Preterm birth affects almost 9–11% of newborns and is one of the leading causes of childhood neurodevelopmental disabilities; the underlying molecular networks are poorly defined. In neurons, retrotransposons LINE-1 (L1) are an active source of genomic mosaicism that is deregulated in several neurological disorders; early life experience has been shown to regulate L1 activity in mice.Very preterm infants were randomized to receive standard care or early intervention. L1 methylation was measured at birth and at hospital discharge. At 12 and 36 months, infants’ neurodevelopment was evaluated with the Griffiths Scales. L1 methylation and CNVs were measured in mouse brain areas at embryonic and postnatal stages.Here we report that L1 promoter is hypomethylated in preterm infants at birth and that an early intervention program, based on enhanced maternal care and positive multisensory stimulation, restores L1 methylation levels comparable to healthy newborns and ameliorates neurodevelopment in childhood. We further show that L1 activity is fine-tuned in the perinatal mouse brain, suggesting a sensitive and vulnerable window for the L1 epigenetic setting.Our results open the field on the inspection of L1 activity as a novel molecular and predictive approach to infants’ prematurity-related neurodevelopmental outcomes.ClinicalTrial.gov (NCT02983513). Registered on 6 December 2016, retrospectively registered.The online version contains supplementary material available at 10.1186/s12916-020-01896-0. Prematurity, which is defined as birth before 37 weeks of gestation, affects 9–11% of neonates globally, and it is the second leading cause of death in children below 5 years of age and the most important in the first month of life . Among pBesides the documented role played by gestational age (GA) at birth and by the occurrence and severity of postnatal morbidities, the impact of early exposure to the hazards of extrauterine life has been recently emphasized. Indeed, during their stay in neonatal intensive care unit (NICU), preterm infants face early environmental stress, mainly represented by deprivation of physiological intrauterine sensory experiences –10, exceIn this framework, developmental care, conceived as a strategy to reduce NICU stressful factors and promote maternal engagement, has been demonstrated to improve brain maturation, as assessed by magnetic resonance imaging (MRI), and neurodevelopmental outcomes , 21. IntMobile DNA elements have the ability to change their genomic position, either by a DNA-based (transposition) or RNA-based (retrotransposition) mechanism. Retrotransposition is one of the main forms of somatic mosaicism in the brain . Among TIn the current study, we sought to document for the first time in humans whether enhanced maternal care could modulate neurodevelopment in preterm infants and whether this clinical aspect could be reflected by L1 methylation levels in perinatal stages. In order to precisely assess the perinatal window in which L1 epigenetic setting is established in the brain, we leveraged on comparative analysis in developing mouse brain and further dissected L1 dynamics (methylation and CNVs).+0 and 29+6 weeks of gestational age (GA) at the same institution were eligible. Exclusion criteria include multiple pregnancy (triplets or higher), genetic syndromes and/or malformations, and infants who developed severe neonatal comorbidities including severe brain lesions. Inclusion and exclusion criteria were designed to enroll a homogeneous cohort of preterm infants. The full study protocol is described in [All the preterm infants consecutively born between 25ribed in . AdherenInfants were randomly assigned either to receive (i) standard care or (ii) an additional early intervention protocol based on maternal care. Standard care, according to the routine clinical protocol of the NICU, included kangaroo mother care, minimal handling, and non-pharmacological pain management. The early intervention protocol included, over routine clinical care, the PremieStart , which iThe randomization was performed using sealed envelopes prepared in groups of 10 through computer-generated randomization. The randomization sequence was concealed until the group allocation was assigned, and the examiners (both biologist and psychologist that performed the follow-up examination) remained blinded for the entire study period.+0 and 29+6 weeks of gestational age (GA), recruited between April 2014 and January 2017. The trial aimed at assessing the effectiveness of an early intervention program, based on early parental involvement in neonatal care, in promoting visual function and neurodevelopment in preterm infants. A positive effect of early intervention on visual function maturation [The present study is a post hoc analysis of a larger randomized controlled trial (RCT) that included 70 very preterm infants born between 25turation  ). Peripheral blood was obtained during blood sampling performed for routine blood examination, according to clinical practice. In healthy full-term infants’ cord blood samples were collected at birth only in infants born by cesarean section after uneventful pregnancies. Each sample consisted of 0.5 mL of cord/peripheral blood.Fresh cord blood samples derived from full-term and preterm infants were subjected to erythrocytes lysis following the manufacturer’s instruction (BD lysis buffer). Nucleated blood cells were then stained with anti CD45 for 30 min at 37 °C, different subpopulations were identified gating on CD45 and SSC as described in , 42. MosNeurodevelopment was assessed as a post hoc analysis of the larger RCT. At 12 months corrected age and at 36 months chronological age the preterm infants underwent the Griffiths Scales to assess neurodevelopment   at 12 moCD-1 mice were housed under controlled conditions for temperature and humidity, using a 12:12-h light-dark cycle. Mice were mated overnight, and females were separated the following morning and checked for vaginal plugs . CD-1 animals deliver pups between day E19 and E20. Cesarean sections (C-secs) were performed at embryonic days E15.5, E18.5, and P0. Pups were sacrificed by decapitation at different time points: at embryonic day E15.5, E18.5, and at postnatal day P0, P3, and P14. At each developmental stage, 4 mice were sacrificed and brains collected to manually microdissect hippocampal, cortical, and cerebellar tissue under a stereomicroscope in sterile conditions. From the same mice, also blood samples were collected at E18.5 and at postnatal day P0, P3, and P14. We excluded E15.5 from blood samples given the low amount of material that was not sufficient for the subsequent molecular analysis; we excluded one sample at P0 because not usable. Blood and microdissected tissues were store at − 80 °C until gDNA extraction was performed.Genomic DNA was isolated with standard phenol-chloroform extraction techniques from human whole cord or peripheral blood that is all the circulating nucleated cells; genomic DNA was isolated from mouse blood, hippocampus, cortex, and cerebellum and was isolated with standard phenol-chloroform extraction techniques.Five hundred nanograms of genomic DNA from each sample were bisulfite-treated using the MethylEdge™ Bisulfite Conversion System  following the manufacturer’s protocol.hL1-5′UTR For: 5′ - AAGGGGTTAGGGAGTTTTTTT – 3′hL1-5′UTR Rev: 5′ - TATCTATACCCTACCCCCAAAA – 3′The methylation analysis of CpG island within the human L1 promoter was conducted as reported in  with minIn each PCR, 40 ng of bisulfite-converted DNA were combined with primers at 0.5 μM final concentration and GoTaq™ Hot Start Green Master Mix (Promega) in a final volume of 50 μL. PCR conditions were as follows: 95 °C for 2 min followed by 30 cycles of 95 °C for 45 s, 56 °C for 1 min and 72 °C for 30 s, followed by a final step of 72 °C hold for 4 min.The product of amplification is 363 bp of length and contains 19 CpGs. The resulting PCR products were checked by agarose gel electrophoresis and then purified by PureLink™ Quick Gel Extraction & PCR Purification Combo Kit . They were then cloned into pGEM-T Easy Vector System I (Promega) using a molar ratio insert: vector of 6:1. Sanger sequencing was performed by GATC Biotech, using the reverse sequencing primer pGEM Seq Rev: 5′-GACCATGATTACGCCAAGCTA – 3′. Resulting chromatograms were examined for sequencing quality using FinchTV software. At least 10 sequenced clones per sample were analyzed in Fig. QUantification tool for Methylation Analysis) software  [To analyze the conversion efficiency and the methylation status of the CpG sites, FASTAQ files were analyzed by QUMA  . For theThe methylation analysis of CpG island within the murine L1MdTf monomer and IAPLTR1a were conducted as reported in  with minEach PCR was performed with 16–40 ng of bisulfite-converted DNA were combined with primers at 0.5 μM final concentration and GoTaq™ Hot Start Green Master Mix (Promega) in a final volume of 50 μL. PCR conditions were as follows: 95 °C for 2 min followed by 30 cycles of 95 °C for 45 s; 56 °C for 1 min and 72 °C for 5 s, followed by a final step of 72 °C hold for 4 min.For L1MdTf monomer the product of amplification is 191 bp of length and contains 13 CpGs while for IAPLTR1a the product of amplification is 205 bp of length and contains 10 CpGs. The resulting PCR products were checked by agarose gel electrophoresis and then purified by Agencourt AMPure XP beads (Beckman Coulter) according to the manufacturer’s instructions. DNA concentration was quantified using a Qubit dsDNA HS Assay kit. All the L1MdTf and IAPLTR1a amplicons were then pooled in equimolar quantities to obtain a final pooling concentration of 2 ng/μL. Library for DNA sequencing was produced on the pooled PCRs. Paired-end 2 × 150 bp sequencing was performed on a HiSeq platform (Illumina) by Eurofins GATC Biotech.https://github.com/dpryan79/MethylDackel). Sample correlation analysis was performed using methylKit [A total of 22,481,000 reads were obtained from bisulfite sequencing and were assigned to samples based on the primers with Illumina barcode index. Briefly, no mismatch was allowed for the barcode index and a maximum of 5 mismatches were allowed for the target primer. None of the reads assigned to IAPLTR1a target aligned on L1MdTf and vice versa. Prior to mapping, reads were trimmed for low quality using Trimmomatic   were used at 37 °C for 30 min and inactivated at 85 °C for 15 min). Efficiency of digestion was proved on 300 ng of gDNA pooled with 300 ng of a 120 bp ssDNA oligonucleotide , 67 mM MgClQuantitative PCR experiments were performed on a StepOne Plus (Thermo Fisher Scientific) with minor modifications to the method reported in . In eachmL1 ORF2 F: 5′ – CTGGCGAGGATGTGGAGAA - 3′mL1 ORF2 R: 5′ – CCTGCAATCCCACCAACAT - 3′mL1 ORF2 Taqman probe: 5′ – TGGAGAAAGAGGAACACTCCTCC - 3′mL1 5S F: 5′ – ACGGCCATACCACCCTGAAC - 3′mL1 5S R: 5′ – AGCCTACAGCACCCGGTATTC - 3′mL1 5S Taqman probe: 5′ – GATCTCGTCTGATCTCGGAAGCTAAG - 3′Standard curves of genomic DNA ranging from 200 ng to 0.2 ng were performed to verify that the 80 pg dilution was within the linear range of the reaction. For CNVs, the quantification includes from five to eight technical replicates. For assays on mouse genome, we adapted a TaqMan probe for the same amplicon reported in , 37. ProClinicalTrial.gov (NCT02983513). Written informed consent was signed by both parents before inclusion in the study (both for preterm and full-term infants). All experimental procedures were performed in compliance with national and EU legislation, and Humanitas Clinical and Research Center, approved by the Animal Care and Use Committee (6B2B3.N.8EK).The present study was approved by the Ethics Committee Milano Area B. The trial is registered at The data from bisulfite sequencing has been submitted in NCBI GEO (GSE136844).t test and Mann-Whitney U test were used in the comparison of continuous variables with normal distribution and non-normal distribution respectively. For the comparison of qualitative data, Fisher’s exact test was used. Shapiro-Wilk test was used to test the normal distribution of the data. To assess the differences between full-term and preterm infants, and between treatment groups in total L1 methylation and on each CpG, unpaired t test and two-way ANOVA model with Tukey’s HSD post hoc tests were used. Linear regression model was used to study the relationship between L1 methylation at NICU discharge and the intensity of care (mean number of massages per week) and independent t test and Mann-Whitney U test were used to assess the difference in neurodevelopmental outcome between standard care and early intervention groups at 12 and 36 months.The present study reports the results of a post hoc analysis of a larger randomized controlled trial (NCT02983513). The power calculation and sample size analysis were performed according to the primary outcome aimed at assessing the effectiveness of an early intervention program in enhancing visual function as a short-term neurodevelopmental outcome in very preterm infants . Demograp < 0.05 was considered significant for all tests. Statistical analyses were performed using R version 3.5.3 .Mouse brain regions’ methylation at different stages of development were analyzed using one-way ANOVA and Tukey’s HSD post hoc tests. All tests were two-tailed, and +0 and 29+6 weeks of gestational age (GA). A schematic representation of the study timeline, sample collection, and of the molecular and clinical analysis performed is provided in Fig. To investigate the effects of preterm birth and early interventions on L1 modulation and neurodevelopmental outcomes, a post hoc analysis was conducted on 34 very preterm infants born between 25One week after birth and only in condition of clinical stability, all the enrolled infants were randomized to either receive standard care or early intervention Fig. . Standarn = 16) and early intervention (n = 17)  Figs. . For nin7) Figs. .Baseline and perinatal characteristics for the standard care and early intervention groups are described in Table In the early intervention group, the massage therapy was started by parents at 31.7 ± 1.8 (mean ± SD) weeks of GA and carried out on average 10.0 ± 2.1 times a week. Visual interaction was proposed from 34.9 ± 0.8 weeks of GA and performed on average 7.1 ± 1.8 times a week.In addition, 20 cord blood samples from healthy full-term infants (named “full-term”) were collected at birth. Mother and infants’ characteristics of the full-term group are presented in Additional file To assess L1 methylation level, we analyzed a region within the promoter of L1Hs, the evolutionary youngest and most active L1 subfamily in the human genome , containn = 20) and preterm (n = 19) infants at birth and in whole peripheral blood of preterm infants at NICU discharge, subjected either to standard care (n = 16) or early intervention (n = 17)  , early postnatal , and late developmental stages P14)  methylation as reported in  see the” sectionWe next investigated whether the reduction in L1 methylation observed postnatally in mouse hippocampus and cortex could correspond to an increased L1 CNVs, as L1 are reported to retrotranspose in the brain . We measOverall, these results suggest that L1 activity is fine-tuned and specifically regulated during hippocampus and cerebral cortical development, and identify E18.5 as a sensitive and “vulnerable” developmental stage for the epigenetic setting of L1 methylation and activity regulation in these brain regions.Based on the hypothesis that L1 modulation in the perinatal period may represent one of the molecular mechanisms involved in the modulation of the infant’s long-term neurodevelopment, we report the results of the post hoc analysis on neurodevelopmental assessment performed with the Griffiths Scales at 12 months corrected age and 36 months chronological age in this subgroup of the larger RCT cohort , 44  and, importantly, this trend is maintained later in childhood (36 months), suggesting a positive long-lasting modulation of their neurodevelopment. These findings were confirmed by the post hoc analysis performed on the whole cohort enrolled in the RCT  in which L1 epigenetic regulation is set Fig. . InteresThus, even without directly aligning the human and mouse neurodevelopmental trajectories, a defined prenatal time window in each species (GA 26–29 for human and E17.5–E18.5 for mouse) that hosts critical events shaping brain architecture and networks maturation has been identified . AlteratImportantly, we further demonstrated that the early intervention program, compared to standard care, enhances neurodevelopment up to 36 months of age, which is considered a crucial milestone in the preterms’ follow-up assessment to detect neurodevelopmental disabilities . HoweverIn conclusion, we are providing evidence that L1 methylation is fine-tuned at prenatal stages in humans and in mouse brain; we can speculate that this developmental window is therefore extremely sensible in preterm infants to the effects of maternal care on the regulation of the L1 epigenetic setting.This work shed light on a possible molecular function for L1 activity in shaping the developing brain connections with potential impact on infants’ neurodevelopment. Besides this mechanistic role of L1, this study supports a so far unexplored translational approach to prematurity-related disorders: L1 methylation analysis on an extended cohort of preterm newborns would be required to define its predictive value as a molecular proxy for neurodevelopmental outcomes.Additional file 1: Figure S1. Early Intervention affects methylation of YY1 binding site in L1 promoter. Figure S2. Granulocytes and lymphocytes isolated from cord blood of full-term and preterm display similar L1 methylation. Figure S3. IAP methylation levels do not change in hippocampus and cortex development while reduce in cerebellum development.Additional file 2: Table S1. Baseline characteristics of the population: descriptive statistics and comparisons between Full-term and Preterm (both Early Intervention and Standard Care) groups. Table S2. Neurodevelopmental outcome at 12 months corrected age and 36 months chronological age of the overall population included in the RCT. Table S3. List of the primer sequences used in L1MdTf and IAPLTR1a NGS methylation analysis in mice.


The search for novel platforms and metamaterials for the enhancement of optical and particularly Raman signals is still an objective since optical techniques offer affordable, noninvasive methods with high spatial resolution and penetration depth adequate to detect and image a large variety of systems, from 2D materials to molecules in complex media and tissues. Definitely, plasmonic materials produce the most efficient enhancement through the surface-enhanced Raman scattering (SERS) process, allowing single-molecule detection, and are the most studied ones. Here we focus on less explored aspects of SERS such as the role of the inter-nanoparticle (NP) distance and the ultra-small NP size limit (down to a few nm) and on novel approaches involving graphene and graphene-related materials. The issues on reproducibility and homogeneity for the quantification of the probe molecules will also be discussed. Other light enhancement mechanisms, in particular resonant and interference Raman scatterings, as well as the platforms that allow combining several of them, are presented in this review with a special focus on the possibilities that graphene offers for the design and fabrication of novel architectures. Recent fluorescence enhancement platforms and strategies, so important for bio-detection and imaging, are reviewed as well as the relevance of graphene oxide and graphene/carbon nanodots in the field. These optical techniques present several important characteristics for detection, identification, and imaging, such as being cost-effective, rapid, non-destructive, and having sub-micron resolution, reaching <10 nm with tip-enhanced Raman spectroscopy (TERS) . I. I99]. IPlasmonic non-interacting Au arrays coated with GO have also been demonstrated to produce more uniform and reproducible electromagnetic enhancement c. MoreovBesides the electromagnetic enhancement due to localized surface plasmon resonances of metallic NPs and the chemical enhancement related to charge transfer or modifications of the molecular electronic structure described in the previous sections, other mechanisms are investigated as the use of excitation wavelengths in resonant conditions for the probe molecule  ..2O3/grap2O3/Gr platforms where the combined enhancement is shown to be very efficient . . 2 layeriameter) b. In add2O3 heterostructure without graphene thus avoiding fluorescence quenching. Large enhancement factors, around 40 for Raman and 50 for PL, were also detected for single-layer MoS2 in a similar Si/Al/Al2O3 platform  and the regions with 2, 3 and 4 layers are well differentiated. On the contrary, MoS2 is almost undetectable (for 1and 2-layer MoS2) on silicon (upper part).While the processes leading to Raman and PL enhancements in the vicinity of metallic NPs are different and generate very different EF; the interference mechanism is identical for Raman and fluorescence except that there is often a larger wavelength difference between the incident and scattered beams in the latter case. This detail has not an important impact since the refractive index variation on the typical wavelength ranges is usually moderate for the involved materials. However, scarce works have reported on the use of light interference for fluorescence enhancement but such platforms may be of interest for fluorescence bioimaging. EF as high as 100 was observed for R6G fluorescence signals a when deplatform b. Moreovplatform c. In FigGraphene, graphene foams, and functionalized graphene (such as GO with different reduction degrees) have a primordial role in novel architectures of SERS platforms to enhance both electric and chemical enhancement mechanisms, to reach single-molecule detection, to stabilize and gain reproducibility, to homogenize the electromagnetic fields when quantification is focused, and to increase the specific area where the probe molecules can be deposited or adsorbed. Finally, graphene can quench undesired fluorescence of the probe molecules and avoids the photo-carbonization of metal NPs thus allowing specific sensing by cleaning up backgrounds in Raman spectra. While pristine graphene offers unique possibilities in the design of enhancement platforms due to its extreme thinness, mechanical properties, and chemical inertness, functionalized graphene and the several variants of graphene and carbon nanodots are priceless for biosensing and bioimaging.The design of plasmonic cavities has great potential for further developments in the field. While graphene has not yet found a relevant role to play in such systems, other 2D materials, with low conductivity, may be of interest to generate ultra-narrow gaps. The design of novel architectures favoring homogeneous and reproducible enhancements is still required to reach reliable quantification of analytes and for adequate imaging.Other enhancement mechanisms of Raman signal such as resonant and interference Raman scattering (RRS and IRS), as well as their integration in SERS platforms, can be further developed. However, both have their specific limitations. RRS can produce enhancements of several orders of magnitude, but the high absorption by the probe molecules at the incident laser energy can provoke their rapid photobleaching. Therefore, an appropriate balance between amplification and stability has to be found. IERS can be combined with SERS but the density of deposited NPs has to be relatively low.The enhancement of fluorescence signals is a very relevant objective in bio-detection, for example, to reduce the threshold limit in the detection of cancer biomarkers, hormones, allergens, or proteins, and in bioimaging using fluorescence markers. While fluorescence is an extremely sensitive technique, improving sensitivity for single-molecule detection requires the development of new designs and approaches. Indeed, plasmonic enhancement of fluorescence still has low efficiency due to the compromise imposed by metal-induced quenching. Graphene and carbon nanodots seem to have an important role to play due to their biocompatibility and much higher stability compared to organic dyes. However, the in vivo biotoxicity of graphene and related nanomaterials has to be further investigated. Finally, interference enhancement-based platforms with uniform and controlled amplification could bring the possibility of obtaining optical images with enhanced sensitivity using either the intensity of a Raman peak of the molecule to be tested, a fluorescence signal, or the white light of a microscope with applications in bioimaging and detection.


Drosophila melanogaster, and zebrafish exhibit dysfunction of locomotor function, defective neuromuscular junctions, and motor neuron defects. There is currently no effective cure for ALS, and the underlying mechanisms of TDP-43 in ALS remain poorly understood. In this study, a genetic screen was performed to identify modifiers of human TDP-43 (hTDP-43) in a Drosophila model, and glutathione S-transferase omega 2 (GstO2) was found to be involved in hTDP-43 neurotoxicity. GstO2 overexpressed on recovered defective phenotypes resulting from hTDP-43, including defective neuromuscular junction (NMJ) boutons, degenerated motor neuronal axons, and reduced larvae and adult fly locomotive activity, without modulating the levels of hTDP-43 protein expression. GstO2 modulated neurotoxicity by regulating reactive oxygen species (ROS) produced by hTDP-43 in the Drosophila model of ALS. Our results demonstrated that GstO2 was a key regulator in hTDP-43-related ALS pathogenesis and indicated its potential as a therapeutic target for ALS.Transactive response DNA-binding protein-43 (TDP-43) is involved in the pathology of familial and sporadic amyotrophic lateral sclerosis (ALS). TDP-43-mediated ALS models in mice,  Drosophila melanogaster, in zebrafish also results in motor neuron defects [Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a fatal and progressive loss of both the upper and lower motor neurons, resulting in muscle weakness, paralysis, and death within 2–5 years of the onset of symptoms . ALS is  defects ,5.Caenorhabditis elegans has been found to increase the resistance to oxidative damage [Drosophila has been shown to provide neuroprotection against H2O2-induced neurotoxicity via the mitogen-activated protein kinase (MAPK) pathway [Drosophila model of Parkinson’s disease (PD) induced by the loss of the parkin gene, GstO2 modulates the mitochondrial ATP synthase activity. The up-regulation of GstO2 restores the disassembly of the mitochondrial ATP synthase induced by the loss of the parkin in the PD fly model. [Oxidative stress plays a critical role in the pathogenesis of neurodegenerative diseases, including ALS. An excess of reactive oxygen species (ROS) or reactive nitrogen species (RNS) and an insufficient antioxidant defense mechanism are attributed to the oxidative stress conditions that give rise to many neurodegenerative diseases . The bioe damage . Besides pathway . Moreovey model. . Human Gy model. ,16. Indey model. ,18. Furty model. . The GSTy model. . Moreovey model. . TherefoDrosophila model. In a neuronal and motor neuron Gal4-upstream activating sequence (UAS) system in Drosophila, the overexpression of GstO2 was found to markedly improve motor neuron function, including neuromuscular junction and axon morphology, in hTDP-43-induced Drosophila larvae and adult flies. Motor neuron function was recovered by overexpressing GstO2, which, in turn, resulted in an increased locomotive activity in larvae and adult flies expressing hTDP-43. Interestingly, GstO2 did not affect the protein expression level of hTDP-43 but acted as a neuroprotector in the hTDP-43-induced Drosophila model. Taken together, our findings suggested that GstO2 was a novel modifier of hTDP-43-related ALS pathogenesis.In this study, we aimed to investigate the neuroprotective function of GstOs in an hTDP-43-associated UAS-hTDP-43 line was gifted by Nancy M. Bonini . The UAS-GstO2 line has been described previously [elav-Gal4, the motor neuron-specific driver, D42-Gal4, and the eye-specific driver, GMR-Gal4 lines were obtained from the Bloomington Drosophila Stock Center. 1118W flies were used as a control.All stock flies were kept under normal conditions with access to standard food at a normal temperature (25 °C) and normal humidity (60%). Crosses between flies were performed according to the standard procedure, and progeny was raised at a normal temperature or 29 °C. The eviously ,22. The The heads of the male flies were glued to glass slides for the imaging of the external eye phenotype. The images of eye phenotype were captured using a Leica MZ10 F stereomicroscope equipped with a Leica DFC450 digital camera. Adobe Photoshop 7.0 imaging software was used to create the final images. To analyze pigment loss in a quantitative manner, a comparable area of eyes for each genotype was selected. Quantification of white regions in the selected area of eyes was analyzed using the ImageJ software.To measure the locomotive activity of the larvae, we prepared 2% grape juice-agar plates. Third instar larvae were carefully washed with PBS to remove any remnants of food medium and dried on filter paper. The larvae of each genotype were placed on a plate at room temperature and allowed to crawl for 60 s. For the quantification of larvae movement, the larvae tracking lines were drawn for the measurement of the moving distance in ImageJ software. At least, 10 larvae of each transgenic line were used to obtain results.To perform the adult climbing assay, the male flies raised at 29 °C were collected and placed in vials . The flies were transferred to an empty vial for 1 h at room temperature to allow for adaption to the environment. The vials were tapped gently for the flies to drop to the bottom of the vial. Then, using the fly characteristic against geotaxis, the number of flies that climbed to the top of the vial within 10 s was counted. This experiment was repeated at least 5 times independently for each genotype.For the analysis of the larval neuromuscular junction, the third instar larvae were dissected on Sylgard plates using PBS and fixed with 4% formaldehyde in PBS for 20 min. The dissected larvae were washed in PBS containing 0.1% Triton X-100 (PBT) and blocked with 3% BSA in PBT. The samples were stained with anti-HRP-FITC  overnight. After mounting the larvae on glass slides, the larval preparations were imaged using a DE/LSM710 NLO Carl Zeiss confocal microscope .4, pH 6.9) for 30 min. Then, the samples were washed in a washing buffer  and mounted on glass slides. Images were acquired using a Carl Zeiss LSM710 confocal microscope . The number of pixels was calculated using ImageJ software for each of the axon areas from femur regions of adult fly legs.To visualize the morphology of the axons, the legs of male flies were dissected in PBS and fixed with 4% formaldehyde in a fixation buffer  sample buffer . The extracted protein was separated using a 4–12% gradient SDS-PAGE gel  and transferred to PVDF membrane . The membrane was blocked with 4% non-fat dry milk in TBS containing 0.1% Tween-20 (TBST) and incubated with primary antibodies overnight. Primary antibodies, including rabbit anti-TARDBP , rabbit anti-, and raThe third instar larvae were dissected on Sylgard plates using PBS buffer. The dissected larvae were incubated with 10 μM 2′,7′-dichlorofluorescin diacetate (DCF-DA)  for 30 min at 37 °C. The signals were observed using an MZ210F Leica fluorescence microscope . Larval brains were outlined manually. The average pixel intensity of the signal in the whole brain area was measured and analyzed using the ImageJ software.t-test. The number of flies used, replicates, and statistical significance  are specified in the appropriate figure legends and methods. Significance was considered by p-values less than 0.05. Data were quantified and represented as the mean ± SEM.Data collection and analysis were acted blindly. All experimental data were organized and analyzed in GraphPad Prism 8.0 using the Student Several studies have suggested that GSTO is a potential novel regulator of neurodegenerative diseases, including AD, PD, and ALS. However, the functions of GSTO in the ALS model are not yet fully understood. To investigate the relationship between GSTO expression and ALS pathogenesis, we performed a genetic interaction study using an hTDP-43-expressing fly model. Using the eye-specific Gal4 driver, glass multiple reporter (GMR)-Gal4, we assessed the genetic interactions between hTDP-43 and Drosophila GstO genes. Our results showed that GstO2, an enzymatic antioxidant, was a genetic suppressor in hTDP-43 expressing flies . The expTo determine whether the expression of GstO2 is able to restore the neurotoxic phenotype of hTDP-43-expressing flies, we generated flies with UAS-hTDP-43, UAS-GstO2 transgenes, and pan-neuronal specific Gal4 driver, elav-Gal4. We investigated the morphology of NMJ in hTDP-43-expressing flies and measured the number of NMJ boutons by staining muscle 6/7 in segment 3 with anti-HRP-FITC. NMJ boutons facilitated the presynaptic terminal transmission of motor neurons. A similar number of the boutons was observed in control larvae, elav-Gal4, and GstO2 single overexpressing larvae a. OverexAxonal degeneration in neuronal cells is the hallmark of neurodegenerative disease. To visualize the axons of motor neurons in adult Drosophila legs, we used flies expressing membrane-tethered red fluorescent proteins (mCD8-RFP) under motor neuron-specific gal4 driver, D42-Gal4. The overexpression of hTDP-43 led to severe degeneration in the axons of motor neurons in legs compared with the control, D42-Gal4 b. Taken The defective axons of motor neurons and NMJs induced by hTDP-43 expression might result in locomotor defects. GstO2 alleviated the hTDP-43-mediated degeneration of the motor neurons and NMJs, which led to the recovery of locomotor defects, as shown in Next, we examined the climbing activity of adult flies, as described in hTDP-43 is the DNA/RNA binding protein predominantly localized in the nucleus. hTDP-43 mutations induce ALS through the mislocalization of hTDP-43 to the cytoplasm from the nucleus. hTDP-43 mutation proteinopathy has been observed in both familial and sporadic ALS patients ,3. ThereWe evaluated the expression level of hTDP-43 protein to determine the stability of the protein. The elav-gal4 and D42-gal4 driver were used to express hTDP-43 and GstO2, specifically in pan and motor neurons. Interestingly, the up-regulation of GstO2 in pan neurons a and motTo examine the protective mechanism of GstO2 on hTDP-43 toxicity, we measured intracellular reactive oxygen species (ROS) in the larval brain by the redox-sensitive fluorophore DCF-DA staining. ROS levels in neurons significantly increased in hTDP-43-expressing flies. Importantly, GstO2-co-overexpressing flies showed a marked decrease in intracellular ROS generation . These rDrosophila resulted in the development of degenerative motor neuronal axons. Moreover, hTDP-43 overexpression was associated with a reduced number of NMJ boutons, as well as the impairment of locomotor activity in the Drosophila model. These results indicated that hTDP-43 was associated with motor neuronal toxicity in Drosophila melanogaster.TDP-43 is a DNA/RNA binding protein encoded by a pathogenetic gene of ALS. There is evidence of the onset of ALS as a result of the presence of cytoplasmic inclusions in TDP-43 mutants in both familial and sporadic ALS patients. Moreover, some research has suggested that wild type and mutant hTDP-43 can result in a shortened lifespan, damaged synaptic morphology, degenerative motor neurons, and defective locomotion. In the present study, we confirmed that overexpressing the hTDP-43 wild type in Identification of the novel suppressor for TDP-43-induced neurotoxicity has important implications in the development of potential therapeutic targets for ALS. Using genetic modifier screening, we identified GstO2 as a novel candidate for the modulator of our hTDP-43-induced ALS fly model. Additionally, the up-regulation of GstO2 was found to directly restore neuronal toxicity, including the loss of NMJ boutons, the destruction of motor neuronal axons, and weakened locomotive ability in hTDP-43-expressing flies. Our findings supported the possibility that GstO2 plays a critical role as a suppressor in the pathogenesis of hTDP-43-induced ALS.The mislocalization of TDP-43 protein, or the misfolding and aggregation of TDP-43 in the cytoplasm, is a hallmark of TDP-43-induced proteinopathy. As mentioned above, TDP-43 mutations have been found to accumulate in the cytosol of patients with ALS. To evaluate the stability of hTDP-43 protein, we quantified the levels of hTDP-43 protein expression. Interestingly, the level of hTDP-43 protein expression did not change with GstO2 overexpression. These results indicated that GstO2 was a motor neuroprotector that did not affect the levels of hTDP-43 protein expression.C. elegans. In addition, GstO1 exerts neuroprotective function against hydrogen peroxide-induced neurotoxicity via the MAPK signaling pathway in Drosophila melanogaster. In the fly model of PD, GstO2 has been found to modulate the activation of mitochondrial ATP synthases. Furthermore, GstO2 exerts dehydroascorbate (DHA) reductase activity and is required for the maintenance of the concentration of ascorbic acid (AsA). Human GSTO1-1 regulates the TLR4 pathway by regulating ROS generators and LPS-induced inflammatory responses in macrophages. In humans, GSTOs play a protective role against oxidative stress, including monomethylarsonic acid (MMA) reductase and DHA reductase activity. Oxidative stress plays a critical role in the pathogenesis of ALS. In the red blood cells and cerebrospinal fluid of ALS patients, the activity of antioxidant enzymes is reduced. Additionally, the levels of the reduced form of glutathione have been found to be decreased in the erythrocytes of ALS patients. Therefore, antioxidant mechanisms are an important therapeutic target against oxidative stress in ALS.GSTs are well-known antioxidant enzymes. They catalyze metabolites, xenobiotics, and endogenous and exogenous toxic compounds by conjugating with glutathione. Among the classes of GSTs, the omega class of GSTs also exerts detoxification effects against various stresses, including oxidative stress. For example, GSTO-1 expression is known to counteract oxidative damage in In our study, GstO2 was found to be a target of antioxidant defense mechanism against oxidative stress induced by hTDP-43. We demonstrated that hTDP-43 expression resulted in motor neuronal toxicity in a fly ALS model and found that GstO2 ameliorated the degenerative and defective phenotypes induced by hTDP-43 overexpression. Taken together, our results indicated that GstO2 could be used as a novel therapeutic mediator for the treatment of neurodegenerative diseases caused by hTDP-43.


Inflammation and immunity play a pivotal but yet unclear role in idiopathic pulmonary fibrosis (IPF), a chronic disorder characterized by progressive damage of lung parenchyma and severe loss of lung function despite optimal treatment. However, the pathophysiological and predictive role of combined blood cell count indexes of inflammation in IPF is uncertain.Seventy-three patients with IPF and 62 healthy subjects matched for age, gender and smoking status were included in this cross-sectional study.We found significant differences in neutrophil to lymphocyte ratio (NLR), derived neutrophil to lymphocyte ratio (dNLR), monocyte to lymphocyte ratio (MLR), platelet to lymphocyte ratio (PLR), systemic inflammation response index (SIRI) and aggregate index of systemic inflammation (AISI) between IPF patients and healthy controls. In logistic regression, all combined blood inflammation indexes, barring PLR, were independently associated with the presence of IPF after adjusting for age, gender, body mass index and smoking status. Furthermore, significant associations between FVC% and NLR, LMR, SIRI and AISI, and between DLCO% and NLR, dNLR, LMR, SIRI and AISI, were observed.In conclusion, our data indicate significant alterations of combined blood cell count indexes of inflammation in IPF. Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrosing interstitial lung disease of unknown aetiology, occurring mainly in adults, that is characterized by a histopathologic and/or radiologic pattern of usual interstitial pneumonia . The pro1 (Forced Expiratory Volume in the 1st second), FVC  and DLCO (Diffusion Capacity for Carbon Monoxide).There is good evidence that indexes derived from calculating the ratios between neutrophils, lymphocytes, monocytes and platelets counts are more strongly associated with chronic inflammation conditions when compared with individual cell populations . Indeed,1, FVC and DLCO, expressed as percentages of predicted values , were performed in accordance with the criteria published by the American Thoracic Society and the European Respiratory Society [d-penicillamine, and colchicine, or oral corticosteroids during the preceding 3 months. A group of 63 healthy controls matched for age, sex and smoking status, with no medical history, was also included.This was a case–control study of 73 consecutive newly diagnosed IPF patients recruited at the Respiratory Unit of the University of Sassari between 2016 and 2019. All patients signed a written consent and the study was approved by the ethics committee of the University Hospital of Cagliari (2262/CE-17/11/2015). IPF was diagnosed according to the current evidence-based guidelines for the diagnosis and management of IPF . High-reFasting blood samples from IPF patients and matched controls were respectively collected at first hospital admission in the Unit of Respiratory Diseases, and the Unit of Occupational Medicine of the University Hospital of Sassari (AOU Sassari); blood cell counts were performed using an automatic blood counter CellDyn Sapphire  in a certified laboratory. The following combined indexes were evaluated: NLR, dNLR, LMR, PLR, SIRI and AISI. The SIRI was calculated by multiplying the number of neutrophils with that of monocytes and dividing the product by the number of lymphocytes . The AISResults are expressed as mean values (mean ± SD) or median values . Variables distribution was assessed by the Shapiro–Wilk test. Statistical differences between groups were evaluated using unpaired Student’s t-test or Mann–Whitney rank sum test, as appropriate. Differences between categorical variables were assessed by Chi-squared test. Correlations between variables were assessed by Pearson’s correlation or Spearman’s correlation as appropriate. Logistic regression analysis was performed to evaluate independent associations between IPF disease and combined blood cell count indexes of inflammation. The association between different indexes and IPF was evaluated using receiver operating characteristics (ROC) curve analysis and selection of optimal cut-off values for sensitivity and specificity according to the Youden Index. Statistical analyses were performed using MedCalc for Windows, version 15.4 64 bit .1 (%) and FVC (%) were significantly lower in IPF patients than in controls . IPF patients had higher WBCs  and neutrophils  and lower lymphocyte values . There were no significant between-group differences in BMI, monocyte and platelet values.Table p < 0.001), d-NLR , PLR , SIRI  and AISI  were significantly higher in IPF patients whereas the LMR was significantly lower . In univariate logistic regression, crude ORs for all inflammation combined indexes were significant, as reported in Table p = 0.0001), d-NLR , LMR , SIRI  and AISI  remained significant after adjusting for age, gender, BMI and smoking status.The combined indexes data are described in Table 1 was negatively associated with NLR , SIRI  and AISI , FVC% was negatively associated with NLR , SIRI  and AISI (rho = − 0.24 p = 0.04) and positively related with LMR , and DLCO% was negatively associated with NLR , dNLR , SIRI  and AISI , and positively associated with LMR . By contrast, barring LMR and SIRI, there were no significant associations between combined blood cell count indexes and functional lung parameters in healthy controls.In IPF patients, combined blood cell count indexes of inflammation were also significantly associated with functional lung parameters Table . In partp < 0.001).ROC curve analysis was carried out to evaluate the sensitivity, specificity, and the diagnostic accuracy of the combined inflammation indexes in distinguishing IPF patients from healthy subjects Table . Except We observed significant differences in neutrophil to lymphocyte ratio (NLR), derived neutrophil to lymphocyte ratio (dNLR), monocyte to lymphocyte ratio (MLR), platelet to lymphocyte ratio (PLR), systemic inflammation response index (SIRI) and aggregate index of systemic inflammation (AISI) between patients with IPF and a group of healthy controls matched for age, gender and smoking status. All combined blood inflammation indexes, except for PLR, were independently associated with IPF after adjusting for age, gender, body mass index and smoking status. Furthermore, there were significant associations between FVC% and NLR, LMR, SIRI and AISI, and between DLCO% and NLR, dNLR, LMR, SIRI and AISI.IPF is the most severe and frequent form of interstitial lung disease, characterized by aberrant deposition of extracellular matrix due to recurrent micro-injuries to the alveolar epithelium. Mediators released by epithelial cells activate fibroblast proliferation leading to accumulation of fibroblasts and highly active myofibroblasts. These cells are resistant to apoptosis and lead to extensive lung remodelling, through deposition of extracellular matrix components such as hyaluronan, fibronectin and interstitial collagens, and consequent alveolar wall thickening and impaired gas exchange , 21. WhiRecent findings suggest that neutrophil elastase (NE) promotes TGF-β activation, fibroblast proliferation and myofibroblast differentiation, all contributing to enhance fibrosis . The samOur results show significant alterations of these indexes in IPF patients, thus supporting the ability of these markers to detect systemic inflammation also in this group. These data agree with previous studies demonstrating that NLR and PLR are reliable indexes of systemic inflammation and predict the prognosis of several chronic inflammatory diseases , 15–17. In ROC curve analysis, the NLR, dNLR, LMR, SIRI and AISI were able to significantly discriminate patients with IPF from healthy subjects. The diagnostic accuracy was higher (AUC > 0.7) with NLR, dNLR and SIRI. NLR was the index which performed better, even than the combined indexes (SIRI and AISI) which included more than two blood cell populations in their calculation. The rationale for the use of such indexes, is based on the fact that multiple blood cell populations participate in systemic inflammation as we mentioned before, and therefore, inclusion of these populations in the calculation of an index should better reflect the inflammatory state and improve its predictive abilities. Indeed, the activation of neutrophils during systemic inflammation, not only increases their number, but stimulates also the production of several mediators (like IL-1a), which in turn stimulate megakaryocytes to produce platelets ; in addiOur study has some limitations. In particular, the relatively small sample size and the single centre nature of the study, even accounting for the low prevalence of the disease, limits its generalizability. Furthermore, the cross-sectional design did not allow establishing a definite cause-effect relationship between the studied indexes, the presence of IPF, and the lung function parameters and no correlations were analysed between these indexes and relevant clinical parameters, such as dyspnoea and the six-minute walking test. These issues notwithstanding, our data show the presence of significant alterations in NLR, dNLR, LMR, SIRI and AISI values in IPF patients when compared to healthy controls, and clinically relevant associations with lung function parameters. Such indexes might be attractive from a clinical point of view as their calculation is relatively simple, inexpensive, and part of routinely available laboratory investigations. Nevertheless, large, prospective studies are now required to investigate their prognostic role in IPF.


In this paper, we presented a study of temperature-dependent Raman spectra of G peak and D’ band at low temperatures from 78 to 318 K in defective monolayer to few-layer graphene induced by ion C+ bombardment under the determination of vacancy uniformity. Defects lead to the increase of the negative temperature coefficient of G peak, with a value almost identical to that of D’ band. However, the variation of frequency and linewidth of G peak with layer number is contrary to D’ band. It derives from the related electron-phonon interaction in G and D’ phonon in the disorder-induced Raman scattering process. Our results are helpful to understand the mechanism of temperature-dependent phonons in graphene-based materials and provide valuable information on thermal properties of defects for the application of graphene-based devices.The defects into the hexagonal network of a sp In this paper, we presented a study of T-dependent phonon properties of G peak and D’ peak at low temperatures from 78 to 318 K in 1LG and FLGs with vacancies and tried to discuss the mechanism of the defective phonon effect and extrinsic T-dependent Raman behavior. Our results are helpful to provide T-dependent information of detects on thermal properties in graphene flakes for applications of devices.To clarify different phonon properties with pristine graphene, we performed a T-dependent Raman measurement of mechanically exfoliated 1LG and FLGs after ion C+ bombardment. Ion beam bombardment has been an effective method to finish graphene cutting and perforation , which c D’ peak  (∼ 1620 2 to obtain 1LG and FLGs. We used the notation NLG to indicate flakes with N layers. The layer number (N) of NLG was estimated by Raman measurements of the Si intensity ratio between the Si peak (I(SiG)) from SiO2/Si substrate overlying graphene flakes and the Si peak (I(Si0)) from bare SiO2/Si substrate [I(SiG)/I(Si0) for NLG flakes deposited on SiO2/Si substrate have been given in the supplementary data of reference [N determined and selected 2 sets of 1LG-4LG, 6LG, and 10LG flakes. Vacancies were introduced intentionally by ion C+ bombardment for one set of samples , with the defect-free set as a contrast. The low energy C+ ions bombarded perpendicularly to the sample surface at room temperature which was performed using an LC-4 type system with the dose and kinetic energy of 2 × 1013 cm−2 and 80 keV, respectively. After ion C+ bombardment, the D band at ∼ 1350 cm−1 and D’ peak at ∼ 1620 cm−1 appeared in the Raman spectra of NLG flakes, as depicted in Fig. −1 before and after ion C+ bombardment, which showed that defects in the samples only broke the symmetry of carbon honeycomb lattice but did not cause evident doping which should make the frequency of G peak upshift. This made subsequent research more straightforward. There was another notable spectral band around 2700 cm−1 before and after ion C+ bombardment, which is referred to as a 2D band [Highly oriented pyrolytic graphite (HOPG) was mechanically exfoliated on the same Si {100} substrates covered with an 89-nm SiOubstrate . The staeference . We prep 2D band  and is a 2D band . The lin 2D band . However−1 spectral resolution. The laser excitation of 532 nm was used. A laser power below 2 mW was used to avoid sample heating. Mapping measurements were performed using a motorized stage. The xy coordinates of each point were previously set in order to find the optimized focus. Mapping images were constructed for each xy coordinate by taking 100 points on the surface of a sample with a 10 × 10 equally spaced array of probing points. In all cases x, y step was 0.5 μm. Raman spectra were measured in the range of 1250–2850 cm−1. The mappings of G peak intensity I(G) as a reference for defects contained in graphene flakes are shown in Fig. I(G) is sensitive to the number of defects [2 atoms in both rings and chains. Moreover, G peak is a phonon originating from a normal first-order Raman scattering process in graphene systems, and its intensity can be enhanced because of the resonance process [I(G) mappings in almost all samples is basically homogeneous over the entire sheet to determine the uniformity of the atomic structure of graphene layers. I(G) in defective NLG flakes is lower than that in defect-free NLG flakes due to the introduction of vacancies. Although the color of some points at the corner in the defective set of samples shows a bit difference, we can identify the uniformity of vacancies in the dominant part of defective samples. In addition, defects can be characterized by the average distance between nearest defects (LD) [LD which is about 4–6 nm in C+-bombarded 1LG based on the intensity ratio between D band and G band, i.e., I(D)/I(G), using the well-known Tuinstra-Koenig relation [LD in C+-bombarded 1LG was shown in Fig. fI(D) is also directly related with the number of defects [I(D) were shown for defective 1LG, 2LG, and 3LG in Fig. fIn order to examine the uniformity of vacancies introduced in graphene structure by ion C+ bombardment, we measured Raman mapping of the samples from the defective set, with the defect-free set as a contrast. The Raman mappings were measured at room temperature in back-scattering with a HR Evolution micro-Raman system, equipped with the unique SWIFT™ CCD, a × 100 objective lens (NA = 0.90). An 1800 g/mm grating resulted in a 0.5-cm defects  at low dcts (LD) , 23. We relation  (the map defects  similar 2) cooled low-temperature Linkam stage equipped with a temperature controller. The programmable cool-stage THMS600 (Linkam Scientific Instruments) covers the temperature range from 78 to 318 K in a N2 gas environment. The Linkam instrument has a temperature stability of ± 0.1 K. Using a grating with a groove density of 1800 g/mm, the achieved spectral resolution was 0.5 cm−1. A long working distance × 50 objective lens (NA = 0.45) was used, achieving a spatial resolution better than 1 μm. All spectra were excited with a 532-nm laser. During all the measurements, laser power has been kept low enough to prevent any sample heating. The integration time of 20 s was adopted to ensure a good signal-to-noise ratio. The T-dependence of Raman modes was measured in the range from 78 to 318 K and recorded at 10 K intervals, for the defect-free and defective sets.For the above prepared samples, we measured the T-dependent Raman spectra near G band (including G peak and D’ band) in both defect-free and defective sample sets of 1LG-4LG, 6LG, and 10LG flakes. The T-dependent Raman spectra were measured in back-scattering with a HR Evolution micro-Raman system, equipped with the unique SWIFT™ CCD. The samples were mounted on an in-house-made sample holder consisting of a thin copper disk with a central pillar and a 500-μm-diameter hole. Measurements were carried out in a liquid nitrogen (LNω(T) = ω0 + χT [ω0 is the peak position of vibrational bands at zero Kelvin temperature and χ represents the first-order temperature coefficient of the modes. The defect-free 1LG exhibits a negative temperature coefficient of − (1.56 ± 0.20) × 10−2 cm−1/K  values in the defective set are larger than those in the defect-free set, which should be another result of ion C+ bombardment.Raman linewidth is another significant quality for uncovering the interactions of electrons and phonons when the crystal structure changes. Figure −1 in C+ bombarded 1LG with a slope of around − (2.37 ± 0.20) × 10−2 cm−1/K  increases whereas Pos(G) decreases; simultaneously, FWHM(D’) decreases whereas FWHM(G) increases as graphene layers become thicker.We then studied the defect-related D’ band. Figure −1 in defect-free 1LG and ~ 1584 cm−1 in defective 1LG at 300 K in variable temperature experiments although their Pos(G) basically stay at 1582 cm−1 in room temperature measurements. The possible reason is the thermal expansion coefficient mismatch between the material and the substrate [−1 in the defect-free set and ~ 1580 cm−1 in the defective set at 300 K, which means that it is increasingly insensitive to substrate effects as graphene layers become thicker. Meanwhile, FWHM(G) significantly sharpens down to ~ 9.2 cm−1 in defect-free 1LG and ~ 10.9 cm−1 in defective 1LG in variable temperature experiments although FWHM(G) of pristine graphene is ~ 13 cm−1 in room temperature measurements. The possible reason is the blockage of the phonon decay into electron-hole pairs [By reviewing the previous work, we come to realize that there are several factors that influence the Raman spectra of the graphene systems. First, the T-dependent Raman study of pristine graphene has been explained by phonon anharmonicity and EPC . Howeverubstrate . Pos(G) le pairs  due to tle pairs  because le pairs . (3) D ple pairs . (4) MorI(G). The T-dependent phonon properties of G peak and D’ band in defective 1LG and FLGs were measured by Raman spectrometer combined with a Linkam cryostat, with defect-free samples as a contrast. At temperatures from 78 to 318 K, defects lead to the increase of negative temperature coefficient of G peak due to the lattice change. D’ mode as a Raman signature for disorder is both interrelated and competitive with G peak under the defect-phonon interaction. The temperature coefficient of D’ band is almost identical with G peak. However, Pos(D’) increases simultaneously as FWHM(D’) decreases with increasing layers, contrary to G peak. In conclusion, the defects in graphene structure by ion C+ bombardment induce a large change of T-dependent properties of phonons; therefore, they have an influence on the physical properties of graphene systems. The introduction of foreign atoms into the hexagonal carbon networks has been a hot topic nowadays for an effective tool for tailoring the intrinsic properties of graphene systems. The corresponding properties should be thoroughly investigated in the future.In this paper, vacancies were uniformly introduced into carbon structures by ion C+ bombardment and characterized by Raman mappings of Additional file 1: Supplementary Materials


Sarcoidosis, a systemic granulomatous disease of unknown etiology, may mimic other conditions at presentation often resulting in delayed diagnosis. These conditions include infections, neoplasms, autoimmune, cardiovascular, and drug-induced diseases. This review highlights the most common sarcoidosis mimics that often lead to pitfalls in diagnosis and delay in appropriate treatment. Prior to invasive testing and initiating immunosuppressants (commonly corticosteroids), it is important to exclude sarcoid mimickers. Jonathon Hutchinson, an English physician, first described sarcoidosis in 1877 . DespiteNearly all infectious causes of granulomas may present similarly to sarcoidosis  3–7) th–7 th3–7)In developing countries with high TB burdens, diagnosing sarcoidosis can be particularly challenging. TB is classically characterized by caseating granulomas whereas sarcoidosis has non-caseating epithelioid cell granulomas. However, when caseous necrosis is not seen and acid-fast staining of biopsy specimens is negative then a patient with suspected TB infection can be mistakenly diagnosed with sarcoidosis . In an eEndobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) to obtain TB-PCR samples makes ruling out TB more certain. In a study by Eom et al., 86 specimens were examined in 46 patients and the sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy were analyzed. EBUS-TBNA TB PCR was found to be 56%, 100%, for sensitivity and specificity, respectively. Positive and negative predictive values were 100%, and 81%. Diagnostic accuracy was 85% .P < 0.001) and coagulation necrosis  are suggestive of TB rather than sarcoidosis. A combination of a positive tuberculin skin test (TST) and either heterogeneous echotexture or coagulation necrosis sign had a specificity of 98% and positive predictive value of 91% for a diagnosis of tuberculosis  drug exposure. An accurate diagnosis of TB prevents exposure to immunosuppressive agents. Concomitant tuberculosis and sarcoidosis is rare .The presence of exudative pleural effusions may favor other diagnoses. Pleural effusions associated with sarcoidosis are uncommon (8.2%) and can be present at the time of diagnosis or at a later time, coinciding with an exacerbation , 16. TheThe presence of an exudative effusion in the setting of sarcoidosis warrants infectious workup e.g., parapneumonic effusion, or empyema. Patients on immunosuppressive agents are particularly susceptible to opportunistic infections –20. The Along with sarcoidosis, granuloma formation is a feature of other chronic interstitial lung diseases such as chronic beryllium disease (CBD) and hypersensitivity pneumonitis (HP).Chronic beryllium disease (CBD) is clinically and pathologically indistinguishable from sarcoidosis. CBD risk is associated with fluorescent light manufacturing and in industries such as nuclear energy, ceramics, aerospace, automotive, electronics, and telecommunications . Non-occSymptoms of CBD include dry cough, progressive dyspnea on exertion, fatigue, and night sweats. The radiological features vary with common chest CT findings including small nodules, ground-glass opacification, mild hilar adenopathy, and septal lines. A diagnosis of CBD is favored over sarcoidosis when there is documented occupational exposure to beryllium. Beryllium exposure should be considered at the time of sarcoidosis diagnosis as well as in chronic corticosteroid-resistant sarcoidosis to ensure they are removed from ongoing exposure to immunosuppressants.A diagnosis of CBD can be aided by the following three criteria: (1) symptomatic disease with a histopathological demonstration of non-caseating granuloma, pulmonary function impairment, and abnormal chest radiographs; (2) proof of beryllium sensitization by two independently positive beryllium lymphocyte proliferation assays (BeLPTs) in the absence of treatment with systemic corticosteroids for preceding 3 months, and (3) proof of beryllium exposure , 44. BerManagement of suspected CBD involves stopping all further exposure and continued clinical surveillance. Therapy with corticosteroids may be beneficial in selected patients, but the response is variable. Oral prednisone (doses ranging from 20 to 40 mg/day) for 3–6 months followed by a gradual taper to the lowest effective dose is deemed acceptable. Unfortunately, CBD is typically a progressive disease and lifelong treatment may be required. BeLPT should be offered to any patient with sarcoidosis who has worked around metal-dust or fumes .Hypersensitivity pneumonitis (HP) has clinical, radiological, and histopathological features that overlap with sarcoidosis. HP pathogenesis results from a combination of immune complex-mediated (type III) and delayed-type (type IV) hypersensitivity reactions to antigen inhalation in a susceptible person. Cryptogenic HP is a subtype of HP in which despite typical HP features an inciting exposure is not identified –48.HP can be categorized by disease duration into acute, subacute, and chronic subtypes . Raghu eThe median age at diagnosis is 65 years which is somewhat older than what is typical of sarcoidosis (20–39 years). Common presenting symptoms of HP include dyspnea, cough, wheezing, and, less frequently, constitutional symptoms such as weight loss. Physical examination may reveal the presence of rales. An acute presentation is more consistent with the non-fibrotic subtype and an insidious presentation is more consistent with fibrotic HP , 62, 63.The radiological features of HP depend upon the subtype. Typical radiologic features of non-fibrotic HP include high-resolution CT (HRCT) showing parenchymal infiltration with ground-glass opacities (GGO), and mosaic attenuation. HRCT may also demonstrate small airway disease, which can be described as ill-defined, small (<5 mm) centrilobular nodules on inspiratory images and air trapping on expiratory images, both in a diffuse distribution. Typical radiological features associated with fibrotic HP include an HRCT pattern of irregular linear opacities and or/coarse reticulation with lung distortion; traction bronchiectasis and honeycombing, all typical of lung fibrosis, and at least one abnormality that indicates small airway disease. The presence of three different lung densities, previously referred by radiologists as “head-cheese sign” and recently being referred to as the “three-density pattern,” is characteristically associated with HP . RadioloBronchoalveolar lavage fluid (BALF) analysis of patients with HP reveals a predominance of lymphocytes, like sarcoidosis. A meta-analysis of 53 studies  by Raghu et al. demonstrated that patients with HP had a higher proportion of BALF lymphocytes than patients with sarcoidosis . This was found regardless of whether the study enrolled patients with non-fibrotic HP , fibrotic HP , or mixed populations with both non-fibrotic and fibrotic HP . For distinguishing non-fibrotic HP from sarcoidosis, BALF lymphocyte thresholds of 20, 30, and 40% yielded decreased sensitivities for HP of 95, 88, and 76%, respectively, but increased specificities of 26, 43, and 61%, respectively, with an area under the curve of 0.71  .A diagnosis of HP requires:A diagnosis of HP can be made with high confidence in patients with an identified provocative exposure and who have a typical HP radiological pattern on HRCT and with BALF revealing predominant lymphocytosis. For less straightforward cases, a multidisciplinary team consisting of a pulmonologist experienced in interstitial lung disease (ILD), a chest radiologist, and when necessary, a pathologist familiar with histopathological features of ILD and HP.Treatment involves prompt and complete avoidance of further exposure to the inducer. Systemic corticosteroids are used in treatment however, their use has no effect on long-term outcomes and is often reserved for patients with more severe symptoms , 68. FibWhen presented with a patient with sarcoidosis and worsening shortness of breath, an assessment for pulmonary embolism (PE) and pulmonary hypertension (PH) is warranted. PE and sarcoidosis associated pulmonary hypertension (SAPH) may often be misdiagnosed as an exacerbation of sarcoidosis.Sarcoidosis, as with other chronic inflammatory conditions, has been associated with an increased risk of venous thromboembolism (VTE) –73. SwigDLCO (<35% predicted) and a 6MWD of <300 m are associated with worse transplant-free survival. Oksana et al. identified preservation of FEV1/FVC ratio as an independent risk factor for worsened outcomes , the hypothalamus and the pituitary gland followed by the meninges, brainstem, spinal cord are less frequently involved –96.Neurosarcoidosis can mimic other neurologic diseases including neoplasm  , infectiCerebrospinal Fluid (CSF) findings in neurosarcoidosis may reveal elevated protein (50–70% of patients), elevated CSF pressure with a lymphocytic pleocytosis (57–72% of patients), and a reduced glucose level (up to 18% patients). None of these abnormalities are specific for neurosarcoidosis. Several studies evaluated the role of elevated CSF angiotensin-converting enzyme (ACE) level for diagnosing neurosarcoidosis , 104. ThIn suspected neurosarcoidosis associated aseptic meningitis, CSF should be sent for routine microbiological studies, fungal, and mycobacterium cultures, mycobacterium TB polymerase chain reaction (PCR). Cytology and flow cytometry should also be considered. Once the infection has been ruled out, corticosteroids can be initiated as first-line treatment. In sarcoidosis, both aseptic meningitis and isolated cranial nerve abnormalities usually respond to steroids. Corticosteroids can be started at 1 mg/kg or as a pulse of methylprednisolone . Steroids may be gradually reduced over the next 6–9 months. In steroid-refractory cases, methotrexate, azathioprine, cyclophosphamide, or mycophenolate should be considered. Radiotherapy has been reported in patients with refractory sarcoid meningitis but has been used infrequently , 108.One of the most challenging presentations is in patients, typically young women, with optic neuritis and finding a few demyelinating lesions on brain MRI. The clinical presentation of both neurosarcoidosis and multiple sclerosis (MS) include both these features. While over 90% of MS patients have oligoclonal bands on CSF analysis, oligoclonal bands can be seen in up to 25–50% of neurosarcoidosis patients making distinguishing sarcoidosis from multiple sclerosis more difficult –112. NeuThe diagnostic workup in a patient with a suspicion of neurosarcoidosis mimicking MS should include chest CT to search for lymphadenopathy and a full-body positron emission tomography (PET) scan to search for other organ involvement that supports the diagnosis of sarcoidosis, e.g., FDG uptake in thoracic lymph nodes, lung, spleen, and bone. PET scans can also direct tissue biopsy for histologic confirmation .Diagnostic criteria proposed by Zajicek et al. categorizes neurosarcoidosis into definitive, probable, and possible neurosarcoidosis . DefinitMRI is the preferred mode of imaging for diagnosis and follow up of neurosarcoidosis as it carries a high sensitivity. Leptomeningeal enhancement favors a diagnosis of sarcoidosis over neoplasm , 129–131Immunosuppression has been the mainstay of treatment for neurosarcoidosis with corticosteroids being the first-line. Other steroid-sparing therapeutic agents include methotrexate, mycophenolate mofetil, azathioprine, cyclosporine, cyclophosphamide, chlorambucil, pentoxifylline, hydroxychloroquine, thalidomide, infliximab, and adalimumab .Peripheral nervous system involvement includes mononeuropathy, mononeuritis multiplex, sensory, sensorimotor, and motor polyneuropathies. Symptoms may be acute, subacute, or chronic. An acute generalized demyelinating motor neuropathy similar to the Guillain-Barré syndrome also has been described . Small fSteroid-induced remissions coupled with frequent relapses observed in patients with neurosarcoidosis can lead to a clinical picture that resembles an inflammatory demyelinating disease. Neurologic involvement may be more difficult to confirm as tissue biopsy may be impractical –138.Harrel et al. first described hypercalcemia associated with sarcoidosis in 1939 and since then abnormal calcium metabolism is considered a key feature of sarcoidosis. Hypercalcemia in sarcoidosis is secondary to an uncontrolled synthesis of 1,25-dihydroxy vitamin D3 by macrophages present in granulomas. The increased 1,25 dihydroxyvitamin D3 leads to increased intestinal absorption of calcium that leads to increased resorption of calcium in the bone , 140. ThIn the kidney, untreated hypercalcemia causes afferent arteriole vasoconstriction and inhibition of sodium-potassium ATPase causing a decrease in glomerular filtration rate and urinary sodium wasting, polyuria, and dehydration. Increased intracellular calcium overload and tubular obstruction by calcium precipitates may lead to tubular necrosis. Renal consequences of hypercalcemia and hypercalciuria are frequently reversible, however, long-standing hypercalciuria may lead to nephrocalcinosis and permanent changes , 142.Once hypercalcemia is detected, serum albumin, ionized calcium, and 24-h urine collection for calcium excretion should be measured. In cases of progressive renal impairment, 24 h creatinine clearance, and abdominal ultrasound should also be performed to exclude nephrolithiasis or nephrocalcinosis. Primary hyperparathyroidism should routinely be ruled out. Histologic confirmation of sarcoidosis may be required to exclude lymphoma as it has the propensity to present with hypercalcemia associated with lymphadenopathy .Asymptomatic and mild hypercalcemia detected in a patient presenting with acute sarcoidosis requires no further assessment, and studies suggest that monitoring the response to corticosteroid therapy is an acceptable practice. Management is aimed to prevent long term renal and bone complications. Patients are advised to maintain a fluid intake of >2 l per day, minimize their exposure to sunlight, and avoid vitamin D. Severe hypercalcemia is fairly responsive to steroids. Corticosteroids achieve normocalcemia by inhibiting the enzyme 1-α-hydroxylase, reducing gastrointestinal calcium absorption, inhibiting osteoclast function, and decreasing the production of parathyroid hormone-related protein (PTHrP) by the macrophages , 144–147Sarcoid-like reaction (SLR) is defined as the presence of non-caseating epithelioid cell granuloma lesions of sarcoidosis without accompanying systemic symptoms. Sarcoid-like reactions are histologically indistinguishable from systemic sarcoidosis .SLRs can be observed in patients with various malignancies. The lesions can be adjacent to the primary tumor, in the tumor itself or adjacent to the local draining lymph nodes , 150. SLBoth SLRs and malignancy demonstrate increased uptake of 18F-fluorodeoxyglucose (FDG) on PET scan, which makes it difficult to distinguish the two. Though PET scans may be useful in selecting possible biopsy sites to search for malignancy, they have no role in differentiating between these two entities. There are no specific markers or radiographic patterns to distinguish SLRs from systemic sarcoidosis or from malignancy –156. SLRper se or a result of an impaired immune system leading to the development of sarcoidosis while on drug therapy (Drug-induced sarcoidosis-like reactions (DISR) can be defined as a granulomatous tissue reaction, indistinguishable from sarcoidosis, that occurs at the same time as initiation of a potential offending drug . DISR ca therapy .Although TNF-α is known to play an important role in the formation and stabilization of sarcoid granulomas, surprisingly DISRs have been reported with the use of TNF-α antagonists therapy. Most commonly reported with etanercept, DISRs occur with any of the TNF-α antagonists. The average time to occurrence of DISRs is 24 months after drug initiation with almost 60% of patients requiring treatment . TreatmeDISRs may mimic other clinical manifestations of sarcoidosis such as infections, drug and autoimmune reactions, and neoplasms. In select cases, DISR can resolve with discontinuation of the offending drug , 167. DISLRs have been reported after joint replacement surgery and after silicone breast implant placement , 181. ThIn 1929, Bernstein was the first to recognize cardiac involvement in sarcoidosis. Granulomas in cardiac sarcoidosis (CS) most often involve the conduction system and can potentially involve any part of the heart including the pericardium and myocardium . DependiA timely diagnosis of CS is often difficult as it may present with clinical features and radiographic findings overlapping with other cardiac disorders. Plain chest radiographs may appear normal. Some of the common mimickers of CS cardiomyopathy include amyloidosis and other infiltrative diseases, non-ischemic and ischemic cardiomyopathy, right ventricle infarction, lymphocytic myocarditis, connective tissue diseases associated cardiomyopathy, vasculitis (Takayasu arteritis and Wegener granulomatosis), Chagas disease, hypertrophic cardiomyopathy, and other infectious causes associated with cardiomyopathy  –192.Echocardiographic abnormalities, present in up to 24–77% of CS patients, range from non-specific findings to more sarcoid associated findings such as thinning of the basal anterior septum, regional wall aneurysm, diastolic dysfunction, or motion wall abnormalities in a non-coronary artery distribution . ImpaireThallium 201 scintigraphy can be used to help distinguish the ischemic disease from sarcoidosis or other infiltrative diseases. The reverse distribution phenomenon on rest and exercise thallium described as defects detected in the resting phase during thallium scanning that disappears or decreases in size during exercise or after dipyridamole infusion . CardiacInfrequently, granulomatous infiltration of the myocardium can result in myocardial thickening which can morphologically mimic hypertrophic cardiomyopathy but unlike CS, which is characterized by inflammation and edema, T2-weighted MRI features not typically seen in hypertrophic cardiomyopathy. Areas of Late gadolinium- enhancement (LGE) in CS are more likely to be patchy and mid myocardial.For cardiac amyloid which is a consideration for unexplained infiltrative disease, both 99mTcpyrophosphate scanning and tissue biopsy (fat pad) showing apple-green birefringence at polarizing light microscopy are distinguishing features –200. In CS can be managed with corticosteroids and when necessary inotropic, vasodilator, and antiarrhythmic medications. Automatic Implantable Cardioverter Defibrillator (AICD) or cardiac pacing may have a role. Cardiac transplantation although rare can be considered in younger patients with advanced end-stage cardiac failure. No difference in outcome has been reported when transplants are done for CS –206.Common variable immunodeficiency syndrome may be complicated by granulomatous lymphocytic interstitial lung disease (GLILD) . The preKey clinical features that should alert one to the diagnosis of CVID include a history of recurrent infections  and a coWith the aid of B cell immunophenotyping, granulomas associated with CVID reveal a severe reduction in switched memory B cells and a high level of CD21 low B cells. Immunoglobulin perfusions are considered the treatment of choice for CVID . MeasureAs illustrated by the numerous diseases that sarcoidosis can imitate, sarcoidosis deserves the reputation of “The Great Mimicker.” The purpose of this review the most common diseases that should be considered when sarcoidosis enters the differential diagnoses. Aside from the time lost for adequate treatment of the disease present, both using immunosuppressive drugs in a state that appears to be sarcoidosis or conversely failing to adequately treat sarcoidosis with its proper treatment regimen will lead to adverse outcomes. The authors herein described common pitfalls in the diagnostic process and how to avoid them in order to provide, to the best of our ability, an accurate diagnosis for a condition that continues to baffle, elude, and mislead clinicians.All authors contributed equally in data collection, interpretation, paper writing, editing, and review.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.