The results provide direct experimental evidence of ripple formation, sp2 to sp3 hybridized carbon transformation, electronic damage, Ar+ implantation, unusual defects and edge reconstructions in FLG, which depend on the irradiation energy. In addition, shadowing effects similar to those found in oblique-angle growth of thin films were seen. Reliable quantification of the transition from the sp2-bonding to sp3-hybridized state as a result of Ar+ ion irradiation is achieved from the deconvolution of the XPS C (1s) peak. Although the ion irradiation effect is demonstrated through the shape of the derivative of the Auger transition C KVV spectra, we show that the D parameter values obtained from these spectra which are normally used in the literature fail to account for the sp2 to sp3 hybridization transition. In contrast to what is known, it is revealed that using ion irradiation at large FLG sample tilt angles can lead to edge reconstructions. Furthermore, FLG irradiation by low energy of 0.25 keV can be a plausible way of peeling graphene layers without the need of Joule heating reported previously.Roughness and defects induced on few-layer graphene (FLG) irradiated by Ar Ion irradiation of materials subjected to different ion energies, ion doses and irradiation geometries such as angle of incidence of the beam, sample tilt angle and sample rotation has been widely studied ,2. In ad2/Si substrate exposed to 30 keV Ga+ ion irradiation pointed out the requirements for developing a more thorough understanding of graphene's ability to withstand prolonged ion irradiation. These include: (1) the absence of cascade collisions due to the 2-D graphene nature, (2) the possibility of C atoms not to be displaced from the lattice as the ion energy is completely transferred to the substrate underneath, (3) graphene open structure which facilitates the ion channeling, hence limiting the number of ion/target collisions and ejected carbon atoms, and (4) implanted ions which might cause a net positive charge to build up in graphene and electrostatically repel the subsequent incoming incident ions. In addition to the above issues, the importance of this study helps to understand the damage production mechanisms and types of defects created by the energetic ions in the sample for the efficient use of ion beams and optimization of the graphene cutting process [ex situ prepared graphene layers [sp2 bonding configurations and affect the graphene carrier mobility, this study shade light on defects associated with this process which are equally important. In this respect, applications and mechanisms based on defects in graphene such ferromagnetism [Experimentally, Lopez et al.  in their process . Further process , eliminae layers  and grapagnetism , creatioagnetism , porous agnetism , atmosphagnetism  and grap+ with energies from 0.25 to 5 keV at incidence angle of 45° and sample tilt angle of 66° to study the irradiation effects in FLG. In addition to defects found on flat irradiated areas, we show that FLG edge defects associated with sp2 to sp3 hybridized carbon transformation are most typical in the low energy range and irradiation geometry used. In addition, we show that the reliable quantification of the sp2/sp3 hybridized state ratio as a function of irradiation energy can be achieved from the deconvolution of the XPS C 1s envelope. This ratio, together with newly developed features in the Auger transition C KVV spectra and π − π* transition behavior as a function of irradiation energy provide insights on the FLG structural and electronic damage. Furthermore, we point out the possibility of a combined effect of ion implantation and ion reflection to be responsible for the low rate of sputtering of FLG.Here, we have employed low energy Ar+ ion irradiation for 30 min for energies ranging from 0.25 to 5 keV at incidence angle of 45° and sample tilt angle of 66° in the ultra high vacuum conditions.FLG samples were obtained by peeling layers from highly oriented pyrolytic graphite (HOPG ZYA) using adhesive tape. They were then fixed on steel substrates by double sided stick carbon tape. These substrates were then subjected to Ar−10 mbar. The chemical composition of the sample was extracted from the wide scan using CASA XPS software . Short scans were recorded at pass energy of 20 eV. In order to avoid charging effect during the scans, an electron gun flooding was used for charge compensation.XPS measurements were carried out using an Omicron Nanotechnology XPS system  using a monochromatic Al Kα radiation . The source voltage and emission current were 15 kV and 20 mA, respectively. The base pressure at which the scans were done was 10The nanoscale images presented were performed using Nanoscope V atomic force microscope (AFM) obtained in tapping mode using ultra high resolution cantilevers made of tungsten having radius of less than 1 nm and force constant of 46 N/m. During the imaging, both the scan rate and the imaging resolution were set at 0.5 Hz and 512 × 512 pixels, respectively.+ ion energies ranging from 0.25 to 5 keV. At first glance, roughness and well-known ripple structures of varying sizes and wavelengths [Ra) and RMS roughness (Rq) reveals that the roughness is not monotonically increasing as a function of irradiation energy; instead, a reduction in the roughness is found at 1.5 keV as indicated in Figure Figure elengths  are visif) with ripple wavelength (λ) of =24 nm. After irradiation at 0.25 keV, the PSD spectrum superimposes to that of the un-irradiated sample, indicating negligible damage of the FLG surface. Interestingly, a decrease in the graphene PSD intensity is observed at energies higher than 0.25 keV for the spatial frequencies between 60 and 120 μm−1 .The evolution of the large scale morphology was probed by two-dimensional power spectral density (PSD) analysis ,17. PSD λ = 72 nm that resulted from sputtering of both the first and the second graphene layers. It should be noted that the irradiation by 1.5 and 5 keV will only affect the first and the second FLG layers; hence, the assignment of ‘second layer’ and ‘first layer’ is used in Figure This suggests strong suppression, smoothing and lateral coarsening of ripple structures  at these energies. At low frequency regime, the spectra show a switching behavior where the PSD intensities are seen to be higher than that of un-irradiated and increase by a factor of 10 and 100 for the 1.5 and 5 keV irradiations, respectively. This increase is a sign of large amplitude ripple formation with long periodicity of K f−β where β and K are the spectral index and spectral strength, respectively [β1 = 6.0 and β2 = 6.5. Since β is related to α by the equation α = (β − d)/2 [d is 2, then α1 = (6–2)/2 = 2 and α2 = (6.5 − 2)/2 = 2.25. Here, α1 attributes to the roughness exponent associated to the first FLG layer and α2 could be assigned to roughness exponent of the second FLG layer.In order to further shed light on the morphology of the two layers after the 5-keV ion irradiation, the roughness exponent α has been determined from the 5-keV PSD spectrum in the high frequency region. It is evident that the spectrum shows bimodal intensity trend /2 , where ts), Ar (2p), and Ar (2s) core level excitations recorded from FLG samples after Ar+ ion irradiation at different energies. It is clear from the XPS spectra and from the inset of Figure + ions are implanted into the FLG and reached the saturation limit at energies ≥ 3 keV.Figure + irradiation might be the reason. In addition, the commonly observed π − π* transition in graphene at binding energy of 290 eV [sp2-bonding to sp3-hybridized state as a result of Ar+ ion irradiation.Also, variation in the effect of ion irradiation can be observed from the newly developed shoulder (shoulder labeled A) in the XPS spectra along with the increasing of the irradiation energy. The origin of shoulder A is unclear at the moment; however, complex defects previously theoretically predicted  to be asf 290 eV  seems to+ at 5 keV for 30 min) of FLG to the Ar+ ion irradiation stems from the combined effect of the Ar+ implantation as evident from Figure + ions build up, they electrostatically repel subsequent incoming incident ions, therefore reducing number of ions involved in collisions. This charge repulsion can be understood from the ion concentration saturation observed in the inset of Figure + ions can also take place and lead to the saturation observed. Auxiliary experiments at constant irradiation energy and at different tilt sample angles were carried out to confirm the effect of the reflection of the incoming ions on the resilience of FLG. Indeed, the results (to be reported elsewhere) obtained from the ion irradiation of FLG samples at constant energy of 1 keV, and different sample tilt angles (Φ) from 0° to 80° show that the amount of implanted Ar+ ions seems to decrease as the tilt angle is increased.It can be concluded that the resilience  XPS spectra of FLG samples for the un-irradiated and irradiated at energies 0.5 and 4 keV. The fitting of the spectra were done by Gaussian-Lorentzian functions with a Shirley background subtraction [sp2 and sp3-hybridized states, respectively [sp2 peak appears to be shifted to higher binding energies at high Ar+ ion irradiation energies.Figure traction . The fitectively . The sp2sp2/sp3 ratio along with the full width at half maximum (FWHM) of the sp2 and sp3 peaks as a function of irradiation energy is shown in Figure sp2 hybridization dominates  - a FLG characteristic of a two-dimensional sheet of sp2 bonded carbon atoms in a honeycomb lattice [sp3 in the un-irradiated sample is attributed either to the edge of individual graphene layers or to the intrinsic sp3 defects reported to exist in the FLG samples [sp2 to sp3 hybridization due to oxygen exposure of FLG [s peak in the XPS spectra.Quantification of the  lattice -27. Howe samples . The wele of FLG  is ruledsp2 to sp3 hybridization as seen in region I of Figure sp2 character due to the enhanced possibility of the damage of the edge of the graphene structure, consequently the formation of dangling bonds. An amorphous stable mixture containing equal sp2 and sp3 hybridizations  is observed (region II) when employing irradiation energy in the range from 1.5 to 4 keV. At high irradiation energies above 4 keV, the number of transformed sp2 bonds seems to be slightly less as depicted in region III of Figure sp2 contribution from this layer and the layers beneath up to approximately 2 nm of the expected XPS sampling depth in the geometry used. In the lateral direction and as seen from the AFM image at 5 keV in Figure sp2 character are expected to exist. The reduced number and the large size of these pits are reflected from the long periodicity of λ = 72 nm and two orders of magnitude increase in PSD intensity found in Figure On the other hand, the initial irradiation of the FLG causes fast transformation of D) between the most positive maximum and most negative minimum obtained from the first derivative Auger transition C KVV spectra recorded from FLG samples, as a function of Ar+ ion irradiation energy.To further account for the effect of the ion irradiation, we adapt the commonly used first derivative of C KVV spectra obtained from XPS data . The selD values are within the reported values of 21 to 23 eV for HOPG (multi-layer graphene) [sp2 to sp3 transition trend similar to that observed in Figure D parameter values obtained from these spectra fail to account for sp2/sp3 hybridization transition, and reliable quantification of sp2/sp3 ratio is only obtained from the deconvolution of the XPS C (1s) envelope as explained before and shown in Figure The raphene) , but notsp2 to sp3 transformation mechanism. For example, irradiation energy will cause lattice displacement in the FLG which affects the FLG layer cross-linking, therefore contributing in the formation of new sp3 bonds. In addition, bonds can be broken to form sp3 due to the collision impact. This is evident from the XPS reduction of π electron (π bond) intensity as the irradiation energy is increased. Furthermore, bending (to be discussed later) and breaking of the graphene planes are likely to contribute to the sp2 to sp3 transformation observed.From all the above findings, it is clear that more than one factor can contribute to the + ion irradiation on the FLG edges. In contrary to the damage found in the flat regions, the edges of FLG are found to be much rougher; with four distinct features as shown in Figure We turn our discussion to the effect of ArIn this case, the edges are seen to be lifted up and shadow the regions behind them; shadowing effects are seen from the cross section profile taken at a position shown by a solid line in Figure + ion irradiation of 1.5 keV. Few holes (enclosed by circles) can be clearly seen, suggesting the possibility of creating nanoporous graphene layers by ion irradiation for filtration applications. Increasing the ion irradiation to 4.5 keV, the peeling of graphene layers in the direction normal to the edge takes place which is associated with triangular-shaped FLG folding (enclosed by a circle) as shown in Figure During the ion irradiation, unusual defects and edge reconstructions were also observed; defects appear only after ion irradiation and were not found in the un-irradiated samples. Furthermore, these defects and edge reconstructions are similar to those reported by Zhang and Feng  during tsp2/sp3 ratio, sp2 and sp3 FWHM values and Auger transition C KVV spectra data analysis reveal that, using low energy of 0.25 keV Ar+ ion irradiation at large sample tilt angle at room temperature, is an alternative way of peeling graphene layers without the need of Joule heating.This type of folding has been observed on the surface of FLG resembling single wall nanotubes or double wall nanotubes due to the single- or double-graphene layer folding back to itself . The fol+ ion irradiation of FLG has been investigated in which ripple structure formation with varying amplitude and periodicity predominantly in the first two graphene layers was observed. In addition, sp2 to sp3 bonding transformation through the quantification of the deconvolution of the XPS C (1s) envelope along with electronic damage as deduced from the gradual disappearance of the π − π* transition in graphene, as a function of irradiation energy, was found. Although the shape of derivative of Auger transition C KVV spectra provided an indication of the ion irradiation effect, the D parameter values obtained from these spectra failed to account for sp2 to sp3 hybridization transition. Among others, energy-dependent edge wiggles, shadowing effects, defects (such as holes and FLG edge reconstructions) and edge folding which depend on the sputtering conditions were all found. The existence of these defects suggests the possibility of creating nanoporous FLG by ion irradiation and the development of high density nanobubble FLG surfaces for various applications. Furthermore, the combined effect of Ar+ implantation and sample tilt angle on FLG irradiation can explain the ion irradiation resilience of FLG. Although we looked specifically at Ar+ irradiation, our results can also provide insights into the response of FLG to irradiation by other noble gasses or other species. Despite the mixing of sp2 and sp3 is not desirable for the electronic properties of FLG, coming up with applications which depend on tuning sp2/sp3 ratio will be essential to fully appreciate the importance of the ion irradiation of FLG. In addition, future investigations at atomic scale for the edge reconstructions and other defects will be essential to support the results of this study.ArThe authors declare that they have no competing interests.SHA contributed in the overall project design and implementation. ME and MA contributed in the data analysis. AK contributed in the atomic force microscopy experiments. TH contributed in the few layer graphene sample preparation and sputtering. MTZM contributed in the X-ray photoemission spectroscopy experiment and manuscript preparation. MRA contributed in the manuscript preparation. All authors read and approved the final manuscript.SHA is an associate professor in Nanotechnology and Surface Science. ME is a professor in Theoretical Physics, doing first principle calculations. MA is an associate professor in Theoretical Physics and Computational Simulation Physics. AK is a masters degree student. TH is a Ph.D. student working on swift ions in materials. MTZM is a Ph.D. student working on nanotechnology. MRA is a professor in magnetism and head of the department.Effect of surface roughness on the nanobubble formation. Figure S1a,b show 2-D and 3-D AFM images of nanobubbles formed on the unsputtered FLG sample. The height and lateral size of the bubbles range from 2 to 5 nm and from 15 to 30 nm, respectively. It is also clear that the bubbles are well dispersed in a random fashion. However, agglomeration and an increase in their size and number density are prominent features of the bubbles grown on the irradiated FLG samples as seen from Figure S1 and , respectively.Click here for fileIrradiation features and roughness amplitudes after 3-keV ion irradiation. Figure S2. (a) 3-D image showing the effect of ion irradiation on the FLG edges after Ar+ ion irradiation of 3 keV. A, B and C denote the irradiated edges, cross sputtered areas at 53° from A direction and shadowing features, respectively. (b) PSD obtained from the image shown in (a) showing the amplitude of the surface's roughness of features A, B, and C as a function of the spatial frequency. FFT of the image shown in (a) revealing the presence of features A, B and C is shown in the inset of (b).Click here for file


Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial pneumonia causing a loss of respiratory surface area due to a proliferative fibrotic response involving hyperplastic, hypertrophic, and metaplastic epithelium, cystic honeycomb change, septal expansion, and variable inflammation. Wnt (wingless) signaling glycoproteins are known to be involved in lung development and tissue repair, and are up-regulated in patients with IPF. Based on previous qRT-PCR data showing increased Wnt7B in lungs of IPF patients, a systematic, quantitative examination of its tissue site distribution was undertaken.Tissue samples from the Lung Tissue Research Consortium (LTRC) of 39 patients diagnosed with mild to severe IPF/usual interstitial pneumonia (UIP) and 19 normal patients were examined for the immunolocalization of Wnt7B.In normal lung, moderate Wnt7B reactivity was confined to airway epithelium, smooth muscle of airways and vasculature, and macrophages. IPF lung showed strong Wnt7B reactivity in fibroblastic foci, dysplastic airway and alveolar epithelium, and in highly discrete subepithelial, basement membrane-associated regions. All reactive sites were sized and counted relative to specific microscopic regions. Those in the subepithelial sites were found in significantly greater numbers and larger relative area compared with the others. No reactive sites were present in normal patient controls.The results demonstrate Wnt7B to be expressed at high concentrations in regions of active hyperplasia, metaplasia, and fibrotic change in IPF patients. In this context and its previously established biologic activities, Wnt7B would be expected to be of potential importance in the pathogenesis of IPF. Idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP) is a debilitating disease characterized by a loss of normal respiratory architecture and replacement with a heterogeneous population of myofibroblast-like cells and excess of fibrous connective tissue restricted to the lung ,2. IPF aThe signaling pathways involved are only partially understood. It is known that there is increased expression of TGF-β and α-smooth muscle actin (α-SMA) in progressive lesions of IPF reflecting the transition of fibroblasts to myofibroblasts with proliferation and collagen maintenance modulated by wingless (Wnt) glycoproteins -12. MousWnt glycoproteins have been found to be expressed at low levels in the normal lung and may be associated with epithelial turnover ,20. For Tissue blocks of formalin-fixed lung tissue samples were obtained from the Lung Tissue Research Consortium (LTRC). The samples had previously been placed into three groups see Table: group 1Sections were treated with citrate buffer for antigen retrieval and treated with a polyclonal goat anti-Wnt7B antibody  at a 1:100 dilution overnight at 4°C, followed by peroxidase-labeled secondary antibodies  and Nova Red . The Wnt7B antibody recognizes both precursor and mature forms of human origin. Control samples substituted normal goat serum for the primary antibody, or were pre-treated with 5% testicular hyaluronidase  for 30 minutes at 37°C to release Wnt7B bound to extracellular matrices , or trea2, 50–100 μm2, and >100 μm2; while their histologic sites were separated into one of three regional designations: 1) epithelium and underlying fibroblasts with or without extracellular matrix (EF), 2) subepithelial fibroblasts and ECM (SE) without epithelial staining (included most fibroblastic foci), and 3) interstitial fibroblasts and ECM (I) that were not directly subjacent to an epithelial surface. The number of sites corresponding to the respective categories was recorded. The total surface area, as defined by the external border of each tissue section, was determined using ImageJ , and the percent of the total area of each reactive site category relative to the total section surface area was calculated  for EF, SE, and I locations. Statistical significance was set at an alpha value of ≤ 0.05.Comparisons of the descriptive statistics of reactive sites were made using Analysis of Variance or Kruskal-Wallis non-parametric test for histologic locations in the same reactive site groups and within lung capacity category using SAS software  or Minitab . For example, for patients in FVC 50-80%, comparisons were made for reactive sites >100  had weak to moderate Wnt7B immunoreactivity in the cytoplasm of airway epithelium (ciliated cells) and smooth muscle of airways and arteries  and strong extracellular reactivity for Wnt7B, with most found adjacent to thickened airways , standard deviations, and minimum, median, and maximum sizes of reactive sites (in μm2) are presented in Tables Patients with FVC > 80% had few quantifiable Wnt7B reactive sites Figurea compareThe initiating cause(s) underlying the pathogenesis of idiopathic pulmonary fibrosis (IPF) is unknown. Much attention has focused on a failed repair of alveolar epithelium, and its impact on subsequent loss of pulmonary architecture. This failure manifests as the inability of epithelial surfaces to proliferate and differentiate effectively resulting in hyperplasia, metaplasia, and/or transdifferentiation into myofibroblast-like cells . Exactly2) that, like fibroblastic foci, had both cellular and extracellular matrix components. The loss of extracellular Wnt7B immunoreactivity after hyaluronidase digestion supports the matrix-associated localization, which is not surprising, as Reichsman, et al., [BMP-4 and Id2[The finding of strong Wnt7B immunoreactivity in the fibroblastic focus of UIP/IPF lungs would seem to fit readily with the above discussion on canonical Wnt signaling involving β-catenin. The demonstration of Wnt7B in dysplastic airway and alveolar epithelium and in myofibroblasts of the fibroblastic foci closely correlates with the site localization of active β-catenin in IPF lungs . However et al.,  have dem et al., . In the 4 and Id2. Further4 and Id2. It may 4 and Id2 where my4 and Id2. Coupled4 and Id2.2 (Figures The notion that Wnt7B may have a potentially contributive if not significant role in the development and/or progression of IPF draws on previous assumptions that the fibroblastic foci are central to the disease process and its prognosis ,40. The  Figures d2a3b-d. These observations draw attention to a specific Wnt signaling ligand, Wnt7B, which by virtue of its established roles in epithelial and mesenchymal proliferation and differentiation, procollagen production, and enhanced gene expression in IPF, would be expected to act as a significant contributor to the pathogenesis of IPF. This is supported not only by the strong expression of Wnt7B in fibroblastic foci but also by the numerous, small subepithelial sites that may represent early stages of developing fibroblastic foci as part of a larger, expanding network of fibrogenic tissue.There are no competing interests to report by any of the authors.TM – performed immunostaining, prepared photographic images, drafting and revision of manuscript; AH - performed immunostaining, KF - developed protocol staining and performed immunostaining; BG - performed immunostaining; JT – developed immunostainig protocol; DN - interpretation of data, drafting and revision of manuscript; SJ – analysis and interpretation of data, manuscript revision; MC - analysis and interpretation of data, statistical analysis; PS – conception and design of study, analysis and interpretation of data, preparation of photographic images, drafting of manuscript and revision; all authors read and approved of the final manuscript.


PD-L1, PD-L2 and other immune-related molecules regulate Th1 and Th2 immune responses. Tumor-expressed PD-L1, when bound to PD-1 or B7.1 on activated T cells, can mediate cancer immune evasion. While inhibiting PD-L1 receptor binding represents an attractive strategy to restore tumor-specific T-cell immunity, other immune-related factors and cell types within the tumor microenvironment affect anti-tumor T-cell responses. MPDL3280A, a human mAb containing an engineered Fc-domain designed to optimize efficacy and safety and to promote Th1-driven responses, is described here with PhI biomarker results. MPDL3280A was administered IV q3w in >300 pts with locally advanced or metastatic solid tumors. ORR was assessed by RECIST v1.1, including u/cCR and u/cPR. PD-L1 and CD8 were measured by IHC. The expression of ≈90 immune-related markers, including PD-L1 and PD-L2, were evaluated at baseline (BL) and on-treatment. BL tumor samples were available for 103 pts, and matched on-treatment samples were available for 26 pts. As of Feb 1, 2013, 140 pts enrolled prior to Aug 1, 2012 were evaluable for efficacy. Responses were observed in multiple tumor types including NSCLC (9/41), RCC (6/47), melanoma (11/38), CRC (1/4) and gastric cancer (1/1). An ORR of 21% (29/140) was observed in nonselected solid tumors with a duration of response of 1+ to 253+ days. Elevated pretreatment PD-L1 expression (IHC) was associated with response to MPDL3280A, and coordinated expression of PD-L1 and CD8+ T cells was seen. The ORR was 36% (13/36) for pts with PD-L1-pos tumors vs 13% (9/67) for pts with PD-L1-neg tumors. 81 pts had tumors evaluable for PD-L2. Median PD-L2 expression was ≈2x higher in PD-L1-pos tumors vs PD-L1-neg tumors. A T-cell gene signature  was associated with treatment response. On treatment, responding tumors had increased PD-L1 expression and a Th1-dominant immune infiltrate, evidence of adaptive PD-L1 upregulation. Nonresponders showed minimal tumor CD8+ T-cell infiltration and an absence of T-cell activation. Additionally, elevated expression of immune suppressors, including FOXP3 and RORC, correlated with lack of response. Both the presence of Th1-related CD8 biology at BL and adaptive tumor PD-L1 enhancement with MPDL3280A treatment correlated with tumor response, as did PD-L1 status. Updated data will be presented.


We conducted a metacommunity experiment to investigate the role of dispersal for bacterial community composition (BCC) and function of freshwater bacteria. Bacteria were dispersed from a common source pool into three different lake communities in their natural lake water. The experiment was conducted in dialysis bags to enable a decoupling between a change in the local environment and dispersal. BCC was determined by terminal restriction fragment length polymorphism (tRFLP) of the 16S rRNA gene. We show that the greatest changes in BCC occurred between 10% and 43% of dispersal of standing stock per day. Functioning, measured as growth rate, was also affected by dispersal in all three communities but the qualitative pattern differed between communities, sometimes showing a hump-shaped relationship to dispersal and sometimes decreasing with increasing dispersal. In all waters, functioning was related to BCC. Our results show that dispersal does affect BCC and functioning but that high dispersal rates are needed. Further, the effect of dispersal on BCC and function seem to depend on the quality of the habitat to which bacteria disperse into. Due to the concern of which consequences the loss of biodiversity will have on ecosystems, it is of considerable interest to evaluate the importance of biodiversity for ecosystem functions e.g. . The mecIn an experimental study, Venail et al. Bacterial communities can serve as practical models in ecology since experiments and field studies can relatively easy (in comparison to larger organisms) be designed over a range of relevant temporal and spatial scales Results obtained from studies of diversity-functioning relationships in bacterial communities have so far yielded disparate results. If different bacteria play different roles in ecosystems, for instance regarding their ability to degrade different organic molecules, changes in community composition (BCC) should lead to an altered functionality e.g. . On the In the present study we are conducting a laboratory experiment in which bacteria were dispersed into bacterial communities in dialysis bags kept in larger containers with natural water. Because the dialysis bags allow transportation of water and dissolved chemicals but not bacterial cells across the membrane, dispersal can be disconnected from a change in environmental conditions. The aim of this study was to evaluate 1) the magnitude of dispersal rates from a metacommunity that are needed to cause changes in local BCC, 2) whether dispersal from a metacommunity affects a functional trait, such as local bacterial growth rate, and 3) whether there is a relationship between BCC and the functional trait.−1, total organic carbon (TOC) = 5 mg L−1, Absorbance at 430 nm, a measure of humic matter content, (Abs430) = 0.073), Lake Fälaren is a humic lake , and Lake Funbosjön is an eutrophic lake . Lakes Fälaren and Funbosjön were sampled on July 27 2009, and Lake Ånnsjön July 25 2009. The experiment started on the 29 of July 2009 prior to which all water was filtered through a 1 µm glass fibre filter (Gelman A/E) to remove larger organisms than bacteria.We sampled water and the natural bacterial communities from three lakes with different characteristics to invoke large differences in environmental conditions between communities. No permits were required to sample these lakes. Lake Ånnsjön is an oligotrophic clear-water lake  = 15 µg LFrom each lake we filled three 10 L buckets (replicates) with filtered water, i.e. in total there were nine buckets. In each bucket we placed five 10 mL dialysis bags  containing the natural bacterial community from that lake . In addition we also added one dialysis bag with equal parts from the three different lakes (Mix) that served as a treatment with no continuous dispersal but where dispersal limitations between communities was eliminated. Thus, for the experiment we had 9*6 = 54 dialysis bags. In addition we also had one negative control dialysis bag of autoclaved MQ water in each bucket. The water in the buckets was mixed by vigorous air bubbling. The dialysis bags were pre-wetted in MQ water for one day before the initiation of the experiment.Three times a day we dispersed bacteria into the communities in the dialysis bags by pipetting. At each dispersal event a metacommunity was first constructed from a mixture of equal volumes of the three original lake bacterial communities. That is, the three natural lake communities were kept in separate 1 L glass bottles under the same conditions as the experiment was run (see below). At dispersal, equal volumes were taken out from the three lake communities and mixed into a metacommunity which we used for dispersion of bacteria into the dialysis bags. See 3, where X is volume transferred in and out of a dialysis bag per dispersal event. Thus, for the 1.7 mL and 3.3 mL treatments the approximate dispersal rates were 43% and 70% per day, respectively. The 0 dispersal and the “Mix” treatment received no dispersal during the course of the experiment, but were opened and closed to mimic the handling of the dispersal treatments.Empirical estimates about how high dispersal rates are needed to affect bacterial communities in lakes are scarce, but there are indications that high dispersal rates are required In nature, lakes exist with water retention times of one day (i.e. 100% replacement per day), and in Sweden the great majority of lakes has a water retention time of less than 100 days The experiment was run for 5 days at 15°C in darkness. At the end of the experiment samples were taken from each dialysis bags for analyses of bacterial community structure, growth rate and abundance. Samples for community composition and abundance were also collected from the three start communities and the three communities making up the metacommunity from which dispersal occurred, at the end of the experiment.2HPO4 (1g L−1). The bag was put in a beaker with deionized water and we recorded conductivity in the beaker for up to 4 hours. The conductivity in this beaker was compared with the conductivity in a beaker where the same amount of salt had been added directly into the same amount of deionised water.Dialysis bags prevent movement of bacteria out of the bag but allow the local environmental conditions in the bags to be close to the conditions in the buckets, independent of dispersal. Compared to previous experiments on microbial communities e.g.  we were HaeIII and HinfI.Bacterial community composition (BCC) was determined by terminal restriction fragment length polymorphism (tRFLP) of the 16S rRNA gene as described by Lindström et al. Peaks (tRFs) in lengths greater than 447 bp were eliminated from the analysis. Peaks with a relative area less than 0.5% were also eliminated from the analysis as these are difficult to separate from noise. The average relative peak area of restriction digest duplicates was used for further statistical analysis. In five samples, among which 4 were experimental negative controls (i.e. autoclaved MQ water), the amount of DNA extracted was too low to enable tRFLP analysis.1 mL samples for bacterial abundance were collected and preserved with 4%  formaldehyde. Bacterial abundance was determined by flow cytometry (Partec) after staining with Syto13 at a final concentration of 1.25 µM Bacterial growth rate was determined using the leucine incorporation method Bray-Curtis (relative peak areas) as well as Sørensen (presence-absence of peaks >0.5%) dissimilarities 2,45 = 15, P<0.001, r2 = 0.33; Sørensen: F2,45 = 26, P<0.001, r2 = 0.46) and dispersal . The importance of lake identity for BCC among communities receiving the same dispersal treatment declined with increasing dispersal rate  showed greater similarity to each other . For botsal rate . Lake idsal rate . The expsal rate . The impsal rate . When th−1) differed depending on dispersal treatments , lakes  as well as on the interactions between them . In the oligotrophic lake (Ånnsjön) and humic lake (Fälaren) the bacterial growth rate peaked at intermediate dispersal, i.e. at 43% per day  and growth rate per cell  in the negative controls were lower than in all other treatments in the oligotrophic and humic lakes, while in the eutrophic lake two of the negative controls had higher growth rate than the two highest dispersal treatments. In the five (of the nine) negative controls where DNA extraction was possible, >84% of the tRFLP peak area corresponded to peaks also found in the start communities or source communities of the respective lakes. Thus, there appears to have been, in some cases, inflow of viable bacteria from the buckets, either over the dialysis membranes or during handling. This may in some cases have had a marginal impact on the growth rate, and may have affected BCC. Thus, this inflow can have exaggerated the role of the local environment for BCC slightly, especially in low dispersal treatments. An important point is though that there was no abundant “alien” bacterium growing within the bags confounding the results.By the end of the experiment water colour at 430 nm was similar in the buckets and the dialysis bags receiving the highest dispersal in the humic  and in the eutrophic  lake. However, in the lake with the clearest water, colour had increased in the highest dispersal treatment by almost 70%, since absorbance was 0.073 in the bucket and 0.124 in the dialysis bag. At the end of the experiment, salt was added in a dialysis bag from the most eutrophic lake (Funbosjön). After 4 h dialysis the salt concentration in the surrounding beaker was 83% of the control beaker. Thus, the diffusion of inorganic ions through the membrane was still efficient at the end of the experiment.Our results show that both the local environment (i.e. species sorting) and dispersal (i.e. mass effects) affected BCC, which in turn altered bacterial functioning. Low dispersal communities (i.e. 0–10% of standing stock per day) were similar to the start communities of the respective lakes, while the high dispersal communities (i.e. 43 and 70% per day) became more similar to the same treatments from other lakes. The Mixed communities, i.e. identical start communities, kept in the different lake environments but receiving no further dispersal, showed variable results. Thus, high and continuous dispersal rates were necessary to consistently change community composition.Clearly, the relative importance of species sorting decreased with increasing continuous dispersal rates. However, even at the highest dispersal treatments the local environment (i.e. the lake water) explained a great share of the variation in community composition, and, consequently, the high dispersal communities in the three environments were not identical. Hence, at least parts of the communities were affected by species sorting processes rather than mass effect also at very high dispersal. This conclusion is not likely to change regarding that organic carbon increased with dispersal in one lake. At the opposite, differences in BCC could have been even larger if the environment would have been kept even more similar to that of the bucket.Results from field studies have shown that not only the immigration rate determines if a community is shaped by mass effect or species sorting. For instance, the position of the habitats of the sink and source communities in the landscape may be of importance In the oligotrophic and the humic lakes the bacterial growth rates peaked at intermediate dispersal levels (43%), i.e. suggesting that also in these lakes variation in BCC imposed by dispersal affected functioning. It should be noted that dispersal increased concentration of organic compounds in the communities from the oligotrophic Lake Ånnsjön, which could have influenced bacterial growth rates. However, since the relationship between dispersal and growth rate was not linear, but rather hump-shaped, this effect did not seem to be a consequence of increased nutrient levels. The close to identical results obtained when using dpm directly or dpm per cell as a measure of growth rate also show that the results were not dependent on changes in cell abundances caused by the dispersal. Thus, we conclude that dispersal appear to have changed growth rate via a change in BCC.Previous studies investigating the role of microbial dispersal for ecosystem function  also found highest biomass Therefore, our results show that dispersal can change community function via a change in community composition but that the exact outcome may differ depending on the characteristics of the habitats studied. Future studies therefore need to address how different environments affect the interplay between dispersal, BCC, and functioning. It should also be fruitful to evaluate the possibility that dispersal and establishment may vary among taxa, which would lead to that different populations are affected by different forces. With the future development of techniques for the study of microbial diversity, i.e. sequencing technologies such as 454-sequencing e.g. , OTU defFuture experimenters should also seek for means to investigate more long-term effects of dispersal, which our design did not allow for technical reasons. The start and end communities of Lake Ånnsjön and Lake Fälaren inocula were similar suggesting that community compositions were close to equilibria. In contrast, the start and end communities of the Lake Funbosjön inocula had shifted over the study period . How thiOur results together with the observations made by Venail et al. Figure S1There were three buckets (replicates) of lake water from each of the three lakes. The source community (metacommunity) was constructed by equal volumes of water from the three lakes. This source community was then dispersed into the six dialysis bags in each bucket, with different rates, three times per day. The dispersal treatments were replacement of 0, 1, 10, 43 and 70% of the dialysis bags volumes per day. The ”Mix” treatment was a mixture of equal volumes of the start communities from each lake, which thereafter received no further dispersal. All dispersal treatments were made in all buckets.(TIFF)Click here for additional data file.


Peripheral blood biomarkers are needed to identify and determine the extent of idiopathic pulmonary fibrosis (IPF). Current physiologic and radiographic prognostic indicators diagnose IPF too late in the course of disease. We hypothesize that peripheral blood biomarkers will identify disease in its early stages, and facilitate monitoring for disease progression.LCO and FVC.Gene expression profiles of peripheral blood RNA from 130 IPF patients were collected on Agilent microarrays. Significance analysis of microarrays (SAM) with a false discovery rate (FDR) of 1% was utilized to identify genes that were differentially-expressed in samples categorized based on percent predicted DLCO >65%) compared to controls and 2790 transcripts were differentially- expressed in severe IPF (DLCO >35%) compared to controls. When categorized by percent predicted DLCO, SAM demonstrated 13 differentially-expressed transcripts between mild and severe IPF (< 5% FDR). These include CAMP, CEACAM6, CTSG, DEFA3 and A4, OLFM4, HLTF, PACSIN1, GABBR1, IGHM, and 3 unknown genes. Principal component analysis (PCA) was performed to determine outliers based on severity of disease, and demonstrated 1 mild case to be clinically misclassified as a severe case of IPF. No differentially-expressed transcripts were identified between mild and severe IPF when categorized by percent predicted FVC.At 1% FDR, 1428 genes were differentially-expressed in mild IPF (DLCO, but not FVC.These results demonstrate that the peripheral blood transcriptome has the potential to distinguish normal individuals from patients with IPF, as well as extent of disease when samples were classified by percent predicted D LCO) and forced vital capacity (FVC) have been utilized as predictive indicators LCO of <35% or a decline in DLCO >15% within a year period are associated with an increased mortality. Similarly, a decline of >10% in FVC over a six month period also indicated an earlier mortality LCO among the placebo arms of the trials indicating there is substantial disease heterogeneity within IPF Idiopathic Pulmonary Fibrosis (IPF) is categorized as an interstitial lung disease (ILD) and is the most common subtype of idiopathic interstitial pneumonias (IIP) comprising nearly 71% of the total cases Rosas and coworkers observed a differential expression of MMP7, MMP1, MMP8, IGFBP1, and TNFRSF1A proteins in the peripheral blood between familial interstitial pulmonary fibrosis and controls LCO and FVC, were used to stratify the cases into severe and mild disease categories. Mild disease is defined as either percent predicted DLCO ≥65% (N = 16) or FVC ≥75% (N = 27). Severe disease is defined as either DLCO ≤35% (N = 15), FVC ≤50% (N = 13). All of these were also compared to age and gender matched non-diseased, healthy controls (N = 27). Eight patients categorized as severe were used in both the DLCO and FVC analysis, whereas the mild disease classified by either DLCO or FVC are all distinct cases. Healthy control subjects are family members who participated in screening for the presence of pulmonary fibrosis, and after evaluation of their medical history, lung function, and chest CT, they were found to have no evidence of lung disease. Individual institutional review boards approved this research. All participants in this study provided written IRB-approved informed consent.One hundred thirty peripheral blood RNA specimens were collected from individuals enrolled in either the Interstitial Lung Disease (ILD) or the Familial Pulmonary Fibrosis (FPF) Programs conducted at National Jewish Health and Duke University. Only one individual case per family was utilized from the FPF repository. Individual samples had a consensus diagnosis of probable or definite IPF, and this was based on the ATS/ERS/JRS/ALAT criteria LCO measurement were obtained according to American Thoracic Society standards and guidelines FEV1 and DPeripheral blood samples were collected in PAXgene RNA tubes . RNA extraction and purification was performed manually utilizing the PAXgene Blood RNA kit  according to the manufacturer’s protocol.Quantification of total RNA was measured via the Nanodrop ND-1000 spectrophotometer . Quality of the RNA was assessed with a RNA 6000 NanoChip  on the 2100 Bioanalyzer  by ratio comparison of the 18 S and 28 S rRNA bands.Agilent Whole Human Genome Oligonucleotide Microarrays , were used to determine gene expression levels in peripheral blood. Total RNA was used as a template for synthesis of cDNA utilizing the One Color Low Input Agilent Quick Amp Labeling Kit utilizing the Spike-In Kit to provide positive controls. The Agilent one-color microarray based gene expression analysis was followed per manufacturer’s instructions, and passed Agilent’s quality control (QC).LCO and FVC. All IPF samples were compared to normal controls to identify differentially-expressed genes. Principal component analysis (PCA) was carried out on genes identified by SAM analyses to identify outliers, and gene-based hierarchical clustering was performed to identify relationships among differentially-expressed genes related to mild or severe disease categorized by DLCO. All microarray data is minimal information about a microarray experiment (MIAME) compliant, and raw data has been deposited into the GEO database (GSE33566).Analysis was performed utilizing the Multi-Experiment Viewer (MeV) software package www.ingenuity.com). The Canonical Pathways Analysis identified the pathways, and the significance of these was determined by Fischer’s exact test. Biomarker Analysis was employed to identify the most relevant molecular biomarker candidates.Data were analyzed through the use of Ingenuity Pathways Analysis  using forty cycles of amplification. All assays were performed in duplicate and data were analyzed by the ΔΔCt method utilizing glyceraldehyde 3 phosphate dehydrogenase (GAPDH) as an endogenous control LCO 77.1% predicted in the mild disease group, and a mean FVC 42.5% predicted and 27.5% predicted DLCO in the severe disease group. The mean age is similar between groups when classified by either % predicted FVC (P = 0.18), or % predicted DLCO (P = 0.87), but the mean age of either disease group is slightly older compared to controls (P = 0.15). The majority of patients are male in both mild and severe disease categories, and similar between both disease categories when classified by either % predicted FVC (P = 0.48) or % predicted DLCO (P = 0.29). There are no significant differences between mild and severe disease categories with respect to prior tobacco use. The diagnosis of IPF was confirmed by surgical lung biopsy in 50.7% of the study subjects , both probable and definite, cluster along the first principal component , both probable and definite, distribute together along the first principal component  was performed to determine outliers in the gene expression data based on disease categorization . The majority of mild cases  compared to normal controls (N = 31) revealed 1,428 differentially-expressed transcripts  of the mild IPF cohort, when categorized by percent predicted Dnscripts , and whenscripts  with <1%controls  comparedcontrols . TherefoLCO (DLCO ≥65% [N = 16] and DLCO ≤35% [N = 15]), demonstrated 13 differentially-expressed transcripts with less than a 5% false discovery rate. LCO. When using a FDR of ≤1%, only defensin A3 (DEFA3) is differentially-expressed between both mild or severe cases compared to controls. SAM revealed no differentially-expressed transcripts with less than a 5% false discovery rate between peripheral blood samples when IPF patients were categorized by percent predicted FVC .Principal component analysis was performed to determine outliers in the data set based on severity of disease categorization. Results demonstrate that 1 severe IPF case appears to be clinically misclassified as a mild case of IPF, while other cases are correctly classified as mild or severe . SignifiLCO for the purpose of discovery common potential biomarkers. This analysis showed that at FDR <5% only 2 differentially expressed transcripts, A3 (DEFA3) and hypothetical protein FLJ11710 , were differentially-expressed between control vs mild, mild vs severe, and control vs severe disease (DLCO ≤35%). There is up-regulation in DEFA3 expression compared to controls for severe IPF, while hypothetical protein FLJ11710 demonstrates a down regulation compared to controls for severe IPF. Between the mild and severe IPF cohort , and between normal and severe disease, there is up-regulation of several other host defense genes including defensin A4 (DEFA4), cathelicidin antimicrobial peptide (CAMP), cathespin G (CTSG), and down-regulation of immunoglobulin heavy chain constant mu (IGHM). The greatest increase found distinguishing controls from severe disease, and mild from severe disease is olfactinmedin 4 (OLFM4). We randomly selected 4 of the 13 differentially-expressed genes for qPCR validation, and qPCR confirmed the differential expression of these genes identified by microarray experiments  software was utilized to identify the intersection or common differentially-expressed transcripts between normal and mild IPF, normal and severe IPF, and mild versus severe IPF when classified by Deriments .LCO  LCO).Four of the 13 genes up-regulated in the current study have functions pertaining to host defense. This raises the question of an association of advanced disease and sub-clinical infection, or host-microbe interactions. All of these patients were stable when blood was drawn without overt signs of infection. Viral infection has been put forth as a possible trigger of acute exacerbations of IPF Differential-expression analysis demonstrates up-regulation of the cathelicidin antimicrobial peptide (CAMP) between the severe IPF subgroup compared to controls, and between mild and severe disease groups. Cathelicidin is an antimicrobial protein of the innate immune system stored in peroxidase-negative granules of neutrophils LCO), but not when correlated with by FVC % predicted. The reason for this difference is unclear. FVC is traditionally used to stage the severity of pulmonary fibrosis. However, it has been recognized that a single measurement of the FVC is not useful for predicting disease progression or mortality of IPF LCO <50% predicts an increased mortality LCO% predicted but not a single FVC% predicted measurement due to the better performance of a single measurement of DLCO compared to FVC. A lack of correlation between FVC and severity of IPF can be due to concomitant emphysema, however review of the subject’s HRCT in this study does not demonstrate significant amounts of emphysema. It is possible the signature is a consequence of impaired oxygen transport, but this seems unlikely since two of the thirteen genes also distinguish controls from mild disease where oxygen transport is not a factor. Also, none of the 13 genes have functions related to oxygen transport, aerobic or anaerobic metabolism, or other pathways that might be implicated in the presence of hypoxemia or impaired oxygenation.There are limitations to this study. Given the minimal disease burden and lack of clinical symptoms, five cases of mild IPF were not appropriate candidates for surgical lung biopsy, and therefore did not have surgical tissue. Due to the minimal disease burden, they did not meet HRCT criteria of definite IPF according to the 2011 ATS/ERS/JRS/ALAT consensus statement. However, in the five cases the mean age was 70, and 4 cases were male supporting the diagnosis of IPF. Furthermore, these cases cluster with definite mild cases along the principal components . TherefoLCO, and distinguish IPF patients from normal. Our data is consistent with a role for MMP-7 interacting with α-defensins, and increased expression of host defense proteins in the peripheral blood being associated with deterioration of DLCO in IPF. The ability to use a peripheral blood biomarker to monitor disease progression for IPF could have a substantial impact on the diagnosis, treatment, and management of this disease, and perhaps be generally applicable to other subtypes of idiopathic interstitial pneumonias.In summary, our results demonstrate that the peripheral blood transcriptome can potentially distinguish extent of disease in individuals with IPF when samples are correlated with percent predicted DTable S1†Definite IPF without surgical lung biopsy is defined by supporting clinical information and HRCT demonstrating sub-pleural and bibasilar predominate reticulation, honeycombing, and traction bronchiectasis without atypical features such as nodules, predominate ground glass opacities, pleural plaques, air-trapping, or lymphadenopathy. ‡Probable IPF without surgical lung biopsy is defined by supporting clinical information and HRCT demonstrating sub-pleural and bibasilar predominate reticulation, traction bronchiectasis without bilateral honeycombing, and without atypical features outlined above. Surgical lung biopsy (SLBx): definite IPF is defined as usual interstitial pneumonia requiring spatial and temporal heterogeneity; subpleurally accentuated microscopic honeycombing, fibroblastic foci without significant parenchymal, airway, or pleural mononuclear inflammation; definite IPF is also advanced honeycombing on lung biopsy with clinical and radiologic features supporting IPF. n/a is not available.(DOCX)Click here for additional data file.Table S2†Definite IPF without surgical lung biopsy is defined by supporting clinical information and HRCT demonstrating sub-pleural and bibasilar predominate reticulation, honeycombing, and traction bronchiectasis without atypical features such as nodules, predominate ground glass opacities, pleural plaques, air-trapping, or lymphadenopathy. ‡Probable IPF without surgical lung biopsy is defined by supporting clinical information and HRCT demonstrating sub-pleural and bibasilar predominate reticulation, traction bronchiectasis without bilateral honeycombing, and without atypical features outlined above. Surgical lung biopsy (SLBx) :definite IPF is defined as usual interstitial pneumonia requiring spatial and temporal heterogeneity; subpleurally accentuated microscopic honeycombing, fibroblastic foci without significant parenchymal, airway, or pleural mononuclear inflammation; definite IPF is also advanced honeycombing on lung biopsy with clinical and radiologic features supporting IPF. n/a is not available.(DOCX)Click here for additional data file.Table S3†Definite IPF without surgical lung biopsy is defined by supporting clinical information and HRCT demonstrating sub-pleural and bibasilar predominate reticulation, honeycombing, and traction bronchiectasis without atypical features such as nodules, predominate ground glass opacities, pleural plaques, air-trapping, or lymphadenopathy. ‡Probable IPF without surgical lung biopsy is defined by supporting clinical information and HRCT demonstrating sub-pleural and bibasilar predominate reticulation, traction bronchiectasis without bilateral honeycombing, and without atypical features outlined above. Surgical lung biopsy (SLBx) :definite IPF is defined as usual interstitial pneumonia requiring spatial and temporal heterogeneity; subpleurally accentuated microscopic honeycombing, fibroblastic foci without significant parenchymal, airway, or pleural mononuclear inflammation; definite IPF is also advanced honeycombing on lung biopsy with clinical and radiologic features supporting IPF. n/a is not available.(DOCX)Click here for additional data file.Table S4†Definite IPF without surgical lung biopsy is defined by supporting clinical information and HRCT demonstrating sub-pleural and bibasilar predominate reticulation, honeycombing, and traction bronchiectasis without atypical features such as nodules, predominate ground glass opacities, pleural plaques, air-trapping, or lymphadenopathy. ‡Probable IPF without surgical lung biopsy is defined by supporting clinical information and HRCT demonstrating sub-pleural and bibasilar predominate reticulation, traction bronchiectasis without bilateral honeycombing, and without atypical features outlined above. Surgical lung biopsy (SLBx): definite IPF is defined as usual interstitial pneumonia requiring spatial and temporal heterogeneity; subpleurally accentuated microscopic honeycombing, fibroblastic foci without significant parenchymal, airway, or pleural mononuclear inflammation; definite IPF is also advanced honeycombing on lung biopsy with clinical and radiologic features supporting IPF. n/a is not available.(DOCX)Click here for additional data file.Table S5Canonical pathway analysis using Ingenuity Pathway Analysis™ tool to identify associated networks. Network functions with their respective P values and the number of differentially-expressed genes identified are presented.(DOCX)Click here for additional data file.Table S6LCO >65%) using SAM at FDR of <1%.Differentially-expressed genes between controls and mild IPF .(XLSX)Click here for additional data file.Table S7LCO <35%) using SAM at FDR of <1%.Differentially-expressed genes between controls and severe IPF .(XLSX)Click here for additional data file.Table S8Validation of selected differentially-expressed genes by Reatime™ PCR. Primers utilized for PCR and differential expression by Realtime PCR using ΔΔ Ct method.(XLSX)Click here for additional data file.


FoxC2) gene are strongly associated with valve failure in saphenous and deep veins of lower extremities. We explored the association of genetic variants of FoxC2 as well as FoxC2 mRNA and protein expression levels with CVD of lower limbs. We systematically sequenced the single coding exon, 5′ and 3′ flanking regions of FoxC2 gene in 754 study subjects which includes 382 patients with CVD and 372 healthy subjects. Four novel and three reported polymorphisms were identified in our cohort. Three variants in 5′ flanking region and one in 3′ flanking region of FoxC2 gene were significantly associated with CVD risk. FoxC2 mRNA in vein tissues from 22 patients was 4±1.42 fold increased compared to saphenous veins from 20 normal subjects (p<0.01). FoxC2 protein was also significantly upregulated in varicose veins compared to control samples. The c.-512C>T (rs34221221: C>T) variant which is located in the FoxC2 putative promoter region was further analyzed. Functional analysis of c.-512C>T revealed increased mRNA and protein expression in patients with homozygous TT genotype compared to heterozygous CT and wild CC genotypes. Luciferase assay indicated higher transcriptional activity of mutant compared to wild genotype of this variant. These findings suggested that c.-512C>T variant of FoxC2 was strongly associated with susceptibility to CVD and also that this variant resulted in FoxC2 overexpression. To obtain a mechanistic insight into the role of upregulated FoxC2 in varicosities, we overexpressed FoxC2 in venous endothelial cells and observed elevated expression of arterial markers Dll4 and Hey2 and downregulation of venous marker COUP-TFII. Our study indicates altered FoxC2-Notch signaling in saphenous vein wall remodeling in patients with varicose veins.Chronic venous disease (CVD) is one of the most prevalent yet underrated disorders worldwide. High heritability estimates of CVD indicate prominent genetic components in its etiology and pathology. Mutations in human forkhead box C2 ( Chronic venous disease (CVD) of the lower extremities is one of the most prevalent diseases worldwide though the prevalence estimates differ extensively due to the different disease evaluation methods. CVD comprises of visible venous disorders which are not associated with an identifiable mechanism of venous dysfunction Several risk factors contribute to the progression of CVD FoxC2 gene  is located 80 kb distant from this marker. It was also reported that homozygous null mice of FoxC2 (foxc2−/−) had abnormal lymphatic vascular patterning and malfunctioned blood vessels A twin cohort study indicated a link between varicose veins to microsatellite marker D16S520 on chromosome 16 FoxC2 gene variants are strongly associated with lymphedema distichiasis (LD) syndrome where majority of patients develop varicose veins FoxC2 gene is also implicated in the pathogenesis of saphenous vein and deep vein reflux FoxC2 genetic variants in patients with varicose veins. We investigated the role of FoxC2 genetic variants in the development of CVD of lower limbs in a case-control study. We quantified mRNA and protein expression level of FoxC2 gene in saphenous vein from patients with varicose veins and healthy subjects. FoxC2 expression was highly upregulated in varicose vein tissues compared to normal control veins. Our results demonstrate significant correlation between c.-512C>T, a promoter variant of FoxC2 and the expression levels of FoxC2 mRNA and protein in CVD of lower limbs. FoxC2 overexpression in vein endothelial cells in vitro led to the upregulation of arterial markers such as Hey2 and Dll4 and the downregulation of venous marker, COUP TFII.The study was approved by the human ethics committees of Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram and all collaborating hospitals. Samples were collected from patients and healthy subjects after obtaining informed written consent.FoxC2 have been reported to result in susceptibility to diabetes mellitus. Blood samples were collected from age and gender matched 352 healthy controls with no known family history for CVD.382 patients with CVD and 372 control subjects were recruited for this study. Whole blood samples (5 ml) were collected from 360 patients (age - 18 to 60 years) with CVD from St.Thomas Hospital, Kerala, India. Diagnosis of CVD was based on physical examination and Doppler ultrasound test. CVD resulting from obstructions such as neoplasm were excluded from the study. Differential diagnosis was performed by an experienced vascular surgeon and presence of distichiasis was ruled out by an ophthalmologist. Patients with type 2 diabetes mellitus were also excluded since genetic variants of For tissue level expression analysis, varicose vein tissue samples were collected from 22 patients admitted for treatment of CVD by operative treatments at Kempegowda Institute of Medical Sciences, Bangalore, India. Saphenous control vein samples from 20 patients who underwent coronary artery bypass graft surgery at Sri Jayadeva Institute for Cardiovascular Sciences & Research, Bangalore, India were also collected for the study. Whole blood samples were also collected from these 22 patients and 20 controls for sequencing assays. Relevant data regarding the clinical characteristics of patients were collected from medical records of the hospitals participating in the study.Demographic data of all study participants and information regarding symptoms such as pain, itching and throbbing sensation in legs and clinical signs such as hemorrhage, lower limb oedema, hyperpigmentation, thrombophlebitis, cellulitis and ulceration were collected for each patient from medical records. Family history, occupational and lifestyle data were collected to examine their influence in aggravating disease manifestation.Disease phenotypes were categorized according to CEAP  classification system Genomic DNA from whole blood samples was extracted using QIAamp DNA blood mini kit  according to the manufacturer's instructions. Genomic DNA and mRNA from vein tissues were extracted by All Prep DNA/RNA/Protein mini kit . Quantification and purity of DNA and mRNA was measured by nanodrop-1000 spectrophotometer  at 260 nm. RNA was further treated with DNase1  for removing any DNA contamination.FoxC2 gene from DNA of patients with CVD and healthy subjects. Nine primer pairs . PCR conditions were as follows: Initial denaturation for 5 min at 96°C, 20 cycles of denaturing at 96°C for 30 sec, annealing at 70°C for 40 sec with a touchdown of 0.5°C per cycle and extension at 72°C for 1.5 min. This was followed by 20 cycles at same conditions except that annealing was at 60°C for 40 sec. PCR products were purified using gel band elution kit . DNA sequencing was carried out on an ABI 3100 DNA analyzer with Bigdye terminator chemistry .A touch-down PCR was performed to amplify the single coding exon, 3 kb of 5′ flanking and 200 bp of 3′flanking region which includes the 5′ and 3′ untranslated regions (UTR) of er pairs  to ampliFoxC2 and GAPDH genes were designed for real time PCR analysis ].Total RNA (500 ng) from each tissue sample was subjected to reverse transcription with oligodT, dNTPs, and M-MLV reverse transcriptase . Primers for analysis . Quantitg for 20 min at 4°C to collect the supernatant. Proteins were estimated by using Bradford reagent . Protein extracts (30 µg) were subjected to 12% SDS–PAGE and electro transferred to a Hybond C Extra membrane  as per the wet transfer procedure (Bio-Rad Mini Protean II). Membranes were blocked for 1 hour at room temperature in PBS containing 0.5% Tween-20 and 5% BSA, and incubated overnight with anti- FoxC2 antibody (1 µg/ml) at 4°C . Membrane was washed with TBS–Tween-20 and incubated with the 1∶10000 dilution of secondary antibody to rabbit IgG - H&L (HRP)  for 1 h at room temperature. Protein band was developed by enhanced chemiluminescence . The membrane was re-probed with anti GAPDH antibody  for normalization of expression. The densities of immunoreactive bands were quantitated by the Quantity One 1-D image analysis software program .Frozen vein tissues were homogenized and incubated in ice-cold RIPA buffer with protease inhibitor cocktail for 90 minutes followed by centrifugation at 15,000 2O2 in methanol by incubating for 30 minutes. Sections were rehydrated through graded alcohols and antigen retrieval was performed by incubating in 10 mM sodium citrate at 90°C for 15 mins. Sections were washed with TBST and then blocked with 3% BSA for 20 mins. Slides were incubated with anti- FoxC2 antibody (Abcam cat # ab65141) diluted with TBS in 1∶100 ratio. Slides were washed thrice for 5 minutes in TBST and incubated for 1 hour with Horse raddish peroxidase conjugated anti rabbit antibody (Abcam cat # ab97051) diluted with TBS in 1∶200 ratio. After washing, slides were incubated with 3,3′-diaminobenzidine tetrahydrochloride (DAB) (Sigma) and immediately washed under tap water after the color development and were counter stained with haematoxylin. Slides were DPX mounted and observed under light microscope .5 µm paraffin embedded tissue sections were de-paraffinized in xylene and endogenous peroxidase activity was quenched with 3% HFoxC2 gene in the promoter activity of gene, a luciferase reporter gene assay was performed. A 664 bp DNA sequence from the −614 bp to +50 bp from the transcription start site of FoxC2 gene encompassing the putative promoter area was amplified from DNA with CC and TT genotype. Primers including KpnI and HindIII restriction sites  and mutant pGL4-FoxC2 promoter (TT genotype) were transfected into cultured cell lines using Lipofectamine . Renilla luciferase construct (phRL-TK) was used as a control for transfection efficiency. After 48 h, each group of cells was lysed and luciferase assay was carried out using Dual Luciferase assay kit  according to manufacturer's instructions and readings were recorded. Technical replicates were performed in triplicate and biological experiments were performed twice.EA.hy926 FoxC2 – pCAGIG construct was made by inserting FoxC2 coding sequence into EcoRI and XhoI restriction sites of pCAGIG mammalian expression vector (Addgene plasmid 11159) FoxC2 -pCAGIG and control empty vector was performed using lipofectamine-2000  according to the manufacturer's recommendation. The concentrations of constructs used were 1 µg per well. After 6 hours of transfection, 20% FBS supplemented DMEM medium was added. The assays were carried out 8 days post-transfection. Transfection was performed in triplicates and repeated twice. GFP positive cells were sorted on FACS Aria (Becton Dickinson) and used for further analysis.FoxC2, GAPDH, Hey2, Dll4, COUP TFII and Ephrin B4 gene expression. Primers sequences are given in table S2. The conditions for amplifying FoxC2 and GAPDH are as described earlier. For assessing Hey2, Dll4, COUP TFII and Ephrin B4 gene expression, the reactions were performed in triplicate in 96-well plates at 48°C, 30 min; 95°C, 10 min; followed by 40 cycles of 95°C, 15 s; and 61°C, 1 min. The real-time PCR products were re-confirmed by electrophoresis on 2% agarose gels. The amount of the target relative to GAPDH mRNA was expressed as 2 −(ΔCt).Total RNA from untransfected and transfected EA.hy926 cells was isolated by Allprep RNA/protein kit . After the reverse transcription reaction as described earlier, cDNA was used for quantitative real time PCR for Hardy-Weinberg equilibrium was tested for a goodness-of-fit using a Chi square test. Chi-square test was used to investigate the possible association between polymorphisms and CVD in case-control studies. Student's t test was used to analyze the difference in luciferase, mRNA transcripts and protein expression levels. Information collected from answered questionnaires and medical records were entered into MS Excel and analyzed using SPSS 16 . Differences between groups were considered significant for p values less than 0.05.Patients with CVD (n = 382) and healthy subjects (n = 372) had a mean age of 48.17 (SD = 13.37) and 37.86 (SD = 13.63) respectively (p<0.001). 53.7% of patients were females and a positive association was seen between female gender and CVD (p = 0.035). A detailed analysis of patients and controls with respect to different age groups and gender is given in FoxC2 gene in patients with CVD and healthy controls are presented in rs78816814, NG_012025.1∶g.4736G>T), c.-512C>T  and c.-1538A>G ] were observed. After adjusting for other confounding factors (age and gender), a significantly increased risk for CVD was found in patients carrying c.-512C>T, c.-1538A>G, c.-2647A>T and c.*126G>A variants. Allelic frequencies of these four polymorphisms also differed significantly between patients with CVD and controls  risk for CVD compared to first group (table S3).To understand the collective effect of these four significant polymorphisms in the disease, we further classified study subjects into two groups. Subjects with none or either one DNA was isolated from vein specimens and sequenced to check any genotypes discrepancy between whole blood samples and tissues of same patients. The genotype profiles obtained were similar in both the DNA samples from same patients.FoxC2 transcript expression was 4±1.4 folds increased in venous tissues from patients compared to normal subjects (p<0.05) (FoxC2 mRNA compared to patients carrying heterozygous CT genotype (p = 0.02) and wild CC genotype (p<0.001) (FoxC2 in tissue specimens (either at mRNA or protein level) was not significantly altered in patients who had all the four polymorphisms compared to four patients who carried TT genotype of c.-512C>T variant alone (p = 0.65) ((p<0.05) . Patientp<0.001) . The upr = 0.65) .FoxC2 protein expression with FoxC2 genotypes revealed significantly higher protein levels in patients carrying TT genotype compared to patients having heterozygous CT or wild CC genotypes (p<0.01)  .To confirm the overexpression of FoxC2 in varicose veins we performed an immunostaining on tissue sections of varicose veins from 3 patients and control vein from 3 healthy subjects. There was a marked overexpression of FoxC2 protein in the tissue sections of varicose veins compared to control veins .From our disease association results and transcript and protein expression analysis it was evident that c.-512C>T variant was related with altered mRNA and protein expression in vein tissues of CVD. We further examined if gene transcription was affected by this promoter polymorphism. As demonstrated in Dll4 and arterial marker Hey2 in the FoxC2 construct transfected Ea.hy926 cells. Expression of venous specific markers such as COUP TFII and Ephrin B4 were also assessed. Ea.hy926 cells were transiently transfected with pCAGIG vector containing FoxC2 construct (FoxC2-pCAGIG) or empty pCAGIG vector and the expression of Hey2 and Dlll4 genes were determined at mRNA levels. FoxC2 activation upregulated (p<0.05) the expression of both these arterial fate specific markers in Ea.hy926 when compared to empty vector transfected cells (FoxC2 overexpression resulted in a significant downregulation (p = 0.003) of COUP TFII, while Ephrin B4 downregulation was not statistically significant.Based on the reports of a potential FoxC2- Notch signaling ed cells . FoxC2 oFoxC2 is a forkhead/winged helix transcription factor coding gene located on chromosome 16q24.1 (NCBI Reference Sequence No: NG_012025.1). FoxC2 is implicated in vascular development especially in arterial and lymphatic differentiation FoxC2 coding sequences were reported in patients with lymphoedema-distichiasis (LD) which is an autosomal dominant form of primary lymphoedema with majority of patients developing varicose veins in lower limbs FoxC2 gene with LD risk and suggested a role of FoxC2 in the pathogenesis of varicose veins et al linked FoxC2 mutations to venous valve failure and reflux using conventional colour Doppler duplex ultrasound in patients with lymphodema distichiasis FoxC2 gene however has not yet been well-defined in patients with varicose veins or CVD. We report for the first time a positive association between genetic variants of FoxC2 and chronic venous diseases and a mechanistic insight on the role of FoxC2 in pathogenesis of CVD.Human FoxC2 polymorphisms and abnormal protein expression have been implicated with insulin sensitivity in patients with obesity and diabetes mellitus FoxC2 polymorphism pattern in patients with CVD alone. Patients with lymphoedema distichiasis were also not included in our subjects.FoxC2 gene from DNA isolated from whole blood samples of 382 patients with CVD and 372 control subjects. DNA sequencing revealed the presence of only two rare synonymous variants, c.354C>T (novel variation) and c.426G>A (rs41312258) with a frequency of 0.02 of 0.04 respectively. Hence these variations were not followed up for further studies. The analysis was extended to approximately 3 Kb 5′ untranslated flanking region of FoxC2 gene as well as 200 bp of 3′ flanking region which also includes the 3′ UTR region of gene. Seven FoxC2 polymorphisms were observed in patients with CVD and normal subjects. Two reported variants (c.-512C>T: rs34221221 and c.-1538A>G: rs4843162) and two novel variants c.-2647A>T and c.*126G>A were found to be significantly associated with risk of disease.We initially sequenced the 1.5 kb single coding exon of http://www.cbrc.jp/research/db/TFSEARCH. html) C.*126G>A variant is located 126 bp downstream to translation termination codon and 35 bp downstream to 3′UTR sequence of FoxC2 gene. C.*126G>A was consistently present in either heterozygous GA or wild GG genotypes but never in homozygous mutant AA genotype in our cohort. Prediction of microRNA/target duplexes for C.*126G>A variant was analyzed by miRNA prediction tools (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid) in silico analysis by RNAhybrid tool gave a very weak binding probability. C.-512C>T variant is present in the highly conserved proximal promoter of the FoxC2 gene Variants such as c.-2647A>T and c.-1538A>G were not further experimentally validated as they lacked the putative binding sites for transcription factors. Transcription factor binding affinity was evaluated by TF SEARCH version 1.3 computer program Click here for additional data file.Figure S2FoxC2 mRNA expression in vein tissues from patients with CVD. Relative FoxC2 mRNA levels in vein tissues from patients (n = 5) carrying all the four variants such as c.-512C>T, c.-1538A>G, c.-2647A>T and c.*126G>A and FoxC2 mRNA levels in vein tissues from patients (n = 4) carrying only TT genotype of c.-512C>T (rs34221221) variant. The difference in mRNA expression in both groups was statistically insignificant (p = 0.65). Data shown are the mean ± SD in each group.(TIF)Click here for additional data file.Table S1Primers used for PCR and sequencing of 5′ flanking region, 3′flanking regions and coding sequence of FoxC2 gene.(DOC)Click here for additional data file.Table S2Primers used for quantitative real time PCR and luciferase reporter assay.(DOC)Click here for additional data file.Table S3Patients and controls categorized as first group with neither or any single polymorphism and second group with 2, 3 or more polymorphisms. Percentages were taken from the column totals. Chi-square test for measure of association was used to derive p value. a Odds ratio and 95% confidence intervals of individual polymorphisms. b Adjusted odds ratio and 95% confidence intervals is obtained adjusting for age group and sex in multiple logistic regression model.(DOC)Click here for additional data file.


Protein-coding genes in eukaryotes are interrupted by introns, but intron densities widely differ between eukaryotic lineages. Vertebrates, some invertebrates and green plants have intron-rich genes, with 6–7 introns per kilobase of coding sequence, whereas most of the other eukaryotes have intron-poor genes. We reconstructed the history of intron gain and loss using a probabilistic Markov model  on 245 orthologous genes from 99 genomes representing the three of the five supergroups of eukaryotes for which multiple genome sequences are available. Intron-rich ancestors are confidently reconstructed for each major group, with 53 to 74% of the human intron density inferred with 95% confidence for the Last Eukaryotic Common Ancestor (LECA). The results of the MCMC reconstruction are compared with the reconstructions obtained using Maximum Likelihood (ML) and Dollo parsimony methods. An excellent agreement between the MCMC and ML inferences is demonstrated whereas Dollo parsimony introduces a noticeable bias in the estimations, typically yielding lower ancestral intron densities than MCMC and ML. Evolution of eukaryotic genes was dominated by intron loss, with substantial gain only at the bases of several major branches including plants and animals. The highest intron density, 120 to 130% of the human value, is inferred for the last common ancestor of animals. The reconstruction shows that the entire line of descent from LECA to mammals was intron-rich, a state conducive to the evolution of alternative splicing. In eukaryotes, protein-coding genes are interrupted by non-coding introns. The intron densities widely differ, from 6–7 introns per kilobase of coding sequence in vertebrates, some invertebrates and plants, to only a few introns across the entire genome in many unicellular forms. We applied a robust statistical methodology, Markov Chain Monte Carlo, to reconstruct the history of intron gain and loss throughout the evolution of eukaryotes using a set of 245 homologous genes from 99 genomes that represent the diversity of eukaryotes. Intron-rich ancestors were confidently inferred for each major eukaryotic group including 53% to 74% of the human intron density for the last eukaryotic common ancestor, and 120% to 130% of the human value for the last common ancestor of animals. Evolution of eukaryotic genes involved primarily intron loss, with substantial gain only at the bases of several major branches including plants and animals. Thus, the common ancestor of all extant eukaryotes was a complex organism with a gene architecture resembling those in multicellular organisms. The line of descent from the last common ancestor to mammals was an uninterrupted intron-rich state that, given the error-prone splicing in intron-rich organisms, was conducive to the elaboration of functional alternative splicing. Spliceosomal introns that interrupt most of the protein-coding genes and the concurrent splicing machinery that mediates intron excision and exon splicing are defining features of gene architecture and expression in eukaryotes Despite the ubiquity of introns in eukaryotic genomes, their biological status is poorly understood. To what extent introns are “junk DNA” as opposed to being functional parts of the genome, remains an open question and the answers are bound to be complicated and multifaceted. There are many reports on the contribution of introns to the regulation of gene expression Given that most unicellular eukaryotes are intron-poor whereas complex, multicellular organisms are intron-rich, it would seem intuitively plausible that introns accumulated in the course of evolution of eukaryotes. However, comparative analysis of the exon-intron structures of orthologous genes of plants and animals revealed a high level of intron position conservation, with the implication that the common ancestor of these organisms was relatively intron-rich Here we employ a probabilistic Monte Carlo model combined with a Markov Chain Monte Carlo (MCMC) method for the inference of ancestral states including robust estimation of confidence intervals to analyze a representative data set of 99 eukaryotic genomes which extensively covered the three supergroups of eukaryotes, Unikonta, Archaeaplastida (Plantae), and Chromalveolata, for which multiple genome sequences are available. The results clearly show that ancestral eukaryote forms were intron-rich, with LECA having a high intron density, on the order of two-thirds of the introns density in human genes. The subsequent evolution was heavily dominated by intron loss, with several episodes of massive intron gain associated with the emergence of some of the major eukaryote groups, in particular, animals.Naegleria gruberi, with a moderate intron density The present analysis of gene structure evolution included an extensive data set of sequenced and annotated genomes from the Unikonta , the Archaeplastida , and Chromalveolata (Heterokonta and Alveolata). Of the five supergroups of eukaryotes Intron loss and gain were modeled using a probabilistic Markov model encompassing lineage-specific loss and gain rates, as well as rate variation across sites. The Markov Chain Monte Carlo (MCMC) method This reconstruction provides a thorough view of the evolution of gene structure across three eukaryotic supergroups and reveal several general trends . Most liSeveral previous studies, performed on much smaller data sets and with less robust reconstruction methods, have suggested that at least some eukaryotic ancestral forms could have possessed intron-rich genes In addition to the Bayesian MCMC estimates, we inferred ancestral densities by using Dollo parsimony The Dollo and ML estimates show a picture of intron-rich eukaryotic ancestors that is qualitatively similar to the MCMC results. Quantitatively, similarly to the case of ancestral molecular sequence reconstruction Dictyostelium and Entamoeba , pelagophytes (A. anophagefferens), diatoms, and other eukaryotes within or outside chromalveolates , Dikarya (“higher” fungi: 3.7–4.7 introns/kilobase), opisthokonts (4.7–5.5 introns/kilobase) and, most importantly, LECA (see below). The uncertainty is larger in ancestors with subsequent turbulent history in the descendants. A case in point is the amoebozoan ancestor. There was extensive intron loss along the branch leading from the intron-rich unikont ancestor to the extant Amoebozoa. It is unclear, however, whether the losses occurred in parallel in multiple descendant lineages, or prior to the split between Naegleria gruberiNaegleria.The gene architecture of LECA is of special interest. Previous estimates of intron density for LECA were very uncertain due to methodological problems with maximum likelihood inference Strikingly, the greatest intron density among all ancestral and extant eukaryotes was inferred for the last common ancestor of the Metazoa, at 120–130% of the human density, with a 95% confidence  and 2.We validated the inference procedures by simulating the evolution of intron sites  offset by a high gain rate.The choice between these routes of evolution in a particular lineage could depend primarily on the intensity of purifying selection that is linked to the effective population size The results of this work, thanks to the extensive data set of analyzed genomes and the robust reconstruction method that yields inferences of ancestral states with minimal uncertainty, seem to close the debate on the gene architecture of ancestors of extant eukaryotes including LECA. It is now clear that the genes of ancestral eukaryotes possessed high intron density, close to the densities in the most intron-rich modern genomes, those of mammals.This finding has substantial implications for understanding the evolution of eukaryotes. It has been noticed that intron-poor genomes typically possess strong, highly efficient splice signals, whereas intron-rich genomes contain mostly weak, error-prone splice signals Remarkably, the present results indicate that the entire line of descent from LECA to mammals was a continuous intron-rich state  that proNaegleria gruberi were selected using the same procedure, but the large evolutionary distance precluded identification of a sufficient number of orthologs and unambiguous alignment of splice sites. Therefore, sequences from N. gruberi were not included them from the ancestral inference.Orthologous genes were identified using a modification of the previously described procedure The intron positions were mapped onto gene sequences using a previously developed computational pipeline . For a formal treatment, define T as the known phylogeny for the terminal taxon set S, i.e., a rooted tree with n leaves that are bijectively labeled by taxa from S. Internal tree nodes correspond to common ancestors. The history of a potential splice site is modeled as a binary labeling of all tree nodes: ξ = . In a Markov model, the labeling is randomly drawn from a distribution for which the parent-child relationships in the phylogeny define conditional independencies. The distribution of ξ at a site is fully determined by the presence probability at the root π = Pr{ξ[root] = 1}, and edge-specific rates uv, labels change with probabilitiesGene structure evolution was modeled mathematically by assuming that the table columns uv as length and rate ratioS] is a random leaf set labeling. The model defines the likelihood Conversely, −1/875. The conversion formula uses human as a reference: 6.946 is the mean number of human introns per 1000 base pairs (kbp) in the coding sequences of the analyzed genes, and 875 is the number of human introns in the data set. The posterior distribution for ancestral intron counts for a given model parametrization is computable without much difficulty Ancestral intron counts were inferred using three methods. Intron count estimates were converted into densities by the formula density = intron count·6.946 kbpIn order to estimate ancestral intron densities and lineage-specific changes in a Bayesian setting, we adapted mutation mapping techniques commonly employed with molecular sequence evolution models  S1. draw random initial parameters θ by their prior distribution P(θ)repeat S1.  S3. propose new random model parameters θ′ by distribution Q(θ→θ′) S4. with probability  S5. generate random ancestral labeling by posterior probabilitiesj by using the so-called conditional likelihoods for labeling node u with x = 0, 1, given the (possibly unresolved) labelings at the terminal taxa uS within the subtree rooted at u:In Line S4, the acceptance probability includes the likelihoods L(θ) at different model parameters, the prior distribution P(θ) of parameters, and a proposed model distribution Q(θ→θ′). In Line S5, random ancestral labelings The conditional likelihoods are calculated by dynamic programming in a postorder traversal by adapting the pruning algorithm of Felsenstein L1. draw random site-specific rate multipliers γ, νuvL2. set L[u∶x] for all nodes u and labels x = 0,1L3. compute conditional likelihoods L4. set for all non-root nodes v in a preorder traversal doL5. L6. set L7. with probability In Line L1, the rate multipliers are drawn from the posterior distribution for the different discretized rate categories using the shape parameters of the respective Gamma distributions. The SAMPLING algorithm generates a Markov chain for pairs of model parameters and ancestral reconstructions. The equilibrium distribution for the chain is the posterior distributionj, and adding the analogous sum for the sampled histories of all-absent profiles. Intron gains and losses on branches were estimated with a similar calculation.In addition to sampling histories of profiles from the input data, we also generated “all-absent” profiles with introns missing at every terminal taxon The prior distribution P(θ) was uniform for every parameter (and thus absent from the formula in Line S4): over the range  for shape parameters and edge lengths, and over the range  for π and the rate ratios. In a typical MCMC proposal, a subset of model parameters was chosen, and then multiplied by a random value; see q of parameters and ancestral intron densities.The convergence and the mixing efficiency were assessed by running 100 chains in parallel see . EstimatIndividual intron site histories were reconstructed using the Malin software Simulations were performed by generating 100 random data sets of a comparable size to the input data set using the MCMC median model parameters, coupled with an erasure procedure simulating missing orthologs, or randomly generated multipliers for simulating heterotachy : see Figure 13 in Supporting Figure S1Posterior distributions of the ancestral intron densities inferred from the sampling chains for all ancestral forms.(PDF)Click here for additional data file.Text S1Detailed methods and results with the illustrating figures and tables.(PDF)Click here for additional data file.Video S1Dynamic representations of the histories of intron loss and gain for the 245 analyzed clusters of orthologous genes.(MOV)Click here for additional data file.


The debate on human rationality goes to the heart of fundamental questions about human existence. When can we say that a decision is correct? What is the basis for the achievements of the human intellect? What is the most important cognitive distinction between humans and non-human organisms? Proposals of rationality have an interesting status as psychological theories. They are not quite theories of decision making in practice—such theories are referred to as descriptive theories, to imply that they describe what goes on. Rather, proposals of rationality are normative theories, to imply theories of how people ought to reason, if they seek decision outcomes, which are deemed to be correct, on the basis of some absolute standard  theory. This approach was established after a major shift in conceptual thinking about rationality. Before (effectively since antiquity), it was believed that the standard for correctness in decision making was classical logic. But this position came under intense scrutiny, with experimental results showing that naïve participants, even in simple tasks, would not reason in a way consistent with classical logic Wason, . One reacorrect association of probabilities to uncertain events. The Dutch Book Theorem  theory in cognitive modeling. By QP theory, we imply the mathematics for assigning probabilities to events from quantum mechanics, without the physics. QP theory is a formal theory of probability, like CP theory. QP and CP theories are based on different axioms and so their predictions can diverge. QP theory is a plausible contender in decision making . Recent work has shown that QP principles can provide the basis for simple, constrained models for empirical findings, which have been persistently problematic from a classical perspective, such as order effects on choice Moore, , the conThe above points attest to the descriptive status of QP theory in cognitive theory, not its normative status. Nevertheless, they motivate a consideration of explanatory concepts from QP theory in psychological debates. Of relevance presently is the idea of incompatibility, in relation to two (or more) questions (or possibilities etc.). According to classical theory, all questions are compatible, which means that the answer to any set of questions can be known concurrently. Following from Tversky and Kahneman's  experimeN dimensions. A classical space for just 10 binary questions requires over 1000 dimensions. The CP theory requirements for representational capacity appear too stringent. Another way to look at this issue is that, regardless of the number of questions considered, classically it is always possible to construct a complete joint probability distribution. But, where would the information come from to construct such a joint probability distribution, especially when considering unfamiliar combinations of questions (such as being a bank teller or a feminist)? Note, the principle of indifference cannot provide a general solution to this issue . For two questions with binary outcomes, we need a four dimensional space. The consideration of each additional binary question increases the dimensionality of the space by a factor of two, so that, for N binary questions, we require 2Thus, we suggest that cognitively it is more plausible to consider some questions, especially ones not typically considered together, as incompatible. Indeed, there have been suggestions that, with practice, some of the decision making fallacies attenuate . Is it possible to prove a version of the DBT for QP theory as well? Superficially, this may appear not to be the case. First, the axioms of CP theory (on the basis of which the DBT is proved) are very different from those of QP theory. Second, verifiably . However, if some of the questions are incompatible, then QP theory will provide more accurate predictions for probabilistic inference. For example, if some questions are incompatible, then order effects may arise in conjunctions (Trueblood and Busemeyer,