In this study, we report the polymer-based graphene foams through combination of bottom-up assembly and simple triaxially buckled structure design. The resulting polymer-based graphene foams not only effectively transfer the functional properties of graphene, but also exhibit novel negative Poisson’s ratio (NPR) behaviors due to the presence of buckled structure. Our results show that after the introduction of buckled structure, improvement in stretchability, toughness, flexibility, energy absorbing ability, hydrophobicity, conductivity, piezoresistive sensitivity and crack resistance could be achieved simultaneously. The combination of mechanical properties, multifunctional performance and unusual deformation behavior would lead to the use of our polymer-based graphene foams for a variety of novel applications in future such as stretchable capacitors or conductors, sensors and oil/water separators and so on. A number of methods, such as hydrothermal and chemical reduction, electrochemical synthesis, template assisted assembly, and chemical vapor deposition have recently been developed to fabricate highly porous nanomaterials-based cellular monoliths2D macrosc45678910111213141516171819202122232425262728e.g. buckling) of graphene adhered on a biaxially pre-stretched polymer substrate, graphene films can be crumpled with improved hydrophobicity and tunable transparency as well as potential in artificial muscles due to the stretchability3637Recently, utilizing the microstructural topology modulation to optimize the functional properties has been developed in flexible and stretchable electrode systems333435363734Inspired by the ideas of introducing buckled structures to enhance both functional performance and flexibility, here we report a novel strategy for the low-cost, scalable fabrication of a new class of 3D multifunctional polymer-based nanomaterial foams based on a triaxially post-buckling microstructure design. The multifunctional foams are first prepared through self-assembly of individual graphene sheets on a 3D commercial polyurethane (PU) skeleton, and then triaxially compressed to introduce microstructural buckles by a simple top-down processing. The resulting polymer-based graphene foams not only effectively transfer the functional properties of graphene, but also exhibit novel negative Poisson’s ratio (NPR) behaviors due to the presence of buckled structure. Our results show that after the introduction of buckled structure, improvement in stretchability, toughness, flexibility, energy absorbing ability, hydrophobicity, conductivity, piezoresistive sensitivity and crack resistance could be achieved simultaneously. The combination of mechanical properties, multifunctional performance and unusual deformation behavior would lead to the use of our polymer-based graphene foams with NPR as stretchable, flexible smart materials in a variety of applications. More importantly, the buckled structure proposed in this work also offers universal, facile way to design a new class of 3D porous template/nanomaterial monolith, which combine ideal properties for potential applications in 3D flexible electronics, stretchable functional materials. Finally, we also applied such post-buckling strategy into fabrication of polymer-based silver nanowires foams, which showed NPR behavior and demonstrated unusual crack resistance as stretchable conductors.Experimental Section for details)2567101518Experimental Section for details). Note that such triaxial compression treatment in our system is completely different with fracture design reported in Ref. e.g. stretchibility). Our triaxially processed foams  were compressed (~33%) in all 3 orthogonal directions of parent PG foams cuboid. The cross-sectional view of as-prepared foams is investigated by scanning electron microscopy (SEM). As shown in 256710256710−1 and 1350 cm−1). Close examination near the edge of a cracked microfiber further reveals the layered structures of the graphene “skin” 34810111217182224283940414243444546As initially mentioned, the bulk physical properties of this 3D PG foam would be affected by the presence of graphene, as well as by the buckling structural geometry of microfibers. The obtained diverse functionality in such multicomponent foams via incorporation of graphene would definitely be tailored by the buckling structure in our systems. Here we present two examples to confirm such effect of buckled structure. First, when the pure PU foam burned in air condition, the pristine polymer frameworks vanish rapidly, whereas the graphene “skin” coated PG foams still remain as an entire framework with apparent structural shrinkage . Compara810Despite of multifunctional performance, many of applications such as flexible supercapacitors, pressure sensors and elastic conductors require that the 3D monoliths possess excellent mechanical performance during deformation. And unlike the functionalities mainly stemmed from the coated nanomaterials, the overall mechanical properties of this 3D porous template/nanomaterial system would be dominated by the cellular structures. As shown in in situ SEM observation in We further explore the influences of buckled structure and its novel NPR behavior on mechanical properties as they are intriguing from an engineering point of view. For example, on the basis of tensile results in e.g. the improved flexibility and stretchability), the buckled structure we designed in A-PG foams would effectively improve their performance as smart materials.To explore the effects of the buckled structure in our A-PG foams on their functional performance, we further undertook electrical characterizations of our sample in response to mechanical deformation. First, the unique buckled structures of A-PG foams give rise to improved electrical conductivity. As expected, the graphene “skin” that modified onto the polymer skeleton confers conductivity upon the whole surface area of the entire micorfibers network, and the buckled structures further increase the contacts or paths between these conductive network as well as decrease the size of the foams. Indeed, the electrical conductivity of A-PG foams is measured to be 0.001 S/m , three t2151Experimental Section for details). Also similar to our testing results of PG and A-PG foams, it is found that as-obtained A-PA foams exhibit prominent improvements in flexibility and stretchability, compared to PA foams. As shown, the PA foams could catastrophically break under less than 30% tensile strain, whereas the A-PA foams with bucked structures showed remarkable tolerance (more than 100%) to tensile deformation. We also integrated LED lights with our samples as simple demonstrations of stretchable conductors in We further tried to apply our compression-induced buckled structure into the 3D porous template/nanomaterial monolith beyond the PU-graphene foams. Here, we chose Ag nanowires as our functional nanomaterials because of their high conductivity and their increasingly uses in the field of electronic conductors. Melamine (PM) foams (or sponges) were chosen as our polymer templates as they are another commercially available templates with the desirable physical properties and thus widely used in recent reports181920212223242526etc. Considering the similarities between these reports and our results in structural geometry of templates as well as in self-assembling process, we suggest that besides basic self-assembly methods, the simple top-down post-compression treatment might be combined, by which buckled topographic microstructures and negative Poisson’s ratio effect could be achieved. Through such structural design not only the potential performance of individual nanomaterials might be more effectively realized in macroscopic architecture , but also many other bulk mechanical properties can be improved as a result of buckled structures such as toughness, flexibility, stretchablity, damage resistance, which are ideal properties for the use of this next generation of cellular solids in potential applications in 3D flexible electronics, stretchable functional materials and biosystem. Additionally, the bucked structure caused NPR effect further makes the 3D nanomaterial monolith of interests because of the novel behaviors they exhibit under deformation. And we expect that the synergies resulting from functions of nanomaterials and NPR effect will be used widely in future applications such as stretchable capacitors or conductors with high flexibility, shock cushion in body armor, more comfortable  sensor with increased sensitivity and measuring range, easy-to-insert-hard-to-extract stoppers for oil/water separators and so on.Recently, extensive works have employed the strategy of incorporation various types of nanomaterails in conventional porous templates to fabricate 3D nanomaterials-based macroscopic monoliths for various novel applications. In addition to aforementioned PU and PM foams, the porous templates also includes Ni foams2O2 solution , followed by centrifugation and careful washing to clean out remnant salt. Colloidal dispersions of individual GO nanosheets in water (3 mg/mL) were prepared with the aid of an ultrasonic cleaner.GO was prepared from purified natural graphite  following our previous method3 nylon vessel. Commercial available PU foams were cut into 30 × 30 × 90 mm3 cuboids and cleaned by ethanol and deionized water (DIW) with squeezing manually, followed by drying at 80 °C for 5 hours. The PU foams were immersed into the mixture by squeezing in an ultrasonic cleaner and then the vessel was sealed and placed in an 90 °C oven for the reduction of GO sheets. After 12-hour reduction reaction, a gel-like product was taken out from the vessel. After cleaning by adequate ethanol and DIW and drying at 80 °C to obtain the polymer based-graphene (PG) foams. The density of the resulting foams is about 15 mg/mL with 2 wt% graphene. The PG foams with 2–7 wt% graphene could be obtained through changing the concentration of graphene oxide solution from 1 mg/mL to 3 mg/mL. Samples with 2 wt% graphene were used in most of characterization and only electrical-performance-related characterizations employed the samples with 7 wt% graphene.In a typical process, 100 mL graphene oxide solution  was added by 300 μL hydrazine hydrate (80%) and stirred for 2 min at room temperature and then the mixture was poured into a 32 × 32 × 100 mm3) (A Teflon mould with void (inner dimensions 20 × 20 × 60 mm3)  was empl3 nylon vessel. Commercial available melamine (PM) foams were cut into 45 × 20 × 20 mm3 cuboids and cleaned by ethanol and deionized water (DIW) with squeezing manually, followed by drying at 80 °C for 5 hours. The PM foams were immersed into the AgNWs solution by squeezing in an ultrasonic cleaner and then picked out and transferred into a dry oven at 60 °C oven for 12 hours. After 12-hour, a gray foam was taken out from the oven. Then cleaning by adequate ethanol and DIW and drying at 60 °C to obtain the polymer based-AgNWs (PA) foams. A Teflon mould with void (inner dimensions 13 × 13 × 30 mm3) was employed to introduce buckling structure into PM foam. The PM foam was compressed in all three orthogonal directions with 33% strain after stuffing it into the Teflon mould with the aid of tongue depressor and steel plate. After leaving the compressed foam in the oven for 40 min at softening temperature (180 °C) and cooling within moulds (2 hours) to preserve this new configuration, PM foams with negative Poisson’s ratio were prepared (A-PM foams). Then follow the step for preparing PA foams to obtain polymer based-AgNWs (A-PA) foams.The method for the fabrication of PA and A-PA foams was quite similar to that of PG and A-PG foams. 20 mL silver nanowires (AgNWs) solution  was put in an ultrasonic cleaner for 30 min at room temperature and then poured into a 50 × 25 × 25 mmThe microstructure and morphology of the as-prepared foams were characterized by SEM (HITACHI S3400). To give an insight of the microstructure, TEM (FEI Tecnai G2 F20 U-TWIN) observations were conducted directly on as-prepared samples. Raman spectroscope (using the 632.8 nm line of a He-Ne laser) was used to examine the presence of graphene on the microfibers of PG foams. For the electromechanical tests, the top and bottom surfaces of the foams were coated with a uniform layer of silver paste and connected by copper wires. During the compression process, the electrical resistance (Keithley 4200 SCS under a bias of 10 mA) was recorded simultaneously.−1. Cyclic strain controlled loading was used to evaluate the dynamic behavior of the PG and A-PG foams with a ramp rate of 200% min−1. Dynamic compression testing in A dynamic mechanical analyzer  was used to evaluate the mechanical performance of these foams. The dimensions of all tested samples for compression were about 2 cm × 2 cm × 1 cm and 0.2 cm × 0.5 cm × 2 cm for tension. All the samples were applied an initial load of around 0.01 N in order to provide uniform contact. Static compression/tension tests in How to cite this article: Dai, Z. et al. Multifunctional Polymer-Based Graphene Foams with Buckled Structure and Negative Poisson's Ratio. Sci. Rep.6, 32989; doi: 10.1038/srep32989 (2016).


Graphene and its derivatives have become the most explored materials since Novoselov and Geim (Nobel Prize winners for Physics in 2010) achieved its isolation in 2004. The exceptional properties of graphene have attracted the attention of the scientific community from different research fields, generating high impact not only in scientific journals, but also in general-interest newspapers. Optical fibre sensing is one of the many fields that can benefit from the use of these new materials, combining the amazing morphological, chemical, optical and electrical features of graphene with the advantages that optical fibre offers over other sensing strategies. In this document, a review of the current state of the art for optical fibre sensors based on graphene materials is presented. Graphene (G), a two-dimensional carbon material with one-atom-thickness, has become a trending topic in different scientific fields, such as physics, chemistry and materials science, since Novoselov and Geim reported its successful isolation in 2004 . Its outIn particular, graphene-based materials  have been widely used for sensing applications in the last few years due to their high specific surface area, high electronic mobility and low electrical noise. A wide range of chemical sensors, biosensors and gas sensors have been developed using graphene materials ,3,4,5,6.Among all other sensing strategies, optical fibre sensors have achieved a high impact in the last decades because they offer several advantages over electronic sensors ,8,9. OneTo date, publications on optical fibre sensors based on graphene materials are limited. However, the unique optical, chemical and morphological properties of graphene combined with the benefits of optical fibre sensing schemes are attracting a growing interest in the scientific community. The increase in the number of publications observed in the last few years is a clear indication of this fact.This review presents a comprehensive summary of the research on optical fibre sensors based on graphene and its derivatives, including experimental and theoretical studies. The document is structured in the following sections: first, a brief introduction to graphene materials is presented, paying special attention to the different synthesis methods: micromechanical exfoliation, epitaxial growth on SiC substrates, chemical vapour deposition, unzipping of carbon nanotubes, liquid phase exfoliation of graphite, and reduction of exfoliated graphene oxide. The next section is focused on the different optical fibre sensors found in the bibliography, classified by sensing technology . Finally, the conclusions of this review are summarized.Graphene is the two dimensional form of carbon in which carbon atoms are arranged in a honeycomb crystal lattice such that each atom is joined to three others by sp2-bonding. The use of three σ-electrons in carbon-carbon bonding results in a system of delocalized π-electrons perpendicular to the honeycomb plane giving rise to graphene's exceptional electrical properties ,16.The name graphene was introduced by Boehm, Setton and Stumpp in 1986 . For sevSince its discovery, graphene has attracted much attention due to its fascinating structural, optical, mechanical and electrical properties ,6,7,8, wDifferent graphene production processes have been reported, which can be classified in different categories depending on the physical or chemical procedures employed. As previously mentioned this is an amazingly simple method developed by Novoselov, et al. . MicromeThe epitaxial growth of graphene on a SiC substrate involves the fabrication of a graphene film by thermal decomposition on a prepared SiC surface in temperature conditions of up to 1450 °C for up to 20 min . This me2/Si wafers or copper foils are ideal substrates for graphene synthesis . Th. Th69]. A similar device using GO as sensitive coating has been recently reported by Dash et al. . When thAn FPI consists of two parallel optical mirrors separated by a certain distance. Interference occurs due to the multiple additions of reflected and transmitted beams at the two mirrors. In the case of optical fibres, FPI sensors can be classified into extrinsic and intrinsic. Extrinsic FPIs use the reflections from an external cavity formed outside the fibre, as shown in Li and co-workers from Beihang University (China) have developed in the last few years a wide variety of sensors based on FPI using a G diaphragm. SPR) is produced [Over the past two decades, surface plasmon resonance (SPR) based sensors have attracted the attention of many researchers due to their potential applications in the field of physical, chemical and biomedical sciences ,82,83,84produced .SPR is obtained in the transmitted spectrum. This λSPR shows a strong dependence on the refractive index of the sensing medium around the metal layer. Using this scheme, a great variety of sensors can be obtained just by depositing onto the metal thin-film a material that is sensitive to the chemical compound or physical property of interest [In the case of optical fibre-based SPR sensors, the device shown in interest ,86.In the last few years, some studies that include graphene materials in optical fibre SPR-based sensors have been published. In these studies, graphene materials can play different roles. Some authors have used them as SPR supporting materials instead of the typically used gold and silver layers or in addition to them. In other cases, the authors have utilized graphene-based coatings as the sensitive material, which reacts to any variation of the target analyte. In the next paragraphs, some examples of these sensors are introduced.Kim et al. used graphene in a SPR sensor as replacement material for gold or silver ,88. A muIn , the autIn , the autMishra and co-authors carried out a detailed study about the fabrication and characterization of SPR-based fibre optic gas sensors using rGO, carbon nanotubes (CNTs) and poly(methyl methacrylate) (PMMA) . Probes A particular case of SPR is localized surface plasmon resonance (LSPR). When a very thin dielectric coating that includes metal nanoparticles  is deposited onto a waveguide, a resonant coupling between the incident electromagnetic wave and the surface of the thin-film is produced. This results in an electromagnetic wave at the metal-dielectric interface that causes appearance of a sharp absorption peak at a determined wavelength in the transmitted spectrum ,96.Nayak et al. developed different refractive index sensors using graphene oxide encapsulated gold nanoparticles (GOE-AuNPs)  and grapB) that can be measured. As strain and temperature have a direct influence on the mentioned parameters, many sensing schemes based on FBGs have been developed to measure these signals [Fibre gratings consist of a periodic perturbation of the properties of the optical fibre, generally of the refractive index of the core . In Fibr signals ,106,107.FBGs can be also used for chemical sensing and biosensing. For this purpose, FBGs are coated with a material whose structure varies in the presence of a particular chemical compound . When the concentration of this analyte changes, the deformation of the coating produces an axial strain. The FBG stretches or shrinks under such strain and therefore the spectral pattern of reflected light changes, producing a shift in reflected wavelength . If the In the last few years, an increasing number of studies about optical fibre sensors based on FBGs have been focused on devices that include coatings made of graphene-based materials. In this section, different examples of these structures are presented.The Sood group from the Indian Institute of Science  has intensively studied this type of sensor. They have developed different sensing schemes based on etched FBGs including gas detectors, physical sensors and biosensors. In particular, they have enhanced the typical sensitivity of bare FBGs to strain and temperature by coating with rGO an etched FBG. These sensors showed a sensitivity to strain of 5.5 pm/με and a sensitivity to temperature of 33 pm/°C (5 and 3 times better than bare FBGs). The resolutions obtained with these sensors were about 1 με for strain measurements and 0.3 °C for temperature measurements .Furthermore, the same group has developed different biosensors based on this configuration. In , they prOther research groups have also exploited the mentioned mechanism. Wang et al. have recently developed a relative humidity sensor consisting of a tilted FBG coated with a GO thin film obtaining a maximum sensitivity of 0.129 dB/%RH in the relative humidity range from 10% to 80% . 3 gas with the concentrations of 0 ppm, ~10 ppm, ~50 ppm, and ~100 ppm. To further demonstrate the enhancement effect by graphene, bare microfibre FBGs (MFBGs) with different diameters were also tested. The results of these experiments are shown in Zhang et al. studied the features of a graphene-coated microfibre FBG (GMFBG) as an ammonia sensor. FBGs/LPGs and optical fibre interferometers require complex fibre optic pre-processing or sophisticated control at the micrometre level respectively. In contrast, optical fibre sensors based on light intensity monitoring, such as transmission, absorption or reflection, are relatively simple to implement. On the other hand, these systems are vulnerable to light intensity variations, light source instabilities, micro and macro bending or external source coupling. In order to overcome these undesired effects these systems use complex detection algorithms and systems with referenced measurements, which are not associated with the light intensity variations produced by the selected analyte ,118.0 and I1 are the excitation and transmitted light intensities and α, the absorption coefficient, can be expressed as α = Cε/loge where C is the molar concentration and ε is the molar absorptivity. The absorbance (A), is proportional to the length of the fibre sensitive region and the analyte concentration and can be obtained from Equation (1) and expressed as:Absorption-based optical fibre sensors rely on the fact that the selected target or transducer must modify the intensity of the light propagated through the optical fibre core as a function of the selected measurand. Here, the intensity loss can be described using the Beer-Lambert law:Optical fibre geometry is generally modified in this type of sensor in order to enhance light interactions with the selected measurand or transducer. Some typical examples are the cases of U-bent, side polished, cladding removed, microstructured, tapered fibres and so on. Concerning these intensity-based measurements, the general idea is based on the fact that the optical properties of the target or transducer are altered as a function of the selected magnitude or analyte concentration. More specifically, the optical absorption of graphene layers can be considered proportional to the number of layers with little or no perturbation between adjacent layers. Graphene also exhibits a quite flat response from 300 to 2500 nm with an absorption peak near 270 nm as shown in The two-dimensional hexagonal shape structure of graphene, large surface area and high electron mobility enable it to adsorb easily different kinds of gas molecules, volatile organic compounds (VOCs) and biological species . MoleculThe next paragraphs will focus the attention on the utilization of thin graphene-based coatings, fabricated onto diverse optical fibre sensing schemes, as transducers for the detection of physical magnitude or property changes such as temperature or UV radiation as well as chemical and biological compounds: relative humidity, ethanol, ammonia, glucose or DNA. These applications, together with the sensing characteristics of the obtained devices are also summarized in Graphene-based thin-films can be exploited for the fabrication of optical fibre sensors. Ambient temperature, humidity or ultra violet light radiation among others have a great impact on the conductivity of graphene, leading to variations of the effective refractive index and having a measurable effect on the transmitted optical power. Fast response all-fibre graphene assisted temperature sensors have been obtained using microfibre  and side110, and heated at 220 °C, GO220) when exposed to gaseous ammonia, ethanol and methanol  ,129,130,anol see . These rHigh to sensitive and selective VOCs detection has been also explored in  by Some Some other works have explored the affinity of graphene and graphene-based materials for the detection of more complex molecules. For example, Qiu and co-workers explored glucose and double strand DNA (ds-DNA) detection by means of the fabrication of a single-layer graphene onto tapered POF (TPOF)  and tapeFluorescence or phosphorescence measuring systems, referred to as fluorescence henceforward, have been established as an important group of optical fibre sensors. This technique has been traditionally used in analytical chemistry or biochemical applications due to its excellent performance, including time saving, fast response, cost effectiveness, high sensitivity and specificity and good reproducibility. The detection mechanisms are based on fluorescence lifetime or intensity measurements using modulated, pulsed or continuous excitation light sources . The fluGO is fluorescent over a broad range of wavelengths, owing to its heterogeneous electronic structure. Blue to green fluorescence emission can be obtained as a function of the light excitation wavelength with a redshift of the fluorescence maximum intensity with the increase of excitation wavelength above 400 nm as shown in In particular, GO fluorescence can be obtained by inducing a bandgap using two preferred routes. One consists of producing graphene ribbons and graphene quantum dots (GQDs) while the other is based on chemical and physical treatments, such as oxygen plasma treatments ,140,141.2+ ions, dopamine (DA) and single-strand DNA (ssDNA) [+ functionalization in Type 3 sensor (to form COO-Na+ bindings). Fluorescence measurement results from the obtained devices are shown in Partially rGO (prGO) with adsorbed fluorescent rhodamine 6G (Rh6G) molecules (Type 1) was coated onto etched MMF for the detection of Cd (ssDNA) . AdditioThe exceptional electrical, mechanical, thermal and chemical properties of graphene offer an ample range of possibilities for the development of optical fibre sensors based on different interrogation schemes, such as resonance, interferometry, fibre Bragg gratings, light intensity modulation or fluorescence. Some application examples for temperature, humidity, UV light and VOCs sensing have been reviewed in the above sections.The use of graphene and graphene-based materials for sensing applications is at a very early stage. It still needs some time and effort to achieve its full potential. In particular, the understanding of the surface chemistry of GO and rGO, the role of functional groups, the modification of graphene structure by means of the introduction of defects and dopants could all contribute to new approaches to enhance the sensitivity and selectivity to gaseous compounds and more complex molecules. These advances combined with novel micro and nanofabrication techniques would enable the fabrication of 3D graphene structures at the nanoscale level opening the door to novel applications. It is also important to improve coating strategies and in-situ reduction of GO to enhance the homogeneity and reproducibility of films. GQDs, which consist of single atom graphene sheets with a thickness in the order of 3~20 nm, have emerged as a good alternative for applications that require high fluorescent activity, robust chemical inertness, long fluorescence lifetime and excellent photostability when compared with other carbon-based nanomaterials . In addi


The observation of large nonlocal resistances near the Dirac point in graphene has been related to a variety of intrinsic Hall effects, where the spin or valley degrees of freedom are controlled by symmetry breaking mechanisms. Engineering strong spin or valley Hall signals on scalable graphene devices could stimulate further practical developments of spin- and valleytronics. Here we report on scale-invariant nonlocal transport in large-scale chemical vapor deposition graphene under an applied external magnetic field. Contrary to previously reported Zeeman spin Hall effect, our results are explained by field-induced spin-filtered edge states whose sensitivity to grain boundaries manifests in the nonlocal resistance. This phenomenon, related to the emergence of the quantum Hall regime, persists up to the millimeter scale, showing that polycrystalline morphology can be imprinted in nonlocal transport. This suggests that topological Hall effects in large-scale graphene materials are highly sensitive to the underlying structural morphology, limiting practical realizations. Nonlocal resistances in graphene Hall bars attributed to neutral current Hall effects have been mainly measured at the microscale. Here, the authors observe consistently strong nonlocal signals in Hall bars with channel length ranging from the micrometer up to the millimeter scale, and explain them by field-induced spin-split edge states. The high-mobility, low spin−orbit coupling (SOC), and linear energy dispersion of graphene has made it a core platform in such quest. In this context, graphene has been explored in magnetic-element-free nonlocal transport using Hall-bar geometries. The large nonlocal signals are observed close to the Dirac point, and have been tentatively related to topological Hall effects such as the spin Hall effect (SHE)10, or long-range chargeless topological valley Hall currents for gapped graphene/h-BN heterostructures12. Spin signals in graphene using nonlocal approaches free of magnetic elements have been reported by applying external magnetic fields13, by using extrinsic sources of SOC14, and by proximity effect to ferromagnetic insulators15. Theoretical predictions have mainly explored spin diffusion in the spin Hall regime, where spin currents are respectively generated and detected by means of the SHE and the inverse spin Hall effect (ISHE)18. Nonlocal signatures related to valley Hall currents have also been predicted to emerge from the different diffraction features of the two valleys in graphene19. The theoretical frameworks in the spin Hall regime consider extrinsic sources of SOC, Zeeman interaction , and proximity-induced strong-exchange bias as possible mechanisms driving such enhancements of nonlocal signals close to the Dirac point23. More recently, spin generation and detection via extrinsic sources of SOC have been questioned by Wang et al.24, Kaverzin et al.25, and Cresti et al.26In recent years, there has been a continuous effort to harvest the spin and valley degrees of freedom of charge carriers for developing innovative information processing as an alternative to complementary metal-oxide-semiconductor technologies15. The micrometer spin and valley relaxation lengths therefore restrict the experimental realizations to such scale. Core in the analysis of nonlocal signals, these approaches rely on comparing the nonlocal signal to the background arising from the classical current spreading (Ohmic) contribution and studying its dependence with the channel geometry to sustain the claim of other-than-charge sources for the nonlocality.All current demonstrations however rely on microscale Hall bars of pristine graphene obtained from micromechanical exfoliation techniques, with natural flakes with dimensions ranging several micrometers, or on microscale Hall bars patterned from chemical vapor deposition (CVD) graphene3 in microscale high-quality pristine graphene devices, and closely follow the same dependence with the device aspect ratio for device lengths that differ by two orders in magnitude. We however exclude an origin based on pure spin and valley currents due to the length scales involved in the transporting channel of the macroscale devices, and conclude that the origin of the intriguing nonlocality for both the microscale and macroscale stems from the same fundamental mechanism. Considering the microscopic details of the fabricated samples and the polycrystalline morphology of the CVD graphene samples, the large nonlocal signals are consistent with a dissipative quantum Hall regime driven by Zeeman-split counter-propagating edge states28. Additionally, the grain boundaries (GBs) shunt the insulating bulk33 induced by the strong magnetic field, and generate a strong asymmetry of the nonlocal magnetoresistance with external magnetic field, a striking feature already observed, but not yet understood3.Here, we demonstrate the persistence of large nonlocal signals at the Dirac point of CVD graphene devices from the micrometer up to the millimeter scale in presence of an external magnetic field applied perpendicular to the surface. These signals exhibit a similar dependence with the magnetic field to those induced via Zeeman spin Hall effect34. The devices studied in this work were fabricated following standard electron-beam lithography procedures on monolayer CVD graphene films wet-transferred onto 1×1 cm2 Si/SiO2(300 nm) substrates (see Methods). Standard local electrical characterization was first employed to ensure the quality of the metal contacts, and that the graphene samples exhibited characteristic transport properties unique to graphene, such as the half-integer quantum Hall effect35 15.In a concise manner, nonlocal measurements in the context of spin Hall currents consist of driving a current in one arm of an H-bar shaped channel and detecting the voltage drop at the other arm see Fig. . Using t37. By injecting a current on the left arm, a net current will reach the detection terminals, with magnitude decreasing exponentially with the distance to the injecting terminals. This Ohmic contribution in the device scheme presented is determined using the formula L > W, is usually approximated as Although nonlocal measurements are used to probe non-charge-related transport, there are classical, charge-related sources of nonlocality that can contribute to the signal detected between terminals 3 and 4 see Fig. . A robusB), and then compared to the Ohmic contribution evaluated under the same conditions. Throughout the manuscript, we keep the same relative orientation of the perpendicular B and of the arms injecting the current and detecting the voltage signal. We explored CVD graphene Hall bars with channel 5, 50, and 500 µm wide, with center-to-center distance between terminals maintaining similar aspect ratios (L/W) of typically 1, 1.4, 1.8, 2.4, and 3.2. The width of the terminals is 1/10th or less of the channel width (see Methods). We report mainly on the results obtained for the extreme cases of 5 and 500-µm-wide samples, using the data of the 50-µm-wide sample to extend the discussion. Further details on the fabrication and electrical measurements are provided in Methods.In our study, the nonlocal resistance at the CNP of graphene is measured for different applied magnetic fields  and without (B = 0) applied magnetic field. Without magnetic field, the expected Ohmic contribution and the measured RNL coincide, following the same dependence with the channel length. Fitting the dependence to an exponential decay, the resulting decay λ = 159.4 ± 1.6 µm closely matches the expected W/π from the Ohmic term RNL follows a similar exponential decay with channel length, but the magnitude of the resistance is greatly enhanced, in some cases being one order of magnitude higher than the Ohmic signal for similar conditions. If the signal were to be interpreted in terms of charge neutral spin currents, , a fitting to the expression λs = 163 ± 19 μm and θSH = 1.6 ± 0.4. Such values are clearly unreasonably high for such a disordered polycrystalline graphene sample supported onto an oxide substrate. Actually, the spin Hall angle determined is one order of magnitude higher than what is typical of metals with strong SOC, such as platinum (θSH ~ 0.2)38.Expanding the study to the different channel lengths and focusing on the side with highest nonlocal signal, Fig. Figure B = 0), and by sweeping the gate voltage, RNL is well described by the Ohmic background, with a decay λ = 1.58 ± 0.03 µm. However, in this case, under an applied magnetic field (B = 6 T), the enhancement of the nonlocal signal is one order of magnitude higher than in the macroscale sample for an equivalent aspect ratio, L/W = 3, with RNL two orders of magnitude higher than the Ohmic background  and without (B = 0) applied magnetic field, Fig. B = 0, following the same dependence with the channel length. In the presence of B, the nonlocal resistance dependence is also clearly described by a similar exponential decay with channel length, but the magnitude of the signals is even further enhanced, from one to two orders of magnitude larger than the expected Ohmic background. An analysis in terms of non-charge currents yields λs = 2.7 ± 1.2 µm, and θSH = 1.3 ± 0.6. We note that this relaxation length is similar to reports that place λs between 1 and 5 μm in CVD graphene on SiO239.Similarly to what was found before, in the absence of an applied magnetic field ; second, the thermal gradient across the detection terminal under B would generate the nonlocal voltage (Nernst effect). Since thermoelectric coefficients in graphene show a strong increase around the Dirac point, the gate dependence should manifest an enhancement at the CNP. But considering both Ettingshausen and Nernst effects, the dependence of RNL with B should be quadratic, RNL ∝ B2. This seems not to be the case in our samples, neither in the microscale sample .From this discussion, the nonlocal signals in our experiment do not seem to originate from spin or valley Hall effects, thermoelectric effects, or purely Ohmic contributions. To further clarify the nature of the observed features, we repeated the experiments for a sample with dimensions in between 5 and 500 μm. With a 50-μm-wide channel, and equivalent aspect ratios between 1 and 3.2, the sample is on the macroscale side, and should exclude spin transport. Figure The nonlocal resistance for the 50-μm-wide sample remains between those obtained for the two other samples, with signals 1−2 orders of magnitude higher than expected from the van der Pauw background. Interestingly, the signal magnitude is similar to the 5-μm-wide sample, where spin signals could conceivably survive a 16-μm-long channel. In this case, with a 160-μm-long channel, we exclude a spin transport origin. The mechanism driving the nonlocal response must be able to generate signals dependent with the magnitude and sign of the external magnetic field, seemingly invariant with scale, and that follow a dependence with channel length similar to the Ohmic background. To explain all these anomalous features, we propose an interpretation based on counter-propagating edge states shunted by GBs.41. The Zeeman splitting of the LL0 couples the charge carrier type to the spin state, so that electrons and holes are now associated with opposite spin polarizations, which triggers a dissipative quantum Hall effect near the CNP, driven by spin-polarized counter-propagating edge states  has already been found in a nonlocal measurement on two-dimensional system with a simultaneous presence of electrons and holes in a 20 nm HgTe quantum well43.When an out-of-plane magnetic field is applied to graphene, the bulk gradually becomes insulating, while charge current increasingly flows at the edges, resulting in a well-defined quantum Hall effect for large fields  are the most likely source of this conduction through the bulk, and as shown in Fig. 45 or by intercepting electron−hole puddles across the device. This manifests as an asymmetry with respect to magnetic field direction due to the Zeeman splitting, which associates each charge carrier type with a different spin orientation. In Supplementary Note ρxx with the sign of B52 or a signature of edge transport in nonlocal magnetotransport measurements55 does not necessarily require a graphene-specific model. In particular, an asymmetry of ρxx with B has been observed in well-behaved semiconductor two-dimensional electron gases, with mechanisms such as conduction paths in the bulk47, carrier density gradients49, hybrid constrictions50, and in-plane electric fields52 driving the effect. Our proposed mechanism, however, differs in that it can explain not only the asymmetry of the nonlocal resistance but also the large nonlocal signals beyond the Ohmic contribution at the CNP. The physics of the LL0 in graphene, with a coexistence of electrons and holes (and thus a sensitivity to electron−hole asymmetries), has no equivalent in other low-dimensional systems. To demonstrate that GBs are at the origin of the B asymmetry of the nonlocal signal, we perform a tight-binding simulation in the nonlocal geometry with a single 558-type line defect connecting the edges (see Methods) of a 40-nm-wide and 85-nm-long graphene channel. As seen in Fig. L/W = 3.2 of the 5-μm-wide sample strongly suggests counter-propagating edge states shunted by GBs to be at the root of the large, asymmetric nonlocality observed in the samples. An extended study over all devices with different aspect ratios of the 5-μm-wide sample shows a strong asymmetry of the nonlocality with the sign of the magnetic field for all devices, further corroborating our analysis  is very general and, in principle, independent of sample size. However, larger samples contain more grains across their width and the asymmetric effects of individual GBs will tend to be averaged out. This will lead to smaller asymmetries with the direction of B, but also smaller nonlocal signals due to a greater number of bulk conduction channels. This picture fits convincingly with the range of effects exhibited by our measured graphene samples from the microscale to the macroscale. Importantly, the appearance of a strong nonlocal signal is independent of spin or valley transport mechanisms arising from, for example, long-ranged coherent spin transport and the SHE/ISHE effects.Due to the random formation of inhomogeneities and GBs during the growth of CVD graphene, samples with individual, random line defects can show nonlocality preferentially for either positive or negative magnetic fields. Electron−hole asymmetry in GB transmissions can arise due to both intrinsic sublattice effects and external doping effects. Other considerations, including the positioning of GBs relative to the probes, the rate of backscattering between counter-propagating states, and the density of electron−hole puddles, can play an additional role in determining the exact nonlocal signature. However, the qualitative effect /SiO2(300 nm) chips with monolayer CVD graphene grown on copper foils were acquired from a commercially available supplier, Graphenea S.A. All samples reported here come from the same batch. The samples were first spin coated with double-layer of Poly(methyl methacrylate) (PMMA) (495/950 kDa) and then baked at 180 °C for 90 s. To define the Hall-bar-shaped graphene, the areas surrounding the Hall bar were exposed with electron-beam lithography, and etched with a chemistry of Ar/O2 (80/5 sccm) in a capacitive coupled plasma reactive-ion etching Oxford PlasmaLab 80 equipment. The remaining resist leftovers were striped in acetone bath at room temperature for 4 h, immersed in isopropanol, and dried with a nitrogen gun. For the definition of the electrical contacts, the samples were again spin coated with double-layer PMMA, and the metal electrodes defined over the Hall bar. The metallization was done on ultra-high vacuum at a base pressure of 10−9 mbar, using electron-beam deposition of Ti (5 nm)/Au (40 nm) at a rate of 0.5 and 1.5 Ås−1. The lift-off was performed with acetone at room temperature. One important feature of the device design was the terminals width of 1/10th or less of the channel width. This comes from a straightforward analysis of the van der Pauw expression for charge diffusive backgrounds, where if the width of the contact is on the same order of the channel width it can lead to edge-to-edge signals ~20 times in magnitude larger than the center-to-center signal.The same fabrication procedures were followed for the fabrication of the 5-, 50-, and 500-μm-wide CVD graphene samples. 1×1 cmThe devices were characterized in a Quantum Design physical property measurement system using standard four-probe direct current measurement methods. The measurements were performed using a Keithley 2182A as current source and a Keithley 6221 as voltmeter. The gate voltage was applied using one channel of the Keithley 2636. In all measurements, the excitation current was 10 μA. Before measuring the devices, we performed an in situ annealing at 400 K for 3 h, with helium flushing cycles to release the sample chamber of evaporated water. The samples would then be cooled down at the maximum rate to 2 K, and any temperature-dependent study performed for increasing temperatures.B = ∇ × A. The transmissions between each set of probes is given by the Caroli formula GR, GA) calculated using efficient recursive techniques. The left and right leads are included via broadening terms (Γ) calculated from the surface Green’s function of semi-infinite nanoribbons, whereas constant broadening terms, representing metallic contacts, are chosen for the top and bottom probes. The Zeeman term is included separately via equal and opposite energy shifts of the spinless transmissions by half the required splitting (10−3 eV). The total transmission is then the sum of the two spin contributions (our model excludes spin-mixing terms). The nonlocal resistance is calculated from the potentials Vp and currents Ip at each probe, which emerge from solving the multi-terminal Landauer−Büttiker relation The system is described by a standard nearest-neighbor tight-binding model with the magnetic field incorporated using the Peierl’s phase approach All relevant data are available from the authors.Supplementary InformationPeer Review File


The same mechanism can occur with thermally generated carriers, which contribute to the high leakage current often observed in graphene/Si junctions.We study the effect of temperature and light on the I-V and C-V characteristics of a graphene/silicon Schottky diode. The device exhibits a reverse-bias photocurrent exceeding the forward current and achieves a photoresponsivity as high as  Graphene is a 2D structure possessing high thermal conductivity , high mo2 insulating layer, forming a metal-oxide-semiconductor (MOS) capacitor I=I0We extracted the Schottky barrier height and the effective Richardson constant from the Richardson plot shown in scussion ,12, can 2 interfacial layer, Assuming that ficiency ,14.The series resistance s method . Taking From Equation (6), when −1 can be neglected, two equations can be derived, both yielding I plot  can be computed from the current and bias voltage measured in the . I plot b, is a s barrier ,17.2/Si MOS capacitor. In forward bias, the positive voltage on graphene causes accumulation of electrons (majority carriers) at the Si/SiO2 interface of the MOS capacitor. These electrons do not affect the forward current of the Gr/Si diode, which is controlled by the injection rate of electrons over the barrier. In reverse bias, the electrons cannot overcome the barrier, and the saturation current is mainly due to minority carriers (holes). The reverse current can thus be increased by enhancing the concentration of minority carriers, e.g., by shining light. Upon illumination, photogenerated holes are attracted by the negative bias of graphene to the Si/SiO2 interface of the MOS region. Their diffusion to Gr/Si junction originates the observed high photocurrent. Indeed, the motion of such holes to the Gr/Si junction is favored by the band bending from the Gr/SiO2/Si to the Gr/Si region, as shown by the band diagrams of We studied the photoresponse of the device by shining light from the top, on the entire device. It has been shown that the same mechanism, when applied to thermally generated minority carriers, contributes to the high leakage of the Gr/Si junctions .To further investigate the carrier distribution in the graphene-controlled Si substrate, we performed a room-temperature capacitance-voltage (C-V) characterization in the dark and under illumination . The cap2 interface forming an inversion channel, which raises the MOS capacitance to a value comparable to the one in accumulation at positive voltage. Otherwise stated, at the lowest biases and under high illumination, the device shows the typical C-V plots of a MOS on p-type semiconductor. This indicates that the photogeneration rate is able to provide enough positive charge to invert the n-Si and that the channel can follow the oscillation of the small ac signal.Focusing on the dark curve (black solid line) in The holes of the inversion channel of the MOS capacitor, injected in the Gr/Si region, cause the high photocurrent of the device, as previously stated.The interpretation of the Gr/Si dominating capacitance in the range 2/Si MOS region has been confirmed by scanning photocurrent measurements. High responsivity, broadband and fast Gr/Si photodetectors were also reported in [A similar model has been recently proposed in , where ported in ,20.2 layer with a thickness of 2 layer was removed from the trench area by a hydrofluoric acid treatment immediately before graphene deposition. This procedure reduced the possibility that oxide will form on the surface. The graphene sheet had been obtained by CVD on Cu foil. In the next step, a graphene sheet of 2/Si MOS capacitor. Ohmic metal contact to graphene was fabricated by evaporating a Ti/Au metal stack through a shadow mask, while the Si substrate was contacted by depositing Ag paste on an area with removed SiO2 and appropriately scratched to produce ohmic contact. For I-V and C-V measurements, the Ti/Au pad was the forcing lead (anode) while the Ag contact on the Si substrate was grounded. Further information about the fabrication process can be found in [The device was fabricated from a lightly doped n-silicon wafer with a resistivity of found in . Both th2/Si MOS capacitor. We used I-V curves at different temperatures to extract the relevant parameters of the Schottky junction. More importantly, we reported a very high photocurrent, which can exceed the forward current. Using C-V characterization, we proved that the MOS capacitor acts as a reservoir of photo-generated minority carriers. Such excess minority carrier injected into the junction region is the origin of the observed high reverse photocurrent. Hence, the parasitic MOS capacitor enhances the photoresponse of the Gr/Si diode.In conclusion, we characterized a hybrid device consisting of a Gr/Si junction in parallel with a Gr/SiO


Interstitial lung disease (ILD) comprises a heterogeneous group of diffuse parenchymal lung processes with overlapping clinical, radiographic, and histopathologic features. Among the most common and deadly ILDs are idiopathic pulmonary fibrosis (IPF) and chronic hypersensitivity pneumonitis (CHP). As the name implies, the cause of IPF remains elusive, but a variety of genetic and infectious risk factors have been identified. CHP results from chronic inhalation of an organic antigen, usually of avian or mold origin, and may occur in patients with a genetic predisposition. While IPF is treated with anti-fibrotic compounds, CHP is generally treated by suppression of the immune system and elimination of the causative antigen. Despite advances in our understanding of IPF and CHP, there exists substantial variability in the diagnosis and treatment of these disease processes. Furthermore, IPF and CHP natural history and treatment response remain far from uniform, leaving it unclear which patients derive the most benefit from disease-specific therapy. While clinical prediction models have improved our understanding of outcome risk in patients with various forms of ILD, recent advances in genomic technology provides a valuable opportunity to begin understanding the basis for outcome variability. Such advances will ultimately allow for the incorporation of genomic markers into risk stratification and clinical decision-making. In this piece, we highlight recent advances in our understanding of the genomic factors that influence susceptibility and outcome risk among patients with IPF and CHP. Genomic modalities used to identify these genomic markers include genome-wide association studies, analyses of gene expression, drug–gene interaction testing, telomere length determination, telomerase mutation analysis, and studies of the lung microbiome. We then identify gaps in knowledge that should be addressed to help facilitate the incorporation of these genomic technologies into ILD clinical practice. The interstitial lung diseases (ILDs) are comprised of a heterogeneous group of diffuse parenchymal lung processes with overlapping clinical, radiographic, and histopathologic features . Among tIdiopathic pulmonary fibrosis is now treated with compounds targeting fibrotic mediators after phase III clinical trials demonstrated efficacy in slowing pulmonary function decline –14. ProsIn this review, we highlight recently identified genomic factors influencing susceptibility and outcomes of patients with IPF and CHP. These include single-nucleotide polymorphisms (SNPs) identified by targeted sequencing and genome-wide association studies (GWAS), gene expression profiling, telomere length testing, and lung microbiome bacterial DNA profiling. We then identify gaps in knowledge that should be addressed to help facilitate the incorporation of these genomic technologies into ILD clinical practice.MUC5B (rs35705950) and an intronic SNP near TOLLIP (rs5743890). MUC5B encodes one of several mucin-producing genes, which facilitate airway clearance and function to maintain immune homeostasis  among the general population  trial (TOLLIP (rs3750920) was associated with a favorable responsive to N-acetylcysteine (NAC)  trial , 27, invne (NAC) . Comparene (NAC) . Those wNo GWAS have been performed in patients with CHP to date, but studies employing targeted genotyping have identified gene variants linked to disease susceptibility and outcomes. Camarena and colleagues conducted targeted genotyping of SNPs within the major histocompatibility complex (MHC) II region and found that SNPs within HLA-DRB1 were disproportionately present in patients with avian antigen-associated CHP compared to control subjects . SubsequWhile GWAS have identified gene polymorphisms that may influence IPF susceptibility, transcriptomic analyses of RNA isolated from lung tissue and peripheral blood have shed important light on gene expression pathways involved in IPF and CHP pathogenesis and outcomes. Selman and colleagues conducted a microarray analysis of RNA obtained from lung tissue in patients with IPF and CHP to determine whether gene expression profiles could differentiate these disease processes. These investigators showed that while patients with IPF had upregulation of genes involved in tissue remodeling, apoptosis and fibroblast signaling, those with CHP displayed upregulation of genes critical to immunologic function, including those T cell signaling and others related to MHC function .Subsequent transcriptomic investigations using lung and peripheral blood specimens from patients with IPF supported the role of genes involved in alveolar epithelial injury and remodeling in IPF pathogenesis , 32. YanIn addition to differentiating IPF from other forms of ILD, investigators have utilized transcriptomic analysis to develop a peripheral blood-based genomic prediction tool to predict IPF survival. Using a two-stage, multi-center derivation and validation approach, Herazo-Maya and colleagues identified a gene signature composed of 52 differentially expressed genes could effectively categorize patients with high versus low mortality risk over a 4-year follow-up period . This geTERT, TERC, RTEL1, PARN, NAF1, TINF2, DKC1)  and improved our understanding of the genetic underpinnings of ILD. To date, there have been seven telomere-related genes that have been implicated in adult-onset FPF  –43. Path2, DKC1) , 45.TERT is the most commonly affected gene and accounts for ~20% of FPF  from patients diagnosed with a variety of idiopathic interstitial pneumonias using 16S-rRNA gene PCR and degenerating gel electrophoresis . This wagression . The retVeillonella, Neisseria, Streptococcus, and Haemophilus spp. in patients with IPF compared to controls. The most striking differences, however, were observed in the bacterial burden, which was increased twofold in IPF patients compared to control subjects. Within the IPF cohort, bacterial burden correlated with disease progression. When stratifying the cohort by this metric, the authors demonstrated a clear increase in mortality risk with increasing bacterial burden (Figure A subsequent prospective study of the lung microbiome compared IPF cases to healthy control subjects and controls with COPD, allowing for direct compassions between health and disease . This inn Figure .MUC5B promoter polymorphism, with individuals carrying the minor allele of this SNP having a lower bacterial burden. Driven by these tantalizing interactions between host and environment, authors of both microbiome studies have attempted to advance the studies of the lung microbiome from merely descriptive and observational to functional (Streptococcus and Pseudomonas and worse survival.These authors also showed few differences in the microbiome between IPF subjects with progressive or stable disease, suggesting bacterial load itself might be more important in driving disease progression. The authors hypothesized a mechanistic link between host and environment and demonstrated an association with bacterial burden and the nctional , 64. IntMUC5B and TOLLIP, influence both susceptibility and mortality risk, few others have demonstrated significant outcome association (While some SNPs associated with IPF susceptibility, notably those within ociation , 28. Givociation , 65, it ociation , 66.The development of a transcriptomic signature to predict mortality has greatly improved our understanding of IPF pathobiology. Now that IPF has two currently approved therapies, it will be important to assess the test performance characteristics of this signature in those treated with prolonged anti-fibrotic therapy and in those without prior exposure to immunosuppressive therapy, as was common practice prior to completion of the PANTHER trial . In addiTERT and TERC suggest that immunosuppression after lung transplant for ILD may be associated with high rates of side effects including bone marrow failure, liver toxicity, and infections (TERT carriers (The presence of telomere-related rare variants or short telomere length predispose to rapid disease progression in both IPF and CHP, however, there is very little data regarding response to specific treatments. The therapeutic strategies for IPF and CHP differ substantially. Immune suppression is often employed for patients with CHP and progressive disease, while immunosuppression is detrimental in IPF . To our fections –69. Thisfections –14, but carriers  but largIdiopathic pulmonary fibrosis is characterized by a distinct respiratory microbiome, with a higher bacterial burden than in health. This is further disturbed during exacerbations of disease . DespiteThe majority of genomic data generated to date has been in patients with IPF, leaving CHP ripe for similar investigation. However, standardization of diagnostic criteria through international consensus is first needed. Once that occurs, GWAS to identify SNPs linked to disease susceptibility and survival would advance our understanding of disease underpinnings and potentially identify novel therapeutic targets for this devastating disease without proven therapy. In addition, determining whether outcome-related transcriptomic signatures derived in patients with IPF informs outcomes in patients with CHP will be of immense clinical value. Finally, determining whether the microbiota makeup of patients with CHP influences disease susceptibility and outcomes has the potential to guide therapy in these patients.In this review, we highlight the most developed genomic technologies informing susceptibility and outcome risk in patients with IPF and CHP. There remain critical questions to be answered to characterize the extent to which these technologies will improve risk stratification. In addition, a cost benefit analysis will be necessary to determine whether individual technologies make sense from a cost utilization perspective. The field of ILD has advanced rapidly over the last 10 years and will continue to do so into the foreseeable future. Clinical genetics represents the logical next step for the field and holds great potential to be a cornerstone of personalized medicine in the field.CN, PM, and JO contributed to the conception and writing of this review. All authors have reviewed and approved the submitted work.CN, PM, and JO have no relevant conflicts to disclose related to the submitted work.


In most advanced cancers, except Hodgkin lymphoma (which has high PD-L1/L2 expression) and melanoma , the objective response rate with anti-PD-1/PD-L1 monotherapy is only ~20%, and immune-related toxicities and hyperprogression can occur in a small subset of patients during PD-1/PD-L1 blockade therapy. The lack of efficacy in up to 80% of patients was not necessarily associated with negative PD-1 and PD-L1 expression, suggesting that the roles of PD-1/PD-L1 in immune suppression and the mechanisms of action of antibodies remain to be better defined. In addition, important immune regulatory mechanisms within or outside of the PD-1/PD-L1 network need to be discovered and targeted to increase the response rate and to reduce the toxicities of immune checkpoint blockade therapies. This paper reviews the major functional and clinical studies of PD-1/PD-L1, including those with discrepancies in the pathologic and biomarker role of PD-1 and PD-L1 and the effectiveness of PD-1/PD-L1 blockade. The goal is to improve understanding of the efficacy of PD-1/PD-L1 blockade immunotherapy, as well as enhance the development of therapeutic strategies to overcome the resistance mechanisms and unleash the antitumor immune response to combat cancer.PD-1–PD-L1 interaction is known to drive T cell dysfunction, which can be blocked by anti-PD-1/PD-L1 antibodies. However, studies have also shown that the function of the PD-1–PD-L1 axis is affected by the complex immunologic regulation network, and some CD8 It is widely known that ligation of programmed cell death protein 1   activate− patients . This article summarizes functional and clinical studies of PD-1/PD-L1 and the resistance mechanisms for PD-1/L1 blockade, and discusses several important questions arising from the disparate data, with the goal of increasing understanding of PD-1, PD-L1, and PD-1/PD-L1 blockade.Despite these discrepancies and unknowns, PD-1/PD-L1 blockade has achieved great clinical success in combating cancers. Durable response could also be achieved in PD-L1patients , 6. None+ T cells  mRNA is mainly expressed in the thymus in vivo, with additional possible distribution in the spleen and lung , and 4-1BB (costimulatory molecule) expression and gained 1,414 activation-related (but not exhaustion-related) accessible chromatin regions (+CD8+ tumor-infiltrating lymphocytes (TILs), but not from PD-1− or bulk CD8+ TILs, showed tumor-reactivity and therapeutic benefit in vivo   tumor-reactive CD8 Ifngr2) . In patipression . Similared TNF-α . In a co T cells .PDCD1 mRNA levels were upregulated in “exhausted” CD8+ T cells with impaired cytokine production and proliferation, but PDCD1 was not upregulated in functional LCMV-specific memory CD8+ T cells during acute viral infection , and probably critical for antigen-specific T cell responses (During chronic infection with lymphocytic choriomeningitis virus (LCMV), nfection . Paradoxhaustion . In factactivity . Similaresponses . Moreoveesponses .hi expression also does not mark T cell exhaustion in patients with autoimmune disease or cancer. In patients with rheumatoid arthritis, PD-1hiCXCR5−CD4+ cells are expanded in pathologically inflamed non-lymphoid tissues and are functionally active (promoting B cell responses)  PD-1lo T cells and PD-1hi “non-exhausted” follicular helper T cells   than PD-1− T cell clones (PD-1sponses) . In follnd IL-4) . Increaslymphoma , 32, lunlymphoma , ovarianlymphoma , or poorlymphoma , 36. Furl clones , and PD-l clones –40. The in vitro but is induced on tumors and in the tumor microenvironment (exceptions include some lymphoma and myeloma cell lines)  , 41. IFNl lines) , is the l lines) . Supportr margin . IFN-γ ar margin –47. JAK2r margin , MEK/ERKr margin  signalinr margin , T cellsr margin , and natr margin , and thir margin –52.in vivo PTEN loss did not always affect PD-L1 expression significantly  and STAT3 signaling , and hypficantly ]. Moreovficantly , and novficantly , 63 decrficantly . Enhanceficantly . The glyficantly .Furthermore, PD-L1 is also expressed under normal conditions in both lymphoid and non-lymphoid tissues on human placental trophoblasts, myocardial endothelia cells, and cortical thymic epithelial cells , 11, 42,in vivo and showed that although all forms of PD-L1 expression contribute to immune suppression in a non-redundant fashion, the relative roles  of immunogenic tumor-derived PD-L1 and host-derived PD-L1 expression in suppressing T cell cytotoxicity and infiltration varied depending on the mouse models used, which had different levels of tumor immunogenicity  mice was as robust as in wild-type mice, which elegantly demonstrated that induced tumor PD-L1 expression directly and sufficiently inhibits antitumor immunity, serving as far more than a marker of an ineffective immune response   or oncoggenicity –76. PD-Lresponse .Similarly designed experiments demonstrated that oncogenic PD-L1 expression in BRAF.PTEN melanoma tumors only slightly inhibited antitumor immunity , whereas+ tumor cells, and the cytolytic function of T cells against other PD-L1− tumor cells with the same antigen is not impaired  mice. TILs from PD-1−/− mice had an increased ratio of CD8+ cells to regulatory T cells (Tregs) and granzyme expression compared with TILs from wild-type mice. In contrast, MC38 tumors (with immunogenic PD-L1 expression) grew similarly robust in PD-L1−/−/L2−/− mice as in wild-type mice; PD-L1−/−/L2−/− mice and wild-type mice had similar CD8/Treg ratios and PD-1, granzyme, and Ki-67 expression levels in TILs  of stimulated antigen-specific CD8+ T cells by cancer vaccines were also regulated by increased PD-1 expression  and had poor recall response upon antigen re-challenge  infection with transient PD-1 expression but were exhausted during chronic (clone 13) infection with stable PD-1 expression . Althougned high . Therefohallenge .+ T cells. In contrast, chronic infection uniquely enriched accessible chromatin regions for NFAT and Nr4a family transcription factors (including enhancers of the PDCD1 locus) but partially lost the accessibility to some regions (such as Satb1 and Il7r loci) in exhausted CD8+ T cells, although exhausted CD8+ T cells and effector CD8+ T cells shared chromatin accessibility at promoter regions of key effector-related genes, including Ifng, Gzma, Gzmk, Fasl, and Prf1, as well at inhibitory receptor genes, including Tim3, Lag3, and Ctla4  in the epigenetic landscape  and dynamic (≥25%) changes in accessible chromatin regions in antigen-specific effector and memory CD8nd Ctla4 . Anti-PDandscape . The epiandscape , suggestin vivo led to tumor regression with increased antigen-reactive T cell infiltrate and increased IFN-γ and TNF-α production upon antigen stimulation ex vivo. However, PD-L1 blockade had only a moderate effect on gene activation and chromatin accessibility in tumor-infiltrating T cells, including upregulation of a few functionally important genes (including granzyme and serpin genes) and dampened accessibility in limited motifs binding NFAT, NFAT:AP-1, TCF, and bZIP:IRF transcription factors. In contrast, 450 accessible regions (including those accessible for Nr4a and NFAT) were gained in “exhausted” T cells compared with “non-exhausted” T cells before the treatment , but not TCR, were the preferred targets of dephosphorylation by PD-1-bound SHP2 in a biochemical reconstitution system . PD-1 coransient .in vitro, ligation of PD-1 on CD4+ T cells inhibited glycolysis ; fatty acid catabolism enhanced in CD8+ T cells  partially preserved antitumor effector functions of CD8+ TILs but upregulated (possibly indirectly) PD-1 expression; PD-1 blockade synergizes (but did not change) this metabolic reprogramming in inhibiting tumor growth   and RGMbin vitro . In contdiabetes .+ T cells was shown to promote survival and effector function of CD8+ T cells during the contraction phase following immunization/antigen stimulation  (+ Th1 cells) (−/− lupus-susceptible (MRL+/+) mice developed autoimmune myocarditis and pneumonitis with increased PD-1+ macrophage and T cell infiltrates in the heart and lung   and expe1 cells) . PD-L1−/and lung . PD-L2−/controls .−/− mice completely suppressed the tumorigenesis of PD-L1+ myeloma cells (−in vitro) in PD-1−/− mice , “deeply exhausted” CD8+ T cells during chronic LCMV infection  with or without CD4+ T cell depletion  of systemic LCMV clone 13 infection also caused vascular permeability and ultimately fatal circulatory collapse , but antepletion . Althougnfection , other sepletion  or Treg epletion  could re−/− mice, anti-PD-1 mAbs exhibited stronger antitumor effects than anti-CTLA-4 mAbs in tumor models   , augmentavidity) , 161, deavidity) , and pro priming , 163 .in vitro with a CT-011 antibody  enhanced natural killer cell trafficking, immune complex formation, and cytotoxicity against PD-L1-bearing multiple myeloma cells  release, antigen presentation, and costimulation owing to NF-κB activation. PD-L1 blockade also increased cytokine release although to less extent  the onset of severe colitis suppressed T cell expansion and Th1 cytokine production and prevented the development of colitis  subset of CD8+ T cells (+CD44hi but not CXCR5−CD44loCD8+ T cells (the former had PD-1loTIM-3lo expression and higher effector function) reduced the viral load in mice chronically infected with LCMV; the therapeutic effect was further enhanced with anti-PD-L1 combination (hiPD-1hiBLIMP-1+T-betlo phenotype (converted from T-bethiPD-1int cells) and increased cytotoxicity but decreased co-production of IFN-γ and TNF-α  have been approved for the treatment of the following cancers as first-line, second-line, third-line, or later-line therapies: melanoma , 187–195However, anti-PD-1/PD-L1 therapies did not work in all cancers . AlthouIn addition, hyperprogression, a new pattern of disease progression after anti-PD-1/PD-L1 monotherapy, that is associated with elderly age and worse overall survival but not specific tumor types, has been identified in ~9% of cancer patients , 233. A + PD-L+ patients and not in PD-1− patients (− patients (most PD-L1− cases are PD-L−) . Furthermore, in some studies of squamous NSCLC and renal cell carcinoma, the efficacy of PD-1 blockade  in PD-L1− patients was similar to or even better than that in PD-L1+ patients .Unlike tumor PD-L1 expression, which has shown predictive value for the efficacy of anti-PD-1 therapy in most studies Table , 236 Ta, 216. Ev+ cells, we would postulate that the association of PD-L1 expression with immune activation status, rather than the correlation with immune suppression strength, may underlie the predictive value of PD-L1 expression for anti-PD-L1 therapy. Interestingly, in NSCLC, anti-PD-1 therapy (pembrolizumab) demonstrated superiority over chemotherapy in patients with ≥50% or <1% tumor PD-L1 scores, but this benefit was absent in patients with 1–49% tumor PD-L1 scores , but not in pre-treatment (median: 3 months prior to treatment), biopsies was highly predictive for response to PD-1 blockade, suggesting the inability to accurately predict the clinical response before anti-PD-1 therapy . In thiseriments , 21, 101gativity , 233.JAK1/2 and APLNR loss-of-function mutations, which result in non-inducibility of tumor PD-L1 expression by IFN-γ, have been associated with primary or acquired resistance to PD-1 blockade in solid tumors; PD-1 blockade was ineffective for these patients even if their mutational load was high . Although a study showed that neoantigen load correlated with T-cell infiltration in colorectal cancers  mutations and clonal neoantigens, as well as T cell clones responding to PD-1/L1 blockade  and mutational burden showed inconsistent significance in studies  within CD8+ TILs  and MHC class II (67%) were found in 108 patients newly diagnosed with classical Hodgkin lymphoma   . High frr virus) . Becauser virus) . In addir virus) , and allr virus) . To enhar virus) , 286–290+ T cells with PD-1 blockade requires CD28/B7 costimulation in a mouse model with chronic viral infection  can result in T cell anergy , impairenfection  and thatnal load . However T cells .+ T cells were CD28−  condition  is important for PD-1 expression in exhausted T cells  and posttranscriptional regulation of PD-1/PD-L1 expression may lead to a more durable response in patients. The transcription factors and pathways positively regulating PD-1 expression include BLIMP-1  , 314, IF T cells .Fourth, insufficient antitumor activity may result from multiple T cell subtypes and subclones (including those with “fixed” T cell dysfunction) that are not responsive to PD-1/L1 blockade. Dysfunction of these T cell subclones may lead to tumor evolution of subclonal neoantigens, which were associated with primary and acquired resistance to checkpoint blockade in patients , 258. Inin vivo , only the minority of cancer patients had durable response to PD-1/PD-L1 blockade monotherapy, and some patients even had disease hyperprogression. Classical Hodgkin lymphoma, which does not have a high mutational burden or MHC class I expression, demonstrated a high response rate to PD-1 blockade therapy.+ and PD-L1+ expression can also indicate T cell activation although PD-L1 and PD-1 may be expressed in different stages of immune response; markers to distinguish PD-1+ T cells with high functional avidity from exhausted PD-1+ T cells are unclear. Both PD-1 and PD-L1 can either dependently or independently drive immune suppression. Whether tumor or host factors dictate immunity remains to be determined. Mechanisms that are not completely understood also include those governing PD-1 expression, molecular pathways underlying PD-1/PD-L1 blockade, the difference in PD-1 signaling upon PD-L1 binding and upon anti-PD-1 mAb binding, metabolic crosstalk between tumor cells and T cells, and functional relationship  between PD-1/PD-L1 expression and cell metabolism. Molecular delineation and critical node identification may also help clarify the inconsistent preclinical results of blocking PD-1 compared with blocking PD-L1.In addition to summarizing these paradoxical results in studies of PD-1/PD-L1 and PD-1/PD-L1 blockade, this review discussed a few open questions from mechanistic and clinical perspectives. As discussed, both PD-1 and PD-L1 are often  associated with T cell dysfunction; PD-1+ and PD-L1− patients. Also uncertain is whether this and other differences between PD-L1 and PD-1  underlie the better predictive value of PD-L1 over PD-1 expression as a biomarker for clinical response. Tumor mutational burden has also emerged as a promising biomarker; however, our understanding of clonal mutations, T cell clonality, and neoantigen-reactive TIL clones responsive to PD-1/PD-L1 blockade may be still in its infancy. In addition, infiltration of immune cells, tumor immunogenicity, strength of TCR signaling and costimulation/co-inhibition, T cell differentiation stage and chromatin flexibility, immune cells and soluble factors in the tumor microenvironment, pharmacologic kinetics of antibodies, and systemic immunity may all affect the efficacy of PD-1 blockade. Future studies in the fast advancing field of immunotherapy may shed light on these intriguing questions, develop algorithms to accurately predict the blockade efficacy, and pave the way for a new era of combination immunotherapy.It is unclear whether PD-1 blockade has different action (antagonist or agonist) in PD-L1ZX-M conceptualized and wrote the manuscript and created the figure. KY contributed to the conception and writing. MZ and JL revised the manuscript. All authors read and approved the final manuscript. The authors thank Erica A. Goodoff from the Department of Scientific Publications, MD Anderson Cancer Center, for her edition of the manuscript.KY receives research support from Roche Molecular System, Gilead Sciences Pharmaceutical, Seattle Genetics, Dai Sanyo Pharmaceutical, Adaptive Biotechnology, Incyte Pharmaceutical, and HTG Molecular Diagnostics.


Programmed death ligand 1 (PD-L1) has emerged as a biomarker that can help to predict responses to immunotherapies targeted against PD-L1 and its receptor (PD-1). Companion tests for evaluating PD-L1 expression as a biomarker of response have been developed for many cancer immunotherapy agents. These assays use a variety of detection platforms at different levels , employ diverse biopsy and surgical samples, and have disparate positivity cutoff points and scoring systems, all of which complicate the standardization of clinical decision-making. This review summarizes the current understanding and ongoing investigations regarding PD-L1 expression as a potential biomarker for clinical outcomes of anti-PD-1/PD-L1 immunotherapy. Novel therapeutics targeting immune checkpoints are leading to a fast and profound paradigm shift in cancer therapy –3. DurabA companion diagnostic is necessary for the safe and efficacious use of a corresponding drug or biological product, whereas a complementary diagnostic identifies, though not essential, a biomarker that can assist in the risk/benefit assessment for a subset of patients who respond to the corresponding drug differentially , 17. CurNotably, PD-L1 IHC 22C3 pharmDx and IHC 28-8 pharmDx are the first two FDA-approved assay systems for qualitative detection of PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) tissue. The level of PD-L1 protein expression is determined by using tumor proportion score (TPS) . The TPSThe third FDA-approved antibody, SP142, is a rabbit monoclonal antibody against PD-L1 , 25. ThiThe fourth PD-L1 antibody, SP263, was just approved as a complementary diagnostic for durvalumab. SP263 is a rabbit anti-human PD-L1 monoclonal antibody directed against the cytoplasmic region of human PD-L1  that hasRecently, studies have also analyzed the PD-L1 expression at the genetic level. PD-L1 and PD-1 protein expression were analyzed in 94 clinical cases of small cell neuroendocrine carcinomas by IHC using two different monoclonal antibodies . RNA-seq was used to profile messenger RNA (mRNA) expression in 43 clinical cases. Results showed that RNA-seq yielded highly comparable results with IHC and even more PD-L1-positive cases than IHC; therefore, RNA-seq is also suitable for detection of PD-L1 expression . This stSo far, analysis of the relationship between PD-L1 expression and response to anti-PD-1/PD-L1 agents has yielded promising results. One pooled analysis summarized data from seven studies with 914 NSCLC patients . PatientAn ongoing challenge to the application of PD-L1 biomarker assays in clinic is that each anti-PD1/PD-L1 agent has its own companion assay. These assays have not been standardized for all the agents and therefore are not interchangeable. However, several groups have examined the degree of agreement between different methods. One large study of 493 samples compared the extent of concordance among three validated, commercially available PD-L1 IHC assays  for NSCLC patients and found an overall percentage agreement of >90% between assays. These assays were also consistent at multiple expression cutoffs, including 1, 10, 25, and 50% tumor cell membrane staining . Most rePD-L1 expression is commonly focal and primarily identified at the tumor–stromal interface  and appeκ = 0.218 [poor agreement]) [κ value of 0.8366, suggesting that there is good concordance and adequate assessment for PD-L1 expression with small samples [One study examined PD-L1 expression with the SP142 IHC assay in both whole surgical tissue sections and matched lung biopsies from 160 patients with operable NSCLC. PD-L1 expression was assayed in both TC and IC cells. The study found that PD-L1 expression between the surgically resected and matched biopsy specimens frequently disagree with each other  . The PD- samples . These r samples –47.In addition to the focal, heterogeneous expression of PD-L1 within a single lesion, separate lesions from the same patient may have different intensities and patterns of PD-L1 expression. One study performed quantitative assessment of the heterogeneity of PD-L1 expression in 49 NSCLC whole tissue sections and a corresponding tissue microarray. Two rabbit monoclonal antibodies (E1L3N and SP142) were used for both conventional IHC and quantitative immunofluorescence (QIF). Results showed that assessment of 588 serial section fields of view by QIF exhibited a discordant expression at a frequency of 25% .In addition, PD-L1 expression varies among TC, IC, and immune stroma in a given patient. Using 67 fully resected, multifocal specimens from 32 NSCLC patients to assess intertumoral heterogeneity, Mansfield et al. reported that there was poor agreement of PD-L1 expression between paired lesions of 20 patients by tumor and immune cells . AlthougPD-L1 expression in TCs and ICs were examined in a separate study which reported that PD-L1-positive TCs were negatively correlated with PD-L1-positive ICs within tumor stroma. In this analysis of 105 patients with resected stage I pulmonary squamous cell carcinoma, tumor PD-L1 expression and increased CD4+ T cell infiltrations in tumor stroma were found to be independent predictors of better overall survival . TherefoThe heterogeneity also exists among primary, metastatic, and transformed tumors. One study reported a case of NSCLC who was found to have also small cell lung cancer (SCLC) transformation in the lung and liver metastasis at autopsy. The PD-L1 protein was partially expressed in tumor cells with adenocarcinoma histology but not in tumor cells from SCLC transformation . AnotherTaken together, these studies suggest that the results of PD-L1 expression assays are affected by a variety of factors, including specimen size, biopsy location, variable components of tumor and immune microenvironment, and tumor transformation. These factors should be carefully considered when employing PD-L1 as a predictive biomarker in clinical practice.p = 0.037) in these patients [Several members of B7 family have been found to have soluble counterparts –54. Usinpatients . It remapatients –62. sPD-Novel therapeutics targeting immune checkpoints are leading to a fast and profound paradigm shift in cancer therapy. PD-L1 expression is a valuable biomarker to guide clinical decisions. PD-L1 expression assays are affected by a variety of factors, including specimen size, biopsy location, variable components of tumor and immune microenvironment, and tumor transformation. These factors should be carefully considered when employing PD-L1 as a predictive biomarker in clinical practice. Future investigations should focus on standardizing detection, developing reliable methods of liquid biopsy, and developing multiparameter quantitative or semi-quantitative biomarker panels to provide clinicians a more comprehensive understanding of the tumor and immune microenvironment. Correlation of PD-L1 expression and tyrosine kinase biomarkers should also be explored .


This study aimed to evaluate the cognitive levels of Multiple Choice Questions (MCQs) & Short Answer Questions (SAQs) and types of Item Writing Flaws (IWFs) in MCQs in Medical Pharmacology internal assessment exams.This descriptive, study was conducted over a period of six months, from December 2015 to May 2016 and evaluated six internal assessment examinations comprising SAQs and MCQs. A total of 150 MCQs and 43 SAQs were analyzed. These questions were administered to third-year medical students in the year of 2015. All SAQs were reviewed for their cognitive levels and MCQs were reviewed for cognitive levels as well as for IWFs. Items were classified as flawed if they contained one or more than one flaw. The cognitive level of the questions was determined by the modified Bloom’s taxonomy.The proportion of flawed items out of 150 items in six exams ranged from 16% to 52%. While the percentage of total flawed items was 28%. Most common types of flaws were implausible distractors 19.69% (26), extra detail in correct option 18.18% (24), vague terms 9.85% (13), unfocused stem 9.09% (12) and absolute terms 9.09% (12). The two-third of MCQs 97(64.67%) were assessing the recall of information, while 29 (19.33%) and 24 (16%) were assessing the interpretation of data and problem-solving skills respectively. The majority of the SAQs (90.7%) were assessing recall of the information and only 9.3% were assessing interpretation of data while none of the questions was assessing the problem-solving skills.The cognitive level of assessment tools (SAQs & MCQs) is low, and IWFS are common in the MCQs. Therefore, faculty should be urged and groomed to design problem-solving questions which are devoid of any flaws. The assessment either summative or formative has a powerful effect on learning and is considered an essential variable in leading the learners for achieving the goal.2From assessment point of view, it is always important that assessment should be reliable and valid and be able to differentiate between satisfactory and unsatisfactory performers.6There are several types of written tests like long essay questions, short answer question, modified essay questions, MCQs, extended matching MCQs, etc. It is mentioned in the literature that essay type questions can be used in the recall of knowledge and higher order cognitive levels questions both.8In Pakistan, Pakistan Medical and Dental Council (PMDC) and Higher Education Commission (HEC) have recommended multiple formats of assessment. Thus all the affiliated colleges of University of Health Sciences (UHS), Lahore, are using various formats like MCQs, SAQs, OSPE/OSCE and Viva. It is also recommended in medical education that medical students should be assessed by various assessment tools.9MCQs are frequently used because of objectivity, elimination of assessor’s favoritism and extensive coverage of the subject in a short period.Item-writing flaws (IWFs) arise when we deviate from the accepted guidelines of making MCQs, and consequently, such MCQs affect the performance of the students in such a way that it might become difficult or easier for the student to answer it.6The designing of a high-quality MCQs is a skill, and like other skills, it needs training and practice without that there is more probability that MCQs would have more IWFs.Our College has faculty with a diverse background, and few are trained and well experienced, while others are new in teaching. So we assume that cognitive level of our written assessment is not up to the mark, and it is likely that our MCQs have several IWFs. Until now in our college, no such study was done to evaluate written assessment tools.Therefore, this study aimed to evaluate the cognitive levels of MCQs & SAQs and types of item writing flaws in MCQs in six Pharmacology and Therapeutics internal assessment exams held in 2015.The descriptive study was conducted on six internal assessment examination comprising SAQs and MCQs from Pharmacology & Therapeutics Department, the University Medical and Dental College, Faisalabad, Pakistan in the year 2015.In each internal assessment exam, there were 25 MCQ and 5- 10 SAQs. A total of 150 MCQs and 43 SAQs from six internal examinations of 2015 were evaluated for their cognitive levels and IWFs. For identifying types of IWF’s standard criteria given by several educationists were used and fourteen 14) frequently occurring violations of item-writing guidelines were selected from literature & were subsequently applied to assess the quality of the 150 MCQs in all six exams. frequentA subject expert and a medical educationist’s were taken in the team from outside the University for analyzing the questions’ cognitive levels and IWFs. A proforma was prepared to evaluate each MCQ, and SAQ and each reviewer assessed the questions individually by the predefined criteria and difference of opinion between the medical educationist, and subject experts were further sorted out for reaching an agreement about the controversial questions.The questions’ cognitive levels were evaluated by the Buckwalter’s modificationItems were classified as flawed if they contained one or more than one flaw. The ethical review committee of the College gave the approval for this study.The SPSS version 21 was utilized to analyze the data and frequencies and percentages were calculated.The descriptive statistics for each internal assessment exam is given in rd year MBBS students. These exams comprised of 150 MCQs and 43 SAQs. Analysis for cognitive levels based on Bloom’s criteria showed that of 60.47% of MCQS were at low cognitive levels. These results are similar to another research that pointed out 46.2 % IWFs in MCQS for nursing assessment, and 90% of the questions were of the low cognitive level.18The current study evaluated the quality of six internal assessment examinations of 3Our results are similar to a study that found the cognitive level of the majority of SEQs (83.33%) was at recall level while very less number of questions 16.67%) were evaluating interpretation of data..67% wereQuantitative analysis of our data indicates that there were 43 IWFs (28%) in 150 MCQs while another study in Pakistan reported 69 IWFs (46%) in 150 MCQs.The types of IWFs found in our study  are similar to numerous other studies.20Additionally, we also noticed that sixth assessment exam of our evaluation had less number of flawed items (16%) and had a greater passing score (70%) as compared to first, second, third and other exams. This finding suggests that flawed item negatively affects the high passing scores which are in agreement with another report.21A recent study by Patil et al., 2016) reported that only 10% of MCQs fulfilled all the criteria for an ideal MCQ.6 reporte22It is essential to design questions that investigate a deeper understanding of the topic/subject, and that stipulate the application of higher-order thinking skills to incorporate Basic Sciences knowledge with relevant, clinically-oriented contexts.24Our study suggests that there is a strong requirement to work on the improvement of assessment tools by initiating faculty development program. The selection or construction of a good MCQ is the most critical step in assessing the knowledge of the student and to differentiate students with different capabilities.27A study by Naeem et al., (2011) at Aga Khan University (AKU) observed that substantial improvement in item quality is dependent on faculty development.30The present study has several limitations; firstly, we analyzed the written tests of only one subject in a medical college. Therefore, our results cannot be generalized and don’t t reflect the quality and IWFs present in other subjects and colleges affiliated with UHS. Secondly, we did not calculate the discrimination and difficulty indices.The cognitive level of assessment tools (SAQs & MCQs) is low, and IWFs are very common in the MCQs.A careful review of the learning outcomes is required and how they are aligned with the assessment. A test blueprint should be developed before the exam, and all questions should be constructed according to the cognitive level mentioned in that test blueprint. The faculty should be trained for the construction of MCQs for higher cognitive levels and developing test blueprint. The properly trained faculty may play an important role not only in minimizing item-writing flaws but also improving cognitive levels.Saba Tariq designed the study, collected the data and drafted the manuscript.Sundus Tariq data entry and analysis, layout design, manuscript writing.Sadia Maqsood data analysis, table’s formulation and manuscript writing.Shireen Jawed contributed in literature review, proof reading and correctionMukhtiar Baig helped in data analysis, drafting, reviewing and editing the final manuscript.