The incorporation of graphene nanoplatelets (GnPs) within a polymer matrix can play an important role in the physical properties and the functionality of the composite material. Composites consisting of low-density polyethylene (LDPE) and GnPs of different concentrations were developed by mixing GnPs with a molten form of the polymeric matrix. The effect of the GnPs content on the morphological, structural, and electrical properties of the composites were investigated. As shown, graphene presence and its concentration significantly modified the polymer matrix properties, a behavior that can be employed for tailoring its applicability in electrical applications. It was found that the increase of the graphene platelets concentration seems to promote the formation of graphene agglomerates, air gaps, and inhomogeneities, while higher dielectric constant/lower dielectric losses can be achieved. Polymers have been proved to be suitable matrices for the development of composite materials due to their ease of production and processing, resistance to corrosion, good mechanical properties, and light weight. In polymer-based nanocomposites, the type and size of the filler, the morphology of the filler network, the thermal properties, and the conductivity have been considered to be important variables influencing its final properties ,2. PolymPresently, graphene, has become one of the most exciting materials , becauseSubstantial amount of research has been carried out in the field of nanocomposites incorporating graphene, some of them regarding electrical applications. In general, the increasing demand of energy is a technical challenge for the generation, transmission, storage, and distribution energy systems. This challenge often requires contradictory features such as increasing voltage levels in combination with more compact designs in urban environments. This leads to an increased electric stress on the insulation systems, which can be addressed by using insulating materials with tunable properties, as well as high dielectric constant and low losses, especially for electric field grading applications. In this particular research field, the reports in the literature on graphene nanocomposites concern, improvement of conductivity in polyethylene (PE) matrices , advanceThis work concerns nanocomposite materials made using commercial widely available ingredients using methods that can easily undergo direct further scale up, to become compatible with the industrial needs. In particular, in this work, low-density polyethylene (LDPE) was chosen as the basic matrix material for the fabrication of graphene nanocomposites, due to its significant importance in various applications such as squeeze bottles, toys, carrier bags, high frequency insulation, chemical tank linings, heavy duty sacks, general packaging, gas and water pipes . In addi3.Nanocomposite specimens were fabricated by employing commercially available LDPE from Sigma Aldrich GmbH, Darmstadt, Germany with average molecular weight—MW of 35,000 determined by gel permeation chromatography (GPC) and average numerical mass—Mn 7.700 GPC. Graphene nanoplatelets were provided by EMFUTUR Technologies Ltd., Hillsborough, Spain, with ~5 μm lateral size, 5 nm thickness, and a bulk density of 0.03 to 0.1 g/cmThe melting method was used since this is rather simple for fabricating polymer nanocomposites consisting of low molecular weight polymer matrices. This method involves melting of the polymer to its viscous form, followed by the addition and the mechanical mixing of the filler within the polymer matrix. Graphene concentration in the composite material was varied in the range 0% (%wt.) up to 5% (%wt.) at a step of 0.5%. For each composition 5 replicas were made and characterized. All the obtained formulations were characterized as follows:2 flow and a temperature increment of 20 °C/min. Finally, the electrical characterization of the nanocomposite samples was conducted by means of Broadband Dielectric Spectroscopy (BDS), using a TH2829C LCR precision bridge  capable of taking measurements in the frequency range of 20 Hz to 1 MHz.SEM characterization was performed using a (FE-SEM) Nova NanoSEM 630 , equipped with an energy-dispersive X-ray spectroscopy—EDX detector , in order to investigate and understand the formation and the structuring of the obtained nanocomposite materials. All samples were examined in the high vacuum mode, low landing energies and fast dwelling time, without any conductive coating in order to preserve the samples in their natural state for further analyses. XRD was also performed using a Rigaku Ultra high-resolution triple axis multiple reflection SmartLab X-ray Diffraction System . Moreover, Raman analysis was done using a WiTec alpha 300S GmbH Germany system , employing an Nd-YAG laser at 532 nm and confocal Raman microscopy . Raman spectra were recorded under ambient conditions, using a 532 nm diode-pumped solid-state laser  with a power of 10% (10 mW) to avoid local drying of the samples. A 20× objective and a 25 μm slit aperture were used in order to obtain quite detailed spectra, while the total acquisition time 0.5, 10 and 20 s (1 s exposure × 20 exposures) and 600 grooves/mm grating was chosen for each Raman spectrum. The only notable difference observed in spectra was a higher overall spectral intensity when the acquisition time increases. Severally spectra, in different points were recorded per sample and a representative typical spectrum for each sample was chosen. The instrument calibrations were performed on a silicon probe prior to measurements to ensure reproducible results. The spectrometer scanning data collection and processing were carried out using the WiTec Project Five software  and appropriate background corrections were performed on all spectra in Origin software . The thermal response characterization was studied using a Diamond  differential scanning calorimeter (DSC) operated at a scan rate of 10 °C/min in order to characterize the thermal transitions of employed polymer nanocomposites. Samples were prepared inside aluminum shielded pans while an empty aluminum shielded pan was used as a reference. Experimental temperature range was set from ambient to 200 °C. For thermogravimetric analysis (TGA), a Diamond  TGA was used under 200 mL/min N2 areas of the sample in order to form a capacitor. Two metallic pins were attached to the silver paste plates, and these pins were connected to an HIOKI 3532-50 LCR HITESTER , through its specialized cables. Impedance Z and corresponding loss angle δ are simultaneously measured, as a function of frequency, in the frequency range 10 Hz–1 MHz. The presented results are representative for the specific kind of samples.For the low energy impedance spectroscopy measurements, samples of a rectangular shape were used, with dimensions 10 mm × 10 mm × 0.5 mm. Conductive silver paste was smeared onto both the 10 × 10 mmSEM characterization of samples was performed to investigate the formation and structuring of the composite materials. Nanocomposite materials with higher than 2% graphene content were found to consist of a quite inhomogeneous bulk, formed by different polymerization domains as well as regions with concentrated graphene. In addition, at high graphene concentrations, large graphene agglomerations were observed in some cases, suggesting that a limit of concentration might exist, above which graphene cannot be fully dispersed in the LDPE molten material.At graphene concentrations higher than 2.5%, the structural inhomogeneity is accentuated and a mix of compact and granular regions as well as “graphene bubbles” were observed, as can be seen in Following the SEM characterization, one can understand the effect of the graphene concentration on the composites structure and morphology. The increase of concentration seems to promote the formation of graphene agglomerates, air gaps, and inhomogeneities being present at the same time. These structural SEM observations, related to the effect of the graphene concentration on the evolution of the bulk morphology, seem to correlate with the dielectric constant measurements, as it will be shown later. Finally, it should be mentioned here that to the best of our knowledge, such detailed morphology study of LDPE-graphene composite and its evolution with graphene concentration has not yet been presented in the literature before.Structural features for the LDPE composites of different graphene concentrations were further investigated using grazing incidence X-ray diffraction (GI-XRD), where the incidence angle was kept to 0.5°. In order to assess the influence of graphene, the attention was focused in the range of 5°–40°. The results can be seen in With increasing graphene concentration, the occurrence of the characteristic diffraction peak for carbon around 26° appeared for graphene concentrations higher than 2% graphene. To get the mean crystallite size τ for LDPE and graphene composites, the Scherrer equation  was empl002 = 3.37 Å, at the highest graphene concentration.The obtained results strongly indicate the embedding of the graphene in the LDPE lattice and suggest an enhancement of its crystallinity at high graphene concentration. At the same time, the interlayer distance of the graphene employed becomes closer to the hexagonal graphene one (2H-polytype), for which dRaman spectra were also recorded, using a 532 nm diode-pumped solid-state laser  with a power of 10 mW. A 20× objective and a 25 μm slit aperture were used in order to obtain quite detailed spectra, while a total acquisition time of 10 s (1 s exposure × 10 exposures) and 600 grooves/mm grating was chosen for each Raman spectrum. The spectrometer scanning data collection and processing were carried out using the WiTec Project Five software.−1, the 2D band is at about 2786 cm−1, while there is also evidence of the so-called disorder-induced D bands around 1345 cm−1. Literature reports similar peaks for graphene, in particular, in pristine-single layer graphene, the G band appearing at 1582 cm−1 (graphite) and a 2D band at about 2786 cm−1 [−1 and 1578 cm−1 respectively, denoted by the D’, while the I2D/IG ratio increases [−1, while peaks near 1420 cm−1 (the CH2 bending mode) are also present and are indicators of crystalline and amorphous PE phases. Literature reports similar results for polyethylene, both low and high density, with Sato et al. reporting four (4) peaks at 1465, 1374, 1174 and 897 cm−1 [−1 respectively [−1 and 2848 cm−1.Examples of Raman spectra collected on melt-fabricated samples corresponding to various concentrations of graphene are presented in 786 cm−1 ,20. Morencreases . Regardi897 cm−1 . Moreoveectively . It can Regarding local homogeneity of the composite material, an example is presented in Detailed characterization of the obtained nanocomposites showed that all different batches, fabricated under similar conditions, resulted in more or less similar inhomogeneous materials exhibiting similar properties. These results indicated that the mechanical mix of GnPs in melted LDPE may not be the best method for the preparation of homogeneous GnPs/LDPE nanocomposites, at least for the case of small quantities, as those used in our work. Another approach considered for obtaining GnPs/LDPE nanocomposites formulations in small quantities could be dry mixing of the LDPE polymer pellets with graphene but, as tested, it was almost not possible, since they did not really mix. Similarly, we found out that extruding the two phases resulted in even worst composite material. For proper mixing of the specific materials hot roll milling would be more proper method but it is not easily available for laboratory small scale trials. Since GnPs are not a monodisperse material, functionalization of GnPs would not be a realistic approach to solve the dispersion problems at the mixing in the mold. In any case, more homogeneous materials are expected in larger scale production, since, in this cases mixing can be done more effectively.TGA was performed to study the mass loss if a thermal event involves loss of a volatile component. To study the phase transitions of the GnPs/LDPE nanocomposites DSC analysis was performed. Two typical DSC analyses of the 2.5% and 5% graphene containing samples compared with pure LDPE are presented in The melting temperature Tm (°C) for pure LDPE was 80 °C while the crystallization temperature Tc—98 °C. All the composite materials show Tm smaller than the pure material and Tc larger. Tm increases as the GnPs increase and Tc slightly increase when GnPs concentration increased.Finally, the dielectric behavior of all fabricated samples was investigated using BDS and its dependence on the graphene content within the LDPE matrix was analyzed. As expected, the dielectric constant significantly depends on the graphene concentration and the structural characteristics of the material. In particular, the dielectric constant can be strongly influenced by the morphology of the composite, especially if this contains air gaps and agglomerations, as was found out in the SEM results. However, at this point, a quantitative correlation between the structural/morphologic parameters and the dielectric parameters variations is not possible due to the non-quantifiable structural evolution of the composite bulk structuring with increased graphene concentration.Compared to existing literature results, Gaska et al. ,24 studiGnP/LDPE composite materials with various graphene concentrations were fabricated using the melting method and were characterized using SEM, XRD and Raman spectroscopy and microscopy in order to understand the formation of the composites and the evolution of structural and morphological characteristics with increasing graphene content. SEM characterization showed that graphene concentrations up to 2.5% lead to more compact structuring, while larger concentrations promote the random formation of a kind of “graphene bubbles”. XRD characterization showed that mean crystallite size increases as the graphene concentration increases, with a value of 26.1 nm at a concentration of 2%, this becoming 40.4 nm at 5%. Moreover, the increasing of the graphene concentration within the polymer matrix indicate an expansion of crystal interplanar distance of the embedded graphene. Regarding Raman characteristic signals of the composite regions, these appeared to increase in intensity as the graphene concentration increases, revealing the formation of a composite material and not a simple component mixing. The dielectric constants of the nanocomposites presented an increased trend, proportional with graphene increased concentration; however, it was strongly affected by the morphology evolution of the material. The random formation of “graphene bubbles” could induce the formation of conductive tunnels within the samples, this phenomenon being encountered at specimens with concentration of 4.5% and 5%, a behavior requiring further studies.


Recent advances in the field of two-dimensional (2D) materials have led to new electronic and photonic devices enabled by their unique properties at atomic thickness. Structuring 2D materials into desired patterns on substrates is often an essential and foremost step for the optimum performance of the functional devices. In this regard, optical patterning of 2D materials has received enormous interest due to its advantages of high-throughput, site-specific, and on-demand fabrication. Recent years have witnessed scientific reports of a variety of optical techniques applicable to patterning 2D materials. In this minireview, we present the state-of-the-art optical patterning of 2D materials, including laser thinning, doping, phase transition, oxidation, and ablation. Several applications based on optically patterned 2D materials will be discussed as well. With further developments, optical patterning is expected to hold the key in pushing the frontiers of manufacturing and applications of 2D materials. Nanomaterials, with at least one geometric dimension at the nanometer scale, have immense applications in miniaturization of existing technology while rendering the intense exploration of fundamental phenomena at nanoscale. 2D materials are a subset of the nanomaterials where geometric length of only one dimension reaches the nanometer regime . Since iFor application-oriented utilization of 2D materials, one often starts with their fabrication or patterning, which might involve control of either thickness, carrier density, or phase of 2D materials to precisely tune the optical, electrical, and thermal characteristics of 2D materials. The patterned 2D materials have led to applications in electrochemistry , biosensin situ patterning of 2D materials through precise digital control of laser beams. The localized nature of high-intensity laser beams offers unique opportunities to develop or pattern 2D materials based on regionally induced physical or chemical modifications. For many micro/nanodevice applications of 2D materials based on their patterned features at micro/nanoscale, the timescales of optical patterning range from a few seconds to minutes. Moreover, laser intensity can be tuned with ease to peel a desired number of atomic layers from the 2D materials [Laser-based fabrication methods provide a promising route to high-throughput, versatile, and aterials  and to caterials . Recent The physical properties of 2D materials are closely dependent on their thickness. For example, it has been reported that 2D materials exhibit thickness-dependent photoluminescence (PL) where PL intensity increases with decreasing thickness . Upon re2. The patterned GO layer was then reduced to graphene via thermal annealing or hydrazine. However, this mechanism usually takes over six hours and must be carried out in a humid atmosphere for water to intercalate between graphene layers. This thinning method could only reach down to three-layer graphene due to the high in-plane thermal conductivity of graphene that reduced the temperature in the perpendicular direction below 500°C.Initial attempts to control the thickness of graphene using high-power lasers are based on oxidative burning and subsequent reduction of graphene oxide films . The irr2 substrate that served as a heat sink for the bottom layer of the graphite adjacent to the substrate, thereby reducing the maximum temperature at the bottom layer while the top layers could reach ~1450°C to enable oxidative etching. −1 corresponds to monolayer graphene [Han et al. proposed that laser thinning of graphite to graphene could be better achieved by similar laser irradiation through substrate engineering . In one graphene . The capgraphene .in situ through four-wave mixing signals. Although throughput is compromised due to low-power multiple scanning, precise quantification of number of graphene layers has been achieved through in situ fast imaging [Despite the prominence of monolayer graphene, multilayered graphene exhibits advantages in certain applications. For instance, hydrogenation of multilayered graphene is more feasible than that of monolayer graphene, which is helpful in tuning physical and chemical properties of graphene for sensing devices . Consequ imaging .2 with patterned feature sizes of 200 nm via laser thinning [2 due to its low sublimation temperature of ~450°C [2 flake before and after laser scanning indicates the thinning of the flake  and NIR detectors. Recently, Nagareddy et al. have proposed a humidity-controlled laser thinning technique where layer-by-layer laser thinning of MoTe2 was achieved for the sample that was maintained at a relative humidity of more than 65-70% [2 result in an uncontrolled bond-breaking at relatively higher laser powers. As relative humidity increases, the water molecules adsorb on all available sites offered by the top layer of MoTe2  has also demonstrated great potential in applications requiring high carrier mobility . HoweverBesides the laser-induced thickness-control procedures in 2 and the optical patterning was imaged through upconversion luminescence [μm [Preliminary work on 2D material ablation was performed on graphene, where a high-powered femtosecond laser was utilized to convert the carbon to CO and COnescence . With a nescence . The ablence [μm . While tence [μm .2 substrate (left panel), and the scanning electron micrographs of transferred graphene nanoribbons with variable widths ranging from 100 nm–9 μm on Si/SiO2 substrates (middle and right panels). Despite its capability of achieving the multiscale and high-resolution graphene patterns on an arbitrary substrate, the ablation-and-then-transfer method has been limited using multiple wet chemical processes, which may cause graphene contamination and compromised pattern alignment.Park et al. demonstrated a procedure that involved laser ablation-based graphene patterning and subsequent transfer of the patterned graphene to an arbitrary substrate . As illuthin layer is peeled off, leaving the patterned graphene/PMMA thin layer on the substrate.To simplify the procedure, Yoo et al. have developed a single-step processing method that enables laser ablation-induced graphene patterning and simultaneous transferring to any substrate . In thisAlthough femtosecond laser ablation has been primarily demonstrated on graphene, it can potentially be employed to pattern many other 2D materials . To achiμm2, which enables localized ablation of 2D materials at the laser spot. The authors examined the feature size of patterned 2D materials under varying laser powers and exposure times  were fabricated, which enabled a photodetector with enhanced performance .  Figurencreases , 76. By hickness . In addihickness  for applhickness , topologhickness , and pulhickness .p-type or n-type doping with high site selectivity and high stability. This can be achieved by laser-induced doping of the 2D materials. As shown in 2 bilayer exhibits an increased source-drain current as a function of back-gate voltage by four orders [p-type characteristics of the WSe2. As another example, p-n junctions in graphene, which are fabricated via direct-laser-writing-induced spatially modulated doping in graphene covered by a pentacene thin film [p-type graphene to n-type at the laser-irradiated sites, which enabled the creation of p-n junctions on graphene with excellent photoresponses.Many 2D applications in electronic and optoelectronic devices such as photodetectors and FETs require the capability of tuning charge carrier type and density of the 2D materials to achieve r orders . The inchin film . Photoox2 to solve the issue of Schottky contact between the semiconducting 2D channel materials and the metallic electrodes. Briefly, a laser beam is imposed on the surface of mechanically exfoliated flake of multilayer MoTe2 in 2H phase to induce phase transition from semiconducting 2H phase to metallic 1T′ phase in the desired area where metal electrode will be located  shifting plates have been utilized to overcome this limitation; the resolution of optically patterned 2D materials is still of the same order , 92. SpaOther methods such as tip-based scanning probe lithography techniques , 99 and in situ measurement techniques based on optical imaging and spectroscopy can be used as a feedback mechanism to control the working laser parameters, as already used in colloidal manipulation and assembly works [So far, optical patterning technologies have mainly been focused on homogeneous 2D materials. Recent reports show the immense potential of van der Waals heterostructures of 2D materials in energy storage , memory ly works –112. Spaly works , 113 andly works .Optical patterning techniques have played a vital role in the tremendous growth of scientific research and applications based on 2D materials. Such techniques enable various physical and chemical changes induced precisely at the required sites of the 2D materials to create functional nanostructures and devices. With their advantages of tunable laser intensity, tunable laser wavelength, and site specificity, optical patterning holds the key to attain versatile high-throughput fabrication of 2D materials with submicron features. Further studies in this field would enable optimized, on-demand and universal fabrication of heterogenous 2D materials with feature sizes down to atomic scale, marking a significant advance in nanoscience and nanoscale industrial revolution.


Celiac disease (CD) is a common intestinal inflammatory disease involving both a genetic background and environmental triggers. The ingestion of gluten, a proteic component of several cereals, represents the main hexogen factor implied in CD onset that involves concomitant innate and adaptive immune responses to gluten. Immunogenicity of some gluten sequences are strongly enhanced as the consequence of the deamidation of specific glutamine residues by type 2 transglutaminase (TG2), a ubiquitous enzyme whose expression is up-regulated in the intestine of CD patients. A short gluten sequence resistant to intestinal proteases, the α-gliadin peptide 31-43, seems to modulate TG2 function in the gut; on the other hand, the enzyme can affect the biological activity of this peptide. In addition, an intense auto-immune response towards TG2 is a hallmark of CD. Auto-antibodies exert a range of biological effects on several cells, effects that in part overlap with those induced by peptide 31-43. In this review, we delineate a scenario in which TG2, anti-TG2 antibodies and peptide 31-43 closely relate to each other, thus synergistically participating in CD starting and progression. The enzyme type 2 transglutaminase (TG2) plays a key role in the pathogenesis of celiac disease (CD), primarily for its enzymatic activity that transforms common food proteins, i.e., gluten proteins contained in cereals, in unhealthy molecules for genetic predisposed individuals . HoweverCeliac disease (CD) is a complex inflammatory and auto-immune disorder triggered by the ingestion of gluten, a proteic component of several cereals, such as wheat, barley and rice . Gluten Gluten is a heterogeneous mixture of seed-storage proteins present in cereals such as wheat, barley, rye and oats . From a 2+-dependent manner, of an isopeptide bond between lateral chains of glutamine and lysine residues present into proteins. Due to this transamidating activity, TG2 acts as a stabilizing enzyme of protein networks, such as those formed by ECM proteins [KB activation; TG2 may also cross-link beclin-1, thus inhibiting the autophagic process [2+ ions. TG2 is inactive at the very low Ca2+ concentration in the cytosol, but is rapidly activated after perturbation of Ca2+ homeostasis. Actually, the modulation of TG2 activity is very complex and involves conformational changes between an open active conformation, bound to Ca2+, and a closed inactive conformation, bound to GTP, a negative regulator of the transamidating activity [The enzymatic deamidation, which is of crucial importance in enhancing gliadin immunogenicity, is only one of several catalytic activities displayed by TG2. This protein belongs to a wide family of cross-linking enzymes with members in all reigns of the living . Well-knproteins ,31. In t process . In the  process . When av process . In the  process . All theactivity ,36. Moreactivity ,38. TG2 activity , a proteactivity , and an activity . In addiactivity ,42.It has been reported that TG2 expression is increased in the inflamed intestinal mucosa of CD patients ,44,45. I2+ mobilization [An increasing interest on possible biological functions of auto-antibodies to TG2 into the organism has been registered in the last years. Since antibodies are not able to over cross the plasmatic membrane, they have to act on the cell surface, where they may interact with membrane-bound TG2 and eventually they can be internalized together with TG2, during the normal turnover of this protein. Studies on the ability of anti-TG2 antibodies to modulate TG2 transamidating activity have given quite contrasting results, thus it remains unclear whether auto-antibodies interfere in vivo with the repairing function of TG2 in ECM or may block those reactions necessary to the breakdown of tolerance to gluten  . In an elization ,57. In alization ,59. In alization . The genlization . It is alization . AuthorsAmong peptides obtained by the in vitro digestion in a multi-compartment system, P31-43 and the longer peptide 31-49 have been detected . Thus, iFrom biophysical studies investigating the secondary structure of P31-43 emerges that this peptide is able to self-organize in solution as a polyproline II conformation, in equilibrium with β-sheets-like structures ,66. AnotSeveral biological/toxic effects of P31-43 are very rapid and some of them may be induced sudden the peptide comes in contact with cell surface. What happens after this contact and how P31-43 enters cells are still debated issues. Experiments performed to elucidate these topics often compare the behavior of P31-43 and of peptide 57-68 (P57-68), the most immunogenic known peptide. Even if these two peptides are of similar length and composition  they are handled by cells in a very different way. In Caco-2 cells, P31-43 is internalized by endocytosis, mainly clathrin-dependent, as methyl-β-cyclodextrin reduces its entrance. P31-43 uptake is unaffected by filipin, indicating that lipid raft/caveolae-mediated endocytosis is not involved ,70. On tIn this scenario, how can anti-TG2 antibodies reduce P31-43 uptake by cells? Maybe antibodies regulate the endocytosis of TG2 itself and of other TG2 membrane partners; P31-43 could merely have undergone a reduced uptake as a consequence of its interaction with membranes near TG2. Another explanation could be that antibodies trigger a signaling that at the end is able to partially block the uptake of the peptide. Further investigations are needed to clarify these issues. In any case, by reducing the uptake of P31-43, anti-TG2 antibodies also attenuate P31-43-induced effects. This observation suggests that P31-43 needs to be internalized by cells to exert its effects and that a protective role towards negative effects induced by P31-43 could be attributed to the auto-immune response to TG2. However, as described in one of the next paragraph, in CD cells this protective role of auto-antibodies seems to fail.2+ ions from intracellular stores and to elicit an endoplasmic reticulum (ER)-stress response in Caco-2 cells [One of the first demonstrations that P31-43 is the main actor in inducing an innate immune response has been reported in the work of Maiuri et al. in 2003 . They fo-2 cells . Finally-2 cells .It is important to underline that several effects due to P31-43 treatment were observed in enterocytes of duodenal biopsies or other cells from CD subjects but not from control subjects ,81,82. FIt has assumed that TG2 participates to CD onset by enhancing gluten immunogenicity, through the deamidation of specific glutamine residues, and causes the auto-immune response toward itself and also towards other self proteins, through its transamidating activity. A less defined role of TG2 in CD has emerging in correlation with the interplay between TG2 and P31-43. In general, P31-43 seems to modulate TG2 activity and expression, even if with important differences in celiac and non-celiac cells; on the other hand, inhibitors of TG2 activity can attenuate some effects induced by P31-43; finally, in some cases, P31-43 has been described as a TG2 substrate.2+ mobilization from intracellular deposits [Regarding the influence of P31-43 on TG2, it has been demonstrated that in CD normalized duodenal biopsies (but not in non-celiac specimens) P31-43 causes an up-regulation of TG2 activity and expression . A possideposits . Prolongdeposits . A recendeposits . TG2 appdeposits . Moreovedeposits . These dNot only P31-43 upregulates TG2 in CD cells, but also TG2 itself seems to affect the biological activity of P31-43. In works investigating how TG2 modulates P31-43 effects , enzymatic inhibitors of TG2 have often been employed . Competitive aminic TG2 substrates  or non specific inhibitors that binds to the TG2 catalytic site, such as cystamine, have often been used ,91. In aIn CD duodenal biopsies, the use of the TG2 inhibitor R283 reduced the T-cells activation induced by the treatment with P31-43, followed by the sequential treatment with P57-68, whereas the clone CUB 7402 exerted a very low protective effect . Similar2+ ions, was able to activate cell surface TG2, probably present in a closed conformation. As a consequence, the hypothesis of a direct interaction between P31-43 and cell surface TG2 was proposed as a way to induce a conformational change able to activate the enzyme [In recent work, TG2 inhibitors have been used to evaluate whether agglutination of K562 cells induced by PT-gliadin was related to the activity of cell surface TG2 . Authorse enzyme . In the e enzyme , thus suA role for P31-43 and TG2 has also been proposed in the context of neurological manifestations of CD, in particular epilepsy . In miceFinally, in a very recent in vivo model, consisting of transgenic DQ8 mice over-expressing IL15 , an elegThus, the use of TG2 sensible and specific inhibitors represents the best tool to investigate TG2 role in mediating the effects of P31-43, or more in general of PT-gliadin, particularly if these compounds are able to inhibit specifically extracellular or intracellular TG2.2+ intracellular chelator BAPTA-AM [P31-43 appears as a good TG2 substrate in both in vitro and in situ transamidating reactions ,74 even BAPTA-AM . Given t2+ ions from the same deposits engaged by P31-43, i.e., ER and mitochondria, and consequently both antibodies and P31-43 activate the intracellular TG2 [The idea of a connection between anti-TG2 antibodies and P31-43 in CD has an old origin. In their work, Picarelli et al. (1999) demonstrated that the exposition to P31-43 was sufficient to induce an auto-immune response: in duodenal biopsies of patients on a gluten-free diet, challenged with P31-43, an accumulation of anti-endomysial antibodies (EMA) was detected, whereas no EMA were observed in cultures of control specimens . More reular TG2 ,89. Finaular TG2  and by Pular TG2 ,107. Affular TG2 ,63. Whenular TG2 , whereasular TG2 . Howeverular TG2 . For a dWhich are the biological consequences into the cell of the simultaneous presence of anti-TG2 antibodies and P31-43 have still been poorly investigated. When Caco-2 cells were treated with both antibodies to TG2 and P31-43, no additive effect was observed in cell proliferation . HoweverFinally, it has to be considered that anti-TG2 antibodies reach all tissues and organs by blood circulation, thus potentially participating in CD extra-intestinal manifestations ,110. IntIt has been well established that TG2 has two main roles in CD onset. First, TG2 enhances the adaptive immune response to gluten, through the reaction of deamidation of specific glutamine residues. Second, the transamidating activity of TG2 causes the generation of a strong auto-immune response to TG2 itself and to other self proteins. Other emerging roles could be related to the different subcellular distribution of TG2 in cells from CD or control subjects and to the influence that P31-43 could have on TG2 function and viceversa, in the context of CD. In addition, anti-TG2 auto-antibodies have the potential to influence the biology of CD cells by interacting with cell surface TG2. Antibodies seem to act on the same pathways engaged by P31-43, thus, antibodies and P31-43 could produce a synergic negative effect in celiac mucosa. In the future, investigations on CD-derived samples (such as biopsy cultures or isolated cells) with the concomitant presence of anti-TG2 antibodies and P31-43 could contribute to clarifying the role of the interplay between these three important “players”, i.e., TG2, anti-TG2 antibodies and P31-43, in CD trigger and progression.


Bacillus licheniformis. Firstly, our results implied that SAM exogenous addition benefited bacitracin production, which yield was increased by 12.13% under the condition of 40 mg/L SAM addition. Then, SAM synthetases and Methionine (Met) synthetases from B. licheniformis, Corynebacterium glutamicum, and Saccharomyces cerevisiae were screened and overexpressed to improve SAM accumulation, and the combination of SAM synthetase from S. cerevisiae and Met synthetase from B. licheniformis showed the best performance, and 70.12% increase of intracellular SAM concentration (31.54 mg/L) and 13.08% increase of bacitraicn yield (839.54 U/mL) were achieved in resultant strain DW2-KE. Furthermore, Met transporters MetN and MetP were, respectively, identified as Met exporter and importer, and bacitracin yield was further increased by 5.94% to 889.42 U/mL via deleting metN and overexpressing metP in DW2-KE, attaining strain DW2-KENP. Finally, SAM nucleosidase gene mtnN and SAM decarboxylase gene speD were deleted to block SAM degradation pathways, and bacitracin yield of resultant strain DW2-KENPND reached 957.53 U/mL, increased by 28.97% compared to DW2. Collectively, this study demonstrated that SAM supply served as the critical role in bacitracin synthesis, and a promising strain B. licheniformis DW2-KENPND was attained for industrial production of bacitracin.Bacitracin is a broad-spectrum veterinary antibiotic that widely used in the fields of veterinary drug and feed additive. S-Adenosylmethionine (SAM) is a critical factor involved in many biochemical reactions, especially antibiotic production. However, whether SAM affects bacitracin synthesis is still unknown. Here, we want to analyze the relationship between SAM supply and bacitracin synthesis, and then metabolic engineering of SAM synthetic pathway for bacitracin production in  B. licheniformis and Bacillus subtilis. Bacitracin biosynthetase gene cluster in B. licheniformis spans over 45 kd of DNA, which containing four genes, bacT, bacA, bacB and back. Gene bacT encodes thioesterase, and the relationship between bacitracin synthesis with bacT is unclear. BacA is responsible for activating and polymerizing five kinds of amino acids  at bacitracin tail, BacB and BacC are responsible for activating and polymerizing seven kinds of amino acids in heptapeptide loop -Ori was applied for gene deletion, promoter replacement and gene integration in B. licheniformis, pHY300PLK was applied for constructing gene expression vector. All primers used in this research were provided in The strains and plasmids used in this research were listed in mbinants , and Esc4)2SO4, 0.6% CaCO3, natural pH), and then cultivated at 37°C, 230 r/min for 48 h. In order to analyze the function roles of Met transporters MetN and MetP, ME medium  was applied (Luria-Bertani (LB) medium was served as the basic medium for strain cultivation, and corresponding antibiotics  were added when necessary. The seed culture was inoculated in a 250 mL flask containing 20 mL LB medium for 6 h, and transferred (1 mL) into bacitracin production medium -Ori at restriction sites SacI/XbaI, diagnostic PCR and DNA sequence confirmed that gene deletion vector was constructed successfully, named T2-metN. Then, T2-metN was transferred into B. licheniformis DW2 via electroporation, and the positive transformants were cultivated in LB medium with 20 mg/L kanamycin at 45°C, and sub-cultured for three generations. Then, transferred into LB medium and sub-cultured for six generations at 37°C. The gene metN deletion strain was attained via homologous double crossover, diagnostic PCR and DNA sequence confirmed that metN deletion strain was constructed successfully, named as DW2ΔmetN.The method for gene deletion in research , and MetSaccharomyces cerevisiae, gene SAM2 mediated by P43 promoter was integrated into the chromosome of B. licheniformis DW2, following to previously reported protocol , equipped with C18 column (ZORBAX SB-C18). Cell biomass was determined by dilution coating method . SAM conlization .P < 0.05 and P < 0.01, and a t-test was applied to compare the mean values using the software package Statistica 6.0, and bars represented the standard deviations, ∗P < 0.05; and ∗∗P < 0.01 indicated the significance levels between recombinant strains and control , and our results in bacT, bacA, bacB and bacC were measured under optimal condition, and our results implied that SAM addition improved transcription of bacitracin synthetase, increased by 1. 43-, 1. 34-, 1. 31-, and 1.29-fold, respectively . In addition, cell biomasses of these strains showed no significant differences, and bacitracin produced by per cell were 1.95∗10–8 U/CFU, 2.00∗10–8 U/CFU and 2.05 U∗10–8/CFU, 7.97%, 10.46 and 13.43% higher than that of DW2/pHY300 (1.81∗10–8 U/CFU), respectively. Furthermore, SAM2 expression cassette was inserted into chromosome of DW2, attaining SAM2 integrated overexpression strain DW2-K. Based on the results of ∗10–8 U/CFU, increased by 6.30% (1.84∗10–8 U/CFU). The concentration of intracellular SAM (36 h) in DW2-K was 26.32 mg/L, 41.96% higher than that of DW2 (18.54 mg/L). Additionally, transcriptional levels of genes bacT, bacA, bacB and bacC were also increased significantly , C. glutamicum (MetH), S. cerevisiae (Met6) were strengthened in DW2-K, attaining strains DW2-K/pHY-MetHBl, DW2-K/pHY-MetHCg and DW2-K/pHY-Met6, respectively. Our results implied that overexpression of MetH from B. licheniformis benefited bacitracin synthesis, however, strengthening Met synthases from C. glutamicum and S. cerevisiae has no positive effect on bacitracin production. After that, the promoter of metH of DW2-K was replaced by bacitracin synthetase cluster promoter PbacA, a proven strong promoter that has been confirmed in our previous research . The bacitracin produced by per cell was 2.23∗10–8 U/CFU, 21.42% higher than that of DW2 (1.84∗10–8U/CFU).Furthermore, the fermentation curves of Bacillus, and low production efficiency limits its promotion and application. SAM is an important cofactor in cell metabolism, which serves as the critical roles in syntheses of multiple antibiotics (Bacillus, especially for bacitracin, is still unknown. In this study, our results confirmed that strengthening intracellular SAM accumulation was beneficial for bacitracin production, and a metabolic engineered strain B. licheniformis DW2-KENPND with bacitracin yield of 957.53 U/mL was attained, via rewiring SAM synthetic and Met transportation pathways.Bacitracin is a widely used veterinary antibiotic that mainly produced by ibiotics . HoweverB. licheniformis (Streptomyces, moreover, our results implied that strengthening SAM supply increased the transcription of bacitracin synthetase gene cluster, and transcriptional levels of genes bacT, bacA, bacB, and bacC were enhanced all under the condition of SAM addition. In addition, the transcriptional level of regulator gene abrB was decreased by 32.54% (bacTABC (Acting as a critical methyl donor, SAM serves as the important role in the biosynthesis of multiple antibiotics , in addiniformis , differey 32.54% . Since r(bacTABC , our resB. licheniformis, C. glutamicum and S. cerevisiae were tested, and SAM2 from S. cerevisiae showed the best performances on SAM accumulation and bacitracin synthesis, consistent with the previous results of our group (E. coli, Streptomyces coelicolor etc., (B. licheniformis DW2, although it has not been identified or annotated. In addition, SAM decarboxylase gene speD was deleted to block synthetic pathway of byproduct, meantime, based on our results, two other harmful by-products, cadaverine and putrescine (Based on the previous researches, Met yield produced by microbial is generally at a high level , while Sur group . Along wur group , couplinur group , increasur group , etc. Inor etc., , also, ttrescine , were altrescine , and thitrescine . Furthertrescine , the ATPC. glutamicum (Bacillus has not been identified until now, although much work has been done on bacitracin microbial breeding and regulation (Engineering transporter has been proven as an efficiency tactic for enhancement production of metabolite. Overexpression of ABC transporter AvtAB benefited avermectin secretion, and further led to the 50% increase of avermectin production . Lys trautamicum . Howevergulation . Here, tgulation , overexpgulation , indicatgulation , LysE, Lgulation , TcyP angulation . The synB. licheniformis DW2-KENPND was attained via rewiring SAM synthetic and degradation, Met transportation pathways. Based on our results, SAM synthetase SAM2 from S. cerevisiae and Met synthetase MetH from B. licheniformis showed the best performance on SAM accumulation and bacitracin syntheses, and Met transporters MetN and MetP were identified as Met exporter and importer in B. licheniformis DW2, respectively. Finally, The concentration of intracellular SAM of DW2-KENPND was 47.53 U/mL, increased by 1.56-fold compared to DW2, and bacitracin yield reached 957.53 U/mL, increased by 28.97%. Taken together, this research demonstrated that SAM served as the critical role in bacitraicn synthesis, and a promising strain B. licheniformis DW2-KENPND was attained for industrial production of bacitracin.S-Adenosylmethionine served as the critical role in antibiotic production, however, whether it affects bacitracin synthesis is still unknown. Here, our results confirmed that exogenous SAM addition benefited bacitracin production, and a metabolic engineered strain The datasets generated for this study are available on request to the corresponding author.DC and SC designed the study. DC, BZ, JZ, and HX carried out the molecular biology studies and construction of engineering strains. DC, BZ, JZ, and PL carried out the fermentation studies. DC, ZW, JL, ZY, XM, and SC analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.JL was employed by Lifecome Biochemistry Co., Ltd., in China. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.


For some decades, the scientific community has been looking for alternatives to the use of fossil fuels that allow for the planet’s sustainable and environmentally-friendly development. To do this, attempts have been made to mimic some processes that occur in nature, among which the photosystem-II stands out, which allows water splitting operating with different steps to generate oxygen and hydrogen. This research presents promising results using synthetic catalysts, which try to simulate some natural processes, and which are based on Au@ZnO–graphene compounds. These catalysts were prepared by incorporating different amounts of gold nanoparticles  and graphene (1 wt.%) on the surface of synthesized zinc oxide nanowires (ZnO NWs), and zinc oxide nanoparticles (ZnO NPs), along with a commercial form  for comparison purposes. The highest amount of hydrogen (1127 μmol/hg) was reported by ZnO NWs with a gold and graphene loadings of 10 wt.% and 1 wt.%, respectively, under irradiation at 400 nm. Quantities of 759 μmol/hg and 709 μmol/hg were obtained with catalysts based on ZnO NPs and commercial ZnO, respectively. The photocatalytic activity of all composites increased with respect to the bare semiconductors, being 2.5 times higher in ZnO NWs, 8.8 times higher for ZnO NPs, and 7.5 times higher for commercial ZnO. The high photocatalytic activity of the catalysts is attributed, mainly, to the synergism between the different amount of gold and graphene incorporated, and the surface area of the composites. If you were asking to identify one of the many challenges that our world is facing right now, you would probably say climate change, energy production, or sustainability. Most of these problems are directly related to the continuous growth of the world population and the use of fossil fuels as our primary energy source .x) or sulfur oxides (SOx) are release to the atmosphere ..2− ions C groups ,42, and Under irradiation at 320 nm, the hydrogen production of ZnO NWs (365 μmol/hg) decreased when compared to that obtained at 280 nm, but then increased again (427 μmol/hg) at 400 nm. This was not expected either since the wide band gap energy of ZnO (3.37 eV for wurtzite) does not favor the production of hydrogen under visible light. Different studies ,43,44 ha2 [2–/SO32–), and the electrons are excited to the conduction band. Electrons transferred from the conduction band of the semiconductor are injected into the graphene because graphene has a slightly lower redox potential than the semiconductor conduction band [+ ions for H2 evolution [Both ZnO NPs and the commercial ZnO obtained similar results in all the wavelengths that were evaluated. At 500 nm, the hydrogen production of the unmodified catalysts was almost zero, with the exception of ZnO NWs that obtained a high value of 350 μmol/hg, showing high catalytic activity. Incorporation of gold and graphene greatly increased hydrogen production in both the UV and visible regions of all the composites. The presence of co-catalysts such as Au and graphene improve the charge separation and suppresses the recombination of excited photogenerated carriers, resulting in a better evolution of H2 . Differe2 ,46,47 ha2 . This ac2 . Further2 ,48. The ion band ,48. Grapvolution ,48.+ ions to form H2. Thus, the synergetic effect between both co-catalysts, plasmonic Au nanoparticles and graphene, can effectively suppress photogenerated charge recombination, enlarge the active adsorption sites and reaction space, and consequently enhance the photocatalytic activity for H2 evolution [2–graphene composites had significantly increased the visible light absorption and enhanced the photocatalytic H2 production activity compared to the Au@TiO2. Luo et al. [2-P25, the hydrogen production via water splitting increased nine times more than bare TiO2-P25.According to other authors ,49, somevolution ,49. In to et al.  found thIn this research, the highest hydrogen production of the Au@commercial ZnO–graphene catalysts was 709 μmol/hg and was obtained by 10%Au@ZnO commercial graphene under irradiation at 400 nm. This enhancement represents a difference of 615 μmol/hg when compared to the highest amount obtained by the unmodified commercial ZnO catalyst (94 μmol/hg), and the fact that the maximum production of the commercial catalyst was obtained at 400 nm (visible light) is an indication that the Au NPs are allowing the use of visible light ,30,37. OIn the case of Au@ZnO NPs–graphene catalysts, the highest hydrogen production measured was 759 μmol/hg, representing a difference of 673 μmol/hg when compared to the maximum hydrogen production of the unmodified ZnO NPs catalyst (86 μmol/hg), and was obtained with the 10%Au@ZnO NPs–graphene catalyst at 400 nm. The highest hydrogen production for 1%Au@ZnO NPs–graphene, 3%Au@ZnO NPs–graphene, and 5%Au@ZnO NPs–graphene catalysts was 537 μmol/hg, 622 μmol/hg, and 728 μmol/hg, respectively. These results confirm once again that the presence of Au NPs allows the use of visible light to produce hydrogen.Au@ZnO NWs–graphene catalysts showed the highest hydrogen production (1127 μmol/hg) with a gold loading of 10 wt.% at 400 nm, representing a difference of 685 μmol/hg when compared to the unmodified ZnO NWs catalyst (442 μmol/hg). The highest amount of hydrogen produced at 400 nm with the 1%Au@ZnO NWs–graphene, 3%Au@ZnO NWs–graphene, and 5%Au@ZnO NWs–graphene catalysts was 701 μmol/hg, 828 μmol/hg, and 944 μmol/hg, respectively.Under irradiation at 500 nm d, the ma2 g−1 for 10%Au@ commercial ZnO–graphene; 117 m2 g−1 for 10%Au@ZnO NPs–graphene; 247 m2 g−1 for 10%Au@ZnO NWs–graphene). Materials with high surface areas can be attained either by fabricating small particles or clusters where the surface-to-volume ratio of each particle is high, or by creating materials where the void surface area (pores) is high compared to the amount of bulk support material [In all cases, the highest amounts of hydrogen reported in this investigation were obtained with the catalysts with the highest surface area  leads to the generation of electrons in the conduction band, and electron holes in the valence band of the semiconductor [2 conduction band leading to the generation of holes in the Au NPs and electrons in the TiO2 conduction band [2 are injected into the reduced graphene in a graphene/TiO2 system because the graphene/graphene redox potential is slightly lower than the CB of TiO2 [2 likely transfer directly to the Au NPs deposited on the surface of the semiconductor by ohmic interconnection or to carbon atoms on the graphene sheets, and the electrons then react with the adsorbed H+ ions to form H2 [In the case of Au/graphene–TiOting see . One of ting see A, a direonductor . The eleonductor . When irion band ,52. Thenion band . The ele of TiO2 . In addi form H2 ,26. This form H2 ,25,26. W form H2 . The wat form H2 ,19,20 ha form H2 . Thus, t form H2 ,19.2 using catalysts based on ZnO and graphene–gold. The materials studied in this research will have to be contrasted by other researchers to establish a much deeper knowledge that allows us to know the complex mechanism of hydrogen production with ternary compounds based on ZnO.Graphene and different amounts of gold nanoparticles were incorporated on the surface of synthesized ZnO supports , and on the commercial form . These catalysts were fully characterized by different techniques, and their photocatalytic activity was determined by measuring the hydrogen produced by water splitting under UV–vis irradiation.The highest amount of the unmodified ZnO support was 442 μmol/hg and was obtained by the ZnO NWs catalyst under irradiation at 280 nm. This unexpectedly high hydrogen production may be attributed to the morphology (nanowires) and possible defects in the crystalline structure. The maximum hydrogen production for the ZnO NPs and commercial ZnO catalysts was 94 μmol/hg and 86 μmol/hg, respectively, at 280 nm.The maximum hydrogen production obtained with the commercial ZnO composites containing gold and graphene was 709 μmol/hg at 400 nm and was obtained with a gold loading of 10 wt.%. The enhancement in the hydrogen production was 7.5 times higher than that reported by the commercial ZnO.The higher hydrogen production for the Au@ZnO NPs–graphene catalysts was 759 μmol/hg at 400 nm and was obtained with 10%Au@ZnO NPs–graphene. The enhancement in the hydrogen production was 8.8 times higher than that reported by the ZnO NPs catalyst.In the case of the Au@ZnO NWs–graphene composites, the higher hydrogen production (1127 μmol/hg) was obtained with the 10%Au@ZnO NWs–graphene under irradiation at 400 nm.The catalysts did not show a reduction in the surface area nor in the hydrogen production with the increment in gold loadings and incorporation of graphene. These results suggest that the best graphene and gold loading for the Au@ZnO–graphene catalysts could be higher than 1 and 10 wt.%, respectively.


Astragalus root, significantly alleviates IPF by inhibiting transforming growth factor β1 (TGF-β1) induced epithelial-mesenchymal transition (EMT). Further investigations into the influence of ASV on lncRNAs expression will be helpful to delineate the complex regulatory networks underlying the biological function of ASV. Here, we found sirt1 AS expression was significantly decreased in BLM-induced pulmonary fibrosis. We further found that sirt1 AS effectively inhibited TGF-β1-meidated EMT in vitro and alleviated the progression of IPF in vivo. Mechanistically, sirt1 AS was validate to enhance the stability of sirt1 and increased sirt1 expression, thereby to inhibit EMT in IPF. Furthermore, we demonstrated that ASV treatment increased sirt1 AS expression and silencing of sirt1 AS impaired anti-fibrosis effects of ASV on IPF. Collectively, sirt1 AS was critical for ASV-mediated inhibition of IPF progression and targeting of sirt1 AS by ASV could be a potential therapeutic approach for IPF.Long noncoding RNAs sirt1 antisense (sirt1 AS) was reported to play crucial roles in the progression of organ fibrosis. However, the roles of sirt1 AS in idiopathic pulmonary fibrosis (IPF) are still unknown. In addition, we have previously demonstrated that astragaloside IV (ASV), a bioactive saponin extract of the  Idiopathic pulmonary fibrosis (IPF) a progressive interstitial pneumonia with poor prognosis . The patLong noncoding RNAs (lncRNAs) are a large class of noncoding transcripts that are>200 nt long and that lack protein-coding capacity . EmerginAstragalus root, could attenuate IPF by inhibiting TGF-β1-dependent EMT [Sirt1, a member of sirtuins family, is suggested to regulate the balance between myoblast proliferation and differentiation . Overexpdent EMT  and ASV dent EMT . We thusFirstly, we examined the expression levels of sirt1 AS in TGF-β1 treated RLE-6TN cells, with the finding that 10 ng/ml TGF-β1 treatment down-regulated sirt1 AS expression in a time-dependent manner . We therTo validate the effect of sirt1 AS in IPF, mice were administrated with bleomycin (BLM) to induce IPF. The expression of sirt1 AS was also found to be down-regulated in lung tissues from IPF mice, and its expression was significantly up-regulated in IPF mice transfected with Ad-sirt1 AS . We founWe then further explore the mechanism how sirt1 AS regulates the IPF progress. A previously studies found sirt1 could control EMT progress of lung fibrosis , and a rOn the base of above findings, we therefore investigated whether sirt1 AS influenced the EMT progress in a sirt1 dependent way. The results of western blot analysis in We previously reported that ASV has inhibitory effects on TGF-β1-induced EMT in IPF, we then investigate whether sirt1 AS is involved in the function of ASV on IPF. We examined the effects of ASV on sirt1 AS expression and found that 40 μg/ml ASV started to up-regulated sirt1 AS expression and 100 μg/ml ASV influenced sirt1 AS expression at most . ThereafTo validate the implication of sirt1 AS in the anti-fibrosis role of ASV on IPF, a loss-of-function approach was employed. Mice treated with sh-sirt1 AS showed significant decrease in sirt1 AS expression when compared with those treated with sh-Scram . KnockdoIn the present study, we characterized that sirt1 AS inhibited TGF-β1-induecd EMT of alveolar epithelial cells in IPF. sirt1 AS was a negative regulator of EMT and has anti-fibrosis function to alleviate the progression of IPF. The results further revealed that sirt1 AS directly bound sirt1 mRNA to increase the stability of its mRNA, therefore to upregulate sirt1 expression; Sirt1 AS suppressed EMT of alveolar epithelial cells in a sirt1-dependent way. Above data suggested that manipulating sirt1 AS represents promising therapeutic strategies for IPF. Moreover, our results indicated the changes in sirt1 AS expression induced by ASV and sirt1 AS silencing could abolish the protective effects of ASV on IPF. These findings demonstrated a new pathway from ASV to sirt1 AS in regulating EMT for the treatment of IPF.Many studies have demonstrated that antisense lncRNAs participates in the process of organ fibrosis –24. HoweNATs alter the recruitment of members of the spliceosome to sense transcript by forming RNA-RNA duplexes with the sense transcript . It has Astragalus membranaceus and reported to have protective effects on IPF [Astragaloside IV (ASV) is one of the active ingredients of s on IPF , 33. Reps on IPF . ASV alss on IPF . These fs on IPF . And we s on IPF . This stOur study has certain limitations. First, we manipulated the expression of sirt1 AS in mice by intratracheal injection of virus vectors. Gene editing, such as CRISP system. may also be the good approaches. Second, although we identified that sirt1 AS alleviated IPF progression by upregulating sirt1, do other regulators, such as miRNAs, transcription factors and splicing factors, participate in the binding process between sirt1 AS and sirt1 mRNA remain unknown. These questions need to be addressed in future studies. In sum, the present study demonstrated that sirt1 AS attenuated IPF through transcriptionally activating sirt1. In addition, our results showed that ASV/lncRNA sirt1 AS/sirt1/Akt/foxo3 signaling pathway participates in modulating TGE-β1 induced EMT and thus alleviated BLM-induced pulmonary fibrosis, providing a novel molecular basis for the application of ASV in the therapy of IPF.th and pulmonary tissue sections were collected for further studies.C57Bl/6J mice were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. and maintained in a 12-hour light-dark cycle and all experimental process were approved by the Ethics Committee of Affiliated Hospital of Shandong University of Traditional Chinese Medicine. The mice of IPF model was achieved by intratracheally injecting with bleomycin  dissolved in a total of 50 microliter sterile saline; the control group were instead with equal amounts of sterile saline. After the administration of BLM for three days, adenovirus vectors named adenovirus-control (ad-NC), ad-sirt1 AS, specific short hairpin RNA for sirt1 AS (sh-sirt1 AS) or sh-NC were intratracheally injected into the mouse pulmonary tissues, as previously described . ASV  were cultured in F12 medium  with 10% Fetal Bovine Serum , penicillin (100 U/mL) and streptomycin (100 μg/mL) in a humidified incubator at 37 °C with 5 % COescribed , treatme5 RLE-6TN cells were plated in six-well culture dishes prior to small RNA transfection. On the following day, cells were washed with serum-free medium and starved for 6 h. Subsequently, sh-RNA/si-RNA and Lipofectamine 2000 (Invitrogen) were separately mixed with Opti-MEM I Reduced Serum Medium for 5 min and the two mixtures were incubated at room temperature for 20 min. The Lipofectamine sh-RNA/si-RNA mixture was added to the cells and incubated at 37 °C for 6 h and fresh medium containing 10% FBS was added. The cells were maintained in culture until subsequent experiments. For the transfection in vivo, mice were intratracheally delivered with adenovirus control, Ad-sirt1 AS + Ad-si-NC, or Ad- sirt1 AS + Ad-si-sirt1 on the third day after BLM treatment.The adenovirus overexpression sirt1 AS (ad-sirt1 AS) were synthesized by Shanghai Genechem Co.,Ltd. Ad-sirt1 AS was used to infect with RLE-6TN cells as previously described . LentiviThe isolated lung tissues were fixed in 4% paraformaldehyde, decalcified, dehydrated, cleared with dimethylbenzene and embedded in paraffin. The 5 μm sections were stained with either Hematoxylin and Eosin or Masson's trichrome staining using standard procedures. Immunohistochemistry was performed to measure the expression of collagen1 or fibronectin1. Sections were incubated with 3% hydrogen peroxide to block endogenous peroxidase activity. Following incubation with the primary antibody at 4°C overnight, the slides were treated with DAB  to visualize the immunoreactivity. The images were obtained using the Leica DM3000 microscope .After treatment, cells were fixed with 4% formaldehyde for 5 mins. Following incubation with anti-α-SMA  or E-cadherin  antibodies and fluorescent-labeled secondary antibodies (Alexa Fluor® 488 or Alexa Fluor® 594), DAPI (Life Technologies Corporation) was used to stain nuclear counter. Images were captured by a Nikon Eclipse 800 epifluorescence microscope with the appropriate filters.RLE-6TN cells were fixed with 4% paraformaldehyde and then incubated with 0.2mol/L HCl followed by proteinase K treatment. After pre-hybridization, cells were incubated with pre-hybrid buffer with 0.1 μg FITC-labeled sirt1 AS probe at 37 °C overnight and washed with 2 × SSC  and 1 × SSC  in order. Cells were then incubated with FITC-labeled streptavidin (1:400) at room temperature, washed with PBS and DAPI (sigma) was used for nuclear staining.−ΔΔCt method.Real-time quantitative PCR (qPCR) was performed to detect the expression of sirt1 AS, and fibrogenesis-related genes. GAPDH was included as an endogenous control. In briefly, total RNA was extracted using TRIzol (Invitrogen). Concentration of the RNA was assessed with NanoDrop-2000 Spectrophotometer , then reverse transcription using a PrimeScript reagent Kit With gDNA Eraser (Takara). The primers of target genes were synthesized by Shanghai bioengineering Co.Ltd. . QPCR waRLE-6TN cells were treated according to the manufacturer’s instructions (Thermo Fisher Scientific) to obtain separate nuclear and cytoplasm parts. RNA extraction and qPCR were then conducted for nuclear and cytoplasm parts, respectively.For mRNA stability measurements, RLE-6TN cells were treated with 2 μg/ml actinomycin D , which suppresses transcription. Total RNA was extracted at indicated time point  to determine the residual mRNAs by using qPCR analysis. β-actin was served as the internal control.Cultured cells and tissues were lysed with RIPA buffer  with phosphatase inhibitor cocktail  and then protein for further western blot following the protocol as describe previously . The ant5/ml. The lower chamber contained 500 μl of F12 medium containing 10% FBS. After 24 h’ culture, the cells that migrated to the other side of the upper chamber were fixed with 4% paraformaldehyde and subjected to crystal violet staining. The number of transmembrane cells was artificially counted.RLE-6TN cells with a cell density of 5,000 cells/well were seeded in 96-well plates. After incubating for 24 h, the cells were treated with varying interventions and then the cells were incubated with CCK-8 solution  for 2 h. A Microplate Reader  was used to measure the absorbance at 450 nm. After transfection, cells were trypsinized, harvested, and resuspended in serum-free F12 medium and seeded in the upper chamber of a Transwell chamber at a cell density of 1 × 10Data are expressed as the mean ± SD. Student’s t-test or ANOVA was used to compare the means of two or three groups. All statistical analyses were conducted using GraphPad Prism v8 . The significance level was set at P<0.05.Supplementary Tables


One-step microwave heating can simplify the reduction step of graphene oxide and provide a convenient, economical, and effective method of preparing graphene composites. The structural analysis shows that LiFePO4/graphene has high phase purity and crystallinity. The morphological analysis shows that LiFePO4/graphene microspheres and micron blocks are composed of densely aggregated nanoparticles; the nanoparticle size can shorten the diffusion path of lithium ions and thus increase the lithium-ion diffusion rate. Additionally, the graphene sheets can provide a rapid transport path for electrons, thus increasing the electronic conductivity of the material. Furthermore, the nanoparticles being packed into the micron graphene sheets can ensure stability in the electrolyte during charging and discharging. Raman analysis reveals that the graphene has a high degree of graphitization. Electrochemical analysis shows that the LiFePO4/graphene has an excellent capacity, high rate performance, and cycle stability. The discharge capacities are 166.3, 156.1, 143.0, 132.4, and 120.9 mAh g−1 at rates of 0.1, 1, 3, 5, and 10 C, respectively. The superior electrochemical performance can be ascribed to the synergy of the shorter lithium-ion diffusion path achieved by LiFePO4 nanoparticles and the conductive networks of graphene.In this study, micro/nanoscale LiFePO Li/Li+). However, LiFePO4 materials have some drawbacks, such as inferior electronic conductivity (ca.10−9–10−10 Scm−1) as well as slow one-dimensional lithium ion diffusion, which is a formidable obstacle to the high performance of lithium-ion batteries  suspension. The above experimental methods are very complicated, and most of them require long-term high-temperature treatment and atmosphere protection, which lead to high energy consumption and cost. Additionally, the graphene and active materials agglomerate easily and distribute unevenly. Therefore, simplifying the preparation technology and obtaining a product with a small and homogeneous distribution remain great challenges for preparing LiFePO4/graphene composites. Microwave heating is a convenient, economical, and environmentally friendly route for the preparation of graphene composites in a way that addresses the deficiency of graphene modification. Microwave heating can simplify the reduction step of graphene oxide, as, due to the microwave-absorbing properties of graphene oxide, microwave irradiation can restore it into graphene directly without any reductive agent and atmosphere.Small particle size can decrease the migration distance of lithium ions from the interior to the surface and increase the diffusion rate , LiOH·H2O (95%), H3PO4 (85%), ethylene glycol, and sucrose were purchased from Sinopharm Chemical Reagent Co. Ltd. Graphene oxide was synthesized from natural graphite powder (325 mesh) using a modified Hummers' method  with Cu Kα radiation operated at 40 kV and 40 mA. The contents of graphene and carbon in the LiFePO4/graphene and LiFePO4/C composites were calculated from TG-DSC , which was carried out from room temperature to 700°C under an air atmosphere at a rate of 10°C min−1. The morphologies of LiFePO4/graphene composites were observed using a scanning electron microscope  and transmission electron microscopy . The Raman spectra of LiFePO4/graphene and LiFePO4/C composites were recorded from 100 to 3,200 cm−1 on a Renishaw Raman microprobe  using a 514.5 nm argon-ion laser at room temperature.The structures of LiFePO4/graphene and LiFePO4/C composites were evaluated with 2,025 coin-type batteries. The cathode electrodes were prepared by mixing 80 wt% active materials (LiFePO4/graphene or LiFePO4/C) and 10 wt% carbon black (TIMCAL) with 10 wt% polytetrafluoroethylene  in isopropyl alcohol solution . A uniform slurry was formed and pasted onto Al foils, dried at 120°C for 12 h, and then cut into circular electrodes with a diameter of 10 mm. Lithium metal  was used as the anode, Celgard polypropylene (Celgard 2400) as the separator, and 1M LiPF6 dissolved in ethylene carbonate and dimethyl carbonate (with a 1:1 volume ratio) as the electrolyte . The cells were assembled in an argon-filled glove box . Cells were charged and discharged at room temperature using a LAND-CT2001A battery cycler  within the voltage range of 2.7–4.2 V (vs. Li+/Li). Cyclic voltammetry (CV) was performed with an Auto Potentiostat 30 system at a scan rate of 0.1 mVs−1 between 2.5 and 4.2 V. Electrochemical impedance spectroscopy (EIS) profiles were obtained at the same open-circuit voltage by applying a 5-mV amplitude of the AC voltage with the frequency ranging from 100 kHz to 0.01 Hz.The electrochemical behaviors of the LiFePO4/graphene and LiFePO4/C composites are here investigated. XRD patterns of the composites are shown in 4/graphene and LiFePO4/C composites. All the sharp diffraction peaks corresponding to the (200), (101), (210), (011), (111), (211), (301), (311), (121), (410), (221), (401), (112), (222), and (123) planes can be indexed to the orthorhombic olivine-type structure LiFePO4 with the Pnma space group (JCPDS card No. 83-2092)  is observed, proving that the graphene oxide has already been reduced into graphene directly without any reducing agent or atmosphere. This is mainly because the graphene oxide with a large amount of oxygen functional groups on the surface that can absorb microwaves easily, and electromagnetic energy is converted into heat and molecular kinetic energy; the reactive oxygen groups are then exfoliated, and, finally, the graphene oxide is restored into graphene. Also, the introduction of graphene has no effect on the structure of LiFePO4. Moreover, the diffraction pattern of LiFePO4/C shows no diffraction peaks corresponding to residual carbon, indicating that the carbon decomposed from sucrose in the sample exists in an amorphous state.The phase constitution and crystal structure of the synthetic LiFePO4/graphene and LiFePO4/carbon composites, as shown in 4 can be completely oxidized to Li3Fe2(PO4)3 and Fe2O3 under air flow, and the total weight gain is about 5.07% in theory  and symmetric (ν1) stretching of the P–O bonds. The modes at 626 and 587 cm−1 correspond to the symmetric (ν2) and anti-symmetric (ν4) bending of the O-P-O angles. The mode at 395 cm−1 corresponds to the lithium cage and oxygen ion breathing cage. The modes in the 100–300 cm−1 range are induced by translation of Fe and coupled translation and vibration of Fe and −1 and a G band at around 1,590 cm−1  is inversely proportional to the degree of graphitization of carbon materials. The ID/IG in LiFePO4/graphene composites is 1.18, while the ID/IG in LiFePO4/C composites is 1.43. This implies that the graphene has a higher degree of graphitization than the carbon decomposed from sucrose. The higher the degree of graphitization, the better the conductivity of the carbon. A high degree of graphitization is favorable for electron transfer and improves the electrochemical performance of the cathode. Additionally, the strong signals of the graphene (D band and G band) weaken and override the bands of LiFePO4 in the high-frequency region.Raman scattering spectroscopy was employed to recognize the chemical structure of the LiFePO4/graphene are shown in 4/graphene composites are composed of micron-scale spheres and blocks with average dimensions of ~2 μm. In 4/graphene microspheres and micron blocks are composed of densely aggregated nanoparticles. This structure forms because the self-heating effect induced by the microwave heating can greatly shorten the reaction time, and the graphene wrapping the surface of LiFePO4 particles can inhibit the growth of grains. Under the action of graphene, the nanoparticles assembled into microspheres and micron blocks. When the highly conductive electrolyte penetrates into the cathode material, the nanoparticles have a high specific surface area, which increases the contact area with the electrolyte. The nanoparticle size can shorten the diffusion paths of electrons and lithium ions and improve the conductivity of the cathode material significantly. Moreover, the micron structure formed by the aggregation of nanoparticles does not collapse during the process of lithium-ion intercalation and deintercalation, which ensures the stability of cathode material in the electrolyte. TEM and HRTEM images of the micron/nanoscale LiFePO4/graphene composite are shown in 4 particles. 4 are highly graphitic. The highly efficient and stable conducting network can give the material desirable electrochemical properties. In the energy spectrum, elements of P, O, Fe, and C are found, as shown in SEM images of the graphene oxide and LiFePO4/graphene composites is illustrated in The formation process of the LiFePO4/graphene electrode dynamics is shown in − and Li+) from their “reservoirs” toward the LiFePO4 particles  is shown as step C. It can be seen that graphene can provide a high-speed channel for the rapid diffusion of electrons and cause the electrons to reach the reactive site quickly, thus increasing the electronic conductivity of the materials. Meanwhile, the nanoparticles can shorten the transport path of Li+ from the surface to the interior of solid active particles and improve the diffusion coefficient of lithium ions. Moreover, the nanoparticles are surrounded by the micron graphene sheets, and the micron structure guards the stability of the material. Therefore, LiFePO4/graphene composites are expected to have excellent electrochemical performance.A schematic diagram of LiFePO4/graphene and LiFePO4/C cathode materials. 4/graphene and LiFePO4/C composites. In the first scan, there is a pair of redox peaks corresponding to the Fe2+/Fe3+ couple (Ding et al., 4/graphene, the potential difference between the oxidation and reduction peaks decreases from 0.27 to 0.26 V, which means that the phase is stabilized in subsequent cycles. 4/C composites. During the second scan, the potential difference increases from 0.31 to 0.33 V, which proves that detrimental polarization becomes more and more serious. The results show that LiFePO4/graphene composites have very high reversibility and better electrochemical activity.Cyclic voltammetry was performed to investigate the electrochemical kinetics of LiFePO4/C electrode is explained by the electron transfer pathway, as shown in + ions intercalate. In contrast, for LiFePO4/graphene, due to the one-dimensional Li+ ion mobility in the framework, the graphene can ensure that electrons reach particles from all directions and alleviate the polarization, as shown in 4/graphene composites, with well-defined peaks and smaller potential difference, have higher electrochemical reactivity.The polarization of the LiFePO4/graphene and LiFePO4/C at progressively increasing C rates from 0.1 to 10 C are shown in 4/graphene composites, the initial discharge capacity is 166.3 mAhg−1 at 0.1 C, and the discharge capacity decreases to 156.1 mAhg−1 with an increase in the discharge rate to 1 C. At a higher discharge rate of 5 C, the cell delivers a capacity of 132.4 mAhg−1. Even at a 10 C rate, the capacities can reach 120.9 mAhg−1, and a good voltage plateau remains above 3 V. For LiFePO4/C, the discharge capacity is 154.8, 133.8, 121.6, 105.9, and 86.4 mAh g−1 at 0.1, 1, 3, 5, and 10 C rates, respectively. The cycling performances of the LiFePO4/graphene and LiFePO4/C from 0.1 to 10 C are shown in 4/graphene, the specific capacity decreases with increasing current rate, the capacity retention remains very good for all of the different rates; the discharge capacity retentions are, respectively, 99.5, 99.2, 99.4, 99.1, and 97.1% at 0.1, 1, 3, 5, and 10 C current rates after being cycled 10 times. While for LiFePO4/C, the discharge capacity retentions are, respectively, 97.7, 96.9, 93.0, 87.1, and 79.5% at 0.1, 1, 3, 5, and 10 C current rates. All of the results demonstrate that LiFePO4/graphene composites have better rate performance and cycling stability. This can be attributed to the excellent electrical conductivity of graphene, which can improve the conductivity and stability of materials.The charging and discharging capacity profiles of the LiFePO4/graphene and LiFePO4/C cathodes. 4/graphene and LiFePO4/C cathodes. The experimental EIS data is simulated by Zview2.1 software according to the equivalent circuit as shown in + ions at the electrode/electrolyte interface and the charge transfer process. Meanwhile, a low-frequency sloping line is related to the Warburg impedance of the lithium-ion diffusion in the electrode (Zhang et al., RS is the internal resistance of the cell and corresponds to the electrodes, electrolyte, and the separator resistance, Rct is associated with the charge-transfer resistance, CPE is associated with the capacitance contributed by the surface of the active material (Guo et al., Rct value of the LiFePO4/graphene cathode is 79 Ω, which is smaller than the 129 Ω value of the LiFePO4/C cathode. The result shows that graphene can reduce the charge transfer resistance of Li-ion insertion and extraction between the electrode/electrolyte and increase the conductivity of the LiFePO4/graphene cathode.Electrochemical impedance spectroscopy was used to investigate the electrochemical behaviors of LiFePO4/graphene composite was successfully prepared as cathode material through one-step microwave heating. The graphene oxide, which has excellent microwave-absorbing properties, can react with microwaves quickly and be restored into high-quality graphene directly without any reducing agent or atmosphere. The introduction of graphene does not impact the structure of LiFePO4, and LiFePO4 nanoparticles are packed into micron graphene sheets. The graphene network, which has a high degree of graphitization, can provide a high-speed channel for the rapid transfer of electrons and thus increase the electronic conductivity of materials. Meanwhile, the nanoparticles can improve the diffusion coefficient of lithium ions. Moreover, because the nanoparticles are surrounded by the graphene sheets, the micron structure guards the stability of the material. The electrochemical analyses reveal that the LiFePO4/graphene composites have excellent high-rate performance and cycling life. The outstanding electrochemical performance, as well as the fast and efficient method, make this technology commercially viable.A LiFePOThe datasets generated for this study are available on request to the corresponding author.SL, PY, and WS performed the experiments. SL, PY, HL, and XZ performed the data analysis. SL and PY wrote the paper. All authors contributed to the theoretical analysis and the general discussion.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Imaging sequences for detection of meningeal and parenchymal lesions are critical in intracranial pathology. Our study analysed FLAIR MRI sequence for evaluating postcontrast enhancement.  FLAIR imaging sequences have been used in evaluation of enhancement in the brain. We conducted a study of FLAIR imaging sequences to better delineate postcontrast enhancement.  In this prospective hospital-based observational study, postcontrast T1 MTC and delayed postcontrast T2 FLAIR and T1 FLAIR images of 66 patients with intracranial pathology were assessed by experienced radiologists from November 2017 to November 2019.  28 cases of meningeal enhancement were identified in delayed postcontrast T2 FLAIR images. Low-grade gliomas included in the study showed postcontrast enhancement on postcontrast T1 MTC images. Multiple sclerosis lesions were better seen on postcontrast T1 FLAIR. In extraaxial lesions of 11 cases of meningioma, brighter enhancement was seen on delayed postcontrast T2 FLAIR images.  We found that delayed postcontrast T2 FLAIR was better in detection of meningeal enhancement in infectious meningitis and in meningitis carcinomatosis than T1 MTC images. In delayed postcontrast T2 FLAIR images, intra-axial parenchyma lesions appeared more conspicuous or similar to T1 MTC images. Delayed postcontrast T1 FLAIR images provided better anatomic delineation of intra-axial lesions. Some., 5. We cIn this prospective hospital-based observational study, postcontrast T1 MTC and delayed postcontrast T2 FLAIR and T1 FLAIR images of 66 patients—32 patients with meningeal enhancement , 23 parenchymal enhancing lesions (7 patients with infection (5 cases tuberculomas and 2 toxoplasmosis), 2 patients with demyelination (multiple sclerosis), 6 patients with primary tumors (4 low-grade gliomas and 2 high-grade gliomas), and 8 patients with metastatic lesions), and 11 patients with extraaxial lesions(meningiomas)—were assessed by experienced radiologists during a study period from November 2017 to November 2019.The study was approved by the institutional ethics committee. Data were collected from all age groups of patients who were referred to the department of radio diagnosis for MRI brain study with contrast, to rule out meningeal or parenchymal lesions.Data were collected from the patients referred to the department of radio diagnosis, for MR imaging of the brain, in the teaching hospitals attached to Kasturba Medical College, Mangalore. MR examinations were performed on 1.5 Tesla (Magnetom Siemens Avanto).Gadolinium dimeglumine (0.5 mmol/ml) was administered.Sequential order of postcontrast sequences was as follows: T1W postcontrast sequence was run prior to delayed postcontrast FLAIR (fluid attenuation inversion recovery) sequences.  TR:9000  TE:92  TI:2500  Slice thickness: 5 mmImaging parameters for delayed (10 min) postcontrast T2 FLAIR imaging:  TR:2000  TI:860  TE:59  Slice thickness: 5 mmImaging parameters for postcontrast T1W FLAIR:  TR:741  TE:17  Slice thickness: 5 mmImaging parameters for postcontrast T1W MTC images:Patients referred for gadolinium-enhanced brain imaging.Patients with intracranial aneurysm clips.Patients with intraorbital metal fragments, any electrically, magnetically, and mechanically activated implants .Patients having renal failure or raised creatinine (1.6 mg/dl) levels.Out of the 28 cases with clinical diagnosis of leptomeningitis, in all of the 28 cases, meningeal enhancement was easily identified in delayed postcontrast T2 FLAIR images. In comparison, in postcontrast T1 MTC images, differentiation of meningeal enhancement from vascular enhancement was difficult and could have been missed easily. CSF studies were positive in all 28 cases. On the contrary, delayed postcontrast T1 FLAIR images demonstrated minimal meningeal enhancement in 26 cases out of the 28 cases of leptomeningitis, and the enhancement was inferior to T1 MTC images in all of these 28 cases .Out of the 23 cases of intra-axial parenchymal lesions included in the study, in 2 cases, postcontrast T1 FLAIR was not done. In rest of 21 cases, delayed postcontrast T1 FLAIR was done, out of which 13 cases showed brighter and thicker enhancement of solid components and peripheral walls of the lesions. It provided better delineation of lesion. These 13 cases included 4 cases of solid cystic parenchymal mass lesions (2 primary and 2 metastatic), 2 cases of peripherally enhancing primary cystic tumors, 2 cases of cystic metastasis, 4 cases of infection (3 tuberculomas and 1 toxoplasmosis), and 1 case of demyelination (multiple sclerosis). In such cases, characteristics of the wall of peripherally enhancing tumors/infectious lesions were better defined by delayed postcontrast T1 FLAIR images as compared to postcontrast T1 MTC.In three out of four low-grade glioma included in the study, subtle postcontrast enhancement seen on postcontrast T1 MTC images could not be well appreciated on delayed postcontrast T2 FLAIR images due to the intrinsic FLAIR hyperintense nature of the lesions . Subtle Postcontrast enhancement could not be well appreciated on delayed postcontrast T2 FLAIR images in two cases of multiple sclerosis as compared to postcontrast T1 MTC images. In patients with multiple lesions delayed T1 postcontrast FLAIR images picked up almost the same number of lesions as compared to postcontrast T1 MTC images.Out of the 11 cases of meningioma, brighter enhancement was seen on delayed postcontrast T2 FLAIR images as compared to postcontrast T1 MTC images. Dural tail was easier to identify on delayed postcontrast T2 FLAIR images in 8 out of 11 cases due to brighter enhancement as compared to postcontrast T1 MTC images. In one case of meningioma, dural tail was less well appreciated than in postcontrast T1 MTC images. CSF cleft was better appreciated on postcontrast T2 FLAIR images than on postcontrast T1 MTC images in 8 out of 11 cases .Dural tail and extraaxial nature of the lesion was better appreciated on postcontrast T2 FLAIR images than postcontrast T1 MTC images.Our results are similar to a report published by Kremer et al.  which coParmar et al.  in an inStudies done by Splendiani et al. and Artzi et al.  show simWe have found that, in delayed postcontrast T1 and T2 FLAIR images, most of the intra-axial parenchyma lesions appeared more conspicuous or similar to T1 MTC images. Flair images show increased signal intensity because the T1 and T2 effects of various tissues supplement each other.In a study done by Hakyemez et al.  in patieHowever, in our study, we found that delayed postcontrast FLAIR images can be helpful in differentiating between metastatic disease and high-grade glioma as a greater degree of perilesional signal change representing edema is more indicative of metastatic disease, as described in another study .In our study, we found that delayed postcontrast T1 FLAIR images were not sensitive for detection of leptomeningeal enhancement. Any leak of gadolinium into the CSF space may shorten the TI of CSF enough so that the longitudinal magnetization recovers before the null point of CSF on T2 FLAIR images; however, this leak of gadolinium is not enough for the recovery of CSF signal intensity on T1 FLAIR images due to the shorter TI time and more T1 weightage of the images as compared to T2 FLAIR images.Postcontrast T1 FLAIR images provided better anatomic delineation, with a better contrast between the gray and white matter and also between gray matter and the lesion, and T2 FLAIR showed delineation between lesion and CSF due to the darker CSF on T2 FLAIR images.We found that delayed postcontrast T1 FLAIR images provided better anatomic delineation with intense enhancement of high-grade tumors, large metastatic, and infectious ring-enhancing lesions .Thickened walls of chronic lesions have increased vascularity which leads to accumulation of gadolinium within the lesions, and high-grade tumors with increased perfusion show increased gadolinium retention in blood pool or within the tumor. Retention of gadolinium results in decrease of TI in CSF so that it is no longer nulled by the inversion pulse and appears bright. The cystic nonenhancing components within the lesion appear even more hypointense (than on T1 MTC) due to the inversion pulse.Increased contrast between enhancing hyperintense and nonenhancing cystic component of the lesions along with the intrinsic high CNR of lesions on T1 FLAIR images provides excellent delineation of lesions on delayed postcontrast T1 FLAIR images. Our findings were consistent with those of Al-Saeed et al. .The overall image contrast was judged to be superior on T1-weighted FLAIR images compared with T1-weighted FSE images by all neuroradiologists  Figure .Delayed postcontrast T2 and T1 FLAIR images showed better enhancement of meningeal and parenchymal lesions and detected more number of lesions as compared to postcontrast T1 MTC. Delayed postcontrast T2 FLAIR images provided better delineation of extraaxial mass lesions with more conspicuous enhancement and better demarcation of dural tail.Hence, we concluded that delayed postcost contrast T2 FLAIR images should be included in the routine protocol for contrast MRI brain study.On the contrary, postcontrast T1 FLAIR had no added advantage over T1 MTC images in detection of meningeal lesions. Although T1 FLAIR images provided better delineation of the extraaxial mass lesions, the degree of enhancement was either inferior or similar to T1 MTC images.Postcontrast T1 FLAIR provides better enhancement and delineation of intraparenchymal high-grade vascular tumors and large tuberculomas than postcontrast T1 MTC.