The water vapor molecular dynamics inside a pore structure control both molecular adsorption and desorption processes and the limit of minimum detection (LOD). Pore morphology design, and a higher concentration of electrolyte-driven anions, in accordance with the kinetics of water vapor molecules, is reported here, as the ultimate answer to extremely low relative humidity (RH) detection. In this report, a series of samples were prepared by anodization in different voltage windows, related to specific electrolyte solutions. The sensing attributes comprised: (i) a LOD of ∼3 RH%, (ii) excellent response time (6 s) and recovery time (54 s), and (iii) a hysteresis loss of ∼0.36%, with sustained stability over the period of one year; all these occurring in a sample with a pore diameter ∼5 nm ±3 nm. Interestingly, the LOD extend towards a lower RH% with a decrease in pore diameter; and a suitable explanation is given for the entire range of humidity level, in terms of the molecular mean free path, loss of kinetic energy due to scattering inside the pores, and subsequent overall loss of Brownian energy of the molecules. It is inferred from the sensing response characteristics that pore morphology and lower detection limit are interrelated; therefore, a further extension in LOD from extremely low RH% to trace levels, needs careful engineering of the pore morphology and parameters related to molecular kinetics. Water vapor molecular dynamics, pore size, and anion concentration within the pores are interdependent and together affect the lower detection limit (LOD) and sensitivity of a humidity sensor. The electrochemical anodization method, on the other hand, has a niche advantage in developing a tailor-made porous anodic alumina (PAA) nanostructure where the pore diameter, pore depth and pore widening may be controlled easily.9 In terms of structural aspects, the incorporation of anions and the design of electrodes work together synergistically to govern the sensing mechanism in a nano-structured PAA.Humidity measurement is playing an ever-increasing role in industrial, laboratory and process control. Over the years, a wide variety of humidity sensing devices have been developed. Most commercially available sensors are based on polymers or ceramics.19–25 Y. Kim et al. investigated the effect of electrode design on humidity sensing,22 L. Yao et al.23 explained the effect of isotropic etching of pores on sensing behavior, and Nahar24 studied the performance degradation of PAA sensors upon exposure to high humidity. S. W. Cheng et al.18 reported sensitivity enhancement by application of a magnetic field. Presently, none of the reported humidity sensors shows any significant change in capacitance/impedance below 30 RH%.19–25 Even when sensing with high sensitivity, the fastest response–recovery, high resolution and stability also arouse great concern for both high- and low-level RH. Again, the quantitative sensing response is a matter of concern, irrespective of sensing level. Therefore, a complete analysis is required to bridge the gap to appreciate the interplay of pore morphology and sensor performance.The measurement of high relative humidity (RH) is not an issue at all, but it certainly is for low RH. Lots of R&D work is underway globally on precursor materials, surface morphology and engineering aspects to check for improvements in the lower detection limit (LOD), sensitivity, long term drift, and hysteresis. Investigations on PAA-based humidity sensors are plentiful and have been reported elsewhere.9 the PAA nanostructure was optimized for humidity sensing applications; and the impacts of various surface pre-processing parameters were discussed in detail. In continuation of that, the present investigation reports a study of the surface morphology dependent humidity sensing response of PAA. For this, a series of samples were prepared by anodization in different voltage windows, related to specific electrolyte solutions. The underlying principles of PAA fabrication, morphology design and internal molecular dynamics, control the adsorption and desorption processes, and the sensing attributes of the sensor.In our previous research article,2.2.1.9 The electropolishing was carried out in galvanostatic mode at 500 mA for 80 seconds at 3 °C to create a reproducible surface finish. The aluminium sheets were then ultrasonicated in acetone in order to remove loosely bound impurities, if any, present on the surface of the Al sheet.Aluminium sheets of high purity  were annealed and electropolished in a 1 : 4 ratio of perchloric acid, and ethanol solution. The process of electropolishing and its optimization were explained earlier in our previous research article.2.1.1.The pore diameter of PAA depends directly on the anodizing voltage. Therefore, samples with different surface morphologies were obtained by anodizing at different anodic polarizations. However, anodization voltage is electrolyte specific; there exists a narrow window of voltage in which the generation of a uniform, symmetrical and self-assembled porous structure with a specific range of pore diameters can occur. It can be observed from After anodization, the samples were treated with alumina etchant solution to widen the nano-pores. The etching rate relies on the chemical composition of the porous oxide layer; that again depends on the anodizing electrolyte and its concentration. Several experiments were performed to optimize the pore-widening step for the three electrolyte solutions. 2.2.Parallel plate capacitive sensors were developed where the anodized film works as the sensing layer. The aluminium at the base acts as the bottom electrode. A porous gold layer of 3 nm in thickness was deposited on the anodized alumina.i.e. the PAA layer. Therefore, careful optimization of the Au film thickness is very necessary to satisfy both the purposes mentioned. The top gold electrode, deposited via a sputtering technique is highly porous and water permeable in nature. We have checked that a gold film thickness of ∼3 nm or slightly thicker proved to be the best for sensing performance. This gold electrode serves the purpose of the second electrode of a parallel plate capacitive sensor. 2.3.via the bubbler route) are combined before the gas is allowed to pass through the sensor chamber. Both the chambers, A and B, of the sample chamber are purged separately for 12 h, in order to achieve a stable baseline capacitance, followed by exposure to different humidity concentrations. Sensing chamber A is connected to a data acquisition system , where a test signal of 30 mV (r.m.s) was used to excite the sensor at frequency 5 kHz with no DC bias.The humidity sensing characteristics of the as-prepared samples were measured in a home-made humidity sensing setup , specialThe moist gas then passes through a commercial RH sensor . The measurement of sensing parameters, such as the humidity sensing response, response–recovery time, hysteresis and long-term stability, is carried out at room temperature (300 K) and at atmospheric pressure (1.03 bar). The flow rate of the carrier gas is kept fixed at 2 lpm (liter per minute) while performing the experiment.3.3.1.25 Depending on the applied voltage, different morphologies with distinctive pore size, interpore spacing, anodic alumina film thickness and electrolyte-driven species, were observed in the PAA nanostructure.31 These parameters are easily tunable, and their effectiveness in sensor performance may be checked.The anodic polarization has a direct influence on the diameter of the pores.Jsat.) is different for each sample. Even for samples anodized in the same electrolyte, the current density is greater for the sample anodized at a higher voltage, than for the sample which is anodized at a low voltage: e.g. Jsat.(SH25) > Jsat.(SH05). Similarly, for the sample anodized in oxalic and phosphoric acid: Jsat.(SO50) > Jsat.(SO38) and Jsat.(SP90) > Jsat.(SP60). e.g. SH25 > SH05, SO50 > SO38 and SP90 > SP60, and affects the overall water vapor adsorption dynamics. The saturation current density (Jsat.) gives an idea of the concentration of incorporated electrolyte anions, viz. SO42−, C2O42− and PO42−, which inherently enter into the anodic oxide layer during anodizing. The plots in e.g. SH25 > SH05, SO50 > SO38 and SP90 > SP60. However, the values for electrolyte-driven anions vary in the ranges: 10–14% in sulfuric acid, 2–3% in oxalic acid, and 6–8% in phosphoric acid solution.252O molecules present in the electrolyte break apart into OH− and O2− anions. The O2− anions assist in the formation of PAA while the OH− anions combine with sulfate anions. Therefore, apart from acid-driven anions, there might be the presence of trapped water molecules producing OH− and O2− anions in the anodic aluminium oxide. The presence of these anions in the anodic oxide structure greatly affects the humidity sensing characteristics, as they influence the water vapor adsorption dynamics.These anions assist the adsorption of water vapor molecules on the porous oxide structure; hence a significant change in capacitance is observed for the samples with a high concentration of acid-driven anions. This is explained and shown later in Section 3.5  and 10 e3.2.The surface morphology of as-prepared PAA samples was observed using FESEM . The samples were generally coated with a thin layer of gold prior to imaging, to avoid any charging effect caused by the insulating nature of PAA. For imaging, the samples were placed on an aluminum stub with carbon tape and positioned inside the sample chamber. i.e. SH05 and SH25, are shown in The micrographs are displayed at two different scales ; emphasi33 However, the overall surface morphology, e.g. the self-assembled long-range ordering and uniformity of the porous structure, also affects the sensing characteristics.26–29 It has been observed that, amongst samples with the same pore diameter but differences in long-range ordering and uniformity, a sample with a better pore arrangement gives better results than a sample with an unordered porous structure, in terms of sensitivity and defining the lower detection limit. Therefore, the aforementioned parameters must be optimized for a better humidity sensing response.The pore diameter plays a major role in deciding the sensor characteristics, especially the lower detection limit and sensitivity.3.3.In the first study, after testing, sensors SH05, SO50 and SP60 were kept at a humidity of 50 RH% continuously for 3 months at room temperature . It can i.e. 3–90 RH% for 12 months at room temperature. In the second study, the developed sensors were tested periodically over the complete RH range: It can be concluded that upon exposure to high humidity that a minor variation in porous structure is observed for samples anodized in sulfuric and oxalic acid solution, whereas no degradation in structure is observed for samples anodized in phosphoric acid solution. Therefore, a sensor developed from anodization in phosphoric acid solution is found to be particularly useful for applications where continuous monitoring of a high humidity level (>50 RH%) is required. On the other hand, a sensor developed from anodization in sulfuric acid or oxalic acid solution is suitable for monitoring low and medium ranges of humidity (0–50 RH%).3.4λ = 1.54 Å. 30,31The phase of PAA fabricated by anodization was determined by carrying out X-ray diffraction measurements  using Cu-Kα radiation, where i.e. α-Al2O3 and γ-Al2O3, are important for humidity sensor fabrication. Most of the reported humidity sensors were found to exist in the γ phase of Al2O3.32 The γ-Al2O3 phase is liable to change to γ-Al2O3·H2O which leads to surface degradation upon exposure to high humidity.23 The phase is mainly amorphous in nature with a small crystalline content.33 The α-Al2O3 is expected to be a promising candidate owing to its high thermal stability. Chen in 1991 reported an α-Al2O3 based humidity sensor fabricated by an anodic spark deposition technique.34Alumina exists in many stable and meta-stable phases; however, only two crystalline phases, 3.5.y) and relative humidity (x) is represented by the equationyo, A, Ro are given in the plots  and recovery time (Tc), the sensor is exposed to a step change in humidity: i.e. 0–50 RH%. The response time is defined as the time taken to go from 10% to 90% of the maximum change in sensor output; whereas the recovery time is defined as the time taken to recover from 90% to 10% of the maximum change in sensor output. The transient curves exhibiting the response and recovery time of the samples are shown in In order to determine the response time  was reduced even further to 3 RH%, an exceptional outcome indeed. Besides, the developed sensor (SH05) was found to be extremely fast with response and recovery times of ∼6 s and ∼54 s, respectively; however, the recovery times of samples SH05 and SH25 were found to be a little longer, which is directly related to the molecular kinetics of vapour molecules in an exceptionally small pore diameter (<11 nm). Sensors prepared via anodization in phosphoric acid solution were found to be useful for monitoring high humidity levels (>50 RH%) whereas sensors developed via anodization in sulfuric or oxalic acid solution are useful for monitoring low and medium ranges of humidity (0–50 RH%). The long-term drift was also investigated and found to show a negligible shift in capacitance value, ensuring the long-term stability of the developed sensor.We have reported a comprehensive as well as transparent understanding of the humidity sensing mechanism in the developed sensors, taking into account the effect of the diameter and uniformity of self-assembled nano-pores, along with electrolyte-driven anions in the grown PAA nanostructure. The underlying principle of PAA fabrication and its impact on sensor performance were thoroughly investigated; and a mechanism to further reduce the minimum detection limit with improved sensing characteristics was suggested. All the prepared samples exhibit an exponential change in capacitance with an increase in humidity, which is given by the equation There are no conflicts to declare.


Ruminants are a critical human food source and have been implicated as a potentially important source of global methane emissions. Because of their unique digestive physiology, ruminants rely upon a symbiotic relationship with the complex and rich community of microorganism in the foregut to allow digestion of complex carbohydrates. This study used 16S rRNA gene sequencing to investigate the composition of microbial communities from three rumen micro-environments of cattle fed identical diets: (1) free fluid, (2) the fibrous pack, and (3) the mucosa. Community composition analysis revealed that while a phylogenetic core including the most abundant and most common ruminal taxa (members of Bacteroidetes and Firmicutes) existed across micro-environments, the abundances of these taxa differed significantly between fluid- and mucosa-associated communities, and specific lineages were discriminant of individual micro-environments. Members of Firmicutes, specifically Clostridiales, Lachnospiraceae, Mogibacteriaceae, Christenellaceae, and Erysipelotrichaceae were significantly more abundant in fluid communities, while members of Bacteroidetes, namely Muribaculaceae and Prevotellaceae were more abundant in mucosa-associated communities. Additionally, Methanobacteriaceae, a family of methanogenic Archaea, was more abundant in fluid-associated communities. A set of four more diverse lineages were discriminant of pack-associated communities that included Succinivibrionaceae, RFP12 (Verruco-5), Fibrobacteraceae, and Spirochaetaceae. Our findings indicate that different ecological niches within each micro-environment have resulted in significant differences in the diversity and community structure of microbial communities from rumen fluid, pack, and mucosa without the influence of diet that will help contextualize the influence of other environmental factors. Ruminant livestock are a critical source of food for humans worldwide, with the population of cattle alone estimated to be over 1.5 billion . CharactThe rumen is a rich and diverse microbial ecosystem largely composed of anaerobic bacteria, protozoa, anaerobic fungi, methanogenic archaea, and phages. Bacteria represent the most abundant and diverse taxonomic group, and it is the bacterial members of the rumen microbial community that primarily drive the degradation and fermentation of plant fibers and proteins into digestible compounds such as volatile fatty acids and microbial proteins , 5. RumiThe microbiota of the rumen as a whole has been well-characterized, but relatively few studies have investigated differences in community structure between ruminal components. While there are a large number of ecological niches available, broadly speaking rumen microorganisms can be free-living or particle-attached in ruminal fluid, attached to the fibrous pack, or attached to the ruminal mucosa . DiffereThis study utilized 16S rRNA gene sequencing to characterize the diversity and composition of microbial communities of different locations within the rumen: within the ruminal fluid, fibrous pack, and those found on the mucosa. It was designed to test if the previously described ruminal phylogenetic core is present across ruminal micro-environments, and to discover which microbial taxa were discriminant of these micro-environments. We hypothesized that core rumen taxa would be present across all three micro-environments, but that the composition of fluid, pack, and mucosa-associated microbial communities would differ.Twelve crossbred feedlot steers  fitted with ruminal fistulas, were utilized in this study. All cattle in this study were adjusted to a high energy finishing diet consisting of ~90% concentrate and 10% roughage , a bolus of the ruminal fibrous pack , and a swab of the ruminal mucosa . The bolus of fibrous material was sampled by collecting 85 g from the center of the solid portion of the rumen, and the ruminal mucosa was sampled by running a sterile swab across the ruminal wall. Fluid was collected by compressing the fibrous bolus and capturing the resulting liquid. After collection, samples were immediately placed on ice, transported to the laboratory and stored at −80°C at the laboratory until DNA isolation.At the completion of the diet adjustment period, samples from three micro-environments within the rumen were collected −1) and assayed for quality (A260/A280) using a NanoDrop spectrophotometer .Genomic DNA was isolated from fluid and mucosal swab samples using a QIAamp PowerFecal DNA Kit  from 0.16 to 0.18 g of material, while a DNeasy PowerMax Soil Kit was used to isolate DNA from 2 to 5 g fibrous pack samples. Following isolation, DNA was quantified .Amplicon library preparation and sequencing was carried out by Novogene Corporation Inc . The V4 region of the 16S rRNA gene was amplified with the 515f (5′ – GTG CCA GCM GCC GCG GTA A – 3′) and 806r (5′ – GCA CTA CHV GGG TWT CTA AT – 3′) primer pair and Novogene's proprietary amplification conditions, which include no template PCR negative controls. Following successful PCR, amplicon libraries were prepared and pooled using Novogene's proprietary process and sequenced on an Illumina HiSeq 2500 instrument  using 2 × 250 bp paired-end chemistry. The number of sequenced reads ranged from 188,276 to 219,029, with an average of 209,279 reads per sample. The average sequencing depth did not differ between the three micro-environments  of 0.1% and a minimum prevalence (proportion of samples the genus was present in) of 80% were used as cutoffs, and the “core” function within the package microbiome was used to calculate and plot a heatmap of core community members.To identify taxa discriminant of each micro-environment , linear discriminant analysis effect size (LEfSe) was performed using the LEfSe tool  with defUnless specified otherwise, R version 3.6.3  was usedAll sequence reads were made available through the BioProject PRJNA749669 at the National Center for Biotechnology Information's Sequence Read Archive.n = 12, p < 0.05). Fluid communities were also more diverse than pack and mucosa communities, which did not differ significantly from each other . However, the phylogenetic diversity of ruminal fluid and pack communities were not different, and both were more phylogenetically diverse than mucosal communities .A comparison of observed ASVs was used to test for differences in richness among the ruminal components, and comparisons of Shannon's index and Faith's Phylogenetic Distance (FPD) were used to test for differences in diversity. The trend was the same for richness, diversity, and phylogenetic diversity, with fluid being the richest and the most diverse followed by pack and then mucosa . HoweverOf the 117 genera above the detection threshold of 0.1% relative abundance, 32 (27%) were present in at least 80% of samples from all three ruminal components across every individual animal, and thus were considered to represent the phylogenetic core . Of the n = 12, p < 0.05). Non-significant PERMDISP tests confirmed that significant PERMANOVA results were the result of differences in community structure and not due to unequal dispersions of variance (p > 0.05). Visualization with NMDS illustrated that microbial communities from each of the three ruminal components were distinct, and that communities became more similar when compared using less weight on abundant lineages . Further, fluid and pack communities appear to be more tightly clustered and distinct from each other, while mucosa communities were slightly more varied , and its designation as mucosa-discriminant was an artifact of the “one-against-all” comparison strategy coupled with a lower abundance in pack communities. A set of four more diverse lineages including RFP12 (Verrucomicrobia), Spirochaetaceae (Spirochaetes), Succinivibrionaceae (Proteobacteria), and Fibrobacteraceae (Fibrobacteres) were discriminant of pack communities  was used to identify taxonomic lineages that were differentially abundant between rumen fluid, pack, and mucosa communities. Results show that six lineages of Firmicutes  were discriminant of fluid communities. Five of the six lineages included members of Clostridiales. Further, Methanobacteriaceae (Euryarchaeota) and RF16  were discriminant of fluid-associated communities . Muribacmunities .n = 12, p < 0.05). Differences in Bacteroidetes were largely the result of differences within the families of Prevotellaceae and Muribaculaceae . RF16  was more abundant in fluid communities , although its relative abundance was far lower than that of Prevotellaceae and Muribaculaceae in all three micro-environments.To further characterize the Bacteroidetes-Firmicutes dichotomy among fluid, pack, and mucosa communities, the relative abundances of each phylum and the families comprising them were directly compared. The abundance of members of Bacteroidetes was higher in mucosa- and pack-associated communities than fluid communities . The differential abundance of Firmicutes between ruminal components was largely the result of differences in the relative abundance unclassified Clostridiales and Lachnospiraceae .The relative abundance of Firmicutes was different among each of the three ruminal components, with the phylum being more abundant in fluid communities than both pack and mucosa-associated communities, and less abundant in mucosa-associated communities then pack , Fibrobacteraceae, and RFP12 then fluid and mucosa-associated communities, which had similar abundances . The family Methanobacteriaceae was in higher abundance in fluid-associated communities than both pack and mucosa-associated communities .With the exception the Methanobacteriaceae, which was discriminant of fluid-associated communities , every dThis study used 16S rRNA gene sequencing to investigate the localization of microbiota within the bovine rumen and to identify microbial taxa discriminant of its micro-environments. Results demonstrated that rumen fluid contained the richest and most diverse microbial communities, while mucosa-associated communities were the least diverse. Importantly, while a large proportion of microbial taxa were present in every ruminal fluid, pack, and mucosa-associated community, the proportions of these taxa differed significantly among ruminal components. Members of the phylum Firmicutes were disproportionately predominant within fluid communities as compared to pack and mucosa-associated communities, while members of the phylum Bacteroidetes were disproportionately under-represented in fluid communities. Further, pack-associated communities contained discriminant lineages from the phyla Proteobacteria, Verrucomicrobia, Spirochaetes, and Fibrobacteres. Diet-induced shifts within rumen microbial communities are well-established –8 and duRumen fluid functions as a transportation and growth medium for digestive microorganisms and as a result it could be expected that that majority of microbial taxa found within the rumen as a whole would be present in the fluid even if in very low abundance. Indeed, fluid communities in this study were the richest, but diversity was similar in fluid and pack-associated communities. Higher richness, but similar diversity suggests that ruminal fluid contains more types of microorganisms, but the additional types are either rare or closely related. Interestingly, mucosa-associated communities in this study were both less rich and less diverse, which contradicts previous research that found mucosal communities to be most diverse . HoweverA large number of studies have used amplicon sequencing to characterize the rumen's phylogenetic core and illustrated the overwhelming dominance of the phyla Bacteroidetes and Firmicutes, and more specifically members of orders Bacteroidales and Clostridiales , 27–29. Despite the most abundant taxa being dominant in all three micro-environments, fluid, pack, and mucosa-associated microbial communities were different when compared with and without weight being placed on abundance, suggesting that both abundant and rare taxa contribute to the differences. Variations in community composition in communities associated with liquid- and solid-fractions of different ruminal micro-environments have been well-described , 12, 13 The primary drivers of the liquid-solid dichotomy in the rumen were members of the phyla Bacteroidetes and Firmicutes. In particular, members of Muribaculaceae (formerly S24-7) and Prevotellaceae (both families of Bacteroidetes) were more abundant in solid attached communities, with Muribaculaceae abundance being the highest in mucosal communities. A study of the Muribaculaceae family showed that its members have high functional diversity with regards to complex carbohydrate degradation, and contain protective mechanisms against benzoate and macrolide antibiotics . Howevervia these fistulas meant that mucosal samples were taken from the surface by swab, and it was not possible to determine whether the mucosa was bathed with ruminal fluid during collection, which could represent a source of cross-contamination between micro-environments. However, it is unlikely that these factors would have been fully responsible for creating systematic differences among the microbiota found in the different ruminal fractions that were study. Another consideration is that fluid samples here were collected from fluid extracted from the pack, which could potentially yield different communities than free fluid found in the ventral rumen. One additional potentially confounding factor in this study was the use of different extraction methods for fluid and solid communities (pack and mucosa-associated), which has been demonstrated to influence 16S rRNA amplicon sequencing and subsequent community composition results in ruminal communities  can be found below: The animal study was reviewed and approved by Colorado State University's (CSU) Institutional Animal Care and Use Committee.KB, PM, TE, JM, RD, MW, and AR participated in and provided oversight of all aspects of the study including securing funding, design, laboratory procedures, data analysis, and report preparation. CW participated in study conduct and laboratory analyses. LP performed the data analysis and wrote the manuscript. All authors read, edited, and approved the final manuscript.This study received funding from MicroBios Inc. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Here, we report a computational investigation of the effect of substituents  on the electronic structure of 9H-adenine and its quartets . DFT calculations were used to show the relationships between the electronic nature of the substituents, strength of H-bonds in the quartets, and aromaticity of five- and six-membered rings of adenine. We demonstrated how the remote substituent X modifies the proton-donating properties of the NH2 group involved in the H-bonds within quartets and how the position of the substituent and its electronic nature affect the stability of the quartets. We also showed the possible changes in electronic properties of the substituent and aromaticity of adenine rings caused by tetramer formation. The results indicate that the observed relationships depend on the A4 type. Moreover, the same substituent can both strengthen and weaken intermolecular interactions, depending on the substitution position.Adenine, one of the components of DNA/RNA helices, has the ability to form self-organizing structures with cyclic hydrogen bonds (A 4-N3 and A4-N7 quartets are more responsive to the electronic nature of substituents than A4-N1.Substituent effects on hydrogen bonds in adenine quartets and aromaticity of adenine rings depend on the quartet type. A Adenine (6-amino substituted purine derivative), one of the building blocks of DNA/RNA helices, can participate in intermolecular interactions which may lead to substantial changes in its electronic structure and, in consequence, to changes in its chemical/physicochemical/biochemical properties. In this perspective, the knowledge of the changes in the electronic structure of adenine caused by well-defined factors, such as introduction of substituents with specific electron donating/accepting properties, is of fundamental importance. It should be stressed that the effects of the intermolecular interactions  on the electronic structure and molecular properties of nucleic acid bases have long been a subject of wide and intensive studies.2 However, the effects of substituents on the properties of nucleic acid bases are much less represented in the literature. It was reported that in the synthesis of deoxyribonucleosides, substituents at the C8 position of purine can greatly affect the rate of deoxyribosyl transfer to the base and the nature of the nucleoside formation.3 An electron-donating substituent directs deoxyribosyl transfer to N9 of the base, while an electron-withdrawing substituent leads to the N3-product. Halogen substituents at the C8 position of adenine do not change the tautomeric preference in the gas phase but induce significant changes in vibrational frequencies and Raman intensities.4 Substituent effects on hydrogen bonding were most frequently studied in structurally-modified Watson–Crick base pairs (adenine–thymine and guanine–cytosine).5–11Purine derivatives play a significant role in medicinal chemistry and they are also important intermediates for the synthesis of biologically-active nucleoside analogues, and frequently exhibit antiviral or antitumor properties.12,13 which occur naturally in telomeres,14 immunoglobulin gene switch regions,15 and certain gene promoters.16 The guanine quartet is the most well-known for a long time, for reviews see 4) have been studied experimentally19–22 and have also been the subject of computational investigations,23–26 the effect of remote substituents on hydrogen bonds in adenine tetramers has not yet been studied. Introduction of additional functionalities to the adenine with subsequent formation of quartet with intermolecular adenine⋯adenine hydrogen-bond interactions is one of the design strategies to enrich chemistry of purine derivatives with ability to bind many metal ions.27 Since the stabilization of such complexes depends on the functional groups, the simulation of adenine quartets with certain substituents of different electronic nature is a good step toward design of synthetic functional materials. Moreover, changes in aromaticity of the adenine rings in the tetramer could be used to fine-tune π–π stacking interactions.One of the interesting features of DNA/RNA bases is their ability to form self-assembled structures (clusters). Among them, the most commonly studied are cyclic hydrogen-bonded quartets (tetramers),2, Cl, F, H, Me, NH2) on electronic structure of 9H-adenine and its quartets  with dispersion-corrected Density Functional Theory. We have demonstrated how the position of the substituent and its electronic nature affect the stability of the quartets. Additionally, we have analyzed the influence of the electronic nature of the substituents on the strength of H-bonds in the quartets and aromaticity of five- and six-membered rings of adenine.In this work, we have investigated computationally the effect of substituents , were inserted into each adenine molecule at the same position within a certain quartet. For A4-N1 and A4-N3 tetramers, it was done either at the C8 or N9 position, whereas in the case of A4-N7 tetramer they were introduced either at the C2 or N9 position, resulting with 33 systems to be analysed. Although N9 substitution is less common, some organic ligands with functional groups can be incorporated into the adenine molecule at the N9 position.27Selected substituents, varying in their electronic properties  in the tetramer geometry.Interaction energy, Eprep, was calculated as the difference between energies of adenine monomers in the tetramer geometry and adenine monomers in equilibrium geometry.Preparation energy, 38 Such parameters as the electron density in bond critical points (BCPs), ρBCP, its Laplacian, ∇2ρBCP, density of the total electron energy in BCP, HBCP, and its two components, potential and kinetic electron energy densities, VBCP and GBCP, respectively, were taken into account.Analysis of intermolecular interactions within the framework of Bader's Quantum Theory of Atoms in Molecules (QTAIM) was performed using the AIMAll program package.39,40 The cSAR(X) value was calculated as the sum of Voronoi charges41 of the atoms of substituent X and ipso C- or N-atom of adenine.Substituent effect was described using cSAR (charge of the Substituent Active Region) concept.para-substituted nitrobenzene derivatives42 and studies of adenine systems substituted in C-X and N-X positions.43The choice of VDD charges is based on our previous research into the electronic structure of the nitro group in 44,45 which is defined as:n is the number of bonds taken into the summation; αi is a normalization constant  fixed to give HOMA = 0 for a model non-aromatic system and HOMA = 1 for the system, in which all bonds are equal to the optimal value Riopt, assumed to be realized for fully aromatic systems  and Ri denotes bond lengths taken into calculation.Electron delocalization was characterized using geometry-based aromaticity index HOMA (Harmonic Oscillator Model of Aromaticity),46 The obtained values of the bonding energies and all substituent effect descriptors are gathered in Tables S1–S4.The local HOMA index was calculated for both five- and six-membered adenine rings. This aromaticity descriptor was previously successfully used to describe the aromaticity changes in adenine rings due to H-bonding and complexation of metal ions.4-N3 and A4-N7 structures with substituents resemble flat geometry in symmetry close to C4h, while the substituted A4-N1 quartets are more distorted and adopt a similar pattern to that described by the S4 point group. The unconstrained optimized geometries of systems with different substituents do not change significantly within one quartet type  and A4-N7 (dN⋯N = 2.93 Å) are close to the experimental values, 3.05 and 2.91 Å for A4-N3 and A4-N7, respectively.20,29 The three quartets have similar stabilities. Our calculations show that unsubstituted A4-N3 tetramer is the most stable followed by A4-N7 at only 0.4 kcal mol−1 and A4-N1 at 2.7 kcal mol−1  have the shortest hydrogen bonds (dN⋯H < 1.9 Å). According to the substitution position, C2/C8-substituted tetramers are considerably more stable than the N9-substituted ones.The optimized Ae Fig. S1. The obt Table S1. However47,48 characterizes how a substituent X affects properties of a fixed group Y  in a substituted system X–R–Y (R – transmitting moiety). In our case, properties of the reaction site – the amino group – can be described either by the cSAR(NH2) value or by the strength of H-bonds.Intramolecular interactions, the so-called substituent effect, can be considered from three points of view, as classical and reverse substituent effects, as well as the influence of the substituent on the properties of transmitting moiety. The classical substituent effect, introduced and quantified by Hammett,2) on cSAR(X) shown in R2 > 0.84, except for C2-substituted A4-N7 series, where very narrow range of cSAR(NH2) values was found. In all cases the slope a is negative meaning that electron-donating properties of the amino group decrease with increasing electron-donating ability (more positive cSAR(X) value) of substituent X. According to the slope a value, the C8 position in monomer is the most sensitive to the electronic nature of substituent X. This is consistent with results for adenine based on other substituent effect descriptors.49An illustrative example of the classical substituent effect is the dependence of cSAR(NH2) values are higher in monomers (triangles) than in quartets (circles) where amino group is involved in H-bond. The reason is accumulation of negative charge on the N atom of proton-donating –NH part of the amino group during formation of hydrogen bond. As can be seen in 2 group is less affected by the tetramer formation in A4-N3 quartets than in the others, because the formed H-bonds in A4-N3 are the longest among all quartets, dN⋯H > 2.0 Å. This observation was also confirmed by direct comparison of cSAR(NH2) of NH2 attached to C6 in monomers and tetramers, see Fig. S2 and Table S5.4-N3, cSAR(NH2) values of monomers and tetramers are comparable, while for A4-N1 and A4-N7, cSAR(NH2) in tetramers are significantly smaller than in monomers. Moreover, A4-N3 quartet is the most responsive to the electronic nature of substituent X either at C8 or N9 position.As expected, cSAR value. More positive cSAR values indicate better electron-donating properties of substituents. According to As a consequence of the substituent effect on the properties of the amino group, H-bond energy in quartets also changes. 4-N3 quartet. The second group is represented by A4-N1 and A4-N7 quartets with the N9-substituents. In the case of A4-N3 and A4-N7 tetramers (R2 > 0.92), the substituents notably change the energy of the intermolecular interactions. The largest changes were found in the N9-substituted A4-N3 system. This is consistent with observation of substituent effect X on properties of the NH2 group  and R2 of these regressions are less than 0.25. Such low coefficients of determination  with positive slope, where H-bond weakens with increase in electron-donating properties of substituent X, and (ii) with negative slope, where H-bond strengthens with the same substituent change. The first group consists of all adenine quartets with the substituents at the carbon atom (either C2 or C8) and N9-substituted AH2 group . On the mination  can be e4-N3 (at the C8 and N9 position) and A4-N7 (at the C2 position) quartets. The electron-donating substituents make H-bonds stronger only in A4-N7 quartet if they are located at the N9 position. Introducing different substituents into A4-N1 quartet has relatively small effect.Summarizing, the strong electron-accepting substituents contribute to strengthening of H-bonds in AdN⋯H, shown in 4-N3 quartets are longer (dN⋯H > 2.0 Å) than in the A4-N7 ones (dN⋯H < 1.9 Å), the bonding energies in A4-N3 tetramers are comparable in most cases with the energies in A4-N7 systems or even stronger. It is worth mentioning that the two systems with the strongest and the weakest H-bonds have a common element, namely the nitro group as a substituent. These terminal cases were found in the A4-N3 and A4-N7 tetramers with the nitro substituent at the N9 position , but also by the presence of additional intermolecular interactions.Some interesting observation was done when considering the relationships between strength of intermolecular interactions and length of the hydrogen bonds, position . Their e4-N3 quartets (2 group and NH2 group (O⋯HN). To characterize these extra contacts quantitatively we performed QTAIM analysis for 3 systems: unsubstituted A4-N3, and N9-NO2-substituted A4-N3 and A4-N7. Particular attention was paid to CH⋯N interactions in A4-N3 quartets. The distance dH⋯N is 2.33 Å in A4-N3, and 2.27 Å in its N9-NO2-substituted analogue, whereas such dH⋯N in adenine–thymine pair is 2.75 Å. The parameters of the corresponding BCPs are collected in 2 and NH2 groups are very week, the CH⋯N contacts in A4-N3 quartets can be considered as weak H-bonds,50 and therefore additionally contribute to their stability.Upon a detailed examination of their structures we found two additional close contacts in Aquartets . One of 42 which describes how the electronic properties of substituents X depend on the properties of the moiety R–Y to which they are attached. Various substituent constant scales51–53 “represent” a fixed electronic structure of substituents or, in other words, its fixed electron-donating/attracting power. In turn, cSAR(X) value characterizes not the fixed electronic properties of substituent X but shows its properties depending on the substituted moiety. The reverse substituent effect can be illustrated by changes in cSAR(X) values for a particular substituent X when it is considered in different adenine quartets  changes the most; these changes correspond to approximately 13 and 20% of the total variation of the cSAR(X) values in the series, respectively.The cSAR concept allows us to study the reverse substituent effect,s Fig. S3. The ranR2 > 0.99 and slopes close to 1.0 indicate a large similarity of interactions between X and substituted moieties in both cases. For N9-substitution in A4-N3 systems, the slope a is slightly larger than in other series. This means that in this case the substituent X is more responsive to tetramer formation most probably due to additional spatial interactions with the amino group of neighboring adenine molecule (2). Only in N9-substituted A4-N7 tetramer, cSAR(X) values become more positive than in monomer due to stronger withdrawal of electronic density to the H-bonding area. Importantly, the H-bond formation may decrease the electron-attracting ability of the nitro group by ∼60%.The effect of tetramer formation on the electronic properties of X can be demonstrated by comparing cSAR(X) in the quartet with the values in the adenine monomer  and is slightly responsive only to the substituents at the C8 position, whereas C2- and N9-substitution does not significantly affect them. Increase in the aromaticity of the five-membered ring by substitution, in general, goes with a reduction of the aromaticity of the six-membered ring. Aromaticity changes by N9-substitution in the five-membered ring translate into small variation in the six-membered ring. Moreover, minor aromaticity changes by C2-substitution are in line with previous studies indicating a high resistance of the π-electron structure to the substituent effect.55 According to the slopes a, the highest HOMA sensitivity to the substituent effect was observed in A4-N3 quartets, most likely due to multiple noncovalent interactions between adenine molecules. Different slopes observed for five- and six-membered rings may depend on the number of bonds between the substituent X and the “reaction site” (NH2). A similar observation is well known for meta and para substituted benzene derivatives, where the substituent effect depends on the number of bonds between X and Y.56The substituent effect on aromaticity of the transmitting moiety can be demonstrated by changes in HOMA index for five- and six-membered rings in the substituted quartets, as presented in Fig. S5 and Table S7.4-N7 quartet become more aromatic than in monomer because their N7 atom participates in the H-bond as a proton-acceptor. Great correlations between HOMA values of five-membered rings for substituted monomers and tetramers  or less sensitive (a < 1.0) to the nature of substituent X in comparison with monomer.Comparison of HOMA values for tetramers with values for monomers of substituted adenine Table S3 clearly 2 group and thus the strength of hydrogen bonds in adenine quartets. According to the findings, to increase the stability of the A4-N3 quartet, it is recommended to introduce a strong electron-accepting substituent into each adenine molecule at the C8 or N9 positions. On the other hand, to stabilize A4-N7 quartet it is necessary to substitute C2 position with electron-accepting substituent or N9 position with electron-donating substituent. Substituent effect in the A4-N1 quartet is not pronounced and cannot be considered as a tool for tuning stability of the quartet.The substituent X, depending on the position, modifies the proton-donating properties of the NH4 type. For C-substituted systems, the largest change in cSAR(X) values is observed for the NH2 group, while for the N-substituted ones the cSAR(NO2) changes the most. Aromaticity of both adenine rings in A4-N3 tetramer is more sensitive to electronic nature of the substituents than in other tetramers. Its “special properties” are most likely due to the fact that additional weak hydrogen bonds occur here as documented by the results of QTAIM analysis. In general, to increase aromaticity of the five-membered rings the electron-accepting substituents should be introduced at the C2 or C8 positions, or the electron-donating substituent at the N9 position. In turn, six-membered rings are highly aromatic (HOMA > 0.9) and slightly responsive to the substituents at the C8 position, whereas C2- and N9-substitution does not affect them.The obtained results show that the electronic properties of the substituent X and aromaticity of five- and six-membered rings are affected by the tetramer formation and are slightly different depending on the AThere are no conflicts to declare.RA-010-D0RA04585C-s001


TERT, TERC, PARN, RTEL1), which are also found in about 10–20% of cases of sporadic IPF. In addition to the Mendelian forms, mutations in the genes encoding for the surfactant proteins  and polymorphisms of genes for the mucin MUC5B and the Toll-interacting protein TOLLIP are other pathways favoring the fibrogenesis that have been thoroughly explored. Moreover, great attention has been paid to the main epigenetic alterations  that are emerging to play a role in fibrogenesis. Finally, a gaze on the shared mechanisms between cancer and fibrogenesis, and future perspectives on the genetics of pulmonary fibrosis have been analyzed.Idiopathic pulmonary fibrosis (IPF) is a rare disease of the lung with a largely unknown etiology and a poor prognosis. Intriguingly, forms of familial pulmonary fibrosis (FPF) have long been known and linked to specific genetic mutations. There is little evidence of the possible role of genetics in the etiology of sporadic IPF. We carried out a non-systematic, narrative literature review aimed at describing the main known genetic and epigenetic mechanisms that are involved in the pathogenesis and prognosis of IPF and FPF. In this review, we highlighted the mutations in classical genes associated with FPF, including those encoding for telomerases ( Idiopathic pulmonary fibrosis (IPF) is a rare, chronic, fibrosing and progressive disease limited to the lung with a largely unknown etiology and a poor prognosis. IPF is associated with a characteristically defined radiological and/or histological usual interstitial pneumonia (UIP) pattern. IPF is characterized by the progressive replacement of lung tissue with fibrotic-scar tissue due to the excessive release of collagen by mesenchymal cells, the myofibroblasts. This process changes the architecture and function of the organ over time and, in the absence of antifibrotic therapy, may lead to death within 3–5 years after diagnosis. IPF is a disease of the elderly, with a mean age of onset of 65 years .IPF is characterized by the presence of progressively worsening dyspnea. Cough and sputum are frequently associated symptoms, especially in more severe patients.According to international guidelines, the diagnosis requires the exclusion of secondary causes of pulmonary fibrosis  and the execution of a high-resolution computed tomography (HRCT) of the chest. Finding a definite usual interstitial pneumonia (UIP) radiological pattern is sufficient to diagnose IPF after the exclusion of any other causes of lung disease. Patients who have a probable UIP pattern on HRCT can receive a diagnosis of IPF after a multidisciplinary discussion (MDD) by at least a pulmonologist, a radiologist and a pathologist. Lung biopsy is today adopted only in limited complex cases, particularly when an IPF diagnosis is strongly suspected but the radiological pattern is indeterminate for UIP or suggestive for other interstitial lung diseases.Bronchoalveolar lavage (BAL) is a bronchoscopic sampling procedure useful in the diagnostic work-up of idiopathic pulmonary fibrosis that helps rule out other fibrosing interstitial diseases that can enter into differential diagnosis with IPF. Although there are no specific cellular patterns for IPF, BAL could help in the diagnosis of other interstitial lung diseases (ILD) such as sarcoidosis, eosinophilic pneumonia, chronic extrinsic allergic alveolitis or nonspecific interstitial pneumonitis ,3.To date, no pharmacological therapy can stop or reverse the fibrosing process. Two drugs, nintedanib and pirfenidone, have been approved for the treatment of the disease, since in clinical trials and observational studies they have been shown to slow down the progression of the disease, without, however, being able to stop or cure it ,5. SeverDespite several advances in understanding the pathogenetic mechanisms of the disease, the origin of IPF remains largely unknown. The main risk factors include cigarette smoking, lung infections , exposure to pneumotoxic inhalants, gastroesophageal reflux, elderly age, diabetes mellitus and genetic predisposition .Intriguingly, forms of familial pulmonary fibrosis (FPF) have long been known and linked to mutations in specific genes . FPF is TERT, TERC, PARN, RTEL1) [TERC and TERT mutations, respectively). The involvement of bone marrow function in the pulmonary fibrosis (PF) phenotype is also reported for patients with ZCCHC8 and RPA1 variants. The familiar forms of PF include ILD2 (OMIM #178500), a disease related to SFTPA2 mutations and characterized by pulmonary fibrosis, interstitial pneumonia and lung cancer.Genes associated with FPF include those encoding for telomerases, responsible for the regulation of telomeres length and therefore involved in cellular aging  ,9,10. TwFinally, pulmonary fibrosis can also be part of the POIKTMP syndrome . FPF can manifest either as IPF, but also as connective tissue disease-interstitial lung disease (CTD-ILD) or unclassifiable interstitial lung disease (U-ILD) ,11.On the other hand, there is little evidence of the possible role of genetics in the etiology of sporadic IPF, although mutations in FPF-causing genes are also found in about 10–20% of sporadic IPF cases.Even if the genetic predisposition to PF is known, the molecular diagnosis of familial cases is probably underestimated, and this could be related to the incomplete penetrance of the disease or the lack of information from the patient’s pedigree. In order to recognize the familial cases, it is important to identify the risk in family members not only for PF but also for possible related conditions, mainly lung cancer and bone marrow failures. In addition to the Mendelian forms, several studies support the existence of a genetic predisposition to pulmonary fibrosis, which is expressed as a complex trait in which low-penetrance genes and environmental factors play a role. In particular, the involvement, even in sporadic forms, of genes related to the production of surfactant proteins, which would alter the functionality of the pulmonary alveoli, or of genes responsible for an increased chronic secretion of mucus, is highlighted .Finally, epigenetic alterations seem to play a role in fibrogenesis. Several studies consider cellular aging as an endogenous causative factor of IPF, which favors the development of genomic instability, the establishment of epigenetic alterations, the shortening of telomeres and cellular senescence, thus laying the foundations for the development of fibrosis .Overall, heterogeneous genetic variants could contribute to the development of an altered broncho-pulmonary tissue, which would become more susceptible to recurrent micro-lesions from various potential environmental factors.In this study, a state-of-the-art review of the genetic and epigenetic bases already explored in IPF and FPF will be presented, along with an overview of their possible implications for therapy. For this purpose, a non-systematic, narrative literature review was conducted. The search engine PubMed was adopted to retrieve the most relevant articles on these topics from international scientific journals.PF can have a strong genetic component in 10–20% of cases, as previously described, or gene variants can modify the risk so that it is affected together with the influence of the environment. In addition, genetic factors might also influence the onset of the disease  Figure .TERT, TERC, PARN and RTEL1, which regulate the telomere length, thus resulting in shortened telomeres, have a recognized causative role in IPF and FPF [Particularly, mutations in the genes of the telomerases, such as  and FPF .SFTPC, SFTPA1, SFTPA2, ABCA3), or polymorphisms in MUC5B, causing overexpression of the mucin 5B, or in TOLLIP (an inhibitor of the Toll-like receptors 2 and 4) together to mitochondrial dysfunction are other genetic mechanisms associated with an increased risk of IPF [A growing number of genetic factors predisposing to pulmonary fibrosis have been explored, also thanks to the possibility of conducting genome-wide association studies (GWAS). Particularly, sequence variants in genes for the surfactant proteins causing surfactant dysfunction , which normally modulate and stabilize the alveolar surfactant tension, can induce an increased stress in the endoplasmic reticulum, which finally leads to epithelial-mesenchymal transitions and apoptosis of type 2 alveolar cells.All the reported causative genes for PF show an autosomal dominant pattern of transmission, leading to a 50% chance of transmitting the pathogenetic variant to the offspring. However, the expression of the phenotype is influenced not only by incomplete penetrance, intrafamilial clinical variability and environmental factors but also by the pathomechanism related to the specific gene involved. For example, for genes involved in telomere maintenance, it is reported that the phenomenon of anticipation, is possibly favored by epigenetic mechanisms triggering telomere shortening .MUC5B, a regulatory mucin, are responsible for mucociliary disfunction with impaired clearance and enhanced mucus production, which predisposes to bacterial overgrowth and infections.Moreover, polymorphisms of TERT) and telomerase RNA component (TERC). Other accessory components act to stabilize TERC, such as dyskerin, NHP2, NOP10 and GAR1 [Telomere dysfunction is one of the main drivers of IPF, as many sequence variants in telomere genes have been described in IPF ,37,38,39and GAR1 . The lenand GAR1 ,44.Among the Mendelian telomere disorders, PF is the most common in the adult age, indeed numerous mutations in telomere-related genes have been described in association with IPF ,39,40,41TERT and TERC genes, these mutations can be found also in 2% of sporadic IPF. TERT and TERC mutations are mainly heterozygous (related to an inherited or de novo mutation), thus showing a dominant transmission. It has been hypothesized [PARN or RTEL1, should be considered as risk factors for the development of earlier IPF onset and more rapid progression and thus the risk of death.Whilst 15% of FPF present mutations in thesized ,27 that TERC KO mice), it was hypothesized that adult stem cells of the lung, the alveolar epithelial type 2 cells (AEC type 2), are the most affected by shortening of the telomeres [Since the decreased ability to regenerate damaged or injured lung tissue was experimentally demonstrated in mice models of IPF with TERC mutations , has been described as the strongest risk factor to predispose to IPF both in sporadic and familial cases [MUC5B, which is indeed associated with the failure of the reparative and regenerative mechanisms of the alveoli, mucociliary dysfunction and pollutants retention, which are the basis for the development of honeycombing cysts and fibroproliferative degeneration [dy clear . Particual cases ,54. Thisneration ,56. Exceneration .The frequency of the T allele in the general population and in cohorts of IPF patients has been thoroughly investigated in several studies across the world. A genome-wide association study (GWAS) has found that in the Caucasian population 41.9% of IPF patients and 10.8% of the healthy control group are carriers of the T allele of the rs35705950 . A recenThe Toll-interacting protein TOLLIP acts as an inhibitor of the Toll-like receptors (TLRs) 2 and 4, which are active in the lung. As a consequence, TOLLIP contributes to the suppression of tumor necrosis factor-α (TNF-α) and IL-6 production . SeveralTOLLIP is mainly expressed in the lung by alveolar type II cells, macrophages and basal cells, where it can prevent damage from reactive oxygen species (ROS) induced by mitochondrial damage. Moreover, it can protect bronchial cells and modulate inflammation and autoimmunity via different pathways  ,66.Consistently, IPF outcome might be influenced by SNPs in the TOLLIP gene. Bonella et al. recently found that IPF patients carrying the TOLLIP rs5743890 minor allele had a worse prognosis, with a median reduction in survival of 20 months compared to IPF patients without this mutation . MoreoveAEC type 2 are responsible for the production of the surfactant. Particularly, the surfactant is composed of phospholipids and the following four surfactant proteins: SP-A, SP-B, SP-C and SP-D. Surfactant proteins are synthesized in the endoplasmic reticulum (ER) of AEC type 2, from which they are transported and subsequently stored in the lamellar bodies. Finally, these proteins are secreted in the alveolar space ,69.SFTPA1, SFTPA2, SFTPC and ABCA3—ATP binding cassette subfamily A member 3) are associated with FPF [In the literature, it has been described that several mutations of the genes encoding these proteins  are associated with ER stress and an increased risk of developing lung cancer in smokers [SFTPC gene (the surfactant protein C) lead to a misfolded SP-C, which thus accumulates in the ER, causing ER stress. Particularly, the BRICHOS domain, critical for the SP-C proper folding, is usually the most affected by SFTPC mutations [In detail, variants in the  smokers ; those iutations ,75.SFTPA2 and SFTPC are dominant, ABCA3 mutations are causative of a recessive form of the disease [Finally, rare variants in the gene encoding for the protein ABCA3 have been described as a risk factor for pulmonary fibrosis. ABCA3 is a protein, which in AEC type 2 acts as a transporter protein for lipids through the plasma membranes ,77. When disease .The possible mechanism at the basis of fibrogenesis in patients carrying mutations in the genes encoding for surfactant proteins might rely on the activation of epithelial-to-mesenchymal transition (EMT) induced by ER stress. Indeed, ER stress is able to activate epithelial cell differentiation in mesenchymal cells and AEC type 2 apoptosis. This causes the production and deposition of collagen and the subsequent fibrosis in an alveolar environment, which is poor for functional surfactant proteins ,81,82.Besides genetics and genomics, which help characterize the molecular landscape of IPF and FPF, a major role is also played by epigenetics. Several studies showed indeed that multiple genes are differentially expressed in IPF lung, mainly involved in pathways such as TGF-β signaling, epithelial to mesenchymal transitions, fibroblast proliferation ,84,85. GAll the biological features characterizing PF could be explained by gene expression dysregulation related to epigenetic modifications. Considering the dynamic nature of epigenetic modifications, they represent an attractive druggable target, since epigenetic marks can be reverted by specific treatments, such as histone deacetylases inhibitors (HDACi) .Moreover, distinct episignatures are, indeed, emerging to be disease-specific and the epigenetic profile may be exploited to support the clinical diagnosis. The identification of methylation changes associated with disease development is particularly important for pathologies strongly associated with environmental exposure, as in the case of PF. Epidemiological studies have, indeed, demonstrated the association between exposure to inhaled environmental agents and IPF development, including cigarette smoke, wood dust, metal dust, silica, textile dust and possibly agriculture, farming and livestock ,88. CigaHuman DNA is enriched in CG dinucleotides, especially in the regulatory regions such as gene promoters. These CpG sites can undergo the addition of a methyl group to the cytosine residue. This methylation process is regulated by specific enzymes, essentially DNA methyltransferases (DNMTs), and the presence of methylated CpGs blocks the binding of the RNA polymerase complex to the promoter region and thus silences gene expression. DNA methylation is an epigenetic mark that triggers conformational chromatin changes leading to different expression levels of genes; the general rule is that hypomethylation in gene promoters should result in increased expression of the related genes, while hypermethylation causes a decrease in expression levels. This rule is not always valid if methylation is located in other regulatory genome regions, such as enhancers .MUC5B, PTGER2 (Prostaglandin E receptor 2) and THY1 (Thy-1 antigen).Several genome-wide methylation studies reported multiple differentially methylated regions (DMRs) between IPF lung and healthy controls . The majMUC5B and the rs35705950 risk allele, Helling and colleagues [MUC5B enhancer region encompassing the rs35705950 variant. This enhancer element dynamically binds the transcription factor FOXA2 as well as RNA polymerase II, suggesting its important role in controlling MUC5B expression. Indeed, the authors indicated that increased methylation is associated with increased expression of MUC5B, IPF and the risk allele T at rs35705950.Regarding lleagues  identifi2 (PGE2) pathways are essential to maintain the correct homeostasis of fibroblasts, guaranteeing the appropriate apoptosis. The lungs of IPF patients are substantially resistant to PGE2, which is conditioned by reduced expression of its receptor, E prostanoid 2 (EP2), due to DNA hypermethylation of its promoter, PTGER2 [2 mRNA transcription and thus the PGE2 responsiveness in fibrotic fibroblasts but not in nonfibrotic controls.Prostaglandin E, PTGER2 . The autTHY1 encodes for the thymocyte differentiation antigen 1 (Thy-1), a cell outer membrane glycoprotein also known as CD90. Studies in human and murine lung specimens demonstrated that the absence of Thy-1 promotes myofibroblast differentiation and that this is due to hypermethylation of the THY1 promoter [THY1 seems to be related to hypoxia [Fibroblastic foci typical of IPF are characterized by abnormal myofibroblasts, in which gene expression is completely altered, especially for several “IPF suppressor genes” ,95,96,97promoter ,98. The  hypoxia , and the hypoxia ,101.These studies suggest that alteration of DNA methylation at genomic loci closely related to the pathogenesis of IPF is a crucial factor in the development of IPF and that DNMT-targeted therapies could be a resource for remodeling this process, although further studies on their specificity and adverse effects are needed. Little is known about the role of epigenetic modifications in the development of FPF; nevertheless, environmental exposure can induce disease-specific methylation changes associated with increased risk of disease development, also in familial cases, thus suggesting a role for epigenetic changes, in addition to the genetic predisposition, in FPF .In addition to DNA methylation, remodeling of the chromatin state is also regulated by post-translational modifications of histones that change the conformation of the chromatin complex, leading to different levels of condensation and thus affecting the gene expression of related loci. Histones can be modified through different mechanisms, among which the two most common are methylation and acetylation. Both types of modifications are dynamically regulated by different classes of enzymes responsible for the addition or removal of methyl or acetyl groups from specific histone residues. Histone methylation is controlled by the following two sets of enzymes: histone methyltransferases (HMTs) and histone demethylases (HDMs), while histone acetylation is the product of the dynamic interplay between histone acetyltransferase (HATs) and histone deacetylase (HDAC).Among the enzyme alterations responsible for histone modifications in the pathogenesis of IPF are increased activity and expression of EP300. EP300 encodes for a histone acetyltransferase involved in cell proliferation and differentiation. Rubio and colleagues  demonstrAberrant HDAC activities are also involved in the epigenetic silencing of Thy-1, an already mentioned “fibrotic suppressor surface glycoprotein”. Indeed Thy-1 silencing is regulated not only by promoter hypermethylation but also by histone modifications in lung fibroblasts  and treaThese studies highlight the close interaction between DNA methylation and histone modification and how treatments against the enzymes responsible for regulating these epigenetic mechanisms could be useful in the treatment of IPF.Other emerging pathways involved in the development of the lung, and which seem to be epigenetically influenced in IPF patients are the Hedgehog pathway GLI1 and the WNT pathway of beta-catenin .Genes expression can also be controlled by microRNAs (miRNA), non-coding RNAs composed of 17–25 nucleotides. They interact with the 3′ untranslated region of mRNAs mediating mRNA degradation and gene silencing.Several miRNAs expression profile studies showed that alteration in the expression of specific miRNAs affects fibroproliferation, epithelial-mesenchymal transition and the TGF-β1 signaling pathway ,108,109.Intriguingly, miR-21 plays a key role in IPF. Indeed, miR-21 can stimulate TGF-β thus upregulating the EMT and impairing earlier the forced vital capacity . MoreoveEmerging studies are focusing on the role of long non-coding RNAs (lnc-RNAs) in fibrogenesis, such as an antifibrotic role for FENDRR  and a prAs for methylation changes, studies on ncRNAs have been mainly focused on IPF. However, the role of these RNAs may be played also in FPF, as an additional mechanism influencing the onset and the severity of the condition associated with exposome .Some genes associated with the onset and progression of IPF might support clonal selection and favor cancer transformation.TGF-β1, interleukin-1 receptor alpha (IL1RN), interleukin 8 (IL-8), Toll-like receptor 3 (TLR3), telomerases, tumor protein 53 (TP53), are also associated with cancer. While TP53 can act as an oncogenic driver, the others can sustain neoplastic transformation but cannot induce it. The TP53 gene was found to be significantly mutated in cases of lung cancer associated with IPF. Mutations in the JAK-STAT signaling pathway were also significantly amplified in IPF-associated lung cancer [Interestingly, genes supporting the inflammation and fibrogenesis in IPF, such as g cancer .As already described, mutations in the telomerase genes are one of the main genetic risk factors for pulmonary fibrosis, but the angiogenic and pro-invasive properties they confer could be adopted by yet-transformed malignant cells . MoreoveTTF-1 is mainly expressed in APC type 2, and it regulates the expression of surfactant proteins in the lungs, which are fundamental for the homeostasis of the alveolar milieu [TTF-1 is an oncogene, which might contribute to cell proliferation in cancer and fibrosis since its expression is enhanced in injured lungs [Intriguingly, the thyroid transcription factor-1 (TTF-1), a highly specific biomarker of primary adenocarcinoma of the lung, seems to be implicated also in the pathogenesis of IPF. r milieu . TTF-1 ied lungs .SFTPA1 and SFTPA2, indeed, predispose to IPF through ER stress and consequent alterations in APC type 2, whilst pathogenetic variants of SFTPA2 are responsible for ILD2, an autosomal dominant condition characterized by a strong genetic predisposition to lung cancer. By promoting cell proliferation and fibrogenesis, these mutations can also induce neoplastic transformation [SFTPC [ABCA3, which is well known for its profibrotic action when mutated, is also implicated in the onset and progression of lung cancer and acts as a mediator of chemoresistance in lung neoplasm [A link between IPF and lung cancer secondary to mutations in the genes of the surfactant proteins has begun to be investigated. Polymorphisms of ormation . Similarn [SFTPC . ABCA3, neoplasm .Even if the mechanisms of their role in lung cancer and pulmonary fibrosis are still not well defined, the peroxisome proliferator-activated receptors (PPARs) nuclear receptors seem to play a part, since their deregulation has been described in several inflammatory conditions, such as cancer and atherosclerosis, but also in IPF. PPARs are involved in the regulation of the metabolism of lipids and in cellular proliferation .MMP1), which shows a high rate of genetic mutations in both of these two pathologies. MMP1 is well represented in neoplasms, promoting the spreading of metastases and their invasive potential, and enhancing the EMT in pulmonary fibrosis, angiogenesis and cell migration. Consistently, high levels of MMP1 have been found in the early phases of lung cancer developed in the context of pulmonary fibrosis [Another link between IPF and lung cancer resides in the matrix metalloproteinase 1  and lung cancer, ranging from mutations and polymorphisms of typical genes involved in the fibrogenesis to alterations in oncogenes with a novel potential profibrotic action. Intriguingly, nintedanib, a tyrosine kinase inhibitor currently approved for the treatment of idiopathic pulmonary fibrosis, has been inherited from oncology, underlying again the common background of pulmonary fibrosis and cancer.A recent study based on next-generation sequencing (NGS) and bioinformatics described some genetic and epigenetic pathways that can be influenced by nintedanib, an antifibrotic therapy .Nintedanib is a tyrosine kinase inhibitor, which exerts its antifibrotic effects by targeting and interfering with fibroblast-derived growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR) and potentially inhibiting the transforming growth factor β (TGF-β) signals to downregulating the ECM . The autE2F1, NPTX1, DDX11, PLXNA4 (downregulated) and SLC25A23 (upregulated).Notably, after nintedanib treatment, the authors identified four downregulated and one upregulated genes, which act on the following miRNA-mRNA interactions: E2F1 is a critical transcription factor in the cell cycle and apoptosis, particularly because it is required for the S phase of the cell cycle. By increasing the levels of E2F1, the proliferating and profibrotic effects of VEGF, epidermal growth factor (EGF) and keratinocyte growth factor (KGF) can be exerted. Nintedanib manages to reduce the levels of E2F1 in the treated fibroblasts, and this might lead to slow fibrogenesis in IPF.NPTX1 belongs to the family of neuronal pentraxin , thus itDDX11 promotes cell proliferation and metastasis in tumoral cells, whilst it might induce the proliferation of fibroblasts in IPF. Nintedanib manages to suppress the DDX11 signaling, thus inhibiting fibroblast’s proliferation.PLXNA4 is also downregulated by nintedanib treatment. Since its action is to promote the vascularization of the tumors, induced by VEGF, its downregulation can decrease the angiogenesis in IPF.Finally, SLC25A23, which, in contrast, is upregulated by nintedanib, induces cell death through oxidative stress. It has been hypothesized that upregulating SLC25A23, nintedanib can reduce fibroblast proliferation, induce their apoptosis and decrease angiogenesis in IPF.Recently, another interesting study focused on the efficacy and safety of nintedanib and pirfenidone in a group of patients with pulmonary fibrosis carrying mutations in telomerases genes. In this work, authors found that both antifibrotic treatments were associated with a reduced decline of FVC, without observing unexpected adverse events .Moreover, another study demonstrated that also pirfenidone can slow the rate of progression of EMT modulating several gene-induced pro-fibrotic pathways . PirfeniSince available drugs are not able to cure IPF, several studies have addressed the use of gene therapies as potential approaches to attenuate a wide range of processes involved in fibrosis. To date, despite the potential benefits of gene therapy, no clinical trials for treating IPF have not been launched. Developing novel drugs to treat IPF is, indeed, challenging due to the complex pathogenesis of the disease and the difficulty of modeling the disease in animals. Currently, available animal models are not specific for IPF but only recapitulate some aspects of pulmonary fibrosis artificially induced by different chemicals . Initial studies exploring the potential application of gene therapies to IPF focused on inducing targeted gene overexpression using both nanoparticles and viral vectors ,130. ThiMore recently, the use of gene silencing by siRNA  for treating PF was investigated in several studies that described the efficacy of multiple siRNAs combined with antifibrotic compounds in treating several aspects of PF . Very feAltogether, these studies support the applicability of gene therapy to suppress the progression of fibrosis. However, no gene therapy approach has demonstrated the capability to reverse established fibrosis.Nowadays, the possibility of exploring the genetic and epigenetic basis of pulmonary fibrosis more in depth represents a clinical and scientific challenge that could, in the near future, help in both the diagnosis and the development of targeted therapy for this devastating disease.Hopefully, genetics might soon support the diagnostic algorithm of IPF, integrating data from radiology and pathology at the molecular level. To this end, it is essential to underline the role the genetic counseling in referring to genetic analysis of the population at risk of familial forms , and in the correct interpretation of the considerable amount of data that can be made available by genome-wide array genetic analysis.Reversing the epigenetic changes capable of influencing the genesis and progression of pulmonary fibrosis might be an interesting research perspective, as well as designing trials for targeted therapy at the genetic level.Since IPF and PFF are rare diseases, it is essential that knowledge of the genetic basis of pulmonary fibrosis should be shared with all other healthcare personnel involved in the management of ILD.An increasing role of genetics and epigenetics in the pathogenesis and prognosis of IPF and PFF is emerging. As described above, specific Mendelian mutations in telomere genes are able to determine the development of familial forms of pulmonary fibrosis, with an earlier onset and often a more severe prognosis. Likewise, gene variations capable of altering surfactant proteins are known to be responsible for the fibrosing disease, some of which affect children. Finally, polymorphisms of mucins, i.e., MUC5B or Toll-like receptor inhibitors (TOLLIP), have been associated with a greater risk of developing pulmonary fibrosis.Many of the genes and pathways involved in pulmonary fibrosis development are today known because of their involvement in the Mendelian form and GWAS studies. Beyond the Mendelian forms, many of the same loci and pathways may be affected by variants or epigenetic modifications that can modify the risk of diseases in the presence of environmental triggers.Although the genetic predisposition to pulmonary fibrosis is known, the molecular diagnosis of familial cases is probably still underestimated, and this could be related to the incomplete penetrance of the disease or the lack of information from the patient’s pedigree. In order to recognize the familial cases, it is important to identify the risk in family members not only for pulmonary fibrosis but also for possible related conditions, such as lung cancer and bone marrow failures. In addition, it is emerging that epigenetic mechanisms can affect the development and prognosis of pulmonary fibrosis. DNA methylation of CpG sites, histone post-translational modifications and non-coding RNA gene silencing are mechanisms that should also be explored in fibrogenesis.Interestingly, epigenetic signatures of disease can have a clinical utility as biomarkers or druggable targets since epigenetic marks can be reverted.Of interest, genes supporting the inflammation and fibrogenesis in IPF, such as TGF-β1, interleukin-1 receptor alpha (IL1RN), interleukin 8 (IL-8), Toll-like receptor 3 (TLR3), telomerases, tumor protein 53 (TP53), are also associated with cancer and genome-wide methylation studies, as well as gene expression studies, showed several parallelisms between the pathogenetic mechanisms that drive IPF and lung cancers. Further studies are needed to better elucidate the molecular mechanisms of the relationship between pulmonary fibrosis and lung cancer.Finally, data are emerging on the molecular pathways, at both the genetic and epigenetic levels, which form the basis of the efficacy of the antifibrotic therapies.


Adhesion molecules regulate cell proliferation, migration, survival, neuritogenesis, synapse formation and synaptic plasticity during the nervous system’s development and in the adult. Among such molecules, the neural cell adhesion molecule L1 contributes to these functions during development, and in synapse formation, synaptic plasticity and regeneration after trauma. Proteolytic cleavage of L1 by different proteases is essential for these functions. A proteolytic fragment of 70 kDa (abbreviated L1-70) comprising part of the extracellular domain and the transmembrane and intracellular domains was shown to interact with mitochondrial proteins and is suggested to be involved in mitochondrial functions. To further determine the role of L1-70 in mitochondria, we generated two lines of gene-edited mice expressing full-length L1, but no or only low levels of L1-70. We showed that in the absence of L1-70, mitochondria in cultured cerebellar neurons move more retrogradely and exhibit reduced mitochondrial membrane potential, impaired Complex I activity and lower ATP levels compared to wild-type littermates. Neither neuronal migration, neuronal survival nor neuritogenesis in these mutants were stimulated with a function-triggering L1 antibody or with small agonistic L1 mimetics. These results suggest that L1-70 is important for mitochondrial homeostasis and that its absence contributes to the L1 syndrome phenotypes. Complex neural functions are achieved by the structural and functional interactions of neural activities, which strongly depend on the functional integrity of the mitochondria ,2,3,4. MMitochondria are not only important for the postmitotic and differentiated neurons, but also for neural progenitor cells during neurogenesis . Thus, dL1 in mice), inhibition of the mitochondrial import of L1-70 or prevention of myelin basic protein-mediated proteolytic generation of L1-70 in cell cultures all lead to reduced mitochondrial Complex I activity, impaired mitochondrial membrane potential, fusion, fission and motility, as well as increased retrograde mitochondrial transport [Because of the need to maintain and even improve mitochondrial function, investigations into mitochondrial heath are essential. A molecule that was recently shown to improve mitochondrial function is the cell adhesion molecule L1, a member of the immunoglobulin superfamily ,23. L1 cransport . Furtherransport . Howeverransport ) and genransport ) showingransport  . On this substrate, the neurite outgrowth and survival of neurons from mutant mice were comparable with those from wild-type neurons A,B. NextWe next determined the consequences of the absence of L1-70 for Schwann cell function, since Schwann cells also express L1, and L1 was shown to be involved in Schwann cell axon interactions ,35. The These results distinguish the neurons and Schwann cells from the L1-70-lacking mice from the cells of the L1-201 mutant, which could be stimulated by function-triggering L1 antibody 557 and L1 mimetics, but were impaired under basal conditions .We next analyzed mitochondrial activity and transport in neurons from L1/858-863, L1/687 and L1-201 mutant mice. In L1-70-lacking L1/858-863 and L1/687 neurons, the velocity and mobility of the mitochondria was normal, but the mitochondria moved more retrogradely than the mitochondria from wild-type neurons A–C. The Mitochondrial functions of L1-201 mutant mice—in which the L1-70 fragment is generated—showed impaired neuronal migration, enhanced cell death and a reduced process length of Schwann cells on the PLL substrate but enhanced process length, and enhanced migration and survival after L1 agonist treatment . InteresComplex I activity and ATP levels were normal in L1-201 mitochondria, whereas the membrane potential was higher in L1-201 mitochondria compared with that of wild-type neurons A–C. FurtOur results show that L1/687 and L1/858-863 mice exhibit lower Complex I activity, reduced mitochondrial membrane potential and reduced ATP levels, whereas their mitochondria move more retrogradely and their velocity and mobility are normal. This indicates that the generation of the L1-70 fragment is important for mitochondrial Complex I activity, production of ATP and generation of normal mitochondrial membrane potential, but not for mitochondrial velocity or mobility. A comparison with mitochondria from L1-201 mice showed that these mitochondria also move more retrogradely, suggesting that the absence of L1-70 is not responsible for the retrograde movement of mitochondria from L1/687 and L1/858-863 mutant neurons, but is indicative of stressed or unhealthy mitochondria in these mutants.Fragments of L1 were previously shown to be imported into the cytosol and from there into intracellular organelles such as the mitochondria and the nucleus ,36,37. IThe mitochondria of neurons in which L1-70 is absent and the mitochondria of neurons with the D201N mutation moved more retrogradely than anterogradely, which indicates that the absence of L1-70 is not responsible for the enhanced retrograde transport but other functions of L1 are responsible, such as L1 signal transduction or the interaction of full-length L1 with its binding partners.Previous studies showed that treatment of wild-type cerebellar neurons with the function-triggering antibody 557 and the L1 mimetics duloxetine, piceid, tacrine, ethinyl estradiol, crotamiton and honokiol enhanced the expression of L1, stimulated the generation of L1-70 and enhanced neuritogenesis as well as neuronal migration and survival ,33. SchwNeurons from mice without L1-70 exhibited reduced neurite outgrowth and neuronal survival and migration when the cells were stimulated with antibody 557 or L1 agonists, but were not affected without stimulation. This feature also distinguishes L1-70-deficient neurons from L1-201 neurons with impaired basal neuronal functions but susceptibility to stimulation by antibody 557 or L1 agonists . These r2+. When mitochondria are aged or damaged, they are retrogradely transported back to the cell soma to be degraded [After showing that L1 plays important roles in the functions of mitochondria , we invedegraded . Throughdegraded . Neuronsdegraded . DeficieWhen their function is impaired, the mitochondria contribute to severe pathologies of the nervous system, not only due to metabolic insufficiency but also to the production of ROS and activation of caspases ,11,39, wGene-edited mice expressing L1 with an arginine-to-alanine exchange at Position 867 in the first FNIII domain (L1/687 mutant mice), with a mutation of the dibasic sequence RKHSKR to SKHSSS at Positions 858–863 in the third FNIII domain (L1/858-863 mutant mice)  and with4 cells/well in 0.01% poly-L-lysine -coated 48-well tissue culture plates  in a defined serum-free medium  supplemented with 2 mM L-glutamine, 4 nM L-thyroxine, 1 mg/mL BSA, 12.5 μg/mL insulin, 30 nM sodium selenite, 100 μg/mL transferrin, 0.1 mg/mL streptomycin and 10 U/mL penicillin ) and allowed to settle for 30 min. They were then treated with 50 µg/mL of L1 function-triggering antibody 557 directed against amino acids 817–835 at the transition between the second and third FNIII-like domains ([6 cells/mL in PLL-coated 48-well tissue culture plates (ThermoFisher Scientific) and maintained overnight in a defined serum-free medium as indicated above [2O2 for 24 h. Live and dead cells were then stained with calcein-AM (ThermoFisher Scientific) and propidium iodide (Sigma-Aldrich) and imaged with a Zeiss AxioObserver.A1 microscope (Carl Zeiss) with a 20× objective (aperture 0.4) and AxioVision 4.6 software (Carl Zeiss) [https://imagej.nih.gov/ij/index.html; RRID:SCR_003070, 8 March 2022). For determining cell migration, cerebellar explants were prepared from two to three 6- to 7-day-old male mice per genotype and experiment as described, and the migration of neurons was determined from 12–15 explants per condition and experiment [Cultures of dissociated cerebellar granule neurons were prepared from 6- to 7-day-old male mice as described . In eachdomains  . Live anperiment . Brieflyperiment . Experim4 cells/well into PLL-coated 48-well tissue culture plates (ThermoFisher Scientific) and allowed to settle for 30 min. Cells were then treated with 50 µg/mL of antibody 557 or 100 nM of duloxetine for 24 h. After fixing and staining with 1% toluidine blue and 1% methylene blue in 1% sodium tetraborate, images of the process were taken, and the lengths of the process from at least 100 cells per condition and experiment were quantified using an AxioObserver.A1 microscope with a 20× objective (aperture 0.4) and AxioVision 4.6 software (Carl Zeiss) [Schwann cells were isolated from male 6- to 7-day-old mice as described . In eachl Zeiss) . Experim® Red CMXRos . A MitoTracker stock solution (1 mM) was diluted to a final concentration of 50 nM in the pre-warmed cerebellar neuron culture medium. Cerebellar granule neurons (density: 1−2 × 106 cells/well of a 6-well plate containing a 20 mm glass coverslip) were prepared as stated above and cultured for two to three days in a medium as indicated above containing 5% horse serum. The medium was then replaced with a serum-free medium, and the cells were incubated with 50 nM of MitoTracker for 30 min at 37 °C. This solution was then replaced with fresh serum-free medium, and the cells were analyzed at 37 °C under 5% CO2 by time-lapse imaging using an upright Nikon Eclipse Ti microscope  combined with spinning disk  live-cell confocal technology (Visitron Systems) and a 100× objective (aperture 1.45). For live cell imaging, a 561 nm argon laser was used at 10% intensity, and images were taken at intervals of 2 s for a duration of 5 min. Videos were acquired using the VisiView 5.0 software (Visitron Systems). To determine mitochondrial velocity, mobility and the direction of mitochondrial transport (anterograde or retrograde), we used the kymograph (time space plot) plugin and the velocity tool from ImageJ . The kymograph represents a time and space plot: the x-axis indicates the distance, and the y-axis shows the time. Vertical lines represent no movement, while diagonal lines show mobile mitochondria. With respect to the position of the cell soma, the direction (right or left) of the diagonal lines indicates retrograde or anterograde transport, respectively [Mitochondria were labeled with MitoTrackerectively . Mitochoectively ,47. For The dual-emission potential-sensitive probe JC-1 iodide  was used to measure the membrane potential of the mitochondria . JC-1 en6 cells/mL onto 22 µm glass coverslips coated with PLL and cultured in a serum-free medium for two days. Afterwards, cells were stained with 3 µM of JC-1 in Hank´s balanced salt solution  for 30 min, incubated with HBSS alone for 15 min and then observed under a spinning disk microscope with a 60× objective (aperture 1.4). For detecting mitochondria with high mitochondrial membrane potential, a rhodamine filter set was used together with the fluorescein filter for detecting depolarized mitochondria with low mitochondrial membrane potential. Experiments were carried out in duplicate and performed independently three times using one mouse per genotype and experiment. The total cell fluorescence intensity was calculated for the green and red channels separately, and the red to green fluorescence intensity ratio was determined from 10 neurons per image and 10 images per group and experiment.To determine the mitochondrial membrane potential, cerebellar granule cells were prepared as stated above and plated at a density of 1−2 × 10® Mitochondrial Complex I Activity Assay Kit . Experiments were carried out in duplicate and performed independently three times using four (L1/858-863), six (L1/687) or nine (L1-201) mice in total per genotype. Only Complex I activity was determined in the present study, since we had found that other complexes’ activities were not altered by the L1-70 fragment [Whole brains were isolated from 6- to 8-day-old male mice, frozen in liquid nitrogen and stored at −80 °C until use. For isolation of the mitochondria, the Mitochondria Isolation Kit for Tissue from ThermoFisher Scientific  was used. For determining Complex I activity, mitochondria were applied to the MitoCheckfragment .® 2.0 Cell Viability Assay  according to the manufacturer´s protocol. In brief, cerebellar granule cells were cultured in a serum-free medium as indicated above at a density of 2.5 × 105 cells/well in 48-well plates for 2 days. Afterwards, cells were scraped off into 200 µL of serum-free medium, 100 µL of the supernatant was transferred into white 96-well plates and 100 µL of BacTiter-Glo was added to the wells. Plates were incubated for 5 min at room temperature with gentle shaking and then the luminescence was measured using a luminometer . ATP concentrations were determined using an ATP standard curve. Experiments were carried out three times independently in triplicate using three (L1/687), five (L1-201) and six (L1/858-863) mice in total per genotype.ATP levels were determined using the BacTiter-Glo2. The medium was then replaced by HBSS without phenol red, and cells were imaged under a spinning disk microscope with a 40× objective (aperture 1.2) and with an excitation wavelength of 488 nm. Experiments were carried out in duplicate and performed independently three times; for each condition and experiment, 10 images were taken for analysis.Intracellular ROS were monitored using the ROS-sensitive fluorescent probe 2′,7′-dichlorofluorescin diacetate , which penetrates into cells rapidly and is hydrolyzed by ROS into a fluorescent green dye. Cerebellar granule cells from 6- to 7-day-old mice (one mouse per genotype and experiment) were prepared as stated above and seeded onto glass coverslips as mentioned above, maintained in the culture for 3 days in a serum-free medium and stained with 10 µM of DCFH-DA for 30 min at 37 °C and 5% COt-test was used to compare two groups, whereas for comparisons of more than two groups, we performed ANOVA followed by Tukey’s post-hoc test for multiple comparisons. Groups containing small sample numbers or data that were not normally distributed were analyzed using the Mann–Whitney U-test for comparing two groups or the Kruskal–Wallis test by ranks followed by Dunn’s multiple comparison test when there were more than two groups. The statistical tests used for comparisons are indicated in the figure legends. Analyses were performed using SPSS , SigmaPlot  or GraphPad  software.All numerical data are presented as group mean values with the standard error of the mean (SEM) or standard deviation (SD); for small sample numbers, individual data are additionally indicated. The normal distribution of the data was determined using the Shapiro–Wilk test or the D’Agostino–Pearson test. For data with a normal distribution, Student’s