In recent years, it has been suggested that host cells exert intrinsic mechanisms to control nuclear replicating DNA viruses. This cellular response involves nuclear antiviral factors targeting incoming viral genomes. Herpes simplex virus-1 (HSV-1) is the best-studied model in this context, and it was shown that upon nuclear entry HSV-1 genomes are immediately targeted by components of promyelocytic leukemia nuclear bodies (PML-NBs) and the nuclear DNA sensor IFI16 (interferon gamma inducible protein 16). Based on HSV-1 studies, together with limited examples in other viral systems, these phenomena are widely believed to be a common cellular response to incoming viral genomes, although formal evidence for each virus is lacking. Indeed, recent studies suggest that the case may be different for adenovirus infection. Here we summarize the existing experimental evidence for the roles of nuclear antiviral factors against incoming viral genomes to better understand cellular responses on a virus-by-virus basis. We emphasize that cells seem to respond differently to different incoming viral genomes and discuss possible arguments for and against a unifying cellular mechanism targeting the incoming genomes of different virus families. Viruses are intracellular parasites and need to reach their site of replication in order to propagate; this requires overcoming several structural and molecular barriers of the host cell. For nuclear replicating DNA viruses, the final destination is the nucleus, but before nuclear import of the viral genome can take place they must pass several subcellular compartments. In addition, each compartment of the host cell is equipped with “sensors” and “effectors” against invading viruses to detect the invading virion for degradation immediately after infection or, if this fails, to target the viral genome to prevent viral gene expression. During virus entry, the first obstacles to overcome are the plasma and/or endosomal membranes. At the cell surface or in the lumen of the endosomal compartment, sensor molecules called toll-like receptors (TLRs) are expressed. These sensors recognize molecules characteristic of pathogens, including their DNA genomes  . PathogeThe most prominent factors in nuclear antiviral responses, which have been implicated in targeting incoming viral genomes, are components of promyelocytic leukemia nuclear bodies (PML-NBs) and the nuclear DNA sensor IFI16 (interferon gamma inducible protein 16). PML-NBs, also called ND10, are distinct subnuclear domains, which can be observed as punctate dots in microscopy analyses ,10. PML-The sensing and detection of incoming viral DNA genomes by nuclear antiviral factors, particularly the involvement of PML-NBs, has been studied with a variety of viral systems, but to date HSV-1 remains the best-studied model in this topic . ImportaMost strategies to analyze the relationship between nuclear antiviral factors and viral genomes rely on biochemical and microscopic techniques. Chromatin immunoprecipitation (ChIP) assays can quantitatively detect how much a given  factor is bound to specific genomic regions providing a single nucleotide resolution when combined with next generation sequencing (ChIP-seq) . AlthougIn contrast, microscopic, imaging-based analyses offer high spatial information  at a single cell level. Localization of cellular and viral proteins can be visualized using specific antibodies in immunofluorescence (IF) analyses or through tagging with fluorescent proteins such as enhanced green fluorescent protein (EGFP) and mCherry. Visualization of viral genomes is technically more challenging. Fluorescence in situ hybridization (FISH) is the most straightforward way to detect specific genomes of interest; but requires harsh denaturing conditions, which may risk the integrity of subcellular structures. To overcome this problem, genome labeling with nucleoside analogs has been used as an alternative method. BrdU (5-bromo-2’-deoxyuridine) is a classical analog widely used for DNA labeling, but requires stringent denaturing for detection using antibodies, similar to FISH. However, when combined with nuclease treatments, incorporated BrdU can be detected with antibodies without denaturing, and this method has been used to visualize viral genomes such as Ad DNAs .Furthermore, a novel genome labeling technique has recently been developed using “clickable” nucleoside analogs such as EdU (5-ethynyl-2’-deoxyuridine) and EdC (5-ethynyl-2’-deoxycytidine) . These nTwo decades ago, the first observations on the fate of incoming viral genomes were published by Ishov and Maul, who showed that PML-NBs are sites for deposition of incoming viral genomes and/or viral DNA replication . As simi+/+ and PML−/− cells is observed during wild type HSV-1 infection [Furthermore, Everett et al. developed a series of recombinant viruses expressing fluorescent protein-tagged ICP4 for live-cell imaging analyses, and demonstrated that PML-NB components are dynamically recruited onto viral genomes to form de novo PML-NB-like structures ,37. Imponfection . Howevernfection . Nonethenfection ,40,41, sConsistent with this, it was shown that simultaneous depletion of PML and Sp100 enhances ICP0-null HSV-1 propagation greater than single depletion of each factor . FurtherThe initial study identifying IFI16 suggested that this nuclear DNA sensor could act against HSV-1 infection . Rapid rSimilar to HSV-1, a member of the alpha-herpesvirus family, modulation of PML-NBs has been shown with the beta-herpesvirus family member HCMV during immediate early phases of infection. Following the original study using HSV-1, Ad, and SV40 , Ishov aThe study of Ishov and Maul demonstrated that IE1 alone is able to and sufficient to disperse PML-NBs, somewhat similar to ICP0 of HSV-1 . In contThe involvement of IFI16 in early infection has also been investigated for several herpesviruses. For instance, knockdown of IFI16 has been shown to greatly enhance HCMV propagation , and ChIIncoming Ad genome is one of the viral genomes found at PML-NBs in the initial study of Ishov and Maul . In thatEven if incoming Ad genomes may not associate with PML-NBs upon infection, modulation of the bodies and inhibitory roles of its components during Ad infection is well established, in common with herpesviruses. The most prominent phenotype is a morphological change of PML-NBs induced by E4orf3, a viral early gene product. E4orf3 is shown to be necessary and sufficient to localize at and reorganize PML-NBs into track-like structures . The groSeveral independent studies under IFI16 knockdown conditions have shown that depletion of IFI16 has no impact on Ad propagation ,47. ThisThe work of Ishov and Maul indicated that DNA replication of SV40, a member of polyomaviruses (PyVs), takes place at PML-NBs . While ePapillomavirus is yet another interesting example of a nuclear replicating DNA virus that behaves differently from the other viruses studied to date. Day et al. reported, using BrdU-labeled pseudoviruses of bovine papillomavirus (BPV), that viral genomes as well as the minor capsid protein L2 localize at PML-NBs immediately after infection . In thisAs shown above, based on the currently existing experimental basis, there does not seem to be a general rule that applies to all different nuclear replicating DNA viruses in terms of the responses of nuclear antiviral factors against incoming genomes. We suggest at least two possible interpretations of the existing data. The first interpretation would assume that host cells have the potential to respond to different viruses using a common mechanism, but each virus has specific countermeasures that prevent genome targeting by nuclear antiviral factors to different degrees, explaining apparent differences in the cellular response. This view has been propagated to some extent in the literature ,6,25. AnThis (non-exhaustive) list of examples shows that many viruses appear to encode antagonists for PML-NB components in their structural proteins, which in some cases seem to be functionally interchangeable. Because they enter cells with the invading virion, they could in principle alter an immediate nuclear response against imported genomes. Still, the amount of antagonistic factors is limited by the amount of invading virions and probably one would expect them to act in a genome-specific way rather than on a global scale. This may be one of the reasons why antiviral effects of nuclear antiviral factors on incoming genomes, at least for herpesviruses, have been best studied at a low multiplicity of infection (MOI) ,41,64,65But what if host cells do not have a “master plan” against different DNA viruses, what could be the determinant(s) for the different responses of nuclear antiviral factors against incoming viral genomes? Currently, the clearest differences in immediate early antiviral responses have been observed between HSV-1 and Ad infections. There are several differences between HSV-1 and Ad, for instance, length of the genomes . More strikingly, Ad genomes remain associated with protein VII forming a chromatin-like structure even after nuclear entry, whereas HSV-1 genomes are likely unprotected at least immediately after nuclear import. Genome-bound protein VII has been reported to be beneficial for viral early gene expression in cells . In addiAnother possible mechanism for the interplay between viral genomes and nuclear antiviral factors is that viruses may actively interact with these host proteins via viral (genome-associated) proteins. A potential candidate for such a role is the L2 minor capsid protein of HPV, which was suggested to be responsible for the viral genome localization at PML-NBs ,89. HCMVAn interesting aspect of studying the immediate early fate of incoming viral genomes is whether nuclear antiviral factors are involved in the establishment and/or maintenance of latent infections. As observed, incoming Ad genomes are not encountered by nuclear antiviral factors and can efficiently initiate a lytic infection cycle, while incoming HSV-1 genomes (and possibly other herpesvirus genomes) are targeted by the restriction factors and frequently establish latency. Based on these observations, it is possible that the lytic-latent decision is made at the time of initial nuclear entry of viral genomes involving the activity of nuclear antiviral factors. It was shown in mice models that HSV-1 genomes locate at PML-NBs during acute phases of infection and during subsequent latent phases in neurons, and that absence of PML affects the expression of latency associated transcripts (LAT) . In contIn this review, we tried to focus the attention of the reader on the existing experimental evidence as well as the possible explanations of how nuclear antiviral factors target incoming DNA viruses immediately upon infection. While it remains possible that nuclear antiviral factors are able to target a variety of nuclear replicating DNA viruses in a similar fashion, we think that this should be experimentally validated in each viral system. The recent work done on HSV-1 and Ad has shown that significant differences in the cellular response exist; this could be explained by individually adapted nuclear responses of the host cell against each virus, rather than an overall antiviral strategy. In this respect, it is important to investigate on a virus-by-virus basis whether suspected immediate early events  are indeed taking place and what could be the driving force(s) behind them. Novel techniques have been developed for viral genome detection that should pave the way to address these questions in greater detail. These techniques allow the visualization of single viral genomes of any virus of interest and thus may help to understand the underlying mechanisms of the confrontation between host cells and incoming viral genomes. In addition, if viral genomes are immediately sensed and repressed upon nuclear entry, viral (immediate) early gene expression should be affected by nuclear antiviral factors in early phases of infection. In many studies, however, depletion effects for cellular antiviral factors have been measured in later phases of infection , thus potentially mixing early and late antiviral  effects. Hence, we believe that a more direct investigation of the immediate early effects elicited by nuclear antiviral factors would help to describe the nuclear response to incoming genomes. Especially since the mechanistic details (and molecules) of initial genome detection remain poorly understood even for well characterized viral systems such as HSV-1 and Ad. Further detailed analyses using these and other viral models would lay the experimental foundation that could help to consolidate or revise the current view for the immediate early fate of incoming viral genomes.


Ultra-thin flexible glass with high transparency is attractive for a broad range of display applications; however, substrates with low optical haze are not ideal for thin film solar cells, since most of the light will go through the semiconductor layer without scattering, and the length of light travelling path in the active layer is small. By simply depositing a layer of TEMPO -oxidized wood fibers (TOWFs), we are able to tailor the optical properties of flexible glass dramatically from exhibiting low haze (<1%) to high haze (~56%) without compromising the total forward transmittance (~90%). The influence of the TOWFs morphology on the optical properties of TOWFs-coated flexible glass is investigated. As the average fiber length decreases, the transmission haze of TOWF-coated flexible glass illustrates a decreasing trend. Earth-abundant natural materials for transparent, hazy, and flexible glass have tremendous applicability in the fabrication of flexible optoelectronics with tunable light scattering effects by enabling inexpensive and large-scale processes. Optically transparent and mechanically flexible electronics have generated significant interest in the past decade due to their ability to meet the increasing technological demands for next-generation electronics1234678910111314151618Using natural materials to fabricate transparent, hazy, and flexible glass without damaging the glass surface by a scalable and simple method is attractive. Wood pulp, primarily constituting cellulose, is an earth-abundant, renewable, light-weight, and biodegradable natural material. Paper made of wood pulp displays a wide range of properties due to the hierarchical structure of wood fiber; it may be transparent or opaque, porous or hermetic, rough or ultra-smooth, flexible or rigid, fragile or tough212224It has been verified that the diameter of the fiber has a significant effect on the optical properties of paper by our groupIn this study, we firstly use TEMPO-oxidized wood pulp to produce a flexible, transparent, and controllable hazy glass through a simple and scalable deposition process. This TOWFs-coated flexible glass presents the ability to manage light propagation while maintaining original optical transmittance. Also, an optically transparent flexible glass with tailored transmission haze is fabricated by mechanically altering the morphology of TEMPO-treated wood pulp. This not only exhibits both high transparency and controllable haze, but also shows strong bonding between the fiber layer and flexible glass, which has great potential for applications in flexible optoelectronics.Glass (Corning® Willow® Glass) with a thickness of ≤200 μm illustrates a high transparency and mechanical flexibility1128To produce highly hazy TOWFs-coated flexible glass without compromising high transparency of flexible glass, a TEMPO/NaClO/NaBr solution was utilized to introduce carboxyl group in the C6 of cellulose to increase the electrostatic repulsive force between neighboring cellulose nanofibers in fiber cell wall. With the assistance of mechanical stirring, the wood fibers tend to be cleaved in the axial direction that reduces fiber length and part of wood fibers break into small fiber debris. The reduced fiber length, dramatic increased fine content (fiber length < 0.2 mm), and the easily crushed hollow structure of fiber due to weak bonds between neighboring cellulose nanofibers lead to form high density of fiber network that contributes to the high transparency of TOWFs-coated flexible glass . To fabricate the coated substrate samples, TOWF suspension was manually deposited on one side of 100 μm thick Corning® Willow® Glass flexible substrate using a pipette to fabricate transparent, hazy, and flexible glass. A variety of solution coating methods are suitable to apply the TOWF layer onto the flexible glass, and the specific process can be chosen based on the overall fabrication and through-put requirements. UV-Vis spectrometer measurements with an integrating sphere (PerkinElmer® USA) were used to measure the optical transmittance and transmission haze of TOWFs-coated flexible glass. Another significant optical property introduced is the high transmission haze after TOWF deposition. As shown in In addition, the influence of the TOWFs morphology on the optical properties of TOWFs-coated flexible glass was also studied. A TOWFs suspension was continually stirred in a magnetic stirrer at a speed of 700 rpm for another 10 h to mechanically cut off wood fibers. The histogram for the length and width distribution of short TOWFs was displayed in Particle coatings are widely used to improve the light scattering effect for solar cell applications3031High light scattering enhances the light trapping in solar cells due to the increase of the path length of light in the active layer, resulting in increased power conversion efficiency (PCE) of thin film solar cells27333435For most solar cell installations, glass substrates are used to encapsulate the cell to protect the device from environmental hazards . A TOWF coating can supply a light scattering effect to the existing encapsulation glass. Moreover, due to the UV filtering ability of glass, TOWFs under glass will have much improved UV-vis lifetime than bared paper film. A schematic showing the potential application of TOWFs-coated flexible glass on the encapsulation of solar cell is shown in Highly transparent substrates with different light scattering properties can be used for various purposes. Highly hazy substrates are suited for the fabrication of photovoltaic devices with enhanced light trapping ability3738404142In conclusion, we demonstrate a uniform coating of one-dimensional TOWF on a flexible glass substrate that dramatically increases the optical haze from <1% to ~56% without decreasing the total optical transmittance. Such modified glass shows a large anti-glare effect useful for displays. When applied to glass encapsulation layers widely used in current solar cell installations, a TOWF coating increases the light scattering, which offers a higher absorption and solar cell efficiency in a broadband. The 1D cellulose based coating shows superior coherent mechanical strength and excellent mechanical binding with glass substrate, which can improve the durability of flexible glass. Highly transparent flexible glass with tailored light scattering behavior also possesses a potential application in solar cells, displays, and OLED light systems. This transparent, hazy, and flexible glass sheds a light to the fabrication of next-generation flexible electronics in combination with earth-abundant and renewable natural materials.A bleached sulfate softwood pulp (Southern Yellow Pine) was used to prepare TEMPO-oxidized wood fibers according to the method proposed in our previous literatures26


Flow diversion (FD)—a young technique using stents with highly increased surface coverage—was introduced to treat complex aneurysms without intra-aneurysmal material placement and has amended the spectrum of endovascular techniques such as stent-assisted coil occlusion considerably. However, ischemic complications, a common side effect in FD, occur more frequently compared with the conventional endovascular approaches and certainly limit the indication of this technique. Our study aimed to investigate the feasibility and efficacy of stent-assisted coiling using low profile self-expandable stents, which exhibit only moderate flow-redirecting properties and therefore represent a combination of hemodynamic endovascular and occlusive endosaccular therapy.39 Patients were included in our retrospective study. Occlusion rates were assessed 6 months after the procedure in a total of 27 cases using the Raymond scale.Complete occlusion (Raymond I) was achieved in 24/27 aneurysms. Small neck remnants (Raymond II) were evident in 3/27 aneurysms. There were no cases with sac remnant or complete persistence of aneurysmal filling (Raymond III and IV).Our study demonstrates interventional treatment of intracranial aneurysms using flow-redirecting stent-assisted coiling to be technically feasible and highly effective in aneurysmal occlusion. We believe that this approach is outstanding in the prevention of long-term aneurysmal reperfusion and exhibits a more acceptable risk profile than highly efficient FD techniques. Although stenting and coiling (SAC) as well as balloon-assisted coiling (BAC) are both longstanding, well established endovascular approaches for the treatment of complex cases of intracranial aneurysms –3, recanLow profile self-expandable (LEO) stents are braided devices composed of a comparatively dense nitinol mesh, exhibiting a smaller pore size than other self-expandable intracranial stents . InteresIn our experience, synergistic treatment of intracranial aneurysms, performing a combination of endosaccular coil occlusion and moderate flow redirection using LEO devices, provides excellent occlusion rates and low therapy-related risk for ischemic complications. Therefore, our retrospective study aimed to explore safety and efficacy of moderately flow-redirecting stent-assisted coiling in incidental and ruptured aneurysms of the intracranial circulation.This retrospective study was approved by the institutional ethics committee .We retrospectively reviewed our patient database to identify individuals with aneurysms of the intracranial arterial circulation which have been treated using flow-redirecting stents in combination with coil embolization from March 2015 to February 2017. Patients with incidental, unruptured aneurysms as well as subjects with ruptured aneurysms and subsequent subarachnoid hemorrhage have been included.Demographic data, localization of the aneurysm, type of stent device, immediate clinical and follow-up angiographic results, as well as complications were recorded.Written informed consent was obtained from all patients scheduled for elective endovascular treatment. Patients with acute intracranial hemorrhage gave written informed consent only in case of good clinical condition. Severely affected patients with acute aneurysm rupture were treated justified as emergency indication without informed consent.Elective patients received pretreatment double antiplatelet loading (ASS 500 mg and clopidogrel 300 mg) followed by standard antiplatelet regimen (ASS 100 mg once per day infinite and clopidogrel 75 mg once per day for 6 months). Clopidogrel resistance was evaluated prior elective intervention. Whenever resistance was present, ticagrelor was administered instead of clopidogrel (90 mg twice per day). In case of acute ruptured aneurysms, ASS premedication (500 mg) was administered during the procedure followed by the double antiplatelet regimen as previously mentioned in elective patients.via the right groin using either a 6- or 8-French introducer sheath. In elective procedures without intracranial bleeding, a bolus of heparin  was administered via the sheath. As a flow-redirecting stent, a LEO device  was inserted via microcatheter access. The selection of stent size was at the discretion of the applying neuroradiologist. Coil embolization of the aneurysms was performed using detachable coil systems in all cases . Immediate treatment success was recorded by biplanar angiographic control series.All endovascular procedures were performed under general anesthesia using a biplanar angiography . Endovascular access was established Patients with bleeding aneurysms were surveilled at the intensive care unit dependent on their clinical course. Furthermore, all elective patients were monitored on an intensive care unit for at least one night after the intervention. Initial angiographic follow-up was scheduled at 6 months. The extent of occlusion completeness was rated according to the Raymond scale .39 patients  with a total of 40 aneurysms were included in our retrospective evaluation. The average patient age was 54.3 years (range 34–79 years). 32 patients were scheduled for elective treatment, 7 patients had ruptured aneurysms with subsequent subarachnoid hemorrhage. 9 patients were already pretreated with aneurysm coiling and were scheduled of elective re-intervention of dome or neck remnants. Vessel localizations of the aneurysms are listed in Table A list of implanted stent devices can be seen in Table via-falsa-puncture had to be treated surgically.Procedure-related complications were observed in 6/39 patients. Acute in-stent thrombosis occurred in two patients in the emergency situation and resolved immediately after local administration of abciximab. Under the assumption that both cases were related to clopidogrel non-response, dual platelet function inhibition regimen was changed to ASA plus ticagrelor (Brilique). Under this regimen, no further thromboembolic complications occurred. One patient scheduled for elective treatment had an iatrogenic intraprocedural coil perforation of the aneurysm with subsequent subarachnoid hemorrhage. In one case of a dissecting vertebral artery aneurysm, acute re-bleeding occurred few days after the initial intervention and was treated successfully by inserting an additional flow diverter. There was one intraprocedural carotid dissection without hemodynamic relevance and no need for specific intervention. Only one groin complication occurred: a femoral arteriovenous fistula resulting from Angiographic follow-up at 6 months was available in 26/39 patients (27 aneurysms). Complete occlusion (Raymond I) was achieved in 24/27 aneurysms. Small neck remnants (Raymond II) were evident in 3/27 aneurysms. There were no cases with sac remnant or complete persistence of aneurysmal filling (Raymond III and IV). One patient presented with an asymptomatic late onset in-stent occlusion.As a consequence of the comparatively small sample size of our preliminary study, especially with reference to the heterogeneity of the included clinical scenarios, predictors for successful treatment can only be based on empirical values in this context. So far, the presented technique using braided LEO stents in combination with coil occlusion has achieved good short-term outcomes when applied in medium-sized or small-sized intracranial branches of the anterior circulation, beginning with the A1 and M1 segments. Owed to the moderate density of the LEO-Nitinol mesh, aneurysms of larger caliber vessels, for example, the internal carotid artery or the vertebral artery, may not be suitable for this approach.In the following, we present predictors of successful treatment, based on our experiences using the LEO-coil approach in medium-sized and small-sized intracranial vessels such as the A1/A2 segments and the M1/M2 segments.In case of initial treatments, complete filling of the aneurysm sac with coils without apparent residual perfusion of the aneurysm dome, exclusive of unsignificant neck remnants, in combination with full coverage of the aneurysm neck by the respective LEO stent, were strongly predictive for persisting occlusion of the aneurysm.In case of subsequent treatments of aneurysms previously treated with solitary coil occlusion, significant down staging (Raymond IV/III to Raymond II/I) was predictable, if coverage of the aneurysm neck was complete and wall approximation of the respective LEO stent was anatomical.Intracranial, especially subarachnoid hemorrhage is a serious complication of aneurysms located at the Circle of Willis. To prevent initial bleeding in incidental aneurysms or re-bleeding in ruptured aneurysms, several techniques for the treatment of intracranial aneurysms have been used with varying success rates in the past.Nowadays, endovascular treatment is favored whenever feasible in the patients specific circumstance . Stents Stents with flow-redirecting properties are able to further enhance aneurysmal thrombosis by diverting blood flow toward the parent vessel and away from the aneurysmal base , 25. FloIn our opinion, coiling in combination with moderately flow-redirecting stent devices represents a worthwhile middle course between pure coiling and the application of flow diverters in the endovascular treatment of intracranial aneurysms. Hereby, increased risk for recurrence, as observed in sole coil occlusion, is avoided, and ischemic complications become less probable compared with treatment with highly efficient flow diverting devices such as the PED and p64. In comparison with the previously mentioned studies, our own results using LEO stent-assisted coiling impressively demonstrate their superiority in long-term occlusion of aneurysms in combination with coiling. On 6-month follow-up, nearly 90% of our patients showed complete aneurysmal occlusion by means of flow-redirecting stent-assisted coiling. We found only three patients (roughly 10%) to have unsignificant neck remnants, of which one was a pretreated aneurysm which sac reperfusion which could have been “down-graded” to Raymond II stage by re-intervention using LEO stent-assisted recoiling. To this end, we believe that this approach is outstanding in long-term prevention of subarachnoid (re)-bleeding.Our study has some inherent limitations. First, this is a retrospective approach so that documentation of the data is not standardized and may vary case dependent. Second, our patient collective was quite inhomogeneous as elective patients were included as well as individuals with acute intracranial hemorrhage. To this end, especially data on complications and outcome have to be seen with cautiousness as they may inherently vary between these two groups. Third, there might be a bias in the follow-up data as not all patients presented for 6-month follow-up. We still included those without half year follow-up, as we aimed to assess not only occlusion rates but also technical feasibility of the procedures. In general, 6 months are a relatively short follow-up period and are only partly a predictor for long-term outcome. Fourth, different coil systems were used upon the discretion of the performing interventional radiologist. Nevertheless, we would not expect significant differences in occlusion rates between the different coil systems in our patient collective.Our study demonstrates interventional treatment of intracranial aneurysms using braided LEO stents with moderately flow diverting features in combination with intra-aneurysmal coiling to be a technically feasible and promising approach. In our experience, medium-sized and small-sized intracranial arteries, exemplarily the M1/M2 segments and the A1/A2 segments, are suitable target vessels. However, further studies validating our results and providing a comparative analysis between the presented method, solitary coil occlusion, stent-assisted coiling, FD, and flow diverter-assisted coil occlusion are necessary to better define the value of this endovascular technique for clinical practice.This study was carried out in accordance with the recommendations of the local IRB with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Ethikkommission der Medizinischen Fakultät (AZ 208-15-010062015).PV, SS, and UQ performed the analysis and wrote the paper. UQ, UN, MK, DW, and DL performed the interventions and were responsible for patients care and management. RJ was responsible for data management and technical assistance during the procedures. K-TH participated in writing the paper and drafting the concept.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Scientific Reports 10.1038/s41598-017-18218-9, published online 10 January 2018Correction to: The original PDF version of this Article contained an error in the order of the Figures and Figure Legends. Figures 1, 2 and 3 were published as Figures 3, 1 and 2 respectively. This has now been corrected in the PDF; the HTML version of the paper was correct from the time of publication.This Article also contained errors in the Results section under subheading ‘Ontogenetic stages and estimated ages’.Eosinopteryx“YFGP-T5197 shows the histocharacteristics of an actively growing juvenile skeleton with high porosity and uniform vascular density in the dominantly primary diaphyseal cortex in most limb bones . Some elements reveal FLC characteristic of fast growth phases. None of the sampled bones shows LAGs or other growth marks. These features indicate the young age of this specimen, probably less than one year old.SerikornisPMOL-AB00200 is categorized as a late-juvenile with more advanced osteohistological maturity  than seen in the bones of Eosinopteryx YFGP-T5197. Primary osteons are more mature. While the humerus and femur lack any growth marks, antebrachial bones reveal a single intracortical LAG close to the bone surface, and D1P1 already shows three asymmetrical growth marks. In most sampled bones, decreasing vascularity towards the periosteal surface  indicates decrease in diametric bone growth rate. However, presence of vascular canals in the outermost cortex suggests that considerable diametric growth would have still been possible, had this animal lived longer. The number and spatial distribution of growth marks in some elements, along with the general histological features in the bones lacking growth lines, imply an age not older than two years. Nevertheless, the diverse presence/absence and number of growth marks in different bones highlight the limits of using LAG counts as a proxy for age (see Methods).AnchiornisYFGP-T5199 represents a sub-adult specimen with histological signs of nearly completed growth. Primary vascularity gradually decreases towards the periosteal surface in most elements , while in others the outermost cortex is avascular and reminiscent of OCL typically seen in fully grown bird bones41. Humerus and antebrachial bones preserve a single intracortical LAG. Although the furcula locally records six closely packed LAGs in an avascular EFS, this element and the entire left wing of YFGP-T5199 are manipulated and the furcula restored from another specimen (U. Lefèvre pers. comm.). Thus, based on the lack of EFS in other elements and the single LAG identified in the longest forelimb bones, this specimen was likely about two years old.Although Aurornis YFGP-T5198 shows poor microstructural preservation, all sampled bones clearly demonstrate its skeletal maturity. Elements reveal a thick avascular outer cortical layer either with avian-like lamellar OCL, or with 1–3 densely spaced LAGs in an EFS . Considering that the general number of LAGs is one within the cortex, and 1–3 in the EFS, this adult may have been two to four years old.JeholornisYFGP-yb2 shows a peculiar interelement histology pattern with the sampled bones showing sharply contrasting ontogenetic tissue traits. While its hand bones are still actively growing with juvenile-like uniformly high cortical vascularity and no detectable sign of growth rate decrease, the rest of the sampled bones reveal a well-developed EFS with 2–4 LAGs, implying the adulthood of this specimen . Based on the bones exhibiting EFS, this adult Jeholornis is estimated to have been three to five years old.”now reads:“Eosinopteryx YFGP-T5197 shows the histocharacteristics of an actively growing juvenile skeleton with high porosity and uniform vascular density in the dominantly primary diaphyseal cortex in most limb bones . Some elements reveal FLC characteristic of fast growth phases. None of the sampled bones shows LAGs or other growth marks. These features indicate the young age of this specimen, probably less than one year old.Serikornis PMOL-AB00200 is categorized as a late-juvenile with more advanced osteohistological maturity  than seen in the bones of Eosinopteryx YFGP-T5197. Primary osteons are more mature. While the humerus and femur lack any growth marks, antebrachial bones reveal a single intracortical LAG close to the bone surface, and D1P1 already shows three asymmetrical growth marks. In most sampled bones, decreasing vascularity towards the periosteal surface  indicates decrease in diametric bone growth rate. However, presence of vascular canals in the outermost cortex suggests that considerable diametric growth would have still been possible, had this animal lived longer. The number and spatial distribution of growth marks in some elements, along with the general histological features in the bones lacking growth lines, imply an age not older than two years. Nevertheless, the diverse presence/absence and number of growth marks in different bones highlight the limits of using LAG counts as a proxy for age (see Methods).Anchiornis YFGP-T5199 represents a sub-adult specimen with histological signs of nearly completed growth. Primary vascularity gradually decreases towards the periosteal surface in most elements , while in others the outermost cortex is avascular and reminiscent of OCL typically seen in fully grown bird bones41. Humerus and antebrachial bones preserve a single intracortical LAG. Although the furcula locally records six closely packed LAGs in an avascular EFS, this element and the entire left wing of YFGP-T5199 are manipulated and the furcula restored from another specimen (U. Lefèvre pers. comm.). Thus, based on the lack of EFS in other elements and the single LAG identified in the longest forelimb bones, this specimen was likely about two years old.Although Aurornis YFGP-T5198 shows poor microstructural preservation, all sampled bones clearly demonstrate its skeletal maturity. Elements reveal a thick avascular outer cortical layer either with avian-like lamellar OCL, or with 1–3 densely spaced LAGs in an EFS . Considering that the general number of LAGs is one within the cortex, and 1–3 in the EFS, this adult may have been two to four years old.Jeholornis YFGP-yb2 shows a peculiar interelement histology pattern with the sampled bones showing sharply contrasting ontogenetic tissue traits. While its hand bones are still actively growing with juvenile-like uniformly high cortical vascularity and no detectable sign of growth rate decrease, the rest of the sampled bones reveal a well-developed EFS with 2–4 LAGs, implying the adulthood of this specimen . Based on the bones exhibiting EFS, this adult Jeholornis is estimated to have been three to five years old.”This has now been corrected in the PDF and HTML versions of the Article.


Nasal mucosal epithelial cells express connexins, the prototypical gap junction proteins, and pannexins, a new family of channel proteins homologous to the invertebrate gap junction proteins. The physiological and pathophysiological roles of these transmembrane proteins in nasal mucosa are largely still unknown.Pannexins participate in ATP release into the extracellular space in various tissues, and ATP plays important roles in mucociliary clearance, especially by regulating ciliary beat activity. Therefore, we focused on the functional relationship between connexins, pannexin-1, ATP release, and mucociliary clearance in nasal epithelia.2O efflux. The synchronized roles of pannexin and connexin may provide a new insight into effective mucociliary clearance systems in nasal mucosa.Connexins participate in the generation of intercellular calcium waves, in which calcium-mediated signaling responses spread to contiguous cells through the gap junction formed by connexins to transmit calcium signaling throughout the airway epithelium. Pannexins in the nasal mucosa may contribute to not only ciliary beat modulation via ATP release, but also regulation of mucus blanket components via H In the upper airway, mucociliary clearance (MCC) serves to remove inhaled particulate matter along with secreted mucus, and thereby contributes to host defense mechanisms. MCC is regulated by appropriate levels of mucus production and ciliary activity, which is usually measured by ciliary beat frequency (CBF). In the nose, mucociliary dysfunction can be a significant clinical problem, and has been reported to occur in patients with various inflammatory diseases including chronic rhinosinusitis (CRS) .The connexin proteins form hemichannels that dock to form gap junctions, which are intercellular communication channels . These jAn intracellular response to calcium influx in nasal mucosal cells induces an increase in CBF and triggers a calcium wave that spreads to neighboring cells through the gap junction channels, such as those containing connexin-43. This wave elicits a calcium influx reaction in these neighboring cells via the same mechanism, leading to the transmission of calcium signaling throughout the airway epithelium , 8. ManyATP is thought to be released into the extracellular space via two distinct pathways: vesicular- and channel-mediated release pathways. Among the many channel-mediated pathways, the pannexin channel is considered a promising candidate mediator of ATP release. Pannexins are a family of transmembrane channel proteins in vertebrates comprising three subtypes: pannexin-1, pannexin-2, and pannexin-3 . PannexiThe pannexin-1 channel forms a homohexameric large-conductance nonselective channel that participates in ATP release into the extracellular space , 16–18. 2O balance as well as an equilibrium between anion efflux and cation influx through the epithelial cell membrane. If the volume of the PCFL is reduced, ciliary movement becomes inefficient because of increased contact between the cilia and the viscous OML. Conversely, when the volume of the PCFL increases beyond its normal volume, ciliary movement also becomes inefficient because the cilia cannot reach the OML to stimulate it. Therefore, PCFL volume is extremely important for ensuring functional MCC.The role of pannexin-dependent ATP release in the nasal mucosa is still a matter of debate. The mucus blanket is divided into a gel-like outer mucus layer (OML) and a liquid periciliary fluid layer (PCFL) . The muc2O homeostasis system for maintaining the appropriate PCFL volume. To address this hypothesis, we examined whether pannexin-1 channels on nasal columnar epithelial cells contribute to H2O efflux. Our recent studies showed that TRPV1-associated, capsaicin-induced ATP release thorough pannexin-1 regulates ciliary beat activity in rat nasal mucosa [These facts led us to hypothesize that pannexin-dependent ATP release may, in part, constitute a Hl mucosa . Therefol mucosa , 29.Application of capsaicin led to shrinking of the columnar epithelial cells. Interestingly, this shrinking occurred only at the apical side but not the basal side. This shrinking was inhibited by ruthenium red or CBX when they were added in combination with the capsaicin Fig. . We coul−, together with H2O efflux to the extracellular space on the apical side of the cell under low-PCFL conditions, because the apical side is more exposed to mechanical stress on the nasal mucosa (Fig. Although we were not able to definitively determine the role of pannexin-1, these results are consistent with our hypothesis described above. One possible explanation for these results is that the pannexin-1 channel is involved in release of the anionic form of ATP, ATPIn conclusion, we believe that pannexin-1 channel-mediated ATP release has a distinct role in MCC from that of connexin channels and hemichannels. Synergetic effects of the pannexin-1 channel and the connexin channel could be the key molecular factors in the regulatory mechanism of the upper airway function. Modulation of these molecules may open a novel therapeutic strategy in the management of upper respiratory disorders.


Long-standing diabetes leads to structural and functional alterations in both the micro- and the macrovasculature. Designing therapies to repair these abnormalities present unique and sophisticated challenges. Vascular endothelial cells are the primary cells damaged by hyperglycemia-induced adverse effects. Vascular stem cells that give rise to endothelial progenitor cells and mesenchymal progenitor cells represent an attractive target for cell therapy for diabetic patients. In this review, we shed light on challenges and recent advances surrounding stem cell therapies for diabetes vascular complications and discuss limitations for their clinical adoption. Diabetes is a serious disease that in 2014 plagued 422 million individuals in total worldwide, with one in 11 adults affected.3Current therapies have revolutionized diabetes, no longer making it a death sentence. The therapies target hyperglycemic states, but even when patients have remarkably good glycemic control devastating complications still occur. This is probably because diabetes is a more complicated pathology than we currently understand. The only curative therapy available is pancreatic islet cell replacement, requiring not only rare donors but also immunosuppressant therapy to reduce rejection, which comes with its own complications. Xenografts have been proposed, but the associated complications beg for better alternative sources. New research advocates the use of pluripotent human embryonic stem (hES) cells as a potential new replacement cell therapy for type 1 diabetes mellitus (T1DM). Several investigators have been able to demonstrate the ability to produce hES that function like β-cells: glucose-sensing and insulin-secreting. For example, among the earliest in the field, Assady et al. have been able to use hES cells to produce spontaneous “lineage specific differentiation” of cells that behave and function like β-cells—producing insulin and secreting the substance based on the level of cell differentiation. Their paper proves that hES cells can be easily coaxed into β-cell forms and are a potential source and basis for future cell replacement therapies in diabetes.4Beyond creating new β-cells, current literature supports the use of stem cells to prevent and treat the varied diabetes complications. Two types of multipotent stem cells exist in our bone marrow: mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs). Hematopoietic stem cells mature to produce all the cells in the bloodline, while MSCs are involved in tissue regeneration and mature to produce various organs such as bone, cartilage, heart, brain, and kidneys. These stem cells may be utilized as injectable progenitors. Recent research has demonstrated that vascular complications including retinopathy, neuropathy, wound healing, and erectile dysfunction can be targeted via HSCs, whereas renal complications can be targeted via MSCs. Induced pluripotent stem cells  are a type of pluripotent stem cell that can be generated directly from adult cells such as human fibroblasts or human renal epithelial cells. The iPSC technology was first reported by Shinya Yamakana’s laboratory in Japan, showing in 2006 that the introduction of four specific genes encoding transcription factors could convert normal adult cells into pluripotent stem cells. He was awarded the 2012 Nobel Prize along with Sir John Gurdon for the discovery that mature adult human cells can be reprogrammed to become pluripotent.5In this report we summarize the current literature about the potential use of stem cells for treatment of micro- and macrovascular complications of diabetes .The range of vascular complications of diabetes is vast, and the exact causes and mechanisms of these complications remain unclear.in vivo and enhancing the healing of ischemic ulcers in diabetic mice.”The discovery of endothelial progenitor cells (EPCs) in peripheral human blood and the characterization of EPCs in diabetic patients have helped the scientific community concretize the role of EPCs in endothelial repair. Endothelial progenitor cells have varying proposed functions throughout the body. Relevant here, however, is their high concentration in subjects with coronary artery disease (CAD), whose vessels require more frequent and intense repair. The current literature suggests that they act via paracrine signaling of “… interleukins, growth factors, and chemokines  that are capable of accelerating vascular network formation ex vivo modification of EPCs can be effectively performed. This would provide better repair mechanisms to the damaged vasculature. A number of studies in mice have shown that diabetic EPCs in healthy mice reduce healthy vascularization. The reverse may be true, but few studies directly address this idea in diabetic cells or models.7The thought is that diabetic individuals with vascular complications may benefit from autologous stem cell transplant therapy if in vivo, MSCs differentiate into renal cells and may repair a damaged segment of a kidney. They have also been shown by Lee et al. to function as reno- and pancreato-protective: in their study MSCs aggregated and prevented further damage to the tissue.not suggest a mechanism by which the damage is reversed. However, the study does offer a few possibilities: the MSCs might have replaced the damaged renal cells; MSCs might scavenge cytotoxic molecules; MSCs might modulate the inflammatory process; or MSCs might promote neovascularization thereby preventing the nephropathy altogether.10Diabetic nephropathy is the main complication in T1DM. Research has demonstrated that, Other reports advocating the renal progenitor iPSC  have demonstrated that iPSCs can easily and quickly be induced into renal cells and that these iPS renal cells have therapeutic significance because they improve acute kidney injury (AKI) in mouse models.12Lam et al. efficiently, and, more importantly, reproducibly, generated kidney lineage cells from hES and iPSCs. First, tubule-like structures were generated by placing them in an environment that expressed intermediate mesoderm markers. With further coaxing using fibroblast growth factor-2 and retinoic acid, these structures began expressing proximal tubule markers. Finally, the cells further differentiated and matured in an environment of fibroblast growth factor-9 and activin until they expressed mesenchyme nephron progenitor cell markers. Importantly, the mesodermal cells spontaneously formed the ciliated tubules that expressed the markers found in mesenchyme nephron progenitor cells. These findings are promising for potential use of bioengineered stem cells in transplantation to alleviate some of the burden of AKI, chronic kidney disease (CKD), and end-stage renal disease (ESRD).11in vitro and in vivo. Further, renal subcapsular transplantation of these iPSCs was able partially to rescue renal injury, measured via urea and creatinine, in mouse models with AKI. These results en-courage the further examination and exploration of iPS renal cells to be used as future regenerative therapies.12A recent study by Toyohara et al. demonstrated the ability to use iPS renal progenitor cells to improve AKI. They were able to use iPSCs to create renal progenitor cells via a differentiation protocol that employed signaling with growth factor signaling molecules like bone morphogenic peptide (BMP) inhibitors and transforming growth factor beta (TGFβ) isoforms to form tubule-like structures Often neuropathy is one of the first noticeable complications in diabetes as 20%–30% of patients are symptomatic. The pathology of diabetic neuropathy is due to metabolic and vascular disturbances in the vasa nervorum, the blood vessels that supply small peripheral nerves.Studies have demonstrated that inducing angiogenesis via secretion of cytokines like vascular endothelial growth factor (VEGF) and basic fibro-blast growth factor (bFGF) ameliorates neuropathic pain by increasing blood supply to the nerves. Below we review studies that demonstrate the use of EPCs, bone marrow mononuclear cells (BM-MNCs), and MSCs for treatment.Previous studies have shown that pharmacologic methods of increasing blood flow to the vasa nervorum vessels, such as anticoagulation and vasodilation, improve neuropathic symptoms.A study by Naruse et al. showed that EPCs derived from human umbilical cord blood reversed diabetic neuropathy in diabetic rats via neovascularization to rescue dying nerves.16As mentioned above, diabetic neuropathy has varied metabolic and vascular causes. On a nerve-level the pathologies include cutaneous endothelium-related vasodilation, C-fiber-mediated vasoconstriction, and epineural blood flow in the sural nerve. Previous animal model studies have demonstrated relief of neuropathic pain using antioxidants like a-lipoic acid, taurine, poly-ADP-ribose polymerase inhibitors, and aldose reductase inhibitors. Further, treatment with C-peptide not only improved neuropathic symptoms but also reduced neurotropic proteins in peripheral nerves.Studies in the past have shown that BM-MNC transplantation improves post-ischemic neovascularization and causes increased blood flow. The leading theory is that the enhancement is due to secretion of pro-angiogenic ligands and cytokines such as fibroblast growth factor (FGF), VEGF, interleukin 1b (IL-1b), and tumor necrosis factor alpha (TNFα). A study by Naruse et al. demonstrated that BM-MNC transplantation alleviates neuropathic pain in diabetic rats as well as increases blood flow to peripheral nerves. This is observed via increased capillary density, with increased nerve conduction velocity. Thus this proposed therapy of BM-MNC transplantation would target not only the debilitating symptom of neuropathic pain but also the underlying cause, improving nerve function altogether.17Shibata et al. transplanted MSCs in diabetic rats and assessed cell homing as well as the effects of the MSCs on diabetic neuropathy. They discovered that the cells settled in the gaps between muscle fibers but were not incorporated into any new structures. The injected muscles had higher levels of VEGF and bFGF mRNA expression, indicating neovascularization and angiogenesis supporting the regeneration of nerve cells. Further, the MSC-transplanted diabetic rats demonstrated improved pain, increased nerve conduction velocity, increased blood flow, and increased capillary density in the MSC-treated limbs. Secretion of VEGF and bFGF was maintained up to 4 weeks after transplantation.19Diabetic retinopathy is the main cause of blindness in the developed world.21Various trials have investigated these ideas in diabetic rat and human models. Using immunohistochemistry, studies were able to demonstrate that intravitreally injected stem cells aggregate and home in on the inner retina; this may improve visual function.Ischemic heart disease is the leading cause of death in the United States. In diabetic patients, cardiovascular complications are the most common cause of death. Current therapies for ischemic heart disease include pharmacologic agents that promote perfusion and/or surgeries to revascularize ischemic areas of the heart. None of these is curative, nor together can they fully reverse the damage—hearts, once injured never work the same as they did before. Thus, the studies discussed in the following section have broader ramifications to both diabetic and non-diabetic patients alike.27A large randomized trial of POSEIDON patients demonstrated that transendocardial injection of MSCs improves heart remodeling in patients with chronic ischemic cardiomyopathy by reducing scar size and improving ventricular functional responses.26The investigators also assessed whether sites of injection of autologous MSCs responded more favorably than other parts of the infarcted tissue. The study concluded that the site of injection of MSCs is important: the response at that point showed (1) scar reduction, (2) improvement in wall thickening, and (3) functional recovery. Segmental ejection fraction was only improved at the site of injection, not distal to it. Previous studies have demonstrated that MSCs home to and differentiate in areas of infarct borders and release paracrine mediators that effect scar reduction, leading to increased transmural functionality. This study confirmed what previous studies have shown, but Suncion et al. were also able to capture improvements in cardiac function metrics such as contractility, lusitropy, and myocardial energetics. Cell dosage of MSCs was also assessed to determine best results with the smaller dose, 20 million cells versus 100 million cells, producing a greater reduction in scar size. Further, exact administration of MSCs with respect to location, dosage, and rate should be customized based on the individual’s infarct details.26Another important paper, by Karantalis et al. on the PROMETHEUS trial, assessed MSC transplantation as an adjuvant treatment for those under-going coronary artery bypass grafting.27The skin’s healing capacity in patients with diabetes is markedly diminished, resulting in a high risk for chronic wounds.34Microvascular, macrovascular, and inflammatory abnormalities in tissues of patients with diabetes present particular hindrances to adequate treatment of the late disease complications. Stem cells offer a promising approach to these complications in the diabetic setting through release of soluble growth factors and cytokines that stimulate new vessel formation and modulate inflammation. Furthermore, autologous cell-based approaches are ideal to minimize immune rejection but may be limited by the declining cellular function associated with diabetes. However, significant hurdles remain in optimizing progenitor cell selection and delivery . As our


We report controllable assembly of silver nanoparticles (Ag NPs) for patterning of silver microstructures. The assembly is induced by femtosecond laser direct writing (FsLDW). A tightly focused femtosecond laser beam is capable of trapping and driving Ag NPs to form desired micropatterns with a high resolution of ∼190 nm. Taking advantage of the ‘direct writing’ feature, three microelectrodes have been integrated with a microfluidic chip; two silver-based microdevices including a microheater and a catalytic reactor have been fabricated inside a microfluidic channel for chip functionalization. The FsLDW-induced programmable assembly of Ag NPs may open up a new way to the designable patterning of silver microstructures toward flexible fabrication and integration of functional devices. Nanopet al reported a layer-by-layer assembly of Ag NPs on the surface of optical fiber tips for the development of remote SERS sensors [et al fabricated Ag plasmonic waveguides by evaporation-induced self-assembly (EISA) of Ag NPs with the help of pillar array templates, which were fabricated beforehand through classical lithography [et al fabricated Ag electrodes by moving the nozzle of Ag NP ink using a three-axis motion-controlled stage [μm. Recently, direct femtosecond laser selective NP sintering (FLSNS) has been successfully developed to fabricate silver patterns [This problem has motivated considerable efforts to develop improved patterning techniques toward nanodevice fabrication. For instance, electrostatic self-assembly –36, Lang sensors . The opthography . In thishography –54. Ahn ed stage . In thispatterns . Femtose2O2 decomposition. The FsLDW-induced controllable assembly of Ag NPs may hold great promise for the fabrication and integration of metal NP-enabled nanodevices.In this work, we report a femtosecond laser direct writing (FsLDW)-induced controllable assembly of Ag NPs for making silver micropatterns. As a designable processing technique, FsLDW enables 3D fabrication, high-resolution prototyping, mask-free patterning, and flexible fabrication on non-planar substrates, revealing great potential in microdevice fabrication . Herein,2.2.1.Ag NPs were synthesized according to a reported method . Typical2.2.In a typical FsLDW fabrication, a tightly focused femtosecond laser beam with central wavelength of 800 nm and a pulse width of 120 fs was introduced into the interface between silver NPs solution and substrates. The tightly focused femtosecond laser would drive and trap the Ag NPs within the focal spot; in this way, assembled silver microstructures formed after sintering of Ag NPs along the traces of laser focus, which was guided by computer programs. In the integration experiment, microfluidic channels were fabricated by photopolymers (SU-8) through lithography. Gold pads for connecting Ag electrodes were deposited by vacuum thermal evaporation.2.3.The Ag NPs were characterized by transmission electron microscopy . Scanning electron microscopy , atomic force microscopy , and optical microscopy  were used to observe the morphology of Ag assembly structures. Voltage applied on a microheater was offered by electrochemical workstation (CHI660E).3.In this work, malate-capped Ag NPs were used for the FsLDW fabrication. To evaluate the quality of the Ag NP suspension, the obtained Ag NPs were characterized by TEM. As shown in figure d ≪ wavelength λ) are much smaller than the wavelength of laser , a gradation force proportional to the polarizability of an NP and the square of optical electric field vector dominates the assembly of Ag NPs, whereas absorption force and scattering force push the Ag NPs along the laser beam direction, leading to the enrichment of Ag NPs in the focal region. Together with the Ag NP deposition, mass transfer was performed by concentration diffusion of the Ag NPs solution. At the same time, due to the extremely high transient power density of the femtosecond laser, the aggregated Ag NPs have been sintered together. Since the near-infrared femtosecond laser beam is not absorbed by the solution, the beam penetrates deeply into the Ag NP solution with very small power loss. By scanning the laser focus along a preprogrammed trace, the desired silver micropatterns could be fabricated accordingly.The obtained Ag NPs have been used for FsLDW directly without the use of any photosensitive reagents. Figure μm. By further magnifying the SEM images, we found that the silver micropattern was constructed by close-packed silver particles whose size grew to tens of nanometers. As compared with the TEM image of the pristine Ag NPs, whose size is below 5 nm, the significant increase in particle size in the silver micropattern could be attributed to the laser-induced sintering.Note that the pulse width of our femtosecond laser is only 120 fs, so thermal relaxation could be effectively suppressed within a small area near the focus. Taking advantage of this feature, high-resolution silver patterns could be fabricated. Figure μm. To get further insight into the surface morphology of these letters, they were characterized by AFM. As shown in figure To illustrate its full potential in flexible patterning, a word (MICRO) was successfully fabricated through FsLDW of Ag NPs. Figures To demonstrate the capability of integrating such silver micropatterns with given microdevices, three kinds of microelectrodes were successfully integrated within a microfluidic chip by FsLDW-induced controllable assembly of Ag NPs figure . As typi2O2 inside a microfluidic channel; the generation of O2 could be observed as soon as H2O2 was injected into the microchannel. Since the Ag catalysts could be flexibly placed at any desired position, the generated oxygen bubble could be potentially used as a pump for directional transport of target objects or to guide the flow inside a microfluidic device [For practical applications, a silver microheater was fabricated by FsLDW-induced controllable assembly of Ag NPs figure . Five pac device .2O2, respectively. As a laser-mediated assembly route, FsLDW-induced controllable assembly of Ag NPs may find broad application in the fabrication of silver-based microdevices. Especially, for microfluidic devices, FsLDW-induced controllable assembly of Ag NPs may hold great promise for flexible integration of functional devices such as SERS substrates, conductive microelectrodes, microheaters, Ag catalysts, and pumps, within a microfluidic chip, contributing to the development of multifunctional microfluidic chips.In conclusion, FsLDW-induced controllable assembly of Ag NPs has been successfully developed for designable patterning of silver microstructures. High-resolution silver micropatterns with the high resolution of 190 nm have been readily fabricated according to preprogrammed models. SEM and AFM images proved that silver micropatterns were constructed by close-packed Ag particles with an average size of tens of nanometers, which indicates the sintering of Ag NPs during laser irradiation. As typical models, three microelectrode patterns have been fabricated within a microfluidic device, revealing its strong capability for post-integration. Additionally, a silver microheater and a silver-catalyst array have been successfully fabricated for localized heating and catalytic decomposition of H


We show this is due in part to misreporting replication cohort-size as a maximum number, rather than per-locus one. In 39 studies accurately reporting per-locus cohort-size for attempted replication of 707 loci in samples with similar ancestry, replication rate matched expectation . In contrast, ancestry differences between replication and discovery  cause the most highly-powered decile of loci to replicate worse than expected, due to difference in linkage disequilibrium.Genome-wide association studies (GWAS) have identified hundreds of SNPs responsible for variation in human quantitative traits. However, genome-wide-significant associations often fail to replicate across independent cohorts, in apparent inconsistency with their apparent strong effects in discovery cohorts. This limited success of replication raises pervasive questions about the utility of the GWAS field. We identify all 332 studies of quantitative traits from the NHGRI-EBI GWAS Database with attempted replication. We find that the majority of studies provide insufficient data to evaluate replication rates. The remaining papers replicate significantly worse than expected  perform both discovery and replication in samples of the same ancestry; and (2) report accurate per-variant sample sizes, replicate as expected. This study provides strong, rigorous evidence for the broad reliability of genome-wide association studies. We furthermore provide a model for more efficient selection of variants as candidates for replication, as selecting variants using cursed discovery data enriches for variants with little real evidence for trait association. The tradeoff between study power per-variant and resources, along with the strategy of testing millions of variants for association, leads to study designs where many associated variants of low effect size , scales inversely with the distance between the observed test statistic and the cutoff applied to variants brought forward to replication. The bias can be solved using any standard zero-finding algorithm  do both terms meaningfully contribute to the bias estimate.Here, ted in C ). Note tTo simplify predictions of replication efficiency, we considered an independent subset of all reported loci. As we lack direct access to the genetic samples used in these studies, we extracted a subset of the variants such that no two variants in a paper are situated within one megabase of any other. This is a very simple modification of the standard clumping protocol used in GWAS studies . To prevThe concept of internal “replication” may be interpreted differently in different reports. We consider three definitions of replication for this study, to observe different characteristics of the data:α = 0.05 [“nominal”]replication at nominal replication at replication within deciles of variants [“deciles”]We specifically only consider methods in which replication is determined from the replication study alone. The nominal and Bonferroni methods are commonly used. We use the decile method to investigate the behavior of the predicted power  to repliα threshold, the number of observed replications is distributed as Poisson binomial with success probabilities equal to each individual variants’ power to replicate (see below). This is a generalization of a Binomial distribution in which each Bernoulli trial is allowed to have a known but variable success rate. We use the implementation of this distribution in R [Given a set of variants and their predicted power to replicate at a given ion in R . We furtα considered, many variants analyzed here have power of effectively, or within machine precision, 0 or 1; with our limited sample size, the convergence properties of our dataset will be undesirable, and thus we use the exact distribution at the cost of computational efficiency. This process may be considered a fitting of the model according to which the WC-corrected discovery data correctly explain the observed replication data.We note that under certain assumptions the number of replications will asymptotically be distributed normally. However, depending on the In several instances, we evaluate the effects of filtering certain subsets of papers based on various criteria, and the extent to which this causes fit criteria to return to null expectation. As this evaluation is potentially confounded by reduced statistical power, in all cases we test whether the change in p-value is significantly different from expectation under random subsampling of variants matched on total power to replicate amongst the observed variants.v for a quantitative trait under the additive model isAssuming the discovery and replication sample of a study are drawn from the same source population with shared expected effect at each variant, the power to replicate a discovered variant α threshold of p is the probability of the variant exceeding the required test statistic from the null, but under the alternative distribution which is noncentral N is the replication sample size and f is the replication allele frequency of the variant. This f should be the actual allele frequency in the replication sample; however, studies sometimes report fhapmap from the closest reference ancestry as a means of protecting patient anonymity. Overall, the predicted number of replications across all variants in a paper is the sum of the power to replicate, as a function of predicted effect size and replication sample size and frequency, across all variants analyzed.In brief, the power to detect a signal at an These papers demonstrate the coverage of population ancestries in the field of quantitative trait genetics. We report and analyze the ancestral coverage of these studies using the simplifying summary statistic of continent of ancestry , tracing generally the ancestries of the original HapMap2 populations. We include a fourth category for African American samples, the largest admixed population nonnegligibly represented in the papers. This geographical partitioning matches the ancestry assumptions used in GWAS methods such as genotype imputation.Ancestry group counts are computed from maximum reported sample size per cohort per paper. In studies where cohorts of different continental ancestry are meta-analyzed, sample sizes are appropriately partitioned to the contributing ancestries. No adjustment is applied for papers reporting on the same cohort. For comparison to what the field’s sample sizes would be under random global sampling, global population estimates are computed , 13.We tested loci for nonrandom annotations. This test is usually conducted with access to the full set of variants tested in an individual study. As in this study design such information is masked, we restricted the analysis to papers using HapMap2 imputation in their discovery data; considered only SNPs present in HapMap2; and restricted the data further to European ancestry discovery data, which includes the majority of papers in the dataset.We annotated all variants in the CEU subset of HapMap2 using ANNOVAR . We compS1 Figα. X-axis: replication α; Y-axis: estimated SNP effect from linear regression. Two simulations with different effect estimates  are shown. The height of the vertical lines corresponds to the average bias (curse) introduced by discovery prioritization.A SNP with fixed frequency, sample size, and phenotypic variance explained was repeatedly drawn at random, and the accompanying standard normal trait was simulated. The apparent effect of SNP on trait was estimated exclusively from simulations exceeding progressively more stringent replication (TIFF)Click here for additional data file.S2 Figp < 0.00001). Generated using the software at [Includes all loci brought forward from discovery, not filtered for independence. Datapoint shapes correspond to approximate continental ancestry: CEU = European, YRI = Click here for additional data file.S3 FigSubset of data from Supp. Fig; includes only loci passing study-specific Bonferroni-corrected replication threshold. Generated using the software at .(TIFF)Click here for additional data file.S4 Figp < 2 ⋅ 10−16. Considers studies with same ancestry in both discovery and replication and correct per-locus sample sizes, as used in the main results.Plotted are receiver operating characteristics (ROC) for raw discovery and WC-corrected estimates of power to nominally replicate variants in individual studies. X-axis: false positive rate, computed as number of non-replicating variants having greater than a given power to replicate; Y-axis: true positive rate, computed as number of replicating variants having such power. AUC: 0.795 (WC-corrected) versus 0.579 (biased); DeLong’s two-tailed (TIFF)Click here for additional data file.S5 FigTop panel: predicted versus expected replication for each paper, with each paper flagged as being within 95% confidence of predicted replication rate under WC-corrected model, greater than or less than expectation. X-axis: predicted number of replications in a given paper, calculated as the sum across all loci of power to replicate based on WC-corrected discovery effect estimates. Y-axis: observed number of replications in the paper. Colors correspond to publication dates binned into six month intervals, from 2008 to 2014. Point shapes to correspondence between observed and expected rates. Replication is defined as a one-tailed replication p-value surpassing a per-paper Bonferroni threshold: (TIFF)Click here for additional data file.S6 FigEach node corresponds to one method of subsetting the data: all papers; papers conducting discovery and replication in the same continental ancestry; papers reporting correct per-locus sample sizes; or papers doing both. Probabilities in nodes correspond to two-tailed Poisson binomial fit test for prediction of nominal replication rates after Winner’s Curse correction. Probabilities along edges correspond to the chance of randomly seeing improvement of fit between connected nodes at least as large as observed due to loss of power exclusively, based on 10000 simulated subsamplings from the source node matched on total predicted power to replicate. Relative position of nodes along vertical axis corresponds to number of loci removed in the subset.(TIFF)Click here for additional data file.S7 FigEach node corresponds to one method of subsetting the data: all papers; papers conducting discovery and replication in the same continental ancestry; papers reporting correct per-locus sample sizes; or papers doing both. Probabilities in nodes correspond to two-tailed Poisson binomial fit test for prediction of Bonferroni replication rates after Winner’s Curse correction. Probabilities along edges correspond to the chance of randomly seeing improvement of fit between connected nodes at least as large as observed due to loss of power exclusively, based on 10000 simulated subsamplings from the source node matched on total predicted power to replicate. Relative position of nodes along vertical axis corresponds to number of loci removed in the subset.(TIFF)Click here for additional data file.S8 FigX-axis: left boundary of bin on replication threshold; Y-axis: percentage of replication within corresponding bin. Tracks correspond to actual data in the replication study, predicted power to replicate using discovery effect estimates, and predicted power to replicate using WC-corrected discovery effect estimates. Loci brought forward to replication in papers were pruned to contain independent signals within (but not necessarily between) individual papers. Error bars correspond to 95% confidence intervals around mean replication rates as estimated across multiple loci. Left panel: including all papers; middle panel: including only papers with accurate per-locus sample sizes; right panel: including only papers with maximal sample sizes per variant. Including only maximum sample size regardless of per-variant missingness will uniformly inflate replication rate estimates.(TIFF)Click here for additional data file.S9 FigX-axis: left boundary of bin on replication threshold; Y-axis: percentage of replication within corresponding bin. Tracks correspond to actual data in the replication study, predicted power to replicate using discovery effect estimates, and predicted power to replicate using WC-corrected discovery effect estimates. Loci brought forward to replication in papers were pruned to contain independent signals within (but not necessarily between) individual papers. Error bars correspond to 95% confidence intervals around mean replication rates as estimated across multiple loci. Left panel: including all papers; middle panel: including only papers with accurate per-locus sample sizes; right panel: including only papers with maximal sample sizes per variant. Including only maximum sample size regardless of per-variant missingness will uniformly inflate replication rate estimates.(TIFF)Click here for additional data file.S10 FigX-axis: left boundary of bin on replication threshold; Y-axis: percentage of replication within corresponding bin. Tracks correspond to actual data in the replication study, predicted power to replicate using discovery effect estimates, and predicted power to replicate using WC-corrected discovery effect estimates. Loci brought forward to replication in papers were pruned to contain independent signals within (but not necessarily between) individual papers. Error bars correspond to 95% confidence intervals around mean replication rates as estimated across multiple loci. Left panel: including all papers; middle panel: including only papers with accurate per-locus sample sizes; right panel: including only papers with maximal sample sizes per variant. Including only maximum sample size regardless of per-variant missingness will uniformly inflate replication rate estimates.(TIFF)Click here for additional data file.S11 FigX-axis: left boundary of bin on replication threshold; Y-axis: percentage of replication within corresponding bin. Tracks correspond to actual data in the replication study, predicted power to replicate using discovery effect estimates, and predicted power to replicate using WC-corrected discovery effect estimates. Loci brought forward to replication in papers were pruned to contain independent signals within (but not necessarily between) individual papers. Error bars correspond to 95% confidence intervals around mean replication rates as estimated across multiple loci. Left panel: including all papers; middle panel: including only papers with accurate per-locus sample sizes; right panel: including only papers with maximal sample sizes per variant. Including only maximum sample size regardless of per-variant missingness will uniformly inflate replication rate estimates.(TIFF)Click here for additional data file.S1 FileContains: Tables A-C, and bibliography for all papers considered from the NHGRI-EBI GWAS Catalog.(PDF)Click here for additional data file.S1 DatasetEach file corresponds to a single PMID as listed in the filename. Only includes data for 100 studies used in analysis. For full citations, see (TGZ)Click here for additional data file.S2 DatasetIncluded: the raw data from (IPYNB)Click here for additional data file.