The abnormal mislocalisation and ubiquitinated protein aggregation of the TAR DNA binding protein 43 (TDP-43) within the cytoplasm of neurons and glia in the central nervous system (CNS) is a pathological hallmark of early-onset neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The pathomechanisms underlying abnormal mislocalisation and aggregation of TDP-43 remain unknown. However, there is a growing body of evidence implicating neuroinflammation and immune-mediated mechanisms in the pathogenesis of neurodegeneration. Importantly, most of the evidence for an active role of immunity and inflammation in the pathogenesis of ALS and FTD relates specifically to TDP-43, posing the question as to whether immune-mediated mechanisms could hold the key to understanding TDP-43’s underlying role in neurodegeneration in both diseases. Therefore, this review aims to piece together key lines of evidence for the specific association of TDP-43 with key immune and inflammatory pathways to explore the nature of this relationship and the implications for potential pathomechanisms underlying neurodegeneration in ALS and FTD. The innate immune system is the body’s first line of defence against pathogens and plays a central yet varying role in both health and disease. Neuroinflammation involves a complex multistage physiological response triggered by cell damaging processes in the brain, including infection, toxins, autoimmunity, trauma, and aberrant responses to altered neuronal activity. Neuroinflammation is driven by reactive resident CNS innate immune glial cells, microglia, and astrocytes, and is accompanied by a dynamic biochemical cascade of inflammatory cytokines and chemokines that modify the CNS microenvironment. The degree of neuroinflammation that exists across the spectrum of neurological conditions varies and is dependent on multiple factors including the duration of the inflammatory response, its course and the circumstances underlying the primary insult. The primary aim of the neuroinflammatory response is to mitigate the triggering factors by invoking CNS immunity to defend from harm and maintain and restore homeostasis. Therefore, neuroinflammation has the capacity to be both beneficial and damaging ,2.The brain has been shown to undergo a process termed ‘inflammaging’, progressively acquiring an increased proinflammatory environment across the lifespan . In addiTARDBP gene and is part of the heterogeneous nuclear ribonucleoprotein (hnRNP) family. TDP-43 was first identified as a host cell protein binding to pyrimidine-rich DNA motifs in a long terminal repeat, referred to as a TAR of human immunodeficiency virus type I [TARDBP mutations have been identified to date [TARDBP transcripts [TDP-43 is a 43 kDa, 414 amino acid nuclear RNA/DNA-binding protein that is widely expressed, but of functional relevance to the CNS. TDP-43 is encoded by the s type I . Structu to date ,11 . In the CNS, PGRN plays a critical role in maintaining physiological functions and is expressed by neurons and microglia within the CNS [GRN is a major cause of familial FTD [GRN mutations are associated with the accumulation of abnormal TDP-43 pathology [GRN carriers present with dysregulated levels of proinflammatory cytokines in serum and CSF, in addition to increased expression of inflammatory genes in leukocytes [GRN knockout mice with PGRN deficiency exhibit an excessive accumulation of activated microglia [GRN knockout mice present with a chronic upregulation of innate immunity and complement expression that increases with age [ the CNS ,89. PGRN the CNS ,91,92. Hlial FTD . GRN mutathology  and sympukocytes . GRN knoicroglia  that proicroglia ,97. Emericroglia . In suppwith age . It has with age .TBK1 encodes a protein kinase with an established role in regulating immune response, autophagy, and neuroinflammation [TBK1 is activated via multiple pathways resulting in phosphorylation and activation of innate immune transcription factors, interferon regulatory factor 3 and 7 (IRF3/IRF7), and activates NF-κB in response to TNFα. Mutations including loss of function, missense, and in-frame deletions of TBK1 are identified as causative of ALS and FTD [TBK1 mutation carriers show TDP-43-positive perinuclear inclusions in temporal lobe neurons, but not in the spinal cord [TBK1-related innate immunity pathway in FTD-TDP patients compared to controls [TBK1 mutations observed in ALS and FTD-TDP patients have previously been shown to reduce the activation of one of the most well-characterised transcription factors involved in innate immunity, interferon regulatory factor 3 (IRF3) [TBK1 mutations could influence the pathogenesis of ALS and FTD given the role of TBK1 in autophagy processes. In support of this, mutations in the autophagy receptors OPTN and SQSTM1 are causal of FTD and ALS and TBK1 promotes autophagy via phosphorylation of OPTN and SQSTM1 therefore enhancing the ubiquitin-binding abilities of both proteins [TBK1 could potentially represent a direct link between neuroinflammation and kinases in the neurodegeneration underlying ALS and FTD.ammation ,100. In  and FTD ,102. Posnal cord . As disccontrols  and TBK13 (IRF3) . TBK1 muproteins ,105. TBKOne of the most prolific inflammatory pathways consistently associated with TDP-43 to date is the nuclear factor kappa light chain enhancer of activated B cells NF-κβ. NF-κβ acts as a transcription activator and modulates hundreds of genes involved in inflammation, innate immunity, cell survival, and cancer. This pathway is identified as a molecular culprit of inflammaging , is invoOf particular interest to this review, TDP-43 and NF-κβ share the same classic nuclear transportation mechanism, therefore it is suggested that they may functionally interact with each other by competing for access to nuclear transportation machinery . In suppIn addition to the role TDP-43’s NLS may play in the interaction with NF-κβ/p65, other key structures of TDP-43 such as the RRM1 domain may also play a facilitatory role. The sensitivity of the RRM1 domain in TDP-43 proteinopathy has been highlighted and oxidation of the RRM1 domain results in cytosolic mislocalisation with irreversible protein aggregation ,27. InteTo therapeutically reduce TDP-43 pathology, Pozzi and colleagues utilised the implicated role of the RRM1 domain in an interaction with the NF-κβ/p65 subunit to generate single chain antibodies targeting the RRM1 domain. The twofold aim was to block TDP-43 and p65 interaction, thus reducing NF-κβ activation and interfering with TDP-43 protein aggregation . Virus mA315T or TDP43G348C resulted in a decrease in the ratio of cytoplasmic to nuclear expression of human TDP-43, partial rescue of large spinal motor neurons at one year of age and improved motor performance and cognition. Neuronal inhibition of NF-κβ therefore rescued TDP-43 proteinopathy and mitigated TDP-43 neurodegeneration [A315T mice [In a separate study, transgenic mice with neuron specific expression of the super-repressor form of NF-κβ (IkBa-SR) were crossed with mice of both sexes expressing ALS-linked gene mutations for TDP-43. Neuronal expression of IkBa-SR in mice expressing TDP43neration . These o15T mice .A315T, where chronic administration of LPS from 6 months of age exacerbated pathological TDP-43 accumulation in the cytoplasm of spinal motor neurons and enhanced levels of TDP-43 aggregation [In the context of neurodegenerative disease TDP-43 has been shown to directly interact with p65 colocalising in the nucleus of neurons in CNS samples from ALS patients , patientregation .A315T fragment to motoneurons alone or to motoneurons cocultured with microglia mediated activation of microglia and triggered a proinflammatory cascade that was toxic to motoneurons. However, in the absence of microglia, the TDP-25A315T fragment was not toxic to motoneurons, suggesting that TDP-43 neurotoxicity is indirect and mediated via proinflammatory microglia [It is clear from multiple studies that a functional interaction between NF-κβ/p65 and TDP-43 exists, although it is yet to be fully understood, there is evidence to suggest such an interaction may be mediated by microglia. In addition to being a coactivator of p65, overexpression of TDP-43 has been shown to produce a hyperactive proinflammatory response after stimulation with LPS and ROS, resulting in microglial sensitivity to immune stimulation, subsequently enhancing the neurotoxicity of neighbouring neurons . Downregicroglia .TBK1, and as discussed above the TBK1 immune pathway has been implicated in both FTD and ALS [The recognition of foreign nucleic acids is one of the key mechanisms utilised by the immune system to detect pathogenic entities to elicit a response. A signal is relayed via the cytoplasmic DNA-sensing cyclic GMP-AMP synthase (cGAS)/stimulator of the interferon genes (STING) pathway (cGAS/STING) following detection of cytosolic DNA, which then induces an immune response. Increased engagement of the cGAS/STING pathway in the CNS results in neuroinflammation and neurodegeneration as recently reviewed . STING c and ALS ,47.The NLR family pyrin domain containing 3 (NLRP3) inflammasome is a member of a family of intracellular innate immune sensors that are integral for cellular defence. NLRP3 is activated by pathogen associated messenger proteins (PAMPs) and danger associated messenger proteins (DAMPs) that signal and activate microglia and are involved in the response to glial production of various inflammatory factors, subsequently promoting an inflammatory response that further engages the innate immune system . NLRP3 iParkin in the hippocampus of mice treated with the neurological toxicant BDE-47 [Parkin plays an important role in mitochondrial activity and integrity and has been shown to link together inflammation, mitochondrial stress, and neurodegeneration with an additional role in restraining innate immunity [Parkin are predominantly linked to PD there is increasing evidence that Parkin facilitates TDP-43 translocation from the nucleus to the cytoplasm and has an essential role in TDP-43 subcellular localisation and toxicity [Parkin protein levels [Parkin cleavage, it has been hypothesised that NLRP3 inflammasome activation may be associated with TDP-43 toxicity [Drosophila studies have suggested that the Parkin pathway may be differentially dysregulated in TDP-43 proteinopathy [TDP-43 inclusions activate the NLRP3 inflammasome in primary microglial cultures resulting in increased production of IL-1β . Similart BDE-47 . Parkin immunity . Althougtoxicity ,133. In n levels . Given Ntoxicity . Drosophinopathy . Q331k ALS mice, with TDP-43 wild-type and mutant proteins able to activate microglial inflammation in a NLRP3-dependent manner. Explicitly, spinal cord gene expression of the inflammasome components NLRP3, caspase 1, and ASC were significantly increased in TDP-43Q331K mice compared to wild type littermates and both wild type and mutant (A315T and Q331K) recombinant forms of TDP-43 protein were able to activate primed microglia to generate the cytokine interleukin 1-beta (IL-1β). However, this was not unique to TDP-43 given aggravated and soluble SOD1G93A also activates NLRP3 in primary mouse microglia. Of particular interest to ALS and FTD, NLRP3 inflammatory activation can be generated by C90rf72 repeat expansions, leading to lysosomal dysfunction, mitochondrial functional impairments, intracellular metabolic imbalances, and intracellular protein aggregation [Deora and colleagues  demonstrregation .Drosophila model. JNK and p38 undertake multiple cellular functions within the CNS including prominent roles in both innate and adaptive immunity and the regulation of activity and expression of key inflammatory mediators as reviewed elsewhere [Stress activated protein kinases (SAPKs) are members of the mitogen activated protein kinase (MAPK) family and are activated by multiple environmental stressors including inflammatory cytokines. Several kinases including p38, JNK, and TBK1 have been associated with ALS-related pathophysiology ,139. Zhalsewhere .Drosophila models, neuroinflammation is demonstrated as a prominent feature of TDP-43 induced neurodegeneration and notably the innate immune response provides a strong phenotypic rescue in TDP-43 transgenic flies, dramatically extending their lifespan [Drosophila motor neurons [Drosophila including Toll/Dif and Imd/Relish have been shown to contribute to TDP-43 neurotoxicity, indicating that immune activation is a critical component of TDP-43 neurotoxicity in Drosophila. Given TDP-43 negatively regulates its own RNA [In lifespan . Both JN neurons . Zhan an neurons . Further own RNA , it has  own RNA . This no own RNA .Although TDP-43 has been shown to induce neuroinflammation, contradicting evidence investigating TDP-43 in the periphery demonstrated that overexpression of TDP-43 reduces the inflammatory response, interfering with the release of inflammatory factors. In a rat model of osteoarthritis (OA), the mechanism of TDP-43 gene expression on inflammatory factors JNK and p38 signalling pathways in ischemic hypoxic stress dependence was investigated. Overexpression of TDP-43 reduced the inflammatory response induced by OA by interfering with the release of inflammatory factors and inhibiting activation of the JNK and p38 signalling pathways via ischemic hypoxia stress . These fQ331K, local complement activation increased expression of C5aR1, part of the terminal complement pathway, which may contribute to motor neuron death and neuromuscular junction denervation. C5aR1 expression was upregulated during disease progression with expression in motor neurons and microglia surrounding regions of motor neuron death [Q331K ALS model.The complement system is a branch of the innate immune system that plays a critical role in development, homeostasis, and regeneration of the CNS throughout life. This dynamic system consists of a group of proteins that work together to destroy foreign invaders, trigger inflammation, and remove debris from cells and tissues. Chronic activation of the complement system is a key mediator of neuroinflammation and complement dysregulation is identified as a key component in neurodegeneration. Notably the complement system has been proposed to drive neurodegenerative mechanisms in ALS ,145. In on death . In addion death . This stAn important role for complement component proteins has been identified in tagging synapses during inflammation and remodelling . Local tGRN knockout microglia is sufficient to promote TDP-43 granule formation, nuclear pore defects, and cell death in excitatory neurons via the complement activation pathway [GRN knockout microglia. Thus, this suggests that GRN knockout microglia may utilise complement-mediated assembly of protein complexes to promote TDP-43 proteinopathy, indicating an important dynamic may exist between resident innate immune glia, the central innate immune complement pathway, and TDP-43.Neurotoxic microglia are shown to promote TDP-43 proteinopathy in PGRN deficiency and the complement cascade may be a mitigating factor. Zhang and colleagues demonstrated that conditioned media from  pathway . Consist pathway . While tThere is increasing evidence demonstrating functional links between TDP-43 and various central innate immune inflammatory pathways as outlined above. In addition, multiple studies discussed above have highlighted the potential therapeutic and pharmacological targets that these innate immune inflammatory pathways could provide in the search for disease modifying therapies in ALS and FTD. However, considerable investigation is required into the underlying mechanisms that can explain such functional interactions. Furthermore, investigation that can determine the contribution of such interactions to the pathogenesis of inflammation-driven neurodegeneration in TDP-43 proteinopathies.GRN mutations had the highest prevalence of autoimmunity and exhibited increased levels of TNFα, representing a unique pattern of systemic inflammation [C9orf72 repeat expansion, and in patients with coinciding FTD/ALS [C9orf72 expansion carriers in a large Finnish cohort study of FTLD patients showed the lowest prevalence of immunological diseases. While still suggesting a role of C9orf72 in immunoregulation, this study did not provide support for the specific association between TDP-43 pathophysiology and autoimmunity [C9orf72, and FTD/ALS is required.GWAS studies report an enrichment of FTD-associated genetic variants in multiple autoimmune disorders . In a reammation . In a si FTD/ALS . In bothimmunity . These dC9orf72 expansion has been the focus of multiple rodent model studies demonstrating that loss or elimination of C9orf72 function profoundly disturbs immune homeostasis and predisposes to autoimmunity, therefore implicating C9orf72 in the regulation of autoimmunity. C9orf72 forms a heterodimer with SMCR8 and ablation of SMCR8 in mouse models results in splenomegaly and autoimmune phenotypes like those observed in mice with C9orf72 deficiency [C9ORF72 and SMCR8 have interdependent functions in suppressing autoimmunity and negatively regulating lysosomal exocytosis [C9orf72 repeat expansions carriers. In addition to C9orf72, GRN mutations have also been linked to autoimmunity with multiple studies reporting prominent upregulation of serum progranulin levels in patients with various autoimmune diseases. PGRN has been identified as a key player in multiple individual autoimmune diseases and antibodies to PGRN have been detected in patients with histories of autoimmune conditions [As discussed earlier, ficiency . C9ORF72ocytosis . Collectnditions ,156,157.Collectively these studies provide further evidence for immune dysregulation in ALS and FTD, specifically altered adaptive immunity that is intrinsically linked to TDP-43 pathophysiology. The mechanisms underlying the overlap between altered adaptive immunity and TDP-43 proteinopathy warrants further investigation, raising the question as to whether adaptive immune dysregulation could provide a novel target for identifying individuals who may be at risk of developing ALS and FTD or may provide an opportunistic target for future therapies that can manipulate adaptive immune pathways, thus potentially altering the trajectory of neurodegenerative processes.Neuroinflammation is a pathological feature of various neurological conditions. TDP-43 proteinopathy is observed in non-neurodegenerative conditions including strokes and TBI in addition to being a feature of traumatic injury related-neurodegeneration in CTE. To further elucidate the link between TDP-43 and immune-mediated pathways, the following discussion explores the presence of TDP-43 pathology in acute and chronic inflammatory conditions not primarily driven by neurodegeneration, but rather involving ischemia and brain trauma.Neuroinflammation plays a key role in the pathogenesis of a stroke, with secondary neuroinflammation post stroke promoting further injury and cell death. Conversely however, neuroinflammation is also shown to be beneficial in promoting recovery in strokes as reviewed elsewhere . In a hiThe dysregulation of TDP-43 expression has been associated with increased microglial activation and innate immune signalling . HoweverFinally in a proteomics assessment of the insoluble aggregated proteome following cerebral ischemia in a mouse model of middle cerebral artery occlusion, ischemia/reperfusion induced the aggregation of RNA binding and heat shock proteins with roles in DNA/RNA processing, stress response, and cell signalling. The largest group of aggregated proteins in ischemia was RNA binding proteins including TDP-43 . This suTBI results in the activation of multiple inflammatory pathways  and is pp-TDP-43 cytoplasmic granules. A single stab injury induced the formation of cytoplasmic TDP-43 granules in wild type animals, which peaked at 3 days post injury (dpi) and began declining by 7 dpi, however a much longer response was observed in mutant TDP-43 mice, who continued to accumulate p-TDP-43 cytoplasmic granules up until 7 dpi. Moreover, the glial and inflammatory response to TBI was significantly more pronounced in TDP-43 transgenic mice, specifically microglial activation was markedly increased at 3 dpi in TDP-43 mutant mice compared to wild type mice [Given that inflammation is demonstrated to induce cytoplasmic TDP-43 translocation , it has ype mice . This suype mice . The autype mice .TBI is linked with the development of CTE, which is characterised by progressive neurodegeneration associated with repetitive head trauma. TDP-43 pathology is present within post-mortem brain tissue in some cases of military related TBI, in sporting athletes who develop CTE and in repetitive head trauma animal models ,168,173.Drosophila models to investigate the link between repetitive brain trauma and ALS show that Drosophila have implicated disruptions to altered protein clearance, autophagy pathways, and nucleocytoplasmic transport [Drosophila expressing C9orf72, repetitive brain trauma showed increased mortality and locomotor dysfunction [C9orf72. In the same study, Anderson and colleagues demonstrated the deposition of ubiquitin, p62/SQSTM1, TDP-43, and stress granule formation within the Drosophila brain following repetitive trauma and, interestingly, Drosophila with repeated brain trauma exhibited highly similar pathology to that observed in ALS patients and ALS mouse models [SQSTM1/p62 is an autophagy receptor and has been identified as a rare genetic variant in both ALS and FTD in addition to being able to activate the NF-κβ pathway [Drosophila brains following repetitive trauma demonstrated that repetitive TBI upregulated nuclear pore proteins, altered nucleoporin stability, nucleocytoplasmic transport proteins, and nucleocytoplasmic transport itself [Research using ransport ,176. In function , which me models . NoteworWhile various studies have investigated TDP-43 proteinopathy across CNS inflammatory disorders outside of neurodegeneration , further research is warranted to investigate the precise mechanisms underlying the highlighted associations to fully explore the paradigm between TDP-43 proteinopathy and acute or chronic CNS insults such as cerebral ischemia and brain trauma. It is important to appreciate that much can be learned from researching TDP-43 proteinopathy in the context of diseases outside of the typical focus of neurodegenerative diseases, material that could be used to further understand TDP-43’s role within the CNS and inform therapeutic targets and pathways that could be manipulated in the context of ALS and FTD.This review established four key lines of evidence for the specific relationship between TDP-43, immunity, and inflammation . This evC9orf72, GRN, and TBK1) in immunity and inflammation.The involvement of various ALS and FTD causative and susceptibility genes Altered adaptive immunity in ALS and FTD that is intrinsically linked to TDP-43 pathophysiology .Investigation into the substantial amount of evidence supporting TDP-43’s relationship with immunity and inflammation that centres around microglia.Determining the manner and involvement of TDP-43 structural and functional sites  with key inflammatory pathways Investigation of the mechanisms underlying TDP-43’s role in triggering cytoplasmic mitochondrial DNA release and activating central inflammatory pathways Deciphering the association between TDP-43 and autoimmunity to determine whether systemic inflammation is a risk factor for TDP-43 proteinopathy or the possibility of common shared mechanisms between TDP-43 proteinopathies and adaptive immune dysregulation.Explore the mechanisms underlying TDP-43’s presence and involvement in strokes, TBI, and CTE to determine whether shared mechanisms exist in the context of neuroinflammation.This review has also identified evidence that warrants further investigation including:In summary, inflammatory pathways and immune-mediated mechanisms present a promising opportunity to explore therapeutic manipulation and biomarkers of inflammation that can inform disease progression during life in ALS and FTD patients expressing TDP-43 proteinopathy. Furthermore, investigation of the role of TDP-43 in other CNS disorders that also exhibit neuroinflammation may provide novel avenues to uncover the mechanisms underlying TDP-43 proteinopathy in the CNS.


Having an insight into graphene and graphene derivatives such as graphene oxide, reduced graphene oxide and graphene quantum dots is necessary since it can help scientists to detect possible properties and features that could be useful when using these carbon materials in preparation of a nanocomposites. In recent years, graphene and its derivatives have attracted a lot of attention and been extensively applied in biosensors due to fascinating properties, such as large surface area, optical and magnetic properties, and high elasticity for the detection of microorganisms as they can be modified with some other materials such as macromolecules, oxide metals and metals to improve the electrochemical behaviour of the biosensor. In this review paper, biosensor design strategies based on graphene and its derivatives (graphene-based nanocomposites in biosensors) are described. Then their application for the detection of microorganisms including prions, viroids, viral and bacterial cells as well as fungi, protozoa, microbial toxins and even microbial sources of antibiotics is reviewed. Biorecognition elements provide specificity, selective and strong affinity to the targets  and could be used as recognition elements in biosensors  region of graphene is able to directly bind to heteroatoms, nanoparticles (NPs), enzymes, antigens, proteins, antibodies, DNA and other specific molecules  applying aptamer-graphene oxide and the results showed a good linearity in the concentration range of about 0.001–1 ng/mL. Zhuang et al. .Since viruses cause many diseases in humans, animals and plants, especially viruses that are detrimental for human health such as HIV , and also a low LOD of 10 CFU/mL for Salmonella sp. Singh et al.  and dormant cysts. Historically, protozoa have been categorized as single-celled species, distinct from phototaxis, single-celled photosynthetic organisms  that are called primitive plants. In both classes, the rank of phylum was commonly granted under the Protista kingdom. Jain Aspergillus ochraceus, Aspergillus carbonarius and Penicillium verrucosum. This toxin may be present in many daily consumed foods. Ochratoxin A may induce apoptosis in several cell types or may increase the incidence of tumours in humans. PVP-coated gold nanoparticles have been reported for the selective determination of ochratoxin A via quenching fluorescence of the free aptamer , reduced GO and graphene quantum dot nanocomposites have fascinating properties, including large surface area, optical and magnetic properties, and high elasticity, which makes it an appropriate basic structure for preparing several graphene-based nanocomposites. They are scaffolds for immobilizing biomolecules and create highly selective biosensors. Based on recent studies, among several detection methods applying graphene-based nanobiosensors, the most common is electrochemical one due to its simplicity and high sensitivity in a rapid assay. Due to these attractive properties and features, these carbon structures can be used in biosensors for the detection of microorganisms such as prions, viroids, viral cells, bacterial cells, protozoa, microbial toxins, fungi and antibiotics from microbial sources, among others.


The current investigation presents a green mechanochemical procedure for the synthesis of a special kind of rubber-compatible organo-montmorillonite (OMMT) for use in the inner liner compound of tires. The compatibility character of the OMMT arises from the mechanochemical reaction of the raw bentonite mineral and gum rosin as some of the organic constituents of the inner liner composition. The monitoring of OMMT synthesis by various characterization techniques reveals that gum rosin gradually intercalates into the montmorillonite (MMT) galleries during milling and increases the interlayer spacing to 41.1 ± 0.5 Å. The findings confirm the simultaneous formation of single- or few-layered OMMT platelets with average sizes from the sub-micron range up to several micrometers during the milling process. The mechanical properties of the OMMT/rubber nanocomposite, such as tensile strength, tear resistance and elongation, present a good enhancement in comparison to the un-modified material. Moreover, the organo-modification of the inner liner composition also leads to a property improvement of about 50%. Bentonite is a swelling aluminum phyllosilicate clay which has attracted tremendous attention because of the layered structure of the montmorillonite (MMT). Besides its low price, the aluminosilicate-layered backbone of montmorillonite makes bentonite a very promising natural candidate for many applications, such as oil-field scale inhibition, pollutant removal, wastewater treatment, rheology control of suspensions, catalysis, composites, packaging, etc. ,3,4,5,6.However, due to the inorganic nature of bentonite, it is not compatible with organic media and, hence, requires being organo-modified ,8,9,10. During the two recent decades, mechanochemical approaches have attracted increasing attention due to the solvent-free, cost effective and environmentally friendly synthesis of a wide variety of nanomaterials ,24,25,26Commercial OMMTs are globally confined to alkylammonium-modified MMTs. On one side, these kinds of OMMTs are potentially toxic, and on the other side, they exhibit a limited compatibility with some polymers ,37,38,3920H30O2), containing conjugated double bonds and a single carboxylic acid. The other residual percentage (about 10%) includes neutral matters with non-conjugated double bonds [Gum rosin is one of those substances widely used in polymers, especially in the tire industry as an adhesion promoter . This nole bonds ,41,42. TIn the present investigation, the objective is to organically modify bentonite powder with gum rosin as intercalating agent through a solvent-free mechanochemical approach. The organo-modified MMT is evaluated for its use in the inner liner composition of tires through investigating the dispersion behavior and the exfoliation of platy MMTs within the rubber composition. Moreover, physical and mechanical properties as well as the gas permeability of the OMMT-modified inner liner composite are also studied.All the chemicals and minerals were used as-received without further treatment. Bentonite with a cation exchange capacity (CEC) of 103 meq/100 g was obtained from the bentonite mines of the South Khorasan province of Iran and supplied by Vivan Industrial Group. Gum rosin WW grade was purchased from Sadkem . The chemical structure of abietic acid as the main constituent of gum rosin is illustrated in A solid-state mechanochemical method was applied to synthesize the OMMT. The reaction mixture was obtained by the addition of 4.1078 g of bentonite powder and 1.9195 g of gum rosin in accordance with the cation exchange capacity of raw bentonite and milled using a planetary ball mill for 4 h at a rotational speed of 250 rpm. The weight ratio of the 10 mm hardened steel balls to the reaction mixture was adjusted to be 30:1.The OMMT/rubber nanocomposites were prepared by the addition of 4 phr and 7 phr synthesized OMMT to the desired amounts of natural and synthetic rubbers, fillers, tackifiers, softeners and antioxidants, as demonstrated in The final inner liner composition of a tire was produced by the addition of appropriate amounts of vulcanizing agents to the master batch on a two-roll mill  for 10 min. The vulcanizing agents consisted of sulfur, MBTS, CBS, vultac and zinc oxide. The resulting final composition was cured in suitable molds at 145 °C under 110 bar pressure for 30 min.In order to investigate the dispersion behavior of the MMT platelets within the rubber mixture, 4 phr of the synthesized OMMT was added to the rubber mixture including all the natural and synthetic rubbers on a laboratory two-roll mill for 20 min. In fact, the other constituents of the master batch inner liner compound were neglected.−1 at a resolution of 4 cm−1. Transmission electron microscopy (TEM) images were obtained using a Philips CM300 instrument  operated with an accelerating voltage of 150 kV to study the extent of intercalation or possible exfoliation of the OMMT. The powdered TEM samples were prepared by suspension of OMMT particles in ethanol followed by sonication in a low-energy ultrasonic bath for few minutes, and then putting a droplet of suspension on a carbon-coated copper grid. For the rubber samples, thin sections (less than 100 nm) were prepared using a cryo-ultramicrotome (Leica EM UC7)  and the sections were supported onto a copper grid for the subsequent analysis. The morphology of the powder was also investigated with a Hitachi S4160 field emission scanning electron microscope (FESEM) , operated at 20 kV. The samples were gold-coated with a thickness of less than 10 nm prior to the analysis. Atomic force microscopy (AFM) images were recorded in air and ambient conditions using a NT-MDT AFM apparatus  in non-contact mode with a NSG10 tip  at 190 KHz resonance frequency and a scanning rate of 0.3 Hz. For AFM sample preparation, a very small quantity of powder was sonicated in ethanol for 10 min, followed by drop casting on a silicon wafer substrate. The measurements were conducted after solvent evaporation and data were collected over 50 particles of three similarly prepared samples. The AFM images were processed by means of the Nova Px 3.1.0  to obtain the numerical data. For thermal gravimetric analysis (TGA), 7.723 and 7.295 mg of gum rosin and the synthesized OMMT were respectively weighed and located at the platinum pans. The measurements were conducted on a TGA-50 Shimadzu thermogravimetric analyzer  using a heating rate of 10 °C/min under air atmosphere.X-ray diffraction (XRD) analyses were conducted by means of a X’Pert PRO MPD diffractometer  with Cu Kα radiation (λ = 1.54060 Å) to determine the phase evolution, intercalation and exfoliation of OMMT during the production process. The chemical structure of the prepared OMMT was characterized using Fourier transform infrared spectroscopy (FTIR) via the KBr pellet technique. The concentration of the sample in the KBr was kept at 0.2 wt.%. Data were recorded with a ThermoNicolet Avatar 370 spectrometer  in the optical range of 400–4000 cm−10 cm3 (STP) cm/cm2 s cmHg), as reported previously [The physical and mechanical properties of the resulting rubber and OMMT/rubber nanocomposites, such as tensile strength, elongation at break (%), modulus 300% and tear resistance, were characterized by a Dynamometer Hounsfield H10KS tensile  testing instrument. In order to obtain reliable results, the average of three samples is reported. The thickness of the samples was tested by the use of a digital thickness tester . The average of at least ten readings was taken for further calculations. Hardness measurements were also performed using a Zwick 3100 model . The vulcanization characteristics of the compounds were checked with an Alpha Technologies MV2000E Mooney viscometer . The air permeability (OTR) of the samples was measured in a permeation cell consisting of two compartments separated by the molded samples. Following the evacuation of the chamber, 2 bar gas pressure was introduced into the upstream compartment of the cell. The variation of the gas pressure in the downstream compartment was recorded. Finally, the permeability coefficient was calculated in Barrer through the equation  on the clay surface. The peaks appearing at around 470 cm−1 and 522 cm−1 are assigned to the bands related to bending vibrations of Si–O–Si and Al octahedral layers (Si–O–Al), respectively [−1 refers to the stretching vibration modes of Si–O bonds, and the other weak peak centered at 918 cm−1 is related to the Al–Al–OH bending vibrations on the edges of the clay mineral [−1 and 836 cm−1 is also respectively attributed to the partial substitution of Fe (Al–Fe–OH) and Mg (Al–Mg–OH) in the octahedral layers [The chemical structures of the MMT, gum rosin and the synthesized OMMT can be elucidated from the corresponding FTIR spectra in tructure ,47,48. Tectively ,47. The  mineral ,48. The −1 arising from C=O stretching bonds of the carboxyl group in abietic acid. Furthermore, the characteristic peaks at 2933 cm−1 and 1385 cm−1 are attributed to the stretching vibration modes of –OH and –C–O bonds of carboxylic acid, respectively [−1 and 2933 cm−1 are respectively associated with CH2 symmetric and asymmetric stretching vibration modes [−1 belongs to the C–H out-of-plane bending, and the one centered at about 1461 cm−1 corresponds to CH2 bending vibrations [The FTIR spectrum of gum rosin in ectively ,50. The on modes . The peabrations .2 bonds to 2934 and 2872 cm−1 in the FTIR spectrum of OMMT points to the gauche conformation of organic molecules within the MMT galleries [It is obvious that the spectrum associated with the organo-modified MMT c is a coalleries .The morphology and structure of the OMMT particles were further investigated by FESEM, TEM and AFM analyses . The FESThe thermal stability of gum rosin and synthesized OMMT was studied by thermogravimetric analysis . The gumA typical TEM image of the OMMT-modified rubber is shown in The modulus 300% is measured to be 5.4 ± 0.1 MPa for the un-modified material; meanwhile, it reaches values of 4.9 ± 0.1 MPa and 5.68 ± 0.1 for the nanocomposites, respectively modified by 4 phr and 7 phr OMMT. The lower modulus for the smaller amount (4 phr) is attributed to the sliding of the rubber chains within the OMMT galleries. For higher amounts (7 phr), the possible aggregation of OMMT platelets may prevent the slipping of rubber chains and, consequently, enhance the modulus. This hypothesis is supported by the high modulus of carbon black-formulated compounds in which the structural networks of carbon black confine the polymer chains and prevent the slipping of the macromolecules. ,66.According to The hardness values are essentially unchanged, showing only a negligible decrease (~1%). As known, most of the surfactants utilized for the organo-modification of MMT, such as amines and quaternary alkyl ammonium substances, decrease the scorch time of the elastomeric composites through decreasing the activation energy and accelerating the vulcanization system . MeanwhiBased on the results of The utilization of an additive of the inner liner composition for the modification of MMT through a facile mechanochemical process gives a promising perspective view for the synthesis of the next generation of OMMTs for further applications in various systems, such as packaging, membranes, films, containers, tires, medicine, etc. This breakthrough approach definitely introduces highly compatible and efficient OMMTs to different industries. Moreover, this concept opens a new window on various layered materials, such as graphene.A solid-state mechanochemical technique was applied to prepare a rubber-compatible OMMT for application in the inner liner layer of tires. To reach a good compatibility, the natural bentonite mineral was milled with gum rosin as one of the organic constituents of the inner liner composition. The results confirm the crucial role of milling on the intercalation of the gum rosin into the MMT interlayers, as well as the subsequent exfoliation of MMT platelets. According to TGA analysis, gum rosin is successfully intercalated into the MMT galleries; however, some amount of the surfactant is adsorbed either on the external MMT surface or between the bentonite pores. XRD and TEM of the organo-modified MMT also indicate the presence of intercalated/exfoliated structures of OMMTs within the rubber matrix when compared to the non-modified rubber mixture. This is due to the good compatibility of the OMMT with the inner liner composition. The resulting OMMT/rubber nanocomposite exhibits improved mechanical and barrier properties. The incorporation of 4 phr OMMT into the inner liner composition shows about 50% improvement compared to the unmodified material.


Our results show that metabolomic defects occur early in ALS motor cortex and establishing NAD+ balance could offer therapeutic benefit to UMNs with TDP-43 pathology.Mitochondrial defects result in dysregulation of metabolomics and energy homeostasis that are detected in upper motor neurons (UMNs) with TDP-43 pathology, a pathology that is predominantly present in both familial and sporadic cases of amyotrophic lateral sclerosis (ALS). While same mitochondrial problems are present in the UMNs of ALS patients with TDP-43 pathology and UMNs of TDP-43 mouse models, and since pathologies are shared at a cellular level, regardless of species, we first analyzed the metabolite profile of both healthy and diseased motor cortex to investigate whether metabolomic changes occur with respect to TDP-43 pathology. High-performance liquid chromatography, high-resolution mass spectrometry and tandem mass spectrometry (HPLC–MS/MS) for metabolite profiling began to suggest that reduced levels of NAD+ is one of the underlying causes of metabolomic problems. Since nicotinamide mononucleotide (NMN) was reported to restore NAD With their multiple important functions, they serve as one of the key organelles for cellular health. Therefore, it is no surprise that mitochondrial dysfunction emerges as one of the common causes of neurodegeneration in many different diseases, and ALS is no exception.Mitochondria are best studied for their ability to generate energy, but they are also essential for lipid homeostasis, ensuring proper Ca9. Even though some patients have mutations in TARDBP, the gene that codes for TDP-43 protein, not all TDP-43 pathologies are associated with the mutation10, and not all protein accumulations occur in the cytoplasm, some are detected in the nucleus—11,12. In fact, most of the patients with TDP-43 pathology do not have mutations in their TARDP gene, and yet have protein accumulations containing phosphorylated TDP-4313, and mostly ubiquitinated protein aggregates15. One of the interesting findings related to the neurons with TDP-43 pathology is the mitochondrial defects observed at a cellular level18. Such mitochondrial defects are also present in the upper motor neurons (UMNs) that are diseased in ALS. Regardless of species, the UMNs of a broad spectrum of ALS patients and corticospinal motor neurons  of well characterized mouse models of TDP-43 pathology, both display prominent mitochondrial defects in a cell-type specific manner16.TDP-43 pathology is one of the common proteinopathies observed in a large spectrum of ALS patients, including familial, sporadic and ALS with frontotemporal lobar dementia (ALS/FTLD)A315T mice, a mouse model which recapitulates many aspects of TDP-43 pathology observed in ALS and ALS/FTLD patients19. An early symptomatic stage of the disease was selected to investigate whether metabolomic perturbations begin to occur early in the motor cortex with TDP-43 pathology.There is an immense effort to reveal the underlying causes of TDP-43 pathology and the downstream cellular events that are perturbed in diseased neurons. Since mitochondrial defects have been broadly observed in many different diseases in which TDP-43 pathology is commonly detected, and because mitochondria are crucially important for maintaining metabolomic homeostasis, one of the key questions that remains unanswered is: “What are the metabolomic changes that occur with respect to TDP-43 pathology?” We investigated the metabolomic perturbations that occur in the motor cortex of prp-TDP-43+, GSH (glutathione), and PEP (phosphoenol pyruvate). We thus tested the hypothesis that increasing NAD+ levels would help restore balance and improve the health of UMNs diseased due to TDP-43 pathology. NAD+ plays a crucial role in several physiological conditions such as neurological disorders, diabetes, aging, oxidative stress and neurodegenerative diseases26. Since NMN (nicotinamide mononucleotide) was previously reported to increase the levels of NAD+ by acting as the precursor for NAD+27, we investigated whether NMN treatment would have therapeutic impact on diseased UMNs.Our findings began to reveal reduced ATP production as well as perturbations in the balance of key metabolites, such as NADA315T-UeGFP mice, a CSMN reporter line in which diseased UMNs in mice that have TDP-43 pathology retain their fluorescence both in vivo and in vitro. Our results not only reveal early metabolomic problems in the motor cortex, but also suggest that maintaining NAD+ balance could offer a therapeutic treatment strategy for diseased UMNs with TDP-43 pathology.In an effort to bring cellular clarity to our analyses, and to investigate UMNs direct response to treatment, we took advantage of the prpTDP-43A315T mice16  of a broad spectrum of ALS patients with TDP-43 pathology16. Since mitochondrial problems begin to occur early and because they are shared among species, we first investigated potential changes in metabolite dynamics, especially in the motor cortex of prpTDP-43A315T mice, which closely recapitulates ALS pathology in patients19. Primary motor cortices of 90 days old (P90) prpTDP-43A315T and healthy wildtype (WT) control mice were dissected out and metabolites were isolated by methanol extraction method followed by high performance mass spectrometry profiling of metabolites  of prpTDP-4328. Heatmap of hierarchical clustering analysis of top 50 highly enriched metabolites revealed differential presence of key metabolites  and converts into GSSH30. Thus, higher GSSG/GSH ratio indicates increased oxidative stress. We find that GSSG/GSH ratio is significantly higher in the motor cortex of prpTDP-43A315T mice even at P90 , in which CSMN express eGFP, and CSMN identity of eGFP+ neurons in the layer 5 of the motor cortex is confirmed by retrograde labeling, molecular marker expression and electrophysiological propertiesA315T mouse model of ALS, generated prpTDP-43A315T-UeGFP mice (diseased) and WT-UeGFP mice . When dissociated cortical cultures are established from these mice, CSMN are distinguished among many different cortical cells and neurons, as they retain their eGFP expression  of both healthy (WT-UeGFP) and diseased (prpTDP-43A315T-UeGFP ) motor cultures in vitro, and CSMN responses to treatment was quantitatively assessed by measuring changes in average axon length and the extent of branching and arborization . In addition, the percent distribution of CSMN based on their axon length also revealed an overall and significant shift towards longer axons, such that about 80% of treated CSMN had axons longer than 300 μm, whereas about 90% of untreated CSMN had maximum axon lenth of 200 μm , on a total of 4 CSMN (untreated (n = 2) and NMN treated (n = 2)). CSMN were evident with their eGFP expression in vitro . However, upon NMN treatment, there were significant improvements in the overall architecture of mitochondria and they had a more intact inner membrane with visible cristae structures. CSMN of prpTDP-43A315T-UeGFP mice had fewer diseased mitochondria . The CLEM results were obtained from 4 independent CSMN isolated from four different mice, revealed the impact of NMN treatment on mitochondrial structure and integrity , exact same cellular problems were detected both in the UMNs of ALS patients with TDP-43 pathology and the CSMN of this mouse model of TDP-43 pathology16.Therefore, we investigated potential metabolomic dysregulation that may occur in the motor cortex of ALS with TDP-43 pathology. We have chosen prpTDP-4310, and this pathology was broadly observed in a wide spectrum of ALS cases44. However, recent evidence also reveals TDP-43 accumulations in the nucleus, not only in the cytoplasm12. Especially in the case of some mutated forms of TDP-43, the protein aggregations are vastly ubiquitinated, but the presence of pTDP-43 is not dominant45. Regardless, patients with TDP-43 mutations have TDP-43 pathology, and the mouse model that have A315T mutation has TDP-43 accumulations mostly in the nucleus, have extensive ubiquitinated protein accumulations in the cytoplasm, and based on the antibody used, pTDP-43 are also detected in the cytoplasm46. Therefore, in line with the current definition of TDP-43 pathology, this mouse model has been considered one of the most reliable models for TDP-43 pathology, especially in the cortex43.TDP-43 pathology was originally defined with the accumulation of proteins that include phosphorylated TDP-43 (pTDP-43) in the cytoplasm of neurons that undergo degeneration54. These studies showed significant alterations in ceramide levels as well as benzoate, creatine and fatty acid metabolisms and sphingomyelins pathways, with respect to ALS progression49, and these changes in metabolite levels correlated with patient’s ALS Functional Rating Scale (ALSFRS-R) scores55. Likewise, alteration in glucose and lipid metabolisms were reported to contribute to neuronal vulnerability in diseased motor neurons50. Components of lipid metabolism such as β-hydroxybutyric acid and medium-chain fatty acids were also found to be dysregulated56.Metabolomic dysregulations in the plasma samples isolated from ALS patients were previously reported+, display significant differences between the healthy and diseased motor cortex with TDP-43 pathology at a symptomatic stage.Metabolomic changes begin to reveal the some of the underlying causes of vulnerability in the motor cortex. In this study, we report that a select set of metabolites, such as phosphoenol pyruvate (PEP), glutathione (GSH), 2-hydroxyglutarate (2-HG), and NAD22. In the motor cortex of ALS patients, glutathione (GSH) levels were reported to be reduced when compared to healthy controls57. Likewise, in hSOD1G93A mouse models of ALS, depletion of GSH resulted in degeneration of spinal motor neurons, dysfunctional mitochondria, and neuron loss58. GSH is critically important for scavenging reactive oxygen species (ROS) generated by mitochondria30, and is also a substrate for antioxidant enzymes that detoxify hydrogen peroxide and lipid peroxides59. Importantly, early studies demonstrate a correlation between GSH deficiency and mitochondrial damage60. GSH normally exists in its reduced from and is converted into its oxidized form, glutathione disulfide (GSSG), due to oxidative stress. To diminish the effects of oxidative stress, the enzyme glutathione reductase reverts GSSG into GSH. Thus, the ratio of GSH/GSSG is a marker of neuronal oxidative stress and is often used as a marker of cellular toxicity30. In resting state, GSSG/GSH ratio were maintained at lower levels, however, in a cell that is experiencing oxidative stress this ratio shifts toward more GSSG29. In our study, the motor cortex with TDP-43 pathology displayed increased GSSG/GSH ratio indicating ongoing oxidative stress.Oxidative stress is a prominent physiological feature of ALS, and mechanisms responsible for reducing oxidative stress are compromised+/NADH ratio indicates energy deficiency and is associated with many diseases, including ALS22. NAD+ accepts H+ released from various substrates and converts into NADH, releasing ATP. A balanced NAD+/NADH ratio is critical for continuous generation of ATP. Metabolic dysregulation leads into depletion of NAD+ and that in turn affects energy homeostasis. NAD+ levels can be restored by compounds such as NR (nicotinamide riboside), NAM (nicotinamide), NA (niacin), and NMN (nicotinamide mononucleotide)61. These precursors convert into NAD+ that is ready to be used in energy generation pathways. Restoration of NAD+ levels had been beneficial and neuroprotective in Alzheimer’s disease and intracerebral hemorrhage65. Moreover, NAD+ enhancement has shown attenuation of astrocyte mediated neurotoxicity in vitro67. Treatment of hSOD1G93A mouse model of ALS with NR slowed down degeneration of spinal motor neurons and reduced neuroinflammation68. In an in vitro model of TDP-43 deficiency in spinal motor neurons, loss of TDP-43 is accompanied by axonal defects. Lack of TDP-43 leads to perturbed transcriptome in axon and dendrites and dysregulated transcripts code for genes that are key component of mRNA translation, cytoskeleton, and mitochondria mediated energy production and metabolism35.Perturbation of NAD+ levels by NAD+ precursor compounds could in fact be a potential therapeutic strategy for improving neuron health. Therefore, we tested whether NMN treatment would help improve the health of CSMN that are diseased due to TDP-43 pathology.Building evidence, including the current study, suggests that mitochondria mediated metabolite disturbances are emerging as prevalent feature in ALS, and restoration of NADA315T-UeGFP mice, in which CSMN are genetically labeled with eGFP expression that is stable and long-lasting, and when in culture retain their CSMN identity and eGFP expression. These diseased reporter mice were exceptionally valuable for directly assessing the cellular responses of healthy and diseased CSMN to treatments in vitro and in vivo71. Here, we utilized them to investigate their response to NMN treatment. We find that acute and short-term treatment of diseased CSMN with NMN not only improve their neuronal health, but also help mitochondria to regain the integrity of their inner membrane. Our results reveal early metabolomic dysfunction in the motor cortex of prpTDP-43A315T mice and begin to suggest that reestablishing the perturbed balance may help UMNs regain their health and stability.In an effort to investigate whether NMN treatment would have an impact on the health of UMNs diseased with TDP-43 pathology, we utilized dissociated motor cortex cultures established from prpTDP-4373, maintaining NAD+/ NADH balance appears to be an important contributor to UMN health, especially the ones that are diseased due to TDP-43 pathology. Therefore, our results suggest the unique importance of understanding the metabolomic problems that occur early in the diseased motor cortex, and encourages for the development of novel therapeutic strategies by reestablishing the perturbed NAD+ levels in the brain.Even though long-term exposure to NMN may not be favorable for axonal stability or neuron health due to its interaction with Sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1), and by modulating its activity+ as a potential converging outcome of altered metabolomic imbalance, NMN treatment is applied to upregulate NAD+ levels and to investigate the relationship between increased NAD+ and CSMN health. Our findings, which utilize a novel CSMN reporter line with TDP-43 pathology, not only reveal the presence of metabolomic defects at an early stage of the disease, but also suggest that proper modulation of metabolomic imbalance could offer therapeutic strategies to improve the integrity of mitochondria, and thus the health of diseased UMNs.Metabolomic defects begin to occur early in the motor cortex with TDP-43 pathology. There is altered energy balance and reduced levels of ATP. Since metabolomic analyses reveal reduced levels of NADA315T , and UCHL1-eGFP . Gastrointestinal obstruction have been reported in the prp-TDP-43A315T mouse line74. However, feeding these mice with high fat jelly diet prevented their gastrointestinal problems and their death was reported to be due to TDP-43 pathology mediated degeneration, and not due to gastrointestinal problems76. In this study, all TDP-43 mouse models were fed with the jelly diet. Hemizygous UCHL1-eGFP females were bred to hemizygous prp-TDP-43A315T males to generate prp-TDP-43A315T-UeGFP mice. All the mice used in this study were on C57/BL6 background and were P90 at the time of analyses. Animals were housed at a controlled temperature (23 °C) and controlled light–dark cycle (12–12 h), with free access to water and food17.All animal experiments were performed in compliance with the standards set by National Institutes of Health (NIH) and were reviewed and approved by the Northwestern University Institutional Animal Care and Use committee . All experiments were conducted in compliance with the ARRIVE guidelines. The following mouse strains were used in this study: prp-TDP-4316. Intact brain was removed and motor cortex was dissected out, weighed and immediately frozen in liquid nitrogen. The tissue was homogenized in 1 ml chilled 80% chromatography grade methanol and vigorously vortexed three times. 200 µL tissue homogenate was mixed into a tube pre-added with 800 µL of ice-cold methanol/water 80% (vol/vol) followed by vortexing rigorously for 1 min, and then centrifuge at ~ 20,160g for 15 min in a refrigerated centrifuge. The metabolite-containing supernatant was separated into a fresh tube and stored at − 80 °C until metabolite profiling was performed77.Mice were deeply anesthetized with intraperitoneal injection of Ketamine g, 4 °C. Supernatant was collected for LCMS analysis77.Extraction solution was dried using SpeedVac. 50% acetonitrile was added to the tube for reconstitution following by vigorously shaking for 30 s. Samples solution was then centrifuged for 15 min @ 20,00077. Briefly, samples were analyzed by High-Performance Liquid Chromatography and High-Resolution Mass Spectrometry and Tandem Mass Spectrometry (HPLC–MS/MS). Specifically, system consisted of a Thermo Q-Exactive in line with an electrospray source and an Ultimate3000 (Thermo) series HPLC consisting of a binary pump, degasser, and auto-sampler outfitted with a Xbridge Amide column . The mobile phase A contained 95% (vol/vol) water, 5% (vol/vol) acetonitrile, 20 mM ammonium hydroxide, 20 mM ammonium acetate, pH = 9.0; B was 100% Acetonitrile. The gradient was as following: 0 min, 15% A; 2.5 min, 30% A; 7 min, 43% A; 16 min, 62% A; 16.1–18 min, 75% A; 18–25 min, 15% A with a flow rate of 400 μL/min. The capillary of the ESI source was set to 275 °C, with sheath gas at 45 arbitrary units, auxiliary gas at 5 arbitrary units and the spray voltage at 4.0 kV. In positive/negative polarity switching mode, an m/z scan range from 70 to 850 was chosen and MS1 data was collected at a resolution of 70,000. The automatic gain control (AGC) target was set at 1 × 106 and the maximum injection time was 200 ms. The top 5 precursor ions were subsequently fragmented, in a data-dependent manner, using the higher energy collisional dissociation (HCD) cell set to 30% normalized collision energy in MS2 at a resolution power of 17,500. Data acquisition and analysis were carried out by Xcalibur 4.1 software and Tracefinder 4.1 software, respectively (both from Thermo Fisher Scientific).Comprehensive metabolomics was performed as described earlierA315T mice were plated on a gridded glass coverslip bottom petridishes (MetTek) and cultured for 3 DIV either in SFM alone or treated with 1 µM NMN (Sigma). Correlative Light Electron microscopy (CLEM) was performed as describes previously78. After fixing with 2% PFA and 0.5% glutaraldehyde for 10 min at room temperature, GFP+ CSMN were identified under epifluorescent microscope, and their location on the grid marked before processing for EM analysis. Cells were further post-fixed in 2% PFA and 2% glutaraldehyde for 10 min at room temperature and 50 min at 4 °C. Cells were washed with 0.12 M phosphate buffer pH 7.4 and treated with 1% osmium tetroxide (EMS) and 1.5% potassium ferrocyanide (Sigma) dissolved in 0.12 M phosphate buffer. Cells were dehydrated by subjecting them to ascending series of alcohol 50%, 70%, 80%, 90% and 100% followed by treatment with Epoxy resin for 24 h. The grids were mounted on resin blocks and cured at 65 °C for 3 days. The blocks were trimmed to contain only CSMN, before proceeding to ultra-thin sectioning. Resin blocks were ultra thin sectioned on a Leica Ultracut UC6 ultramicrotome . 70 nm thin sections were collected on 200-mesh copper–palladium grids. Ultra-thin sections were counterstained on a drop of UranyLess solution  and 0.2% lead citrate. Grids were examined on FEI Tecnai Spirit G2 TEM , and digital images were captured on a FEI Eagle camera.Mixed cortical cultures from prpTDP-43A315T–UeGFP mice, dissected, dissociated, and cultured on glass coverslips  coated with poly-L-lysine . Neurons were cultured in serum-free medium  in a humidified tissue culture incubator in the presence of 5% CO2 at 37 °C as previously described28. NMN (1 µM) was added at the start of the culture. Cultures were fixed after 3 days in vitro.P3 motor cortices isolated from WT-UeGFP, and prpTDP-43Anti-GFP  antibody was applied in blocking solution  for 2 h at room temperature. After incubation, and washes with PBS, appropriate secondary fluorescent antibodies  were added to the blocking solution at room temperature for 2 h in the dark. Nuclei were counterstained with DAPI.A315T-UeGFP mice (n = 3) were culture in vitro for 3 days. Immunocytochemistry was performed with eGFP to enhance visualization of GFP+ neurons. Images were acquired using epifluorescent microscope. Neurite length was measure using FIJI ImageJ software. CSMN processes were traced using the Simple Neurite Tracer plugin from FIJI (NIH), which enables semi-autonomous tracing of neuron morphology. The longest neurite was selected to measure the length of the axon in µm.CSMN from WT-UeGFP, and prp-TDP-43The aggregation of the neurite tracings centered at the soma generates a profile available for Sholl analysis to quantify number of intersections at 5 µm radius intervals for each neuron included in analysis. Minimum 15 neurons per treatment from 3 independent experiments were used for analysis.16. For each quantification at least n = 3 mice were used for each genotype and group. D’Agostino and Pearson normality tests were performed on all data sets. Student’s t test was used to determine statistical differences between two experimental groups depending on the genotype, experimental conditions and the disease group. One way ANOVA was used for analysis of more than two groups. Data are shown as mean ± SEM of at least three replicates and are representative of three independent experiments unless otherwise stated and statistically significant differences were taken at p < 0.05, and p values are reported in the text.Statistical analyses were performed using Prism software Supplementary Information 1.Supplementary Information 2.


Herein centimetre-scale CVD graphene/polymer nanolaminates have been produced by using an iterative ‘lift-off/float-on’ process and have been found to outperform, for the same graphene content, state-of-the-art flake-based graphene polymer composites in terms of mechanical reinforcement and electrical properties. Most importantly these thin laminate materials show a high electromagnetic interference (EMI) shielding effectiveness, reaching 60 dB for a small thickness of 33 μm, and an absolute EMI shielding effectiveness close to 3·10 The properties of graphene/polymer composites are usually limited by the use of discontinuous graphene flakes. Here, the authors report a fabrication method to realise continuous cm-scale graphene/polymer nanolaminates with enhanced electromagnetic interference shielding effectiveness, conductivity and mechanical properties. An increasingly growing field of application of such composites is electromagnetic-interference (EMI) shielding, which has become crucial in aerospace, automotive, and portable electronics7. To date, graphene in the form of nanoparticles, such as nanosheets of graphene oxide (GO), reduced graphene oxide and graphene nanoplatelets (GNPs), has often been employed for the production of polymer composites9. However, the full exploitation of the properties of graphene is limited in the discontinuous, particle-reinforced composites11, and many issues have to be overcome to make these materials attractive for industrial applications. Typical limitations of discontinuous graphene composites are the small lateral size of graphene flakes that prevents efficient  stress transfer from the surrounding matrix8, the difficulties with particle dispersion and the limited control of flake thickness. Thus high filler loadings are normally required for the attainment of decent electrical and thermal conductivities and for a moderate EMI shielding effectiveness (EMI SE)15.Since its discovery, graphene has attracted a lot of attention for the development of light-weight, multifunctional composite materials owing to its exceptional mechanical and electronic properties16, which exhibit physical properties not often encountered in man-made materials. In light of that, the incorporation of large-size graphene sheets grown by Chemical Vapour Deposition (CVD) in nano-laminated structures of alternating polymer and graphene layers, can be a smart strategy in order to overcome the typical drawbacks of nanoparticle fillers mentioned earlier.Nanolaminates, consisting of assemblies of continuous, well-oriented, nano-thin layers, are an emerging class of nanocomposite materials potentially able to exploit the unique multi-functional properties of two-dimensional materials such as graphene. Such structures are designed with different stacking sequences and layer thicknesses, and mimic in effect materials found in nature17 and in polycarbonate (PC)18 by stacking and folding approaches has been proposed, resulting in a significant increase of elastic modulus and electrical conductivity and giving basic evidence of the potential of continuous graphene nanolaminates. However, the maximum graphene content that could be achieved by stacking method was <0.2%, since the manipulation of centimetre-scale ultra-thin layers may be rather tricky. Also, characterisation of mechanical and electrical properties was presented only for one17 or very few compositions18, therefore a full understanding of prevailing trends in these materials is still limited.Recently, the inclusion of CVD graphene monolayer into poly(methyl methacrylate) (PMMA)23. This is leading to a pressing demand for novel and lightweight shielding materials combining small volume and high frequency operation, in order to effectively reduce or eliminate wave interference in tiny and delicate environments. By casting ultra-thin polymer films and combining an iterative ‘lift-off/ float-on’ process with wet depositions, a large number of polymer/CVD graphene laminae are progressively deposited to construct the nanolaminates at the macro-scale. The process is semi-automatic, scalable and allows for the production of laminates of maximum dimension of 7 cm × 7 cm. We have produced free-standing nanolaminates from CVD monolayer polycrystalline graphene with layer numbers ranging from 10 to 100 and volume fractions of 0.04–0.5%. Moreover, supported nanolaminates with higher graphene content (1 vol%) have also been produced revealing the potential of this class of material. A systematic investigation of the mechanical, electrical and EMI shielding properties of these nanolaminates is presented herein. Herein we report on the development of centimetre-size CVD graphene-polymer nanolaminates that have the potential to outperform the current overall state-of-the-art graphene-based composite materials in both mechanical and electrical properties per graphene content, and that show high absolute EMI shielding effectiveness in a full frequency decade approximately centred at 1 THz. The fast-growing development of sources, devices and systems presently renders the terahertz region of the electromagnetic spectrum the new frontier in different fields such as data communication, signal modulation, high resolution imaging, and molecule sensing25. By using the proposed all-fluidic manipulation /I(G) (Raman 2D-peak to G-peak) is ~2, which are typical values for CVD graphene monolayers26. However, a slight increase of the intensity ratio I(D)/I(G) (Raman D-peak to G-peak) in the Gr/PMMA nanolaminates indicates that some structural defects are inadvertently induced by the lamination process.The semi-automatic, iterative ‘lift-off/ float-on’ process combined with wet depositions proposed herein is schematically depicted in Fig. 32. This can be ascribed to the efficient reinforcement provided by graphene sheets in the nanolaminate configuration . This proves that our production method leads to regular incorporation of graphene monolayer sheets which are uniformly distributed in the final composite. Moreover, in discontinuous PMMA-graphene composites, the effective Young’s modulus of graphene ranges from 15 to 350 GPa32, depending on particle type, lateral size, composite production process and functionalization. This is not surprising since, as mentioned earlier, in discontinuous composites the maximum reinforcement is limited by the small lateral size of the filler and aggregation/orientation issues11. Also, the Gr/PMMA nanolaminates present a significant improvement of strength (around 100%) compared to neat PMMA laminate. Nevertheless, it is worth adding that, for graphene content over 0.22 vol%, an embrittlement of the nanolaminates is observed which could be ascribed to defects originated at random locations in the non-uniform thickness of the ultra-thin polymer/graphene layers35. Actually, optical microscopy and AFM images , the contribution of such morphological defects contributes to the premature failure of the entire nanolaminate under tensile loading. Furthermore, the increased number of depositions in the lamination procedure (up to 100 in the laminate with 0.5 vol% graphene) may also introduce ruptures and tears in graphene that can affect its strength, in agreement with the increase of the Raman D/G intensity ratio mentioned earlier. Production of nanolaminates by using higher quality graphene grown on Cu foil with lower roughness is in progress and is expected to improve this behaviour, as shown by preliminary tests reported in Supplementary Fig. The mechanical performance of the produced nanolaminates vis-a-vis the PMMA control has been assessed by means of uniaxial tensile testing and representative stress strain curves are shown in Fig. tion see , an effe17) to 95 S/cm (for 0.5 vol%) which, to our knowledge, is one of the highest values reported for polymers filled with graphene or graphene-related materials38. Actually, electrical conductivity of PMMA filled with graphene particles can be several order of magnitudes lower than those measured for nanolaminates with the same graphene content , which may suggest the occurrence of further mechanisms contributing to charge transport. Actually, the aforementioned inhomogeneity of the polymeric layer thickness due to the use of a rough sacrificial substrate probably causes interconnection or bridging between adjacent layers. At this stage, therefore, it is possible that the local thickness of the PMMA spacer is comparable with the cut-off distance for electron jumping between two parallel graphene sheets insulated by polymers (<5 nm48) and hopping electrons are triggered thus enhancing the micro-current in the layered package of graphene. Under these conditions, the conduction mechanism could be ascribed to the combination of migrating and hopping electrons49. It is important to underline that the current maximum for free-standing graphene-polymer nanolaminates produced in this work is achieved at 0.5 vol%; however, there is still room for improvement for higher graphene volume fractions by further reducing the thickness of PMMA interlayer. In fact, several studies demonstrated that stable ultra-thin PMMA films with thicknesses of 20–30 nm can be produced by spin coating50; therefore, in principle, the proposed iterative ‘lift-off/ float-on’ process enables the fabrication of laminates with even higher content of graphene and hence higher values of electrical conductivity. In order to prove that, model nanolaminate specimens with 1 vol% of graphene were fabricated by depositing a limited number of Gr/PMMA layers on quartz substrate  and an average in-plane electrical conductivity of 250 S/cm has been measured  reflection, and internal multiple reflections, strongly dependent on the nanolaminate geometrical parameters such as the number of layers and interlayer distance56 59. The SSEt values at 1 THz for the freestanding Gr/PMMA nanolaminates are plotted in Fig. t, with the potential to achieve values close to 3 × 105 dB cm2 g−1. As shown in Fig. t reported for polymer filled with carbon-based fillers63, graphene foams64 and other non-metallic shielding materials4. Therefore, with only a tiny amount of large-size graphene, the produced Gr/PMMA nanolaminates can be placed among the most efficient EMI shielding material systems known to date, including the recently developed Ti3C2Tx films and foams65. This finding is notable since in our sample several commercial requirements for an EMI shielding product are engrained in a single material, such as high EMI SE, low density, small thickness and mechanical integrity. Thanks to this unique combination, the nanolaminates presented herein clearly surpass the performance of the light-weight graphene-based foams as best candidates in EMI shielding, since the latter suffer from difficulties in achieving small thicknesses and from the mechanical fragility due to the large and disordered pore structures. Furthermore, unlike state-of-the-art materials which have reached the limit of their potential , the shielding performance of the CVD graphene nanolaminates presented herein still has room for improvement by either varying graphene content and/or increasing the number of layers.In order to assess the EMI shielding performance of different materials per unit weight and thickness, we have divided the EMI SE by the specimen density and thickness, obtaining in such a way the absolute shielding effectiveness of the material  and 67. After spin coating, the copper was etched away using a 0.1 M aqueous solution of ammonium persulphate (APS). Afterwards, the floating PMMA/Gr layer was rinsed with deionized-double distilled water inserted in the transfer device until full replacement of the APS solution. By slowly reducing the water level, the floating PMMA/Gr layer was deposited on another PMMA/Gr layer on a copper foil, which represents the substrate for subsequent depositions. After each deposition, the multi-layer was firstly dried at 40 °C for some hours under vacuum to remove the excess of water and then, in order to improve adhesion between subsequent layers, a post-bake process was performed at 150 °C for 5 min on a hot plate. In order to scale up and accelerate the production of nanolaminates, the etching/ lift-off steps were simultaneously performed in several transfer devices. The cycle was repeated until the desired number of PMMA/Gr layers was reached  in a commercially available CVD reactor . Then, continuous graphene laminates were prepared by semi-automatic, sequential transfer of Gr/PMMA layers onto the top of the previous layer using an iterative ‘lift-off/ float-on’ process combined with wet depositions. In the process, we exploit the Cu foil adopted for graphene growth also as sacrificial layer and specific transfer devices were designed in order to control the flow of the fluids during each deposition. The transfer system consisted of conical polytetrafluoroethylene (PTFE) tanks equipped with control flow devices to regulate the fluid inlet and outlet. The production of each PMMA/Gr layer was done by spin coating a PMMA solution in anisole  on the graphene grown on copper foil of dimensions 35 mm × 35 mm. This step is fundamental for the control of the graphene volume fraction mentclass2pt{minimmentclass2pt{minimmentclass2pt{minim−1 (corresponding to a test specimen strain rate of 0.008 min−1). Stress and strain were calculated based on the measured machine-recorded forces and displacements. The Young’s modulus was estimated through a linear regression analysis of the initial linear portion of the stress–strain curves (~0.4% strain). Average results of 10 test specimens are reported for each sample.Tensile test was performed using a micro-tensile tester  equipped with a 5 N load cell. Prior to the test, samples were cut into stripes having an overall length of 35 mm, a gauge length of 25 mm and a width of 1 mm. For each specimen, the thickness was determined as the mean of 10 measurements along the gauge length with a digital micro-metre with a resolution of 0.1  μm . All test specimens were secured onto paper testing cards using a two-part cold curing epoxy resin  to avoid damages to the gripping area. The specimens were subsequently loaded in tension with a crosshead displacement speed of 0.2 mm min2, by acquiring spectra at steps of 3 μm. A Renishaw Invia Raman Spectrometer with 2400 and 1200 grooves/mm grating for the 514 nm laser excitation and a 100× lens were used for the evaluation of graphene quality on macroscale nanolaminates.Raman mapping has been performed on an area of 100 × 100 μmThe electrical resistance was measured in a four-point scheme on stripes cut from the nanolaminates, and the conductivity was calculated from specimen dimensions.w0 at the focus point is approximately 1.5 mm, the area under illumination is at most only 10% of the sample effective surface, making negligible any scattering given by edge or wedge effects. Since (k0w0)2 is much larger than unity in the investigated frequency range (k0 being the wavevector in free space), we can safely assume that the plane wave approximation holds for all measurements that have been carried out within a few percent relative error68. The THz beam impinges on the sample surface under normal incidence. The holder is placed on a kinematic mount for fine adjustment and the sample surface parallelism with respect to the beam wavefront is controlled by a three-points calibration method. Therefore, paraxial error is considered to be very small. The electric field versus time was acquired separately upon transmitting through the sample (The EMI shielding response of the nanolaminates was investigated in the range 0.2–2 THz by using a time domain spectrometer (TeraK15 from Menlo Systems) driven by a femtosecond laser fibre-coupled to photoconductive antennas both for THz emission and detection. Data acquisition was realised by means of a lock-in amplifier coupled with electronics and computer software. A standard setup with four polymethypenetene lenses was used to collimate and focus the beam first impinging onto the sample plane and then received by the detector. Computer aided motion controller was utilised for accurate target positioning. All samples have been tested under dry conditions (<0.1% relative humidity) in a nitrogen environment to avoid spurious effects caused by moisture. Samples are placed on an Al sample holder having a hole of radius 8 mm. Since the THz beam waist Supplementary InformationPeer Review FileSupplementary Movie 1


Many granulomatous diseases can mimic sarcoidosis histologically and in terms of their clinical features. These mimics include infectious granulomatous diseases, granulomatous reactions to occupational and environmental exposures, granulomatous drug reactions, vasculitides and idiopathic granulomatous conditions. It is important to distinguish sarcoidosis from these mimics, as a misdiagnosis of these diseases may have serious consequences. This manuscript reviews numerous sarcoidosis mimics and describes features of these diseases that may allow them to be differentiated from sarcoidosis. Distinguishing features between sarcoidosis and its mimics requires a careful review of the medical history, symptoms, demographics, radiographic findings, histologic features, and additional laboratory data. Understanding the clinical characteristics of sarcoidosis and its mimics should lead to more accurate diagnoses and treatment of granulomatous disorders that should improve the care of these patients. As the diagnostic criteria of sarcoidosis are not standardized, it is possible that some of these sarcoidosis mimics may represent varied clinical presentations of sarcoidosis itself. Sarcoidosis is a multisystem granulomatous disease of unknown cause. The diagnosis of sarcoidosis is arbitrary , not staFor the purposes of this discussion, we will discuss only granulomatous diseases that mimic sarcoidosis. We will not address diseases that may mimic sarcoidosis in terms of presenting symptoms , radiographic findings  or laboratory findings  that are not associated with granulomatous inflammation.The diagnosis of sarcoidosis is based on three major criteria: a compatible clinical presentation, identifying non-necrotizing granulomatous inflammation in one or more tissues, and the exclusion of alternative causes of granulomatous disease . CurrentSarcoidosis is associated with many different occupational and environmental exposures  and has Table 3. Table 3 and There are certain radiographic features that are typical for sarcoidosis, and these are listed in Mycobacterium tuberculosis (MTB) is the most common infectious granulomatous lung disease in the world  and various fungi. Because NTM and fungi are common infectious causes of granulomatous inflammation , 27, 28,Various environmental and occupational exposures may cause granulomatous disease. Hypersensitivity pneumonitis (HP) is a granulomatous lung disease induced by a wide variety of organic bioaerosols and airborne chemicals. These aerosols induce an allergic type 3 (antigen-antibody) and type 4 (granulomatous) response within the lung in susceptible individuals. HP has three clinical presentations: acute, subacute and chronic; the latter two are associated with more chronic antigen exposure and granulomatous inflammation. A more recent classification of HP partitions the disease into non-fibrotic and fibrotic forms based on the presence or absence of significant fibrosis on lung imaging .HP may be confused with sarcoidosis. The presenting symptoms of HP include cough, dyspnea, fatigue and weight loss , 41 thatChronic beryllium disease (CBD) is a granulomatous disease caused by exposure to beryllium. It may be indistinguishable from sarcoidosis both radiographically and histologically. CBD occurs in approximately 2 to 5 percent of beryllium exposed workers . IndividLymphomas have been associated with granulomatous inflammation in lymph nodes and visceral organs that have been involved with lymphoma , 61. GraSarcoid-like reactions have been described not only with lymphoma but with various solid organ malignancies , 69, incBecause vasculitides are systemic disorders, commonly affect the lung and may be associated with granulomatous inflammation, they are occasionally confused with sarcoidosis. However, the clinical presentations and histologic findings of granulomatous vasculitides are usually sufficiently discordant from sarcoidosis that the two diseases can usually be easily differentiated.Granulomatosis with polyangiitis (GPA), formerly known as Wegener's granulomatosis, is a vasculitis of small to medium-sized vessels that usually affects the upper airway, ear/nose/throat, airways, lung parenchyma and kidney. Common extrapulmonary presentations include otitis media, hearing loss, sinusitis, epistaxis, septal perforation, saddle nose deformity, hemoptysis and renal dysfunction . AlthougEosinophilic granulomatosis with polyangiitis (EGPA), formerly known as Churg-Strauss syndrome is a vasculitis of small to medium-sized vessels that presents with the triad of asthma, eosinophilia and vasculitis . The astRheumatoid nodules are most commonly found subcutaneously at points of pressure , and mayInnumerable foreign substances may penetrate the skin and induce a foreign body granulomatous reaction. These substances include particles used in tattoos and cosmetic fillers as well as substances that enter the skin from cutaneous trauma . In addiAspiration pneumonia may result in foreign body granulomas from aspirated material . AlthougA drug-induced sarcoidosis-like reaction (DISR) is a systemic granulomatous reaction that is indistinguishable from sarcoidosis and occurs in temporal relationship with initiation of an offending drug . DISRs rDiffuse granulomatous reactions to foreign substances differ from localized granulomatous foreign body reactions in that they are systemic and not localized to the site of the foreign material. Sarcoidosis like reactions may result from an autoimmune inflammatory syndrome induced by adjuvants (ASIA) , 98 thatMore than 30 years ago, a syndrome was described with prolonged fever; epithelioid granulomata in the liver, bone marrow, spleen, and lymph nodes; a benign course; and a tendency for recurrence. Unlike sarcoidosis, pulmonary granulomatous involvement is rare and usually confined to mediastinal lymph nodes . This enCommon variable immunodeficiency (CVID) is a primary immunodeficiency that may occur at any age and is characterized by an inability to mount specific antibody responses to ubiquitous exogenous antigens and low levels of IgG , 112. CVNecrotizing sarcoid granulomatosis (NSG) is a granulomatous disease consisting of granulomas that are often confluent, necrotic, and associated with a granulomatous and lymphocytic vasculitis . It is cBlau syndrome is a granulomatous disease affecting almost exclusively the skin, joints, and/or uveal tract of the eye . The disOrofacial granulomatosis (OFG) is often used as a term to describe all conditions with oropharyngeal swelling and histologic evidence of granulomatous inflammation . The lipSarcoidosis and Crohn's disease are granulomatous disorders with overlapping features. Distinguishing these two entities may be problematic. Common presentations of Crohn's disease include bloody and non-bloody diarrhea. As gastrointestinal tract sarcoidosis usually causes no symptoms , the disSarcoidosis may be confused both clinically and histologically with primary biliary cirrhosis. The granulomas of sarcoidosis are often situated in the portal triads resulting in bile duct obstruction . This maAlthough sarcoidosis is referred to as a systemic granulomatous disease of unknown cause, sarcoidosis obviously has a cause. In the future, once the cause of sarcoidosis is determined, it may become less problematic to distinguish sarcoidosis from its granulomatous mimics. At that point, we may deduce that several mimics of sarcoidosis are actually forms of sarcoidosis. Until that time when the immunopathogenesis of sarcoidosis becomes clear, we must rely on our understanding of the clinical features of sarcoidosis and alternative granulomatous diseases to distinguish these entities. Such a distinction requires understanding of the medical history, demographics, potential exposures, radiographic manifestations, histologic features, and laboratory findings associated with these disorders. Distinguishing sarcoidosis from its mimics is not an academic exercise, as minimizing the misdiagnosis of these diseases will result in improved patient care and less patient suffering.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.