Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease. Recent studies have identified the key role of crosstalk between dysregulated epithelial cells, mesenchymal, immune, and endothelial cells in IPF. In addition, genetic mutations and environmental factors  have also been associated with the development of IPF. With the recent development of sequencing technology, epigenetics, as an intermediate link between gene expression and environmental impacts, has also been reported to be implicated in pulmonary fibrosis. Although the etiology of IPF is unknown, many novel therapeutic targets and agents have emerged from clinical trials for IPF treatment in the past years, and the successful launch of pirfenidone and nintedanib has demonstrated the promising future of anti-IPF therapy. Therefore, we aimed to gain an in-depth understanding of the underlying molecular mechanisms and pathogenic factors of IPF, which would be helpful for the diagnosis of IPF, the development of anti-fibrotic drugs, and improving the prognosis of patients with IPF. In this study, we summarized the pathogenic mechanism, therapeutic targets and clinical trials from the perspective of multiple cell types, gene mutations, epigenetic and environmental factors. Idiopathic pulmonary fibrosis (IPF) is a progressive, life-threatening, interstitial lung disease of unknown pathogenesis. IPF has a familial and sporadic onset with a poor prognosis, and death usually occurs within 2–5 years of diagnosis due to secondary respiratory failure . CT imagThe current paradigm suggests that IPF occurs as a result of epithelial injury and dysregulation of the epithelial/mesenchymal crosstalk, which continuously activates multiple interconnected downstream profibrotic pathways, ultimately leading to an abnormal repair response and decreased lung function .In the next sections, we provide a brief overview of the pathogenic mechanisms of IPF . Damage There are AECs, alveolar capillary endothelial cells, immune cells, fibroblasts, and mesenchymal progenitor cells near the alveoli. These cells maintain the homeostasis of the alveolar environment under normal physiological conditions. However, in the pathophysiological process of IPF, the intercellular crosstalk leads to reprogramming of cell phenotype. AECs, endothelial cells, and immune cells work together through multiple signaling pathways to regulate fibroblast phenotype. Then, fibroblasts recruitment, proliferation, differentiation, and secretion of extracellular matrix directly lead to fibrosis and exhaustion of pulmonary function. therefore, it is necessary to clarify the roles of various cells and extracellular matrix in the development of IPF.Alveolar epithelial cells/endothelial cells participate in IPF via various ways, including unfolded protein response (UPR), Epithelial-mesenchymal transition (EMT), coagulation cascade, angiogenesis, and the secretion of a variety of signaling factors (such as TGF-β) . In thisThe UPR of AEC is one of the underlying mechanisms for the development of IPF. As an important site for cellular protein synthesis, the ER (endoplasmic reticulum) must maintain relative homeostasis. Under pathological conditions , misfolEMT refers to a process in which AECs gradually lose their epithelial characteristics under specific stimuli and conditions, with the subsequent appearance of interstitial cells characteristics. During this process, AECs are reprogrammed with changes in secretory phenotype, cytoskeletal proteins, intercellular junctions, and cell polarity . The conHowever, in recent years, the central role of EMT in the pathogenesis of IPF has been questioned. The localization of type 2 epithelial cells by markers showed that the conversion of epithelial cells into myofibroblasts was incomplete  and thatUnder pathological conditions, the coagulation cascade and angiogenesis are important driving forces for the promotion of pulmonary fibrosis. Because tissue factor can activate PAI 1, PAI 2, fibrinolysis inhibitors and protein C inhibitors through the coagulation cascade, resulting in a local pro-coagulation microenvironment, it inhibits the degradation of the ECM in this microenvironment and promotes the differentiation of fibrocytes . IPF patIn addition to the above pathways, AECs also participate in pulmonary fibrosis through the secretion of a variety of mediators, including growth factor , matrix metalloproteinases (MMP1/MMP2/MMP7), chemokines (CCL17/CCL2/CXCL12), pigment epithelium-derived factor, autotaxin, sphingosine-1-phosphate, neuregulin (NRG) 1α, growth and differentiation factor 15 (GDF15), transmembrane protease serine 4 (TMPRSS4), tumor necrosis factor-alpha (TNF-α), osteopontin, and angiotensinogen. Mareike Lehmann et al. summarized the possible roles of these cytokines in detail in the occurrence and development of IPF .A large number of studies have demonstrated that immune cells play a role in IPF. However, anti-inflammatory therapies, i.e., TNF-α monoclonal antibodies  and glucWith the deepening of research in the field of immunization, the role of immune factors in IPF has received increasing attention. Although the diagnostic criteria for IPF require the exclusion of autoimmune diseases as the underlying pathogenesis, many IPF patients still have unexplained elevated autoantibodies, and some autoantibodies are associated with acute exacerbations of IPF (AE-IPF) . To inhiHeukels conducted a detailed review on the role of each immune cell in IPF and the correlation of each cell type with the development of fibrosis . Neverth+ cells under IFN-γ/IL-12 induction. CXC chemokine receptor 3 expressed by Th1 cells can recognize interferon-inducible T cell a chemoattractant (I-TAC), interferon g-inducible protein of 10 kD (IP-10), and monokine induced by interferon gamma (Mig) (Th1 (helper T cell type 1) cells are helper T cells produced by CD4ma (Mig) . The maima (Mig) .+ cells under IL-4 induction. Th2 cells that express CC chemokine receptor 4 can recognize thymus- and activation-regulated chemokines (TARCs) and macrophage-derived chemokines (MDCs) (Th2 (helper T cell type 2) are helper T cells produced by CD4s (MDCs) . The mais (MDCs) .Th2 polarization has been observed in IPF. In the BALF of IPF patients and in systemic circulation, the levels of Th1 cells and their secretion of IFN-γ are relatively low, and the levels of Th2 cells and their secretion of IL-4/IL-5/IL-13 are relatively high . In addiThe inhibition of Th2 polarization is a possible direction for the treatment of IPF. Many factors can cause Th2 polarization. Galectin-1 and prostaglandin E2 promote Th2 polarization by inducing Th1 apoptosis and reducing the synthesis of Th1 inducers , and supIn addition to the Th1/Th2 balance, the balance of macrophage subpopulations also plays an important role in the pathogenesis of IPF. Because cytokines secreted by Th1/Th2 cells greatly affect the differentiation of M1/M2 macrophages, Th1/Th2 cells and M1/M2 macrophages interact with each other to jointly shape the type 1 and type 2 immune microenvironments .The main function of M1 (type I macrophages) is to respond to lipopolysaccharide (LPS), IL-1, and IL-6. They can secrete type 1 immune factors such as IL-12, induced nitric oxide synthase (iNOS), TNF-α, IL-1β, IL-23, IL-6, and CXCL10 , therebyM2 polarization is one of the important links in the occurrence and development of pulmonary fibrosis. The M2-mediated type 2 immune response is an important component of pulmonary fibrosis. M2 macrophages provide an important microenvironment for pulmonary fibrosis by secreting profibrotic substances such as CCL18, IL-10, TIMP1 , TGF-β, FGF, PDGFα, IGF1, and VEGF . Macrophin vitro environment, CD45+/CD34+ primary bone marrow fibrocytes were stimulated by the ECM of IPF patients; then the hematopoietic surface antigens CD45 and CD34 rapidly disappeared, and mesenchymal markers, i.e., α-SMA, rapidly increased. The fibrocytes transformed into fibroblasts with a contractility like that of smooth muscle cells and a strong ability to synthesize ECM fibroblasts, fibrocytes, epithelial cells , mesenchymal stromal cells, and pericytes. The vast majority of interstitial and fibrillar collagens (mainly collagen I and collagen III) are secreted by (myo)fibroblasts. These collagens constitute the main frame of the ECM and increase the mechanical stiffness of fibrotic tissue . CurrentThere are relatively few studies on ECM glycoproteins and proteoglycans. In a radiation-induced pulmonary fibrosis model, the glycoprotein glectin-3 produced by type I AECs increased dramatically . In receECM regulators include serine protease inhibitors, cystatins, TIMPs, MMPs , a disinECM contains a variety of signal molecules secreted by cells; therefore, the ECM is a transfer station for signal exchange. In IPF, the signaling molecules from different cells constitute a complex signaling network through the bridge function of the ECM. Upstream signals are modified and regulated by positive feedback in the ECM; this feedback, e.g., the TGF-β pathway and Rho/ROCK signaling, can affect the transcription and translation of ECM-related genes and eventually lead to pulmonary fibrosis .Although there have been many studies on active fragments, the roles of these active products in IPF are still not clear. In recent years, the ECM has shown extraordinary clinical and scientific value in the fields of anti-infection, anti-angiogenesis, and wound healing . TherefoA growing number of studies have shown that genetic mutations are associated with susceptibility, diagnosis, progression, prognosis, and adverse effects of treatment in IPF. The proportion of patients carrying genetic mutations in IPF may be underestimated. A cohort study showed that up to 36% of patients with IPF have familial genetic mutations . In addiNotably, the majority of existing studies still remain to verify the correlation between mutations and IPF susceptibility, and there are only a few studies related to the causality and pathogenesis of IPF. Moreover, further research needs to focus on how to cross the gap between genetic testing and clinical treatment. Drugs targeting histone modifications and DNA modifications have also shown potential in preclinical studies, so we will also cover the role of epigenetics in IPF in the next sections.Telomeres are a TTAGGG repeat sequence at the end of human chromosomes, whose function is to stabilize the chromosome, prevent fusion of chromosome ends, protect structure of chromosomes, and determine cell life span, but telomerase activity in adult human cells is extremely low .Telomeres are protected by the shelterin complex, which consists of the protein components TRF1, TRF2, RAP1, TIN2, TPP1, and POT1. The ends of telomeres are bonded with telomerase, which is comprised of the catalytic subunit of TERT, the telomerase RNA component of TERC, and accessory proteins. The telomerase exerts its effects by adding telomeric sequences to the telomeric ends . TelomerThe causal relationship between mutation-mediated telomere shortening and the development of IPF is inconclusive because not all individuals carrying a known telomere defective gene develop telomere shortening and IPF . FirstlyThe mechanism by which telomerase gene mutations lead to IPF is unclear. Available data suggest that cellular senescence or death of alveolar stem cells induced by telomere dysfunction is implicated in pulmonary fibrosis . KnockdoTelomere length and specific telomere gene defects are instructive for susceptibility, diagnosis, progression, prognosis, and early warning of adverse effects in IPF. Shorter telomere length as an independent predictor is associated with worse survival and more pronounced imaging changes in IPF patients . PirfeniDrugs that target telomeres have also shown therapeutic potential. GRN510, a telomerase agonist, reduces inflammatory infiltration and collagen deposition in a mouse model of bleomycin-induced pulmonary fibrosis . PAP-assIn summary, details remain unclear in the complex field of the role telomeres play in the pathogenesis of ILDs and no clinical trials employing gene therapy have been initiated in patients with telomeropathies. Hopefully, adeno-associated virus-9 (AAV9)-mediated gene therapy has been reported to be successful in mouse models , and perPhospholipids and four surfactant proteins  can be secreted by AEC II. The process of surfactant secretion is shown . ATP-binThe MUC5B promoter variant, rs35705950, as the strongest genetic risk factor and common variation, was found in up to 30% of IPF . The rs3Genetic mutations in cytokines have also been reported in patients with IPF. IL1RN gene polymorphisms may be associated with susceptibility to fibrosing alveolitis, a disease characterized by interstitial lung fibrosis . MesenchIn recent years, the launch of pirfenidone and nintedanib has witnessed breakthroughs in the signaling pathways of cytokines and growth factors in the treatment of IPF, however, studies focusing on cytokine and immune-related gene polymorphisms need to be further developed.In recent years, with the availability of next-generation sequencing technologies, more and more novel mutations have been identified in IPF patients. The mutations of the gene DSP  and DPP9DNA methylation is a chemical modification process in which a specific base on the DNA sequence acquires a methyl group by covalent bonding with S-adenosyl methionine (SAM) as the methyl donor. DNA methylation is catalyzed by DNA methyltransferase (DNMT). In humans, DNA methylation occurs mainly at the fifth carbon atom of cytosine in CpG dinucleotides. It is generally believed that DNA methylation can inhibit the transcription and expression of the gene.ARF, IP-10  was associated with decreased mRNA expression . In ordeF, IP-10 , SFRP1/4F, IP-10 ], but alF, IP-10 ], which F, IP-10 . DecitabF, IP-10 . PGE2 haF, IP-10 .Nucleosome, the basic unit of chromatin, is comprised of 146–147 base pairs of DNA and a histone octamer with one H2A-H2B tetramer and two H3-H4 dimers .The N- and C-terminal tails of H3 and H4 are the most frequently modified regions. The types of modifications include methylation, acetylation, phosphorylation, SUMOylation, and ubiquitination. The histone mark is named according to the histone, the modification site of these tails, and the type of modification. Histone modifications can change the electronic charge and structure of DNA-bound histone tails, thus altering chromatin state and gene expression . The effHistone methylation is catalyzed by the histone methyltransferase (HMT), which uses S-adenosyl methionine (SAM) as the substrate to transfer methyl groups onto the lysine residues of histones. Methylation of H3K4, H3K36, and H3K79 is thought to be associated with gene activation, while H3K9, H3K27, and H4K20 are thought to be associated with gene inactivation . InhibitHistone acetyltransferases (HATs) promote histone acetylation, while histone deacetylases (HDACs) reduce histone acetylation. Histone acetylation is thought to be associated with gene activation . In addiInhibition of histone deacetylation may have anti-pulmonary fibrosis effects. HDACi consists of a zinc-binding-group, a hydrophobic group for protein recognition and interaction, and a linker connecting both of them . A largeEnvironmental factors such as asbestos, metal dust, stone dust, wood dust, chemotherapy and allergen exposure are important causes of IPF . EpidemiSmoking is a relatively special environmental factor. Both direct smoking and indirect smoking are independent exposure factors, and the correlation between the direct/indirect smoking and IPF increase with the increase of smoking in dose . InteresAccording to epidemiological surveys, advanced age is one of the most relevant demographic factors for IPF . The incThe main manifestations of lung senescence are decreased lung tissue elasticity, decreased effective lung volume, the thickening of intercellular substances, and decreased lung function. Cell senescence mainly manifests as cell morphology and structure disorder, cell dysfunction, and cell growth/proliferation arrest .The mechanism of pulmonary fibrosis caused by aging is not very clear, but there are quite a few theories to explain the mechanism of senescence in IPF. Cell senescence can disrupt of a variety of processes and generate imbalances , resulting in increased SASP, myofibroblast resistance to apoptosis, and stem cell depletion, ultimately manifesting as abnormal repair at the lung injury site  . Aging hBecause senescent cells that resist apoptosis can continuously produce profibrotic cytokines, which in turn aggravate pulmonary fibrosis, the induction of apoptosis in senescent cells  or selective antagonism of profibrotic senescence-related factors  may help to alleviate IPF . Many anPirfenidone and nintedanib are two drugs approved by the FDA for the treatment of IPF. They can delay lung function deterioration and prolong patient survival in IPF patients. Although the launch of Pirfenidone and nintedanib has demonstrated the feasibility of drug treatment for IPF, they do not reverse pulmonary fibrosis and their efficacy in patients with end-stage IPF is not fully clear. Therefore, there is a need to develop additional anti-pulmonary fibrosis drugs. The development of drugs for some specific targets is currently underway, which will be described in the following sections. In addition, drug combinations for antifibrotic therapy are worth investigating. Immune checkpoint inhibitors (ICIs), either as single agents or in combination regimens, have shown great success in clinical treatment . A case In this section we briefly describe the current therapeutic targets in clinical trials and the latest clinical trial developments. We summarize the emerging therapeutic targets and representative mechanisms in the development of IPF .TGF-β plays an important role in the pathogenesis of IPF by stimulating the activation and proliferation of fibroblasts . Both anChemokines and interleukins are also involved in recruitment of pro-fibrotic cells and formation of pro-fibrotic microenvironment, as we discussed in our previous paper . HoweverLeukotrienes have profibrotic effects by inducing fibroblast migration, proliferation, and matrix protein synthesis . MN-001/As lipid proinflammatory mediators, The ATX-LPA-LPAR axis and SPHK1-S1P-S1PR axis have also been reported as targets of IPF. LPA and S1P have been demonstrated to promote the development of fibrosis . BMS-986PTX-2, a ligand for the Fcγ receptor, can downregulate monocyte and macrophage activity  . PRM-151Multiple cellular signaling pathways play a crucial role in the development of IPF. JAK/STAT signaling , receptoThere are also drugs in clinical trials targeting oxidative stress , GPR40/GWe searched clinical trials in recent years and summarized the anti-IPF drugs undergoing clinical trials and their mechanisms of action . We alsoThe research on the pathogenesis of IPF has made considerable progress. After years of basic and clinical studies, the pathogenesis of IPF has changed from simple inflammation to abnormal epithelial-mesenchymal crosstalk and other common pathogenic mechanisms. Based on this pathological mechanism, many studies have systematically studied the key roles of fibrocytes/(myo)fibroblasts, epithelial/endothelial cells, and ECM in the pathogenesis of IPF. With the progresses in understanding the key role of genetics and epigenetics in IPF, researchers have found that an increasing number of genetic loci and their apparent modifications are related to the maintenance of lung homeostasis although the effects of gene mutations and epigenetics on IPF still need further study.The advent of pirfenidone and nintedanib is undoubtedly a sensational event in the field of IPF treatment, but they also have some major limitations, such as insufficient curative effect and poor pharmacokinetic properties. In addition, combination therapy based on them needs to be further studied. The number of clinical trials for therapies targeting cytokines, growth factors, and their signaling pathways is increasing rapidly. Furthermore, drugs targeting MMPs, telomerase activity, and epigenetic modifications need to be further developed.Although IPF is idiopathic by definition, this deadly disease may become less mysterious with the progress in the understanding of the pathogenesis of IPF. The number of observed therapeutic targets is increasing, as well as the number of new drugs entering clinical trials. For patients with IPF, these advances described in this review show that slowing the IPF progression, prolonging their lives, and improving their quality of life might be possible in the future.


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


Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which motor neurons in spinal cord and motor cortex are progressively lost. About 15% cases of ALS also develop the frontotemporal dementia (FTD), in which the frontotemporal lobar degeneration (FTLD) occurs in the frontal and temporal lobes of the brain. Among the pathologic commonalities in ALS and FTD is ubiquitin-positive cytoplasmic aggregation of TDP-43 that may reflect both its loss-of-function and gain-of-toxicity from proteostasis impairment. Deep understanding of how protein quality control mechanisms regulate TDP-43 proteinopathies still remains elusive. Recently, a growing body of evidence indicates that ubiquitinating and deubiquitinating pathways are critically engaged in the fate decision of aberrant or pathological TDP-43 proteins. E3 ubiquitin ligases coupled with deubiquitinating enzymes may influence the TDP-43-associated proteotoxicity through diverse events, such as protein stability, translocation, and stress granule or inclusion formation. In this article, we recapitulate our current understanding of how ubiquitinating and deubiquitinating mechanisms can modulate TDP-43 protein quality and its pathogenic nature, thus shedding light on developing targeted therapies for ALS and FTD by harnessing protein degradation machinery. Amyotrophic lateral sclerosis (ALS) is among the most prevalent and fatal neurodegenerative diseases in which motor neurons in spinal cord and motor cortex are progressively lost. Patients diagnosed with ALS suffer from the gradual respiratory dysfunction, having the mean survival time of 3–5 years due to no effective therapeutic treatment . ApproxiA 414-amino acid TDP-43 protein contains an N-terminal domain with a nuclear localization signal (NLS), two RNA recognition motifs (RRM1 and RRM2) with a nuclear export signal (NES), mitochondria localization signals (M1-M5), and a glycine-rich C-terminal domain where multiple ALS-associated mutations were reported  Guerrer. This febona fide protein  interaction networks. Above certain threshold that can be held by protein quality control mechanisms–such as ubiquitin-proteasome system (UPS), heat shock response, or autophagy-lysosomal degradation pathway, TDP-43 proteopathies can become runaway or even aggravated by faulty proteostasis pathways. Moreover, aberrant accumulation of pathological TDP-43 species sequesters the critical UPS components, directly impairs the proteasome activity, induces the accumulation of insoluble polyubiquitinated proteins, and also depletes free ubiquitin pool, thereby leading to perturbed protein and ubiquitin homeostasis , and organelle homeostasis (as in mitochondria) . Under peostasis . In conseostasis ; 2) the eostasis . Accumuleostasis . In addieostasis .TARDBP gene (encoding TDP-43) have been associated with ALS and FTLD-TDP , E2 (ubiquitin conjugating enzyme), and E3 (ubiquitin ligase) enzymes . By geneUBQLN, VCP, p62, and OPTN  can exclusively reverse the ubiquitination process and thus are capable of stabilizing the target proteins. Besides, DUBs also play essential roles in ubiquitin recycling and ubiquitin chain editing, which may lead to altered subcellular localization of substrates and various responses to cellular signaling . Althougin vivo fly model. Thus, in this case, UBPY may serve as a protective factor against TDP-43-induced neurotoxicity (M719V), which was genetically identified from ALS and FTLD disease locus, exhibits significantly elevated K63-linkage specific deubiquitinase activity and caused the impairment in autophagosome-lysosome fusion pathway. This CYLDM719V mutant also increased cytoplasmic mislocalization of TDP-43 and reduced the axonal length (USP14, one of the major DUBs on the proteasome, can deubiquitinate and stabilize the protein substrates that are ubiquitinated at multiple sites . Given ttoxicity . DUBs mal length . Similarl length .Aberrant stress granule dynamics represent another pathological feature of TDP-43 proteinopathy . NotablyWhile ubiquitin-enriched cytoplasmic inclusion of TDP-43 is considered to be the proteopathic signature of ALS and FTD, the pathophysiological meaning of the ubiquitination has been a long-standing question. Dramatic alteration of TDP-43 localization, stability, and post-translational modifications under the pathological conditions may disturb its genuine functional interaction networks by losing nuclear functions or gaining cytoplasmic toxicity. On top of TDP-43’s cytoplasmic mislocalization and aggregation-prone property, the initial pathogenic cue may trigger ‘butterfly effect’ and eventually the TDP-43 proteinopathy would prevail. Reduced proteasomal or autophagic activity and free ubiquitin pool depletion will badly influence the quality control of TDP-43 protein, or rather aberrant or undesirable ubiquitination of TDP-43 from proteostasis failure may only further exacerbate the disease phenotype. In fact, ubiquitination-rich pathogenic inclusions or liquid condensates, if the modification is not cleared properly, may act as avid and irreversible absorbent chambers to sequester many important proteolysis components such as ubiquitin binding proteins and proteasome. In this sense, pharmacological modulation of the pathogenic TDP-43-specific ubiquitination or the protein turnover itself may provide promising therapeutic opportunities to cope with TDP-43 proteinopathies. Remodeling or editing the ubiquitin chains or fine control of TDP-43 half-life may favor its proteolysis flux or antagonize the disease progression. It should be also noted that the total depletion of TDP-43 is not a viable option because of its essentiality. Indeed, pharmacological activation of the UPS or lysosomal pathways have been shown to bear the promise for TDP-43 clearance. For example, as noted above, a small-molecule inhibitor targeting proteasome-bound USP14 was reported to accelerate TDP-43 turnover by enhancing the proteasome activity . Similar