-epidemic), and their interaction in our model. For the microsatellite analyses, we considered locus-specific metrics of allelic diversity and heterozygosity for each population as independent data points in our model.We defined rare MHC variants as those that were only present in a single individual in each population . To compare the proportion of rare MHC variants in each population, we standardized for the differences in sample size among populations  by repeaWe distinguished the 30 codons that likely make up the peptide-binding region (PBR) st values across our four populations  via Mantel tests. For the genetic structure analyses, we predicted that the MG epidemic would result in significant genetic structure between pre- and post-epidemic eastern populations whereas we would not detect genetic structure between control populations sampled over the same time period (pre-control versus post-control western).We used Arlequin 3.1 n = 39, 2r = 0.34; effect tests: SSCP diversity t = −3.18, p<0.001; SSCP diversity*SSCP diversity t = 2.59; p = 0.01). The model including MHC diversity as a second-order relationship was a significantly better fit when compared to the model including MHC diversity as a first-order relationship alone . Although not statistically significant, higher microsatellite heterozygosity tended to be associated with lower disease severity , consistent with prior results in this system House finches with intermediate to high multi-locus class II MHC diversity, as measured by the number of segregating SSCP bands, showed the lowest disease severity in response to experimental infection with MG . These results reflect a ∼23% loss of MHC diversity as a result of the introduction. Individual-level MHC diversity increased significantly following the MG epidemic in eastern house finches but did not change over an equivalent time period in control, western populations . As a result, eastern and western house finches did not differ in individual-level MHC variability following the MG epidemic . We also tested whether our MHC results held true when we limited the analysis to the Tompkins Co., NY population, for which we had a large number of both pre- and post-epidemic samples (n = 72). We detected a significant increase in MHC diversity consistent with our broader meta-population patterns within Tompkins Co., NY , suggesting that the detected increase in MHC diversity across the eastern range is not biased by the inclusion of multiple populations.Prior to the emergence of MG, eastern house finch populations had significantly lower individual-level class II MHC diversity than native western house finches . Pre- and post-epidemic or control house finch populations did not differ significantly in allelic richness . Mean heterozygosity across loci was statistically equivalent for all populations and over time . However, consistent with prior work (32), mean heterozygosity at both time periods was lower for introduced, eastern populations  than for western, native populations .As detected previously (33), western native house finches had significantly higher microsatellite allelic richness than eastern, introduced populations at both time periods examined (n/ds), with high values (>1) signifying positive molecular selection s values, rendering the result difficult to interpret.Sequence-based indices tracked the SSCP diversity patterns : Tajima'n = 8 for both), western house finches harboured 15 rare variants, 1.53 times that of eastern populations (9.8 rare variants). These differences were similar post-epidemic (n = 14 for both), where western populations (15 variants) harboured 1.47 times the number of rare variants as eastern populations. Overall, these results reflect a ∼35% loss of rare MHC variants as a result of the introduction bottleneck.Western, native populations of house finches harboured, on average, 1.5 times the number of rare MHC variants  than that of eastern, introduced house finches both prior to and following the epidemic: in our pre-epidemic subsample which standardized for sample size across populations . Differences between pre- and post-control western populations accounted for only 0.31% of detected variation  while differences between pre- and post-epidemic eastern populations explained 1.35% of the total molecular variance .The analysis of molecular variance revealed that the majority (98.6%) of molecular variance for our MHC sequences was detected within populations , where low MHC variant diversity is associated with increased susceptibility to infection with both tapeworms and microsporidians Borrelia spp. bacteria M. gallisepticum is known to rapidly alter expression of its surface antigens during infection in poultry Balancing selection likely resulted in this system because the emergence of MG selected for house finches capable of responding to as many foreign antigens as possible . Harboring a larger suite of receptor-variants increases the probability that at least one of those variants is capable of initiating a T-cell response in response to one or more Mycoplasma epidemic  that may have acted in this system due to the unknown locus identity for our amplified variants. Identifying single-locus primers in passerines such as the house finch may be possible with the recent characterization of the zebra finch MHC epidemic B. The dSeveral alternative mechanisms that may also explain the detected patterns cannot be ruled out by the present study. Individual-level diversity at the examined MHC exon may not directly mediate house finch immune responses to MG antigens, but may instead be linked or correlated with an unmeasured gene(s) important for resistance. Given that the microsatellite loci distributed throughout the genome did not change in diversity following the epidemic, it is unlikely that MHC diversity in this study reflects a general measure of genetic diversity but rather a more specific response, either at the exon examined or closely-linked immune loci. Indeed, MHC regions are known to show high levels of linkage disequilibrium The technique we employed for measuring individual-level, multi-locus MHC diversity—SSCP segregation — is appropriate for passerine class II MHC loci, where gene conversion has resulted in suites of highly similar loci whose alleles are often indistinguishable via standard PCR techniques The multi-locus technique employed here allowed us to sample a broad but not exhaustive set of functionally equivalent loci. However, the inability to pinpoint locus identity, and consequently, the inability to measure locus-specific heterozgosity, remains a strong drawback. Recently, Rakus et al. methods for details). Finally, although we pool a number of geographic areas in our pre- and post-epidemic samples, our restricted analysis of Tompkins Co., NY alone indicates that it is unlikely that population genetic structure, a potential selective mechanism underlying MHC polymorphism The use of museum DNA for the majority of our pre-epidemic samples raises an alternative hypothesis that detected increases in MHC diversity reflect increasing amplification likelihood of rare variants in higher quality DNA. We used two types of controls to eliminate this possibility. First, if MHC diversity increased over time due to amplification likelihood alone, we would expect to see this pattern in our control population. In contrast, pre-control samples from western house finch populations show equal levels of diversity to post-control samples despite equal potential for degradation of museum DNA in this population. Second, we included tissue samples wherever possible in our pre-epidemic samples, and used direct side-by-side comparisons of tissue and toe-pad samples for six individuals to demonstrate comparable amplifiability of freshly collected blood and 10–20 year old museum specimens  Mycoplasma epidemic (The extent to which population bottlenecks compromise functional genetic variability for pathogen resistance in threatened vertebrate populations continues to be of strong concern epidemic B. A comIn conclusion, the unusually high fitness consequences of the house finch-MG epidemic, in combination with the discrete time period and geographic range over which the epidemic occurred, make it a particularly valuable system for directly linking pathogen-mediated selection with changes in MHC class II genes in a natural vertebrate population. In so doing, the house finch system provides direct evidence for Haldane's long-standing yet largely untested hypothesis that pathogens contribute to the maintenance of the tremendous levels of genetic variation detected in natural populations of vertebrates


Intrinsically disordered proteins (IDPs) are characterized by substantial conformational plasticity. Given their inherent structural flexibility X-ray crystallography is not applicable to study these proteins. In contrast, NMR spectroscopy offers unique opportunities for structural and dynamic studies of IDPs. The past two decades have witnessed significant development of NMR spectroscopy that couples advances in spin physics and chemistry with a broad range of applications. This article will summarize key advances in basic physical-chemistry and NMR methodology, outline their limitations and envision future R&D directions. In contrast to their stably folded counterparts, IDPs feature a rather flexible nature. The efficient sampling of a vast and heterogeneous conformational space endows them with enormous potential to interact with and control multiple binding partners at the same time and it was thus proposed that this structural plasticity and adaptability allows IDPs to efficiently engage in weak regulatory networks (such as transcription regulation). The inherent structural flexibility of IDPs mandates the use of new experimental methods since X-ray crystallography, which is by far the most utilized tool in structural biology, cannot access these proteins in the completeness of their native states. NMR spectroscopy has been developed into a powerful structural biology technique complementing protein X-ray crystallography. In particular, it offers unique opportunities for structural and dynamic studies of IDPs. A fundamental problem in the structural characterization of IDPs is the definition of the conformational ensemble sampled by the polypeptide chain in solution. Often the interpretation relies on the concept of ‘residual structure’ where the observation of structural preferences and deviations from an idealized random coil devoid of any structural propensity are interpreted as prevalence of residual structures.2Over the last decade an NMR based methodological framework has emerged to characterize the structural dynamics of IDPs. Hydrogen exchange rates, NMR chemical shifts and residual dipolar couplings (RDC) can be used to evaluate local transient secondary structure elements with atomic resolution, whereas paramagnetic relaxation enhancement (PRE) reports on transient long-range contacts 2.113C, 15N and 1H nuclei. Applications of these efficient techniques to IDPs are hampered because of severe spectral overlap and due to significant chemical exchange with bulk water that reduces 1HN signal intensities leading to low signal-to-noise (S/N) ratios. 15N–1H HSQC spectra obtained for different IDPs. While the latter can be partly overcome by measurements at low temperature and/or low pH, signal overlap problems required the development of novel NMR techniques. Capitalizing on improved instrumental sensitivities substantial improvements have been achieved due to: (i) non-uniform sampling technologies allowing for high-dimensionality experiments 13C) using cryoprobe technology and thereby circumventing problems of exchange broadening NMR signal assignment is well established for globular proteins. Typically, a suite of triple-resonance experiments is used to find sequential connectivities between neighboring residues. These experimental strategies rely on coherence transfer steps involving backbone 1H–15N experiments have been developed, that either detect 1H–15N correlations of residues following a proline (Pro-HNcocan) or preceding a proline (Pro-iHNcan) Since NMR spectroscopy of IDPs (due to their favorable relaxation properties) is typically not limited by sensitivity but rather spectral resolution, relaxation-optimized detection schemes lead to further improvements. Recently, for example, a 3D BEST–TROSY-HNCO experiment has been described following this approach Given the availability of this powerful and robust NMR methodology spectral assignment of complex IDPs has been almost become a routine task and it can thus be anticipated that even larger and more complex IDPs will be amenable to this suite of NMR experiments.2.2Chemical shifts are known to be exquisite reporters of backbone conformation and therefore considerable efforts have been made to exploit this information to probe local structural propensities of IDPs . Conversely, positive 1DNH values are found for α-helical segments Dissolving proteins in anisotropic media leads to restricted overall reorientation, thus dipolar coupling interactions no longer average to zero leading to residual dipolar couplings (RDCs) that are experimentally observable in NMR spectra 2.41H–1H nuclear Overhauser effects (NOEs) are characterized by pronounced distance dependence conventional NOESY experiments are not sensitive enough to probe distances beyond approximately 6 Å, particularly, as the effective populations of compact sub-states are generally rather small in IDPs. To study paramagnetic relaxation enhancements the protein under investigation is chemically modified by attaching paramagnetic spin labels at defined positions. Typically, the thiol groups of Cys residues (introduced via site-directed mutagenesis) are used to covalently attach the spin label. It has to be noted that the introduction of paramagnetic spin labels into the protein affects both chemical shifts  and/or signal intensities via dipolar relaxation between the unpaired electron and the 1HN and 15N nuclei R2 depending on the inverse sixth power of the distance (1/r6) between the unpaired electron and the observed nucleus. For the quantitative analysis of PRE data two approaches have been proposed. In the first approach PRE data are converted into distances using well-established methodology Undoubtedly the most relevant experimental approach to probe transient long-range contacts in IDPs employs the measurement of paramagnetic relaxation enhancements (PREs) Despite the popularity and the robustness of the PRE approach applications to IDPs are still far from trivial. Firstly, the identification of suitable spin label attachment sites without prior knowledge of the compact structure is not a trivial task as the introduction of the spacious spin label at positions that are relevant for the compact tertiary structure will inevitably perturb the structures. In the worst case, as observed for globular proteins, single point mutations can have detrimental effects on the structural stability of proteins. Thus, additional, entirely primary sequence-based analysis tools are needed for the reliable definition of attachment sites. Secondly, it has been shown that the pronounced distance dependence of PREs can lead to significant bias in the derived ensemble, although this can be partly improved by invoking independent, complementary experimental data (e.g. SAXS) 33.1Recent studies provided some insight into the molecular details of the conformational ensembles populated by IDPs in solution and call for a reassessment of the binary description scheme proposed for proteins lacking a stable tertiary structure 13Cα chemical shift index, with positive values for α-helices and negative values indicating the presence of an extended conformation. It has already been shown that this novel approach is very useful for the analysis of IDPs The meta-structure of the protein is quantified by two sequence-derived parameters, compactness and local secondary structure. Residue-specific compactness values quantify the spatial embedding of individual residues within the 3D protein structures. Residues in the interior of a structure exhibit large compactness values while residues located on the surface and exposed to the solvent display small  values. The meta-structure derived secondary structure parameter is defined in analogy to the well-established NMR Here another application of meta-structure data (e.g. compactness) is proposed. Despite the ease of experimental implementation, PRE applications are limited due to uncertainties in the identification of suitable spin label attachment sites without prior knowledge of the compact structure. In order to overcome these limitations, it was therefore suggested to use meta-structure derived compactness data to identify suitable sites of spin label attachment 3.21H–1H spin diffusion or NOE effects, when a selective inversion pulse (Hsat) is applied on aliphatic protons before the start of the pulse sequence. In this experiment, two data sets are recorded with (Isat) and without (Iref) the inversion pulse Hsat. The intensity ratio (λNOE = Isat/Iref) depends on spin diffusion effects and quantitatively probes the structural dynamics of proton spin networks λNOE ≪ 1, while in loosely folded proteins  λNOE ≈ 1. In BASP1 (Brain Acid Soluble Protein 1) a significant decrease of λNOE was observed upon lowering pH (0.75–0.60) corroborating the predicted structural compaction of BASP1 under acidic conditions. Given its ease of implementation and reliability of quantitative analysis the SOFAST-HMQC technique will be important for future studies of IDPs’ structural adaptations under varying experimental conditions.In addition to their innate conformational flexibility (plasticity) IDPs are also sensitive to changes of environmental parameters . For example, it was shown that although the thymic hormone Prothymosin-α and α-Synuclein remain natively unfolded under acidic conditions, local secondary structure propensities in proximity to acidic residues change upon variations in pH and the conformational ensemble becomes enriched in compact structures with pronounced local rigidity of the protein backbone. In a recent study, we showed that intrinsically disordered human proteins fold under acidic conditions into more compact structures with higher α-helical content largely due to reduced electrostatic repulsion of negatively charged side chains 1H–1H cross-relaxation (NOE) during adiabatic fast passage (AFP). The proposed experiment involves adiabatic fast passage radio-frequency (RF) pulses with a parabolic phase modulation leading to a linear frequency sweep through a considerably large spectral window. In addition to its well-established applications for broadband spin inversion and/or decoupling, the original AFP concept has been used to measure heteronuclear spin lock relaxation rates σeff will change sign dependent on the applied radiofrequency field. At weak ω1 longitudinal cross-relaxation (NOE) dominates the effective spin-lock cross-relaxation rate, while at strong ω1 transverse cross-relaxation (ROE) prevails and, thus, leads to the characteristic zero crossing of the spin-lock cross-relaxation rate for large molecules, where NOE and ROE cross-relaxation rates cancel. For a rigid macromolecule zero crossing occurs at an effective tilt angle of θeff = 35.26°. Enhanced internal mobility leads to zero crossing at smaller tilt-angles, while spin diffusion effects  lead to zero passages at larger tilt angles.The acid induced compaction of BASP1 was additionally probed by a novel NMR technique that measures 1H–15N-HSQC experiment with the exception that the AFP pulse replaces the NOESY mixing time and that the initial element recording 1H chemical shift evolution is replaced by a 13C-filter element to restrict NOE/ROE measurements to the dipole-interaction between aliphatic, 13C-attached protons and amide protons. In contrast to a conventional INEPT element, the delay Aτ is chosen so that 2Aτ = 1/JCH and, thus, leads to a selective inversion of protons bound to 13C-labeled carbons. Experiments are performed twice, with and without JCH scalar coupling evolution (1H inversion). Signals stemming from 13C-bound protons are selected by proper combination of sub-spectra. All other contributions, amide proton to amide proton as well as solvent to amide protons are thus largely suppressed. The results are given in 1H–1H cross-relaxation dynamics. Increasing the AFP spin lock strength  processes and are, therefore, of great interest to medical and pharmaceutical research 15N NMR relaxation data to analyze the interaction between the IDP Osteopontin (OPN) and heparin (manuscript in preparation). 15N relaxation parameters (15N-T2 and 1HN–15N NOE). As can be seen OPN exhibits distinct changes of long-range contacts upon heparin binding. In particular, spin labels at C108 and C188 experience displacements from the core region . Changes of motional dynamics were probed by 15N relaxation parameters. The regions containing most of the heparin binding site (140–170 and 180–200) display a decrease in 15N T2 due to local rigidification of the backbone upon binding , paralleled by larger 1HN–15N heteronuclear NOE values indicating reduced fast, picosecond timescale backbone motions. In contrast, the region encompassing residues 90–120 exhibits increased backbone flexibility . Interestingly, isothermal titration calorimetry (ITC) measurements provided evidence for significant enthalpy entropy compensation. More details will be given elsewhere (manuscript in preparation). Summing up, it was found that upon binding to heparin, OPN largely retains its disorder and undergoes compensatory  adaptations largely mediated through electrostatic interactions. These results indicate the relevance of dynamical adaptations in IDP complexes for thermodynamic compensations and the control of rapid substrate binding and release events in IDP interaction networks.Naturally, a comprehensive analysis has to address both structural and dynamical changes. In a recent NMR analysis we have employed both PRE and 44.11H(i)–15N(i)–13C′(i) dipolar–CSA interference can be efficiently used to discriminate between type-I and type-II β-turns in IDPs Although NMR chemical shifts are very powerful to probe local structures in proteins additional NMR parameters are desirable to define dihedral angle distributions along the polypeptide chain of IDPs. Cross-correlated NMR relaxation (CCR) has attracted substantial interest in the past as a powerful tool to study structural dynamics of proteins in solution It has to be noted, however, that an experimental single cross-correlation rate does not provide unambiguous geometrical information but is consistent with several dihedral angles. To overcome this limitation a method has been suggested exploiting the simultaneous analysis of different complementary cross-correlation rates for the extraction of unambiguous and reliable dihedral angles along the protein backbone 4.2r−3. However, the established analysis tools fail in the case of IDPs as a consequence of the rather broad pair-distribution functions between the two spin labels of a double mutant. Therefore the observed non-modulated DEER data were analyzed through an effective modulation depth, Δeff, that is an approximate measure of the average interspin distance for broad P(R)s. Δeff values were measured as a function of urea concentration  and analyzed using well-established theoretical frameworks  time scale motions are probed by ated OPN . It can 4.4Despite their annotation as unstructured/disordered there is growing NMR experimental evidence that IDPs sample heterogeneous conformational spaces comprising both extended marginally stable conformations as well as stably, eventually even cooperatively folded compact states with distinct arrangements of side-chains In this context it is important to note that a similar conclusion was made for the unfolded state of proteins 5IDPs display significant structural plasticity and undergo large structural rearrangements of the time-averaged conformational ensemble. Therefore, they seriously challenge classical structural biology that, historically, emphasized only structural aspects of proteins, the spatial arrangements of atoms in proteins and their mutual interactions resulting from a unique conformation. Proteins are characterized by a funnel-like energy landscape and thus do not exist in single conformations but exchange between many different conformational isomers (substates). Fundamental processes or interaction events such as crystallization, protein domain exchanges (swapping), conformational adaptations (e.g. induced-fit) and broad-range binding can be explained as conformational switches  resulting from the ruggedness of the energy landscape. Most importantly, this conceptual view provides a unified physico-chemical description for both globular and IDPs. While stably folded, globular proteins display a smooth bottom with only few  minima, IDPs have very rugged energy surfaces with low barriers and a large number of accessible minima. The problem of characterizing IDPs has a parallel in the history of polymer science where the introduction of quantitative statistical mechanics models allowed for the successful explanation of the dependence of physical properties of polymeric materials on molecular weight distributions


CD8 cells may contribute towards an autoimmune process in COPD. Down regulation of T cell receptor (TCR) signalling molecules occurs in autoimmune diseases with consequent T cell dysfunction. We hypothesise that TCR signalling is abnormal in COPD pulmonary CD8 cells. Micro-array gene expression analysis of blood and pulmonary COPD CD8 samples was performed and compared to pulmonary CD8 cells from smoker controls (S). We focused on the TCR signalling pathway, with validation of key findings using polymerase chain reaction and immunofluorescence. TCR signalling molecules in COPD pulmonary CD8 cells were down regulated compared to blood CD8 cells (CD247: fold change (FC) −2.43, Q = 0.001; LCK: FC −2.25, Q = 0.01). Micro-array analysis revealed no significant differences between COPD and S pulmonary CD8 cells. However, PCR revealed significantly lower gene expression levels of CD247  and LCK  in COPD compared to S pulmonary CD8 cells. CD247 down regulation in COPD CD8 cells was confirmed by immunofluorescent staining of bronchoalveolar lavage cells: Significantly fewer COPD CD8 cells co-expressed CD247 compared to healthy non-smoker CD8 cells  There is down regulation of TCR signalling molecules in COPD pulmonary CD8 cells. This may cause T cell dysfunction. Chronic obstructive pulmonary disease (COPD) is characterised by poorly reversible airflow obstruction and airway inflammation that develop as a result of an abnormal inflammatory reaction to the inhalation of noxious particles, most commonly cigarette smoke Historically, there has been much focus on the role of the innate immune system in COPD The T cell receptor (TCR) is responsible for recognising antigens from pathogens such as viruses, in order to generate an immune response In order to further elucidate the role of CD8 cells in COPD, we undertook molecular phenotyping of COPD CD8 cells, using microarray analysis. This revealed decreased expression of key components of the TCR signalling pathway in pulmonary CD8 cells. It was subsequently confirmed using specific polymerase chain reaction (PCR) analysis and protein quantification by immunofluorescence that TCR components including CD247 are down regulated in COPD pulmonary CD8 cells compared to controls.2 separate cohorts of patients were recruited for this study – see PBMCs were obtained from venous blood by Ficoll-paque centrifugation . CD8 cells were isolated from PBMCs using positive selection CD8 microbeads .Pulmonary CD8 cells were isolated from explanted human lung. Briefly, lung tissue was homogenised in a blender .BAL was obtained using a flexible bronchoscope whilst volunteers were under conscious sedation. The bronchoscope was wedged sub-segmentally and 240 ml of 0.9% saline was instilled and aspirated from each upper lobe. BAL fluid was filtered  and resuspended in RPMI-1640  at a concentration of 1×10Cells were lysed in RLT buffer  +1% β-mercaptoethanol (Sigma-Aldrich) and stored at −80°C. Samples were homogenised with Qiashredder (Qiagen) and ribonucleic acid (RNA) was extracted using RNeasy mini kit (Qiagen) with DNAse I treatment (Qiagen) according to manufacturer’s instructions. RNA was quantified and quality assessed using Agilent 2100 bioanalyser.RNA was amplified, converted to double stranded cDNA and biotin labelled according to Affymetrix protocols . Biotinylated cDNA was hybridised to Human U133 plus 2.0 chips (Affymetrix). The gene chips were scanned using an Agilent gene array scanner 3000 and fluorescence intensity was analysed using gene chip operating software (Affymetrix). Quality control of the gene chips was checked using AffyQCreport R software (Bioconductor.org). The full datasets have been submitted to arrayexpress (Accession number E-MTAB-1688).50 ng of RNA was reverse transcribed using the Verso 1-step QRT-PCR mix kit  according to manufacturer’s instructions. Genomic DNA contamination was monitored with no-RNA control reactions. TaqMan Reverse transcription PCR was performed in duplicate for each sample using 1 µl of cDNA in a 25 µl reaction in step 2 of the Verso 2-step QRT-PCR mix kit (Thermo-scientific) containing 0.5 µl of premade ABI TaqMac gene expression assays for CD247 , Linker of activated T cells , LCK , VAV2 guanine nucleotide exchange factor  and VAV3 guanine nucleotide exchange factor  and the endogenous control was 18s ribosomal RNA . Gene expression levels were calculated using the comparative CT method normalised to 18s.Cells were fixed in frozen methanol (Sigma-Aldrich), washed in phosphate buffered saline and then blocked with 1.5% goat serum . Cells were then incubated overnight at 4°C in a cocktail of mouse anti-CD8  and rabbit anti-CD247  diluted in goat serum. Cells were washed and then incubated with a cocktail of alexa-488 conjugated goat anti-mouse secondary antibody  and alexa-568 conjugated goat anti-rabbit secondary antibody (Invitrogen) for 90 minutes at 37°C. Cells were washed, nuclei counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted with vectashield (Vector laboratories). CD8 positive cells and dual-labelled CD8+, CD247+ cells were counted using a Nikon eclipse 80i microscope . Alexa-488 (green) and Alexa-568 (red) conjugated secondary antibodies were visualised using exciter filters with band widths of 465–495 nm and 540–580 nm respectively. For dual-label images, fluorescent images from the same field were captured and digitally merged to identify CD8 staining cells (green) that co-stained with CD247 (red). Images were obtained of all identified CD8 cells. Cell counting was performed by an individual blinded to the patient information until after cell counting was complete.Differential gene expression analysis was performed by linear modelling using the lmFit and eBayes functions of the Limma software package Principal component analysis (PCA) was performed to compare COPD pulmonary samples with paired blood samples and also COPD pulmonary samples with S pulmonary samples. Arrays were plotted on a 2-dimensional image of principal components to show trends in gene expression.The list of genes differentially expressed between paired blood and pulmonary COPD samples  was inputted into the Database for Annotation, Visualisation and Integrated Discovery (DAVID) The genes identified within the cluster of interest were input into the Search Tool for Retrieval of Interacting Genes/proteins (STRING). This database creates networks of genes based upon known or predicted protein-protein interactions Due to the small group sizes it was not possible to accurately test all datasets for normality. Therefore data was analysed using non-parametric tests. Gene expression levels measured by PCR were compared using Mann-Whitney U test. Immunoflourescence data were compared between groups using Kruskal-Wallis test, with subsequent pairwise comparisons using Dunn’s multiple comparisons test if Kruskal-Wallis p<0.05. For PCR and immunofluorescence data, p<0.05 was considered statistically significant. Statistical analysis was performed using Graphpad inStat .The procedure to isolate CD8 cells from COPD and S lung tissue resulted in a near-pure population, with 90.2% and 89.8%  CD8 cells positive for CD8 by immunofluorescence. The isolation of CD8 cells from COPD blood samples resulted in marginally higher CD8 purity (mean 95.1%).PCA showed a clear separation between clusters relating to COPD pulmonary and peripheral blood CD8 samples. PCA showed no significant variation between COPD and S pulmonary samples .There were 2938 probe sets accounting for 1924 genes that were differentially expressed between paired COPD pulmonary and peripheral blood samples, and the most highly regulated genes are shown in DAVID was used to identify clusters of genes specifically associated with T cell activation or signalling; one cluster was identified that was composed of genes specific to T cell function. There were a total of 51 genes within the entire cluster  comprisiNatural killer (NK) cells can co-express CD8 but do not express TCR. NK cell markers  were either similar between blood and lung or significantly higher in the blood samples, ruling out the possibility of reduced TCR gene expression in pulmonary CD8 cells due to a higher proportion of NK cells in the pulmonary preparations .There were no statistically significant differences between the gene expression profile of the isolated cells from COPD and S pulmonary samples in the microarray analysis. However, the expression levels of the TCR genes which were down-regulated in pulmonary compared to blood samples revealed fold changes showing decreased expression of CD247  and LCK  in COPD pulmonary CD8 cells compared to S pulmonary CD8 cells, but Q values that were non-significant.Quantitative real time PCR was performed for CD247, LCK, LAT, VAV2 and VAV3; these 5 genes relate specifically to TCR signalling and were differentially regulated between COPD pulmonary and blood CD8 cells in the microarray analysis. PCR gene expression levels were significantly different for CD247 , LAT  and VAV2  between COPD pulmonary and blood cells . The difAlthough analysis of the microarray dataset did not reveal any statistically significant differences between COPD and S pulmonary samples, this may be related to the relatively high false negative rate associated with multiple testing in micro-array analysis. Therefore, we analysed the 5 gene panel  by applying PCR to the COPD and S pulmonary samples; CD247  and LCK  were both expressed at significantly lower levels in COPD compared to S pulmonary CD8 cells . There wCD247 was chosen for protein analysis in a separate cohort of patients as this protein was significantly differentially expressed between blood and lung COPD CD8 cells and also down-regulated in COPD compared to S pulmonary CD8 cells. Furthermore, CD247 is known to be down-regulated in other autoimmune diseases BAL cytospins from 7 COPD patients, 5 S and 8 HNS were dual stained for CD8 and CD247 . A mediaOur understanding of the phenotype of CD8 cells within COPD lung remains limited. We now show that components of the TCR in COPD pulmonary CD8 cells, including CD247, LCK, LAT and VAV3, have reduced expression compared to circulating CD8 cells in the same individuals. These changes in CD8 gene expression may be due to local factors unique to the lung microenvironment. We also demonstrate down regulation of CD247 and LCK in COPD compared to S pulmonary CD8 cells using qRT-PCR and immunofluorescence. It appears that pulmonary CD8 cells from COPD patients have down-regulated expression of key components of the TCR signalling pathway compared to controls.CD247 (or TCR-ζ) is a transmembrane protein with a number of immunoreceptor tyrosine-based activation motifs (ITAMs) that plays a key role in both transduction of TCR signalling and also in anchoring the TCR to the cell membrane The onset of COPD is associated with an increase in CD8 numbers There were no statistically significant differences found between COPD and smoker control pulmonary CD8s in the microarray data. The nature of the statistical analysis of large microarray datasets means that correction for multiple testing must be made. In a limited sample size, it is inevitable that false negative findings will occur using this approach. The PCR and immunohistochemistry analysis performed did not require such stringent correction for multiple testing, and so were able to demonstrate significantly lower levels of CD247 and LCK in COPD compared to S pulmonary CD8 cells.LCK is a member of the Src family of tyrosine kinases which controls the initiation and amplification of TCR signalling. Antigen binding to the TCR promotes LCK recruitment to the TCR complex where it phosphorylates ITAMs in the CD247 complex thus beginning the TCR signalling cascade Natural killer cells can co-express CD8 but do not express the T cell receptor. Increased numbers of NK cells within the pulmonary samples compared to the peripheral blood samples could be an explanation for the key findings within the current studies. However, there were no significant differences in the microarray data of levels of NK cell markers such as CD56 suggesting no difference in NK cell levels between the samples.The results reported here concern the expression of CD8 genes, with validation of CD247 by protein analysis. We have not studied the functional consequences of the observed changes in gene expression. It will be important to understand whether the reduction in the expression of TCR associated components in COPD CD8 cells are associated with a loss of function. This may be important clinically; for example, when considering the ability of CD8 cells to mount an antigen specific response to pathogens.We used immunofluorescence to evaluate CD247 expression using samples that had been historically collected for research purposes. An alternative method to evaluate CD247 in bronchoscopy samples is flow cytometry, which is potentially more sensitive as more cells are counted. However, flow cytometry requires fresh samples to analyse immediately after collection, which was not possible with our historic samples. Nevertheless, the immunofluorescence analysis for CD247 provided results compatible with the reduction in expression observed by PCR in COPD patients.The gene expression study using lung surgical tissue had a limited sample size, but was sufficiently large enough to identify candidate genes by microarray that were validated using PCR and immunohistochemistry. The limited sample size meant that the effect of clinical variables such as smoking pack year history and severity of disease could not be accounted for. Furthermore, the practicalities of obtaining lung tissue from patients undergoing cancer surgery preclude the collection of accurate COPD phenotype data such as emphysema assessment by high resolution CT scan, as the patients are usually “fast tracked” to surgery leaving insufficient time to perform extensive research procedures. Furthermore, the cancer itself can impact on COPD symptoms such as exacerbation rates or breathlessness. Nevertheless, the relationship between COPD clinical characteristics and CD8 phenotype is of interest, but should be addressed in studies that obtain cells from COPD patients without lung cancer.It would have been preferable to include a group of never smokers in the gene expression analysis, but no such patients underwent surgery for lung cancer during the course of the study. Only a small number of never smokers develop lung cancer, which made it practically very difficult to include such a group. We used historically collected samples from bronchoscopy to evaluate CD247 protein, and were able to include a group of non-smokers in this analysis.In the gene expression study, it should be noted that all the COPD patients and controls were ex-smokers; this ensured that there was no confounding effect of current cigarette smoking on gene expression. In the bronchoscopy study, the COPD patients were all current smokers, and it is possible that current smoking may have an effect on CD8 gene expression. Nevertheless, there was a difference in CD247 expression in COPD patients compared to HNS, while this was not observed between S and HNS; this suggests the downregulation of CD247 occurs because of COPD and not current cigarette smoking.It has been assumed that an increase in the pro-inflammatory and cytotoxic activity of CD8 cells is the principal mechanism by which these cells contribute to the pathophysiology of COPD Table S1Most significantly regulated genes between pulmonary and peripheral blood samples.(DOCX)Click here for additional data file.Table S2Annotation terms within Functional Annotation Cluster.(DOCX)Click here for additional data file.Table S3Expression of natural killer cell markers in blood and lung samples.(DOCX)Click here for additional data file.Methods S1Detailed methods for the isolation of pulmonary lymphocytes from explanted human lung.(DOCX)Click here for additional data file.


Idiopathic pulmonary fibrosis exhibits differential progression from the time of diagnosis but the molecular basis for varying progression rates is poorly understood. The aim of the present study was to ascertain whether differential miRNA expression might provide one explanation for rapidly versus slowly progressing forms of IPF.AGO1 and AGO2  were lower compared with either slow or normal lung biopsies and fibroblasts.miRNA and mRNA were isolated from surgical lung biopsies from IPF patients with a clinically documented rapid or slow course of disease over the first year after diagnosis. A quantitative PCR miRNA array containing 88 of the most abundant miRNA in the human genome was used to profile lung biopsies from 9 patients with rapidly progressing IPF, 6 patients with slowly progressing IPF, and 10 normal lung biopsies. Using this approach, 11 miRNA were significantly increased and 36 were significantly decreased in rapid biopsies compared with normal biopsies. Slowly progressive biopsies exhibited 4 significantly increased miRNA and 36 significantly decreased miRNA compared with normal lung. Among the miRNA present in IPF with validated mRNA targets were those with regulatory effects on epithelial-mesenchymal transition (EMT). Five miRNA  were significantly increased in rapid compared with slow IPF lung biopsies. Additional analyses of rapid biopsies and fibroblasts grown from the same biopsies revealed that the expression of These findings suggest that the development and/or clinical progression of IPF might be the consequence of aberrant miRNA processing. Idiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disorder characterized by excessive, irreversible scarring of the lungs Although a high degree of variability in IPF progression has been observed in patients Dicer1 is required for further processing There is growing evidence that the regulation of gene transcription in health and disease involves several non-redundant mechanisms. One such mechanism involves microRNA, which are 22 nucleotide non-coding RNA molecules that exert a biologically important effect on post-transcriptional gene expression MicroRNAs have been implicated in cancer, heart and neurodegenerative diseases, diabetes, and inflammation AGO1 and AGO2 (the core components of the RISC) were expressed at lower levels in rapidly progressive IPF biopsies and/or fibroblasts grown from the same biopsies compared with both normal and slowly progressive IPF biopsies and fibroblasts. Together, these data suggest that the development of IPF and/or the pace of its clinical progression might be a consequence of abnormal miRNA generation and processing.The aim of the present study was two fold: 1) to determine whether miRNA biosynthesis differed between IPF and normal lung tissues; and 2) to determine whether miRNA expression differed between IPF patients exhibiting varying speeds of disease progression. Herein, we report that the miRNA levels in diagnostic lung biopsies markedly differ in both rapidly progressive and slowly progressive IPF patients compared with normal individuals, albeit the large majority of the miRNA identified were significantly lower in the IPF biopsies compared with the normal biopsies. Among the miRNA present in IPF with validated mRNA targets were those with regulatory effects on epithelial-mesenchymal transition (EMT), a process of emerging importance in IPF CO) decrease of ≥15%. IPF patients that did not meet these criteria over the first year of follow-up were clinically diagnosed as slowly progressive . Samples taken from disease-free patient autopsies were used as normal controls . A University of British Columbia Institutional Review Board approved the procurement and analysis of these tissues.All surgical lung biopsies (SLBs) were obtained from the patient at the time of diagnosis of IPF. Patients were diagnosed with IPF using a multidisciplinary approach involving a clinicians, radiologists, and pathologists 2 qPCR-Grade miRNA Isolation Kit . Concentration and purity were again measured after miRNA isolation using the NanoDrop 1000 photospectrometer and samples were screened for an A260∶A280 ratio greater than 1.8. Conversion of miRNA to cDNA was carried out using 100 ng of miRNA according to manufacturer's instructions for the RT2 miRNA First Strand Kit .Left side upper and/or lower lobe surgical lung biopsies were stored at −80°C and thawed on ice immediately prior to RNA isolation. Approximately 1 ml of Trizol  was added to each biopsy. Each biopsy was homogenized using the Tissue-Tearor  and total RNA extraction was performed according to the manufacturer's instructions. Concentration and purity of each lung sample was determined using a NanoDrop 1000 UV-Vis photospectrometer . 1 µg of total RNA from each biopsy was converted into cDNA using Murine Moloney Leukemia Virus Reverse Transcriptase . MiRNA was isolated and purified from 10 µg of total RNA according to manufacturer's instruction for the RT2 miRNA PCR Array Human miFinder . Plates contained 88 of the most abundantly expressed and best-characterized miRNAs found in humans as well as relevant housekeeping miRNA. Samples were analyzed by quantitative real time polymerase chain reaction (qRT-PCR) using the ABI 7500 Real Time PCR System  following the manufacturer's protocol. A dissociation curve was run to factor out wells with nonspecific amplification and primer dimers. Samples were grouped as normal, slowly progressive, and rapidly progressive and analyzed using the PCR Array Data Analysis Excel Template with the ΔΔCt method . Data were normalized using the small nuclear RNA housekeeping panel: SNORD 47, SNORD 44, and U6.MicroRNA expression values for each sample were measured using a RThttp://mirecords.biolead.org/) MiRecords  were analyzed using a preloaded custom TaqMan Array Microfluidic Card . Data were normalized to an internal standard, GAPDH in the 96 well format and to 18s in the card. Samples were grouped into either slowly progressive IPF or rapidly progressive IPF, and compared with normal using the ΔΔCt method.Circular DNA (cDNA) made from total RNA was analyzed using quantitative real time polymerase chain reaction (qRT-PCR) using the ViiA 7 Real-Time PCR System . Pre-mixed primers and probes were purchased from Applied Biosystems and used to detect human 2O2 solution to permeablize tissue sections and block endogenous peroxidase activity. Tissue sections were additionally blocked for with 5% BSA/ 1% rabbit serum, Avidin Block , and Biotin Block . The sections were incubated overnight at 4°C in anti-AGO1 rabbit polyclonal antibody  or anti-AGO2 rabbit polyclonal antibody . In-house non-specific rabbit polyclonal IgG was used as a concentration matched negative control. Sections were incubated with biotinylated anti-rabbit secondary antibody  for 1 hour then with HSS-HRP Streptavidin Peroxidase  for 30 minutes. Tissue was stained with DAB chromogen, counterstained with hematoxylin, and photographed with an Olympus BX40 microscope and IP Lab Spectrum software .Five-micron thick paraffin-embedded tissue sections were cut from formalin-fixed normal, slowly progressive, and rapidly progressive SLBs. Each tissue sections was de-parafinized and rehydrated by washing with xylene and a decreasing ethanol gradient. Sections were heated in 10 mM citric acid (pH 6.0) for 12 minutes for antigen retrieval and incubated for 30 minutes in a 10% methanol/ .4% H2 cell culture flasks with media. The media consisted of DMEM  with 15% fetal bovine serum , 2 mmol/L glutamine, and 1× Penicillin-Streptomycin-Amphotericin B . Cells lines were cultured in media at 37°C in a 7% CO2 incubator and were serially passaged 4 times to yield pure populations of adherent fibroblasts. All primary fibroblast cell lines from each patient group were used between passages 6 and 11. Prior to an experiment, fibroblasts from each group  were plated into a six-well tissue culture plate with 5×105 cells per well and activated for 4 hours with media alone. Trizol reagent was added to each well to terminate the experiment and mRNA was isolated and analyzed as described above.To culture primary human fibroblasts, histologically normal and IPF SLBs were finely minced and the dispersed tissue pieces were placed into 150 cmwww.R-project.org). The “Ward” method, which derives spherical clusters has been used as the agglomeration algorithm for forming clusters.Student's T-test or Mann-Whitney test for non-normally distributed data were used to determine statistical differences among the analyzed groups. Calculations were performed using PRISM 5.0 software for Macintosh . P≤0.05 was considered statistically significant. The statistical analysis performed on the EMT-associated genes used qRT-PCR generated dCT values following the (Ct of gene of interest (GOI) – Ct of housekeeping gene (HKG)) calculation. Data were clustered using the absolute value of correlation coefficients (distance measure) using hierarchical clustering to show the genes that are strongly related in each group  . These data revealed that many transcripts relevant to fibrosis are validated targets of the miRNA present in the normal and IPF biopsy samples including many EMT-related genes (Supporting Table S1).We next hypothesized that a decrease in miRNA expression might explain, in part, the increased expression of fibrosis-related mRNA transcripts observed in IPF biopsies compared with normal or non-fibrotic lung biopsies. Unfortunately, predicting miRNA gene targets is challenging because each target prediction method predicts thousands of mRNA transcripts per miRNA. Since each target prediction program's methodology differs, these programs also predict discordant mRNA transcripts for a given miRNA. To lessen the discordance in miRNA target prediction, we focused on compiling only experimentally validated miRNA targets after an analysis of interactions. A list of experimentally validated targets was compiled using the miRNA that differed between slowly progressive and rapidly progressive IPF biopsies compared to normal biopsies  mRNA levels were significantly decreased in slowly progressive biopsies and rapidly progressive biopsies in comparison to normal tissue  was apparent in rapid IPF versus slow IPF.We next determined whether EMT-related genes differed in the three groups of lung biopsies using TaqMan Array Microfluidic Cards, and these data are summarized in HMGA2Further statistical analysis of miRNA expression in biopsies from both groups of IPF patients revealed that 5 miRNAs were increased in rapid IPF versus slow IPF, and one miRNA was decreased in rapid IPF versus slow IPF. A summary of these miRNA and their validated targets is shown in AGO1, which is a core component of the RNA induced silencing complex (RISC) Supporting Table S1). Argonaute proteins function to bind the miRNA and correctly position it to recognize the appropriate mRNA targets AGO1 and AGO2 were analyzed at the transcript and protein level in IPF and normal biopsies. Compared to normal biopsies, AGO1 transcript levels in slow and rapid IPF biopsies were significantly lower compared with levels in normal biopsies  targeted AGO1 revealed very little regardless of the antibody dilution or biopsy group, perhaps due to the lack of a suitable antibody for formalin-fixed tissues. However, AGO2 was detected in both slowly progressive and rapidly progressive sections when this antibody was used at a dilution of 20 µg/ml  in IPF patients. Thus, the findings from the present study coincide with other published findings regarding the differential expression of miRNA in clinical and experimental fibrosis.Our findings are consistent with a number of recently published studies directed at the characterization of miRNA expression and function in pulmonary fibrosis. First, a recent study by Pandit DICER1 enzymatic activity is necessary for miRNA biogenesis and loss of DICER1 expression through gene silencing and knockout approaches has been shown to cause aberrant or decreased endothelial cell proliferation DICER1 is also associated with familial pleuropulmonary blastoma DICER1 caused overall reductions in mature miRNA levels, which contributes to the loss of transcript regulation. However, analysis of DICER1 expression revealed no differences in the amounts of this transcript amongst the biopsies and fibroblast lines analyzed, leading us to presently conclude that changes in the expression of DICER1 do not appear to explain the differential miRNA levels in IPF versus normal.ZEB1 and SIP1, which repress E-cadherin and promote mesenchymal marker expression. As expected CD44, COL1A2, VIM, and FOXC2 were significantly increased in rapidly progressive IPF over normal and/or slowly progressive IPF biopsies. CD44 is a membrane associated cellular adhesion receptor that plays a role in remodeling the lung COL1A2 has been previously described in IPF Vimentin is a definitive marker for the meshchymal cells derived from epithelium VIM were significantly increased only in rapidly progressive IPF. These data suggested that levels of a particular miRNA might be less important than the processing efficiency of a particular miRNA. Together, these data highlight that transcripts associated with EMT were significantly elevated in IPF, particularly the rapidly progressive form of this disease. The impact of differential miRNA expression on EMT during IPF progression is presently not clear and requires further investigation.EMT is the transition of differentiated epithelial cells into motile mesenchymal cells, and this process is prominent in both experimental and clinical pulmonary fibrosis AGO2 is the catalytically active “slicer” that cleaves mRNA transcripts Argonaute proteins bind miRNA and position it in a conformation that promotes mRNA target recognition In summary, our results demonstrate that miRNA profiling in diagnostic surgical lung biopsies differentiates normal from IPF, and rapidly progressive IPF from slowly progressive IPF. Further analysis of miRNA levels in circulating cells as a means of ascertaining IPF disease progression is certainly warranted given the present findings. Alterations in circulating miRNAs have been detected in other diseases such as cancer. Finally, our results indicate that aberrations in the miRNA processing pathway might be the cause of altered transcript expression leading to the fibrotic phenotype in IPF and the differing speed of progression of this disease.Table S1This table is a list of the experimentally validated gene targets compiled using the miRNA species that differed between slowly progressive and rapidly progressive IPF biopsies compared with normal lung biopsies.(DOCX)Click here for additional data file.