The potential predictive role of programmed death-ligand-1 (PD-L1) expression on tumor cells in the context of solid tumor treated with checkpoint inhibitors targeting the PD-1 pathway represents an issue for clinical research.Overall response rate (ORR) was extracted from phase I-III trials investigating nivolumab, pembrolizumab and MPDL3280A for advanced melanoma, non-small cell lung cancer (NSCLC) and genitourinary cancer, and cumulated by adopting a fixed and random-effect model with 95% confidence interval (CI). Interaction test according to tumor PD-L1 was accomplished. A sensitivity analysis according to adopted drug, tumor type, PD-L1 cut-off and treatment line was performed.p<0.0001) according to tumor PD-L1 expression was found in the overall sample with an ORR of 34.1% (95% CI 27.6-41.3%) in the PD-L1 positive and 19.9% (95% CI 15.4-25.3%) in the PD-L1 negative population. ORR was significantly higher in PD-L1 positive in comparison to PD-L1 negative patients for nivolumab and pembrolizumab, with an absolute difference of 16.4% and 19.5%, respectively. A significant difference in activity of 22.8% and 8.7% according to PD-L1 was found for melanoma and NSCLC, respectively, with no significant difference for genitourinary cancer.Twenty trials  were identified. A significant interaction , and in the context of advanced melanoma and NSCLC. The concept that the immune system plays a critical role in controlling and eradicating cancer and that immune response, driven by T-lymphocytes, is closely regulated thorough a complicated and delicate balance between inhibitory checkpoints and activating signals is well established –4. CytotWith regard to the PD-1 immune-checkpoint pathway, a series of specific inhibitors are currently under investigation and in clinical development such as nivolumab, a fully human IgG4 PD-1 immune checkpoint inhibitor antibody, pembrolizumab (formerly known as MK-3475 or lambrolizumab), a high affinity humanized IgG4 monoclonal antibody targeting PD-1, and MPDL3280A, an engineered IgG anti-PD-L1 antibody [The most promising and revolutionizing data in terms of activity of these immune checkpoint inhibitors derive from advanced melanoma, where pembrolizumab and nivolumab have been recently approved by the U.S. Food and Drug Administration (FDA) for patients with unresectable or advanced melanoma progressing after ipilimumab and a BRAF inhibitor, if BRAF V600 mutation positive , 10. In Overall, the immune checkpoint inhibitors targeting PD-1 and its ligand PD-L1 have shown unprecedented rates of durable clinical responses, with an activity range from 10% to 45% in the context of unselected populations affected by advanced solid tumors , 16–19.In order to identify the proportion of patients most likely to benefit from the immunotherapy and thus to optimize their therapeutic index, the investigation of potential predictive biomarkers represents a relevant aspect of the more recent clinical research.In this regard, the investigation of the PD-L1 assay (which can be constitutively or inducible expressed on either the surface of tumor cells or upon cancer-infiltrating T-cells) with different methods as a potential biomarker, represents one of the most challenging strategy . PD-L1 iWith respect to the predictive value of the tumor PD-L1 expression, explored in the majority of cases by immunohistochemistry (IHC), it emerged as a potential predictor of response to immune checkpoint inhibitors in a series of clinical trials: one of the first phase I study which enrolled patients with advanced melanoma, NSCLC, castration resistant prostate cancer, RCC and colorectal cancer to receive nivolumab, demonstrated an objective response rate (36%) exclusively in patients with PD-L1 positive tumors .Nevertheless, this finding did not constantly emerge in all studies, thus allowing to speculate about the reliance and reproducibility of the different methods adopted for the detection and the quantification of the biomarker. Indeed, clinical activity was demonstrated in patients with PD-L1 negative tumors , 24. On In order to explore the potential differential activity of nivolumab, pembrolizumab (targeting PD-1) and MPDL3280A (targeting PD-L1) according to the PD-L1 expression on cancer cells in melanoma, NSCLC and genitourinary cancers we conducted a sensitivity analysis of phase I, phase II and phase III trials.The analysis was conducted according to 4 pre-specified steps: 1) definition of the outcomes; 2) definition of the trial selection criteria; 3) definition of the search strategy; and 4) detailed description of the statistical methods used , 26.The analysis was conducted to determine the differential activity of nivolumab, pembrolizumab and MPDL3280A in terms of overall response rate (ORR), according to tumor PD-L1 expression for the treatment of advanced melanoma, NSCLC and genitourinary cancers. In presence of significant differential activity, a sensitivity analysis was subsequently performed in the overall sample and according to the following factors: 1) the adopted drug ; 2) the tumor type ; 3) the PD-L1 expression cut-off (1% and 5%); 4) the treatment line .th, 2015 in which patients affected by advanced melanoma, NSCLC and genitourinary cancers (RCC and bladder carcinoma) were assigned to receive anti-PD-1/PD-L1 antibody therapy including nivolumab, pembrolizumab and MPDL3280A as single agent or in combination with other immunotherapies such as vaccines or checkpoint antibodies were considered. As inclusion criteria, eligible arms for the current analysis had to report the ORR, assessed by the Response Evaluation Criteria in Solid Tumors (RECIST), according to PD-L1 expression at IHC evaluated in cancer cells with the cut-off more frequently represented in the selected trials (1% and 5%). Other antibodies targeting the PD-1/PD-L1 axis in less advanced stage of clinical development, such as those including pidilizumab, MDX-1105, MEDI4736, MSB0010718C, AMP-224 were excluded. Studies employing nivolumab, pembrolizumab or MDPL3280A in combination with target therapies or chemotherapic agents and trials with PD-L1 expression evaluated in tumor infiltrating immune cells, or different assay from IHC, were excluded as well.All phase I, phase II and phase III clinical trials published in peer-reviewed journals or presented at the American Society of Clinical Oncology (ASCO), the European Society for Medical Oncology (ESMO), the World Conference of Lung Cancer (WCLC), and the Chicago Multidisciplinary Symposium in Thoracic Oncology (CMSTO) meetings up to February 15th, 2015. Updates of trials were gathered through Medline (Pubmed: www.ncbi.nlm.nih.gov/PubMed), ASCO (www.asco.org), ESMO (www.esmo.org), WCLC (www.iaslc.org), and CMSTO (www.thoracicsymposium.org) searches. Keywords used for searching were: PD-L1, PD-1, nivolumab, pembrolizumab, MPDL3280A, melanoma, NSCLC, bladder carcinoma, RCC, phase I, phase II, phase III. In addition to computer browsing, review and original papers were also scanned in the references section to look for missing trials. Furthermore, lectures at major meetings having immune checkpoint blockade for advanced solid tumor’ as the topic were checked.Deadline for trial publication and/or presentation was February 15Data for selected outcomes were extracted: the last available update of each trial was considered as the original sources. Data for each explored outcome were extracted from trials according to the following criteria: 1) adopted drug; 2) tumor type; 3) PD-L1 expression cut-off; 4) treatment line. All data were reviewed by 3 investigators  and separately computed by 4 investigators  .Overall response rate according to RECIST were extracted from papers and/or presentations and 95% confidence intervals (CIs) were derived –29. DataThe analysis to test for interaction (Cochrane-Q) according to the PD-L1 categorical expression  was accomplished. The sensitivity analysis was conducted in the context of: 1) the adopted drug ; 2) the tumor type ; 3) the PD-L1 expression cut-off (1% and 5%); 4) the treatment line  , 31. In Calculations were accomplished using the licensed Comprehensive Meta-analysis , and MedCalc  software.Twenty trials  were identified , 32–46. With regard to tumor type, 7 studies 636 patients)  patients, 39 inclp<0.0001) according to tumor PD-L1 expression was found in the overall sample with an ORR of 34.1% (95% CI 27.6–41.3%) in the PD-L1 positive population and 19.9% (95% CI 15.4–25.3%) in the PD-L1 negative population .With regard to the adopted drug, ORR was significantly higher in patients with PD-L1 positive tumor in comparison to PD-L1 negative tumor for nivolumab and pembrolizumab, with an absolute difference of 16.4% (95% CI 10.0–22.7) and 19.5% (95% CI 8.1–27.8) respectively . Resultsp = 0.809).For what concerns the tumor type, patients with PD-L1 positive tumor significantly achieved more responses than PD-L1 negative, with an absolute difference of 22.8% for melanoma (95% CI 12.5–30.3) and 8.7% for NSCLC (95% CI 1.1–15.5) as shown in p = 0.108) . No significant difference is seen for trials where the cut-off was 1% (= 0.108) , Panel Ap = 0.0512); conversely, the difference was statistically significant in the context of second or subsequent and mixed lines with an absolute difference of 10.7% (95% CI 4.6–16.8) and 19.5% (95% CI 8.7–29.4), respectively  and its ligand PD-L1 (MPDL3280A) provide a significant differential effect in terms of activity according to PD-L1 expression on tumor cells status, which translates into a higher ORR for PD-L1 positive advanced tumors in comparison to PD-L1 negative tumors, with an overall absolute difference of 14.2% .The secondary objective of the analysis was to evaluate if the differential effect remained robust even in the context of the different adopted drugs, tumor types, PD-L1 cut-off expressions and treatment lines.With regard to the sensitivity analysis according to the adopted drug, PD-L1 status seems to predict the chance of response for nivolumab and pembrolizumab , while dWhit respect to disease setting, our analysis shows that a significant higher ORR in PD-L1 positive tumor than PD-L1 negative tumor is documented for melanoma and NSCLC while a significant effect is not documented in the context of genitourinary cancer, despite an absolute difference of 8.1%. Across the tumor types, although we cannot perform a comparison between the different disease settings, the most overall ‘immuno-sensitive’ disease seems to be melanoma (as expected) where, even in the worst case (PD-L1 negative tumors) an activity of 26.2% is documented .The PD-L1 IHC positivity on tumor cells was defined by a 1% or 5% threshold in most of the selected studies. In these studies, besides the various cut-off, various methodologies were adopted in the context of IHC such as different anti-PD-L1 antibodies, staining techniques , the definitions of ‘positive’ tumor (cell surface versus cytoplasmic expression), the definitions of PD-L1 ‘positive’ patients  and the sample used for the assay (primary tumor or metastatic lesion).In the more recent release of the KEYNOTE-001 trial, which evaluated pembrolizumab in advanced NSCLC, a cut off of 50% was adopted to define the IHC PD-L1 positivity: patients were divided into three groups, based on whether they had membranous PD-L1 expression in their tumor cells of ≥50%, 1%-49%, or <1%. Patients’ survival significantly differed between patients with high PD-L1 (1-year OS >50%) in comparison with those patients with a PD-L1 lower than 50%, who did not overcome a 1-year OS of nearly 28% .The use of multiple proprietary PD-L1 IHC assays, whose comparative outcomes are not well established, and the lack of a clear definition of ‘positive’ tumor-PD-L1 represent a limit for the interpretation of data from clinical trials according to biomarkers .According to our results, despite the heterogeneity, the best cut-off able to discriminate between ‘responders’ and ‘not responders’ seems to be 5% , Panel AIn addition, patients with PD-L1 positive tumor had a significantly higher chance to respond to treatment regardless of the treatment line , Panel BBesides the heterogeneity of the PD-L1 expression assessment, another limitation of the analysis is the heterogeneity of the included studies (few phase II with ORR as primary objective were available). The hypothesis that a differential effect according to the biomarker analyzed exist seems to be reasonable regardless of the anti-PD-1 drugs, tumor types (melanoma and NSCLC), and treatment lines. With this promise, how much this differential activity in terms of response according to the tumor PD-L1 expression status translates into a significant outcome benefit for the patients? This questions represents an interest and recent matter of research.On the basis of the available phase III trials, the KEYNOTE-066 study, demonstrated an overall survival benefit with pembrolizumab in patients with advanced melanoma, as compared to ipilimumab, across all subgroups except for the small subgroup of patients (less than 20%) with PD-L1 negative tumors .Robert et al to determine whether nivolumab, as compared with dacarbazine, improves overall survival among previously untreated advanced melanoma patients, demonstrated a survival benefit with nivolumab regardless of tumor cells PD-L1 expression status. However, these results are still preliminary as in the nivolumab group the median overall survival was not reached in either PD-L1 subgroup. Interestingly, in the dacarbazine group, the median overall survival was slightly longer in the subgroup with positive PD-L1 status than in the subgroup with negative or indeterminate PD-L1 status (12.4 vs. 10.2 months), thus opening the possible role of PD-L1 as prognostic factors, which actually still remains to be determined. Overall, the authors concluded that, given the magnitude of the clinical benefit observed in patients receiving nivolumab, PD-L1 status alone, does not seem to be useful in the selection of patients for nivolumab treatment [The CheckMate 066 study conducted by reatment .In this regard, the reason why even patients with PD-L1 negative tumor respond and why the majority of patients with PD-L1 positive tumor do not response to PD-1 pathway blockade represents an area of ongoing research. Recent studies demonstrate that besides the PD-L1 expression by tumor cells, the expression of PD-L1 on immune cells infiltrating the tumor is a potential predictor of clinical response .Herbst et al the association of tumor infiltrating immune cell PD-L1 expression with treatment response to MPDL3280A in several solid tumor types appears stronger than that with tumor cell PD-L1 expression [Powels et al in the context of metastatic bladder cancer treated with MPDL3280A [Furthermore, in the study of pression . SimilarPDL3280A . ConversPDL3280A .Overall, these results are in agreement with our sensitivity analysis data, where the predictive value of PD-L1 on tumor cells seems to be consistent just for anti-PD-1 antibody. Despite still unclear, several other mechanisms and immune regulatory pathways seem to be involved in the response to PD-1/PD-L1 pathway blockade such as the PD-L2 expression, a second known ligand for PD-1, the PD-1 expression on T-lymphocytes, and the discovery of immunogenic neo-antigens, encoded by gene mutations called ‘passenger’ that do not trigger the cancer development but play an important role in immunogenicity , 51–53.Snyder and colleagues in the context of advanced melanoma treated with CTLA-4 blockade demonstrated that a high mutational burden providing a greater likelihood of the development of specific tumor neo-antigens, recognized by the T-cells, is associated with a long-term clinical benefit from CTLA-4 blockade; conversely the absence of mutation-derived neo-antigens is associated with a minimal benefit or no benefit [In this regard, even the results reported by  benefit , 55. Ver benefit . Another benefit .Despite the overall heterogeneity, the non-prospective comparison according to PD-L1, and the fact that ORR according to this biomarker was not determined in all treated patients, the results reported herein show that patients affected by melanoma, NSCLC and genitourinary with positive PD-L1 on tumor cells may have a higher chance of response to nivolumab, pembrolizumab, and (with limited confidence) MPDL3280A in comparison to PD-L1 negative tumor. Besides the pure significant statistical effect, the magnitude of the benefit may be clinically significant in some clinical context as well. For example, with respect to the second or subsequent line treatment for metastatic NSCLC, where the maximum activity of the chemotherapy is around 10–15%, the identification of a predictive biomarker able to improve ORR over 20% in the PD-L1 positive tumor should be considered clinically meaningful . The preIn conclusion, the PD-L1 expression may potentially represent a reasonable candidate biomarker for the patients’ selection in order to optimize the treatment strategy with immune checkpoint antibodies. Nevertheless, the analytical validation and standardized definition of PD-L1 expression on tumor cells or infiltrating immune cells according to a shared cut-off to define positivity are warranted. In addition, the clinical validation of this potential biomarker as a predictor of response requires further phase III trials stratified according to tumor PD-L1 status and prospective studies in large cohorts of patients with PD-L1 positive or negative disease. Clinical trials aiming to understand the complex tumor immune microenvironment and to identify additional predictive markers or gene signature of response or resistance to immunotherapy, are currently needed.S1 Checklist(PDF)Click here for additional data file.


Approaching the Dirac point, the sub-harmonic gap structures due to multiple Andreev reflections display a strong Fermi energy-dependence and become increasingly pronounced, while the normalized excess current through the superconductor-graphene interface decreases sharply. Our observations are in qualitative agreement with the long standing theoretical prediction for the emergence of evanescent transport mediated pseudo-diffusive transport in graphene.In Dirac materials, the low energy excitations behave like ultra-relativistic massless particles with linear energy dispersion. A particularly intriguing phenomenon arises with the intrinsic charge transport behavior at the Dirac point where the charge density approaches zero. In graphene, a 2-D Dirac fermion gas system, it was predicted that charge transport near the Dirac point is carried by evanescent modes, resulting in unconventional “pseudo-diffusive” charge transport even in the absence of disorder. In the past decade, experimental observation of this phenomenon remained challenging due to the presence of strong disorder in graphene devices which limits the accessibility of the low carrier density regime close enough to the Dirac point. Here we report transport measurements on ballistic suspended graphene-Niobium Josephson weak links that demonstrate a transition from ballistic to pseudo-diffusive like evanescent transport below a carrier density of ~10 In graphenear the charge-neutral Dirac point, the evanescent mode conductivity reaches a quantum limited value of In the last decade, there has been an increasing interest in the transport properties of Dirac materials such as graphene, topological insulators, topological Dirac semimetals1234578Zitterbewegung” of relativistic particles121315161718νF ≈ 106 m/s is the energy independent Fermi velocity, EF is the Fermi energy and L is the length of the graphene channel. Therefore the potential fluctuations (δEF) near the neutrality point (NP) should be small, a few meVs, even for a micrometer long channel. In addition, charge carrier scattering should largely be eliminated, so that the transmission of the carriers reflects the intrinsic nature of the transverse modes. This requires the devices to be ballistic. Due to the strong substrate-associated disorder, previous observations were marred by the presence of large potential fluctuations  and short mean free path . More recently, monolayer graphene/h-BN hetero-structures have demonstrated ballistic transport2122Evanescent transport at the charge neutral Dirac point is a phenomenon unique to Dirac materials. For graphene, several long standing theoretical proposals have been made as to how pseudo-diffusive charge dynamics should manifest itself in transport characteristics, based on the nature and distribution of charge transmission in the conduction channels. For example, in a short and wide ballistic graphene strip, the fluctuations in electrical current (shot noise) are found to be enhanced and have strong energy dependence in the evanescent transport regime, a phenomenon analogous to the “n < 1010 cm−2), where the evanescent mode transport starts to dominate over the conventional propagating modes, the multiple Andreev reflection related features known as sub-harmonic gap structures (SHGS) become more pronounced. We also find that the normalized excess current (IexcRN), which remains constant at high carrier densities, becomes suppressed rapidly at low carrier densities near the NP. Both observations are in contrast with previous experimental observations in disordered graphene superconductor junctions where both SHGS and IexcRN show no significant gate dependence1924In this letter, we present our study on charge transport in ballistic suspended graphene-Niobium(Nb) superconducting weak links. When approaching the NP  ~ 9 with width W = 5.5 μm and length L = 0.6 μm. Such geometry minimizes any effect from the edges of graphene and complies with the theoretical prescription W/L ≥ 410.The devices used in this study are suspended graphene–Nb Josephson weak links fabricated on Si/SiOTc) and 1.5 K, are presented in T ~ 9 K the device resistance shows a very strong and sharp gate-dependence. The excellent quality of the device is evident from the quantum Hall (QH) measurements. As shown in T = 1.5 K, and in a low magnetic field of B = 300 mT, pronounced magneto-oscillations are already observed. At B = 500 mT, the sample displays fully developed anomalous quantum hall plateaus at ν = ±2, ±6… where n)-gate voltage (Vg) relation: n = 1.84 × 1010 × (Vg − VNP)[Volt] cm−2 where VNP = −0.9 V is the gate voltage at NP. This is consistent with the estimation using the geometrical capacitance considering 285 nm SiO2 in series with 220 nm (thickness of the PMMA spacer) of vacuum. On the higher mobility electron side, we also observe additional oscillatory features in resistance, R (Vg), at ν = 1, 4, 8…, etc., which may be attributed to the onset of broken symmetry states. The resistance at the NP displays diverging behavior with increasing field, starting at a low B ~ 0.3 T. All the features observed here suggest that the sample is of extremely high quality, with long mean free path and minimal potential fluctuations.After cooling down to ~10 K, the device was current-annealed through which Joule heating removes the surface contaminant from the fabrication process. The gating curves of the device after current annealing, measured at two different temperatures, 9 K  model in presence of thermal fluctuationsC = 0.9 pF and an Andreev reflection reduced resistance of R = 100 Ω, we estimate a critical current of Ic = 220 nA. The strong impact of thermal fluctuations accounts for the absence of a real zero resistance state, especially near the Dirac point where critical current is low. Hence the Josephson energy is small compared to the thermal energy: Vbias) taken at κ ~ 39). From the curve we obtain the superconducting gap at the SN interface (Δ) ~0.34 meV. This value is significantly smaller than the BCS gap of Nb due to the Andreev reflection process. Evaluating the value of the normalized excess current Z ~ 0.5. The SHGS are very weak, consistent with the theoretical prediction for ballistic channels with high transmission30Next, we study the superconducting proximity effect at  reduced . At largVbias obtained at various gate voltages. While the (x-axis) bias voltage values of the SHGS remain gate-independent, significant gate-modulation of the (y-axis) line-shape can be observed. When Vgis ramped further towards the NP, i.e., for Vbias. All the observed features appear at n = 1, 2, 3… as expected for multiple Andreev reflection processes and their positions in Vbias are independent of Vg. The higher order features are within noise. The observed features are sharpest at NP where the SHGS at n = 1, 2 and 3 are all easily resolvable. WithVg ramped across the NP and into the hole side (κ = −1.9), the SHGS begin to weaken again. The dip at n = 1 shows the most prominent response to the gate voltage. In conjunction with the appearance of the pronounced SHGS, the overall shape of the normalized differential resistance curve transforms from a “V”-shape to a shallower profile. We note that the observed gate-dependence of the Tc the “background” Now we focus on the behavior of the differential resistance as we approach the NP. bias / ΔV close to and away from NP of a diffusive device with similar Ti/Pd/Nb contacts (2 and has a mean free path R vs. Vg dependence around the Dirac point), indeed, the SHGS becomes almost completely gate-independent as demonstrated in previous work with diffusive graphene Josephson links with Ti/Al contactsFor a comparison with the ballistic device  and an ‘excess’ current (excI) due to the superconducting proximity effect. Compared to the Josephson current, excess current is much more robust against the influence of the electromagnetic environment, and hence provides a reliable parameter for characterizing the proximity effect. In S-G-S junctions the abundance of nearly ballistic modes at large Fermi energies leads to large excess current. However, in the vicinity of the NP, the number of highly transmitting propagating modes decreases and the charge transport becomes increasingly evanescent thereby decreasing the excess current. IexcRN in our device  and following previous theoretical worknth transverse mode at the Fermi level: nth mode n = 0, 1, 2, … labels the modes. The discrepancy between the theory and our observation, especially in the values for normalized excess current, may be attributed to the following factors. First, the theory assumes an ideal SN interface (Z = 0), whereas in our device Z ~ 0.5. Secondly, our measurements were carried out at a base temperature of ~1.5 K, while the theory does not consider a finite temperature. Both these factors contribute to the reduction of the normalized excess current. In addition, for lowest values of κ, the theory does not consider the presence of the electron hole puddles that exists in an actual device. This and due to the smearing from finite temperature, we observe a slight broadening in the excess current dip with an onset at κ ~ 9 compared to the theory (κ ~ 4).Further comparison with theoretical predictions for pseudo-diffusive transport can be made by characterizing the excess current in the S-G-S device. Generally in a SNS junction when Besides evanescent transport, we also consider other possible mechanisms for the observed sharp reduction of excess current near the Dirac point. One possible scenario in S-G junctions which may happen at the Dirac point is specular Andreev reflection (SAR)32We also consider the possible impact of an imperfect S-G interface that may also result in Fermi energy modification of transmission coefficients without any direct consequence of the Dirac fermionic nature of graphene. A careful study of our Nb/Pd/Ti-graphene contacts has been carried out using non-suspended devices with different channel aspect ratios. These measurements reveal a contact resistance which is at least 10 times lower that the two-terminal resistance measured in this work, throughout the gate-tunable range. Based on Landauer formalism this indicates that negligible reflection happens at the superconductor-graphene interface due to the presence of “classical” contact resistance.p-n junction that imposes the charge carrier reflections34p-n junction is the electron-hole asymmetry in the R vs. Vg dependence. As the gate voltage is swept across the NP, the S-G interface changes from p-n to n-n and the asymmetry in the R vs. Vg dependence can be associated with the transmission probability across the S-G junction. While the presence of such junctions may affect the SHGS and excI, we expect its gate voltage dependence to be gradual with no particular energy scale. In addition, such contact-doping associated reflection should give rise to asymmetry in the quasi-particle current-voltage characteristics that persists up to large gate voltages on both the electron and hole sidesIexcRN vs. EF dependence for EF < 8 meV, and the qualitatively symmetric behavior with respect to the NP.Another potential complication arises from the doping of the metal contacts which can extend into graphene, forming a In summary, we have studied the transport properties of Dirac electrons extremely close to the charge neutrality point in ultrahigh quality superconductivity induced suspended graphene. We observe a clear transition from ballistic to pseudo-diffusive behavior of Dirac electrons. This transition is reflected in the Fermi energy dependence of sub-harmonic gap structures and excess current in the vicinity of the neutrality point. Our results unambiguously demonstrate long standing theoretical predictions for the emergence of evanescent transport mediated pseudo-diffusive transport in graphene. The devices developed in this work also opens up the opportunities for future studies of low charge density physics of Dirac electrons, such as electron-electron interactions and microwave/photo-responses very close to the Dirac point, where correlated and non-Fermi liquid behaviors may become strong.et al.2/Si substrate. Here the PMMA is spin coated and hot baked at 180 °C for 90 s to form ~220 nm thick layer. An imaging resist layer of the copolymer methyl methacrylate (MMA) is then spin-coated at 3000 rpm on top of the graphene/PMMA stack and baked at150 °C, for definition of the electrical contacts using electron beam lithography. By controlling the electron beam exposure dose, we develop the 3-dimensional surface profile necessary to suspend and support the graphene channel over the SiO2/Si substrate. After e-beam lithography, the sample is exposed to UV ozone for 1.2 minutes before loaded into a metallization chamber with a base pressure below 1 × 10−8 Torr. For electrical contacts, the sample is metalized with the buffer layers Ti and Pd, each of thickness ~1.0 nm. Without breaking the vacuum, ~60 nm of the superconducting Nb film  is then DC Magnetron sputtered on top of the buffer layers in Argon plasma, at a rate of ~1 nm/s. The sputtering parameters and thickness of the metal films are determined to reduce the stress of the sputtered film on graphene and to establish a transparent interface. The lift off process is performed in two successive baths of hot (80 °C) acetone and isopropyl alcohol. The sample remains in the liquids during the process, and is finally transferred to the low surface tension hot (~60 °C) hexaneFor fabrication of the suspended graphene devices we use the method similar to work presented by Mizuno All the measurements were performed in an Oxford Instruments Variable Temperature Insert (VTI). To minimize microwave heating and lower the electron temperature, electromagnetic radiation is filtered at various stages, including a filter assembly of ferrite beads, RF chokes and pi-filters at room temperature, and cryogenic two-stage RC filters (1000 Hz cut-off frequency) at 4.2 K. For differential resistance measurements, a Keithley 6221 current source is used to supply a 10 nA AC current while ramping the DC offset current. The AC voltage response is measured with a Stanford Research SR830 lock-in amplifier, while the DC bias voltage is measured using a Keithley 2182A nanovoltmeter.ϕ is the macroscopic phase difference between the two superconducting leads; Ic is the critical current in the “pure” Josephson junction; R is the normal resistance which takes into account the Andreev reflection effect; and C is the effective capacitance between the superconducting leads, coupled by the conducting backgate.We describe the Josephson switching behaviors of the graphene-Nb devices using the resistively and capacitively shunted Josephson junction (RCSJ) model. For a current-driven device, the total current through the junction splits into the “pure” Josephson junction, a “normal state” resistor and a shunt capacitor: To solve the equation numerically, we change the differential equation to the difference equation:t is chosen to be much (~1000 times) smaller than the period of the Josephson oscillations. From f is the bandwidth used in our simulation. For each generated Johnson noise, we calculate an IV curve based on the above method. The IV curves are averaged 100 times over randomized Johnson noise currents. The final results are compared with the experimental data.Here ΔMore details of the RCSJ model simulation including its validation and the impact of damping, thermal fluctuation and skewed current-phase relation are reported in our previous work on the study of ballistic supercurrent in grapheneHow to cite this article: Kumaravadivel, P. and Du, X. Signatures of evanescent transport in ballistic suspended graphene-superconductor junctions. Sci. Rep.6, 24274; doi: 10.1038/srep24274 (2016).


Saccharomyces cerevisiae genome has undergone extensive intron loss during its evolutionary history. It has been suggested that the few remaining introns (in only 5% of protein-coding genes) are retained because of their impact on function under stress conditions. Here, we explore the possibility that novel noncoding RNA structures (ncRNAs) are embedded within intronic sequences and are contributing to phenotype and intron retention in yeast. We employed de novo RNA structure prediction tools to screen intronic sequences in S. cerevisiae and 36 other fungi. We identified and validated 19 new intronic RNAs via RNA sequencing (RNA-seq) and RT-PCR. Contrary to the common belief that excised introns are rapidly degraded, we found that, in six cases, the excised introns were maintained intact in the cells. In another two cases we showed that the ncRNAs were further processed from their introns. RNA-seq analysis confirmed that introns in ribosomal protein genes are more highly expressed when they contain predicted RNA structures. We deleted the novel intronic RNA structure within the GLC7 intron and showed that this region, rather than the intron itself, is responsible for the cell’s ability to respond to salt stress. We also showed a direct association between the in cis presence of the intronic RNA and GLC7 expression. Overall, these data support the notion that some introns may have been maintained in the genome because they harbor functional RNA structures.The  Saccharomyces cerevisiae genes contain introns  regulates its export from the nucleus . Tiling arrays and deep RNA sequencing (RNA-seq) have revealed the existence of many novel intronic transcripts, most of which have no known function  and cryptic unstable transcripts (CUTs) . Genes orthologous to intron-containing genes from S. cerevisiae were identified in 36 fungal genome sequences  using TBLASTX, with the coding gene sequence as the query and with the following settings: -E e-6 -qframe 1 -hspsepsmax 1000 -topcomboN 1. We collected the sequences of putative orthologs with at least 65% query coverage, to which 1000 bp and 300 bp of flanking sequence were added to the 5′ and 3′ ends of each hit. The putative ortholog gene sequences were then searched for the presence of the orthologous intron using BLASTN with options: -E 0.1 -W 3 -hspsepsmax 1000. The best hit  was retained for each intron in each species.Sequences of intron-containing genes were extracted from the r > 5 and folding free energy of < −5 kcal⋅mol−1 were considered as putative positives. For RNAz and EvoFold predictions, intron sequences were first aligned with mLAGAN. Structure prediction by RNAz was performed according to the manual . The, rnazWindow.pl script was used to slice the alignments with the following options: –max-gap=0.25 –min-id=30 –max-seqs=6. RNAz was then run on the forward strand of the gapped alignments (options: –forward –g –p 0) and sequences with probability P > 0.5 were considered as putative positives. For EvoFold predictions, the required phylogenetic tree containing the species present in the intron alignments was derived by pruning the tree presented by File S1. To extend the phylogenetic range of the RNA predictions, BLAST and INFERNAL 1.0.2 (Three structure prediction programs were used: CMfinder (v 0.2), RNAz (v 2.0), and Evofold (v 7b). Sequences of orthologous introns were used for predictions with CMfinder as described in AL 1.0.2 .MATa/α, his3Δ1/his3Δ1, leu2Δ0/leu2Δ0, lys2Δ0/LYS2, MET15/met15Δ0, and ura3Δ0/ura3Δ0), BY4742  and BY4741  parental strains and maintained in YPD medium containing 2% (w/v) yeast extract, 1% (w/v) peptone, and 2% (w/v) glucose. The transformants were plated on solid YPD medium with 300 mg/ml geneticin (Gibco BRL) for kanMX selection and on 10 mg/ml phleomycin (Invitrogen) for pCre-ble selection. Mineral salts medium (F1 medium) used was of the following composition (grams/liter); (NH4)2SO4 (3.13), KH2PO4 (2), MgSO4⋅7H2O (0.55), NaCl (0.1), CaCl2⋅2H2O (0.09), and 2 ml of trace element solution per liter was added to it. The trace element solution used was of the following composition (grams/liter): ZnSO4⋅7H2O (0.7), CuSO4⋅5H2O (0.1), H3BO3 (0.1), and KI (0.1). The F1 was supplemented with 2% (w/v) glucose and 1.65 ml vitamin stock before use. The synthetic minimal SD medium  and F1 medium were also supplemented with required amino acids appropriate for the parental strain (Intron sequence replacement strains were engineered from the BY4743 (S. cerevisiae strain BY4741 was grown in 500 ml rich media (YPD) in 30° with shaking at 200 rpm to an absorbance of 0.5 at 600 nm. The RNA was extracted using Trizol (Invitrogen), precipitated in lithium chloride (Ambion), washed twice with 70% ethanol, and the pellet resuspended in dH2O. RNA concentration and quality was evaluated by measuring absorbance at 260 nm on a NanoDrop spectrometer ND-1000 (Thermo Scientific). The low molecular weight enriched RNA sample was obtained from total RNA as described in The File S2. The reaction mix was composed of 4 pmol of each primer and 20 ng of total or low molecular weight cDNA for each 10 μl of total reaction mixture. The cycling conditions were an initial denaturation for 5 min at 95°, 35 cycles of denaturation , annealing , and elongation , followed by a final elongation for 5 min at 72°. Amplification of both cDNA using snR44 primers was used as a positive control. Genomic DNA extracted from BY4741 with Wizard Genomic DNA Purification Kit (Promega), according to the manufacturer’s protocol, was used as a positive control and water was used as a negative control. The PCR products were visualized by ethidium bromide staining on 1.2–3.5% agarose gels. For each predicted RNA structure, at least two independent PCR reactions with genomic DNA, total cDNA, low molecular weight cDNA, and snR44 positive control primers were performed in order to confirm expression.Complementary DNA (cDNA) was synthesized from 2 μg RNA of either total or low-molecular weight RNA using QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s protocol. Fragments of cDNA corresponding to the predicted intronic RNA structure (ncRNA), whole intron of interest (intron), and exons surrounding the intron (mRNA) were amplified by PCR with BIOTAQ DNA Polymerase (Bioline) according to the supplier’s guidelines. The list of all primer sequences used can be found in GLC7 were loaded in RNA loading dye (Fermentas) onto separate lanes of a denaturating gel containing 36.5 mM MOPS, 9.1 mM sodium acetate, 0.9 mM EDTA, 2 M formaldehyde, 0.5 µg/ml ethidium bromide, and 1% agarose. RNA transfer, UV cross-linking, and Northern blotting were performed as previously described (32P]-ATP end-labeled mixtures of two sense (to detect transcription from antisense strand) and two antisense oligonucleotides  were used as probes.A total of 10 μg of the total RNA and 10 pmol of the oligonucleotides mimicking the intronic region of escribed . [32P]-AGLC7 gene in the intron replacement mutant and BY4742 wild type grown in F1 media and in F1 + 0.9M NaCl were assessed by quantitative real-time PCR using the QuantiTect real time PCR kit . cDNA was extracted using QuantiTect Reverse Transcription Kit  according to the manufacturer’s manual. Real-time primers are listed in File S2. The PCR reactions were performed in triplicate for two independent biological replicas, as described previously (ACT1 as a reference gene.The expression levels of the eviously . RelativS. cerevisiae genome (SacCer3) using Bowtie with settings –m 1 –v 2 ] [in reads per kilobase per million mapped reads (RPKM)], reads for each intron or CDS were summarized using featureCounts  under accession no. GSE58884 (File S3). We considered genes with an average percentile rank with the top 10% to be preferentially bound by the exosome protein components.The cross-linking and analysis of cDNA  data presented by S. cerevisiae BY4743 strain was transformed with 1 μg of each PCR product according to In order to generate deletions of intron fragments or insertions into introns, the Cre-loxP system was used with a kanMX cassette flanked by loxP sites (GLC7 ncRNA deletion mutant) and 140-bp (GLC7 control deletion mutant) intronic regions with 139 bp of the loxP scar with the fragments of the transformation vector. We also constructed an insertion mutant with the 139-bp remnant of the cassette inserted into the middle of the predicted RNA structure without deleting any intron bases. All primers used in the creation of mutant strains are listed in File S2.The effect of the loxP deletion was to replace the 204-bp  GLC7 ncRNA sequences were amplified and inserted in both sense and antisense orientations into a modified version of the pRS315 yeast shuttle vector , containing the constitutively expressed TDH3 promoter and LEU2 auxotrophic marker. The pRS315 TDH3 ncRNA recovery plasmids were transformed into BY4724 and the ncRNA deletion strain, following the method described in To perform the rescue experiment of the Growth properties of the BY4742 strain, the intron mutants, and rescue mutants were assessed by time course growth profiles obtained using a FLUOstar optima microplate reader. Cells were cultured to stationary phase in YPD or F1 medium. The OD was measured at 595 nm and the cultures were diluted to an OD at 595 nm of 0.1 with prewarmed YPD media, F1 media, or F1 media containing 0.9 M NaCl. Each of the 96-well plates was filled with 240 μl of diluted culture or media control. Absorbance measurements were taken every 5 min immediately after 1 min shaking. Growth curves were plotted and analyzed using R according to a modified version of the method specified previously (5 cells/ml) of a mutant strain and the BY4742 reference strain, which had the HO gene replaced with kanMX as a marker to facilitate selection between strains. The GLC7/reference competition was performed in F1 media containing 0.9 M NaCl. The cultures were grown at 30° and maintained in log phase by diluting each culture to 2 × 105 cells/ml in fresh media every 12–24 hr until the generation number 37 ± 1 or 50 ± 2 was attained for F1 + 0.9 M NaCl or YPD media, respectively. The number of generations was calculated as described by vs. reference strains.Competitive growth assays were performed in 8 ml media by adding an equal number of cells  and the average percentile from exosome targets data (CRAC) for each gene. This article reanalyzed two publically available data sets by Strains are available upon request. S. cerevisiae introns in other fungi. We searched for orthologs of intron-containing host genes in fully sequenced genomes and then for corresponding introns in those genes. In 36 fungal genomes, we were able to identify at least two orthologs for 281 introns and at least one ortholog for 305 introns .Orthologous intronic sequences were used to predict novel RNA structures using three independent computational methods: CMfinder, RNAz, and EvoFold. Each of these approaches yielded a very different number of predicted RNA structures in introns. RNAz, CMfinder, and EvoFold identified putative conserved RNA structures in 17, 54, and 175 introns, respectively. We found 14 structures in the intersection of all three approaches, within the only intron of each of f RPS22B , File S1i.e., introns in RPL22A and RPL7B), and three introns with high prediction scores in at least two of the three approaches . We used the INFERNAL software . A similar pattern was also observed for MPT5, although its expression was very low . The RT-PCR corresponding to snR191 in NOG2 displayed a pattern indicative of complete splicing of the host mRNA, but also showed the maintenance of the complete intron and the predicted ncRNA. We refer to these sequences as “introns maintained” in the cell after splicing as opposed to “introns retained” in the pre-mRNA. We observed a similar pattern of correct splicing with intact intron and ncRNA maintenance for our predicted structures in the introns of RPS13, RPS9B, RPL7A, RPL22A, and in the 5′ UTR intron of RPS22B . These data show that a mixed population of mature ncRNAs and spliced but unprocessed introns are present in the cell. We also found that the mRNA transcripts of IMD4 (containing snR54 in its intron), PSP2, RPS9A, RPL28, RPL22B, RPL18A, RPL18B, and YRA1 were present in both spliced and unspliced forms, supporting the existing evidence that intron retention is the most common case of alternative splicing in yeast . We found no evidence of splicing of HAC1 under the specific experimental conditions we tested . As negative controls, we conducted the same RT-PCR analysis on six intron-containing genes that had no predicted RNA structures. None of the six displayed patterns consistent with ncRNAs processed from the introns. The genes YBR219C and BMH2 did not appear to be expressed and the four ribosomal protein genes RPL27A, RPS27B, RPS16A, and RPS19B showed the intron-maintained pattern . RNA-seq data show higher expression of introns in ribosomal protein genes that contain predicted RNA structures.If a S. cerevisiae   genes, we next measured the relative expression levels of maintained introns in RP that contain or do not contain predicted structures. Since the mRNA levels of the genes hosting predicted intronic structures appeared to be greater than the average mRNA amount for the entire set of intron-containing genes, we also normalized the intron expression levels by the level of their host gene transcript. Interestingly, expression of RP introns with predictions was significantly higher compared with all RP introns before and after normalizing for host gene expression  .in vivo RNA cross-linking (CRAC) of exosome components to show that noncoding RNAs, snoRNAs, pre-tRNAs, and pre-mRNAs are the most prominent exosome targets. The in vivo cross-linking experiments to the tagged protein components of the exosome. When we normalized the number of reads of each intron-containing gene from GSE40046 by the average number of reads for this intron-containing gene from GSE58884, we observed that the set of genes with both known snoRNA host genes and our novel RNA predictions appears to be targeted by the exosome machinery more than expected   . This suGLC7 was recently shown to play a role in the cellular response to osmotic pressure  and the intronic region corresponding to the ncRNA (GLC7 ncRNA deletion mutant). For the alteration, we inserted 139 bp in the middle of the predicted ncRNA to modify its structure . Previously it was shown that replacing the GLC7 gene with its cDNA decreases the cell viability in NaCl stress (GLC7 intron in F1 medium and in F1 medium containing 0.9 M NaCl (GLC7 ncRNA deletion and insertion mutants compared with the WT (one-way ANOVA with Dunnett’s multiple comparison test: for both mutants P < 0.0001) or the negative control deletion  and the final biomass after 48 hr (A) are decreased, whereas for the negative control deletion mutant those parameters are the same as the WT . We attempted to restore the fitness of the GLC7 ncRNA deletion mutant by inserting the ncRNA expressed under the strong constitutive promoter . There was no significant recovery in fitness from the addition of the sense or antisense orientation ncRNA overexpression plasmids . This suggests that the intronic ncRNA is exclusively cis-acting.To study the phenotype of the ncRNA, we used two approaches: deletion and alteration of the ncRNA structure, the latter of which increases the distance between the two predicted hairpins. We deleted two regions in the intron of 9 M NaCl . In F1 sGLC7 deletion mutant were grown together in F1 medium supplemented with 0.9 M NaCl, and the composition of the population was analyzed after 37 generations. A significant drop in relative amount of the mutant strains was detected with just over 25% of total cells being GLC7 deletion mutant, instead of the expected 50% . Taken together, these data further indicate that the deletion of the putative ncRNA in the GLC7 intron is responsible for the observed impairment of cell growth rate and competitive fitness during salt stress, whereas deletion of the upstream intronic region overlapping the CUT568 expressed from the opposite strand has no impact on the phenotype in salt stress.We further verified the phenotypic difference of the mutant using one-to-one competition experiments. The WT BY4742 strain and the GLC7 via real-time PCR in WT, control deletion mutant, and GLC7 ncRNA deletion mutant. The data show that the deletion of the ncRNA or the control region in GLC7 intron does not impair GLC7 expression in F1 media. In fact, the GLC7 mRNA level in ncRNA deletion mutant appears to be slightly elevated compared to that of WT or control deletion (mean ± SEM: 1.20 ± 0.01). However, in salt stress the GLC7 mRNA level of the GLC7 ncRNA deletion mutant is reduced to 0.57 ± 0.03 of that of the WT . We suggest that the phenotypic effect of the ncRNA knockout under salt stress may be due to the decreased expression or a splicing defect of GLC7 in the GLC7 ncRNA deletion mutant.We then looked at the expression of f the WT , whereasTRM10 mRNA (RPS22B (RPL18A (Rnt1p (RPS9A and RPS9B (RPS13 were shown to regulate the expression of the host gene or its paralogous copy, change cell sensitivity to drugs, or alter the competitive fitness when deleted. Our RNA-seq data analysis shows that introns in RP genes containing RNA structures are significantly more expressed than the introns in RP genes lacking a predicted ncRNA. Among the seven non-RP proteins with high-scoring predictions, only the PSP2 introns have no previously suggested function. HAC1 and YRA1 introns contain structures that regulate their own splicing and IMD4 and NOG2 contain snoRNAs that guide chemical modification of other RNAs, whereas GLC7 and MPT5 introns appear to be required for stress tolerance (ncRNAs can be present in intergenic regions ( (RPS22B  and in t (RPL18A . Both innd RPS9B , and thend RPS9B . In anotGLC7 intron was previously shown to mediate the response to salt stress (cis. RNAs functioning in cis are common among eukaryotes and originate from their own transcriptional unit (Our experimental characterization and follow-up analysis have allowed us to generate hypotheses regarding the mechanism of function of specific sequences. For example, the t stress . Furthernal unit . There iin vivo RNA cross-linking to protein complexes coupled with next-generation sequencing, can aid the discovery of novel pathways involving all types of ncRNA, including those encoded in introns. Our analysis of exosome target data presented by New techniques, such as S. cerevisiae introns do not appear to contain classical intronic ncRNAs. Although the function of most intronic RNAs in higher eukaryotes is still unknown, the evidence of tissue-specific expression (GLC7 intronic RNA structure in an intron-poor single-celled fungal species.With the exception of a few snoRNAs, Xenopus tropicalis embryos showing that 90% of introns are maintained in the nucleus postsplicing (ANKRD52 (Our work, and that of others mentioned here, raises interesting questions about the general nature of intron stability postsplicing. Introns removed by splicing have been thought to be rapidly degraded and the main focus of intron biology has concerned elucidation of splice sites and the arrangement of the splicing machinery. For example, it was observed that the deletion of the debranching enzyme promotes the accumulation of lariat introns , so it wsplicing . Linear S. cerevisiae genome in order to identify introns with undiscovered function. Comparing intron sequences of related yeast species, we found at least 19 introns contain putative conserved RNA structures. By RNA-seq and RT-PCR, we show that several of the intronic sequences containing secondary structures are not degraded after removal from pre-mRNAs. Furthermore, we show that RNA structures embedded in introns can be directly responsible for regulating gene expression and maintaining phenotype in the intron-poor yeast. For example, a small portion of the GLC7 intronic sequence, representing a novel RNA structure, plays an important role in the cellular response to salt stress. More generally, the cellular abundance of intron sequences from RP genes with predicted intronic RNAs is significantly higher than for those lacking such predictions. Overall, our data support the possibility that the presence of functional RNA structures in introns has contributed to selective intron retention in the Saccharomycetes.We undertook a systematic approach using the well-studied


Previous studies investigating a genetic basis for idiopathic pulmonary fibrosis (IPF) have focused on resequencing single genes in IPF kindreds or cohorts to determine the genetic contributions to IPF. None has investigated interactions among the candidate genes.To compare the frequencies and interactions of mutations in six IPF-associated genes in a cohort of 132 individuals with IPF with those of a disease-control cohort of 192 individuals with chronic obstructive pulmonary disease (COPD) and the population represented in the Exome Variant Server.SFTPA2), and C (SFTPC), the ATP binding cassette member A3 (ABCA3), telomerase (TERT), thyroid transcription factor (NKX2-1) and mucin 5B (MUC5B) and compared the collapsed frequencies of rare , computationally predicted deleterious variants in each cohort. We also genotyped a common MUC5B promoter variant that is over-represented in individuals with IPF.We resequenced the genes encoding surfactant proteins A2 (SFTPA2 (n=1), SFTPC (n=5), ABCA3 (n=4) and TERT (n=5)). No individual with IPF had two different mutations, but one individual with IPF was homozygous for p.E292V, the most common ABCA3 disease-causing variant. We did not detect an interaction between any of the mutations and the MUC5B promoter variant.We found 15 mutations in 14 individuals (11%) in the IPF cohort: (SFTPA2, SFTPC and TERT are collectively over-represented in individuals with IPF. Genetic analysis and counselling should be considered as part of the IPF evaluation.Rare mutations in  SFTPA2, SFTPC, (surfactant proteins) and TERT (telomerase) were found in individuals with idiopathic pulmonary fibrosis (IPF) but not those with chronic obstructive pulmonary disease.Rare mutations in Mutations associated with childhood interstitial lung disease are present in adults with IPF supporting that these mutations predispose to a spectrum of interstitial lung diseases manifesting at all ages.Genetic analysis and counselling should be considered as part of the IPF evaluation.Idiopathic pulmonary fibrosis (IPF) is the most common chronic interstitial lung disease (ILD) in adults.3TERT, Gene ID 7015) and in the surfactant-associated genes, surfactant protein A2 , surfactant protein C  and the ATP-binding cassette member A3 .MUC5B, Gene ID 727897) promoter is significantly more prevalent in individuals with both sporadic and familial IPF.SFTPC, ABCA3, and the thyroid transcription factor gene  are associated with childhood ILD.SFTPC, disease penetrance varies in severity, age of onset, histopathology and clinical characteristics suggesting that childhood ILD and IPF in adulthood may be part of the same spectrum of disease.18–21Previous studies investigating genetic predisposition in sporadic and familial pulmonary fibrosis have identified rare mutations in the gene encoding telomerase  or in population-based cohorts, and to determine if interactions among these genes and the Individuals with IPF were included if they had a usual interstitial pneumonia (UIP) pattern on lung explant or biopsy, or had a clinical diagnosis of IPF validated by the research team using American Thoracic Society/European Respiratory Society 2011 criteria.Archived DNA samples from individuals with COPD through the COPD SCCOR Project at Washington University/Barnes-Jewish Hospital (5 P50 HL084922) from patients recruited from 2005 to 2011 were used as a disease-based control. Through permission for sample sharing, we obtained the DNA and linked de-identified clinical information including age, gender, race and smoking history from existing databases through a waiver of consent. The clinical characteristics of each cohort are summarised in http://evs.gs.washington.edu/EVS/) were used to provide race-stratified, population-based frequencies of variants in candidate genes (accessed 1 July 2013).Data from the NHLBI Exome Sequencing Project  and genes associated with both childhood ILD and IPF . Mutations in ABCA3 are expressed in a recessive manner, while mutations in SFTPA2, SFTPC, NKX2-1 and TERT are dominantly expressed. The presence of either one or two copies of the MUC5B promoter variant is associated with IPF, however, exonic mutations in MUC5B have not been studied. Therefore, we also interrogated MUC5B, though the mode of inheritance is unknown. Although mutations in the telomerase RNA component gene  are also associated with IPF, the mutations occur in the part of the gene that encodes the RNA component and we therefore could not apply our definition of ‘mutation’ based on alteration in protein function.23Based on our hypothesis that childhood ILD and IPF may be part of a spectrum of disease, we selected genes that were known at the time we designed the study between associate with IPF only  and the presence of significant repetitive sequence that prevented standard sequencing approach), NKX2-1, SFTPA2, SFTPC and TERT were amplified, approximately 45 kb of sequence per individual. Equal amounts of amplified product were pooled for next generation sequencing using the Illumina Genome Analyzer/MiSeq platform. Negative and positive controls were inserted into the pooled sample to optimise detection of rare variants with high specificity and sensitivity.25We performed DNA purification on preserved saliva samples obtained from the patients with IPF using Oragene DNA self-collection kits and purification protocol . The DNA from each specimen was quantified using the Qubit Fluorometer and equimolar amounts of each patient's DNA were pooled. Using this pooled DNA, all exons of http://evs.gs.washington.edu/EVS/ ). We then validated using an independent genotyping strategy through Sanger resequencing or Taqman Genotyping Assays. Taqman genotyping was also used for the MUC5B promoter polymorphism. The validation of variants allowed us to confirm the frequency of each variant and to link it to an individual.We used SPLINTER (Short IN/DEL Prediction by Large deviation Inference and Non-linear True frequency Estimation by Recursion) to identify rare single nucleotide variants and small insertions/deletions.Study data were collected and managed using REDCap electronic data capture tools hosted at Washington University School of Medicine's Institute for Clinical and Translational Studies.NKX2-1. There were no differences in age of onset, sex or tobacco use in the IPF individuals with or without a mutation  previously identified in children with ILD but not in adults with IPF  and SFTPA2 (p.A91P) were present in similar frequencies in the IPF and COPD cohorts compared to the general population, as were the common synonymous variants in ABCA3  (see online supplementary table S1). The frequency of SFTPA2 p.Q223K was less than the ESP frequency, which could be due to sample size. The frequencies of the common TERT variant p.A279T, previously associated with aplastic anaemia, were similar in IPF and COPD cohorts and lower than the 3% population-based frequency in the ESP, suggesting that this mutation is not contributing to pulmonary disease (see online supplementary table S1).18To determine if the individuals in the IPF and COPD cohorts were representative of the general population, we also examined the frequencies of common variants in these genes. Although none of these common variants are predicted to be deleterious, we cannot exclude the possibility that some of these variants could be in linkage with our functional mutations, but our sequencing methodology did not permit identifying on which allele these variants resided. The common non-synonymous variants in SFTPA2, SFTPC and TERT. The remaining 23 individuals (88%) did not have a mutation in any of these candidate genes that explained their family history.Of the 132 individuals in the IPF cohort, 26 (20%) reported a family history of IPF. In those 26 individuals, only 3 (12%) had a mutation identified in one of the 6 genes, one each in Interestingly, the other 11 individuals in the IPF cohort with an identified mutation did not have a family history of IPF, suggesting (1) that the mutations were spontaneous in these individuals, (2) the mutations were inherited but not penetrant in the parent, (3) in the case of novel mutations, they were not disease causing or (4) family history of pulmonary fibrosis had not been fully ascertained or recognised through family member recall.MUC5B were similar in the IPF and COPD cohorts and in the ESP database. Consequently, mutations in the coding sequence did not appear to be associated with attributable risk for IPF and were analysed separately from mutations in the other genes  of the individuals in the IPF cohort had mutations in IPF-associated genes. This frequency may be either overestimated or underestimated based on our definition of a ‘mutation’ which relied on a previous association of the gene with disease, or computationally predicted effects on protein function. We used a stringent definition for altered function by requiring that 2 of 3 algorithms predicted functionality. However, only testing in a relevant model system and replication in an independent cohort can confirm the functional significance of these mutations. Additionally, our IPF cohort was weighted toward individuals who were referred for lung transplantation which could bias towards younger individuals, and thus, could also be enriching the cohort for genetically-based disease. The significant gender difference between the IPF and COPD cohorts is consistent with the demographics of each disease and mutations were identified in individuals of both genders in each cohort but any possible gender affect on rare variant discovery would have to be investigated with a larger patient population.SFTPC in one individual with familial IPF (4%) and four individuals with sporadic IPF (4%). Previous studies have demonstrated the prevalence of SFTPC mutations to be as high as 25% in familial pulmonary fibrosis  and as low as 0.7% in sporadic IPF.SFTPC mutations identified in our cohort have previously been identified in children and adults with ILD and/or IPF. Two of these mutations, p.I73T and p.L110R, result in aberrant prosurfactant protein C protein products in vitro.SFTPC in the COPD cohort, and they are not present in the general population, this enrichment in the IPF cohort further strengthens the evidence that these dominantly expressed mutations are disease-causing and demonstrates that even ‘sporadic’ IPF may have a genetic basis due to spontaneous mutation.As anticipated from other studies, we found mutations in SFTPA2 have been identified in kindreds with familial IPF and lung cancer.Dominant mutations in ABCA3 mutations cause neonatal respiratory failure and childhood ILD, and single ABCA3 mutations (p.E292V and p.D123N) interacting with SFTPC p.I73T have been reported in two families, one with childhood ILD and one with IPF.ABCA3 p.E292V adds to the recent identification of a kindred with pulmonary fibrosis due to a p.G964D thus further supporting the possibility that adult-onset fibrotic lung disease due to homozygous or compound heterozygous mutations in ABCA3 may occur.ABCA3 mutations in newborns with RDS, suggesting a developmental interaction that increased risk or severity of disease, we did not find the frequency of single ABCA3 mutations in the IPF cohort to be greater than the 3–5% in the general population. This is consistent with a prior study showing single E292V mutations were not a major risk factor for pulmonary disease in the general population, and also suggests that interactions with other modifiers are less likely to increase risk for disease.35–37Homozygous recessive or compound heterozygous NKX2-1 are extremely rare in the general population, the lack of identifiable mutations in NKX2-1 in our IPF and COPD cohorts may simply be a function of the limited sample size of our cohort, thus making it difficult to know the true prevalence of NKX2-1 mutations in patients with IPF.Since mutations in TERT and the RNA component of telomerase (TERC) have shown heterozygous mutations to be present in 8–15% of familial IPF cohorts.TERT were only seen in the IPF cohort, solidifying the link between this gene and predisposition to IPF. That the p.A279T mutation was present in IPF and COPD cohorts with similar frequencies makes it unlikely that it contributes directly to the pathogenesis of IPF. Complex functional assays will be necessary to identify any mechanisms whereby these mutations exert an effect.Previous studies of MUC5B to determine if mutations in the coding sequence of the gene might also associate with IPF. Although we found individual rare deleterious variants in IPF and COPD cohorts, the similar collapsed frequencies in both cohorts makes it unlikely that mutations in coding regions of this gene contribute appreciably to the risk of IPF.Since the rs35705950 promoter polymorphism is over-represented in individuals with IPF, we resequenced all but the largest exon of MUC5B promoter polymorphism was approximately 30% in our IPF cohort which is similar to previous reports.ABCA3, SFTPA2, SFTPC or TERT.The MAF of the The presence of childhood ILD-associated gene mutations in adults with IPF supports the hypothesis that these mutations predispose to a spectrum of fibrotic lung diseases manifesting from infancy to adulthood. Therefore, it is possible that childhood ILD and adult IPF represent variable expression of a common underlying gene-based pathogenic mechanism. While these mutations increase disease risk, the environmental modifiers that cause injury, disrupt repair and ultimately result in disease remain to be identified, along with the full complement of gene mutations predisposing to fibrotic lung disease. While possible that exome sequencing may only capture coding region variants and disregard non-coding elements of genome leading to an underestimate the genetic contribution of a gene to IPF, further investigation of genetic susceptibility to IPF using exome sequencing of affected and unaffected family members could elucidate not only the relationship between childhood ILD and adult IPF, but also the unidentified genetic factors leading to disease expression.


Noncoding RNAs are key players in the maintenance of genomic integrity, particularly in silencing the expression of repetitive elements, some of which are retrotransposable and capable of causing genomic instability. Recent computational studies suggest an association between L1 expression and the generation of small RNAs. However, whether L1 expression has a role in the activation of small RNA expression has yet to be determined experimentally.; Here we report a global analysis of small RNAs in deep sequencing from L1-active and L1-silenced breast cancer cells. We found that cells in which L1 expression was silenced exhibited greatly increased expression of a number of miRNAs and in particular, members of the let-7 family. In addition, we found differential expression of a few piRNAs that might potentially regulate gene expression. We also report the identification of several repeat RNAs against LTRs, LINEs and SINE elements. Although most of the repeat RNAs mapped to L1 elements, in general we found no significant differences in the expression levels of repeat RNAs in the presence or absence of L1 expression except for a few RNAs targeting subclasses of L1 elements. These differentially expressed small RNAs may function in human genome defence responses. Drosophila germline cells. These repeat-associated siRNAs are now known as piRNAs [Small RNAs have recently emerged as being important in the control of gene expression in almost all biological processes. Many different types of small RNA, including microRNAs (miRNAs), endogenous siRNAs (endo-siRNAs), small nucleolar RNAs (snoRNAs) and more recently, Piwi-interacting RNAs (piRNAs), are expressed in a variety of tissues and exert diverse effects on cellular functions -3. The ds piRNAs , since ts piRNAs , 11.The human genome contains large numbers of repetitive sequences and retrotransposable elements both within and between genes. These repeat elements, accounting for nearly 45% of the genome, are grouped into four classes: LINEs (long interspersed elements), SINEs (short interspersed elements), LTRs  and DNA transposons. The most active retrotransposons are the LINE-1 or L1 elements  which arDrosophila and in mouse oocytes have shown that endo-siRNAs regulate retrotransposons and protein-coding genes that are complementary to the endo-siRNAs [Several studies carried out in somatic cells of o-siRNAs , 18, simo-siRNAs . Intereso-siRNAs . In relao-siRNAs . Overexpo-siRNAs . This racis-regulatory sequences [Nearly 25% of human promoters have been reported to contain retrotransposon-derived sequences including many equences , which aequences , 24, whiequences , which aequences . Moreoveequences . Despiteequences . Here, wp=0.001) and 78 ± 5.8% (p=0.003) for ORF1 and ORF2 mRNA, respectively . Finally, we mapped the reads to the deepBase repeat-associated small RNA database  [To investigate whether the activation or silencing of L1 elements plays any role in contributing to small RNA expression, we used the Illumina GAIIx high-throughput sequencing platform to sequence small RNA libraries from the endo453 (L1-silenced) and scrambled (L1-active) cells. Since L1 expression was controlled by the introduction of exogenous (synthetic) endo453, the introduced sequences were bioinformatically removed from the files of Illumina reads prior to mapping in order to identify unbiased expression of small RNAs in the L1 silenced library. We then used the miRanalyzer small RNA mapping pipeline  to map te.fasta)  to detecTable 1let-7a in the L1-silenced library compared to 149,428 reads in the L1-active library, accounting for ~41% of the difference in the unnormalized counts of the mapped sequence reads. This large difference remained even after normalizing the read counts in both libraries using the DESeq package which is incorporated into the standard miRanalyzer pipeline [The distribution of lengths of the L1-silenced small RNAs was similar to that of the L1-active library, with peaks at 22–23-nt Figure . After rpipeline . DESeq upipeline , 33.-7 family members; let-7a, let-7b, let-7c, let-7e and let-7f were all up-regulated in the L1-silenced cells, in some cases, very markedly  using the miRanalyzer pipeline , which ay Figure . This regulation . In addiThe canonical miRNA biogenesis pathway generates an miRNA duplex that consists of an miRNA and miRNA* strand. Several miRNA* species have been recently found in humans and some of them have beenimplicated in differentiation of cancer cell states . In the piRNAs are a distinct class of small RNAs that interact with Piwi proteins and are involved in epigenetic and post-transcriptional silencing of retrotransposons and other genomic repeats in germline cells. Unlike miRNAs, which are in the size range of 21-23 nt, piRNAs are 24-31 nt in length. piRNA sequences do not have a precursor with a stem-loop and are not processed by Dicer. While there have been few reports of the presence of Piwi proteins in human cancers , one rec−7. BLAST searches with the sequences of hsa_piR_001101 and hsa_piR_020450 also gave hits to C/D box snoRNAs residing in the introns of genes. These observations suggest that such piRNAs may have a role influencing gene expression. Although the functions of piRNAs are quite diverse and largely unknown, some of the differentially expressed piRNAs observed here may be involved in regulating cell activities and further studies in this area should reveal the functions of these piRNAs.The most highly differentially expressed piRNAs as assessed by GFOLD were hsa_piR_000586 (fold change = 22x), hsa_piR_001101 (14x) and hsa_piR_020450 (16x) which were all up-regulated in the L1-silenced cells Figure . The extRepeat-associated RNA refers to a class of noncoding small RNAs that includes transcripts from genomic repeats. Although it is unclear how they are processed and their biological functions are largely unknown, repeat-associated small RNAs map to repetitive elements such as LINEs, SINEs and LTRs. Like siRNAs, most repeat-associated small RNA sequences are in the size range of 18-23-nt. Until recently, all repeat-associated small RNAs were grouped together based on their genomic locations as rasRNAs . While aUsing Unix command-line tools and Perl and R scripts (available on request), we extracted the RepeatMasker  annotatiThe number of sense and antisense reads in each repeat RNA family and their relative fold changes between the two libraries are summarized in Figure Drosophila and their biogenesis is at least in part, Dicer-dependent [Drosophila endo-siRNAs in somatic cells [A summary of the small RNA mapping results for the L1 active and L1-silenced libraries is given in Figure ependent . Thus itic cells . Becauselet-7 family of miRNAs, and increased expression (above a GFOLD threshold of 2.0) of several other miRNAs including hsa-miR-196a, hsa-miR-30a/d, hsa-miR-191 and hsa-miR-200c following silencing of L1 expression. We also observed increased expression of a number of specific piRNAs, but overall, a decrease in total piRNA expression in the L1-silenced cells. The most highly up-regulated piRNAs were hsa_piR_000586, hsa_piR_001101 and hsa_piR_020450. Remarkably, these piRNA sequences showed a perfect match to C/D box snoRNAs residing in the introns of protein-coding genes, suggesting that expression of such piRNAs may influence gene expression.In light of the accumulating data reporting a connection between the expression of L1 elements and the generation of retrotransposon-derived small RNAs, we analyzed changes in the expression profile of miRNAs, piRNAs and repeat-associated small RNAs before and after silencing the expression of L1 elements in breast cancer cells, using the published L1-specific endo453 sequence that specifically silences endogenous L1 expression . We founConsidering the repeat-associated small RNAs that mapped to repetitive elements, in general we found no significant differences in the levels of the repeat RNAs in the presence or absence of L1 expression except for a few antisense RNAs potentially targeting subclasses of retrotransposons. The antisense repeat RNAs that were most differentially expressed between the L1-active and L1-silenced cells mapped to active L1 elements , and the MIRc element. The majority of the repeat RNAs analyzed in this study are 18-23 nt in length suggesting that they are endogenous siRNAs as opposed to longer piRNAs, as would be expected in somatic cells. Since the mechanisms of biogenesis of small RNAs derived from repeat elements are not yet known, it is difficult to say whether these repeat RNAs are functional siRNAs, and answering this question would require a functional analysis. The alternative possibility is that leaky transcription from genomic repeats could produce a range of fragmented small RNA sequences, which may or may not be involved in the control of repetitive elements. It is also unclear why the genome expresses sense and antisense repeat RNAs against the oldest and inactive L2 elements. It seems more likely that the L2-derived repeat RNAs are an exaptation of L2 sequences rather than a defense against them. Interestingly, our study shows that most of the repeat-associated small RNA reads mapped to the L1 family and only a small proportion mapped to SINE and LTR elements. The high proportion of repeat RNAs expressed against L1 elements is not unexpected as L1 is the only known active retrotransposon in the genome.bona fide human miRNAs that can target L1 elements. siRNAs can arise from dsRNA precursors, which are processed by the RNAi machinery to generate functional siRNAs against genomic repeats and retrotransposons [The expression of L1 elements poses a substantial mutagenic threat to genomic stability. To control L1 expression, cells have developed a variety of silencing mechanisms. Several types of small RNAs, including miRNAs, endo-siRNAs and piRNAs, have evolved as defense mechanisms against L1 activity. However, to date, there has been no report of nsposons , 4. In cnsposons . In addinsposons . piRNAs nsposons  and in tlet-7 miRNAs in L1-active and L1-silenced cells is an intriguing finding; similar to the differential expression of let-7 seen in normal and cancer cells whereby the expression of let-7 is post-transcriptionally inhibited in cancer cells [let-7 and L1 elements, there is evidence indicating that cancer cells, which normally express high levels of L1 elements, have globally reduced levels of small RNAs, and in particular, miRNAs, compared to their normal counterparts [Recent genome-wide studies have revealed that L1 expression and retrotransposition occurs at a high level in the cancer genome, resulting in large-scale restructuring of the genome . Consister cells . Althougterparts .At this stage, the underlying mechanism of the activation of the small RNAs in L1-silenced cells is unknown. One possibility is that silencing of L1 expression substantially reduces the restructuring of the genome and the transcriptome that occurs in cancer. One could therefore argue that the relative expression of other RNAs should change in L1-silenced cells. Strikingly, it has been shown that inhibition of L1-encoded reverse transcriptase in several cancer cell types modulates cell growth and differentiation by affecting global gene expression . AnotherT47D (ATCC HTB-133) human breast cancer cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 2 mM L-glutamine and 10% FCS under standard cell culture conditions. An shRNA that mimicked 22-nt of the L1-specific endo453 and a scrambled control shRNA were synthesized as described in reference  and clonTotal RNA from control and endo453-transfected cells was extracted using TRIzol (Invitrogen). Low MW small RNA was enriched by adding 50% PEG-8000 and 5M NaCl to a final concentration of 5% and 0.5M, respectively, followed by gel purification in a 15% urea-PAGE gel (Invitrogen). Small RNAs ranging from 18-30 nt were gel purified and ligated to 3' adaptor and 5' adaptor oligonucleotides as described in the Illumina Small RNA Kit. Briefly, the 3'-RNA adaptor (5'/5rApp/ATCTCGTATGCCGTCTTCTGCTTG/3ddc/), which specifically ligates to RNAs that contain a hydroxyl group at their 3'end, was ligated to 1 μg of low MW small RNAs using T4 RNA ligase (NEB). The resulting products were subsequently ligated to the 5'-RNA adaptor (5'-GUUCAGAGUUCUACAGUCCGACGAUC-3'). cDNA was synthesized with SuperScript II Reverse Transcriptase (Invitrogen) and subjected to 12 cycles of PCR amplification with high-fidelity Phusion Polymerase (NEB) using primers as published by Illumina. Each library was loaded on a single Illumina lane at 20 pM and underwent 36 cycles of sequencing on an Illumina GAIIx Genome Analyzer.http://bioinfo2.ugr.es/miRanalyzer/miRanalyzer_tutorial.html#x1-110004.1.1 In order to calculate differential expression, the mapped read counts were normalized and fold changes between L1-active and L1-silenced cells were calculated with the R/Bioconductor DESeq package [Singleton reads and reads with a 3'-adapter substring <6 nt or trimmed sequence length <17 nt were removed from the analysis carried out by the miRanalyzer pipeline. Trimmed reads were aligned to the miRBase database of human miRNAs (version 16) using Bowtie as implemented in the miRanalyzer pipeline (with the following Bowtie options: minimum score of 30 and minimum identity of 90%) . Many mi package  which isTo identify differentially expressed piRNAs, we selected the trimmed reads in each library with lengths greater than 23 nucleotides using the filter function in CLC Genomics Workbench 5.5.1 and mapped these subsets of reads against the human piRNABank database  using CLTo identify differentially expressed RNAs mapping to repetitive elements, we selected the trimmed reads in each library with lengths less than 23 nucleotides using the filter function in CLC Genomics Workbench. Repeat RNAs were identified and annotated by alignment with the known rasRNA sequences in the deepBase database (version GRCh37). Alignme