Multiphoton absorbing upconversion nanoparticles are emerging as bioimaging materials but are limited by the low quantum yield of their visible fluorescence. This article contains colloids of graphene quantum dots (GQDs), Neodymium, and Cobalt doped Graphene Quantum dots (Co-GQDs and Nd-GQDs) surrounded by carboxylic acids are synthesized which especially are suitable for bio applications; in this way, carboxylic acid groups exchanged by Amoxicillin as an antibiotic with bactericidal activity. The XRD diffraction method, TEM microscope, UV–Vis, and photoluminescence spectroscopies characterize the synthesized materials. The synthesized Quantum dots (QDs) exhibit upconversion properties and their emission is centered at 480 nm, but a red shift was observed with the increase of the excitation wavelength. In the emission spectra of synthesized QDs that can be related to the defect levels introduced by passivation of the QDs in the structure, the results show that with the interaction of the surface QDs with more carboxylic groups, the redshift is not observed. As the results indicate an increase in the intensity of upconversion emission is recorded for Co-GQDs and Nd-GQDs. The absolute quantum efficiency (QY) for Co-GQDs and Nd-GQDs were determined to be 41% and 100% more than GQDs respectively. DFT calculations indicate a strong bond between graphene and cobalt and Neodymium atoms. In doped materials, there are trap levels between the band gap of the GQDs which are responsible for increasing the intensity of the upconversion phenomenon. One of the important features of these nanoparticles is having high fluorescence properties that can be used for biological imaging and optical sensing. GQDs have attracted much attention as promising biomaterials due to their exceptional advantages of low cytotoxicity, excellent solubility, stable photoluminescence (PL), biocompatibility, and low cost59.In recent years, graphene quantum dots (GQDs) have attracted much attention due to their unique structural and optoelectronic properties and their great potential in various applications in drug delivery63. Recently, the rapid development in nanomedicine has opened a new way to find such materials, and nanomaterials have become a very important topic in biomedical applications due to their great potential for the formulation of anticancer drugs. Engineered nanomaterials that have the effect of permeability, biocompatibility, and low toxicity can be used in biomedical applications, including biomedical diagnosis and disease treatment65.Conventional imaging probes, such as fluorescent dyes and fluorescent proteins, have attracted much attention. However, they face poor diagnostic sensitivity, instability, and high toxicity and have no therapeutic effect. It was difficult to find a dual-purpose material that could act as an imaging probe and a drug delivery at the same time. As an effective drug delivery, the fluorescent matrix should meet the following criteria: (1) effective drug delivery, (2) good biocompatibility, and (3) good circulation stability in vivo67. Lanthanide-doped materials are among the most well-known UCNPs in which trivalent lanthanide ions are dispersed as guests in a suitable host network with dimensions less than 100 nm. Lanthanide impurities are photoactive centers that emit light after excitation with low-energy photons. Through a suitable selection of lanthanide dopants, UCNPs can exhibit selective wavelength (color) conversion such as NIR to shorter NIR, visible , or UV68.Up conversions, nanoparticles (UCNPs) have anti-Stokes luminescence that can convert near-infrared (NIR) light into visible light. UCNPs have high chemical stability and can be used as fluorescence probes in a variety of complex organisms and facilitate the bioassay process. In addition, UCNPs can lead to surface conjugation with specific targeting ligands  and can be used as probes to identify and target specific cells with high sensitivity and selectivity73. In addition, as a new generation of imaging agents, UCNPs are widely used in PDT, MRI, X-ray CT imaging, photoacoustic imaging (PA), NIR thermal imaging, phototherapy (PTT), chemotherapy, and radiotherapy. Increasingly studies have focused on their application in PDT because they can act as a photosensitizer in photodynamic therapy79. Also, they can be used as drug carriers for bioimaging therapeutic drugs or genes  or light-sensitive materials. More importantly, UCNP in antibacterial photodynamic therapy (aPDT) has played an essential role in the treatment of bacterial infectious diseases, such as periodontitis and Staphylococcus aureus infections, which are incurable and difficult to eradicate80. The development of nanomaterials that exhibit both good upconversion luminescence (UCL) performance and good performance is undoubtedly a challenge for materials scientists, physicists, and chemists.UCNPs are an improved alternative to traditional optical imaging materials due to several other advantages, such as weak photobleaching, low background fluorescence, deep tissue penetration, and minimal photodamage81. For example, surface modification of UCNPs is important for loading some hydrophobic drugs, and surface charge for the adsorption of small molecules82. However, different needs make different changes, and careful design and optimization of these aspects are necessary before using UCNPs for bioanalytical applications83.Surface modification of nanoparticles is usually necessary to create a suitable surface composition for biomedical applications84, thiols85, carboxylic acids86, aldehydes, and amine groups.Bioconjugation of UCNP materials with biomolecules is very important in therapeutic and diagnostic applications. Recent research has shown that surface-modified ligands on UCNPs can be transformed into a new functional group to influence the subsequent bioconjugation step. Ligands commonly used for coupling are maleimides60. The large surface area of UCNPs can prolong the shelf life of topical drugs and enhance the use of drug targeting in tissues88. Even better, UCNP-based composites used as drug delivery systems allow real-time tracking and evaluation of drug release efficiency90.Compared with traditional medicine, UCNP-based drug delivery has obvious advantages such as small particle size, which facilitates the endocytosis of cells to obtain a good therapeutic effect93.Graphene-based nanomaterials have been designed to help deliver or target drugs more effectively. They are being investigated for therapeutic applications, especially for cancer treatment, as well as for the development of new diagnostics and nanosensors, and are expected to contribute to molecular imaging for diagnosis and treatment, especially in the development of therapeutic strategies in oncologyIn this article, colloidal graphene nanoparticles doped with cobalt and neodymium cations were designed and synthesized with the aim of biological imaging. The surface of nanoparticles is decorated by carboxylic groups that can be bioconjugated with biomolecules. Here our goal is to increase the quantum yield of the upconversion property of the graphene quantum dots by doping cobalt and neodymium ions in their structure. The results show a 41% increase for cobalt and a 100% increase for neodymium-doped graphene in the intensity of the upconversion phenomenon. The mentioned structures were simulated using DFT calculations and their electronic properties were obtained.All chemicals purchased from Merck Company and used as received.−1 NaOH solution, under vigorous stirring. After neutralizing to pH 7.0 with NaOH, the aqueous solution of GQDs was obtained94. For synthesis, Nd-doped Graphene cobalt acetate was substituted by Neodymium acetate. The obtained synthesized materials were centrifuged and washed with water for 1 h at 12,000 rpm and redispersed in deionized water.The GQDs and Co-doped GQDs were prepared by the thermal decomposition of citric acid (CA). In a typical procedure of GQDs preparation, 2 g CA and, 0.1 g of the cobalt acetate was put into a 50 mL beaker and heated to 200 °C using a heating mantle. About 5 min later, the CA was liquated. Subsequently, the color of the liquid was changed from colorless to pale yellow, and then orange in 30 min, implying the formation of GQDs. The obtained orange liquid for preparing GQDs was added drop by drop into 100 mL of 10 mg mL0.5 g of Amoxicillin was solved in 10 CC of deionized water and dropwise added on 20 CC of the synthesized colloids and stirred for 2 h. The obtained exchanged materials were centrifuged and washed with water for 1 h at 12,000 rpm and redispersed in deionized water..The size and shape of synthesized nanoparticles were measured at ×200–×300 magnifications using TEM microscopy imaged by a Zeiss-EM10C TEM microscope at 80 kV accelerating voltage and ~ 2 nA beam current. The EDX analysis was employed to identify the elemental composition of the nanoparticles. UV–Vis absorption spectra were recorded employing a PG Instruments Ltd T70 UV/Vis spectrophotometer. PL spectra were taken by a Perkin Elmer LS-45 spectrophotometer equipped with a 500 W Xenon lamp. For lower powers, we used an optical filter in front of the instrument’s source. ICP analysis was recorded by ICP-OES SPECTRO ARCOS. Powder X-ray diffraction patterns were recorded by XRD Bruker D8 advance2O molecules. B3LYP/6-311G  basis sets and LDA approximation were used in the geometry optimizations. In the CASTEP, calculation, the number of plane waves included in the basis was determined from cut-off energy (Ec) of 500.0 eV. The summation over the Brillouin zone was carried out with k-point sampling using a Monkhorst–Pack grid. Geometry optimization under applied hydrostatic pressure with parameters of 2 × 2 × 5 was used to determine the modulus of a material (B) and its pressure derivative, Bʹ = dB/dP95.The Gaussian 03 package and materials studio CASTEP model was applied for DFT calculation. The electronic structures were established based on Graphene nanoclusters containing 54 carbon atoms passivized by carboxylic groups and bonded with Cobalt acetate surrounded by HFigure X-ray diffraction shows that no peak related to cobalt and Nd ions can be seen and only there are small shifts in the appeared peaks in the related patterns, which indicates the doping of ions in the structure of the graphene quantum dot. The peaks appeared at 2θ = 24° along the (002) orientation and 2θ = 43° according to (102) orientation indicating synthesizing GQDs  with δE = 1.1 eV. All these changes come from the surface passivation of nanoparticles (in our case citrate ligands) that strongly influence the optical properties of the GQDs and trap states created by surface defects of the structure.To further investigate the optical properties of the synthesized GQDs, a detailed PL study was performed using different excitation wavelengths. In general, the PL spectrum of graphene nanoparticles depends on the excitation wavelength. In other words, the PL peaks shift to longer wavelengths of maximum intensity because the excitation wavelength is a bathochromic shiftThe photoluminescence emission spectrum of the synthesized Co-GQDs and GQDs at 350 and 400 nm excitation wavelengths are presented in Fig. As the figure shows, the intensity of photoluminescence in Graphene is relatively higher than Co-GQDs because of inserted trap levels by doping Co atoms (it will be described more in the mechanism section).To prove and correct this phenomenon, the surface of nanoparticles was modified with excess citric acid . As Fig. 4G7/2 + 2K13/2 and 2G7/2 + 4G5/2 to 4I9/2 respectively98. As the figure shows an increase in the intensity of PL spectra is recorded for Co-GQDs and the absolute quantum efficiency (QY) for Co-GQDs, and Nd-GQDs were determined to be respectively 41% and 100% more than Graphene QDs. As Fig. In addition to the PL features, significant upconversion is observed for the synthesized Co-GQDs and Nd-GQDs. Figure The upconversion effect of nanoparticles was examined by different power sources Fig. . The obtFigure d orbitals of Cobalt ions and f orbitals related to Neodymium, allowing the absorption of photons with lower energy. It means that absorbing multi-photons with lower energy can excite electrons in multi-steps, which increases the intensity of upconversion emission in cobalt-doped graphene. These trap levels can also reduce the intensity of photoluminescence emission (excitation wavelengths: 350 and 400 nm) in doped quantum dots and increase non-radiative transitions in this structure.A simplified energy diagram of optical transition states for cobalt and Neodymium doped graphene structure to illustrate the upconversion mechanism is shown in Scheme d,p) theoretical level. HOMO/SOMO/LUMO orbitals of the Cobalt doped graphene sheet, show highly symmetric configurations indicating the homogeneity of electronic distribution through these orbitals either in the ground states on carbon atoms of the graphene sheet. However, they present asymmetric distribution for SOMO and LUMO, and orbitals are nearly concentrated around the Graphene's added Cobalt atoms and carboxylic acid groups.Figure Calculated band structures for GQD, Co-GQD, and Nd-GQD are presented in Fig. The density of states (DOS) in Fig. p orbitals of C and O atoms are attributed to the electronic transitions of GQDs.As described above, there are carboxylic groups around Graphene QDs and we calculated the partial density of states for C and O atoms. As Fig. p orbitals of the C and O atoms also, the p and d orbitals of the Co atoms have the most contribution to electronic transitions of Co-GQD. For Nd-GQD (Fig. p orbitals of C and O atoms also f orbitals of Nd atoms have a high contribution to the electronic transitions.Figure GQD Fig. , the p oIn this article, the colloids of Graphene, Graphene-cobalt, and Graphene-Neodymium quantum dots decorated by carboxylic groups were synthesized for bio-imaging purposes and their physical properties were characterized. X-ray diffraction results show that cobalt and Neodymium were doped in the graphene structure and DFT calculations confirm that a strong bond was formed between doped atoms and graphene passivized by citrate. Also, the PL spectra indicate the existence of trap levels between HOMO and LUMO levels of Graphene QDs created by doped atoms in the structure. In this condition, multi-photons with lower energies can excite the electrons and the upconversion transitions can be done with higher intensity. As the results show with doping cobalt and neodymium atoms in the structure of graphene, upconversion intensity can be increased to 41% and 100% more than un-doped graphene. By exchanging the carboxylic groups on the surface of synthesized nanoparticles with Amoxicillin as an antibiotic molecule the upconversion property remains and therefore they can use as drug delivery materials in bio-applications.


Caenorhabditis eleganscan be used to phenocopy ALSin vivo. Since disrupted locomotion is a strong readout of toxicity, we examined multiple motor phenotypes of aC. elegansmodel expressing human wild-typeTDP-43(hTDP-43) pan-neuronally. Our data reveal that impaired locomotion includes more than the common deficits in crawling capacity and the presence of early-onset paralysis. We show that reduced thrashing, abnormal coiling, and decreased pharyngeal pumping are also observed, in a temperature-dependent fashion.Transactive response DNA binding-protein 43 (TDP-43) is a conserved RNA/DNA-binding protein with a role in RNA metabolism and homeostasis. Aberrant TDP-43 functioning has been considered a major culprit in amyotrophic lateral sclerosis (ALS). Caenorhabditis elegansmodel expressing humanTDP-43pan-neuronally (hTDP-43 worms)  we investigated the functional consequences of these molecular features by assessing global motor function . Movement phenotypes like crawling and thrashing have proven to be auxiliary in studies concerning the pathology of muscles and neuronsC. elegans . In addition, rhythmic movements that are dependent on interactions between the nervous system and muscles can also be studied by assessing the pharyngeal pumping rateC. elegans After observing TDP-43 pathology in aFigure 2B-D). The slow movement of hTDP-43 worms was accompanied by a distinctive ‘uncoordinated’ phenotype as previously describedFigure 2E-G). Here, impaired ‘thrashing’ coincided with an ‘coiler’ phenotype , a posture that is often observed in mutants with abnormal cholinergic signalingFigure 2I). Impaired locomotion early in life progressed to complete paralysis at adulthood day 9 (20 °C) or day 6 (25 °C) . Noteworthy, the mechanical stimuli provided to assess paralysis revealed a clear ‘shrinker’ phenotype characteristic for GABAergic dysfunctionFigure 2C-I).Using automated tracking we found that the crawling capacity of hTDP-43 animals, expressed as average and maximal translocation speed, was impeded  with the WF-NTP platform. Determination of crawling speed and generation of crawling maps was performed using the WF-NTP softwareParalysisOP50at 20 °C. At day 1 of adulthood, 50 worms per condition (unless stated differently) were transferred to 6-cm NGM plates containing FUdR (10 worms per plate). Plates were kept at 20 °C or 25 °C, as described in the figures. Animals were tested for paralysis every day by tapping their nose/tail with a platinum wire as described previouslySynchronized worms were cultured on standard NGM plates seeded withPharyngeal pumpingWorms were freely moving on freshly seeded NGM plates and were examined under an optical microscope to determine pumping rates by visual observations. One pharyngeal pump was defined as a complete forward and backward movement of the grinder in the pharynxTable 1: Strains used


In this paper, we present a study of the sub-monolayer gold intercalation of graphene on Ir(111) using scanning tunnelling microscopy (STM). We found that Au islands grow following different kinetics than growth on Ir(111) without graphene. Graphene appears to increase the mobility of Au atoms by shifting the growth kinetics of Au islands from dendritic to a more compact shape. Graphene on top of intercalated gold exhibits a moiré superstructure, with parameters significantly different from graphene on Au(111) but almost identical to graphene on Ir(111). The intercalated Au monolayer shows a quasi-herringbone reconstruction with similar structural parameters as on Au(111). Whig metals . In ordeg metals  is used g metals , Cu [13,g metals , and alkg metals ,16, at td Ni bands is entirely recovered upon gold intercalation [11 holes cm−2), which is lower only for graphene on Ir(111). Such a charge transfer leads to a shift of the Dirac point of 0.24 eV above the Fermi level [Gold surfaces are known for their weak interaction with graphene , but alscalation . Gold hacalation ,23. The mi level ,18,23. IGraphene intercalated by gold on Ir(111) has been studied only for thick gold films which exhibit moiré super-structures with a periodicity of 17.0 Å due to the 14.7% lattice mismatch between graphene and Au(111) .In this paper, we present an STM study of graphene on Ir(111) intercalated by a sub-monolayer coverage of gold. We studied the structure of the intercalated gold in terms of the possible influence of graphene on its interaction with the underlying iridium surface.−10 mbar. The setup was used for the characterization of a clean, graphene-covered Ir(111) surface and Au intercalated graphene on Ir(111). The STM measurements were performed on Aarhus STM (SPECS) at room temperature (R.T.). The STM images were recorded while scanning the surface by an electrochemically etched tungsten tip, in constant current mode, with a bias voltage applied to the sample. The STM was calibrated by measurements on the HOPG sample and the STM images were processed with WSxM software 5.0. The Ir(111) sample was a crystal 6 mm in diameter, of 99.99% purity, polished with roughness <0.03 μm and with an orientation accuracy <0.1°. The sample was heated by e-beam heating using a hot filament at a negative potential near the grounded sample. The sample temperature was calibrated by the C-type thermocouple attached to the sample with respect to the heating time and power used and, additionally, values were measured by a K type thermocouple attached to the sample plate. The C-type thermocouple was then removed from the sample to enable multiple graphene preparations and transfer from the sample preparation manipulator to STM.The STM and low energy electron diffraction (LEED) experiments were performed in an ultra-high vacuum (UHV) setup operating at a base pressure of 5 × 10−7 mbar at 1100 K, and a final 5–15 min annealing in UHV at 1150 K. After five such cycles, typically a sharp LEED pattern and large-scale STM images confirmed a clean surface.The Ir(111) sample was cleaned by using cycles consisting of 50 min of 1 keV argon-ion sputtering followed by 10–25 min of annealing in oxygen partial pressure of 1 × 10−8 mbar for 60 s and flash to 1400 K) followed by chemical vapour deposition . This TPG + CVD procedure growth led to a uniform orientation of graphene (referred to as Gr/Ir(111)) with the lattice aligned to the substrate lattice (R0) and at full monolayer coverage. Gold was deposited by resistive heating of a tungsten basket filled with pure gold (purity: 99.999%) heated by the direct current through the W wires, while the sample was kept at the R.T. The intercalation was carried on immediately after Au deposition by sample post-annealing at 800–900 K.The graphene monolayer on Ir(111) was prepared by a temperature programmed growth cycle  before and after Au intercalation by means of LEED. As can be evidenced from the inset in The intercalation of graphene by Au produced a visible impact on the STM images of the Gr/Ir(111) system . They exThe bright areas with enhanced contrast and clear hexagonal structure correspond to the moiré superstructure of non-intercalated graphene. The area characterised by weak moiré modulation is associated with the intercalated graphene regions. From A comparison of the images in We measured the moiré periodicity across the area with intercalated and non-intercalated graphene see . The linSTM images, given in The fact that the gold monolayer between gr and Ir(111) exhibits the same reconstruction with similar structural parameters as the Au(111) surface layer indicates that the structure of the gold overlayer is dominated by the inter-atomic interaction over the adsorbate–substrate one. However, the herringbone pattern is not registered in the Au monolayer on Ir(111), suggesting a noticeable influence of graphene on the observed reconstruction. On the other hand, graphene on Au(111) had no impact on the reconstruction of the surface layer . Having Regarding the metals weakly interacting with graphene, intercalation can change the growth mode, e.g., from cluster to smooth films (ref. ). HoweveTo correctly interpret the emergence of the quasi-herringbone pattern in sub-monolayer Au intercalated between graphene and Ir(111), a theoretical investigation of electron transfer and the potential energy landscape of the system is required.Finally, The graphene grows on Ir(111) in a continuous layer over macroscopic distances and wrinkles, together with multi-atom vacancies are regarded as preferential penetration channels for graphene intercalants which interact weakly with graphene ,38,39,40In conclusion, we have studied the sub-monolayer gold intercalation of graphene on Ir(111). We found that Au islands grow following different kinetics to the growth on Ir(111) without graphene. It appears as if graphene increases the mobility of Au atoms, shifting the growth kinetics of Au islands from dendritic towards a more compact shape. Graphene on top of intercalated gold shows a moiré superstructure with parameters significantly different to graphene on Au(111), but almost identical to graphene on Ir(111). The intercalated Au monolayer exhibits well-known herringbone reconstruction, with similar structural parameters as on Au(111). The explanation of the emergence of this reconstruction requires a theoretical investigation of electron transfer and the potential energy landscape of the system, and is beyond the scope of the present work. Nevertheless, the observed effects can provoke interest in further theoretical calculations, e.g., the DFT of the intercalated gr/Au/Ir(111).


Sarcoidosis is a systemic inflammatory disease of unknown etiology, which mainly affects the lungs and lymph nodes, as well as extrapulmonary organs. Its incidence, and prevalence rate, and disease course largely vary with regions and populations globally. The clinical manifestations of sarcoidosis depend on the affected organs and the degree of severity, and the diagnosis is mainly based on serum biomarkers, radiographic, magnetic resonance, or positron emission tomography imaging, and pathological biopsy. Noncaseating granulomas composing T cells, macrophages, epithelioid cells, and giant cells, were observed in a pathological biopsy, which was the characteristic pathological manifestation of sarcoidosis. Angiotensin-converting enzyme (ACE) was first found in the renin–angiotensin–aldosterone system. Its main function is to convert angiotensin I (Ang I) into Ang II, which plays an important role in regulating blood pressure. Also, an ACE insertion/deletion polymorphism exists in the human genome, which is involved in the occurrence and development of many diseases, including hypertension, heart failure, and sarcoidosis. The serum ACE level, most commonly used as a biomarker in diagnosing sarcoidosis, in patients with sarcoidosis increases. because of epithelioid cells and giant cells of sarcoid granuloma expressing ACE. Thus, it serves as the most commonly used biomarker in the diagnosis of sarcoidosis and also aids in analyzing its therapeutic effect and prognosis in patients with sarcoidosis. Sarcoidosis is a multisystem inflammatory disorder disease, mainly affecting the lungs and lymph nodes, as well as extrapulmonary organs, such as the skin, heart, eye, and nervous system , 2. It mDiagnosing sarcoidosis is difficult due to its slow onset and atypical clinical manifestations, which is mainly based on serological, imaging and pathological biopsies, including noncaseating granulomas and excluding nodular reactions to other granulomatous diseases such as tuberculosis, lymphoma, cancer, and other idiopathic granulomas. The most commonly used serum biomarker in sarcoidosis is ACE, with high specificity . In addiThe level of serum biomarkers and corresponding imaging examination are different with the different organs involved in sarcoidosis. Furthermore, the clinical diagnosis of sarcoidosis must be confirmed by histological biopsy, that is, non-caseous nodular granuloma composed of T cells, macrophages, epithelioid cells and giant cells. However, the diagnosis of granuloma is insufficient without consistent radiological findings . As the ACE  was first found in the renin–angiotensin–aldosterone system (RAAS), which was earlier known as hypertension invertase , 17. NorHumans have two forms of ACE: somatic ACE (sACE) and testis ACE . sACE is composed of two domains: C-terminal and N-terminal, while tACE only has C-terminal . sACE exACE is encoded by a single gene located on chromosome 17q23, comprising 26 exons and 25 introns, with a length of 21 kb . sACE, wSarcoidosis is also a polygenic disease without specific causative pathogenic genes, and its pathogenesis is results from granulomatous inflammation triggered by the interaction between genes and the environment . The epiThe serum ACE activity is affected by the site of sarcoidosis lesions and the ACE gene I/D polymorphism. Significant differences in the serum ACE level may be found among different ethnic groups, and the normal range of serum ACE is defined according to different genotypes regardless of their level of involvement in human organs , 41. ACECorticosteroid prednisone, immunosuppressant MTX or azathioprine, or biological agent infliximab should be given when sarcoidosis has obvious pulmonary symptoms or affects extrapulmonary organs, including heart, eye, skin, and nervous system . After tAlthough sarcoidosis can involve multiple organs, the most frequently involved are the lungs and lymph nodes. Sarcoidosis can be divided into five stages based on the chest X-rays: radiograph showing the normal chest (stage 0), nodal enlargement only (stage I), nodal enlargement and parenchymal opacity (stage II), parenchymal opacity without adenopathy or evidence of fibrosis (stage III), and lung fibrosis (stage IV). Radiographs of about 5%–15% of the patients showed stage 0, 45%–65% stage I, 30%–40% stage II,10%–15% stage III, and 5% stage IV . The natSkin is the second most common organ affected in sarcoidosis impacting about one-third of patients. This isolated cutaneous sarcoidosis has multiple erythemas, plaques, or subcutaneous nodules as the main manifestations. The histological examination can result in the absence of characteristic nodular granulomas, which refers to non-specific cutaneous sarcoidosis. Nodular erythema is the most common nonspecific lesion, occurring in up to 25% of patients . The treClinically, about 3%–39% of patients with systemic sarcoidosis have cardiac involvement, while the incidence of isolated CS in patients with CS is about 27%–54% . About 567Ga) scanning was positive, the sensitivity of serum ACE increased to 100%, and the specificity was 73% (Ocular sarcoidosis (OS) is reported in about 25%–50% of patients with systemic sarcoidosis. It is mainly characterized by anterior uveitis involving young patients or total uveitis in middle-aged and elderly patients . The sen was 73% . The ser was 73% . About 8 was 73% . The cli was 73% . In pati was 73% .The serum ACE level increased in patients with sarcoidosis, dropped to normal with improvement in condition during treatment, and increased again after the recurrence of sarcoidosis. Serum ACE level is related to the systemic lesions of sarcoidosis, which is lower in patients with isolated sarcoidosis than in those with non-isolated sarcoidosis. In addition, the level of serum ACE is also affected by ACE gene I/D polymorphism, so clinically, ACE genotypes should be detected first for patients with elevated serum ACE level, and the relationship between elevated serum ACE level and sarcoidosis can be determined according to the ACE genotypes. Most laboratories detect the level of serum ACE, which also increased in other diseases such as tuberculosis and tumor, besides sarcoidosis. The serum ACE level was also affected by ACE gene I/D polymorphism and use of drugs such as ACEIs, while the level of ACE protein did not change. We should take ACE protein level as a serological marker for the diagnosis of sarcoidosis, which has more clinical significance. At the same time, we should clarify the changes of ACE protein level in different types and different stages of sarcoidosis to help us diagnose sarcoidosis and understand its pathogenesis better. Therefore, re-evaluating the ACE level in sarcoidosis enhances the understanding of sarcoidosis in clinic.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.


Despite the potential of graphene for building a variety of quantum photonic devices, its centrosymmetric nature forbids the observation of second harmonic generation (SHG) for developing second-order nonlinear devices. To activate SHG in graphene, extensive research efforts have been directed towards disrupting graphene’s inversion symmetry using external stimuli like electric fields. However, these methods fail to engineer graphene’s lattice symmetry, which is the root cause of the forbidden SHG. Here, we harness strain engineering to directly manipulate graphene’s lattice arrangement and induce sublattice polarization to activate SHG. Surprisingly, the SHG signal is boosted 50-fold at low temperatures, which can be explained by resonant transitions between strain-induced pseudo-Landau levels. The second-order susceptibility of strained graphene is found to be larger than that of hexagonal boron nitride with intrinsic broken inversion symmetry. Our demonstration of strong SHG in strained graphene offers promising possibilities for developing high-efficiency nonlinear devices for integrated quantum circuits. Nanostructured platforms for efficient nonlinear optics are the building block of next generation integrated photonic devices. Here the authors provide a straightforward method to engineer the lattice symmetry of monolayer graphene, boosting the SHG signal at low temperature. Among many nonlinear processes, second-order χ(2) nonlinear processes such as second-harmonic generation (SHG)4 and spontaneous parametric down-conversion (SPDC)7 are most frequently used in many applications owing the high χ(2) susceptibility. The χ(2) processes are only possible in materials with broken inversion symmetry. Some of these materials, such as beta barium borate (BBO)8, are extensively used in various applications as they are commercially available. However, these traditional materials have crucial limitations, including low nonlinear susceptibility and phase-matching issues9. Most importantly, those materials only exist in the form of bulk crystals, thus prohibiting them from being scaled down10. Towards the realization of miniaturized, integrated nonlinear photonic devices, the search for materials that can exist in nanometer dimensions with uncompromised nonlinear properties has become an important research topic.Nonlinear optics is a branch of modern optics with many essential applications ranging from material analysis to quantum computing2)11 and hexagonal boron nitride (hBN)12 intrinsically possess broken inversion symmetry. In addition to their strong χ(2) responses, the atomically thin nature of these 2D materials allows overcoming the phase-matching restriction, which has otherwise been a major challenge in developing nonlinear optical devices. Among all the 2D materials, graphene is the most researched material and has thus far attained the greatest amount of attention owing to its superior mechanical13, electrical14, and optical properties15. For example, graphene’s excellent carrier mobility and universal absorption make it an unparalleled material for many optoelectronic applications19. Over the past decade, there has been a vast amount of research works on nonlinear optical responses in graphene23, including saturable absorption20, four-wave mixing22, and third harmonic generation (THG)23. Unfortunately, however, graphene does not allow any intrinsic χ(2) processes because of its preserved inversion symmetry. To break the inversion symmetry in graphene, researchers have recently devised various methods such as applying external electrical bias27, inducing in-plane optical fields28, and exploiting interlayer interaction in multilayer graphene30. However, all these methods do not provide any insight into breaking the lattice inversion symmetry within one layer of graphene, which is crucial in understanding how graphene’s lattice can be further engineered at the most fundamental level. Furthermore, the second-order response created by the in-plane photon momentum is considered weak since the SHG intensity is only proportional to the photon momentum31. Lastly, the requirements of supplying continuous electrical energy and using oblique incidence of optical pumping27 makes these methods unsuitable for making integrated graphene devices.Recently, atomically thin two-dimensional (2D) materials have shown great promise since many of them such as tungsten diselenide (WSeχ(2) response when the temperature of the graphene is reduced to 4 K, which can be explained by resonantly enhanced optical transitions between pseudo-Landau levels (pLLs). Moreover, the experimental benchmarking of our strained graphene against a well-investigated monolayer hBN with an intrinsically broken inversion symmetry allows us to conclude that our strained graphene possesses a 30% larger second-order nonlinearity than hBN. As a result, our approach utilizing unique non-uniform strain and sublattice polarization presents new possibilities for fashioning the χ(2) responses in graphene by fundamentally engineering the material’s symmetry properties. Additionally, our work provides a very convenient, stable, and scalable way to generate the second-order nonlinearity for integrated graphene devices, which holds the promise of practical optoelectronic applications. We believe that this work opens new opportunities for graphene as a promising material for the on-chip nonlinear optoelectronic devices.Herein, we report an experimental observation of a new type of SHG from monolayer graphene deposited on our unique strain-engineering platform. The non-uniform strain in graphene induces a strong polarization between graphene’s two sublattices that are originally balanced. This polarization breaks the sublattice symmetry in graphene, thus enabling a strong second-order response in graphene. The experimentally observed SHG in strained graphene exhibits unconventional features including a significant temperature dependence that is not previously reported graphene. In particular, we observe a nearly 50-time enhancement in graphene’s 33. The two sublattices possess an equal local density of states (LDOS)33. Therefore, the sublattice polarization becomes zero and the sublattice symmetry is preserved. In the strained region, contrastingly, a strong non-uniform strain at the edge of the nanopillar can significantly deform the carbon-carbon bonds and enables one sublattice (dark green dots) to obtain a significantly higher LDOS than the other (light yellow dots), as illustrated in the right ellipse35. This imbalance of the LDOS between the two sublattices results in a non-zero sublattice polarization that breaks the sublattice symmetry. While the existence of this sublattice polarization in graphene has previously been confirmed by scanning tunneling microscopy (STM)35, the influence of which on the optical properties of graphene has not been experimentally studied yet. Here, we exploit this sublattice polarization to observe a strong and unconventional SHG (green beam) upon the excitation of femtosecond laser pulses (red beam) .Figure Figure − and 2D+. We observe a Raman peak shift of 82.6 cm−1 between the 2D peak of unstrained graphene and the 2D− peak of the deformed graphene, yielding a strain of ~1.26%36 . By considering the theoretical lattice deformation38, we calculate the maximum local atomic strain of ~2.4%, which is larger than the measured value of ~1.26%. The discrepancy is ascribed to the limited spatial resolution of the Raman spectroscopy due to a large laser spot size compared to the sharp edges of our graphene nanopillars39.To quantitatively obtain the amount of the induced strain in graphene, we performed Raman spectroscopy in strained graphene on the nanopillar  measurements, we confirmed that the graphene layer used in our study is close to intrinsic  that can generate discrete pLLs44. Here, we develop a new theoretical framework that provides a relationship between non-uniform strain, sublattice polarization, and PMF (See Supplementary Note P to be:a0 (~0.14 nm) is the bond length of pristine graphene, dn is the bond length along the direction δn after strain. δn refers to the three nearest neighbor vectors of a carbon atom  is a constant and ∇ · P is the divergence of P. Figure P and Bps, respectively. P 48 and graphene-based heterostructures31 was theoretically predicted to activate a strong and resonant SHG when the SHG energy matches with inter-Landau level separation. Here, we theoretically extend this phenomenon of the resonant SHG to the case of strained graphene possessing a strong PMF. We show that the resonant SHG via inter pLLs in our strained graphene sample has a strong temperature dependence. First, by using rigorous tight-binding simulations, we calculate the distribution of density of states (DOS) in pristine and strained graphene  because of the sublattice polarization-induced χ(2) nonlinearity. Also, the magnitude of SHG can be strong owing to the resonant optical transitions between pLLs. It should be noted that in pseudo-Landau-quantized graphene, the resonant SHG can be considered as two successive one-photon transitions. Each of these transitions must obey the optical selection rule of li and lf are the pLL index of the initial and final states respectively. As shown in Fig. The PMF generated at the edges of the nanopillar creates discrete pLLs in graphene, which can strongly modify the electrical and optical properties of grapheneDOS Fig. 37. In thχ(2) strength in strained graphene based on a well-reported method of nonlinear bubble diagrams31 (see Supplementary Note χ(2) at 300 K and 4 K, respectively, assuming that the excitation laser wavelength is 1035 nm. At 300 K, local χ(2) at the edges of the nanopillar attains a considerably high value compared to areas away from the nanopillar, owing to the strong sublattice polarization and the resonant SHG enabled by pLLs at these locations. The distribution of χ(2) becomes more interesting at 4 K, where χ(2) becomes extremely large at only certain locations along the edges of the nanopillar  value significantly only at low temperatures (see Supplementary Note χ(2)). Meanwhile, we also consider the temperature-dependent phonon-assisted scattering events which mediate the efficiency of the SHG emission. When the temperature decreases, the scattering rate between the carriers and phonons decreases, leading to a more dominant SHG process, therefore favoring a stronger SHG emission  value over an entire area shown in Fig. Lastly, by adding up the In summary, we have demonstrated the experimental observation of SHG from strained graphene on nanopillar structure. The SHG arises from the sublattice symmetry breaking in graphene due to the non-uniform strain induced by the nanopillars. At a low temperature of 4 K, the SHG intensity is almost 50 times higher than that at room temperature. The drastic enhancement of SHG intensity is explained by the inter-pLL resonance when the strain-induced PMF is present. At a low temperature, the DOS at each pLL is significantly increased owing to a reduced thermal broadening, which strongly enhances the resonant inter-pLL optical transitions and SHG when the energy separation between pLLs matches the SHG energy.49 . This shows that the strain-induced sublattice polarization can be used to modify the point symmetry of graphene and allows us to design new anisotropic SHG patterns. Additionally, we also observe that the intensity of SHG increases when the average height of the nanopillar array increases (see Note 11 of Supplementary Information). This can be explained with the fact that a large PMF forces the LDOS of pseudo-Landau levels to be sharper, and therefore enables a stronger resonance between the pseudo-Landau levels and produces a large SHG intensity. Lastly, we also observe that the SHG intensity in our strained graphene can be tuned by the back gate voltage (see Note 12 of Supplementary Information). Due to the effect of Pauli blocking53. The study of strain-induced nonlinear characteristics in graphene can also be further extended to third harmonic generation or other nonlinear processes, which can be of great use in other nonlinear optoelectronic devices. In addition, circular polarization-dependent or valley-dependent nonlinear processes could be possible in pseudo-Landau-quantized graphene system55, which adds another dimension to the study of nonlinear processes. Interestingly, our simulation result reveals that the sublattice polarization can significantly suppress the LDOS at the zeroth pseudo-Landau level of one sublattice while that of another sublattice remains very large   for 3 min 30 s, forming an array of nanopillars. The square pillars have 500-nm side length and 85-nm height. The whole chip was coated with a 20-nm Al2O3 layer by atomic layer deposition before graphene dry transfer.The nanostructured substrate was patterned using e-beam lithography (EBL) and a wet chemical approach. Patterns for the square arrays were firstly defined in a 380 nm spin-coated polymethyl methacrylate (PMMA) layer. Subsequently, using the patterned PMMA as an etch mask, the SiOGraphene flakes were obtained with Scotch tape by mechanical exfoliation of a small piece of Kish graphite (Graphene Supermarket). The tape with thinned graphene flakes was pushed down onto a polydimethylsiloxane (PDMS) film and then detached. The monolayer flake on PDMS was found under an optical microscope, and then the number of layers was confirmed using Raman spectroscopy. The graphene flake was then aligned and stamped on a nanostructured substrate with an all-dry transfer method using our home-built dry transfer setup.The structural characteristics and morphology of the fabricated sample devices were characterized by field-emission scanning electron microscopy (SEM) (Apreo S FESEM). The electron acceleration voltage in SEM was 5 kV.58. The maximum measured strain is estimated to be 1.26% by assuming a strain-shift coefficient of 65.4% cm–1/%36 between the 2D peak of unstrained graphene and the 2D– peak of the strained graphene.The thickness of graphene flakes and strain distribution in graphene on nanopillars were characterized using a WITec Raman setup with 100× objective lens (NA = 0.9) and 600 lines/mm grating. We used a 532-nm laser at a low laser power of <1 mW to avoid any heating effect. We confirm the graphene sample to be monolayer based on the observation that the 2D peak intensity is ~2 times the G peak intensity2 on the sample. Supplementary Fig. The measurement on graphene’s SHG was performed in the reflection geometry. The sample was placed in an open-cycle cryostat (Janis ST-500) cooled by liquid helium. The temperature for our measurements varies between 4 K and 300 K. The excitation pump pulses were generated from a 1035-nm femtosecond laser with a 120-fs pulse duration and a repetition rate of 76 MHz . The laser was defocused using a focal lens to cover a spot-size area of approximately 15 × 15 μmSupplementary Information


Interstitial lung diseases (ILDs) are a large group of pulmonary disorders characterized histologically by the cardinal involvement of the pulmonary interstitium. The prototype of ILDs is idiopathic pulmonary fibrosis (IPF), an incurable disease characterized by progressive distortion and loss of normal lung architecture through unchecked collagen deposition. Acute exacerbations are dramatic events during the clinical course of ILDs, associated with high morbidity and mortality. Infections, microaspiration, and advanced lung disease might be involved in the pathogenesis of acute exacerbations. Despite clinical scores, the prediction of the onset and outcome of acute exacerbations is still inaccurate. Biomarkers are necessary to characterize acute exacerbations better. We review the evidence for alveolar epithelial cell, fibropoliferation, and immunity molecules as potential biomarkers for acute exacerbations of interstitial lung disease. Interstitial lung diseases (ILDs) are pulmonary disorders characterized histologically by the involvement of the pulmonary interstitium. Clinical manifestations of these include dyspnea, coughing, hypoxia, and impaired lung function. Several of these conditions are associated with underlying systemic conditions, such as connective tissue diseases (CTDs), asbestos exposure, drug exposure, or allergen exposure, as in hypersensitivity pneumonitis. It is nevertheless unclear in many cases what causes ILDs . IdiopatPatients with ILDs have unpredictable clinical outcomes. There is often a slow but steady progression of the disease in many patients. In some patients, the ILD remains stable, while other patients experience a rapid clinical deterioration. Acute worsening of the ILD (known as acute exacerbation) can also occur. Acute exacerbations may be fatal or may lead to significant worsening of symptoms ,2.Acute exacerbations of interstitial lung diseases (AE-ILDs) refer to a sudden aggravation of symptoms in patients with ILDs. The exacerbations of chronically present ILD were initially recognized in IPF, but they can occur in almost all ILDs. These exacerbations are characterized by increased dyspnea and cough and decreased oxygen saturation. Acute exacerbation of interstitial lung diseases (AE-ILD) is an acute and often morbid deterioration of the patient’s respiratory function leading to hospitalization.-A previous or concurrent diagnosis of IPF.-Deterioration within 30 days.-New bilateral ground glass opacities and/or consolidation on a background of reticular or honeycomb pattern consistent with usual interstitial pneumonia (UIP).-No evidence of pulmonary infection by endotracheal aspiration or bronchoalveolar lavage.-Exclusion of alternative causes, including left heart failure, pulmonary embolism, or an identifiable cause of acute lung injury.The Idiopathic Pulmonary Fibrosis Network (IPFnet) had proposed in 2007 the following definition of an acute exacerbation of IPF :-A previThe above-mentioned criteria were revised in 2016 .-Previous or concurrent diagnosis of IPF.-Acute worsening or development of dyspnea, typically less than one-month duration.-Computed tomography with new bilateral ground-glass opacity and/or consolidation superimposed on a background pattern consistent with the usual interstitial pneumonia pattern.-Deterioration not fully explained by cardiac failure or fluid overload.AE-IPF is defined as an acute, clinically significant respiratory deterioration characterized by evidence of new widespread alveolar abnormality. The revised diagnostic criteria are :-PreviouThe exact causes of AE-ILDs are thought to be related to a variety of factors, including viral infections, environmental exposures such as air pollution, drug toxicity, procedures such as bronchoscopy or VATS, and microaspiration. Regarding the effect of smoking as a risk factor for acute exacerbation of IPF, Song et al. reported that never-smoking status and low FVC were risk factors for AE-IPF . In the Among patients with all forms of ILD with acute exacerbations, 52% of admissions for decline of respiratory function were idiopathic, 20% due to infection, 15% due to subacute progression or end-stage disease, 6% due to heart failure or severe pulmonary hypertension, 4% due to venous thromboembolism, and 2% due to diffuse alveolar hemorrhage or perioperative exacerbation .Further research is necessary to understand the causes of AE-ILDs better and to develop more effective treatments for these exacerbations. Treatment options for AE-ILDs include oxygen therapy, corticosteroids, and antibiotics, but there are no targeted treatments that have been proven to be effective in treating these patients .Diagnosis of AE-ILD can be challenging. The clinical condition of these patients is so severely impaired due to the disease exacerbation that even minimally invasive diagnostic procedures, such as bronchoscopy with bronchoalveolar lavage, can have detrimental effects on the clinical course of the patients. Moreover, invasive lung biopsies are associated with increased procedure-associated mortality, which often makes them an unacceptable diagnostic tool in the clinical setting of an AE-ILD ,9. HenceA biomarker is a biological molecule or genetic feature that can be used to identify the presence of a disease or condition. Biomarkers can be used to diagnose, monitor, and evaluate treatment outcomes for a variety of diseases, including interstitial lung diseases ,11. IdeaThe focus of the present review will be placed on the use of biomarkers for the diagnosis and prognosis of patients with AE-ILDs.Currently, there are no established biomarkers in clinical practice that can predict the onset or outcome of AE-IPF. However, several biomarkers have been proposed as potential predictors of AE-IPF.p = 0.002). In the multivariate analysis, baseline serum KL-6 (both continuous and at a cut-off level of >/= 1300 U/mL) was an independent predictive factor for AE-IPF [The Krebs von den Lungen 6 (KL-6) glycoprotein is a mucin-like, high-molecular-weight protein expressed on the surface membrane of alveolar epithelial cells. Proliferating, stimulated, or injured alveolar epithelial cells release KL-6 into blood vessels. Increased levels of KL-6 in the serum have been found in patients with ILDs . It has r AE-IPF .p = 0.0001). Patients with both initial serum KL-6 >/= 1000 U/mL and serial increase in serum KL-6 had the steepest decline, suggesting a role for KL-6 predicting a worse lung functional status [In another study, IPF patients with increased serum KL-6 levels during follow-up had a significantly steeper decline in FVC than those without increased KL-6  reduce alveolar surface tension and prevent lung collapse. They are synthesized and secreted by alveolar epithelial cells (AEC) II. Patients with IPF, as well as those with other ILDs, have significantly elevated levels of SP-D and SP-A in their serum and BALF ,21.Increased levels of SP-D were observed in patients with AE-IPF compared to those with stable disease . In a meIn the normal process of cell replication, telomeres protect chromosome ends from progressive shortening . AbnormaGrowth differentiation factor 15 (GDF-15), also named macrophage inhibitory cytokine-1 (MIC-1), belongs to the transforming growth factor-β (TGF-β) superfamily. There is an increase in the expression and upregulation of GDF-15 in epithelial cells in IPF. GDF-15 is a useful biomarker for epithelial stress associated with poor IPF outcomes. In a study of 47 patients with AE-IPF and 61 patients with stable IPF, serum GDF-15 levels, as well as the protein and mRNA expressions of GDF-15 in the lung, were significantly elevated in AE-IPF patients compared with stable IPF cases. A serum GDF-15 level above 989.3 pg/mL was a risk factor for AE occurrence, and the level above 1075.76 pg/mL was an independent predictor for survival in IPF cases .mucin 5B (MUC5B) genotype, revealed an independent association between MMP-7 and survival in IPF patients [Matrix metalloproteinases (MMPs) are endopeptidases with zinc in their structure that degrade proteins of the extracellular matrix. Development, morphogenesis, tissue repair, and remodeling rely heavily on the timely degradation of extracellular matrix. By regulating extracellular matrix turnover, MMPs contribute to the pathogenesis of fibrosis ,31,32. Mpatients . It has Biomarkers released by matrix metalloproteinase-mediated degradation of collagen have also been examined in AE-IPF; such a degradation product, versican, was found to be associated with mortality in a study of 68 patients with AE of idiopathic interstitial pneumonias, including 28 patients with AE-IPF .A disintegrin and metalloproteinases (ADAMs) are believed to be involved in the pathogenesis of many fibrosis-related diseases. ADAM-17 was increased in IPF and other ILDs, such as CTD-ILD . ADAM-17Human epididymis protein 4 (HE4) is widely used as a biomarker in ovarian and endometrial cancer. HE4 inhibits the function of various proteases, such as MMPs and serine proteases, suppresses the degradation of type I collagen, and plays a crucial role in the pathogenesis of fibrosis. In a retrospective study of 59 patients with stable IPF and AE-IPF, the expression of serum HE4 was elevated, especially in AE-IPF patients, as was KL-6. In the multivariate analysis, HE4 levels, as well as GAP index, were associated with survival. Histologically, HE4 could be found in the mucosal epithelium of dilated bronchi .The levels of Lysyl oxidase-like proteins (LOXL) have also been associated with increased mortality and progression of IPF; however, a direct association with AE-IPF has not been shown .Circulating fibrocytes are progenitors of fibroblasts and myofibroblasts. IPF patients with stable disease have been shown to have increased circulating fibrocytes , and patNon-structural extra-cellular matrix protein, periostin, promotes mesenchymal cell proliferation and fibrosis through its expression at sites of injury or inflammation. In a recent study, serum monomeric periostin levels were prospectively measured from onset of AE in 37 patients with AE of chronic interstitial pneumonia to day 14, and its association with outcome was evaluated. Serum monomeric periostin levels were significantly higher in patients with AE compared to the ones with stable IPF and decreased significantly in AE survivors compared to AE non-survivors. Furthermore, a decrease of serum monomeric periostin levels in patients with AE was associated with better survival in 3 months after AE (OR 12.4) . ImmunohAn important cytokine involved in tissue repair is osteopontin (OPN). It has been shown that OPN can activate MMP-7 and stimulate fibroblast migration and proliferation . IPF patTLR3 L412F in viral- and bacterial-mediated AE death [The innate immune response to infection and tissue injury is mediated by toll-like receptors (TLRs) . IPF patAE death .TOLLIP have also been reported to be associated with the development and/or prognosis of IPF [TOLLIP rs5743890 was associated with worse survival and a more rapid progression of IPF, which could be useful for stratifying IPF patients at baseline [The toll-interacting protein (TOLLIP) is an inhibitor of the toll-like receptors (TLRs) 2 and 4, which are active in the lung, thereby suppressing tumor necrosis factor-a (TNF-a) and interleukin 6 (IL-6) production. Genetic variants of s of IPF . In IPF baseline . Moreovebaseline .Defensins are proteins secreted by neutrophils which exert antimicrobial activity . Acute eC-X-C motif chemokine ligand 13 (CXCL13) facilitates B-cell trafficking in lymphoid aggregates and inflammatory foci. The presence of elevated CXCL13 protein levels in patients with IPF has been linked to increased mortality, and CXCL13 levels were highest in IPF patients with AE-IPF .S 100 calcium binding proteins 8 and 9 (S100A8 and S100A9) are released mainly by activated neutrophils, which promote inflammation and decrease vascular endothelial cell integrity, consequently inducing further neutrophil extravasation. In ILDs, S100A8 and S100A9 are more often produced in patients with IPF with chronic-phase disease than in patients with other diseases, such as sarcoidosis or CTD-ILD. In a retrospective study of 37 patients with AE, patients with higher levels of S100A8 or S100A9 showed significantly worse 3-month survival than those with lower levels. In the multivariate analysis, the serum levels of both S100A8 and S100A9 were significant prognostic factors of AE-IPF . In anot14 CD36hi CD84hi CCR2− monocyte population. The same phenotype was found in patients with progressive IPF [Immune cells, particularly macrophages and lymphocytes, have been implicated in the pathogenesis of ILDs. CD4+ cells in IPF are in a highly activated status . Decreassive IPF .Increased levels of circulating pro-inflammatory cytokines have been reported during AE-IPF. Interleukin 1b (IL-1b) upregulates interleukin 8 (IL-8), which is critical for neutrophil recruitment. IL-8 is produced by phagocytes. Higher concentrations of IL-8 have been found in the BALF of patients with IPF and fibrosis associated with collagen vascular diseases . PatientIL-1b levels have been reported to be higher in patients with AE-IPF than in stable IPF . IncreasInterleukin 6 (IL-6) is a pro-inflammatory, pro-fibrotic cytokine. Increased serum levels of IL-6 above 25.20 pg/mL have been reported to be diagnostic of AE-IPF and to be an independent risk factor for mortality . An incrInterleukin 35 (IL-35) is a cytokine which is considered to have anti-fibrotic and anti-inflammatory effects by affecting interleukin 17 (IL-17) production. IL-35 also prevents the phosphorylation of Smad3, a downstream cascade of transforming growth factor (TGF-b) signaling, thus blocking TGF-b binding to its receptor and preventing the differentiation of T helper 17 (Th17) cells . In a reProteobacteria, Campylobacter spp., and Stenotrophomona spp., while a significant decrease was found in Veillonella spp. and Campylobacter spp [Klebsiella pneumonia accounted for 26%, Mycobacteria tuberculosis for 21%, and Acinetobacter baumannii for 10% of the total strains [Lungs have been traditionally considered sterile of bacteria. In part, this notion was due to the inability of traditional culture techniques to isolate bacteria from lung specimens. The introduction of sequencing of the 16S rRNA gene technique allowed recognition of the fact that bacteria not only exist within the human lung, but they are also altered in lung disease. A higher microbial burden is associated with a worse prognosis in stable IPF. It has been shown that patients with AE-IPF have a higher microbial burden in their lungs, which has been associated with higher morbidity and mortality rates. A significant increase was noticed for cter spp . In sput strains . Althoug strains ,72.miR-25-3p and let-7d-5p in plasma were differentially expressed between AE-IPF and stable IPF, suggesting that a combination of these two mi-RNAs may be a potential biomarker for AE-IPF from IPF [Non-coding RNAs represent a type of epigenetic change, i.e., a gene expression change that does not involve a change in the DNA . Long nofrom IPF .miR-302c, miR-423, miR-210, miR-376C, and miR-185 mi-RNAs were found to be related to the severity of IPF, differentiating fast and slow progressors [In a study, the gressors .In aging-related lung diseases, such as IPF, mitochondrial dysfunction is critical in regulating programmed cell death in alveolar epithelial cells . MitochoHistologically, AE-IPF is characterized by diffuse alveolar damage (DAD) overlying IPF fibrosis. AE-IPF was associated with alveolar hemorrhage in ca. 25% of the patients and with pulmonary thromboembolism in ca. 20%, suggesting that capillary injury and thrombosis play a significant role in AE-IPF . A studyp = 0.036). The NLR before death was also significantly higher than that at admission in patients with AE-IPF who did not survive (p = 0.014) [NLR is a ratio calculated from the absolute number of neutrophils and lymphocytes in the blood. Elevated NLR has been found in IPF patients with acute exacerbation, and it has been proposed that measuring NLR could be used to predict AE-IPF. In a multicenter study using discovery and validation cohorts, high values of NLR (>/=2.9 vs. <2.9) were associated with an increased risk of mortality in IPF (HR 2.04). This was confirmed in the validation cohort. NLR correlated with GAP stage and GAP index, which are widely used scores combining gender, age, and pulmonary function to predict survival in IPF . These r= 0.014) . It is n= 0.014) . Another= 0.014) . NLR is Apart from NLR, other blood cell ratios have been shown to be associated with prognosis in IPF. Such a marker is the PLR . In a poSystemic inflammation response index (SIRI) and aggregate index of systemic inflammation (AISI) as indexes of inflammation have also been reported to correlate with IPF and worse functional status .LCK), IL-2 inducible T-cell kinase (ITK) as part of a 52-gene RNA signature [Multiple biomarker signatures can be used more effectively than a single protein in the diagnosis and prognosis of IPF. The combination of serum MMP-1 and MMP-7 levels, for example, provided higher positive and negative predictive values for the diagnosis of IPF than either protein on its own . Serum bignature , a compoignature , and theignature  have beeignature .2/FiO2 ratio, diffuse HRCT pattern, and serum C-reactive protein (CRP) were significantly associated with 3-month mortality in patients with AE-IPF [PaOh AE-IPF . In anoth AE-IPF .Proteomic comparative analysis of BALF in stable IPF patients vs. AE-IPF revealed that proteins involved in the propagation of β-catenin WNT transduction signal, proteins upregulated in lung carcinogenesis , as well as acute phase proteins involved in protease–antiprotease imbalance (such as a1 antithrypsin fragments) correlated to AE-IPF .One type of idiopathic interstitial pneumonia (IIP) is nonspecific interstitial pneumonia (NSIP). Besides being idiopathic, NSIP can be associated with HIV infection, connective tissue disease, various drugs, and hypersensitivity pneumonitis. In addition, it can occur in combination with other IIPs. The nonspecific nature of NSIP is due to the absence of the histopathologic characteristics of usual interstitial pneumonia (UIP), desquamative interstitial pneumonia (DIP), respiratory bronchiolitis-associated interstitial lung disease (RB-ILD), or acute interstitial pneumonia (AIP) . NSIP isThe development of acute ILD may occur with any CTD, as a de novo acute interstitial pneumonia (AIP) or as an acute exacerbation of an ILD that already exists. Acute exacerbations have been described in rheumatoid arthritis (RA), polymyositis/dermatomyositis (PM/DM), systemic sclerosis (SSc), systemic lupus erythematosus (SLE), and mixed connective tissue disease (MCTD). Common underlying pathology patterns are that of NSIP and UIP . Common Autoimmune biomarkers are particularly useful for the diagnosis of the underlying CTD in the case of a novo presentation of ILD . HoweverAs a major extraarticular feature of RA, lung involvement affects 10–60% of patients. The prevalence of ILD ranges from 5 to 58%, with clinically significant RA-ILD occurring in less than 15% of cases. Antibodies to citrullinated protein antibodies (anti-CCPs) and rheumatoid factors (RFs) are frequently found in the serum of patients with RA. RF levels greater than 90 international units/mL may identify patients at higher risk of ILD . It has Most of the biomarkers tested in IPF have also been tested in RA-ILD . HoweverAge, sex, smoking history, positive RF, and anti-CCP, along with a combinatorial signature of serum biomarkers, including MMP-7, pulmonary and activation-regulated chemokine (PARC), and SP-D, could predict RA-ILD .KL-6 and SP-D have been examined in RA patients with ILD or AE-ILD; no significant difference in their levels in stable RA-ILD and AE-ILD or de novo acute ILD has been reported . A reasoIt has been suggested that the metabolites mannosamine, alliin, kynurenine, and 2-hydroxybutyric acid could successfully discriminate acute ILD in the setting of RA from the stable condition .Radiomic analyses have also been reported to predict mortality in RA-ILD, and its use has been proposed as a digital biomarker .The reported prevalence of ILD in SSc is 50–60% . ILD is In a recent meta-analysis, KL-6, SP-D, and IL-8 in blood and BALF were the best biomarkers of SSc-ILD . PatientThe above-described biomarkers are predictive of progressive ILD and mortality. Progressive disease is a risk factor for AE. It is unclear whether these biomarkers can also depict the development of AE-ILD in SSc-ILD.Hypersensitivity pneumonitis (HP) is an ILD that results from inhalation of organic antigens in susceptible individuals. HP is classified into fibrotic or nonfibrotic , and acuPeriostin is an established biomarker of Th2 immune response and fibrogenesis. Serum periostin has been associated with AE of HP. In a study of 63 patients with HP, 13 with IPF, and 113 controls, a significant positive correlation was found between periostin levels and serum KL-6 levels, CD4/CD8 ratio in bronchoalveolar lavage fluid, and fibrosis score on HRCT, and a significant negative correlation was found between periostin levels and carbon monoxide diffusing capacity. Periostin concentrations exceeding 92.5 ng/mL or 89.5 ng/mL were significantly associated with a worse prognosis and a higher frequency of acute exacerbations in chronic HP patients. An independent prognostic factor in the multivariate analysis was a high serum periostin level (92.5 ng/mL or higher) .Small studies have reported several biomarkers that are associated with AE-ILD in mixed ILD populations. Most of the publications included patients with AE-IPF. The examined biomarkers are the following:A meta-analysis of KL-6 in ILDs reported that ILD patients with severe and progressive disease had higher KL-6 levels. The KL-6 level of patients with severe ILD was 703.41 U/mL higher than in mild ILD. The KL-6 level in the progressive ILD group was 325.98 U/mL higher than that in the non-progressive ILD group. In addition, the KL-6 level of patients in AE of ILD was 545.44 U/mL higher than stable ILD. The higher KL-6 levels in ILD patients predicted poor outcomes. The KL-6 level in patients who died due to ILD was 383.53 U/mL higher than in survivors of ILD. The pooled HR (95%CI) of elevated KL-6 level predicting the mortality of ILD was 2.05 .Most data are derived from patients with IPF but reduced survival has also been reported in patients with SSc-ILD  and dermIn a retrospective study of a mixed population of patients with ILD, elevated D-dimer levels were associated with a ten-fold increase in risk of developing an AE three months after the D-dimer measurement .The soluble mediator IL-6 plays a pleiotropic role in inflammation, immune responses, and fibrosis. In a retrospective study of 83 patients with ILD, high levels of IL-6, along with lower baseline saturations of peripheral oxygen, were independent risk factors for AE. Similar results have been reported in patients with AE-IPF ,67.HBP was significantly higher in patients with AE-ILD at the early stage compared to patients with ILD at the stable phase, and this increase was found in the serum and BALF. With the remission of the disease, there was a linear trend of progressive decline. HBP identified ILD patients who had co-infections .BALF lymphocytosis has been associated with a better prognosis, and BALF neutrophilia with worse prognosis, in a study of 71 patients with AE-ILD. BALF lymphocyte and neutrophil count ≥25% and <20%, respectively, predicted favorable survival after AE .In a recently published study, proteomic analysis was performed in a discovery cohort of 385 patients with various ILDs, including CTD-ILDs, HP, and unclassifiable ILDs. The validation cohort comprised 204 patients. A total of 31 biomarkers were associated with progressive fibrosing ILD in the discovery cohort, with 17 maintaining an association in the validation cohort. Validated biomarkers showed a consistent association with progressive fibrosing ILD irrespective of ILD clinical diagnosis. A proteomic signature comprising 12 biomarkers was derived by machine learning, predicting a progressive fibrosing ILD. Use of this biomarker signature showed not only that progressive fibrotic ILDs, irrespective of aetiology, share common pathogenetic mechanisms but also overcame the modest use performance of other single biomarkers used for prediction of the clinical course of ILDs .Acute exacerbations are detrimental in the clinical course of ILDs. Early diagnosis before the onset of severe respiratory failure and early recognition of the patients who are more susceptible to AE are of paramount importance for therapeutic decisions in those patients. Despite the fact that treatment of AE in most ILDs is supportive and the use of immunosuppressive treatment—at least in AE-IPF—lacks significant therapeutic effect, early recognition of AE could lead to earlier hospitalization, lower clinical threshold for antibiotic therapy, and faster and probably more extensive diagnostic work-up and decisions for palliative treatment in the right clinical setting. Biomarkers can help in this direction, probably in combination with clinical parameters, lung function indices, and HRCT. Equally important is decision-making, when the diagnosis of an ILD must be first made in the setting of an AE and when the patients are in the intermediate care or intensive care station. Decisions regarding mechanical ventilation, ECMO, and transplantation are often based on the underlying pathology pattern of the ILD.While the understanding of ILD pathogenesis has grown substantially and biomarker research has progressed, the use of biomarkers for AE-ILD in clinical practice remains limited.In case of a de novo diagnosed ILD presenting as an AE, diagnostic tests that are currently used are autoantibodies, which can help to differentiate between idiopathic ILDs and ILDs associated with collagen vascular diseases, and the precipitating antibodies supporting the diagnosis of hypersensitivity pneumonitis. In certain cases, BALF lymphocytosis might support an intensive immunosuppressive treatment for AE-ILD, contrary to a neutrophilic BALF.Despite many biomarkers being studied in AE-ILD , most ofKL-6 and MMP-7 are promising and well-studied biomarkers in IPF that might find their way into routine clinical practice in the future. The same applies for simple markers, such as the NLR. However, it seems that multiple biomarker signatures, combined with clinical data, may provide more accurate information regarding AE-ILDs. Simple biomarkers are, however, of particular interest, since they can be used in medium- and low-resource health systems as well.In prospective clinical trials with large numbers of patients, biomarker analyses in combination with genotyping of these patients may allow the identification of predictors of disease progression and subgroups of patients with varying disease courses ,152,156.