Idiopathic pulmonary fibrosis (IPF) is a type of interstitial lung disease (ILD) characterized by the proliferation of fibroblasts and aberrant accumulation of extracellular matrix. These changes are accompanied by structural destruction of the lung tissue and the progressive decline of pulmonary function. In the past few decades, researchers have investigated the pathogenesis of IPF and sought a therapeutic approach for its treatment. Some studies have shown that the occurrence of IPF is related to pulmonary inflammatory injury; however, its specific etiology and pathogenesis remain unknown, and no effective treatment, with the exception of lung transplantation, has been identified yet. Several basic science and clinical studies in recent years have shown that statins, the traditional lipid-lowering drugs, exert significant antifibrotic effects, which can delay the progression of IPF and impairment of pulmonary function. This article is aimed at summarizing the current understanding of the pathogenesis of IPF, the progress of research on the use of statins in IPF models and clinical trials, and its main molecular targets. Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and fibrotic interstitial lung disease (ILD) characterized by alveolar epithelial cell damage and abnormal repair mechanisms . IPF is Pirfenidone and nintedanib have been approved by the US Food and Drug Administration in the treatment of IPF; they are efficacious in delaying the decline of lung function and reducing mortality . HoweverStatins, which act as 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, are widely used to lower lipid levels by inhibiting cholesterol synthesis. They are the first-line treatment option for hyperlipidemia and coronary heart disease. In recent years, a growing body of research has demonstrated that statins can suppress inflammatory responses, reduce the expression of cytokines, attenuate oxidative stress (OS), inhibit myofibroblast differentiation, prevent pulmonary parenchyma remodeling, and block the key processes associated with fibrosis. Furthermore, several clinical studies have reported that statins could play an active role in the treatment of IPF, which may delay the decline of lung function and reduce mortality in patients with the disease. In this review, we focus on the progress of research studies investigating the use of statins in models of IPF and clinical trials, as well as the main molecular targets of the drugs, to provide a theoretical basis for the clinical application of this class of pharmacological agents.β) [The etiology of IPF is unclear, and its pathogenesis has not been fully elucidated; however, there is sufficient evidence to demonstrate that the disease is related to inflammatory injury of the immune system, mediated by a variety of signaling pathways . Some geβ) . Conversβ) .α), and interleukin-1β (IL-1β) to combat pathogens [β, which can induce epithelial-mesenchymal transformation (EMT) through TGF-β/Smad2 signaling pathway and promote the proliferation of fibroblasts [α, which is produced by macrophages, is a proinflammatory factor that is closely related to the pathogenesis of IPF [α can inhibit the role of fibrogenic macrophages, thereby slowing the progression of fibrosis and promoting the degradation of fibrotic lesions [α has been shown to activate the nuclear factor kappa B (NF-κB) transcription factor, leading to myofibroblast differentiation of lung resident mesenchymal stem cells, which ultimately contributes to the deterioration of pulmonary fibrosis in the murine model of bleomycin-induced pulmonary fibrosis [β [α2 receptor, thereby inducing the production of TGF-β in the alveolar macrophages [β1 signaling pathway, and the number of mast cells has been found to increase in pulmonary fibrosis and be positively correlated with disease severity [In the histologic analysis of IPF patients, inflammation was the most common histological feature, observed in approximately 25% of patients with IPF . Followiathogens . M2 macrroblasts . The sturoblasts . TNF-α, s of IPF . Studies lesions . Howeverfibrosis . Interlefibrosis . Immunohfibrosis . Neutropfibrosis . Animal rosis [β . One sturosis [β , and therophages . IL-13 crophages . Mast ceseverity .Inflammatory cells and cytokines play an important role in pulmonary fibrosis. It is believed that damage to and abnormal repair of AECs are the basis of IPF. During lung tissue repair, normal reepithelialization cannot occur, resulting in alveolar injury. During this process, inflammatory cells aggregate and cytokine secretion increase, mediating various factors leading to pulmonary fibrosis.β is a multifunctional cytokine that regulates cell growth and differentiation and induces fibrosis and scar formation [β is produced by a variety of cell types in the lung tissue, including alveolar macrophages, AECs, fibroblasts, and myofibroblasts, and it is temporarily stored in the ECM [β is an important factor in the regulation of cellular activities; however, the pathway through which TGF-β becomes activated is not well understood, although it has been reported to be associated with certain conditions, including an acidic environment [αvβ5 and αvβ6) [β receptors, including TGF-β receptors of type I (TβR-I), type II (TβR-II), and type III (TβR-III), are expressed in almost all tissues, although only the former two subtypes are involved in cell signaling [β is activated by the large latent complex (LLC), which comprises latent transforming growth factor β binding proteins (LTBPs) and latency-associated peptide (LAP) before binding to its respective receptors. TGF-β recruits TβR-I into the glycine-serine-rich (GS) domain, where activated TβR-I phosphorylates its downstream targets after binding to TβR-II. Smad proteins, which are important regulatory molecules of the TGF-β superfamily of signaling proteins, are the downstream transmembrane receptors of TGF-β that are precisely regulated at different levels [β binds to the receptors on the surface of the cell membranes of fibroblasts and phosphorylates Smad2/3 proteins that bind to Smad4. Subsequently, the Smad complex translocates into the nucleus, acting as a marker of activation of the Smad2/3 signaling pathway, thereby regulating and driving EMT and the deposition of ECM [TGF-ormation . TGF-β i the ECM –33. TGF-ironment  and the ironment  and intend αvβ6) . TGF-β rignaling . TGF-β it levels . Activatn of ECM –41.β signal transduction could also affect Smad-independent signaling pathways, including those involving extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). TGF-β is activated by binding to TβR-I; it then recruits and phosphorylates the ShcA domain on tyrosine and serine residues to induce the formation of the ShcA/growth factor receptor-bound protein 2 (Grb2)/Son of Sevenless (Sos) complex and further activates the Ras-Raf-MEK-ERK1/2 signaling pathway [β-activated kinase 1 (TAK1), a member of the MAP kinase 3 (MAP3K) gene family, is activated by binding to TAK1 binding protein 1 (TAB1) and has been confirmed to be involved in the TGF-β signaling pathway. The activation of TGF-β promotes TGF-β-induced expression of collagen and the differentiation of fibroblasts through either the TAK1/TAB1-MKK3-p38 MAPK or TAK1/TAB1-MKK4-JNK signaling pathway [In addition, TGF- pathway . TGF-β-a pathway , 44.β [β, mediating fibroblast activation and ECM synthesis. Researchers have found high expression levels of YAP/TAZ in the nuclei of fibroblasts in the lung tissues of patients with IPF [CTGF is an effective profibrotic mediator capable of inducing the proliferation of fibroblasts and the secretion of ECM, driving tissue fibrosis alongside TGF-β . During with IPF , 47. Undwith IPF .β and α-smooth muscle actin (α-SMA), inducing the proliferation and activation of fibroblasts. In addition, fibroblasts can produce stem cell factors and nourish mast cells, which are processes that play a role in tissue fibrosis [γ) was observed in AECs and alveolar macrophages of the lung tissues [γ expression leads to pulmonary fibrosis remains unclear, and PPAR-γ activation can inhibit TGF-β-induced transformation of fibroblasts into myofibroblasts as well as the deposition of ECM [Upon stimulation by factors released from the inflammatory cells, mast cells degranulate and activate TGF-fibrosis . Fibroblfibrosis . In a mo tissues . The mecn of ECM .β is a pleiotropic cytokine that binds to receptors to regulate cell proliferation, differentiation, and apoptosis, and it plays an important role in ECM synthesis and trauma repair. In summary, TGF-β activates Smad and Smad-independent signaling pathways, leading to fibroblast proliferation and excessive accumulation of ECM, which causes pulmonary fibrosis ; ROS can directly oxidize LAP to activate TGF-β, and TGF-β can also induce the production of ROS [β and ROS is one of the causes of pulmonary fibrosis. It has been reported that destruction of the integrity of the epithelial barrier and apoptosis of AECIIs are important events in the pathogenesis of IPF, and these processes may be related to the continuous release of ROS [In vivo, ROS can directly bind to lipids on cell membranes, causing oxidation reactions that eventually lead to lipid peroxidation. The end product of lipid peroxidation is malondialdehyde (MDA), which damages mitochondrial DNA (mtDNA) and induces apoptosis [2+ release [Other studies found that TGF-n of ROS , and ROSn of ROS . As descn of ROS . This ine of ROS . In vivopoptosis . MDA conpoptosis . In addipoptosis –67, suggpoptosis . One stupoptosis . Another release . Some st release , 72.β activation, upregulated collagen expression, and induced fibrosis in the lung fibroblasts through the ROS-mediated p38 MAPK signaling [There is evidence to show that p38 MAPK is activated in response to the fibrosis induced by single-walled carbon nanotubes, a process that requires the participation of ROS . The samignaling .In addition to their lipid-lowering properties, statins have antioxidative properties. For example, many studies have confirmed that statins can inhibit NOX activity and reduce ROS production –76. ERK,In addition to being associated with apoptosis, ROS are also involved in autophagy , an intrThe expression of ROS is significantly upregulated in the lung tissues of patients with IPF, a change that may be related to external stimuli, pharmacological factors, and the continuous release of inflammatory cells. Increased apoptosis and inadequate autophagy have also been observed in the lung tissues of patients with IPF, and the changes are associated with the altered activity of ROS-mediated signaling pathways see . Thus, sβ/CTGF interactions, and specifically override the potent induction of CTGF by TGF-β [β-induced CTGF expression, and loss of Rho protein can inhibit the expression of CTGF [In 2004, Watts et al. found that simvastatin, which blocks Rho prenylation and its subsequent signaling, can significantly inhibit CTGF gene expression in normal human lung fibroblasts IMR 90 cells) and IPF-derived lung fibroblasts , modulate TGF-by TGF-β . Geranylby TGF-β . Statinsby TGF-β . Rho pro cells anα in the bronchoalveolar lavage fluid of mice, and it could also inhibit the expression of TGF-β and CTGF and reduce the accumulation of collagen in a murine model of bleomycin-induced pulmonary fibrosis [β in the bronchoalveolar lavage fluid of rats. However, they found that simvastatin did not reduce the levels of hydroxyproline in the lungs of bleomycin-treated rats [β1-induced EMT and inhibited Smad2/3 phosphorylation [α (MIP-1-α) and significantly reduced the number of fibrocytes and the degree of collagen deposition in the lung tissues of mice. They also found that compared with that of the placebo group, mice in the lovastatin group exhibited a 45% lower risk of death, which represented a significant survival advantage [β, and p38 MAPK interact with each other to jointly affect the degree of tissue fibrosis. In a study that investigated the effects of statins on acute hypoxia-induced fibroblast proliferation in the lungs and systemic arterial system and on p38 MAPK activity, Carlin et al. found that fluvastatin was effective in inhibiting both processes [β; following degranulation, the components can promote the proliferation of fibroblasts, and alveolar macrophages can also secrete TGF-β, thereby promoting the enhancement of collagen fiber synthesis and leading to the deposition of ECM [γ in alveolar macrophages and inhibited the pulmonary fibrotic response [Ou et al. confirmed that simvastatin could reduce the number of neutrophils and lymphocytes, and inhibit the expression of TNF-fibrosis . The stufibrosis . For exated rats . Yang etrylation . Zhu et rylation . Hamblindvantage . As discdvantage . ROS, TGrocesses . In anotrocesses . El-Moharocesses . Mast cen of ECM . In 2017response . The benresponse . Studiesresponse . For exaresponse . The mevresponse . Simvastresponse , 105.β can induce senescence of the human bronchial epithelial cells, secrete excessive IL-1β, and promote the transformation of fibroblasts into myofibroblasts in the IPF lung samples [β, interleukin-6 (IL-6), and MMP-12, which regulated pulmonary fibrosis, and deletion of senescent cells reduces pulmonary expression of these factors [β, IL-6, and TGF-β [In addition to these factors, researchers have found that there is a close relationship between cell senescence and IPF. Senescent cells can secrete a variety of cytokines, chemokines, growth factors, and MMPs, which constitute senescence-associated secretory phenotype (SASP) . Minagaw samples . Schafer factors . Accordi factors . Liu et nd TGF-β . This inCollectively, the basic science trials described above (and summarized in P = 0.895) after adjusting for comorbidities due to the large variability in the baseline measurements between the two groups. The statistical results showed that the use of statins did not improve the median survival time of patients with IPF. Concurrently, researchers also found that the prevalence of coronary heart disease and the number of smokers were significantly higher in the statin group than in the control group, and the number of participants in the statin group was lower, which may explain the negative results of that study.The earliest clinical trial to investigate the association between IPF and the use of statins was published in 2004. A total of 477 participants diagnosed with IPF were included in the study, among whom 35 (7%) were treated with statins, 18 were with lovastatin, nine were treated with simvastatin, six with pravastatin, and two with fluvastatin; the remaining 442 (93%) participants were not treated with statins. The mean age of the patients in the statin group was 66.6 ± 8.7 years, whereas that in the nonstatin group was 70.9 ± 8.9 years. The study comprised 29 men (83%) in the statin group and 302 men (68%) in the nonstatin group. Nadrous et al.  retrospeP = 0.05), which suggested that the use of statins could prolong the median survival time of patients with IPF while also reducing the all-cause mortality.Despite the lack of effect, these findings did not discourage researchers from investigating whether statins are effective in the treatment of IPF. For example, in 2015, Vedel-Krogh et al.  retrospeHowever, the results of a prospective cohort study did not show reduced mortality in patients with IPF with the use of statins. A total of 462 patients (mean age: 76.5 ± 9.1 years) diagnosed with IPF and who had started long-term oxygen therapy were enrolled, including 312 men (68%) and 150 women (32%). There were 122 (26%) patients in the statin group and 340 (74%) in the nonstatin group. Ekstrom et al.  found noP = 0.1135), all-cause mortality (multivariate HR: 0.54 (95% CI: 0.24–1.21); P = 0.1369) or absolute forced vital capacity (FVC) decrease ≥ 10% (multivariate HR: 0.71 (95% CI: 0.48–1.07); P = 0.1032). However, there were statistically significant differences between the group in terms of a decrease in the 6-minute walk distance of >50 m (multivariate HR: 0.69 (95% CI: 0.48–0.99); P = 0.0465), all-cause hospitalization (multivariate HR: 0.58 (95% CI: 0.35–0.94); P = 0.0289), respiratory-related hospitalization (multivariate HR: 0.44 (95% CI: 0.25–0.80); P = 0.0063), and IPF-related mortality (multivariate HR: 0.36 (95% CI: 0.14–0.95); P = 0.0393). The findings suggested that the use of statins could reduce the hospitalization rates, delay the decrease in the 6-minute walk distance, prolong the survival time, and improve the quality of life in patients with IPF.In a post hoc analysis of statin treatment for IPF, 624 patients diagnosed with IPF were included, including 276 (44%) in the statin group and 348 (56%) in the nonstatin group. There were 465 men (75%) and 159 women (25%) in the study. The baseline characteristics were similar between the groups, except that the mean age was higher in the statin group than in the nonstatin group , and the statin group had a higher risk of cardiovascular disease. Kreuter et al.  analyzedP = 0.1065), although the difference was not statistically significant. There was no statistically significant difference between the two groups in the decline in FVC% of ≥5% predicted or death (HR: 0.81 (95% CI: 0.62–1.06); P = 0.1235), decline in FVC% of ≥10% predicted or death (HR: 1.01 (95% CI: 0.72–1.41); P = 0.9700), time of the first acute exacerbation of IPF (HR: 0.76 (95% CI: 0.33–1.73); P = 0.5144), or the St. George's Respiratory Questionnaire (SGRQ) total score (difference: 1.22 (95% CI: -1.65–4.09); P = 0.4039). Studies have shown that statins do not improve IPF-related mortality or the first acute exacerbation, and although they may delay the decline in FVC, they cannot completely prevent the progressive decline of FVC. In 2018, Kreuter et al. also investigated whether using statins at baseline increased the antifibrotic effect of nintedanib; the results showed that compared with nintedanib alone, the use of nintedanib in combination with statins had no significant effects on FVC, SGRQ score, disease progression, or acute exacerbations in patients with IPF.Kreuter et al.  publisheP < 0.001), as was the decrease in annual diffusing capacity of the lungs for carbon monoxide (DLCO), P = 0.013), but statins did not improve survival in patients with IPF (multivariate HR: 0.82 (95% CI: 0.50–1.35), P = 0.428). Lambert et al. also found that statins combined with nintedanib or pirfenidone did not improve lung function in patients with IPF compared with statins alone .A recently published study included 323 patients diagnosed with IPF between January 2013 and December 2017, with an average age of 70.8 years at the time of IPF diagnosis. The study included 247 men (76.5%). In total, 171 patients (53%) were treated with statins  and 152 (47%) were not treated with statins during the study period. Lambert et al.  reportedThe six clinical trials described above  and synthetic compounds   see Fig. The meceductase . The stueductase  and increductase . Lovastaeductase . Lovastavastatin . The stuvastatin . Pravastvastatin . The stuvastatin . Atorvasvastatin . Patientvastatin . Rosuvasvastatin . Patientvastatin . DiffereOn reviewing the literature, we noted that Xu et al. reported that statins can aggravate pulmonary fibrosis. In their regression analysis, researchers found that statin use was associated with interstitial lung abnormalities among smokers and that statins enhance bleomycin-induced lung inflammation and fibrosis in mice through a mechanism involving enhanced NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation, which appears to be related to the increase in mitochondrial ROS associated with the administration of statins . Similarβ/CTGF, inhibit the Rho signaling pathway and the phosphorylation of Smad2/3, suppress the activity of YAP/TAZ, and attenuate pulmonary fibrosis in basic science studies, including in vitro assays or animal models. In clinical trials in humans, Kreuter et al. in 2018 and Lambert et al. in 2021 showed that statins could slow the decline in FVC in patients with IPF, and Lambert et al. also found that statins could delay the decline of lung diffusion capacity. Vedel-Krogh et al. in 2015 and Kreuter et al. in 2017 showed that statins extended the median survival time and reduced mortality in patients with IPF, with the study by Kreuter et al. also showing that statins significantly reduced IPF-related hospitalizations and delayed the decrease in the 6-minute walk distance. Collectively, these clinical trials demonstrated the possibility that statins could be efficacious in the treatment of IPF. However, some findings have been negative or contradictory; for example, Kreuter et al. in 2017 showed that statins did not delay the decline in FVC in patients with IPF, and Nadrous et al. and Ekstrom et al. found that statins did not prolong the median survival time of patients with IPF. Lung function is closely related to age and individual baseline conditions, such as cardiovascular diseases and hyperlipidemia, which may lead to impaired lung function [It has been demonstrated that statins can inhibit the inflammatory response after lung tissue injury, reduce the number of inflammatory cells, decrease the expression of TGF-function . The negβ and inhibits the proliferation of fibroblasts [P = 0.001) and was more effective at 24 weeks compared with the placebo group, but there was no difference in the percentage of predicted DLCO or dyspnea [To date, only two drugs have been approved for the treatment of IPF, including nintedanib and pirfenidone. Nintedanib (formerly known as BIBF1120) is a potent intracellular inhibitor of tyrosine kinases that competitively binds to the kinase domains of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) . In a raroblasts . In a cl dyspnea . In anot dyspnea . AccordiTo date, basic science and clinical trials that have assessed the treatment of IPF with statins have predominantly focused on simvastatin and atorvastatin. The effects of various other types of statins on the treatment of IPF are unknown, and it is unclear whether different types of statins or those with a particular structure could have different benefits in patients with IPF. To date, no prospective, randomized, controlled, double-blind trials have been conducted to evaluate the efficacy of statins in the treatment of IPF. More clinical studies are required to determine the efficacy of statins alone in the treatment of IPF and its related comorbidities; in addition, we hope to determine whether combination treatments with statins and the currently available antipulmonary fibrosis drugs such as nintedanib and pirfenidone would benefit patients with IPF compared with the administration of statins alone. We hope to focus on the research and development of molecular targeted therapies to delay the progression of the disease and improve the survival rate and quality of life in patients with IPF.


Alzheimer’s disease (AD) is a neurodegenerative disorder for which there is currently no effective treatment. Despite advances in the molecular pathology of the characteristic histopathological markers of the disease (tau protein and β-amyloid), their translation to the clinic has not provided the expected results. Increasing evidences have demonstrated the presence of aggregates of TDP-43 (TAR DNA binding protein 43) in the postmortem brains of patients diagnosed with AD. The present research is focused on of the study of the pathological role of TDP-43 in AD. For this purpose, immortalized lymphocytes samples from patients diagnosed with different severity of sporadic AD were used and the TDP-43 pathology was analyzed against controls, looking for differences in their fragmentation, phosphorylation and cellular location using Western blot and immunocytochemical techniques. The results revealed an increase in TDP-43 fragmentation, as well as increased phosphorylation and aberrant localization of TDP-43 in the cytosolic compartment of lymphocytes of patients diagnosed with severe AD. Moreover, a fragment of approximately 25 KD was found in the extracellular medium of cells derived from severe AD individuals that seem to have prion-like characteristics. We conclude that TDP-43 plays a key role in AD pathogenesis and its cell to cell propagation. Alzheimer’s Disease (AD) is a neurodegenerative disorder marked by progressive impairment of cognitive ability, generally in later life, affecting more than 50 million people worldwide . Age is The disease begins with a decline in cognition followed by a number of other changes in brain functioning, including impairments in language and visual-spatial skills, and disorientation. This impairment in cognitive functions is due to anatomical atrophy of AD brains, which correlates with severe neuronal loss . In partamyloid precursor protein (APP), presenilin-1 (PSEN-1), presenilin-2 (PSEN-2) [Based on age of onset, AD can be divided into early onset AD, and the more common, late-onset AD, in people over 65 years old, accounting for more than 90% of all AD cases . Most ca(PSEN-2) . On the AD is considered a multifactorial disease associated with a number of risk factors other than advanced age and genetic factors, such as environmental factors, diet, cardiovascular diseases, and diabetes, head injuries, and obesity among others ,11,12. RAD is characterized by two main neuropathological hallmarks: extracellular amyloid-β (Aβ) deposition in senile plaques and intracellular accumulations of hyperphosphorylated microtubule-associated tau, called neurofibrillary tangles (NFT) ,15,16.Based in the density of NFTs in various brain regions, AD cases have been classified in three different subtypes hippocampal-sparing, with lower NFT in hippocampus, limbic-predominant with lower cortical NFT, and the more frequent typical AD forms . In addiTDP-43 was originally found to associate with frontal temporal lobar degeneration with ubiquitin inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS) . These nThe manner in which TDP-43 pathology induces neuron degeneration is not yet completely understood, and the role of TDP-43 in cognition remains elusive, since conflicting results had been reported ,29. AppaTDP-43 is a predominantly nuclear protein, although it can shuttle between the nucleus and cytosol. It plays a variety of roles in RNA metabolism, including transcription, splicing, mRNA transport, mRNA stability through recruitment into stress granules (SGs), and microRNA biosynthesis ,32. UndeThe present work was undertaken to better understand the role and significance of TDP-43 in AD. To accomplish this goal, we assessed and compared pathological characteristics of TDP-43 of control and AD-derived immortalized lymphocytes. These lymphoblastoid cells lines had been extensively characterized in our laboratory, demonstrating that they display disease specific signature molecules ,37,38, aThe results presented here indicate an increase in TDP-43 fragmentation, as well as increased phosphorylation and aberrant localization of TDP-43 in the cytosolic compartment of lymphoblasts of patients diagnosed with severe AD. Moreover, a fragment of approximately 25 KDa was found in the extracellular medium of cells derived from AD individuals, that appears to be transported by extracellular vesicles (EVs) inducing TDP-43 pathological features and cytoskeletal changes in control cells. The data may suggest a role of these EVs containing fragmented TDP-43 in contributing to the propagation of AD disease in a prion-like manner.RPMI 1640 culture medium , DMEM culture medium , Fetal Bovine Serum (FBS) , penicillin/ streptomycin , polyvinylidene difluoride (PVDF) membranes for Western blots , enhanced chemiluminescence (ECL) system , Pierce BCA Protein Assay kit , protease inhibitor complete mini mixture .Antibodies used in this study were from Santa Cruz Biotechnologies , Cell Signal , Thermo Fisher , Molecular Probes (Thermo Fisher), Bio-Rad  or Proteintech,  and are listed in Healthy controls and patients were recruited from Hospital Doce de Octubre, Madrid Spain. The clinical diagnosis of probable AD was based on the criteria of the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA) , and diaBlood samples (approximately 8 mL) were obtained through antecubital vein puncture in EDTA-treated tubes. Peripheral blood mononuclear cells (PBMCs) were isolated on Lymphoprep™ density-gradient centrifugation according to the instructions of the manufacturer .Establishment of lymphoblastoid cell lines (LCLs) was performed in our laboratory, by infecting peripheral blood lymphocytes with the Epstein Barr virus (EBV) .v/v) fetal bovine serum (FBS), and 1% penicillin/ streptomycin. U2OS cells were obtained from the American Type Culture Collection and grown in DMEM media, supplemented with 10% (v/v) fetal bovine serum and 1% penicillin/streptomycin. All cell lines were grown at 37 °C in a humidified 5% CO2 atmosphere.Lymphoblastoid cells were grown in suspension in T flasks in an upright position, in approximately 10 mL RPMI 1640 medium that contained 10%  and the associated software using the manufacturer’s recommended conditions. Each reaction was performed in biological triplicates with 20 ng of cDNA by using FastStart Essential DNA Green Master . The sequences of the forward and reverse primers used are the following: for hTDP-43 FW: 5′-GAGAAGTTCTTATGGTGCAG-3′and RV: 5′-TGGCTTTGCTTAGAATTAGG-3′, for hRPS17 FW: 5′-CCATTATCCCCAGCAAAAAG-3′ and RV: 5′-GAGACCTCAGGA ACATAATTG-3′. Data analysis was based on the ΔΔCT method with normalization of the raw data to hRPS17 housekeeping gene.Total RNA extraction, cDNA synthesis and quantitative polymerase chain reaction (PCR) was done as detailed in the previous article . Quantit6 cells × mL−1 and 24 h later, cells were harvested, washed in PBS and then lysed in ice-cold RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 15 mM MgCl2, 0.5% (vol/vol) sodium deoxycholate, 0.5% (vol/vol) NP-40 and 0.1% (vol/vol) SDS), containing 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonylfluride (PMSF), 1 mM sodium pyrophosphate and protease inhibitor mixture. The protein content of the extracts was determined by de Pierce BCA Protein Assay kit.To prepare whole cell extracts the lymphoblasts were seeded at initial density of 1 × 1050-µg of protein from cell extracts or 35 µL of extracellular medium were fractionated on an SDS polyacrylamide gel, transferred to PVDF membrane and blocked with 5% non-fat milk. The antibodies used are listed in 2B4O7·10H2O 15 mM, pH 8.4) overnight at 37 °C.Immunofluorescence analysis was performed on cells grown on coverslips. For the attachment of lymphoblastoid cells, the coverslips were previously coated with a solution of 0.025% Gelatin  for 30 min at room temperature followed by a solution of 1 mg/mL poly-L-lysine  diluted 1:50 in Borax buffer  following manufacturers’ recommendations. EV-containing pellet was resuspended in phosphate-buffered saline and stored at −20 °C until further analyses.To generate conditioned medium for extracellular vesicles isolation, lymphoblasts were grown in RPMI medium with 10% of exosome-depleted FBS , instead of normal FBS. After 72 h, conditioned medium was recollected by centrifugation at 2000× EV characterization was performed by nanoparticle tracking analysis (NTA) and Western Blot. Size distribution and quantification of EVs preparations were analyzed by measuring the rate of Brownian motion with a NanoSight LM10 system with a 630 nm laser , equipped with fast video capture and particle-tracking software. EVs samples were diluted 1:1000 in Hank’s balanced salt solution (HBSS), disaggregated and injected in the sample cubicle of the NanoSight. The mean of the number of particles acquired per milliliter was compared between AD and control cells and the measurements of EVs concentration (particles/mL) were normalized with the cell number from which conditioned medium containing EVs was recollected. For Western Blot analysis 20 µL of EVs were lysed with Laemmli buffer and denaturalized at 95 °C for 5 min. Western Blots were performed in parallel in EVs samples and cell lysates and the antibodies used are described in t-test for statistical comparisons between groups. A “p-value < 0.05” was considered statistically significant.Statistical analyses were performed using Graph Pad Prism software version 6 . Data are presented as means ± standard deviation (SD) of the mean. Statistical significance was estimated using one-way ANOVA followed by the Bonferroni test for multiple comparations, or, two-tailed Student’s To examine TDP-43 pathology in human lymphoblasts, we first analyzed the expression of TDP-43 mRNA and protein levels in immortalized lymphocytes from AD patients as a function of disease severity. Interestingly, a longer exposure of TDP-43 blots revealed the presence of a TDP-43 fragment of approximately 25–30 KDa perhaps due to TDP-43 cleavage, in most of the AD cases but not in control samples B.We then, analyzed the phosphorylation status of TDP-43 in control an AD  patients, using a phospho-specific antibody by Western blotting and confocal laser microscopy. As shown in Increased phosphorylation of TDP-43 had been associated with mislocalization of the protein in TDP-43 proteinopathies such as ALS and FTD . For thiMoreover, immunofluorescence staining for filamentous actin (F-actin) with Alexa Fluor-568 phalloidin, revealed cytoskeletal abnormalities in AD lymphoblasts A such asIt has been documented that full-length and fragmented TDP-43 may be released in extracellular vesicles or exosomes in different neurological diseases. For this reason, we first preformed Western blot analysis looking for TDP-43 protein in the extracellular medium of control and AD lymphoblasts. Furthermore, CM from AD lymphoblasts induces also TDP-43 pathological changes, mislocalization and generation of TDP-43 aggregates in osteosarcoma U2OS cell line .Finally, we isolated extracellular vesicles (EVs) from CM of control and AD cells, and NTA analysis was used to identify their size and concentration. As shown in Over the past decade, TDP-43 deposition has been associated with an increasing number of neurodegenerative diseases  being thIn this study we have examined some TDP-43 features in peripheral cells from AD patients as a function of disease severity. For this purpose, a comparative study on TDP-43 expression levels as well as TDP-43 phosphorylation pattern, protein fragmentation and subcellular localization was performed in lymphoblasts derived from control subjects and mild, moderate and severe AD patients.The results herein presented show the presence of pathological features of TDP-43 in these easily accessible patient-derived cell lines, that could be considered systemic manifestations of the disease. Increased TDP-43 phosphorylation, protein cleavage, and cytosolic TDP-43 accumulation are more evident in lymphoblasts from severe AD patients. Moreover, higher expression of TDP-43 mRNA was observed in moderate and severe AD cases, which may be the result of increased self-regulation of TDP-43 expression, in order to maintain homeostasis in late stages of AD . These oTDP-43 pathological changes observed in AD lymphoblasts are similar to those found in lymphoblastoid cell lines derived from FTLD-TDP and ALS patients ,40, despNowadays, there is an expanding field of research trying to elucidate how TDP-43 pathology may be mechanistically related with AD, especially with the limbic-predominant subtype, in which TDP-43 deposition is more frequent . It is nTDP-43 may participate in various pathogenic mechanisms underlying AD, such as amyloid β deposition, tau hyperphosphorylation, mitochondrial dysfunction, and neuroinflammation . TDP-43 TDP-43 pathological features, such as increased phosphorylation, cleavage, aggregation and cytoplasmic accumulation are believed to impaired neuronal function . It is sSince TDP-43 protein contains a prion-like domain in its C-terminus, it was hypothesized that TDP-43 toxic species could be transmitted intercellularly . TherefoFinally, it is worth highlighting that fragmented TDP-43 (25 KDa) and, in less amount, full-length TDP-43 were detected in EVs isolated from AD lymphoblasts. EVs have been shown to participate in the communication and transfer of cargo among different cell types within the CNS , modulatWe confirmed that TDP-43 pathology is present in peripheral cells from AD patients, being exacerbated with disease severity. Thus, protein kinase inhibitors able to modulate TDP-43 emerge as promising agents for AD including those patients in more advanced stages. Furthermore, the addition of these treatments to those able to decrease amyloid-β may open new hopes for AD therapy. A prion-like disease propagation of TDP-43 pathology via actin protrusions and secretion of fragmented TDP-43 in EVs is in line with the finding that conditioned medium from AD cells provoked TDP-43 phosphorylation and cytoplasmic accumulation in control cells. Although further work is warranted for a better understanding of mechanisms involved in disease dissemination and to elucidate the possible interactions of pathological TDP-43 with other AD-associated molecules, amyloid-β and tau, for patient stratification and development of novel therapeutic strategies, this last discovery support TDP-43 potential therapies as disease-modifying agents able to modulate the neurodegeneration.The results of this study are somehow limited by the scarce number of patients included in the analysis. The patient-sample set should be increased to validate these results. We believe that the analysis of the complex interactions among TDP-43, amyloid-β, and tau may open new areas of therapeutic interest for the treatment of this devastating disease, mainly based in multitarget approaches. Research aiming to study the influence of protein kinase inhibitors able to recover TDP-43 homeostasis, in pathogenic mechanisms underlying AD pathology and spreading is ongoing in our laboratory.


GAP-43 is a vertebrate neuron-specific protein and that is strongly related to axon growth and regeneration; thus, this protein has been utilized as a classical molecular marker of these events and growth cones. Although GAP-43 was biochemically characterized more than a quarter century ago, how this protein is related to these events is still not clear. Recently, we identified many phosphorylation sites in the growth cone membrane proteins of rodent brains. Two phosphorylation sites of GAP-43, S96 and T172, were found within the top 10 hit sites among all proteins. S96 has already been characterized , and here, phosphorylation of T172 was characterized. In vitro (cultured neurons) and in vivo, an antibody specific to phosphorylated T172 (pT172 antibody) specifically recognized cultured growth cones and growing axons in developing mouse neurons, respectively. Immunoblotting showed that pT172 antigens were more rapidly downregulated throughout development than those of pS96 antibody. From the primary structure, this phosphorylation site was predicted to be conserved in a wide range of animals including primates. In the developing marmoset brainstem and in differentiated neurons derived from human induced pluripotent stem cells, immunoreactivity with pT172 antibody revealed patterns similar to those in mice. pT172 antibody also labeled regenerating axons following sciatic nerve injury. Taken together, the T172 residue is widely conserved in a wide range of mammals including primates, and pT172 is a new candidate molecular marker for growing axons. Growth-associated protein of 43-kDa  is a neuron-specific protein in vertebrates and is related to axon growth and regeneration –4. Thus,2+-independent manner , 5 μM of SB203580 (Sigma-Aldrich) as a p38 inhibitor, or 5 μM of U0126  as a MEK1/2 inhibitor that at this concentration works as an ERK inhibitor ) for reducing background staining for 10 min. They were then permeabilized with 0.1% Triton X-100  in CSB for 10 min, were blocked with 3% bovine serum albumin  for 30 min, and reacted with primary and secondary Abs in 3% BSA. To visualize F-actin, the cells were incubated with rhodamine-phalloidin . Quantitative distribution of the region of interest (ROI) of the growth cone area was measured by fluorescence intensity along a line plot from the filopodial tip to display a two-dimensional graph of pixel intensity for pT172 and F-actin images using ImageJ software Fiji (http://rsb.info.nih.gov/ij). The x-axis represents the distance along the line, and the y-axis is the normalized gray value.Mouse E15 cortical or hippocampal neurons were differentiated into stage 3, when each neuron has an axon , and immFluorescent images were acquired with a confocal laser scanning microscope  as previously described . For linThe ratio of the fluorescence values of GAP-43 and pT172 in the longest neurite defined as an axon and the second longest neurite defined as a dendrite was obtained using Fiji. The average fluorescence intensity of each ROI was measured.Human induced pluripotent stem cells (1210B2), which were established from peripheral blood mononuclear cells  at the Center for iPS Cell Research and Application, Kyoto University  by an integration-free reprogramming method, were induced into neural progenitor cells using the dual SMAD-inhibition method with dorsomorphin plus SB431542 as previously described , 66. hiPThe shotgun proteomics method was described previously , 13. BriThe gel slices were reduced with 10 mM dithiothreitol, carbamide methylated with 55 mM iodoacetamide, and subjected to in-gel trypsin digestion to improve recovery of the digested peptides. The peptides from each sample were finally dissolved in 15 μl of 0.3% trifluoroacetic acid. A 50-kDa-level gel sample (3 μl of 1/5 dilute solution) and a 10-kDa-level gel sample (4 μl) were injected into a nano-flow-LC  coupled with a tandem MS .Analysis was conducted in triplicate for each sample under direct injection mode with an analytical column of 75 μm × 150 mm . Mobile phases A and B were 0.1% formic acid and 0.1% formic acid in acetonitrile, respectively. Peptides were eluted using 40 (10 kDa) or 60 (50 kDa)-min gradients from 2 to 32% B at 300 nl/min. MS spectra (250 ms) followed by 10 MS/MS spectra (100 ms each) were acquired in data-dependent mode. The dynamic exclusion time was set at 5 s. Autocalibration using 50 fmol of tryptic peptides of bovine serum albumin was performed. Protein identification was carried out using Mascot version 2.6.0 (Matrix Science) as a search engine  protein database was used to search for amino acid sequences in human, cynomolgus monkey, marmoset, ferret, rat, and mouse GAP-43. The GAP-43 protein sequence of ferret was that of the predicted one. Multiple sequence alignment analysis was displayed using these search results by Snap gene software .t test and one-way analysis of variance (ANOVA) were used for statistical analysis, as appropriate, and a p-value < 0.05 was considered statistically significant. Significance testing using one-way ANOVA was further performed followed by a post hoc test using Bonferroni methods. All statistical analyses were performed using GraphPad Prism 8 software.All data are shown as the means ± standard deviation (SD). Paired Student’s Additional file 1: Table 1. Antibodies used for immunological detection in this paper.Additional file 2: Figure S1. Intracellular localization of phosphorylated GAP-43 T172 and its responsible kinase. (a) As well as pan-GAP-43 Ab (left), the immunoreactivity of pT172 Ab on full-size membranes (right), showing a wider range of molecular masses, indicated that this Ab mostly recognizes a single band of mouse GAP-43 (mGAP-43) in the total proteins derived from the lysate of cultured mouse cortical neurons (DIV3). (b) In vitro phosphorylation of GAP-43 by activated JNK. Myc-GAP-43 was immunoprecipitaed using anti-myc and used as the JNK substrate. Phosphorylation experiments were performed following the manufacturer’s manual of “JNK1, Acitve” . (c) Immunostaining of cultured hippocampal neurons using pT172 Ab (blue) with rhodamine-phalloidin (red) and Tuj-1 Ab (green). Note that pT172Ab labeled the tubulin-positive area of the axon more intensely than the actin-positive area. Scale bar: 20 μm. (d) pT172Ab immunoreactivity in the growth cone was colocalized with GAP-43  at ROI (white box). Scale bar: 10 μm.Additional file 3: Figure S2. pT172 and GAP-43 are regulated in mouse developing brain. (a) The distribution of GAP-43, pS96, and pT172 persists throughout life . GAPDH: positive control. The frontal cortical lysates of a C57BL6N mouse were used. (b) Densitometric quantitation of (a). Values are represented as the means ± SD for three experiments. Although some GAP-43 expression was maintained throughout life, the immunoreactivity of pT172 and pS96 decreased rapidly with development. One-way ANOVA followed by a post hoc test using the Bonferroni method. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (c) Immunoblot for pT172Ab on full-size membranes. Note that this Ab mostly recognizes a single band of GAP-43 in the total protein of cortical lysate.Additional file 4: Figure S3.. Expression pattern of pT172 in the developing mouse brain. (a) Negative control immunostaining of E15 thalamocortical pathway. pT172 pAb (a-1), secondary Ab alone , and nonspecific IgG . Each concentration of proteins was 0.26 µg/ml. Scale bar: 200 µm. (b) Microscopic images of sagittal sections derived from various brain regions were DAB-stained using pT172. Boxes in (b) represent the regions enlarged in (c-g). pT172 succeeded in labeling nerve fibers under a growing state. Scale bar: 500 µm in (b), 200 µm in .  Z-stack maximum-intensity projection images of basal nuclei (h) and OB (i). pT172 was more closely colocalized in the developing axons labeled by L1 than pan-GAP-43 in OC, LOT, and STR. GAP-43 and pT172 were detected in neurons of MCL and ONL (i). CP: cortical plate; LOT: longitudinal olfactory tract; MCL: mitral cell layer; OB: olfactory bulb; OCh: optic chiasm; ONL: olfactory nerve layer; Teg: longitudinal tegmental tracts; STR: striatum; PON: pons; EGL: external granular layer, PK: Purkinje cell layer.Additional file 5: Figure S4. pT172Ab immunoreactivity in regenerating axons after neural tissue injury in young adult mice.  pT172Ab immunoreactivity detected in injured sciatic nerve (day 1 (a) and day 3 (b) after crush injury). Black-arrowhead: damaged point; white-arrowhead: the farthest point of immunoreactivity. (c) pT172Ab immunoreactivity was not present in uninjured sciatic nerves, while GAP-43 reactivity itself was detected there. Scale bar: 500 μm. (a-c). See also Fig. 3.Additional file 6: Figure S5. Amino acid sequences of GAP-43 surrounding the residue corresponding to S96 of rodents, in various mammalian species. S96 position of rodent GAP-43 is shown in red. See Discussion.Additional file 7: Figure S6. Phospho-specificity of GAP-43 pT181 in the P1 marmoset. (a) Negative control immunostaining of P1 Marmoset visual cortex. pT172 pAb (a-1), secondary Ab alone , and nonspecific IgG . See also Fig. S3. Scale bar; 500 µm. (b) Immunoblot reactivity of pT172 Ab on a full-sized blot membrane. These results show the specificity of pT172Ab. (c) Phosphoproteomics procedure for the mid-brain including pons of P1 marmoset. The band corresponding to GAP-43 was cut out after SDS-PAGE of the sample, trypsinized in-gel, and subjected to MS analysis. *, molecular mass marker. Note that the GAP-43 immunoreactivity band (arrow in red) did not consist of the GAP-43 amino acid sequence by LC/MS/MS analysis, suggesting that it may be a non-specific reaction. The analyzed results are shown in Fig. 5p.Additional file 8: Figure S7. Stress-dependent JNK activation phosphorylates human GAP-43 T181 and is recognized by pT172Ab. Extracts from the transfected U251G cells were prepared at 30 min after osmotic stress (0.5 M NaCl) for JNK activation and immunoblotted using pT172, GAP-43, GFP, pJNK, JNK, and GAPDH Abs by the methods of Kawasaki et al. (2018).


An assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) has become an increasingly popular method to assess genome-wide chromatin accessibility in isolated nuclei from fresh tissues. However, many biobanks contain only snap-frozen tissue samples. While ATAC-seq has been applied to frozen brain tissues in human, its applicability in a wide variety of tissues in horse remains unclear. The Functional Annotation of Animal Genome (FAANG) project is an international collaboration aimed to provide high quality functional annotation of animal genomes. The equine FAANG initiative has generated a biobank of over 80 tissues from two reference female animals and experiments to begin to characterize tissue specificity of genome function for prioritized tissues have been performed. Due to the logistics of tissue collection and storage, extracting nuclei from a large number of tissues for ATAC-seq at the time of collection is not always practical. To assess the feasibility of using stored frozen tissues for ATAC-seq and to provide a guideline for the equine FAANG project, we compared ATAC-seq results from nuclei isolated from frozen tissue to cryopreserved nuclei (CN) isolated at the time of tissue harvest in liver, a highly cellular homogenous tissue, and lamina, a relatively acellular tissue unique to the horse. We identified 20,000–33,000 accessible chromatin regions in lamina and 22–61,000 in liver, with consistently more peaks identified using CN isolated at time of tissue collection. Our results suggest that frozen tissues are an acceptable substitute when CN are not available. For more challenging tissues such as lamina, nuclei extraction at the time of tissue collection is still preferred for optimal results. Therefore, tissue type and accessibility to intact nuclei should be considered when designing ATAC-seq experiments. The completion of the equine genome assembly  has enabAn important component of gene expression and regulation is chromatin accessibility. Active genes and regulatory elements are typically found within or near regions of the DNA accessible to transcription factors. Therefore, identifying open chromatin regions is a crucial step to identify and categorize tissue specific regulatory elements in order to advance our understanding of complex traits in the horse. An assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq)  is commoTo address these gaps of knowledge in the applicability of ATAC-seq in snap-frozen horse tissues, and to provide a guide for future ATAC-seq studies to assess chromatin accessibility, we compared data from CN prepared from fresh tissue to that of nuclei extracted from snap-frozen tissues collected from the two mares from the initial equine FAANG biobank study . In ordeLiver and lamina tissues from two mares (AH2 and AH1) were collected as described in Both snap frozen tissues and CN were stored at −80°C for 3 years until shipped on dry ice overnight to two commercial laboratories (L1 and L2) for library preparation. Nuclei were extracted from frozen tissues using each laboratory’s internally optimized protocol see . Extracthttps://github.com/SichongP/FAANG_ATACseq.Read QC was carried out using FastQC . Adapter1 under accession PRJEB35307. Histone marks were determined according to Histone ChIP-seq data were downloaded from FAANG data repositoryATAC-seq peaks called by HMMRATAC and MACS2 were validated using histone ChIP-seq data following  with modReal positive set (RP): peaks from either H3K4me1 or H3K4me3 that overlap H3K27ac peaksReal negative set (RN): peaks from H3K27me3 dataThen, following metrics were calculated for each dataset:Increasing quality scores as produced by MACS2 or HMMRATAC were used as the cutoff score to filter peaks before the remaining peaks were used to calculate above metrics. Changes in the metrics as the cutoff score increased were used to identify the thresholds at which to filter final sets of open chromatin peaks.PRJEB26787. Briefly, RNA was isolated from liver or lamina tissues using Trizol chloroform phase separation followed by a column cleanup using Zymo Research Direct-Zol Mini columns. TruSeq mRNA libraries were prepared at Minnesota Genomics Center  and sequenced at 125 bp paired-end. These reads were quantified against Equcab3 Ensembl annotated genes  overlapped with identified ATAC-seq peaks. These genes were then compared to their RNA abundance estimated using FAANG data.Libraries prepared by two laboratories (L1 and L2) using nuclei isolated from snap-frozen tissues (FTDN) or cryopreserved from tissues at time of collection (CN) from liver and lamina of two animals  were sequenced at PE75 on an Illumina HiSeq 4000 (L1) or PE42 on an Illumina NextSeq 500 (L2). ATAC-seq libraries are expected to present a laddering pattern that corresponds to different nucleosome-bound fragments. Since libraries from L1 and L2 were sequenced at different lengths , we trimmed longer reads from L1 from 3′ down to 42 bp and compared read alignment statistics to those obtained using full length reads (75 bp), after appropriate quality trimming. There were no significant changes in read alignment statistics, with less than 0.02% fewer reads aligned and less than 0.3% fewer reads identified as duplicates for each library after length trimming. Therefore, we proceeded with data analysis using original full length reads from both laboratories.Overall, liver libraries have higher mitochondrial contamination than lamina libraries, likely due to higher metabolic activities in liver . Among lTo assess which part of the ATAC-seq protocol contributed more to library variations and complexities, we compared genome coverage and enrichment . PrincipTo identify accessible chromatin regions, MACS2  and HMMRTo better assess the quality of peaks, we used histone mark ChIP-seq data generated from the same samples as described in Despite higher quality from L2 in liver AH2 CN library, L1 produced the only libraries from laminae with high quality peaks . TherefoA cutoff score, where the precision and recall lines intercept, was used for each sample set to filter peaks identified by HMMRATAC. Final peak counts are shown in in silico validation of the results, peaks were overlapped with Ensembl gene annotation for EquCab3  from the equine FAANG project for ATAC-seq library generation, using two nuclei extraction methods. Nuclei extracted and cryopreserved immediately after tissue collection and nuclei isolated from snap-frozen tissues were used to determine suitable methods for performing ATAC-seq to identify accessible chromatin regions in a wide variety of equine tissues for functional annotation. Similar to what was identified by We also showed that the frozen tissue protocol is more prone to variations introduced at the library preparation step. Specifically, FTDN liver libraries generated at two different laboratories only have a moderate correlation (0.68 for AH1 and 0.76 for AH2). Our analysis suggests that, similar to RNA-seq experiments, library preparation can introduce large variation that will impact subsequent data quality, specifically peak detection for ATAC-seq studies. However, since the two commercial laboratories used different internally optimized protocols, it is impossible to determine whether the variation was protocol-specific or lab-specific. Nonetheless, it is advisable for all ATAC-seq library preparations to be performed at a single site using the same protocols to minimize variability in datasets when trying to integrate information.During library preparation, the CN aliquot from AH2 was partially thawed twice by L1 (first for an optimization experiment (data not shown) and then a second time to perform the data collection). The nuclei obtained during the second partial thawing were used in this study. Due to the precipitation of nuclei and contaminating mitochondria, this was likely the cause of low quality observed in that library preparation. The effect of different read lengths used by two laboratories was investigated and deemed to have no significant impact on read alignment. Our analysis suggested a detrimental impact on data quality by this practice and resequencing of this particular library also did not improve data quality nor was this resequencing effort able to identify more peaks. Therefore, it is advisable to avoid repeated partial thawing of CN aliquots.Library fragment size screening using gel electrophoresis proved to be predictive of final fragment size distribution in sequencing results and data quality. As indicated in We identified 20–33,000 accessible chromatin regions in lamina and 22–61,000 in liver, largely in line with observations of liver ATAC-seq from studies in other species . As a prIn this study, we demonstrated the feasibility of using snap-frozen tissues for ATAC-seq experiments for the equine FAANG project. For acellular tissues, more optimization is required for ATAC-seq experiments. We also showed that significant variation can be introduced during library preparation. This study provides important guidelines for planning future ATAC-seq experiments using equine FAANG tissues. We will use the guidelines established here to conduct ATAC-seq experiments on six other prioritized tissues in the mares. Furthermore, following these guidelines should enable the most meaningful integration of datasets across studies thus building a reliable functional tissue specific atlas of the equine genome which would advance our understanding of complex traits in the horse.PRJEB41317. RNA-seq data from the liver and lamina tissues of the same two animals used in this study can be found from the European Nucleotide Archive under the accession PRJEB26787.ATAC-seq data used in this study are available from the European Nucleotide Archive under the accession CF and SP were responsible for the conceptualization, methodology, investigation, formal analysis, resources, and writing of the manuscript. CF, RB, JP, and TK were responsible for the funding acquisition. All authors have reviewed the final manuscript.The cost of library preparation and sequencing was partially covered by two core laboratories as part of collaboration to optimize ATAC-seq protocol using horse tissues.


Idiopathic pulmonary fibrosis (IPF) is a progressive, lethal fibrotic lung disease that occurs primarily in middle-aged and elderly adults. It is a major cause of morbidity and mortality. With an increase in life expectancy, the economic burden of IPF is expected to continuously rise in the near future. Although the exact pathophysiological mechanisms underlying IPF remain not known. Significant progress has been made in our understanding of the pathogenesis of this devastating disease in last decade. The current paradigm assumes that IPF results from sustained or repetitive lung epithelial injury and subsequent activation of fibroblasts and myofibroblast differentiation. Persistent myofibroblast phenotype contributes to excessive deposition of the extracellular matrix (ECM) and aberrant lung repair, leading to tissue scar formation, distortion of the alveolar structure, and irreversible loss of lung function. Treatments of patients with IPF by pirfenidone and nintedanib have shown significant reduction of lung function decline and slowing of disease progression in patients with IPF. However, these drugs do not cure the disease. In this review, we discuss recent advances on the pathogenesis of IPF and highlight the development of novel therapeutic strategies against the disease. Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease of unknown etiology and with a poor prognosis. IPF primarily occurs in middle-aged and elderly adults. In the United States, median age of newly diagnosed patients is 62 years, and 54% of them are male . The epiThere is a growing portfolio of treatment options for IPF. Two drugs, nintedanib and pirfenidone, have been approved for treatment of patients with IPF. Nintedanib is a tyrosine kinase inhibitor, while pirfenidone is an oral pyridine that has anti-inflammatory, antioxidant, and anti-fibrotic effects. The two drugs have demonstrated reduction of lung function decline and slowing of disease progression, but they were also associated with some side effects and tolerability issues . Lung trGenetic factors play an important role in the development of IPF . GeneticThe genome-wide association studies (GWAS) found that a SNP (rs35705950) in the promoter region of mucin 5B (MUC5B) greatly increases the risk of IPF . MUC5B cEnvironmental exposure and genetic predisposition may have a synergistic effect in the development of IPF . In both+ channel (ENaC) through ubiquitination, which is essential for proper mucociliary clearance of inhaled irritants and pathogens. Recent study has shown that expression of NEDD4-2 is reduced in IPF lung tissue. NEDD4-2 promotes fibrosis remodeling through regulating the expression of proSP-C, Smad2/3 and TGF-β signaling pathway  play a potential role in the pathogenesis of IPF . Compare pathway .Aging is a pathological feature of both human IPF and experimental lung fibrosis in animals. The major characteristics of aging lungs include telomere mutations, epigenetic changes, loss of protein homeostasis, mitochondrial dysfunction, and cellular senescence . TelomerIncreasing evidence demonstrated that under the influence of environmental factors and aging, epigenetic changes play an important role in IPF . EpigeneX axis of human lung fibroblasts in vitro could reduce fibrotic hallmarks  in IPF lungs compared with healthy lungs . Methylaallmarks .Studies of histone modifications of IPF lungs found that when treated with histone modification-related drugs , the level of surfactant protein C (Sp-C), the activation and proliferation of fibroblasts are all significantly affected. Additionally, histone modifications can also affect the anti-apoptotic ability of fibroblasts by inhibiting anti-fibrotic genes such as FAS and caveolin 1 (Cav-1) . LigrestmiRNA microarray analysis showed that expression of miR-21 and miR-199a-5p was increased in IPF lungs, while expression of miR-31, let-7 and miR-200 was decreased . Among tCurrently, the prominent initiation of IPF is widely considered to be repeated lung epithelial cell damage and repair dysfunction. Under normal circumstances, the damage of alveolar epithelial cells will lead to recruitment of inflammatory cells, fibrosis and matrix deposition in order to repair the damaged cells. This stage is temporary and then normal pulmonary homeostasis will be restored through activation of apoptotic pathways and phagocytosis of macrophages during the injury repair stage . However+ or Lgr5+ can participate in alveolar homeostasis by stimulating Wnt signaling (Axin2+) located in the alveolar compartment. These subset of fibroblasts are involved in promotion of alveolar growth and maturation, and preferentially differentiate into myofibroblasts after lung epithelial injury  . Studiesl injury .In IPF, genetic and environmental factors may cause damage to AT1s, while dysfunction of AT2s makes it difficult to repair the damaged AT1s. AT2s, serve as the predominant epithelial progenitor in alveoli, play an important role in maintaining lung homeostasis . AbnormaIn IPF, pro-fibrotic mediators secreted by activated fibroblasts continue to act on fibroblasts to form a positive feedback, which leads to production of ECM and myofibroblast differentiation . TGF-β iIn the normal wound healing process, unwanted fibroblasts are eliminated by activating the apoptotic pathway. The elimination mechanism of fibroblasts limits the ongoing matrix deposition and fibrosis . In IPF,An important feature of epithelial cell remodeling in IPF is the expansion of distal basal cells, which can serve as stem/progenitor cells of the pseudostratified epithelium of the lung. In the cellular area of IPF, secretory sensitized basal cells (SPB) are enriched, and the formation and secretion function of its subpopulations are regulated by Notch signal. Specifically, NOTCH2 restricts the differentiation of basal cells, while NOTCH3 can inhibit secretory differentiation . In addiThere is overwhelming evidence in support of a key role of TGF-β in the pathogenesis of IPF . TGF-β pCTGF, also known as cellular communication network factor 2 (CCN2), is an important mediator of organ fibrosis in human body . It is cPDGF is widely expressed in macrophages, platelets, endothelial cells and fibroblasts . Highly Insulin-like growth factor (IGF1) is a key molecule that regulates cellular senescence . As mentThe massive deposition of extracellular matrix in IPF is mainly involved in changes in two families of proteins: MMPs and tissue inhibitors of metalloproteinases (TIMPs) . StudiesMatrix stiffening is a prominent feature of lung fibrosis. Compared with healthy lung scaffolds, IPF scaffolds increase tissue stiffness, density, ultimate force, and differential expressions of matrisome proteins . The colIn previous study, we demonstrated that matrix stiffness regulates the ability of fibrotic lung myofibroblasts to invade the BM, by increasing α6-expression, mediating MMP-2-dependent pericellular proteolysis of BM collagen IV. Genetic ablation of α6 in collagen-expressing mesenchymal cells or pharmacological blockade of matrix stiffness-regulated α6-expression protects mice against bleomycin injury-induced experimental lung fibrosis. Studies found that a mechanotransduction pathway involving Rho/Rho kinase (Rho/ROCK), actin cytoskeletal remodeling, and a mechanosensitive transcription factor, megakaryoblastic leukemia 1 (MKL1), that coordinately regulate myofibroblast differentiation, and pharmacologic disruption of this pathway with the ROCK inhibitor fasudil induced myofibroblast apoptosis through a mechanism involving downregulation of BCL-2 and activation of the intrinsic mitochondrial apoptotic pathway . RecentlEndoplasmic reticulum stress (ERS) occurs when there is an imbalance between cell’s demand for protein synthesis and the ability of endoplasmic reticulum to synthesize, process, and package proteins. As ERS occurs, cells activate an UPR, which attempts to restore normal function of the endoplasmic reticulum. When ERS is persisting or severe, it triggers cell apoptosis . It has 1 adrenoreceptors  to exacerbate fibrosis . Elevateeceptors . CCL2 aneceptors . Furthereceptors .Autophagic pathways (including macroautophagy and mitophagy) in IPF lung epithelial cells and fibroblasts are reduced, aggravating inflammation and fibrosis . AutophaRecent research on pathogenesis of IPF has promoted notable advances in pharmacotherapeutic treatment. There are currently two recommended antifibrotic drugs, nintedanib and pirfenidone, have been shown to delay the progression of pulmonary fibrosis and reduce mortality, but there is still no cure for IPF . TherefoPirfenidone (PFD) is a pharmacological compound for IPF treatment . PFD treNintedanib is a triple tyrosine kinase inhibitor with anti-fibrotic effects. In IPF treatment, Nintedanib can reduce the decline of forced vital capacity (FVC) and inhibit progression of pulmonary fibrosis . In prevAs mentioned above, pulmonary fibrosis is a complex pathological progression in which pathogenic factors activate complex fibrotic pathways in various cells . TherefoIn a recent clinical phase 2b trial, patients with advanced IPF and pulmonary hypertension were treated with pirfenidone plus sildenafil for up to 52 weeks. Unfortunately, there was no therapeutic effect. . FurtherDue to adverse effects of currently drugs for IPF and there is no effective cure, IPF research is increasingly focused on developing new molecular targets and treatment options. As mentioned above, CTGF is an important pro-fibrotic growth factor associated with extracellular matrix secretion and abnormal tissue repair . A recenIdiopathic pulmonary fibrosis is an interstitial pulmonary disease with high mortality. It is associated with a large economic and healthcare burden. Genetic and epigenetic changes are important factors in the pathogenesis of IPF, although the definite cause of IPF has yet to be clarified. Our understanding of the pathogenesis of IPF have significantly improved in the past decade. In the last few years, the research progress of idiopathic pulmonary fibrosis has highlighted the important role of stem cell dysfunction and extracellular matrix in mediating lung pathological remodeling and promoting the process of fibrosis . Unfortu


Three-dimensional graphene-based aerogels (3D GAs), combining the intrinsic properties of graphene and 3D porous structure, have attracted increasing research interest in varied fields with potential application. Some related reviews focusing on applications in photoredox catalysis, biomedicine, energy storage, supercapacitor or other single aspect have provided valuable insights into the current status of Gas. However, systematic reviews concentrating on the diverse applications of 3D GAs are still scarce. Herein, we intend to afford a comprehensive summary to the recent progress in the preparation method (template-free and template-directed method) summarized in Preparation Strategies and the application fields  illustrated in Application of 3D GAs with varied morphologies, structures, and properties. Meanwhile, some unsettled issues, existing challenges, and potential opportunities have also been proposed in Future Perspectives to spur further research interest into synthesizing finer 3D GAs and exploring wider and closer practical applications. Aerogel, generated by the replacement of liquid inside a gel with gas by freeze-drying or supercritical drying technique, was first presented by S. Kistler in the 1930s ,3,4. The2/g), chemical stability, and high electron mobility, as well as excellent mechanical and optical properties, which has great application potential in many fields [As the most important component of two-dimensional (2D) carbon-based material, graphene possesses superior thermal conductivity (~5000 W/m·K), high specific surface area , hydrazine vapor, Fe (II), sodium hydrogen sulfite (NaHSO3), sodium sulfide (Na2S), and vitamin C and hydrogen iodide (HI) [3O4 aerogel via in situ self-assembly of graphene with NaHSO3 as the reducing agent in the presence of Fe3O4 nanoparticles. The capture of nanoparticles was similar to a fishing process, wherein nanoparticles acted as the fish and graphene/GO nanosheets served as the fishing net that was formed through the self-assembly of GO during reduction. Similarly, Li et al. [ide (HI) ,41. The ide (HI) ,42, Yan ide (HI)  preparedi et al.  proposedi et al. , moving i et al.  developeHydrothermal reduction is another facile and effective strategy to prepare graphene from GO. The reduction degree is determined by reaction temperature and time. Generally speaking, the higher the temperature, the higher the reduction degree. However, there is no consensus on the required time and temperature of the heat-treatment for complete reduction of GO [2-graphene aerogel (SGA), which involved the synchronous processes of hydrothermal-induced reduction of GO, self-assembly of graphene sheets, and in suit growth of SnO2 nanoparticles, presented in on of GO ,48. Moston of GO . Li and on of GO  proposedElectrochemical reduction is an appealing strategy used to form 3D GA. Unlike other self-assembly methods that rely on the unique shape of graphene sheets and features of the second components and whose materials are bulky monoliths and unsuitable for electrode modification, electrochemical reduction-induced self-assembly approach is believed to be the most promising means to fabricate a 3D structure applied in electrode material. Chen et al. [n et al.  proposedThe abundant oxygen-containing functional groups on their basal planes and edges make GO sheets sensitive to the experimental conditions, and only assemble into 3D microstructure under certain conditions . The obtPhysical cross-linking refers to linking GO sheets and a crosslinker mainly by hydrogen bonding, as illustrated in 3O4 nanoparticles and GO nano-sheets).Chemical cross-linking refers to linking GO sheets and a crosslinker by covalent bonding, which has the characteristics of high uniformity and stabilization. Ye et al. [e et al.  developeDuring the synthesis process, hydrogen bonding and/or covalent bonding between crosslinker and GO sheets accounts for forming the cross-linking sites, which contribute to the generation of hydrogel with a 3D network. Such aerogels usually show significantly improved mechanical properties. However, the introduction of cross-linkers inevitably sacrifices the electrical conductivity of GAs to a certain extent. So, there is a fundamental tradeoff to grasp. Utilizing conductive polymer as a crosslinker to fabricate highly compressible and electrically conductive 3DGAs might be a new attempt.The template-directed strategy, including the emulsion technique, breath figure method, and ink-printing technique, is one of the mainstream approaches employed in the formation of 3D GA with ordered and hierarchical structures . It usesHard templating strategy, using solid nanoparticles, porous metal foams, and specific movable nozzles as a guide is conceptually the simplest method to synthesize 3D graphene aerogel with the desired microstructure and performance . In geneCompared with the hard template approach, the soft template method with the emulsion droplet, vesicle/micelle, and gas bubble as templates is easier due to the unnecessity of removing the templates . AlthougThe carbon aerogels  prepared by various methods generally have lower density than the graphene/GO-based hybrid (other nanoparticle or polymers) aerogels. Moreover, for the same GO aerogels obtained by the two-stage preparation procedure involving subsequent microwave irradiation (MWI) or thermal treatment, which further eliminates functional groups after freeze-drying, are ultralight and their density came down to a lower scale compared with those prepared via one-step. However, in any case, all the as-prepared GA possess an interaction network and hierarchical pores ranging from micropores to mesopores and macropores, which can be characterized by SEM and the nitrogen adsorption/desorption isotherm . The porGraphene aerogel, with characteristics of low density, high surface area and porosity, and good electrical and thermal conductivity, has attracted the attention of researchers and flourished in the recent decades . They poWater contamination caused by harmful chemicals, particularly oils and soluble dyes and phosphate, has become an issue of serious global concern. Various technologies including chemical precipitation ,78, bioln-decane adsorption as an example, 8.5 mg of aerogel can completely adsorb 1.4 g of oil within 6 s. The average absorption rate (27 g/g·s) is much faster than that of pure graphene (0.57 g/g·s). The capacity for absorption is up to 120–250 g/g, depending on the liquid densitY.S.milarly, Gao et al. [n-hexane, crude oil, toluene, motor oil, vegetable oil, ionic liquid, and phenixin)  d, fallin3), high surface area (214.75 m2/g), paramagnetic property (26.59 emu/g), excellent adsorbent efficiency (93%) for cationic malachite green (MG) dye through the Yoshida H-bonding, dipole-dipole H-bonding, π-π interaction, n-π interaction, electrostatic attraction, and physical adsorption, as shown in 4), once adhered to the graphene or GO, will show great application potential in capturing phosphates in water, Losic et al. [3O4 nanoparticles, respectively. The prepared aerogels showed superior capacity of 350 mg/g at an initial phosphate concentration of 200 mg/L under acidic conditions and the adsorption process followed the second order model and Freundlich isotherm  with high energy density and voltage, which can store and supply electricity, have a wide range of applications with the development of modern electronic devices. Currently, the biggest challenge is to develop durable, nontoxic, and inexpensive materials for electrodes.3O4 [3O4 [3O4 [2 [2O4 [3O4 aerogel via in situ self-assembly of graphene in the presence of Fe3O4 nanoparticles. The graphene/Fe3O4 composite aerogel showed a high capacity and cyclic stability (a remaining capacity of 1100 mA h/g after 50 cycles of charge and discharge), meaning it can work well as an anode for LIBs, as shown in 3O4 nanoparticles working as spacer and graphene nanosheets, as both sides can adsorb lithium ions [2-graphene aerogel (SGA), which displays a well-defined and interconnected network porous microstructure and possesses higher capacity retention and discharge capacity (602 mA h/g after 60 cycles) compared to other SG composites , with mechanical stability under dynamic tension and compression deformations, can find potential applications in strain sensors, stretchable components, and lightweight mechanical devices.Another approach is to enhance the interconnection of aerogels to produce a hierarchical structure by adopting freeze-shaping, 3D ink-printing, and other synergistic assembly techniques ,99. Guo Based on the major advances made in the design of flexible 3D graphene aerogels, the trend is to push their structure towards a smarter and more well-controlled system , in ordeThe existing commercial fire-warning equipment, including temperature, smoke, and infrared flame detectors, is usually unsatisfactory , as theyWith the increase of temperature, the electrical resistance of GO decreases dramatically, which endows it an attractive application prospect in fire-warning materials ,104,105.The GO modified aerogel with excellent flame retardancy, thermal isolation, and intrinsic fire warning performance broadens its application territory to cover the drawbacks in delayed response and restricted application scenarios of traditional fire detectors. Therefore, the aerogel that can be triggered in the precombustion stage to offer favorable opportunities for firefighting and emergency rescue is endowed with enticing prospects in chemical industries, pipeline transportation, and high-rise buildings.2 reduction [A 3D network structure provides multidimensional electron transport pathways and large accessible surface area, which is conducive to improve the separation efficiency of photogenerated electron-hole pairs and the adsorption of reactants. Such intrinsic hierarchical porous structure characteristics and properties make GAs endowed with potential as promising and efficient photocatalysts for practical applications in solar energy conversion , such aseduction , and cheeduction .VI, showing that after visible light irradiation for 8 min, the MO was completely degraded by AgBr/GAs compared with the 65% of degradation rate by AgBr  and reducing CO2 to CH4 via acquiring 8-electrons process (CO2 + 8H+ + 8e–→CH4 + 2H2O) [2, with higher catalytic efficiency compared to other semiconductors. Tong and co-workers [3N4/GO aerogel (CNGA) by the hydrothermal induced self-assembly method, in which g-C3N4 acted as the efficient photocatalyst, and GO was responsible for supporting the 3D framework. The as-prepared GAs could reduce CO2 into CO with a high yield of 23 mmol/g (within 6 h), which was about 2.3-fold increment compared to pure g-C3N4  . The 3D -workers  designede g-C3N4 . That isThe 3D GAs also play an important role in catalyzing some chemical reactions, such as the selective oxidation of alcohol to carbonyl, the reduction of nitroaromatic compound to amino compound, and the synthesis of ammonia . Yang etThere is no doubt that considerable advances have been achieved in the fabrication of 3D GA materials in the past few decades. Various preparation methods involving a series of template-free and template-guided methods are proposed to fabricate 3D GAs with integrated properties of graphene and other unique characters. The excellent properties make them attractive and promising in potential applications in varied fields, which brings us closer to practical applications. In this review, we summarized the advancement of literature, historical progress of synthesis of 3D GAs, and introduced the main application in absorption, anode material, mechanical device, fire-warning material, and catalysis aspects.p-conjugation compared to pure graphene, which causes the improvement of electrical correlation performance of 3D GAs to often be restricted. Therefore, rational utilization of graphene with superior electrical conductivity to advance the 3D GAs still needs continuous efforts. Secondly, the widely used drying methods of preparation of aerogel are mainly freeze-drying and supercritical drying [Currently, although dramatic progress has been realized, the performance and application filed of 3D GAs has not been fully exploited. There are still many challenges toward the preparation and mechanism elaboration of highly efficient 3D GAs. Firstly, most of the 3D GAs are derived from the reduction of GO, so their electrical conductivity and charge carrier mobility are remarkably decreased due to the disruption of l drying . Freeze-l drying . Therefol drying . TherefoWith the output of constant efforts, we believe that substantial breakthroughs for the finer structural control and practical applications of 3D GAs would be expected in the near future.