Nanoparticle-on-mirror systems are a stable, robust,and reproduciblemethod of squeezing light into sub-nanometer volumes. Graphene isa particularly interesting material to use as a spacer in such systemsas it is the thinnest possible 2D material and can be doped both chemicallyand electrically to modulate the plasmonic modes. We investigate asimple nanoparticle-on-mirror system, consisting of a Au nanosphereon top of an Au mirror, separated by a monolayer of graphene. Withthis system, we demonstrate, with both experiments and numerical simulations,how the doping of the graphene and the control of the gap size canbe controlled to tune the plasmonic response of the coupled nanosphereusing nitric acid. The coupling of the Au nanosphere and Au thin filmreveals multipolar modes which can be tuned by adjusting the gap sizeor doping an intermediate graphene monolayer. At high doping levels,the interaction between the charge-transfer plasmon and gap plasmonleads to splitting of the plasmon energies. The study provides evidencefor the unification of theories proposed by previous works investigatingsimilar systems. The NPoM approach retains the sameconfinement effects as with two nanoparticles coupled together butinvolves the coupling of a nanoparticle with its mirror image.13The precise control ofthe separation between two metal nanoparticlesis of high interest due to the many applications involved exploitingthe ability to trap light at the sub-wavelength scale.18 Another advantage of utilizing 2D materials in these systems isthat their thicknesses are well known and constant at the monolayerlimit. This constant gap thickness can be useful in keeping the gapsize fixed while monitoring the other changes in the plasmonic system.19It is interesting to combinetwo-dimensional (2D) materials withthe NPoM geometry. This is because the light is confined within dimensionssimilar to the material of atomic thickness. This gives rise to apathway for much stronger light–matter interaction for 2D materials,allowing much deeper exploration into their optical properties.21 Graphene has many unique properties, including itshigh electrical conductivity and its high chemical stability.25 Graphene has a tunable dielectric response that can be modifiedby doping both chemically and electrically. This can be manipulatedfor a wide variety of applications, including hyperlenses, metacouplers,photovoltaic devices, and super-resolution imaging and sensing.27 Many techniques have been developedto allow for the growth of a large surface area and a high-qualitymonolayer film, such as chemical vapor deposition, flame synthesis,and pulsed laser deposition.28 Many studieshave explored the coupling of graphene to plasmonic structures, mostlyin the infrared region. It has been demonstrated that graphene canbe used to modulate the plasmonic response by modifying the dielectricproperties.37 This can be achieved by both electrical gating and chemical doping.When the graphene layer overlaps with the electric field hot spot,it can have remarkable effects on the modulation of the plasmonicspectra, even in the visible wavelength range.38A well-explored 2D material is graphene,the thinnest of the 2Dmaterials, with a thickness of 0.34 nm.20 andMertens et al.,16 investigated a systeminvolving an Au nanosphere on top of an Au film, with an intermediatelayer of graphene. These systems demonstrated optical modulations,tuned by graphene, in the visible to near-infrared spectrum. Thismakes these works particularly interesting as most systems involvingthe tuning of plasmonic energies with graphene are limited to theinfrared region. Both works showed the formation of three differentpeaks in the scattering spectra. Shao et al. attributed the threepeaks to the octupolar, quadrupolar, and dipolar modes. Mertens etal. attributed the three peaks to the transverse plasmon and the couplingbetween the charge-transfer plasmon (PCTP) and gap plasmon(PGAP).Twoseparate groups, Shao et al.2 layer on an Si substrate. Several plasmon modes are visiblein the scattering spectrum of this system. Nitric acid is employedto modulate these modes by two different mechanisms. It serves todope the graphene and also to reduce the gap size in between the Aunanosphere and film. Our results demonstrate the unification of thetheories presented by both Shao et al. and Mertens et al. and theregimes in which each is observed.In this paper, we present evidence to showthat both of these theoriesare correct, and we demonstrate the conditions under which each isthe dominant effect. Our system involves a Au nanosphere of 150 nmin diameter, on top of a 100 nm thick Au film, with an intermediatelayer of graphene. The Au film is on top of a 100 nm thick SiO225 This method was chosen due to its high monolayercoverage. The CVD graphene was transferred onto the target substratesusing a polymer-mediated etching and transfer process,40 utilizing poly(methyl methacrylate)(PMMA) as the handling polymer and ammonium persulfate as the copperetchant. The PMMA coating was removed by heating to 150 °C andwashing in acetone. The gold layer was deposited on the SiO2/Si substrates by electron beam evaporation with a Temescal. TheAu nanospheres were synthesized with a wet chemistry approach usingthe method of Zheng et al.41 This methodwas chosen to obtain a high yield of nanospheres that were uniformin size and highly spherical.The monolayergraphene was grown by chemical vapor deposition (CVD)on commercial polycrystalline copper foils using methane as the hydrocarbonprecursor, similar to the technique employed by Li et al.Figure S1. Theproduct was diluted in ethanoland sonicated for ≈3 min before drop-casting onto the substrates.The 150 nm Au nanospheres were drop-cast onto the Au film with a graphenemonolayer on top, the Au film without a graphene monolayer, the SiO2/Si substrate with a graphene monolayer on top, and the SiO2/Si substrate without a graphene monolayer. The substrateswere heated for 10 min in acetone at 40 °C after the depositionto reduce the CTAB (cetrimonium bromide), [(C16H33)N(CH3)3]Br layer surrounding the particles.32 This also helped to reduce the amount of residualPMMA remaining on the graphene. The measurements of scattering spectrawere taken at the single particle level under white light excitationwith a dark field microscope using a 100× objective lens of 0.9numerical aperture. The spectra were recorded with an Andor CCD cameraand an Andor 230i spectrometer. Approximately 50 measurements weretaken for single nanospheres drop-cast onto each of the four differentsubstrates considered.A SEM image of a sample of synthesizednanospheres is shown in 42 A thickness of 0.5 nm was used for the graphenelayer, which was modeled according to Stauber et al.43 This model was chosen as it is valid for visible wavelengths.The doping of the graphene layer was modeled by changing the chemicalpotential energy (Fermi level) from 0.1 to 1.2 eV. The change of theFermi level shifts the electron valence band level in the Dirac cone-approximatedband structure of graphene.44 The gap betweenthe nanosphere and graphene layer was modeled with a permittivityof 1.5 to approximate any residual polymer and the CTAB layer.45FDTD simulations were carried out usingthe commercial software,Lumerical solutions. These were used to illustrate how the gap sizeand changes to the permittivity of the graphene layer modulate theenergy of the plasmon modes. The nanospheres were modeled with a diameterof 150 nm and approximated as perfect spheres. The dielectric functionof Au was taken from a fit of the points measured by Johnson and Christy.20 The octupolar peak is visible as a shoulder to the quadrupolar peak.The electric field map from a simulation of this system is shown in Figure S2, demonstrating a clearer dipolar mode when thereis no polymer layer for the charge to leak into.Example experimental scattering spectraof four different nanospheresare shown in 2/Si substrate, with andwithout a monolayer of graphene, are shown in 2/Si substrate.An experimental scattering spectrum of the samesystem, withoutthe graphene monolayer, is shown in 3Gap plasmons have a very high sensitivity to their environment.Therefore, a myriad of factors can cause slight discrepancies betweenthe experimental results from different particles, including the particleshape, size, gap size, and surface roughness of the Au film. Due tothese small differences between different particles, the simulationsemployed in this paper are used only to show global trends. The systemscorresponding to the spectra in 30 When the dopinglevel is as high as 1.2 eV, the dipolar mode splits in two in The effect of the doping of the graphene layeris shown in 46 with more multipolar modes appearing as thegap size is brought to just 0.25 nm. A schematic illustrating theeffect of the sphere being brought closer to the gold film is shownto the right of this plot. The PMMA layer modeled as a medium witha refractive index of 1.5 has the effect of some extra small peaksforming in the spectra due to the charge leaking described in Figure S3, with the PMMA modeled asair instead. These plots give a clearer view of the trends occurringdue to the change of gap size and chemical potential.Another difference between the spectra in S6 and the simulated spectra in Anotherfeature evident in 49 With the formation of this neck, it is no longer possible for themultipolar modes to form because the capacitor effect cannot occurwithout the gap region between the Au sphere and film. Simulationsof this effect are shown in 49When a gold nanoparticle is brought into contactwith a gold substrate,a neck is formed between them by the rearrangement of the atoms ofthe nanoparticle touching the sphere.43). Thenitric acid was used as a tool to effectively p-dope the graphenelayer and also etch away the residual CTAB and PMMA between the Ausphere and film, reducing the gap size between the two. The sampleswere immersed in 5, 10, 20, 30, 40, 50, 60, and 70% nitric acid for5 min each. The nitric acid had the effect of p-doping the graphene.50 Raman spectra were taken of the graphene aftereach immersion in various concentrations of nitric acid. The resultsare shown in Figure S4 and can confirma high level of doping across the entire graphene monolayer.51 The doping is estimated to be between 0.9 and 1.1 eV after beingimmersed in 70% nitric acid, with slight variations across the graphenesample. This is evidenced by the Raman spectra presented in Figure S4 and the gap plasmon (PGAP) interacting.The charge-transfer plasmon is the dipolar resonance of the wholesystem. The gap is conductive and gives rise to an equal and oppositecharge in the Au sphere and the Au mirror film, as demonstrated in We have seen that the peaks in the scattering spectraof the NPoMsystem correspond to the multipolar modes as described in Shao etal.GAP increases, causing thedipolar mode to split into two new modes, P+ and P–. This is supported by both experimental results andsimulations (see As the doping gets larger, Pions see 3 and 4. ions see 3a,b. Thi+ and P–. P– occurs as a result of PCTP and PGAP actingin opposite directions. This has the effect of reducing the energyof the plasmon and also distorting the charge distribution. This givesit the appearance of an octupolar mode, despite being lower in energy.P+ occurs as a result of PCTP and PGAP acting in the same direction. This has the effect of enhancing thedipolar effect and also increasing the plasmon energy.Further simulations were carried out to ensure that the chargedistribution matched this theory. A 150 nm sphere on an Au film, separatedby a monolayer of graphene with a chemical potential of 1.2 eV, wassimulated with a gap of 1 nm between the sphere and graphene layeras in 6We have investigated asingle Au nanoparticle on an Au mirror filmseparated by a monolayer of graphene. We have demonstrated with bothnumerical simulations and experimental results how sensitive the plasmonmodes are to both the gap size and the level of doping in the graphenelayer, even at visible wavelengths. The coupling of the nanosphereand film gives rise to multipolar modes which can be tuned to blue-shiftby doping a monolayer of graphene in between them or by altering thegap size. At high doping levels, the plasmons are seen to split intotwo new energy levels. Our results serve to explain how differenttheories in the two previous papers by Shao et al. and Mertens etal. are both correct under different levels of doping and differentgap sizes. These results will be useful for the design and fabricationof future devices employing the NPoM system separated by a 2D material.They give a deeper understanding of the interactions between the differentplasmonic modes and how they can be controlled.


Idiopathic pulmonary fibrosis (IPF) is a chronic, rare progressive lung disease, characterized by lung scarring and the irreversible loss of lung function. Two anti-fibrotic drugs, nintedanib and pirfenidone, have been demonstrated to slow down disease progression, although IPF mortality remains a challenge and the patients die after a few years from diagnosis. Rare pathogenic variants in genes that are involved in the surfactant metabolism and telomere maintenance, among others, have a high penetrance and tend to co-segregate with the disease in families. Common recurrent variants in the population with modest effect sizes have been also associated with the disease risk and progression. Genome-wide association studies (GWAS) support at least 23 genetic risk loci, linking the disease pathogenesis with unexpected molecular pathways including cellular adhesion and signaling, wound healing, barrier function, airway clearance, and innate immunity and host defense, besides the surfactant metabolism and telomere biology. As the cost of high-throughput genomic technologies continuously decreases and new technologies and approaches arise, their widespread use by clinicians and researchers is efficiently contributing to a better understanding of the pathogenesis of progressive pulmonary fibrosis. Here we provide an overview of the genetic factors known to be involved in IPF pathogenesis and discuss how they will continue to further advance in this field. We also discuss how genomic technologies could help to further improve IPF diagnosis and prognosis as well as for assessing genetic risk in unaffected relatives. The development and validation of evidence-based guidelines for genetic-based screening of IPF will allow redefining and classifying this disease relying on molecular characteristics and contribute to the implementation of precision medicine approaches. Idiopathic pulmonary fibrosis (IPF), the most common type of interstitial pneumonia, is a chronic and progressive disease in which healthy tissue in the lung parenchyma is progressively replaced by an altered extracellular matrix leading to the loss of lung function , 2. ThisMUC5B  is coined when at least two members from the same biological family are affected with IPF . In bothMUC5B .Despite the important recent advances in understanding the genetics underlying disease risks, the current IPF guidelines do not recommend the use of genetic testing for diagnosis . In thisGenetic factors play a significant role in the development of IPF. The known genetic variants associated with IPF are classified into two broad categories: common SNPs that are broadly found in the general population , and rare damaging variants in the spectrum of allele frequency below <1% which are typically not recurrent in the general population . Next-geThe genetic studies in IPF have provided significant advances in the understanding of disease pathophysiology. The genes encoding proteins which fall in the same pathway are expected to disrupt the same mechanism and, therefore, can be targeted similarly. This fact reinforces the importance of disclosing the molecular causes underlying disease risk.Telomeres are specialized structures at the end of chromosomes. In humans, they typically consist of the tandemly repeated TTAGGG nucleotide sequence and the associated protective proteins known as the shelterin complex. Their main functions are to protect genome integrity and prevent degradation and chromosomal end-to-end fusion . As the TERT and TERC) were the first to be described in families with IPF and no history of dyskeratosis congenita  tails from the precursor RNA including that of TERC, and RTEL1, which encodes a helicase that unwinds the G-quadruplex and the T-loop secondary structures at the telomeric end (DKC1 (ZCCHC8 (NAF1 (TINF2 (ACD (TERT and TERC being the most frequent (8–15%)  , 28. Rars so far , 34  in a pre-protein form and then transported to lamellar bodies where they are stored until they are secreted to the alveolar space. Rare pathogenic variants in SFTPC and SFTPA1-2 have been associated with both adult-onset and pediatric forms of IPF . Since then, numerous studies have identified more than 40 coding and non-coding rare pathogenic mutations in the gene. These mutations are described in pediatric and adult patients, predisposing to a range spectrum of fibrotic lung diseases which segregate in an AD pattern of inheritance , expressed in AEC2 lamellar bodies where it participates in lipid transportation , 62. Earhood ILD  , heterozygous and homozygous of the risk allele (T) of the polymorphism were estimated in as much as 6.8  and 20.8  for FPF, and 9.0  and 21.8  for sporadic IPF  than in unaffected controls irrespective of genotype  cohorts have replicated the association of the variant with IPF risk , 93–95. MUC5B variant has been also associated with the presence of interstitial lung abnormalities  for the first time in IPF showed that the disease is highly polygenic and there are potentially hundreds of, as yet unidentified, variants associated with IPF susceptibility  relative to mutation carriers in TERC (mean of 58 years), RTEL1 (mean of 60 years), and PARN (mean of 65 years)  .TERT, RTEL1, PARN, and TERC, one consistent phenotype is progressive deterioration , suggesting that telomere-related mutations are predictive of disease progression independently of the clinical diagnosis , unclassifiable ILD (8–12%), and other idiopathic interstitial pneumonias (14–18%) are described , 106. Hoiagnosis  , which increases the IPF risk, has been associated with better IPF progression and survival . TeSLA (Telomere Shortest Length Assay) and TRF  analyses have shown to be as reliable as other available options  and flow-FISH , PolyPhen2, Mutation Taster, Variant Effect Predictor (VEP), or the Combined Annotation Dependent Depletion (CADD) score can help in this step, especially for missense mutations  cumulative evidence from other cases sharing the same variant and phenotype, submitted to public databases; (3) segregation analysis of the variant between affected cases but within the same family; (4) existence of local population-level data to provide a more accurate allele frequency of the variant; and (5) improvement and updates in genetic variant annotation  and the Association for Molecular Pathology (AMP) in five categories: pathogenic, likely pathogenic, uncertain significance, likely benign, or benign . A positnotation . Altogetnotation , 149–151notation . HoweverIt should be noted that the workflow described above is specific for single nucleotide variants (SNVs) and indels (short insertions or deletions). Structural variants (SV) are defined as large regions (affecting more than 50 base pairs of sequence) that could involve changes in number of copies of a sequence , orientation (inversions), or alterations of the chromosomal location (translocations) . They haFamily history ascertainment is crucial for identifying familial cases of IPF. Therefore, the construction of a pedigree and the collection of the medical history from relatives within at least three generations are essential . ImportaDifferent extrapulmonary manifestations may be considered as part of telomere-related syndrome and suggestive of a telomere disorder. Accelerated telomere shortening manifests in a broad spectrum of symptoms involving tissues with high proliferation rates, such as epithelium or the hematological system . IPF patClassically, IPF is an aging disease that usually affects patients older than 60  years . TherefoGenetic testing should also be considered when there is a suspicion of a genetic syndrome such as Dyskeratosis congenita, Hermansky-Pudlak syndrome, Coats plus syndrome, Høyeraal–Hreidarsson syndrome, and Revesz syndrome . In all SFTPC, SFTPA1, SFTPA2, and ABCA3 are included as consensus in the analysis . Biallelic variants are usually related with syndromic forms such Dyskeratosis congenita, Høyeraal–Hreidarsson syndrome, and Coats plus syndrome . Re. ReSFTPCs of IPF , 158. Mos of IPF . Borie eanalysis , 160. Whanalysis . More resyndrome   because of the influence in the natural history of the disease in other family members . Thus, pThe current real situation is that there are wide differences between countries regarding aspects related to genetic counseling such as laws, health systems, or culture . FurtherIn recent years, the application of high-throughput genomic technologies has led to the conclusion that a great inter-individual variation exists regarding the mechanisms that contribute to the disease. This variability is expected to impact its treatment, monitoring and diagnosis. In this review, we have summarized some important genetic aspects of IPF and how the current knowledge is starting to guide the molecular diagnosis of patients and relatives. However, this field is continuously evolving, and new approaches are becoming available and are expected to contribute to the implementation of precision medicine in the healthcare system.Common and rare variants, as well as TL information can inform about disease risk and outcome of IPF patients. In addition, unexplored types of genetic variants of interest for IPF, such as SVs or variants affecting non-coding regions, are expected to be discovered as larger cohorts are included in genomic research studies . DespiteTERT c.2005 T mutation in a Dutch cohort estimates the origin in a founder individual who lived in The Netherlands 300 years ago  in IPF is variable and should be carefully considered and explained when the subject receives the related information before being tested , 160. Thears ago . For allMAFK and SMAD2. Both genes are transcription factors that are related to IPF by regulating target genes which are differentially expressed in specific IPF cell types of affected patients  and co-financed by the European Regional Development Funds, “A way of making Europe” from the European Union; the agreement OA17/008 with Instituto Tecnológico y de Energías Renovables (ITER) to strengthen scientific and technological education, training, research, development and innovation in Genomics, Personalized Medicine and Biotechnology; and by Cabildo Insular de Tenerife (CGIEU0000219140). AA-G was supported by Ministerio de Universidades  and ET-H was supported by fellowship from Agencia Canaria de Investigación, Innovación y Sociedad de la Información del Gobierno de Canarias (TESIS2021010046).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: The pathogenesis of sarcoidosis involves T cells and B lymphocytes that produce autoantibodies. We compared the expression of different T and B cell subsets in sarcoidosis and three B-mediated rheumatic diseases that can affect the lungs in an attempt to identify similarities and differences that distinguish these diseases. Methods: The study included patients referred to Siena University Hospital’s respiratory disease and rheumatology units. Patients were enrolled prospectively and consecutively. Healthy volunteers were also included. Multicolor flow cytometry was performed on phenotype T and B cell subsets. Multivariate analysis was carried out to reduce the dimensionality of the data. Results: Fifteen patients had a diagnosis of sarcoidosis, fourteen idiopathic inflammatory myopathies (IIM), five granulomatosis with polyangiitis (GPA), ten microscopic polyangiitis (MPA), and seven were controls. Thirty-five T and B cell subsets were phenotyped, 15 of which were significantly different in sarcoidosis, B-mediated rheumatic disorders, and controls. Principal components analysis distinguished the four groups of patients with a total explained variance of 54.7%. A decision tree was constructed to determine which clustering variables would be most useful for distinguishing sarcoidosis, IIM, MPA, and GPA. The model showed regulatory T helper cells (Th-reg) > 5.70% in 91% of sarcoidosis patients as well as Th-reg ≤ 5.70 and Th17 > 43.27 in 100% of MPA. It also showed Th-reg ≤ 5.70, Th17 ≤ 43.27 and Tfh-reg ≥ 7.81 in 100% of GPA patients, and Th-reg ≤ 5.70, Th17 ≤ 43.27 and Tfh-reg ≤ 7.81 in 100% of IIM patients. Conclusion: The immune cell profile sheds light on similarities and differences between sarcoidosis and B-mediated rheumatic diseases. Sarcoidosis and autoimmune diseases show similar patterns of cellular immune dysregulation, suggesting a common pathogenic pathway that may provide an opportunity for further understanding autoimmunity and exploring biological therapies to treat sarcoidosis. Diagnoscoidosis . SeveralAmong autoimmune rheumatic disease (ARD), there are ANCA-associated vasculitis (AAV) and idiopathic inflammatory myopathy (IIM), both of which are B-mediated rheumatic disorders. A loss of immune tolerance leads to the activation of the immune system against self-tissues and organs. These disorders can be systemic or organ-specific. ANCA-associated vasculitis in the form of granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) affects the respiratory system, kidneys, and upper and lower extremities . A necroThree factors influence the pathogenesis of the abovementioned ARD: genetic predisposition (associated with major histocompatibility antigens and other immunoreactive molecules), environmental exposure to physical and chemical agents, and infection. Hormonal factors and stressful life events may also play a part in its development. Autoimmune disorders exhibit an inflammatory milieu due to leukocytosis and acute-phase proteins . Together, these findings emphasize the need for comparative studies on sarcoidosis and B-mediated rheumatic diseases for insights into the failure of peripheral tolerance linked to the development of these diseases. However, only one article reported the expression of immune checkpoint molecules  on T- and NK-cells that negatively regulate the T-cell immune function in granulomatous diseases including sarcoidosis and AAV .No data are available on adaptive immune cell profiles shared by ARD and sarcoidosis, despite emerging evidence that these conditions are related in terms of cellular immune dysregulation. Here, we used the relative distribution of different T and B cell subset percentages to find similarities and differences between sarcoidosis and three B cell-mediated rheumatic conditions that can affect the lungs. Adaptive immune profiles were also compared with those of a group of healthy volunteers.Patients referred to Siena Regional Referral Centre for sarcoidosis and to the rheumatology unit of Siena University Hospital were prospectively and consecutively enrolled in the study. The diagnosis of sarcoidosis was confirmed by multidisciplinary discussion according to American Thoracic Society/European Respiratory Society/World Association of Sarcoidosis and Other Granulomatous Disorders (ATS/ERS/WASOG) guidelines. IIM patients were diagnosed according to the 2017 EULAR criteria and/or Peter and Bohan criteria for dermatomyositis, anti-synthetase positivity, and typical clinical features of anti-synthetase syndrome, while AAV patients, including granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA), were diagnosed according to the 2022 ACR/EULAR classification criteria. Patients diagnosed with polymyositis were excluded because no specific diagnostic criteria are available for this disorder which is currently diagnosed by exclusion . Peripheral blood samples were collected from patients for immune profiling. Their demographic, clinical, and radiological data were entered into a prestructured electronic database. Written informed consent to participate in the study was obtained from all patients and healthy controls who had no history of concomitant pathologies, were not on medication, and had normal lung function. The study was approved by the regional ethical review board of Siena, Italy , and complied with the Declaration of Helsinki.Multicolor flow cytometric analysis was performed, and the gating strategy is reported in the The second tube was to detect follicular T cells and included mAbs: CD4-APC-Vio770, CD45RA-PE-Vio770, CCR6-APC, CXCR3-VioBright FITC CXCR5 PerCP-Cy5.5, and CCR4-PE, all from Miltenyi Biotec .® 647, BD, San Jose, CA, USA), CXCR5 PerCP-Cy5, CD8 horizon450, and CD3 APC-Cy7 (OKT3) .The third tube was to phenotype regulatory T cells according to the BD Human Regulatory T Cell Cocktail (CD4-FITC (SK3), CD25-PECy7 (2A3), CD127 Alexa FluorGating was carried out using Kaluza software 2.1 : details are shown in the All values were expressed as the mean, interquartile range (IQR), or standard deviation, as appropriate. The Shapiro–Wilk test was used to check for normal distribution of values. ANOVA  and the Dunn test for multiple comparisons were performed using a non-parametric method. For categorical variables, Fisher’s exact or Chi-squared tests were used to compare proportions between groups. Spearman tests were conducted to determine correlations between clinical and immunological parameters. p-value less than 0.05 was considered statistically significant. Statistical analysis was performed with GraphPad Prism 9.4  and XLSTAT 2021 software .Multivariate analysis was conducted on cell subsets  that differed in a statistically significant way between sarcoidosis, IIM, MPA, and GPA groups. A two-dimensional representation of multidimensional data was used to identify trends in immunological features by supervised principal component analysis (PCA). Four groups  were formed to identify the best thresholds for group classification. Based on the Gini criterion, a classification and regression decision tree were constructed to identify optimal clustering variables. We evaluated the accuracy of binary classifiers by confusion matrix, creating a series of test/training partitions. A Forty-four patients were enrolled consecutively and prospectively at diagnosis; none of them were being treated at the time of sampling. Fifteen patients had sarcoidosis (mean ± standard deviation age 55 ± 11.3 years): nine were women, and there was a prevalence of never-smokers (57%). All sarcoidosis patients were in Scadding radiological stage II, indicating thoracic lymph node enlargement and sarcoid involvement of the respiratory system. Fifteen patients had a diagnosis of AAV . Ten out of fifteen AAV patients had MPA, and 5 out of 15 had GPA. Fourteen patients had a diagnosis of IIM . Five out of fourteen IIM patients had dermatomyositis (3 TIF1gamma-positivity and 2 MDA5-positivity), and 9 out of 14 had anti-synthetase syndrome (6 Jo1-positivity and 3 PL7-positivity). One patient with polymyositis was excluded from the analysis. Eleven healthy volunteers  were also enrolled in the study.p = 0.0035). Follicular Th regulatory cells (Tfh-reg) were less numerous in sarcoidosis and IIM than in MPA  and GPA samples . T effector cells were also less numerous in sarcoidosis than in MPA (p = 0.0259). T naïve cells were more numerous in IIM than in sarcoidosis (p = 0.0136). T helper regulatory cells were more numerous in sarcoidosis than in IIM (p = 0.0019), MPA (p = 0.0033), and GPA samples (p = 0.0029). Follicular Th 17.1 were more numerous in sarcoidosis than in IIM (p = 0.0450). Th1 cell percentages were higher in GPA than in IIM (p = 0.0227) and sarcoidosis (p = 0.0129). They were also higher in MPA than in IIM (p = 0.0127) and sarcoidosis samples (p = 0.0126). Th17 cell percentages were higher in GPA than in IIM (p = 0.0353) and sarcoidosis (p = 0.0435). Immature B cells were more numerous in sarcoidosis than IIM samples (p = 0.0062), MPA (p = 0.0035), and GPA samples (p = 0.0297). T cytotoxic regulatory cells were more numerous in sarcoidosis than in IIM (p = 0.0456), whereas CD8-positive cell percentages were lower in sarcoidosis than in IIM samples (p = 0.0152). Follicular T cytotoxic-17 (Tfc17) cell percentages were higher in GPA (p = 0.0439) and IIM (p = 0.0492) than in sarcoidosis samples.T cytotoxic naïve cells were less numerous in sarcoidosis than in IIM (p = 0.0182). CD3+ cells were less numerous in sarcoidosis than in control samples (p = 0.015). They were also more numerous in MPA (p = 0.0237) and GPA (p = 0.0329) than in control samples. Th effector cells were more numerous in sarcoidosis and MPA than in control samples . Tfh-reg were more numerous in MPA than in control samples (p = 0.0203). Th-reg cells were more numerous in sarcoidosis than in control samples (p = 0.0048) but less numerous in MPA than in control samples (p = 0.0133). Th1 cell subsets were more numerous in GPA and MPA than in control samples . Th17 cell percentages were higher in GPA than in control samples (p = 0.0308). Immature B cells were more numerous in controls than in sarcoidosis patients (p = 0.038). Tc naïve cells were less numerous in the control samples than in MPA samples (p = 0.0151). CD8+ cells were more numerous in the control samples than in the sarcoidosis samples (p = 0.0302). Tc1 cell percentages were lower in controls than in MPA (p = 0.0474) and GPA patients (p = 0.0363). Tc effector cells were more numerous in sarcoidosis than in control samples  was conducted to identify the best clustering variables that can distinguish between the groups of sarcoidosis, IIM, MPA, and GPA . The modAn important objective of the present preliminary study was to determine the differences and similarities in immune profiles between sarcoidosis patients and patients with IIM and AAV. We also compared all profiles with those of healthy controls. Multivariate analysis of the data reduced the number of dimensions, and a total variance of 54.7% was obtained based on T and B cell subsets. The total variance was determined by a classification and regression tree, showing good clustering variables for sarcoidosis, IIM, MPA, and GPA. Treg cell percentages clustered 91% of sarcoidosis patients with a cut-off of 5.7%, while 100% of IIM, MPA, and GPA patients were clustered on the basis of Treg, Th17, and Tf-reg cell percentages.Sarcoidosis and ARD patients often have lymphopenia. We found lower CD3 and CD8 cell percentages in sarcoidosis than in IIM and control samples; T cells were also below the normal range of controls in MPA and GPA patients. + population in circulating memory CD4 T cells since this strategy is often conducted prior to analyzing subsets of Tfh1, Tfh2, or Tfh17 [The immune response to inflammatory diseases is generally regulated by B cells . In lineor Tfh17 .The helper and cytotoxic subsets of T follicular cells (Tfh and Tfc) expressed the main Th17-cell chemokine receptors implicated in chronic inflammatory conditions. This population of cells is referred to as Tfh17 and Tfc17. Autoimmune diseases typically have a preponderance of Tfh17 cells . AlthougAlthough they are transcriptionally and phenotypically distinct from other CD8 T cell subsets, Tfc cells may play a role in antibody-mediated autoimmune disease progression. There is limited information available about Tfc distribution in sarcoidosis and acute lung diseases. Recently, Zhai et al. demonstrated elevated Tfc cell percentages in patients with primary Sjogren syndrome, especially between those with lung involvement and controls . A wide variety of effector subsets are generated by T cells. They include Th cells, T follicular cells (Tfc), and regulatory T cells (Tregs). T cells, Tfc, and Tregs stimulate target cells, whereas Th cells, Tfs, and Tregs suppress them . As a subpopulation of T cells, Tregs control the immune system, maintain tolerance to self-antigens, and prevent the occurrence of autoimmune diseases. The role of Tregs in sarcoidosis pathogenesis has long been suspected, although research findings are inconsistent . CD127 (IL-7Ra) is highly expressed by naive T cells, which are precursors of effector and memory T cells . T cellsThe expression of IL-7Rα on CD4 T cells implies the proliferation of T cell receptors. Indeed, its lacking and expression of CD25 result in anergic and regulatory CD4 T cells . Since tARD has been associated with significant reductions in Th-reg levels and the production of autoantibodies . The proThe innate immune system is activated in sarcoidosis and ARD, leading to T reg cell dysfunction and elevated Th-17 cell count in peripheral blood . ŻabińskDespite the value of this new data on the immunological pathways involved in sarcoidosis and B-mediated rheumatic diseases, our study has several limitations: (1) the IIM cohort was small and heterogeneous; (2) our findings are in need of validation for other autoimmune rheumatic disorders by multicentric prospective studies such as rheumatoid arthritis in order to consider in differential diagnosis; (3) the study population was too small to detect any correlations between immunological features and disease activity or severity; (4) there was a lack of lymphocyte subset total counts.The etiology and pathogenesis of sarcoidosis remain largely uncharacterized despite extensive research. Autoantigen-specific T cells and autoantibody-producing B lymphocytes may be a sign of autoimmune inflammatory reactions in sarcoidosis due to a number of triggers that contribute to the development of the disease. Our immune cell profiles shed light for the first time on the similarities and differences between sarcoidosis and B-mediated rheumatic diseases. The observation of similar cellular immune dysregulation patterns in sarcoidosis and autoimmune diseases suggests common pathogenetic pathways that may offer an opportunity for further study of autoimmunity and the development of biological therapies to treat sarcoidosis .


Deep mutational scanning (DMS) is a high-throughput experimental technique that measures the effects of thousands of mutations to a protein. These experiments can be performed on multiple homologs of a protein or on the same protein selected under multiple conditions. It is often of biological interest to identify mutations with shifted effects across homologs or conditions. However, it is challenging to determine if observed shifts arise from biological signal or experimental noise. Here, we describe a method for jointly inferring mutational effects across multiple DMS experiments while also identifying mutations that have shifted in their effects among experiments. A key aspect of our method is to regularize the inferred shifts, so that they are nonzero only when strongly supported by the data. We apply this method to DMS experiments that measure how mutations to spike proteins from SARS-CoV-2 variants  affect cell entry. Most mutational effects are conserved between these spike homologs, but a fraction have markedly shifted. We experimentally validate a subset of the mutations inferred to have shifted effects, and confirm differences of > 1,000-fold in the impact of the same mutation on spike-mediated viral infection across spikes from different SARS-CoV-2 variants. Overall, our work establishes a general approach for comparing sets of DMS experiments to identify biologically important shifts in mutational effects. Deep mutational scanning (DMS) is a high-throughput experiment that measures the effects of thousands of mutations to a protein , 2. It hHowever, when comparing DMS experiments, a key challenge is determining whether observed differences in mutational effects are due to real biological signal or the noise inherent in any high-throughput experiment. Previous studies have addressed this challenge by separately inferring mutational effects in each experiment, and then trying to identify mutations with differences between experiments that are significantly larger than the experimental noise . Howeverlasso — for inferring compressed or structured models , receptor-binding domain (RBD), and regions involved in conformational dynamics and inter-protomer packing. These sites tend to cluster near each other in spike’s 3D structure, but are often far away from non-identical sites that differ in amino-acid sequence between homologs, suggesting that many shifts are due to long-range epistasis. We experimentally validate a subset of the inferred mutational shifts, identifying some mutations that differ in their effect on spike-mediated viral entry by > 1,000-fold between homologs.We assume the DMS data report an experimentally measured functional score for each protein variant from each homolog or condition, where a variant corresponds to a unique protein sequence covering the entire mutagenized region. This requirement is usually satisfied by DMS experiments that either sequence the entire length of the mutagenized gene, or use barcoding to link full gene sequences to barcodes , 30. Eacreference experiment — and shifts in mutational effects in each of the other experiments relative to the reference , and a lasso regularization term that encourages the inferred shifts to be zero. Specifically, for each shift parameter, the lasso term adds a penalty that scales linearly with the absolute value of the shift. If a shift in the effect of a specific mutation is strongly supported by the experimental data, then inferring a nonzero shift for this mutation will decrease the loss term enough to overcome the regularizing effect of the lasso term. How strongly a shift is supported by the data is influenced by multiple factors, including whether the shift substantially minimizes the loss term for an individual variant, an example of which is illustrated in https://github.com/matsengrp/multidms for the code; see https://matsengrp.github.io/multidms/ for the documentation.We implemented the model described above in a Python package called multidms. See We applied the above approach to infer shifts in mutational effects between three homologs of the SARS-CoV-2 spike protein: Delta, Omicron BA.1, and Omicron BA.2. The Delta and BA.1 homologs are separated by 43 amino-acid mutations and indels (97% identity), while the two Omicron homologs are separated by 27 mutations (98% identity). Given the high percent identity, we expected most mutations to have similar effects among homologs.As input to our analysis, we used previously published DMS data on how mutations to the spikes of Delta and BA.1 affect spike-mediated viral entry in the context of pseudotyped lentiviruses , as wellWe fit a single multidms model for the three homologs, using just one of the DMS experiments for each homolog. We used BA.1 as the reference because it had the lowest level of noise , but fouWe analyzed the inferred mutational effects, focusing on the 5,934 mutations seen at least once across all three homolog DMS experiments. Because we used BA.1 as the reference, the inferred Both https://matsengrp.github.io/SARS-CoV-2_spike_multidms/spike-analysis.html#shifted-mutations-interactive-altair-chart for an interactive heat map that enables more detailed analyses of the mutational shifts.Sites with shifted mutational effects occurred across the length of spike . SeveralWe experimentally validated the inferred shifts in mutational effects using spike-pseudotyped lentiviral particles . SpecifiAs expected, some mutations inferred to have undergone large shifts in their effects caused large changes in viral titer in some homologs . In geneThe most striking shift was for mutation A419S, which is highly deleterious in BA.1 and BA.2 , but is nearly neutral in Delta. The mechanistic basis of this shift is easily understood: A419S introduces an Nlinked glycan at 417 in BA.1 and BA.2 (which have N417), but not in Delta (which has K417). This glycan greatly reduces ACE2 affinity , 17, makThe other validated mutations showed a similar pattern, where each mutation was highly deleterious in at least one homolog and substantially less deleterious in at least one other, with specific patterns differing by mutation. In nearly all cases, the inferred shifts in latent phenotype matched the experimentally measured shifts in effects on viral titer. These mutations occur in multiple regions of spike. D142L is in one of multiple loops in the NTD that form an antigenic supersite  and helpThe validated mutations were often deleterious in BA.1 and considerably less deleterious in Delta due to large positive shifts. In general, shifts in Delta tended to be positive , and theIn an effort to further validate the inferred shifts, we compared our inferences to ones that we computed from other studies, including DMS experiments of RBD homologs measuring mutational effects on ACE2 binding and RBD expression on the surface of yeast , 17, andSites with strongly shifted mutations tended to cluster in three-dimensional space  and B. WWe describe a general method for jointly fitting a single model to multiple DMS experiments to identify mutations that have shifted effects across homologs or selective conditions. Algorithmically, the method is essentially an extension of globalepistasis models  to multiSI Appendix and online documentation for multidms suggest strategies for normalizing functional scores between experiments to help make them comparable.Our method does make several assumptions. First, the joint-modeling approach assumes that most mutations have similar effects between experiments. This approach would not make sense if many mutations are expected to have large shifts, which can occur when comparing highly divergent homologs , 19. SecSI Appendix). Further, we developed an open-source software package with comprehensive documentation so that others can easily use our method. Altogether, this method could greatly accelerate future use of DMS to identify mutations with biologically interesting shifts in effects.Despite the above assumptions, we envision that our method could be applied to many future studies comparing DMS experiments. Most DMS experiments have appreciable levels of noise, necessitating a method to account for noise when comparing them. Our method enables the use of global-epistasis models to analyze libraries with multiple mutations per variant, but is also compatible with libraries that only have a single mutation per variant .The multidms Python package is available via the Python Package Index (PyPI). It provides tools for processing functional scores from DMS data, fitting a multidms model to the data, and generating plots for analyzing the results. The core models and optimization algorithms are implemented using the JAX and JAXopt packages, enabling automatic differentiation and just-in-time compilation for high performance on CPU and GPU , 48. TheFor the full analysis pipelines used to generate functional scores from raw DMS data for a given spike homolog, see:https://dms-vep.github.io/SARS-CoV-2_Delta_spike_DMS_REGN10933/Delta : https:/https://dms-vep.github.io/SARS-CoV-2_Omicron_BA.1_spike_DMS_mAbs/BA.1 : https:/https://dms-vep.github.io/SARS-CoV-2_Omicron_BA.2_spike_DMS/BA.2 (from this study): https://github.com/matsengrp/SARS-CoV-2_spike_multidms) with all code used to curate the above DMS data, fit multidms models to these data, and make all figures in the paper. The code, as well as a step-by-step explanation of the analysis pipeline, is in a single Jupyter Notebook, an HTML version of which can be viewed at https://matsengrp. github.io/SARS-CoV-2_spike_multidms/ . For instance, variants with stop codons tended to have more negative functional scores in the BA.1 and BA.2 experiments compared with Delta. To help make scores more comparable between homologs, we truncated all functional scores from all experiments at a lower bound of −3.5 and an upper bound of 2.5 . The lowThe DMS experiments were performed with at least two biological replicates per homolog, where each replicate experiment used an independently synthesized barcoded variant library. Each of the Delta and BA.2 replicate experiments were performed with two technical replicates, and we combined all functional scores between pairs of technical replicates into a single dataset. For variants associated with multiple unique barcodes in a single biological replicate dataset, we averaged the variant’s score across all barcodes. This averaging step increased the speed of model fitting without substantially changing the final results (data not shown).Some sites in the spike protein were mutated in one or two of the homolog DMS libraries, but not all three. For instance, due to indels, some sites that are present in one homolog are completely missing in another. Since it is not possible to compute shifts across all homologs at such sites, multidms automatically discards all variants with mutations at any of these sites.We fit a single multidms model to one biological replicate dataset per homolog, using BA.1 as the reference, and using 30,000 proximal gradient iterations to allow the Huber loss term to converge . We theneplicate .The Results section reports data from multidms models fit using a regularization weight of λ=5×10-5 . In contWe also compared weights based on the model’s ability to predict experimentally measured functional scores in the training data, as quantified by the loss function used to train the model, not including the lasso term . As expe−5. This pattern is consistent with the hypothesis that the shift parameters from each replicate are overfit to their corresponding datasets at low weights, and that increasing the weight tends to reduce overfitting, leading to a higher correlation. Of note, at the highest tested weights, mutations that we experimentally validated to have large shifts were inferred to have shifts near zero, indicating that these weights are too strong. The correlation of We also quantified the correlation of shift parameters between the replicate model fits as a function of lasso weight . In eachrameters .In all, the above lines of evidence suggest that a lasso penalty https://github.com/dms-vep/SARS-CoV-2_Omicron_BA.2_spike_DMS/tree/main/library_design/plasmid_maps. Pseudoviruses were generated using a method described previously (Spike genes with desired mutations were introduced using PCR with overlapping mutation-carrying primers followed by HiFi assembly. Plasmids used as Delta, BA.1 and BA.2 spike templates can be found at eviously  with theSupplement 1


The main types of PD-L1 in the blood include soluble PD-L1 (sPD-L1), exosomal PD-L1 (exoPD-L1), and PD-L1 in circulating tumor cells (CTCs). However, the predictive and prognostic values of these three indicators in patients with non-small cell lung cancer (NSCLC) undergoing immune checkpoint inhibitor (ICI) therapy are unclear, warranting a systematic meta-analysis.P < 0.05 was regarded as significantly different.A systematic literature search was performed in the PubMed, Cochrane Library, and Embase databases. The pooled hazard ratio (HR) and 95% confidence interval (CI) values were extracted from the included studies to investigate the correlation between the three PD-L1 indicators and overall survival (OS) or progression-free survival (PFS). The Newcastle–Ottawa Scale (NOS) was used to examine the quality of the included studies. Subgroup analyses were employed to investigate the heterogeneity. The publication bias of the included studies was assessed using Begg's and Egger's tests. P < 0.001) and PFS . However, dynamic changes in sPD-L1 after immunotherapy were not statistically significant for OS  or PFS . Meanwhile, the upregulated pre-treatment exoPD-L1 levels were significantly associated with poor PFS , whereas the post-treatment dynamic upregulation of exoPD-L1 was significantly correlated with superior PFS  and OS . For PD-L1 in CTCs, the pooled results indicated that PD-L1 expression in CTCs was not significantly correlated with OS  and PFS .The pooled results revealed that high pre-treatment sPD-L1 levels were significantly associated with inferior OS (HR = 2.32, 95% CI = 1.68–3.18, Blood-based PD-L1 analysis is a potential strategy for predicting treatment efficacy and prognosis in patients with cancer.The online version contains supplementary material available at 10.1186/s12957-023-03215-2. Among the malignant tumors, lung cancer, a global public health issue, is the most frequent cause of tumor-related deaths globally . Small cImmune checkpoint inhibitors (ICIs) have improved the survival outcomes of patients with cancer . The US + CTCs) after immunotherapy were significantly and positively correlated with progression-free survival (PFS) [The use of blood-based biopsies to analyze PD-L1 expression in patients with cancer can overcome the challenges associated with the use of solid tumor biopsies. Compared with tissue biopsies, blood-based biopsy techniques have several advantages, including ease of sampling and reproducible monitoring of the dynamic biological changes in vivo . Some stal (PFS) , 27. Howal (PFS) . The rolhttps://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42023402077.The recommended reporting criteria for the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines were followed to perform the meta-analysis. The study protocol was prospectively registered at PROSPERO (CRD42023402077) and is available at Relevant literature in the English language published up to February 23, 2023, was retrieved from the PubMed, Embase, and Cochrane Library databases. The following keywords were used: "non-small cell lung cancer," "lung cancer," "lung tumor," "lung neoplasm," "immune checkpoint inhibitor," "PD-1," "PD-L1," "anti-PD-1," "PD-L1," "programmed death 1 receptor," "programmed death ligand 1," "cytotoxic T lymphocyte antigen 4," "CTLA-4," "blood," "circulating tumor cell," "PD-L1," "soluble PD-L1," "exosomal PD-L1," "circulating PD-L1," "PD-L1 in CTCs," "sPD-L1," and "exoPD-L1." The references in the reviews were manually searched to identify additional related articles.Studies on patients with pathologically diagnosed primary or metastatic NSCLC; studies on patients undergoing ICI mono-therapy or ICI com-therapy (chemotherapy or targeted therapy); studies evaluating the correlation between PD-L1-based blood markers  and OS/PFS of patients with NSCLC; studies reporting the hazard ratio (HR) and 95% confidence interval (CI). If the HR was not directly available from studies, it was extracted from the Kaplan–Meier curve. When the univariate and multivariate analysis results were provided simultaneously, only the multivariate analysis results were extracted.Case reports, reviews, meta-analyses, conference abstracts, and letters; studies in which the full text was not available for download; studies that included animal experiments; studies in non-English language.Based on selection, comparability, and outcomes, the quality of the included studies was evaluated using the Newcastle–Ottawa scale (NOS). The NOS quality score ranged from 0 to 9. Studies with NOS scores of ≥ 7 and < 7 were categorized as high-quality and low-quality studies, respectively .Information, such as the name of the first author, the publication year, the region, the sample size, the age, the gender, the cancer stage, the cut-off value, the time point, the study design, and the outcome, was retrieved from each included study. Two authors (Qian Cui and Dong Wang) independently extracted data from univariate and multivariate analyses with HR and 95% CI for OS or PFS. Any controversy was addressed via discussion.I2 combined values were applied to analyze the heterogeneity of the results [P > 0.10 and I2 < 50% indicated low-level heterogeneity [P < 0.05. All statistical analyses were performed using Stata 15.1 software.The HR and 95% CIs were extracted to assess the association between these three blood-based PD-L1 indicators and survival outcomes of patients with NSCLC undergoing ICI therapy. Additionally, the association between clinicopathologic features and survival outcomes was evaluated by the HR and 95% CI. The HR values of > 1 and < 1 were considered adverse and favorable prognoses, respectively. Cochran's q-test and  results . P > 0.1ogeneity . SubgrouThe HR and 95% CIs were extracted to assess the association between these three blood-based PD-L1 indicators and survival outcomes of patients with NSCLC undergoing ICI therapy. Additionally, the association between clinicopathologic features and survival outcomes were evaluated by the HR and 95% CI.Literature searched in the PubMed, Embase, and Cochrane Library databases yielded 521 studies. Of these, 490 studies were excluded as they were duplications, animal studies, commentaries, and incomplete full texts. The complete texts of the remaining 36 articles were comprehensively evaluated. Of these, 22 studies were excluded, and 14 studies were included in this meta-analysis , 31–38. As shown in Table P < 0.001), and a significant heterogeneity was not observed among studies . As shown in Fig. P < 0.001), and these studies had low-level heterogeneity .As shown in Fig. P < 0.001; Table P < 0.001; Table P > 0.05; Table P > 0.05; Table I2 = 43.3%, p = 0.171).Subgroup analysis was performed based on the correlation of sPD-L1 with OS and PFS according to the time point, sample size, and region. No significant heterogeneity was observed in most subgroups Table . StratifP = 0.170). A moderate level of heterogeneity was observed among studies . As shown in Fig. + CTCs appeared to have a longer PFS than those with negative PD-L1 expression in CTCs, but this difference did not achieve statistical significance . A low level of heterogeneity was observed between studies .As shown in Fig. P > 0.05; post-treatment: HR = 0.83, 95% CI 0.35–1.98, P > 0.05; Table P > 0.05; post-treatment: HR = 0.95, 95% CI 0.50–1.80, P > 0.05; Table +CTCs were associated with superior OS when the sample size was ≤ 30 , and there was a high level of heterogeneity . The relationship between the post-treatment dynamic upregulation of exoPD-L1 and OS in patients with NSCLC undergoing ICI therapy was reported only by Yang et al. In particular, the fold-increasing changes of exoPD-L1 were significantly associated with longer OS .Two studies demonstrated the relationship between exoPD-L1 and prognosis in patients with NSCLC undergoing ICI therapy. Of these, one reported the correlation of exoPD-L1 with both OS and PFS, while another involved the correlation with only PFS. As shown in Fig. P < 0.001; Fig. P < 0.001; Fig. As studies on the correlation of pre-treatment exoPD-L1 with OS were not retrieved, subgroup analysis was performed with data on exoPD-L1 and PFS after different treatment durations. The results of the subgroup analysis revealed that the difference in treatment duration was the source of heterogeneity. The heterogeneity decreased to 0% in both the pre-treatment and post-treatment subgroups Fig. b. High pFive studies , 31, 33 P > 0.05)  and PFS . The subgroup analysis results revealed that high pre-treatment levels of sPD-L1 were significantly associated with poorer OS  and PFS . However, after immunotherapy, the upregulation of sPD-L1 was not significantly correlated with OS  or PFS . This result suggests that high pre-treatment levels of sPD-L1 can be considered a biomarker to predict the prognosis of NSCLC patients receiving ICIs. These findings are consistent with those of Han et al., who reported that lower pre-treatment sPD-L1 levels predicted improved PFS and OS [The analysis revealed that high levels of sPD-L1 were significantly correlated with worse OS  or PFS . Furthermore, pre-treatment and post-treatment subgroup analyses did not reveal significant differences. These results suggest that PD-L1+ CTCs may not be correlated with the prognosis of patients with NSCLC undergoing ICI therapy. Of the six included studies, three were pre-immunotherapy studies. Guibert et al. demonstrated that the PD-L1+ CTCs before immunotherapy were not significantly correlated with prognosis, but this correlation was significant in patients with treatment failure and disease progression [+ CTCs before immunotherapy and PFS [n = 17). However, Zhang et al. demonstrated that pre-treatment PD-L1+ CTCs were positively correlated with PFS and OS in patients with NSCLC treated with immunotherapy plus chemotherapy [+ CTCs have significantly longer PFS after immunotherapy [Recently, several studies have proposed that high expression of PD-L1 in CTCs is associated with a poorer prognosis in patients with various cancers treated with other therapies, such as chemotherapy or surgery , 49, 50.gression . Dhar et and PFS . This caotherapy . The resotherapy , 37. Howotherapy .P < 0.001; Fig. P < 0.001) and OS . In conclusion, the role of exoPD-L1 in determining the prognosis was reversed with the increase in treatment duration. Similarly, one study on solid tumors treated with anti-PD-1 therapy reported that the high pre-treatment exoPD-L1 levels predicted a short PFS [In this meta-analysis, only two studies related to exoPD-L1 were included. The pooled results showed that elevated pre-treatment exoPD-L1 levels were significantly linked to poorer PFS (HR = 4.44, 95% CI 2.87–6.89, hort PFS –56. Tumohort PFS . Additiohort PFS . Chen ethort PFS . Circulahort PFS . Howeverhort PFS , 26, 59.hort PFS . During hort PFS . This fihort PFS . Exosomehort PFS . Notablyhort PFS , 64.This meta-analysis has several limitations. First, the number of included studies and the sample size were small. Small-sample studies may overestimate the results of efficacy and prognostic evaluations. Second, the pooled results revealed that post-treatment sPD-L1 levels and PD-L1 expression in CTCs were not correlated with the prognosis of patients with NSCLC undergoing ICI therapy. This can be attributed to the high heterogeneity among the studies. Most studies have no definite consensus on the cut-off values of biomarkers and utilize various assay platforms, which may have contributed to the high heterogeneity of these studies and biased outcomes. Therefore, a criterion threshold must be established through a uniform and standardized protocol. In the future, the correlation of PD-L1 expression in CTCs and post-treatment sPD-L1 levels with the prognosis of NSCLC patients receiving ICI therapy needs to be validated using large-sample and multi-center clinical trials. Third, only three indicators were included in the analysis. EVs and some immune cells in the blood also contain PD-L1, such as dendritic cells and myeloid-derived suppressor cells , which wThe comprehensive meta-analysis of three blood-based PD-L1 indicators suggested that pre-treatment sPD-L1 and exoPD-L1 serve as unfavorable prognostic factors in patients with NSCLC undergoing ICI therapy. In contrast, the dynamic upregulation of post-treatment exoPD-L1 levels indicates a favorable prognosis. However, post-treatment sPD-L1 levels and PD-L1 expression in CTCs were not correlated with the prognosis of patients with NSCLC undergoing ICI therapy. The blood-based PD-L1 analysis can provide clinicians with a convenient biomarker and complement tPD-L1 analysis to improve the clinical application of ICIs. It is also a potential strategy for predicting treatment efficacy and prognosis in patients with cancer.Additional file 1. PRISMA Checklist.Additional file 2. Full search strategies.Additional file 3: Table S1. Correlation of OS/PFS and clinicopathological characteristic of NSCLC patients. Table S2. Results of Begg's and Egger's tests of 3 indicators related to PFS/OS. Fig.S1. Results of Begg's and Egger's tests of 3 indicators related to PFS/OS a, Beeg’s and Egger’s between sPD-L1 and OS; b, Beeg’s and Egger’s between sPD-L1 and PFS; c, Beeg’s and Egger’s between PD-L1 in CTCs and OS; d, Beeg’s and Egger’s between PD-L1 in CTCs and PFS; e, Beeg’s and Egger’s between exoPD-L1 score and PFS.


Aim: We aimed to evaluate the performance of Chat Generative Pre-trained Transformer (ChatGPT) within the context of inflammatory bowel disease (IBD), which is expected to become an increasingly significant health issue in the future. In addition, the objective of the study was to assess whether ChatGPT serves as a reliable and useful resource for both patients and healthcare professionals.Methods: For this study, 20 specific questions were identified for the two main components of IBD, which are Crohn's disease (CD) and ulcerative colitis (UC). The questions were divided into two sets: one set contained questions directed at healthcare professionals while the second set contained questions directed toward patients. The responses were evaluated with seven-point Likert-type reliability and usefulness scales.Results: The distribution of the reliability and utility scores was calculated into four groups (two diseases and two question sources) by averaging the mean scores from both raters. The highest scores in both reliability and usefulness were obtained from professional sources . The ranking in terms of reliability and usefulness, respectively, was as follows: CD questions (4.70±1.26 and 4.75±1.06) and UC questions (4.40±1.21 and 4.55±1.31). The reliability scores of the answers for the professionals were significantly higher than those for the patients . Conclusion: Despite its capacity for reliability and usefulness in the context of IBD, ChatGPT still has some limitations and deficiencies. The correction of ChatGPT's deficiencies and its enhancement by developers with more detailed and up-to-date information could make it a significant source of information for both patients and medical professionals. Inflammatory bowel disease (IBD) is a group of disorders that affect the gastrointestinal system and are characterized by chronic and complex clinical conditions. Crohn's disease (CD) and ulcerative colitis (UC) constitute the two main disease categories within IBDs] . These dDue to the recent advancements in technology and artificial intelligence (AI), numerous potential solutions are being targeted to improve the outcomes of many health problems. AI is defined as a technology that possesses the ability to solve complex problems akin to human thinking and to mimic human cognitive processes . AI consIn recent years, large language models (LLMs) have shown promise in addressing human-like language understanding and respond to research questions due to their training on extensive textual data . Chat GeIn this study, our aim was to evaluate the performance of ChatGPT within the context of IBD, which is expected to become an increasingly significant health issue in the future. In addition, the objective of the study was to assess whether ChatGPT serves as a reliable and useful resource for both patients and healthcare professionals.This article was previously posted to the Research square preprint server on August 18, 2023.For this study, specific questions were identified for the two main components of IBD, which are CD and UC. A total of 20 questions were selected, with 10 questions for each disease. The aim was for the first five questions to be the most frequently asked questions by patients for both diseases, CD and UC. In this context, for each disease, separate Google Trends searches were conducted on June 24, 2023, to identify the top five most commonly searched keywords related to these diseases. Trends in search terms were identified based on global results between 2004 and the present day in the health subcategory. In the results, the "most relevant" option was selected in the "relevant questions" section. According to the search results, the most searched keywords on Google for both diseases were identified. Repetitive keywords with similar meanings were excluded. Questions were prepared based on these keywords, covering aspects, such as the nature of the disease, its causes, symptoms, treatment, and diet. The next five questions for each disease were planned to be directed toward healthcare professionals. These questions were generated by a committee of four gastroenterologists, led by an expert gastroenterologist with over 15 years of experience in the field of IBD. All of the researchers were working in different clinics. The expert gastroenterologist has authored numerous articles and has more than 1000 follow-up IBD patients. These questions were related to the classification, diagnosis, activity, poor prognostic indicators, and complications of the disease. The questions devised for both diseases were inputted into the "prompt" section of the ChatGPT AI chatbot. During the conversation, the next question was rewritten by different users in separate sessions. This approach was implemented to ensure that the response given to each question in the conversation section was not influenced by the previous question or response. Each answer given was recorded in a separate file. The answers given by ChatGPT-4 were obtained from the March 14th premium version.Each chatbot was graded according to a scale of 1-7  across two categories for reliability and usefulness . These sThe study adhered to the ethical standards outlined in the Helsinki Declaration and complied with national regulations in the respective field. Since the study did not involve the use of human or animal data, ethics committee approval was not necessary.Statistical analysisThe statistical analyses were performed using Statistical Package for the Social Sciences  software package. The inter-rater compliance was assessed with Cronbach's α and 95% confidence intervals (CI). According to intraclass correlation coefficient results, positive values ranging from 0 to 0.2 indicate poor agreement; 0.2 to 0.4 indicate fair agreement; 0.4 to 0.6 indicate moderate agreement; 0.6 to 0.8 indicate good agreement; and 0.8 to 1 indicate very good agreement. The variables were evaluated using the Shapiro-Wilk test to determine whether or not they exhibited a normal distribution. In the descriptive statistics, the data were expressed as mean ± standard deviation. Independent t-test was used to compare two groups' difference, and statistical significant difference among the groups was performed by an analysis of variance test. The significance level for this study was set at p<0.05.In total, 20 different questions were presented to the OpenAI chatbot. The first five questions were for patients and caregivers for UC and CD, and the next five questions were highly specific for medical professionals for both diseases. The Supplementary Material of the study include an extensive list comprising all the questions and their corresponding answers.We had two experts evaluate the responses given by ChatGPT on UC and CD. The results of the evaluation for questions related to reliability and usefulness are shown in Table The inter-rater Cronbach's α values for both reliability and usefulness scores showed very good agreement . The question topics were rated using Likert scores ranging from 3 to 7. In terms of topics, the highest reliability score (both raters: point 7) was for CD (classification) and the highest usefulness score  was for UC (poor prognostic factors) and CD (classification). Both raters assigned the lowest reliability score (point 3) to questions related to UC (causes and diagnosis) and CD (treatment). Similarly, they rated questions about UC  and CD (treatment) with the lowest usefulness score (point 3).The total scores of the topics and their evaluation by each rater are shown in Table The reliability total scores of the answers according to patients and professionals and their evaluation by each rater are shown in Table The distribution of the reliability and utility scores was classified into four groups  by averaging the mean scores from both raters, as shown in Figure The main finding of this study is that OpenAI's ChatGPT conversation bot, which is an LLM, exhibits partial reliability and usefulness in the field of IBD, with some limitations. Another significant finding from our study is that ChatGPT could be a more reliable source for healthcare professionals compared to patients.In the study, the authors assigned the highest validity score of seven points to the CD classification section. Because this response is consistent with the British Society of Gastroenterology (BSG) guideline, it received the same rating from both raters . In our In a recently published study evaluating gastrointestinal diseases in patients, using a scale of 1 to 5 for rating, the accuracy and clarity of CD treatment were rated 4 and 3, respectively, while the treatment for UC received a score of 5 for both accuracy and clarity. In the supplementary file of that study, we also identified that ChatGPT's response to the same question was different from the response in our study . This siİn terms of usefulness, response to the related poor prognostic indicators were rated with the highest score for UC, whereas for CD disease, response to classification was rated with the highest score. To provide an example, it was observed that the Montreal criteria  for CD cThere are studies in the literature that indirectly assess the performance of ChatGPT in IBD by evaluating it among all gastroenterological conditions. In a study where the four parameters were evaluated with scores ranging from 1 to 5, it has been reported that ChatGPT responded to IBD with an average mean score close to 5 in the relevant and clarity parameters . This stAnother research focus of our study was to compare the proficiency among users of the LLM. Some of the studies in the literature related to ChatGPT have examined the responses given to typical patient questions ,11, whilThe first limitation of this study was the restricted number of evaluators to assess ChatGPT's performance and the use of subjective evaluations for ratings. An evaluation by a committee with a larger number of experts specializing in the field of IBD could provide a clearer assessment of ChatGPT's performance in this disease. The second limitation, even though we categorized the questions in the study as general questions from Google sources and detailed questions from guideline sources to differentiate between patients and medical professionals, is likely that every user might ask any question.Despite its capacity for reliability and usefulness in the context of IBD, ChatGPT still has some limitations and deficiencies. The correction of ChatGPT's deficiencies and its enhancement by developers with more detailed and up-to-date information could make it a significant source of information for both patients and medical professionals. Developers should definitely carry out the necessary updates and enhancements in this regard, paying attention to ensuring that the information they provide to users comes from reliable sources.


Graphene nanoplatelets (GNPs) were prepared using the electrolytic exfoliation method on graphite foil in an ammonium sulfate solution. A series of experiments were conducted in order to optimize the production of the flakes by varying the pH of the solution, applied voltage and current, duration of electrolysis, temperature in the electrolytic system, and type and duration of the ultrasound interaction. The quality of the produced graphene nanoplatelets was analyzed using X-ray diffraction, Raman and IR spectroscopy, and TEM. These σ bonds are responsible for the high mechanical strength of the material [Graphene is a two-dimensional 2D) material that is one atom thick and was first isolated by Geim and Novoselov in 2004 . Since tD materiamaterial . It is wmaterial ,6,7. Thematerial . A membrmaterial ,9. Anothmaterial . In addimaterial . The elematerial .N,N-dimethylformamide (DMF)) [8. The KC8 was then reacted with ethanol in order to form potassium ethanolate, thereby causing exfoliation and resulting in the formation of a carbon monolayers. The byproduct of this process was hydrogen, which helped to separate the single graphene layers from each other. A similar method based on the switching voltage technique and eutectic molten salts of NaOH and KOH was applied for graphite exfoliation by Alshamkhani et al. [42− anions [Graphene can be prepared in various ways, differing in price, ease of fabrication, and quality of the obtained product. The most common synthesis methods include chemical vapor deposition (CVD) ,14,15, me (DMF)) . Anothere (DMF)) . The scii et al. . The thii et al. . Comparei et al. ,29. Thisi et al. . The advi et al. . On the i et al. ,33. The − anions ,35.In the presented study, a combination of two types of liquid-phase exfoliation was used to produce graphene: electrochemical and ultrasonic extraction. In order to obtain a product of the best possible quality, parameters such as process duration, applied current and voltage, pH of the solution, time and type of the ultrasound interaction, and electrolyte temperature were modified. To the best of our knowledge, this is the first paper where systematic research has been undertaken to define the influence of the mentioned factors on the quality and quantity of the obtained graphene during electrolysis process.4)2SO4, Sigma Aldrich, ≥99.0%), pure graphite blocks, and graphite foils  were used as starting materials. In order to change the pH of the electrolyte, nitric acid , ammonia solution , and sodium hydroxide  were used. The solutions were made with the use of distilled water with a conductivity of 0.51 μS/cm and pH = 7. The graphene nanoplatelet reference material was produced by ACS MATERIAL . Aqueous solutions of acetone  and ethanol  were used in the ultrasonification stage. Hard filters type 380 with pore sizes in the range of 3–5 µm were used for filtration.Ammonium sulfate  were measured using a Nicolet iS50 infrared spectrometer using a KBr pellet method. The spectral resolution was 2 cm−1. The morphology of the flakes was studied using a Philips CM-20 SuperTwin transmission electron microscope with an accelerating voltage of 200 kV and a resolution of 0.24 nm. Scanning electron microscopy (SEM) FEI NovaNanoSEM 230 (USA) was used to reveal the graphene nanoplatelet thickness obtained at different voltages. The oxygen content in the GNPs was measured using an FESEM FEI Nova NanoSEM 230 scanning electron microscope equipped with an EDS spectrometer (EDAX Genesis). The zeta potential of the aqueous suspension of graphene nanoplatelets was measured and analyzed under the same conditions  using the a Zetasizer Nano ZS . The materials were dried in a FD-10-R Vacuum Freeze-Dryer from LABFREEZ .In the electrolysis process, a laboratory power supply from STAMOS (Model: S-LS-38) with a voltage regulated in the 0–30 V range and a current regulated in the 0–20 A was used to adjust the current flow through the electrolytic system. Liquid-phase exfoliation was obtained by the TF-900N sonicator by Tefic Biotech and the Proclean 3.0DSP ultrasonic cleaner by Ulsonix. XRD measurements were carried out on an X-ray diffractometer  with Cu Kα monochromatic radiation (λ = 1.5406 Å), scanning the diffraction angles (2θ) between 10° and 55° at room temperature in Bragg–Brentano geometry. The measurements of Raman spectra were performed with a Renishaw inVia Microscope Raman spectrophotometer equipped with a CCD detector. The tests were performed at room temperature using the 514 nm line of the argon laser. The spectral range was 200–3250 cmThe electrolytic system consisted of two cathodes, being two blocks of pure graphite with dimensions of 7 cm × 2 cm × 1 cm, and one anode, which was cut out of graphite foil into dimensions of 9 cm × 2 cm × 0.1/0.2 cm. The electrodes were 2 cm apart from each other and separated by spacers to counteract electrophoresis in the solution . Electro42− ions. The next step was the reduction of intercalated SO42− and the self-oxidation of water, the products of which were gas bubbles (SO2 and O2). The released gas destroyed the weak bonds, detaching graphite layers one from another. During the electrolysis process, it was possible to observe the release of hydrogen at the cathode and oxygen at the anode. Sediment accumulated at the bottom and part of the exfoliated material floated on the surface of the solution. The current increased during the process due to acidification of the electrolyte and the increase in graphene content in the solution. In order to obtain optimal electrolysis conditions, the following parameters were modified: voltage (from 6 V to 12 V), temperature of electrolyte (from 5 to 25 °C), time of the process (from 45 min to 90 min), and pH (from 3.0 to 10.3) of the electrolyte . The XRD results of the samples with the highest efficiency of graphene to graphite formation for a particular factor are presented in In order to investigate the purity of the prepared graphene nanoplatelets, X-ray diffraction analysis was performed. The results obtained for the GNPs were compared to those obtained for commercial samples and are presented in 2 planes were used for the calculations. The ratio of the area under the graphene band and the graphite peak allows determining their share in the particular sample [C) calculated for all samples is presented in To be able to compare results for samples obtained under different conditions, the content of graphene in the final product was determined on the basis of the XRD measurements. The area of the peak assigned to the graphite and a broad band related to the reflections of graphene spr sample . The gra42− to SO2 and a consequent absence of bubbles that exfoliate the graphene flakes. Another factor, the duration of the electrolysis process, was determined experimentally by running the process for the period of 45, 60, and 90 min. The analysis of the diffraction patterns showed that the highest graphene content in the final product was obtained for the 60 min process (4(SO4)2 standard solution used as the electrolyte in the exfoliation process was 5.3. To check the impact of pH on the course of reaction, the electrolyte was modified using nitric acid (HNO3) and ammonia water (NH3·H2O). The XRD analysis showed that an increase in the pH of the electrolyte had a positive effect. The content of graphene in the obtained product was increased at higher pH values  allowed for an assessment of the obtained graphene nanoplatelet size and quality and the presence of byproducts that formed during the synthesis . The in-depth analysis of the TEM images showed that the size distribution graphene of flakes was quite wide and depended on the synthesis conditions . The anaThe morphology of the samples obtained at different voltage was checked using scanning electron microscopy . The ana−1, 1580 cm−1, and 2680 cm−1, respectively. The presence of the G band is related to transverse vibrations, while D and 2D bands are related to longitudinal vibrations in the graphene plane. The difference between them consists of the conservation of momentum of the reflected photon. In the case of the D band, momentum is lost due to encountered defects; in the case of the 2D band, the defects are not involved. The analysis of both types of vibrations by comparing the ratio of the respective bands (D to G band) allowed determining the degree of deformation of the obtained carbon structure. By analyzing the full width at half maximum (FWHM) of the 2D band, the number of carbon layers could be determined [Raman spectroscopy was used for the qualitative analysis of the obtained graphene flakes. The Raman spectra of graphene were characterized by the presence of three primary bands denoted as D, G, and 2D. Their positions were at about 1350 cmtermined . The deptermined .D) of the obtained graphene flakes, as the ratio of the D-band intensity to the G-band intensity, was determined for the analyzed samples.The degree of deformation . The deformational vibrations of δOH and δNH contributed to the band observed in the range of 1500–1750 cm−1. The band slightly below 1500 cm−1 derived from υC–OH deformational vibrations and δCH deformation vibrations. The bands observed about 1120 cm−1 could be attributed to υCO and/or υCN stretching vibrations.Because of the high scattering of light in the measured samples, graphene was ground with KBr and then formed into a pellet; thus, the results obtained in the IR analysis were evaluated qualitatively rather than quantitatively . Interfe−1 could have arisen from the hygroscopicity of KBr. Any of the other modifications resulted in the formation of the same bands below 1500 cm−1, indicating the appearance of similar amounts of characteristic functional groups. The use of organic modifiers, such as acetone, resulted in characteristic absorption near 3000 cm−1, suggesting the presence of CH groups.The changes in IR spectra depending on the method used were small, but allowed some very general conclusions to be drawn. The sample that was not modified (10 V) contained the fewest types or no functional groups on the surface, as the observed bands above 1600 and 3000 cmThe zeta potential is a measurement of the repulsive and attraction forces between particles suspended in liquids. The investigation was performed on selected samples from previous experiments. For the experiment, freeze-dried flakes were suspended in water using ultrasound. It is known that a lower zeta potential value indicates a higher stability of the particle dispersion in solution. Stable colloids are formed when the absolute value of potential is higher than +30 or lower than −30 mV . The val3·H2O would increase the price of the process due to the need of using large amounts of it. Considering the ratio of product quality to the price, it was decided to continue the research without changing the pH of the electrolyte. On the basis of the conducted measurements, it was found that the most favorable power and operating time of ultrasound process were 630 W and 15 min, respectively. It is also recommended to use an ice bath during the operation of ultrasound. Lowering the temperature prevents the separated graphene layers from reassembling, which ensures better quality. Mass analysis showed that, with an increase in acetone concentration in the electrolyte, the amount of the product obtained dropped sharply. The average relative weight of graphene obtained with a 33% acetone solution was halved, while the difference was up to 80% with a 50% solution. The experimental work aimed to obtain the largest possible amount of good-quality product with minimal financial and time consumption. As such, sonification of graphene should be conducted in water or 33% acetone solution. On the basis of the analysis of data obtained with the use of XRD, TEM, Raman/IR spectroscopy, and zeta potential measurements, the following parameters were determined as optimal to give the best quality, highest mass, and the lowest process price of the product: constant voltage of the system equal to 10 V, process temperature reduced by an ice bath, 5.3 pH of the electrolyte (0.1 M solution of (NH4)2SO4), and process time of 60 min. The solution of the electrolysis product should be prepared using pure water  and then subjected to ultrasound (630 W) for 15 min. To obtain dry graphene nanoplatelets, the sonicated product should be frozen and evaporated using a freeze-dryer.In order to optimize the process of obtaining graphene nanoplatelets by combining the electrochemical exfoliation and exfoliation methods in liquid phase using ultrasound, the following parameters were investigated: applied voltage, process duration, temperature, and pH of the electrolyte. The influence of acetone content in the solution of graphene obtained after electrolytic exfoliation and interaction of this solution with different power and duration of ultrasound was also examined. By selecting the optimal voltage (10 V), it was possible to obtain a significantly larger mass of the product while also providing a slight difference in its quality. Another important factor that increased the yield of the process was the temperature of the reaction environment. At 5 °C (ice bath), the mass of the graphene nanoplatelets could even be even doubled as compared to the exfoliation in 15 °C (water bath) and tripled if the temperature was not controlled. A change in the electrolyte pH had a positive effect on the quality of the obtained graphene. However, the use of NH