The exceptional properties of two-dimensional (2D) solids have motivated extensive research, which revealed the possibility of controlling many characteristics of these materials through strain. For instance, previous investigations demonstrated that compressive deformation could be used to direct the chemisorption of atomic hydrogen and oxygen. Still, to our knowledge, there is no work detailing how strain affects the adsorption isotherms of 2D materials and the adsorption properties of materials such as the graphynes, which are monolayers composed of sp and sp The graphynes, which are carbon monolayers composed of atoms with sp and sp5, but the first member of this family, the graphdiyne, was only synthesized in 20106. Since then, the synthesis of 7, and other experimental works proposed using 8 and supercapacitor applications9. Regarding the electronic properties of the graphynes, theoretical investigations revealed that some are semiconductors with moderate gaps10, while others feature Dirac cones in their electronic band structure11. And, in the case of 11. Other simulations also characterized the mechanical properties of various graphynes, particularly under uniaxial strain.The isolation of graphene12. Deformation can also control the bandgap of other TMDs and phosphorene13. However, the magnitude of the bandgap change depends on the type of strain considered 13. Furthermore, the magnetic14 and thermal properties15 of certain 2D materials can be controlled through deformation.We mentioned above that strain can modify the electronic properties of 16. Similarly, biaxial strain increases the size of graphenylene pores, allowing the separation of different gas mixtures at particular pore sizes17. And by manipulating pore size using biaxial strain, it is possible to control water permeability in porous 2D materials while blocking the diffusion of salt ions dissolved in the water19. Finally, it is possible through deformation to change the adhesion in a covalent organic framework, and through this effect, it is possible to pick up and drop off other 2D materials20. This control of a material’s properties using strain has been termed strain engineering13, and here we investigate the effect of deformation on the adsorption properties of graphene and Of relevance to the present work, strain in porous 2D materials can also modify the size and shape of their pores. For instance, by applying uniaxial deformation, it is possible to control diffusion rates in porous graphene, with potential applications in gas separation21. At the same time, theoretical studies have determined the most stable configuration for the adsorbed H22, determined its binding energy curve23, and the adsorption isotherms24. Regarding the adsorption of CO21, while combined experimental and theoretical investigations examined the adsorption of CO25. Concerning methane, its adsorption on graphene was investigated experimentally at temperatures ranging from 253.15 to 293.15 K and pressures ranging from 0 to 8 MPa26. The authors found similar adsorption characteristics for CH27. Finally, note that various studies have also considered doping or decorating graphene with other elements or molecules to (1) improve its H29 and (2) CO31.The adsorption of the gas molecules considered here on graphene has been examined experimentally and theoretically. Experimental investigations on the adsorption of H32. Graphtriyne sheets have also been considered, and the results indicate that single layers of this solid could be used to separate CO33. Meanwhile, investigations on graphynes decorated with other elements are more common, aiming to improve: (1) hydrogen storage38, (2) methane storage39, and (3) CO43.On the other hand, the adsorption of gases on graphyne sheets has received less attention, and to our knowledge, there is no experimental work on this topic. However, theoretical studies have obtained the adsorption energies, preferential adsorption sites, and the change transfer for H44. Next, a DFT study proposed applying compressive strain to create ripples in graphene, creating convex regions where hydrogen preferentially adsorbs, allowing for the control of the bandgap45. Experimental studies then confirmed the preferential adsorption of atomic hydrogen on convexly curved areas in graphene grown on SiC and showed that this uneven adsorption does not occur in graphene bilayers47. Following simulation studies investigated the structure and electronic properties of pristine and hydrogenated corrugated graphene, intended to mimic graphene on SiC48. Regarding oxygen chemisorption, the simulation proposal was to use strain to introduce ripples, leading to preferential O adsorption on regions with high local curvature, followed by oxidative cutting of the resulting structures to produce graphene nanoribbons49. The effect of strain on adsorption properties has also been considered for transition metal dichalcogenides (TMDs) monolayers. Theoretical studies have introduced biaxial deformation to control the interaction of different TMDs with hydrogen50, NO52, and CO53. Sensor applications have been proposed for this effect52.Some studies have already considered applying strain to graphene layers to alter their adsorption properties, but these have focused on atomic hydrogen or oxygen chemisorption. Initial theoretical proposals introduced out-of-plane deformation in graphene to control the hydrogen-graphene binding energy. The authors discovered that convex regions are favorable for atomic hydrogen adsorptionHowever, to our knowledge, there is presently no work detailing how strain affects the adsorption isotherms of 2D materials and no study examining how deformation modifies the adsorption properties of the graphynes. Here, we investigate the adsorption properties of three gases on relaxed and strained monolayers of graphene and graphyne. For each combination of solid and gas, we calculate adsorption isotherms and enthalpies of adsorption. We also examined the structural changes and obtained the mechanical properties of all considered monolayers under biaxial strain.The methodology used to determine the effect of strain on the adsorption properties of mentclass2pt{minim57. Table 58 and Zhang et al. in the case of 59. Overall, graphene is the stiffest and strongest material, with a biaxial modulus of 1020 GPa and enduring stress values of up to 75.8 GPa. On the other hand, the Let us begin the discussion of the results by examining the mechanical properties of graphene and the graphynes. Figure mentclass2pt{minim60. Two of those studies considered the variation of these properties with the temperature. They found that the biaxial modulus55, the ultimate strain, and the ultimate strength of graphene decrease as the temperature increases57, in agreement with our findings.To avoid thermal perturbation on the stress–strain curve and to compare with values reported in the literature, we have also applied equal biaxial deformation at cryogenic temperatures  varies continuously with the deformation, intermediate values might be useful to separate hydrocarbon gases. For instance, at Inspecting the obtained pore diameters, we find that the triangular pores of see Fig. . On the One final note regarding the passage of gases through the graphyne membranes is that we compared energy profiles obtained using classical potentials with profiles obtained using Density Functional Methods in the Supplementary Information Figs. , S24. Wh63 and CCSD64. From Fig. The material deformation may also affect their affinities with gases. To investigate that, we performed grand-canonical Monte Carlo simulations to predict the adsorption isotherms of COon Figs. , S3.Furthermore, we compute the adsorption enthalpy at zero-loading, which is a measure of the heat released during the molecule adsorption and therefore provides a guide to the energy interaction between guest and host. Comparing the enthalpy values in Table entclass1pt{minimasee Fig. . A similFor HFor CHExcept for CHNow, let us consider the effect of the material’s deformation on the adsorption of COon Figs. , S5. To We have presented all isotherms up to now in units of mol of gas per kg of material, and there is a methodology issue behind this choice. Consider Fig. zLet us now carefully analyze the gas-monolayer interaction and the adsorption mechanism. Figure z < 2 Å . These probability values do not affect the results. In Fig. 78 and the enthalpy of adsorption at infinite dilution is given by 79.The COz-direction by computing the histograms of the height position of the adsorbed molecules. The histograms were constructed using bin width equal 1 Å and slider width equal 0.1 Å. To increase the number of adsorbed molecules and improve the statistics, we performed GCMC simulations at a higher pressure of 50 bar for the histogram calculations. We also mapped the interaction between a gas molecule at a given height and a monolayer using the LAMMPS code. To achieve this, we fixed the z position of a gas molecule and varied its x and y positions to scan the surface of the 2D material. And we recorded the interaction energy as we changed the molecule’s position. For each interaction energy map, we scanned an area with dimensions 20 Å For post-simulations analyses we obtained the density profile along Supplementary Information.


In recent years, the field of conductive fabrics has been challenged by the increasing popularity of these materials in the production of conductive, flexible and lightweight textiles, so-called smart textiles, which make our lives easier. These electronic textiles can be used in a wide range of human applications, from medical devices to consumer products. Recently, several scientific results on smart textiles have been published, focusing on the key factors that affect the performance of smart textiles, such as the type of substrate, the type of conductive materials, and the manufacturing method to use them in the appropriate application. Smart textiles have already been fabricated from various fabrics and different conductive materials, such as metallic nanoparticles, conductive polymers, and carbon-based materials. In this review, we study the fabrication of conductive fabrics based on carbon materials, especially carbon nanotubes and graphene, which represent a growing class of high-performance materials for conductive textiles and provide them with superior electrical, thermal, and mechanical properties. Therefore, this paper comprehensively describes conductive fabrics based on single-walled carbon nanotubes, multi-walled carbon nanotubes, and graphene. The fabrication process, physical properties, and their increasing importance in the field of electronic devices are discussed. Traditional textiles were created to protect people from the elements, such as cold and rain, and to serve as covering material. The two most important qualities associated with clothing are their ability to provide protection and their aesthetics. Throughout history, advances in smart materials and electronics have contributed to a unique potential that has led to the emergence of a new field called “smart textiles”. Smart textiles, also called intelligent textiles or e-textiles, are a type of intelligent materials that can detect and respond to changes in their environment . The stiAlthough recent advances in the field of smart textiles are extremely interesting, several obstacles still need to be overcome to make them suitable for commercial and economic use . UnfortuThis article is about the fabrication of smart textiles using carbon-based materials, especially SWCNTs, MWCNTs, and graphene, and is organized as follows: The smart materials, discussing the structure, physical properties, and potential applications of carbon nanotubes, SWCNTs and MWCNTs, and graphene. Then, there are three major sections focusing on the smart textiles fabricated with SWCNTs, MWCNTs and graphene, respectively. Meanwhile, the fabrication method, physical properties, especially electrical properties, factors affecting these properties, and potential applications of smart textiles are described.Materials that are described as “smart” or “functional” are usually part of a “smart system” that can sense and respond to its environment. If they are truly intelligent, they have a significant impact on the performance of smart textiles . In thisCarbon nanotubes (CNTs) belong to the fullerene family, which includes carbon allotropes whose atoms are connected in cage-like configurations, such as a hollow sphere, an ellipsoid, or a cylinder ,20,21,22CNTs are generally classified by the number of carbon layers into single-walled (SWNTs) or multi-walled (MWNTs) carbon nanotubes, as shown in CNTs were used in wide range of applications due to its small and lightweight, which makes them suitable for a . They caGraphene is a flat monolayer of carbon atoms densely condensed into a two-dimensional (2D) honeycomb crystal structure ,59,60,61The exceptional properties of graphene can be exploited in numerous applications, including biomedicine , membranConductive fabrics are usually made from various substrates, such as cotton , polyest2 stretching cycles with a minimum sheet resistance of 65 Ω/□ at 35% tensile load. Recently , conduct−1 was prepared by immersing cotton leaves in SWCNT ink [−1. This approach is characterized by its speed, simplicity, robustness, low cost, and easy scalability. The fabrication of conductive Lycra fabric has also been investigated using the conductive materials SWCNTs and polyaniline, using the immersion drying process [In another study, SWCNT ink was printed on a stretchable substrate using inkjet printing technique . It was ng study , a piezoWCNT ink . The resWCNT ink . The resWCNT ink  fabricat process . The res process , conduct process . The res−1 with a sheet resistance of 1.67 kΩ/□ after four times of immersion. In addition, the thermal conductivity of the fabric was also increased by 70%. MWCNTs dispersed in DMF were used in the recent study presented by Alamer et al. [−2. The results also showed that the conductive cotton treated with HNO3 resulted in a reduction of sheet resistance to 1.5 kΩ.cm−2 which was attributed to the interaction between MWCNTs and cellulose through glycosidic bonds.As discussed in the previous section, conductive fabrics made of SWCNTs have excellent electrical properties; however, SWCNTs are expensive, purification is difficult, and dispersion in liquid is also difficult. Therefore, many researchers focused on fabricating conductive fabrics using MWCNTs instead of SWCNTs because they are cheaper, can be produced in large quantities, and are more stable compared to SWCNTs. Rahman et al.  fabricatr et al.  to prepar et al. . The amo−2, had a specific capacitance of 8.01 F g−1, a high energy density of 6.30 Wh kg−1, and a cyclability of 5000. In the study presented by Nafeiea et al. [−3 S cm−1, which is ten times higher than the conductivity of the wool fabric treated with MWCNTs. In the study presented by Kowalczyk et al. [−1, and the sample exhibited good stability up to 3000 cycles. The conductive yarns based on MWCNTs were fabricated in the study presented by Abbas et al. [The conventional dyeing method was used to deposit synthetic MWCNTs on the surface of cotton fabrics . The depa et al. , conductk et al. , MWCNTs k et al. , where tk et al.  fabricats et al. ,124, in −1 at a graphene concentration of 14% by weight and under continuous mechanical stress.Incorporating graphene into textiles not only imparts conductivity to the textiles, but also enables the production of multifunctional textiles due to the excellent physical properties of graphene, as we discussed in −1 by using graphene oxide nanosheets as a conductive material and a coating reduction method. Briefly, the untreated silk fabric was immersed in bovine serum albumin, which generates a positive charge on the surface of the fabric and increases the absorption of the conductive material when the fabric is immersed in the graphene oxide nanosheet solution. Then, a hydrazine vapor reduction method was used to reduce the graphene oxide on the fabric. In the study presented by Zulan et al. [−1. In another study [−1 was prepared using reduced graphene oxide and a dip coating and reduction method. The results also showed that the conductive cotton yarn exhibited mechanical stability up to 1000 cycles and absorbed UV irradiation of about 1.0 mA/W under bending deformation. Yarns from Calotropis gigantean [−1 at high concentration and was shown to be resistant to washing, which was due to the hydrogen bonding formed between the fiber and graphene during the dyeing process.Lu et al.  fabricatn et al. , the coner study , a flexiigantean , which h2 for the untreated fabric to 23.15 Ω. cm2 for the fabric coated with three layers of reduced graphene oxide. In another study [Molina et al.  fabricater study , the knier study  were use−1 at a graphene concentration of 6 wt.%. Another interesting method to improve the bonding between graphene and cotton fabric and to fabricate a scalable conductive fabric with a length of 150 m and a sheet resistance of about 11.9 Ω/□ was presented in the study by Afroj et al. [4 S m−1. The biocompatible conductive fabric sensor was fabricated using graphene nanoplatelets dispersed in a water-based ink and screen-printed onto the fabric surface [−1 at a graphene concentration of 3.8 wt%.A conductive stretch-sensitive fabric was fabricated using graphene oxide nanosheets and a reducing deposition method . Brieflya et al.  found a j et al. . The conj et al.  was usedj et al. , and the surface . The res−1, and this value was stable up to five washing cycles. In the study by Sahito et al. [−1, which is not affected by bending and washing cycles. In another process [In addition, Yapici et al.  also fabo et al. , the suro et al. . A new m process , UV lighConductive fabrics based on SWCNTs have been prepared by various fabrication methods: chemical vapor deposition, filtration technique, dyeing and drying method, inkjet printing method, dipping and drying method, and drop-casting method. It has been shown that the electrical conductivity of conductive fabrics and sheet resistance has a wide range from low to high values, and depends on various factors, such as: deposition times, dopants, content of SWCNTs in the fabric, stretching cycles, number of coating layers, treatment of the fabric with plasma, and mixing of SWCNTs with other carbon-based materials, such as MWCNTS and graphene, or with conductive polymers, such as polyaniline and PEDOT:PSS. These SWCNTs based fabrics have been used in various applications such as: UV light shielding, piezoresistive sensor, supercapacitor, antenna, and metal thread.Conductive fabrics based on MWCNTs were prepared by various fabrication methods: Dipping and drying, drop casting and drying, dipping, impregnation and drying, dipping and drying, and tape casting. The fabricated conductive fabrics exhibited a wide range of electrical conductivity, which was influenced by several factors: Size of MWCNTs, number of dipping operations, type of organic solvents, content of MWCNTs, temperature, dopants, repetition of the fabrication process, use of anionic and cationic surfactants, and use of enzymes. These conductive fabrics based on MWCNTs have been used as electrodes in supercapacitors, and as electrodes in dye-sensitised solar cells and block UV light.Conductive graphene-based fabrics have been fabricated using graphene, graphene nanoribbons, graphene oxide, graphene nanosheets, natural graphite flakes, and graphene ink. Various methods were used in the fabrication process: wet coating, trapping method, chemical reduction method, vacuum filtration—hot press method, dipping and gamma ray reduction method, cationization—dip coating, coating and reduction method, dyeing and reduction method, dipping and reduction method, reducing deposition method, brush coating, pad dry curing method, screen printing method, chemical vapor reduction method, electrostatic self-assembly—low temperature reduction process, UV reduction method. The electrical properties of graphene-based materials are influenced by several factors: repetition of the manufacturing process, graphene content, graphene oxide concentration, tensile stress, cationization process, number of coating layers, number of dipping cycles, exfoliation agent, and number of printing cycles. Thus, the conductive fabrics produced have been used in various applications: electrode for a dye-sensitive solar cell, strain sensor, portable supercapacitors, UV blockers, heat-resistant gloves, electrocardiogram electrodes, and portable heaters. Finally, the field of smart textile fabrication from carbon-based materials is developing rapidly but needs further development to bring these applications from small scales (in research laboratories) to large scales . Therefore, more basic research is needed to enable the next wave of smart textile products.This review article summarizes the method of designing and fabricating electrical, flexible, and lightweight conductive fabrics with embedded SWCNTs, MWCNTs, and graphene, and their applications in the field of smart textiles. The development of smart textiles was carried out in three stages: the first stage is to impart conductivity to the textiles, the second stage is to fabricate the smart textiles, and the final stage is to functionalize the conductive yarns. Carbon-based materials, particularly SWCNTs, MWCNTs, and graphene, are discussed from their structure, physical properties, and potential applications to their use in the design and fabrication of conductive fabrics with a wide range of electrical conductivity and interesting physical properties that make them suitable for various wearable electronic applications. We come to the following conclusions:


Τ/τP-181 combination. Hence, TDP-43 combined with tau proteins could be a useful tool for the diagnosis of genetic FTD with TDP-43 underling histopathology, supplementing clinical, neuropsychological and imaging data.Cerebrospinal fluid (CSF) biomarkers, namely total tau, phospho-tau and amyloid beta peptides, have received much attention specifically regarding Alzheimer’s disease (AD), since they can detect the biochemical fingerprint of AD and serve as a diagnostic tool for accurate and early diagnosis during life. In the same way, biomarkers for other neurodegenerative disease pathologies are also needed. We present a case series of six patients with genetic frontotemporal dementia (FTD), with TDP-43 underlying proteinopathy, in an attempt to assess TDP-43 as a novel biomarker alone and in combination with established AD biomarkers for this specific patient group, based on the principles of personalized and precision medicine. Our results indicate that genetic TDP-43-FTD is characterized by increased CSF TPD-43 and increased TDP-43 × τ A common neuropathological feature of neurodegenerative diseases is cytoplasmic protein aggregates. Cytoplasmic mislocalization and aggregation of TAR-DNA binding protein 43 (TDP-43) is found in the vast majority of patients with amyotrophic lateral sclerosis (ALS), and in approximately half of patients with frontotemporal dementia (FTD) with ubiquitin inclusions .C9orf72 gene involving a hexanucleotide repeat expansion (GGGGCC) in the first non-coding intron of the gene, responsible for both familial and sporadic FTD and ALS, but more frequently for the combined FTD-ALS phenotype [Recently, great progress in the field of neurogenetics has resulted in the recognition of several gene mutations related to the FTD-ALS spectrum, especially with ubiquitin-TDP-43 predominant histopathology, thus highlighting a new brain proteinopathy. The most recent, but more common discovery, is a genetic mutation in the henotype .GRN) can also lead to FTD with TDP-43 inclusions [VCP) gene constitute another rare cause of FTD, which has been recognized to present with the clinical syndrome of “IBMPFD” , often in combination with ALS in certain families. [TARDBP) are also an extremely rare cause of the FTD phenotype [Heterozygous loss of function mutations in the progranulin gene (clusions . GRN mutamilies. . Mutatiohenotype . T) and phosphorylated (τP), as well as amyloid-β peptides (Aβ) expressing the pathological features of the disease, have been recognized as biomarkers with high diagnostic accuracy and are now included in research diagnostic criteria for AD [T, tau phosphorylated at the threonine 181 site (τP-181) and decreased Aβ with 42 amino acids (Aβ42) in the CSF, has currently been established as the biochemical signature of the disease in vivo [In recent years, cerebrospinal fluid (CSF) has proven to be an excellent material for biomarker discovery for neurodegenerative diseases . Due to a for AD ,13,14,15 in vivo . Τ/τP-181 may increase sensitivity and specificity (>80%) [P-181/τT ratio has been proposed as an indirect indicator of a non-tauopathic pathology [Accordingly, the detection of TDP-43 could be a specific biomarker for TDP-43 proteinopathy. Attempts to quantify TDP-43 in CSF have been successful ,18,19,20athology ,23.Since almost all the aforementioned studies lack pathologic confirmation, we isolated and present herein a case series of six patients with genetic FTD and a presumed TDP-43 underlying proteinopathy. We aimed to investigate how the above-mentioned biomarkers perform in a personalized setting, based on the principles of a precision medicine clinical approach. We also include the clinical and imaging data of this group of patients.A total of six patients were included in this case series study. All patients were hospitalized at the 1st Department of Neurology of the National and Kapodistrian University of Athens at Eginition Hospital during the years 2011–2018. The study was performed in accordance with the ethical guidelines of the Declaration of Helsinki, and had the approval of the local Ethical and Deontology committee of our hospital (244/5-7-2010). All study subjects and/or their relatives gave informed consent for inclusion in the study. Patients underwent detailed clinical, neuropsychological, biochemical and neuroimaging examination, to exclude secondary causes of dementia and establish an FTD diagnosis, based on the criteria of either Rascovsky et al.  for probT, τP-181 and Aβ42 and Aβ40 were measured in duplicate by commercially available enzyme-linked immunosorbent assay (ELISA) kits  according to manufacturer’s instructions, and blind to clinical diagnosis. The above biomarkers are measured routinely in the Neurochemistry and Biomarker Unit of our department, as part of an everyday diagnostic approach, as described elsewhere [The CSF levels of τlsewhere . TDP-43 T = 511.6 pg/mL, normal τP-181 = 28.7 pg/mL, decreased Aβ42 = 504.2 pg/mL, but normal Aβ42/Aβ40 = 0.1 and decreased τP-181/τT = 0.056 ratio. TDP-43 = 4.46 pg/mL and the formula TDP-43 × τΤ/τP-181 = 79.5 were increased. Genetic testing revealed a c.463-2A > G splice site variant—heterozygous mutation in the GRN gene. The patient also had more than a 2-fold decrease in plasma progranulin levels, as described elsewhere [A 61-year-old female patient was admitted to our department for progressive language impairment over the past 1–2 years. She had word-finding difficulty, with intact comprehension, an absence of phonological speech or grammar errors, no other significant cognitive/behavioral disturbances, and preserved activities of daily living. Neuropsychological assessment revealed mild cognitive impairment, with an MMSE score of 25/30 and a FAB score of 12/18. Her writing skills and phrase and sentence repetition were preserved, too. On magnetic resonance imaging (MRI), moderate cortical atrophy in the frontal and temporal lobes, with left predominance and relative preservation of the hippocampus, was apparent . CSF bioT = 565.12 pg/mL, normal τP-181 = 41.9 pg/mL, Aβ42 = 640 pg/m and Aβ42/Aβ40 = 0.11, while τP-181/τT = 0.074, was decreased. TDP-43 = 5.92 pg/mL and formula TDP-43 × τΤ/τP-181 = 79.9 were increased. Genetic testing revealed a c.934-1G > A splice site variant—heterozygous mutation on the GRN gene, and similarly, a decrease in plasma progranulin levels, as in case 1.A 60-year-old male patient, with a family history of dementia on his mother and maternal grandmother’s side, presented with language impairment over the last 10 years. Initially, he had difficulty naming objects and writing. Three years later he developed effortful speech with sound errors, decreased fluency, and memory complaints and disorientation. At the age of 55, a dysexecutive syndrome as well as behavioral disturbances (overeating and irritability) appeared. Gradually, he became almost mute, while bradykinesia and tremor on the right hand appeared. At the time of his admission, he was completely mute, and unable to eat. Neurological examination revealed diffuse muscle atrophies without fasciculations, bilateral Parkinsonian syndrome (more severe in the right limbs), pyramidal syndrome with spasticity, hyperreflexia, and Babinski sign bilaterally. Neuropsychological assessment was impossible to perform. MRI performed at the age of 57 revealed significant cortical atrophy in the frontal and temporal lobes with left predominance A–C, withT = 316.16 pg/mL and τP-181 = 27.2 pg/mL, decreased Aβ42 = 501.51 pg/mL but normal Aβ42/Aβ40 = 0.14 and low τP-181/τT = 0.086 ratio. TDP-43 levels were 5.74 pg/mL and TDP-43 × τΤ/τP-181 = 66.7, both increased. The genetic testing revealed a pathologic hexanucleotide repeat expansion in the C9orf72 gene.A 72-year-old male patient, with a family history of motor neuron disease (brother), gradually developed language  and memory impairment, bradykinesia and stereotypical movements of the right hand over the last two years. Three months before admission, his symptoms aggravated. Neurological examination revealed bilateral akinetic-rigid Parkinsonian syndrome, mild pyramidal signs (mainly on the right lower limb with hyperreflexia and extensor plantar response) and apraxia. Neuropsychological assessment revealed severe diffuse cognitive deficits . On MRI, severe cortical atrophy in frontal and temporal lobes was observed . CSF bioT = 250.3 pg/mL, τP-181 = 35.45 pg/mL and Aβ42 = 935.95 pg/mL, while τP-181/τT ratio was 0.14. The levels of TDP-43 = 2.48 pg/mL and TDP-43 × τΤ/τP-181 = 17.5 were also normal. Genetic testing, however, revealed a hexanucleotide repeat expansion in the C9orf72 gene.A 54-year-old female patient, with a medical history of depression with psychotic features was referred for evaluation. She had a family history of dementia (mother) and symptoms of motor neuron disease . The patient presented with apathy, inertia, disinhibition, loss of empathy and sympathy, unusual stereotyped and perseverative behaviors, and hyperorality with overeating of carbohydrates during the last two and a half years. One and a half year before her admission, she also developed inappropriate crying and a language impairment. Neurological examination revealed severely impaired non-fluent speech with single-word answers, severe disorientation in time and space, and frontal gait disorder. Neuropsychological assessment revealed moderate cognitive deficits with an MMSE score of 15/30 and a FAB score of 6/18. On MRI, some T2 hyperintense lesions attributed to chronic small vessel ischemia (Fazekas II), and mild cortical atrophy of the temporal lobes was observed . CSF bioVCP-associated syndrome was raised. Genetic testing  revealed a pathogenic heterozygous missense mutation p.R159H (c.476G > A) in the VCP gene, confirming the diagnosis of IBMPFD [A 62-year-old male patient, with a medical history of arterial hypertension and an operation for a herniated lumbar disc at the L4-L5 level, was referred for evaluation of neuropsychiatric symptoms. He had a family history of “presenile dementia” in his mother, ALS of his older brother, who died at the age of 53, and behavioral symptoms of another brother. The patient presented with progressively deteriorating steppage gait 20 years ago. A year before admission, the patient was unable to walk independently. Additionally, he developed obsessive behavior, aggression, palilalia and apathy. His neuropsychological evaluation revealed severe cognitive impairment, suggestive of predominant frontal lobe dysfunction, MMSE: 2 and FAB: 0 (not applicable). Brain MRI showed mild frontal lobe atrophy . EMG shof IBMPFD .T = 356.75 pg/mL, τP-181 = 32.77 pg/mL, low Aβ42 = 431 pg/mL, but normal Aβ42/Aβ40 = 0.1 and reduced ratio τP-181/τT = 0.091, while TDP-43 levels = 5.27 pg/mL, and TDP-43 × τΤ/τP-181 = 57.3 were increased.Lumbar puncture and CSF biomarker analysie were performed one year later and revealed normal τT = 328.05 pg/mL, τP-181 = 32.07 pg/mL, decreased Aβ42 = 397.1 pg/mL, with normal ratio Aβ42/Aβ40 = 0.17 and decreased τP-181/τT = 0.098, while TDP-43 levels 5.74 pg/mL and the formula TDP-43 × τΤ/τP-181 = 57.66 were increased. Genetic testing finally revealed a hexanucleotide repeat expansion in the C9orf72 gene.A 41-year-old male patient, with a medical history of dyslipidemia was admitted to the neurology department for delusions of religious content over the past 12 years. Moreover, apathy, a change in eating habits with carbohydrate craving and weight gain gradually developed, while dysarthria, executive deficits, stereotypical behaviors and inappropriate laughter appeared over the last two years. Finally, difficulty in swallowing liquids was noticed six months before admission. Neurological examination revealed pseudobulbar dysarthria, an absence of pharyngeal reflex, bilateral pyramidal syndrome and fasciculations in tongue and back muscles. Neuropsychological assessment revealed mild to moderate frontal cognitive deficits with an MMSE score 28/30, and a FAB score 15/18. Neurophysiological examination could not confirm lower motor neuron involvement. On MRI, significant cortical atrophy in frontal, parietal and temporal lobes was observed . CSF bioP-181 and only two out of six cases had increased τT. Four out of six cases had decreased Aβ42; however, all had a normal Aβ42/Aβ40 ratio. Five out of six patients had increased TDP-43 and formula (TDP-43 × τΤ/τP-181) values, while τP-181/τT was abnormal in all.C9orf72 gene; two had a novel mutation in the GRN gene which led to more than a 2-fold decrease of plasma progranulin levels, as expected [VCP gene. Although small in number, this group of patients is indicative of the most common mutations found in the Greek population [Herein, we present six patients with clinical, neuropsychological and imaging data fulfilling the criteria of Rascovsky et al.  and Gornexpected ,30; meanpulation ,31.T, τP-181, Aβ42 and Aβ40) and a novel emerging (TDP-43) biomarker were analyzed. Combinations of these biomarkers as the formula TDP-43 × τΤ/τP-181 and the ratio τP-181/τΤ were also calculated, as previously suggested [In this group of well-characterized genetic FTLD patients, established CSF biomarkers for AD diagnosis  system proposed by the National Institute of Aging and Alzheimer’s Association (NIA–AA) Research Framework group, AD biomarkers are divided into those with molecular specificity for AD, namely τent case . All pre42 was reduced, but the use of Aβ42/Aβ40 ratio restored normality, ruling out amyloidopathy (A). The Aβ42/Aβ40 ratio has been suggested to better reflect the presence or absence of amyloid pathology than Aβ42 alone. Furthermore, the Aβ42/Aβ40 ratio correlates better with amyloid load in PET (positron emission tomography) [In four out of the six presented cases, Aβography) , as it hography) ,34,35,36Τ/τP-181 formula. TDP-43 is a protein, which may be suggestive and specific of TDP-43 proteinopathy. Additionally, the τP-181/τT ratio was decreased, which in non-AD (FTD) patients could be considered as an indirect indication for a non-tauopathic pathology [The main findings of our study, however, were the increase of CSF TDP-43 levels  in five out of six cases, and the increased values of the TDP-43 × τathology ,23.C9orf72, GRN, VCP, and TARDBP genes for FTLD and the FTD-ALS spectrum [TDP-43 aggregation appears in approximately 50% of all FTLD cases  and in 97% of all ALS cases . Mutatiospectrum . TDP-43 spectrum . Abnormaspectrum .Although thought to exist as an intracellular/intranuclear protein, recent studies using ELISA and Western blot techniques suggest that TDP-43 can also be detected in extracellular fluids such as plasma and CSF .C9orf72 pathological repeat expansion, represents an exception. Nonetheless, the proposed mechanisms through which C9orf72 mutations cause neurodegenerative changes are three: C9orf72 loss-of-function through haploinsufficiency, RNA toxic gain of function, and gain of function through the aggregation of toxic dipeptide repeat proteins [The results of the present study indicate that CSF TDP-43 levels were increased in five out of six patients with TDP-43 histopathology-associated mutations. Case 4, with a proteins . The firproteins . As a reproteins . These Dproteins ,45. The TDP-43 abnormalities have also been observed in other neurodegenerative diseases as well, mainly in ALS, the other end of the ALS-FTD spectrum, but also in AD, limbic-predominant age-related TDP-43 encephalopathy (LATE), cerebral age-related TDP-43 with sclerosis (CARTS), and some other even rarer conditions ,46,47. RThere are also other biomarkers that can be used to differentiate FTD patients from patients with other neurodegenerative diseases, such as NfL . HoweverThe current study has certain limitations. TDP-43 pathology can only be presumed from genetic mutations, since it is an ante-mortem study. Another disadvantage is the small number of patients, coming from a single-center study that included only patients of Greek origin; however, the mutations that the patients harbored are indicative of the most commonly observed in TDP-43-related FTLD found in the Greek population ,31.Our study also has some important advantages. We describe patients that have undergone extended clinical, neuropsychological and imaging examinations, fulfilling the criteria of Rascovsky et al.  and GornBased on the results of the present personalized case series study, which is nonetheless indicative of the mutational profile of the population of Greece, the following conclusions can be drawn:Established AD biomarkers are reliable indicators for the exclusion of AD pathology in genetic FTLD.P-181/τΤ ratio may be useful as an indirect marker for non-tauopathic pathology.A decreased τTDP-43 alone and combined with tau proteins, could be a useful tool for the diagnosis of genetic FTD patients with TDP-43 underlying pathology.Future studies with larger and well characterized patient cohorts are needed to further explore the diagnostic potential of these biomarkers in differentiating TDP-43 proteinopathy from non-TDP-43-related pathologies.


Group II introns are extraordinarily versatile self-splicing ribozymes and retrotransposable elements widespread in bacteria and in bacterial-derived organelles (mitochondria and chloroplasts) of fungi, algae, plants, and of some early-branching metazoans . These lDespite their bacterial origin, it is now widely accepted that group II introns have been repurposed by natural evolution into the eukaryotic spliceosomal introns and the spliceosome . Both grIn bacteria, most group II introns encode a multifunctional reverse transcriptase (RT) and invade genomes through “retrohoming”, a highly site-specific mobility process operated both by the ribozyme and its encoded RT enzyme . RetrohoNatural group II introns are remarkably diverse regarding the structural organization of their ribozyme moiety, self-splicing pathways and the strategies they use to invade genomes. Here, I discuss recent findings regarding the splicing and mobility activities of group II introns that further emphasize the structural plasticity of these elements and their ability to be remodeled by molecular evolution into unique RNA-based machines, including the eukaryotic pre-mRNA splicing system.Group II introns possess a single catalytic center that positions two metal ions responsible for catalysis of the transesterification reactions . This orThe small terminal domain VI harboring the unpaired branchpoint adenosine , has lonRecent crystal structures of an intron lariat captured just after splicing completion revealed the crucial role of the 2′-5′ branch structure in promoting proper assembling of the second-step active site . UnexpecMore recently, cryo-electron microscopy (cryo-EM) structures of a bacterial subgroup IIB1 intron in complex with its reverse transcriptase and a DNA target allowed visualization of the large-scale toggling undertaken by domain VI between the two splicing steps . InteresBacillus genus that contain a large insertion of about 53/56 nucleotides (sometimes longer) located precisely between the end of domain VI and the intron 3′ splice site  is much more flexible than previously recognized and this plasticity could have an impact on 3′ splice site selection. Interestingly, some natural group II introns that deviate from the typical 6-nt/7-nt length of the bpA-3′ss spacer support this hypothesis. A remarkable example is provided by a number of closely related IIB introns found in bacteria of the ice site . These iice site . In anotirst one . Both 3′The ability to directly invade DNA by reverse splicing and the high specificity of target recognition make mobile group II introns unique retrotransposons. The DNA target is primarily recognized by extensive arrays of Watson-Crick pairings (∼12–14 bp) between the intron EBS sites and the complementary IBS sequences lying on the top strand of the target . ImportaThe studies discussed above highlight the flexibility of group II introns and their ability to acquire novel structural features with specific functionalities in splicing and mobility. Over the course of evolution, this remarkable plasticity not only allowed the great diversification of group II introns, but it certainly played a key role in the molecular process that repurposed group II intron self-splicing into one of the most complex cellular function of eukaryotes: spliceosomal pre-mRNA splicing. It is hypothesized that during this evolutionary pathway, fragmentation of ancestral group II intron RNPs gave rise to the small nuclear RNAs (snRNAs) U2, U5 and U6 that constitute the active core of the extant spliceosome . Concomi


This paper explores the capabilities that contribute to community transformation and the common pathways followed by communities in the 100 Million Healthier Lives SCALE  initiative in their transformation journeys towards a “Culture of Health”.Funded by the Robert Wood Johnson Foundation (RWJF), from 2016 to 2020, between 18 to 24 community coalitions nationwide participated in SCALE, the goal of which was to co‐design, implement, test, and scale up a model called the Community of Solutions (COS) Framework, that built community capacity around a set of skills and behaviors to advance culture change and create sustainable improvement in health, well‐being, and equity. We adapted and applied two qualitative research techniques, meta‐ethnography and participatory action synthesis, to evaluate SCALE initiative data.Eight concepts emerged that represent the knowledge, capabilities and practices commonly acquired and utilized across the communities. Overall, these concepts emphasize individual and team leadership, quality improvement skills, an intentional focus on equity, and partnerships for spread and sustainment. Concepts were linked into lines of arguments which were unique storylines explaining the transformation pathways. Three stories of the transformation process emerged from the data. Causal Loop Diagrams (CLDs) were created to represent non‐linear system relationships and visually capture some of the most important dynamics of the process of transformation. Even with vast heterogeneity among the SCALE communities and the diversity of activities that the communities undertook, our analysis showed there were a few basic principles that undergirded the process of building capability for transformation.The knowledge from our findings should be useful to expand further research and practice in community learning systems. The need for systems changes at the community level, and the urgent imperative to focus on equity has never been more apparent than during the devastation from COVID‐19, where a thousand different versions of the pandemic have been manifested in a thousand different communities. Public health analysis and responses to COVID‐19 have not adequately considered the effect of inequities arising from the intersection of race, gender, and class on an individual or community's experience of the pandemic.The COVID‐19 pandemic has merely reinforced what has been known for a long time. Health and well‐being are intimately connected to community level contexts and require collaborative, transformational solutions that are built on communities' capacity to learn, in real‐time, how to improve what matters most to them.1.2Community of Solutions (COS) Framework that built community capacity around a set of skills and behaviors to advance culture change and create sustainable improvement in health, well‐being, and equity.Convened by the Institute for Healthcare Improvement (IHI), 100MLives was a global movement and collaboration from 2014 to 2020 in support of Robert Wood Johnson Foundation's Culture of Health (COH) Strategy.1.3What are the key contributors to achieving capability for community transformation?What knowledge, capabilities, practices, and relationships did communities acquire and utilize?What common pathways did communities follow in their transformation journey?What are the mechanisms through which communities brought about change?Our description of SCALE in this paper focuses on the following four questions:22.1The theory of change for SCALE is the COS model shown in Figure 2.2The primary activities based on the COS model is the development—co‐designed with communities—of skills of community coalition members in five areas  through intense training, coaching and support activities. The key activities are shown in Table 2.3processes of change in the complex environments within which the communities operated, rather than attempting to assess the extent to which these processes resulted in the achievement of specific outcomes. The evaluation was intentionally designed as a participatory partnership between the funder, the implementation partners, the communities, and a multidisciplinary evaluation team led by the Universities of North and South Carolina. Recognizing the heterogeneity in coalition structures, community contexts, and areas of improvement emphasis, the evaluation approach was designed to be flexible, adaptive, and process focused. The evaluation also needed to be sensitive to the fact that much of the knowledge about the implementation was generated by and existed within the communities, and that their interpretation of the data was an important part of the knowledge synthesis process. The approach was related to learning or developmental evaluationThe primary intent of the evaluation was to advance knowledge about the 2.4inquiry, observation, and reflection were used to obtain data about the implementation to capture as much contextual information as possible. Together, these methods provided a multifaceted view of the SCALE implementation process that was synthesized and periodically shared with the implementation team and the communities to stimulate improvements to the program design and its execution.The formative evaluation approach is described in detail in Scott et al.2.5The summative evaluation was designed in the latter half of the project after the theory of change and the primary components of the program design had stabilized. This evaluation focused on the behaviors component of the theory of change through a longitudinal analysis of each community's traversal through SCALE. Since one of the goals of the summative evaluation was to generate insights from a plurality of voices, a collaborative bottom‐up methodology called ToAST (Telling our Amazing Stories Together) was developed.http://www.communitycommons.org/entities/ac745b94-1b33-4143-acc3-6df3e0217b3cDetails of the ToAST approach are described in Reed et al.improvement science and measurement, COS skills , spread and scale up, race, racism and equity and engaging people with lived experience. Across communities, the topical sub groups identified similar ideas that were categorized into themes. Subsequently, the subgroups contextualized the topic‐specific themes into community‐specific concepts representing the workgroup's interpretation of the particular elements of each community's journey through SCALE. For communities that had complete data on all the topical areas, the community‐level concepts were then assembled into overarching concepts representing the most common mechanisms employed by all communities to bring about change. Descriptions were developed for each of these concepts and were sent back to the coalition members to validate and revise if needed. Communities were also invited to create lines of arguments. In meta‐ethnography, these are new story lines or overarching explanations arising from the synthesis of the date. To create lines of argument, communities linked the overarching concepts visually into transformation pathways. The evaluation team used these to develop causal loop diagrams (CLDs) to highlight the nonlinear nature of the transformation process. Table To implement the ToAST approach, a synthesis workgroup consisting of volunteer participants from the SCALE communities and selected implementation and evaluation team members was assembled. The synthesis workgroup systematically worked through the seven phases of meta‐ethnography, working in four topical areas that roughly aligned with the skill areas in the theory of change: 3We describe results from both the formative and summative evaluations, organized by the questions of interest presented earlier in this paper.3.1robust theory of change that could serve as a common roadmap for all communities despite heterogeneity in contexts and areas of improvement focus; (b) a structured improvement process using a well‐known quality improvement method called the Model for Improvementimplementation support system for coaching communities through the process of improvement and change; and (d) an evaluation approach that provided ongoing data for learning and improvement. The existence of these areas was necessary but not sufficient. There needed to be a process that could facilitate the adaptation of these areas in response to changes in context or to new and emergent priorities. The process established for feedback of the formative evaluation results provided the mechanism to do this. An example of change to each area based on recommendations from the formative evaluation is shown in Table The formative evaluation revealed four key areas that were critical drivers for achieving the goals of SCALE: (a) a 3.2individual and team leadership, quality improvement skills, an intentional focus on equity, and partnerships for spread and sustainment. The first two concepts (nos. 1 and 2) related to individual leadership skills that gave the SCALE coalition members the confidence to approach complex problems and to engage in sensitive and difficult conversations. The next two (nos. 3 and 4) pertained to capabilities related to team leadership and practices that brought together those with local context specific knowledge needed to bring about change. Concepts 5 and 6 involved the acquisition of technical skills that were needed to complement local knowledge. A systematic approach to improvement based on a change theory, and accessing specialists  were practices utilized by the communities. Finally, as indicated by concepts 7 and 8, each community made equity and racism a central focus of their work and built deep relationships with other SCALE communities to support and sustain their work.The overarching concepts from the ToAST process represent the knowledge, capabilities, and practices commonly acquired and utilized across the SCALE communities. These are shown in Table These eight cross community concepts emerged in Phase 6 of the ToAST process as the output of multiple levels of synthesis shown in Table 3.3The transformation pathways shown in Figure It took longer than expected to develop the infrastructure of the “Quarterback Organization” and to provide engagement opportunities with activities. [Our SCALE community] was focused on fixing the lake and not fixing the fish, which involves more long‐term changes.The “build the core” story was reported by the coalitions that had strong existing connections with people with lived experience in their communities but first focused on strengthening their internal capacity by learning and internalizing improvement and relationship building tools and then used these to expand and deepen their relationships with the community to spearhead honest and authentic conversations about race and inequity. A core team member of a SCALE community that followed this pathway stated:We had a discussion on racism at every coalition and staff meeting. We created a safe and open space to discuss personal &/or patient experiences with racism or current events dealing with racism.The “create a shared language” story was followed by community organizations that began with using community partners with deep connections within the community to develop a shared language that facilitated mutual communication and dialog. Initial conversations to strengthen the ability to communicate was seen as especially important by these communities as was the need to create safe spaces for open and honest discussions and sharing experiences. These efforts accelerated trust and relationships in these communities. A coalition member stated:While all coalitions had a community champion from the outset, the “integrate people with lived experience (PLE) early” story emphasized developing community leaders as the first component of building the core team's ability to engage with the wider community. They also used the power of the local social networks to build relationships to build relationships quickly to advance equity work.A focus on authentic engagement of partners, building trust and relationships was foundational to the ability to scale up and sustain work. SCALE helped us offer more leadership development opportunities for community champions and staff members. This was valuable for building local capacity to improve authentic engagement and peer mentorship opportunities to lead civic engagement and service leadership.As one core team member remarked:3.4While the lines of argument are a convenient visual representation of how change happens, their sequential structure does not capture the complexity of the change process. CLDs allow us to better represent nonlinear system relationships. Using the data from Figure input into the system is the available external funding that influences the extent to which SCALE activities can be supported. The output from the system is the SCALE goal, that is, the capability for community transformation. As seen from the picture, there are three direct drivers of the output represented by the lines leading to the community transformation box: (a) the ability of communities to create collaborations with broad multisector leadership (including community members); (b) creating opportunities for dialog about addressing racism and equity and (c) engaging people with lived experience.In Figure SCALE program activities were intended to strengthen these drivers. The two places in the system where SCALE interventions were focused (leverage points to change the system) are indicated by the two arrows L1 and L2. L1 indicates that all SCALE training and support activities revolved around strengthening collaboration within the community. This enabled communities both to create leaders from within the community (loop R1) and facilitate dialog around difficult questions about inequity and race, without which true change is not possible. At the same time, to ensure that collaboration did not exclude those who needed to be at the center of transformation activities, SCALE required the intentional engagement of people with lived experience in leadership roles (leverage point L2), which strengthened the capability for transformation in multiple ways.As SCALE progressed and the community coalitions took on more complex projects, and attempted to spread what they had learned across the region, there was an increasing need for implementation support and technical assistance as indicated by the loops R2 and R3. These required increased resources, which were limited, by the available resources as shown in loop B1 . SCALE communities needed to do their best with fixed resources that were stretched thin, and these served as a limit to what the communities could ultimately achieve.4Even with vast heterogeneity among the SCALE communities and the diversity of activities that the communities undertook, our analysis showed there were a few basic principles that undergirded the process of building capability for transformation. A planned approach to transformation based on a theory of change, devoting time and attention to skill and relationship building and centering equity, ensuring the active engagement and leadership of community members and creating space for dialog and conversation were common ingredients across all communities. Trust within and across SCALE communities, and the ability to interact and learn were vital. Fostering collaboration and trust needed sustained coaching and technical assistance resources, both to build leadership and improvement skills and to recruit and engage community champions. The reliance on grant funding to support these activities was a limitation.4.1What does it mean to be in a community system that is truly engaged in learning to advance health, well‐being, and equity in sustainable ways? Results from our evaluation align with the existing literature related to multisector coalitions. Well‐known frameworks such as Collective Impactprocesses through which these capacities are developed. Even though these pathways were inductively constructed from the unique experiences of individual communities, we discovered enough commonality across communities to have confidence that Figures Our evaluation results also advance the knowledge about how community coalitions can build capacity to bring about change. The CLD and the transformation pathways extend the literature beyond just the factors that influence community capacity for change to hypotheses about the 4.2Our findings from SCALE about the dynamics that affect community change also allow us to set a direction for future research. Future efforts must focus on supporting community coalitions to reduce their reliance on external funding and technical assistance for their transformation initiatives. One possibility could be through strengthening regional hubs of excellence with local expertise that enable communities to learn from and support each other. SCALE began to build the infrastructure for regional change by requiring communities to spread the capabilities they had acquired, but in an environment where the communities had competing priorities, limited resources for implementation and staff turnover, there were limitations to what could be achieved. Part of the national and research conversation about shifting power to advance equity and dismantle racism should include ways to shift research and implementation resources to communities.Future research should investigate how to convert the constraints shown in Figure 5Our evaluation approach was designed to make the best use of existing project data, which was recorded in a variety of systems with varying degrees of completion. Consistent availability of high‐quality data to meaningfully document progress and learning was an ongoing challenge. The SCALE implementation teams introduced multiple measurement plans and mechanisms for data collection over the duration of the initiative, but in the end, there were still gaps in data quality and completeness. The ToAST process used the collective knowledge of the various stakeholder groups and triangulated data from various sources to develop the concepts and transformation pathways, but there were gaps in data that might affect the final results and conclusions. Also, our results have demonstrated progress in skills and behaviors, which are the first two components of theory of change. Given the range and complexity of the projects undertaken by the SCALE communities, there was not enough time for communities to demonstrate how this progress resulted in the achievement of the COH outcomes related to health, well‐being and equity before the funding ran out. This is not unusual, and our inability to evaluate whether the capabilities acquired by the communities resulted in improved health and well‐being is a serious limitation. The second CLD we have presented describes the scenario where communities have sustained financing to continue their transformation efforts. If this scenario were realized, it would be possible to follow communities over time to evaluate the impact of their work. Financial sustainability should be emphasized as a required component for future grants to facilitate this scenario.6The United States has invested trillions of dollars in health, well‐being and equity with little to show for these efforts. Simply investing more money and resources will not likely result in measurable improvement. Complexity‐informed approaches that approach well‐being through the lens of learning rather than through the lens of technical program implementation are needed. SCALE is an example of such an approach. Communities learned how answers to complex questions about change are not apparent at the start of the work, and that practices of learning and testing with meaningful measures to guide progress will help uncover solutions. Learning in a context of complexity requires a willingness to “fail forward,” in an environment where failures are opportunities for learning and growth rather than for judgment. Our results validate the relevance of the elements of the SCALE COS Theory of Change as the key components of skills and behaviors needed to bring about change. The extent to which the acquisition of these skills and behaviors drive sustainable health, well‐being and equity outcomes they aim to achieve should be a priority for future research.The authors have no conflicts of interest to declare.


Introduction: Several neurodegenerative conditions are associated with a common histopathology within neurons of the central nervous system, consisting of the deposition of cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43). Such inclusions have variably been described as morphologically and molecularly ordered aggregates having amyloid properties, as filaments without the cross-β-structure and dye binding specific for amyloid, or as amorphous aggregates with no defined structure and fibrillar morphology.Aims and Methods: Here we have expressed human full-length TDP-43 in neuroblastoma x spinal cord 34 (NSC-34) cells to investigate the morphological, structural, and tinctorial properties of TDP-43 inclusions in situ. We have used last-generation amyloid diagnostic probes able to cross the cell membrane and detect amyloid in the cytoplasm and have adopted Raman and Fourier transform infrared microspectroscopies to study in situ the secondary structure of the TDP-43 protein in the inclusions. We have then used transmission electron microscopy to study the morphology of the TDP-43 inclusions.Results: The results show the absence of amyloid dye binding, the lack of an enrichment of cross-β structure in the inclusions, and of a fibrillar texture in the round inclusions. The aggregates formed in vitro from the purified protein under conditions in which it is initially native also lack all these characteristics, ruling out a clear amyloid-like signature.Conclusions: These findings indicate a low propensity of TDP-43 to form amyloid fibrils and even non-amyloid filaments, under conditions in which the protein is initially native and undergoes its typical nucleus-to-cell mislocalization. It cannot be excluded that filaments emerge on the long time scale from such inclusions, but the high propensity of the protein to form initially other types of inclusions appear to be an essential characteristic of TDP-43 proteinopathies.Cytoplasmic inclusions of TDP-43 formed in NSC-34 cells do not stain with amyloid-diagnostic dyes, are not enriched with cross-β structure, and do not show a fibrillar morphology.in vitro from pure TDP-43 do not have any hallmarks of amyloid.TDP-43 assemblies formed  Frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U or FTLD-TDP) and amyotrophic lateral sclerosis (ALS) are two highly widespread neurodegenerative conditions associated in most cases with a common histopathology within neurons of the central nervous system, consisting in neuronal cytoplasmic inclusions (NCIs) of TAR DNA-binding protein 43 (TDP-43) in these cells . In ALS,per se, where TDP-43 is hyperphosphorylated, polyubiquitinated and partially proteolyzed pyrimidin-4-one, respectively. Coverslips stained with Amitracker 630 were also incubated in PBS containing DAPI for 15 min at 20 °C to detect cellular nuclei. Fluorescence emission was detected after excitation at 405 nm and 514 nm for coverslips stained with DAPI and Amitracker 630, and 488 nm for those labelled with compounds 3 and 4j, respectively, by the TCS SP8 scanning confocal microscopy system (Leica Microsystems) equipped with an argon laser source, using a Leica Plan Apo 63× oil immersion objective. The confocal microscope was set at optimal acquisition conditions, e.g. pinhole diameters, detector gain, and laser powers. Settings were maintained constant for each analysis.NSC-34 cells were plated and transfected with either vehicle or the pCI-neo plasmid expressing TDP-43 and analyzed after 40 h, as reported above. NSC-34 cells were also transfected with 0.15 µM native BSA or 2 µM Aβescribed . The tracker 630  . After 40 h, they were then pelleted (200 g for 6 min) and washed twice with PBS between successive centrifugations. For a single Raman measurement, 2 μl of the cell pellet was drop-casted onto a gold mirror support . Raman experiments were carried out on a micro-Horiba Xplora coupled to a 532 nm and a 785 nm wavelength laser for the excitation. The microspectrometer used a 1200 grooves mm-1 grating with a confocal microscope in backscattering geometry and a 2 D-CCD camera. The backscattered light was collected by a 100× microscope objective with 0.9 NA, which generated a ≈ 2 μm large laser beam waist. An integration time of 5 s and a laser power value of 1 mW on the sample were employed for Raman measurements on the cells. For each sample at least five individual cells were inspected, each in three areas enriched with fluorescent tdTOMATO protein, while carefully avoiding cell dehydration. The Raman spectra of each sample were averaged and baseline-corrected. To monitor only the cells overexpressing tdTOMATO protein, fluorescence emission in the 550–700 nm range was measured in the areas selected for Raman experiments, by exciting at 532 nm with a 10× (0.25 NA) objective.NSC-34 cells were plated in 12-well plates containing coverslips at 75,000 cells/well density and transfected with 80 µg of wtTDP43tdTOMATOHA plasmid (Addgene) coding for human TDP-43 C-terminally fused to the fluorescent tdTOMATO protein, using 3.5 µl of Lipofectamine 3000 Reagent, 5 µl of 5 mg/l transferrin, and 2.5 µl of P3000 reagent in DMEM for 3 h in a 5% CO−1, containing the amide I band, was isolated and analyzed with a fitting procedure using the sum of five Lorentzian functions, one for the 1580–1590 cm−1 peak, one for the 1610–1625 cm−1 peak and the remaining three for the α-helical component (1655–1665 cm−1), β-sheet component (1665–1675 cm−1), and unstructured component (>1675 cm−1):iA and ia are parameters related to the height and width of the Lorentzian function i, respectively, and 0ix is its wavenumber of maximum intensity (peak). The 15 parameters of the five Lorentzian functions  were left free to float in the fitting procedure simultaneously, but their 0ix values were restrained within their corresponding 10 cm−1 range. Moreover, since the α-helical, β-sheet, and unstructured components are known to be 36, 22, and 42% in the human and mammalian proteome [a ± 50% range around these percentage values.Each Raman spectrum from 1500 to 1800 cmproteome ,50, thei2 window and dried at room temperature for 30 min. FTIR spectra were then measured in transmission mode by the infrared microscope Varian 610-IR coupled to the Varian 670-IR FTIR spectrometer , equipped with a mercury cadmium telluride nitrogen-cooled detector, using the following settings: variable microscope aperture set at ∼100 × 100 μm, spectral resolution 2.0 cm−1, scan speed 25 kHz, 512 scan co-additions, and triangular apodization. The absorption spectra were normalized at the amide I band area, for comparison, and the second derivative analysis was performed, after a binomial 13-point smoothing of the normalized absorption spectra, by the Savitzky-Golay method , using the GRAMS/32 software .NSC-34 cells were plated in 12-well plates containing coverslips at 75,000 cells/well density and transfected with either vehicle or the pCI-neo plasmid expressing TDP-43. NSC-34 cells were also transfected with vehicle (control cells). After 40 h, they were then pelleted (200 g for 6 min) and washed twice with 0.9% NaCl between successive centrifugations and finally deposited onto a BaF4 for 1 h, 1% tannic acid for 30 min and en bloc stained with 1% uranyl acetate for another hour. Then samples were dehydrated through a graded ethanol series and flat embedded in epoxy resin  for 24 h at 60 °C. Ultrathin sectioning (50 nm) was performed with Leica ultramicrotomes . Flat-embedded cells were cut parallel to the substrate and counterstained with 5% uranyl acetate in 50% ethanol. Ultrastructural analysis was performed with a Hitachi 7800 120 kV electron microscope (Hitachi) operating at 100 kV using a Megaview G3 digital camera and Radius software (EMSIS). Electron micrographs were taken using the Multiple Image Alignment (MIA) montage and screenshot tools.Primary neuronal cultures from PS19 mice expressing P301S mutant human tau and cultured NSC-34 cells transfected with vehicle or human TDP-43 expressing plasmid were fixed, 40 h after transfection, with 2.5% glutaraldehyde (Electron Microscopy Science) in 0.1 M cacodylate buffer for 1 h at room temperature, post-fixed in 1% OsOEscherichia coli BL21(DE3) cells and purified as previously described [ca. 1 mg ml−1 and stored at −20 °C. Protein concentration was determined by optical absorbance spectroscopy using a molar extinction coefficient at 280 nm (ε280) of 46,410 M−1 cm−1. The final purified protein had the sequence stretch MHHHHHHSSGVDLGTENLYFQS at the N-terminus before Met1, contained 436 residues, and had a molecular weight of 47,292.53 Da.Human full-length TDP-43 was recombinantly expressed in escribed . In brieescribed . The lasThe purified TDP-43 sample was thawed and large aggregates were spun down at 18,000 g, 4 °C for 15 min. TDP-43 was labelled with Tetramethylrhodamine-5-maleimide (TMR) (Invitrogen) in a 1:10 ratio (dye:protein) and then subjected to desalting through a Sephadex G15 resin column . Protein concentration was monitored with NanoDrop One (ThermoFisher Scientific). Aggregation was induced by diluting the protein down to 5 μM into 20 mM acetate buffer, 150 mM NaCl, 5% (w/v) PEG8000, 2 mM TCEP, pH 5.0, to reach a final pH of 6.0, and incubating under shaking at 560 rpm on a TS-100 Thermo Shaker (Kisker) at 25 °C for 4 or 10 days. Then, 40 µl of the protein aggregation sample was deposited onto an 8-well Chamber Slide and TMR was excited at 550 nm, and emission was collected at 564–599 nm. Confocal and STED microscopy images were acquired by the SP8 confocal microscope described above, equipped with the Leica HC PL APO CS2 100×/1.40 oil STED White objective and the HyD (hybrid detector). A series of optical sections (z-stacks) was taken through the aggregate’s depth; images were then de-convolved with Huygens Professional software version 18.04 (Scientific Volume Imaging B.V.), and the maximum intensity projection of confocal z-stacks was obtained by superimposition. The Leica Application Suite X (LAS X) software equipped with the 3D Projection Tool (Leica Microsystems) was used to generate depth coding profiles of 3D reconstructions from de-convolved images and movies from the z-stacks.TDP-43 aggregates were prepared as described in the previous subsection, with the addition of 25 μM Thioflavin T (ThT). The resulting fluorescence was recorded from 450 to 600 nm (excitation 440 nm) at 25 °C using a Cary Eclipse Fluorescence Spectrophotometer (Agilent). A small volume 3 × 3 mm quartz cell was used and the blank was subtracted from the spectrum. Other details are as previously described .TDP-43 aggregates were prepared as described above. The far-UV circular dichroism (CD) spectrum was collected over the 200–260 nm wavelength range at 25 °C using a Jasco J-810 Spectropolarimeter, averaged from 8 scans, blank-subtracted and normalized to mean residue ellipticity. A quartz 1 mm pathlength cuvette was used. Other details are as previously described .Data were expressed as means ± SEM.in vivo under pathological conditions [We transiently transfected human full-length TDP-43 in NSC-34 cells, which is a motoneuron/neuroblastoma hybrid cell line that expresses several motor neuron features . To thisnditions ,6,14,15.3 [4j [in vitro, compounds 3 and 4j were found to be able to discriminate between amyloid and amorphous aggregation/native protein forms by increasing the fluorescence signal [42) peptide as a positive control, we observed a dramatic increase in the intracellular dye-derived fluorescence with all three probes, confirming the ability of the probes to detect specifically amyloid assemblies present in the cytoplasm , as reported . It is kgregates , cyt-c igregates ,54, explca. 1220–1260 and 1630–1700 cm−1 correspond to the amide III and amide I bands, respectively [Methods section, and the amide I components emerging from the analysis . The enrichment of α-helical structure in the cytoplasmic inclusions of TDP-43 expressing cells may have many explanations, such as structural rearrangement of TDP-43 in the aggregates , accumulation of chaperones in these areas , accumulation of cyt-c , etc.The bands at ectively . Both apanalysis  were assanalysis . The anaanalysis . The anaanalysis . This in−1 arising from β-sheet structure is missing and does not emerge as an evident shoulder in the TDP-43 or difference spectra relative to the control spectrum.As a further inspection we also analyzed the amide I difference spectrum, obtained by subtracting the spectrum of control cells from that of TDP-43 expressing cells . The difin situ, we also used FTIR microspectroscopy, which has been successfully used for other systems to detect in situ the cross-β structure within amyloid deposits [−1) of control cells and TDP-43 expressing cells indicate the absence of an increased absorption in the 1630–1615 cm−1 range in the second spectrum relative to the first ), ruling out an enrichment of intermolecular cross-β structure in TDP-43 expressing cells. The enrichment of α-helical structure at the expense of unordered structure, detected with Raman microspectroscopy in the TDP-43 expressing cells, remains undetected with FTIR spectroscopy because the absorptions of the two secondary structure types overlap closely in the amide I band ). As a positive control, we show the spectrum obtained with the same procedure and technical apparatus of FTIR microspectroscopy as those used here of a cardiac tissue section of a human patient affected by light chain amyloidosis (AL) containing amyloid fibrils with cross-β structure of an immunoglobulin light chain ).To have an independent measurement of the secondary structure of the TDP-43 cytoplasmic inclusions deposits ,56. In tUltrastructural analysis of TDP-43 expressing cells using TEM (40 h after transfection) revealed that the cytoplasmic inclusions appear in most cases localized within the lysosomal compartments, indicating an active autophagy process , arrows.in vitro in a buffer solution mimicking physiological conditions, we purified human full-length native TDP-43 and diluted it from ca. 35 µM of its storing solution, in which it was soluble, to 5 µM in 20 mM acetate buffer, 150 mM NaCl, 5% (w/v) PEG8000, 2 mM TCEP, pH 5.0, to reach a final pH of 6.0, and incubated under shaking for 4 days at 25 °C.To assess the intrinsic propensity of TDP-43 to form amyloid fibrils ca. 485 nm with a value of ca 50a.u. (The ThT fluorescence spectrum in the absence of protein (blank) had a maximum at ca. 204 nm and a shoulder around 222 nm, which reveals the presence of α-helix and random coil secondary structure, particularly the former (The far-UV circular dichroism (CD) spectrum of the TDP-43 aggregates formed after 4 days showed a prominent negative peak at e former . The CD e former . This suca 10 µm (Supplementary Movie S3). To assess whether the large non-filamentous aggregates analyzed after 4 days reorganize into filamentous species upon prolonged incubation under the same conditions, we acquired confocal images of the same sample after 10 days, which is a time length sufficiently high for proteins to form amyloid fibrils when they have the propensity to form such assemblies [ca 20 µm, yet in the absence of a defined ordered fibrillar morphology .Confocal microscopy images of the same TDP-43 sample, but pre-labelled with the TMR fluorophore, revealed large aggregates of TDP-43 with no defined ordered morphology and a size of semblies . In thisca 5-fold, our results suggest that TDP-43 aggregates formed in vitro under these conditions did not show typical amyloid characteristics. Our lab has previously identified these characteristics in many protein aggregates formed in vitro from at least seven different protein systems, therefore acting as positive controls of amyloid [Overall, since amyloid aggregates generally have an orderly and rigid fibrillar morphology, with the formation of cross-β structure that can induce an increase of ThT fluorescence emission by over  amyloid .in vitro self-assemblies. Using TEM and super-resolution STED microscopy, the cytoplasmic inclusions do not show any obvious fibrillar morphology, with a few fibrils appearing only occasionally within the inclusions.In this work, we have expressed human full-length TDP-43 and observed its self-assembly in the immortalized NSC-34 cell culture model, which recapitulates many of the characteristics of motor neurons and is the model cell line of election for ALS-related studies ,40,66. Win situ. To this purpose, we used three amyloid-diagnostic dyes that have the ability to cross biological membranes and stain amyloid in the cytoplasm [The investigation of the TDP-43 cytoplasmic inclusions was carried out ytoplasm , Raman aytoplasm ,55,56, aytoplasm ,26–30,35ytoplasm .in vitro assemblies and that detected in post-mortem NCIs is the absence of a clear filamentous texture in our two conditions of analysis. This may result from the shorter time scale of the aggregation process in our cellular and test-tube conditions, as opposed to the slow process occurring in pathology where more ordered filaments can form upon slow conversion of the early amorphous inclusions or be seeded by rarely and occasionally formed filaments that then propagate rapidly. Filaments were not observed, however, in vitro from pure TDP-43, even after 10 days of incubation, which is a time largely sufficient for purified proteins to form amyloid fibrils when they have the propensity to form such assemblies [in cell and in the test tube, TDP-43 exhibits a very high propensity to form solid-phase aggregates in both environments, under conditions of pH and temperature that do not otherwise promote aggregation of stable proteins.An important difference between the morphology of our cellular and semblies . Similarsemblies . By contin cell and in vitro shows that this does not yet occur in the initial steps of TDP-43 aggregation.Another important difference with the cryo-EM post-mortem filaments is the enrichment of α-helical structure at the expense of random coil structure. This probably results from the collapse of the CTD that is initially largely unfolded in the native protein but may present the characteristic α-helical enrichment of early-formed protein aggregates . Later oin vivo (see below).One of the advantages of our experimental approach is the possibility to assess the intrinsic propensity of the TDP-43 protein to form any given inclusion type spontaneously, on a time scale that is otherwise sufficient for amyloid to form. Another advantage is offered by the opportunity to detect and analyze the early inclusions that may eventually lead to filaments, which is a step that escapes detection when analyzing only post-mortem specimens. Finally, our analysis has been carried out in the absence of TMEM106B amyloid fibrils that may mislead and confound the analysis of natural specimens in vitro, in a non-cellular context, is not entirely novel. Early reports published in 2009–2014 showed that full-length TDP-43 can form filaments that were not able to bind ThT or Congo red [in vitro into aggregates that presented both fibrils and amorphous aggregates with, again, a weak ThT fluorescence increase [The identification of non-amyloid TDP-43 assemblies formed ongo red , or induongo red ,74, whicongo red ,58. Overongo red . A laterongo red , which wongo red . Anotherongo red . Similarincrease . Finallyincrease .in vitro under appropriate conditions [Histopathologists also imaged, in post-mortem ALS and FTLD cases, TDP-43 NCIs in the absence of ThT/ThS or Congo red binding ,35. The nditions . Such obin vitro with the TDP-43 CTD or even shorter CTD peptides, that have by contrast amyloid characteristics [Much of this debate has been resolved by the recent cryo-EM structural elucidation of the TDP-43 filaments from two distinct patients, both with a history of ALS and FTLD-U . Both steristics . In addieristics , suggestca. 250–350 residues has been proposed as a threshold beyond which amyloid formation is rarely observed in pathology [ca. 150 residues) and full-length TDP-43 (414 residues) have shown the formation of amyloid and non-amyloid assemblies, respectively. The finding by Bigio et al. of a marked ThS positivity in ALS and FTLD-U specimens probably originated from the chemically harsh treatment of the tissue sections, based on the sequential use of potent oxidants, reductants, acids, and bases [in vitro [in cell (present work), and in vivo inclusions [We also believe that this debate can be reconciled following the knowledge accumulated on the fundamentals of amyloid fibril formation over the past  25 years. Small peptides and protein fragments are well known to have a high propensity to form amyloid-like fibrils through a mechanism and pathway independent of the corresponding full-length proteins . Large pathology . In thisnd bases , all knoin vitro ,67,72–78clusions ,35,38, ain vitro from the purified native protein under conditions close to physiological. In both cases, the inclusions do not bind to ThT or derived fluorophores specific for amyloid, do not possess a significant β-sheet structure, and do not appear fibrillar in morphology. We, therefore, propose that self-assembly of TDP-43 starts with the mislocalization of the protein from the nucleus to the cytoplasm [The data presented here show the absence of a clear amyloid signature in both the TDP-43 inclusions formed in NSC-34 cells following overexpression of the protein and in the TDP-43 aggregates formed ytoplasm , where iytoplasm . Later oytoplasm  or absenytoplasm ,38 of suClick here for additional data file.Click here for additional data file.