Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are neurodegenerative disorders that exist on a disease spectrum due to pathological, clinical and genetic overlap. In up to 97% of ALS cases and ~50% of FTLD cases, the primary pathological protein observed in affected tissues is TDP-43, which is hyperphosphorylated, ubiquitinated and cleaved. The TDP-43 is observed in aggregates that are abnormally located in the cytoplasm. The pathogenicity of TDP-43 cytoplasmic aggregates may be linked with both a loss of nuclear function and a gain of toxic functions. The cellular processes involved in ALS and FTLD disease pathogenesis include changes to RNA splicing, abnormal stress granules, mitochondrial dysfunction, impairments to axonal transport and autophagy, abnormal neuromuscular junctions, endoplasmic reticulum stress and the subsequent induction of the unfolded protein response. Here, we review and discuss the evidence for alterations to these processes that have been reported in cellular and animal models of TDP-43 proteinopathy. Frontotxpansion .TARDBP, coding for mutant TDP-43 proteins, are a rare cause of ALS and FTLD [TARDBP, with most being missense mutations that affect the C-terminal domain [A key characteristic of neurodegenerative diseases is the identification of abnormal protein aggregates in neurons . In up tand FTLD . Four pel domain . Mutatiol domain ,15. Of nl domain ,17,18. Tl domain . The aggl domain . TDP-43 l domain , axonal l domain , mitochol domain , and autl domain . Thus, wWhile TDP-43 pathology is identified in almost all cases of ALS and non-tau-related FTLD, the mechanisms underlying pathogenicity are varied and incompletely understood. Both conditional knockout  and TDP-A315T [A315T in the primary cortical neurons of a distinct transgenic mouse did not significantly affect dendrite or axon outgrowth [sterile alpha and TIR motif-containing 1. Deletion of this gene in a TDP-43Q331K mouse model of ALS-FTLD reduced the degeneration of motor axons and the denervation of NMJs, thus implicating Wallerian-like pathways in the axonal degeneration observed in ALS [Q331K transgenic mice, defective synaptic transmission at the NMJ is observed at age 3 months, preceding the motor dysfunction that was observed at age 10 months [N390D mouse [A315T in mouse cortical neurons decreased the localization of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropinionic acid (AMPA) glutamate receptor subunit GluR1 at dendritic spines, which was associated with a reduction in the presynaptic marker synaptophysin [Axon outgrowth is essential for neuronal communication and innervation of action potentials in neuromuscular junctions (NMJs). TDP-43 has been implicated in axon outgrowth and the denervation of NMJs, causing defective synaptic transmission, and impacting cell survivability and conductivity. The overexpression of wild-type or mutant TDP-43 (p.M337V or p.Q382T) in mouse primary neurons caused impaired axon outgrowth. This was shown to be dependent on the C-terminal domain . FurtherA315T . Howeverutgrowth . A criti0 months . This fi0 months . Further0D mouse . ExpressTDP-43 is primarily a nuclear protein . Its traWhile TDP-43 is autoregulated at a transcriptional level, it is also degraded by both the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway, hereafter autophagy . It has When expressed in HEK293 cells, a 35 and a 25 kDa CTF formed cytoplasmic aggregates in 25% and 75% of cells, respectively . ExpressA315T mutant protein is fragmented into persistent CTFs that resist proteolytic degradation [cystic fibrosis transmembrane conductance regulator gene [Translocation of TDP-43 out of the nucleus causes loss of function see , while tradation . The p.Qradation , p.G348Cradation , p.M337Vradation , p.A382Tradation  mutant pradation ,55,56. Aradation ,57. The radation ,59,60. Aradation . Sasagurtor gene . Thus, CAcetylation is an enzyme-mediated process whereby an acetyl group is moved from one molecule to another to alter intracellular localization, protein–protein interactions or tag proteins for degradation. Various TDP-43 RNA regulatory functions are impaired due to acetylation. Specifically, RNA binding is inhibited by post-translational acetylation at lysine residue 145 (Lys-145), which sits within the RNA-binding domain . This leSanna et al. (2020) show that TDP-43, via its RRM1 and RRM2 domains, interacts with histone deacetylase 6 (HDAC6). The RRM domains contain the two major acetylation sites in TDP-43 Lys-142 and Lys-192 . The autAttachment of ubiquitin to target proteins is a well-known signal for protein degradation. TDP-43 ubiquitination was one of the first signature modifications to be associated with ALS and FTLD ,42. A muThe small ubiquitin-like modifier (SUMO) pathway involves enzymatic PTM, resulting in the covalent attachment of SUMO proteins to a target protein’s lysine residues . SUMOylaIn addition to PTMs such as ubiquitination, oxidation and acetylation, phosphorylation, the generation of CTFs, cytoplasmic accumulation and mutations are all factors that affect TDP-43 aggregation . VariousA315T was mainly present in the insoluble fraction, whilst overexpressed wild-type TDP-43 was primarily soluble [Expression of mutant TDP-43  appears to increase the propensity of TDP-43 to aggregate in response to oxidative stress . Indeed,TARDBP mutations, it is important to understand the role of aggregated and mislocalized wild-type TDP-43 in disease pathogenesis. Transfection of SH-SY5Y cells with purified bacterially expressed wild-type TDP-43 aggregates caused decreased cell viability due to an increase in reactive oxygen species (ROS) and exacerbated caspase-3 activation [A315T transgene developed progressive and fatal neurodegeneration, with selective vulnerability of spinal cord neurons and neurons from the frontal cortex. However, TDP-43 aggregates were not present, reinforcing that altered TDP-43 function is required for neurodegeneration to occur, independent of aggregation [Given that the majority of ALS and FTLD cases with TDP-43 proteinopathy are not caused by tivation . Overexptivation . Furthertivation ,27. Thertivation ,87, whiltivation . A mouseregation . TDP-43 is primarily located in the nucleus of cells. However, it can also be present in the cytoplasm. This nucleocytoplasmic shuffling is mediated by nuclear localization and nuclear export signals. The loss of nuclear TDP-43 due to abnormal cytoplasmic mislocalization is thought to lead to loss-of-function effects and contribute to disease pathogenicity in ALS .TDP-43 has key roles in the metabolism, processing, alternative splicing and transport of RNA . TDP-43 Rac1 [mRNAs are trafficked in dendrites and axons as ribonucleoprotein (mRNP) granules that consist of mRNAs, RNA-binding proteins (RBPs), ribosomes and translation factors ,94. mRNPRac1 . Chu et Rac1 . When thRac1 ,99,100.fused in sarcoma/translocated in liposarcoma (FUS/TLS), progranulin and other genes associated with neurodegeneration. Using splicing-sensitive microarrays, TDP-43 was found to be important for the maintenance and splicing of more than 1000 mRNAs. The RNAs that were most reduced by TDP-43 knockdown encoded proteins that are important for synaptic activity [TDP-43 has a critical role in repressing splicing to protect the transcriptome . Using aactivity . While iactivity .Q331K, which develop adult-onset ALS symptoms, exon exclusion in some pre-mRNAs was enhanced compared to mice expressing human wild-type TDP-43. Of note, TDP-43Q331K increased the normal splicing of some TDP-43 targets, while other targets exhibited a loss of splicing function that mimicked changes observed due to reduced TDP-43 activity [TARDBP mutations near the RRM domains  disrupt RNA binding and also cause sequestration of wild-type TDP-43 in cytoplasmic inclusions, suggesting that RNA binding may be linked to TDP-43 solubility [TARDBP mutation (p.A315T) drosophila model of ALS [N-ethyl-N-nitrosourea (ENU) mutagenesis, Fratta et al. (2018) were able to identify a mutation-associated (p.F201I) novel splicing event that involves the skipping or inclusion of constitutive exons, referred to as “skiptic” exons, to alter gene expression [CTFR and Sortilin-1 genes, while 47 skiptic exons were differentially regulated in the p.F210I strain. The skiptic exons identified in the p.M323K strain were absent in the p.F210I strain. Of note, the p.F210I mutation had similar effects transcriptome wide as shRNA-mediated TDP-43 knockdown. The p.M323K mutation had opposing effects and led to the skipping of constitutive exons in a distinct set of 44 genes, including 7 genes from the UPS [Tardbp mRNA levels. By contrast, the p.F210I loss-of-function mice did not display a motor phenotype when aged up to 2 years [In mutant mice expressing high levels of TDP-43activity . Some exactivity . ALS-asslubility . Of notel of ALS . Using tpression . The mut the UPS . Importa 2 years .M337V, which did not show a motor phenotype, mRNA splicing was deregulated, including that of Kcinp2, Sort1, Sema3f [The causes of TDP-43 translocation out of the nucleus are unknown but may include genetic (mutation) or environmental factors. A p.M337V transgenic pig model of ALS that exhibits a severe phenotype was used to investigate the role of cytoplasmic mislocalization on RNA splicing events . TDP-43 , Sema3f . Thus, ecacophony, a voltage-gated calcium channel. This affected neuromuscular junctions. Of note, expression of cacophony rescued the locomotion phenotype indicating that changes to splicing due to TDP-43 are functionally significant in ALS [TDP-43 deficiency in mouse embryonic stem cells decreased the splicing of non-conserved cryptic exons, leading to the incorporation of these exons into the mRNAs, translation and subsequent nonsense-mediated decay. Reduced TDP-43 repression of these exons was also observed in cases of ALS and FTLD . The autt in ALS .C9orf72 repeat-positive ALS cases, Tank et al. (2018) observed a severe destabilization of RNA transcripts. This destabilization was mirrored by overexpression of TDP-43. The authors show that energy production may be impeded, while protein synthesis was increased, potentially as compensatory mechanisms. Reduced transcripts for oxidative phosphorylation genes and ribosome protein-coding RNAs were also observed in the spinal cord from ALS patients [TDP-43 promotes mRNA instability by interacting with Caf1, and thereby facilitates RNA deadenylation, which is the rate-limiting step of mRNA decay . Depletipatients . Recognipatients . Using cpatients . TDP-43 patients .A315T in mice led to upregulation of SYNGR4 and downregulation of PLEKHB1. These changes were also observed in an additional mouse model expressing TDP-43Q331K [SYNGR4) or impact G protein-coupled signaling and protein trafficking via interaction with myosin (Pleckstrin homology domain-containing protein family B member 1) [Camta1, Mig12 and Dennd4a mRNAs. Camta1 and Dennd4a are associated with neurodegeneration, thus TDP-43 regulation of these transcripts may be involved in ALS and FTLD pathogenesis [Expression of p.Q331K or p.M337V mutant TDP-43 in mice leads to a motor phenotype ,116 and -43Q331K . The funember 1) . In NSC-ogenesis .Atg7. A mutant TDP-43 lacking the RRM1 domain had impaired ability to bind to and stabilize Atg7 mRNA, leading to impaired autophagy [TDP-43 binds to the 3′ untranslated region to promote instability of mRNA transcripts of tau, vascular endothelial growth factor A and progranulin, all implicated in ALS and/or FTLD ,119,120.utophagy . TDP-43 utophagy . A furthutophagy . Thus, Tutophagy . Decreasutophagy .TDP-43 and has a regulatory role in gene expression and silencing and the stress response via interaction with G-quadruplex (G4)-RNA. G4 is a structure found in DNA or RNA that are rich in guanines. The structure of G4 is important for long-distance transport and translation of mRNA in neurons. Ishiguro et al. (2020) investigated the effects of 10 randomly selected ALS mutants  on interaction with G4-RNA in HEK293 cells. All mutant proteins showed reduced G4-RNA binding and decreased trafficking of G4-RNA . As all met. Disrupting the initiation complex from binding to ribosomes permits the binding of T-cell intracellular antigen 1 (TIA-1) to the 48S ribosomal complex [Stress granules are protective cytoplasmic granules that form in response to cellular insults to protect and sequester non-essential mRNA transcripts  is the phenomenon that drives the formation of membrane-less organelles and protein aggregation . The lowThe liquid–liquid phase separation of TDP-43 is regulated through the low complexity domains. The C-terminal domain of TDP-43 is a 160-residue domain that contains very low sequence complexity that contains only a single alpha-helix between residues 321 and 330 . The majNEAT1 promoted the LLPS of TDP-43 [Phase separation and the formation of stress granules present a possible precursor to cytoplasmic aggregates of TDP-43 . TDP-43 f TDP-43 . Importaf TDP-43 . Thus, wf TDP-43 . Li et af TDP-43 .Axonal transport functions to deliver and distribute cellular growth factors, organelles, synaptic cargoes and cytoskeletal factors to increase the efficiency of trafficking within neuronal cells . ImpaireSOD1 mutations in ALS patients [Mitochondrial dysfunction has been implicated in the disease progression since the observation of abnormal mitochondrial morphology and the identification of patients . SOD1 ispatients . Reactivpatients . ALS patpatients . As neurpatients . The brepatients . Mitochopatients . There is growing evidence that early mitochondrial dysfunction within upper motor neurons leads to their vulnerability to TDP-43 proteinopathy . TDP-43 A315T [A315T under the prion promoter, mitochondrial defects are observed as early as postnatal day 15 indicating that they may contribute to motor neuron vulnerability [Electron microscopy of patient samples revealed mitochondrial impairment. Similar impairments, including the presence of smaller mitochondria with swollen and degenerated cristae, or the complete lack of cristae, were observed in cell and animal models of overexpressed TDP-43A315T . This waA315T . In a ceA315T . In micerability . The mitrability . In cellrability . The numrability . M337V mice could be alleviated with the use of an inhibitor that prevented TDP-43 localization to mitochondria [Parkin transcripts, but not that of PINK1 [Q331K mice. The increase in cleaved PINK1 caused mitochondrial dysfunction [Overexpression of wild-type TDP-43 decreased the length and density of mitochondria in primary motor neurons. This was enhanced in cells expressing mutant (p.Q331K or p.M337V) TDP-43 . Importachondria . The intchondria . In addichondria . Overexpof PINK1 . Howeverfunction . n repeat sequence expansions, the most common cause of ALS and FLTD, impairs mitochondria and the calcium balance within neurons. Low calcium levels correlate with a low response in motor neurons through depolarization, decreasing recovery times and neuronal function [SOD1 mutant neurons in addition to a disruption of cellular calcium stores, impaired mitochondria generate activated caspase-1, -3, -7 and -9. This demonstrated a link between mitochondrial imbalance and ALS [Mitochondrial involvement in the progression of ALS may be mediated via apoptotic signals in calcium buffering and the activation of caspases. Calcium signaling is an essential mechanism involved in normal neuronal function . Signal function . In SOD1 and ALS . Like SO and ALS . Thus, TReactive oxygen species (ROS) are produced due to electron leakage that occurs during oxidative phosphorylation within the mitochondrial energy production pathway. Irregularities in the regulation of ROS and reactive nitrogen species (RNS) can cause oxidative stress. Oxidative stress can be defined as the imbalance between the production and the clearance of ROS/RNS that may result in molecular damage . OxidatiQ331K, increased levels of ROS were observed, along with increased DNA strand breaks and apoptosis [The cytoplasmic mislocalization and aggregation of TDP-43 has been associated with oxidative stress . Irregulpoptosis .M337V to the cytoplasmic poly (A)-binding protein and eukaryotic initiation factor 4A-I, essential proteins for initiation of stress granule formation, was observed and may be linked to the impaired stress granule formation that is observed in the TDP-43M337V motor neurons [The functional connectivity of motor neurons has been investigated in an oxidative environment (induced by arsenic) using embryonic stem cells from mouse models expressing either wild-type or p.M337V TDP-43 . In comp neurons . Thus, o neurons .Proteostasis refers to the maintenance of protein homeostasis and balances the production of nascently translated proteins with the degradation of misfolded or aggregated proteins . Protein2+) are stored in the ER. It plays a role in the ER mitochondrial calcium cycle, which couples mitochondrial energy production and ER protein processing with neuronal synaptic activity [2+ homeostasis. Activation of GSK-3β and disrupted Ca2+ homeostasis are observed in ALS [2+ signaling from the ER was reduced 50% compared with cells expressing wild-type TDP-43 [2+ in wild-type TDP-43-expressing cells mimicked the cellular phenotype of mutant TDP-43 expressing cells, indicating that TDP-43 pathology may be linked with reduced Ca2+ [G348C [A315T in SH-SY5Y cells increased cytotoxicity by activating ER stress-induced autophagy, with cell death reduced by autophagy inhibition [ER stress is a characteristic of ALS and FTLD ,168. Wheactivity . Interacactivity . Howeveractivity . Overexpd in ALS . In celle TDP-43 . Decreasced Ca2+ . In smal+ [G348C . Expresshibition . Thus, tIf the ER is unable to refold a misfolded protein, it is shuttled to the UPS for degradation . The UPSTDP-43, either full-length or truncated forms, can be degraded via both the UPS and autophagy ,179,180.Dysfunctional autophagy is often observed in neurodegenerative disorders. Autophagy is a cellular mechanism for the removal of aggregated proteins and damaged organelles, via selective or non-selective (bulk) processes. It can be divided into microautophagy, macroautophagy or chaperone-mediated autophagy . Unless Atg7 [N390D mouse model. This was due to a progressive increase in TDP-43 levels, which upregulated autophagy regulator Bcl-2, with the mouse showing an ALS phenotype [While TDP-43 aggregation and autophagy dysregulation have been highlighted in ALS and FTLD pathology for some time, the role of TDP-43 in autophagy regulation was unknown. However, knockdown of TDP-43 led to decreased expression of the critical autophagy gene Atg7 . This caAtg7 . A role Atg7 . The micAtg7 . Autophahenotype . TDP-43 henotype . Howeverhenotype . A statehenotype . In ALS In immortalized motor neurons and stabilized myoblasts the overexpression of TDP-25 resulted in impaired autophagy and TDP-25 was instead cleared by the proteasome . In a NeA315T motor neurons showed significantly reduced neurite outgrowth and fewer branching points [The expression of mutant TDP-43 within non-neuronal muscle cells indicated non-cell-autonomous effects of these proteins. Wächter et al. (2015) showed that expression of TDP-43 p.A315T within muscle cells had a degenerative effect on motor neuron cells . Healthyg points . Serio eg points . A315T. When doxycycline is added to the feed expression of the transgene is suppressed. Expression of TDP-43A315T induced a neurodegenerative phenotype that included pronounced activation of astrocytes. Importantly, when the expression of the transgene was suppressed motor and behavioral deficits were improved [A315T mouse, Bargsted et al. (2017) observed a significant loss of motor neurons, axonal degeneration and activation of astrocytes and microglia. Primary astrocytes isolated from another transgenic hTDP-43A315T mouse had impaired L-glutamine uptake and abnormal accumulation of ATP [M337V in astrocytes in rats led to the activation of both astrocytes and microglia and motor neuron loss [Glial cells are three times more abundant than neurons and work to support neuronal function . Gliosisimproved . In a ded et al. 7 observed et al. 7 observeron loss . Howeverron loss . Yet, whron loss .Prion diseases involve the propagation of infectious particles of proteinaceous origin, leading to neurodegenerative diseases . The proAMP-activated protein kinase (AMPK) is an energy metabolism and stress pathway regulator. AMPK senses imbalance between ATP and AMP in the cell, often caused by oxidative stress or nutrient deprivation, and responds by activating glucose uptake, and autophagy, while decreasing protein translation and glycogen synthesis . ActivatExpression of phosphatases, which dephosphorylate AMPK, was assessed in the TDP-43A315T mice. Protein phosphatase 2A (PP2A) was significantly increased in TDP-43A315T mice and correlated with reduced activation of AMPK. In NSC-34 cells expressing TDP-43Q331K and TDP-43M337V AMPK activation was reduced to 50% and 70%, respectively, when compared with cells expressing wild-type TDP-43 . ConcurrTDP-43 is the primary pathogenic protein observed in almost all cases of ALS and the majority of non-tau-related FTLD. Expression of mutant proteins or overexpression of wild-type TDP-43 decreased axon outgrowth . Mutant CTFs are observed in tissues from ALS and FTLD patients ,18,45. CTARDBP mutation leads to cytoplasmic mislocalization, or in sporadic cases where an unknown trigger causes translocation, the normal RNA regulatory functions of TDP-43 may be severely reduced and contribute to pathogenicity. Therefore, strategies to reduce TDP-43 nuclear depletion are an attractive approach to the effective treatment of ALS and FTLD. As deficiency and TDP-43 overexpression have profound effects on neuron viability and function [TDP-43 regulates the metabolism, splicing and transport of 1000s of mRNAs . Expressfunction ,26,29,34Overall, the literature suggests that TDP-43 proteinopathy occurs due to the translocation of TDP-43 out of the nucleus into the cytoplasm. This may occur due to stressors that induce ROS generation, or the presence of a gene mutation. Either may lead to a loss of nuclear function, including altered splicing and translation of mRNAs, and a gain of toxic functions in the cytoplasm, including dysregulated proteostasis, impaired mitochondria, activation of ER-induced cell death and impaired stress granule formation. The major unresolved questions are the following: are PTMs or TDP-43 misfolding a cause of disease or a result of pathogenesis? And do they induce TDP-43 translocation out of the nucleus? In answering these questions, we can tackle the problem of how to prevent PTMs and mislocalization from occurring. This will help to identify therapeutic targets, which may include chaperones involved in the folding of TDP-43.


Human papillomavirus (HPV) infection is the major etiological factor for cervical cancer. HPV prophylactic vaccines based on L1 virus-like particles have been considered as an effective prevention method. However, existing recombination vaccines are too expensive for developing countries. DNA vaccines might be a lower-cost and effective alternative. In this study, a plasmid (pcDNA3.1-HPV16-L1) and a co-expressing plasmid (pcDNA3.1-HPV16-L1-siE6) carried by attenuated Salmonella were constructed and their prevention and treatment effect on cervical cancer were observed, respectively. The results showed that pcDNA3.1-HPV16-L1 carried by attenuated Salmonella could induce the production of HPV16-L1 antibodies, IL-2 and INF-γ in mice serum, which presented its prevention effect on HPV. Subsequently, E6 and E7 gene silencing by pCG-siE6 inhibited the growth of cervical cancer both in vitro and in vivo. Furthermore, L1 up-regulation and E6/E7 down-regulation caused by co-expressing plasmid (pcDNA3.1-HPV16-L1-siE6) contributed to a significant anti-tumor effect on the mice. This study suggests that pcDNA3.1-HPV16-L1-siE6 carried by attenuated Salmonella has a synergistic effect of immune regulation and RNA interference in cervical cancer treatment. Despite of the widespread screening and vaccine implementation, there are still approximately 54,000 and 11,000 cases each year in Europe and USA, respectively3. The standard treatment of advanced cervical cancer is radical surgery or chemoradiation, but the quality of life in multiple cases is still poor4. Hence, the exploration of specific strategy for the prevention and early treatment of cervical cancer is urgently needed.Cervical cancer is the predominant cancer in developing countries among women5. HPV16 is identified as the most prevalent type and detected in more than 50% of cervical cancer cases6. The genome of HPV consists of a circular double-stranded DNA including non-coding control regions (NCR), early region (E) coding E1, E2, E4, E5, E6 and E7 genes, and late region (L) coding L1 and L2 genes8. L1 and L2 proteins are structure proteins for HPV capsid. Under specific conditions, L1 protein can self-assemble to virus-like particles (VLPs) with a strong immunogenicities without infectious and carcinogenic abilities5. Currently, three HPV prophylactic vaccines based on L1 VLPs have been widely used in developed countries and showed a desired reduction of 38% in high grade dysplasia9. However, the developing countries with high incident rates are not able to implement these vaccines due to the high cost and requirement of multiple injections10. Thus, control measures against HPV around the world still require lower-cost prophylactic vaccines and therapeutic alternatives11.Cervical cancer is widely considered as the outcome of high-risk human papillomavirus (HR HPV) infections12. Many studies showed that silencing E6 and/or E7 gene by small interference RNA (siRNA) can significantly inhibit the development of cervical cancer in vitro or in vivo15. However, the absence of vectors that can stably transmit siRNA into target cells limits their clinical applications16. Attenuated Salmonella can easily accumulate and proliferate in the tumor microenvironment17. Moreover, alive attenuated Salmonella can induce mucosal immune response10. Therefore, attenuated Salmonella is considered as a promising vaccine vector for cervical cancer prevention and therapy. A previous study testified that a HPV16-L1 expressing plasmid carried by attenuated Salmonella (Ty21a) successfully induced HPV16 neutralizing antibodies in serum and genital secretions in mice model10.E6 and E7 proteins play a crucial role in cervical carcinogenesis through disrupting important cell pathways such as ubiquitin-mediated degradation of tumor suppressor protein P53In this study, pcDNA3.1-HPV16-L1 carried by attenuated Salmonella (Ty21a or PhoP/PhoQ) was initially constructed and significantly induced the production of HPV16-L1 antibody in serum and genital secretions of mice through intranasal dripping. After the anti-tumor effect of pGC-siE6 had been verified, pcDNA3.1-HPV16-L1-siE6 plasmid carried by attenuated Salmonella (PhoP/PhoQ) was further conducted, and its effect of therapy on cervical cancer was observed.pcDNA3.1-HPV16-L1 was constructed  both in phoP/phoQ (pcDNA3.1-HPV16-L1) and Ty21a (pcDNA3.1-HPV16-L1) groups as compared to control groups, respectively  compared to control and pGC-Scramble groups  contributing to the virus and tumor therapy through cellular immunity27. Furthermore, the expression of IL-2 and IFN-γ up-regulated by antigen presenting cell (APC) could enhance the immune response induced by DNA vaccine30. In this study, pcDNA3.1-HPV16-L1 carried by attenuated Salmonella presented well features as a vector with increased anti-HPV16-L1 antibody, IL-2, and IFN-γ. Hence, we supposed that after this DNA vaccine was engulfed by APC, HPV16-L1 protein was expressed and stimulated B lymphocytes to mature and produce anti-HPV16-L1 antibodies. Simultaneously, the activated APC also promoted the release of IL-2 and IFN-γ, which might play an important role against viruses and tumors. The data above implied that pcDNA3.1-HPV16-L1 carried by attenuated Salmonella might be an effective DNA vaccine for HPV prevention. Interestingly, we also found this pcDNA3.1-HPV16-L1 carried by attenuated Salmonella could inhibit cervical tumor growth in vivo, which implied the DNA vaccine might be a potential treatment strategy. However, this treatment effect has rarely been observed in the other VLP vaccines. We just cautiously supposed the features that attenuated Salmonella carrying pcDNA3.1-HPV16-L1 was easier to accumulate and express HPV16-L1 protein in tumor tissues compared with other VLP vaccines contributed to this anti-tumor effect.As an intracellular parasitic bacterium, attenuated Salmonella can express natural mucosal immune adjuvant and exogenous antigen that effectively elicits immune cells activation, cytokine secretion, cytotoxic lymphocyte (CTL) production and specific antibody formation12. In non-infected cervical cells, cellular ubiquitin ligase, E6AP (E6 associated protein) does not affect P53 who triggers cell cycle arrest to induce apoptosis32. However, with continuous HPV16 infection, E6 exerts its ubiquitin-mediated p53-degration effects by interacting with E6AP in cervical cells33. Bax protein and Caspase family proteins are crucial mediators and symbols of cell apoptosis regulated by P5334. c-Caspase9 and c-Caspase3 can trigger the caspase cascade and process of apoptosis35. The results in this study showed that the expression of E6 and E7 was down-regulated by pGC-siE6 in vitro or in vivo, resulting in a marked anti-tumor effect. Additionally, E6 gene silencing inhibited the development of cervical cancer through activating p53-mdiated apoptosis pathway including the up-regulation of Bax, Caspase9, and c-Caspase3. These results identified that pGC-siE6, one part of the recombination vaccine, restrained the degradation of P53 protein and then initiated the apoptosis pathway, which finally inhibited the development of cervical cancer.It is well established that the continuous expression of E6 and E7 proteins in cervical cells triggers and promotes the carcinogenesis of cervix36. Therefore, we also suggested that the APC cells of tumor-bearing mice were activated by engulfing the co-expression plasmid vaccine and then up-regulated the levels of IL-2 and IFN-γ both in serum and genital secretions. Collectively, the vaccine stimulated both of the humoral immunity and cellular immunity through inducing HPV16-L1 expression in the murine tumor tissues and increasing the levels of IL-2 and IFN-γ in serum and genital secretions. It is therefore possible to inhibit the tumor size through activating immune pathway and down-regulating the E6 and E7 expression in the tumor tissues in vivo.In order to expand the range of application and enhance the anti-tumor effect of the vaccine, a co-expression plasmid vaccine, pcDNA3.1-HPV16-L1-siE6 carried by attenuated Salmonella was constructed. After being vaccinated with the co-expression plasmid vaccine through nasal dripping, HPV16-L1 gene expression was induced whereas E6/E7 gene expression was silenced in the tumor tissues. Due to the expression of HPV16-L1 induced by the co-expression plasmid vaccine, B lymphocytes produced anti-HPV16-L1 antibodies and precisely target the cervical tumor in mice. Previous studies had shown that secretion of pro-inflammatory cytokines, including IL-2 and IFN-γ, is observed in macrophage activation and then induced anti-tumor activity on human cervical cancer cells37. The reasons for the undesirable outcome might as follows: (a) Most of metastatic lesions of the patients were detected by fine-needle aspiration (FNA) whose sensitivity might have been limited by focal tumor colonization or bacterial concentration. (b) The hypoxia environment and necrosis of tumor might also influence the colonization of attenuated Salmonella typhimurium. However, the metastatic melanoma lesions of the patients in that study might lack of hypoxia environment and necrosis as compared with that in primary lesion. The effect of attenuated Salmonella typhimurium on primary solid tumor remains unclear. (c) The main pathway of infection of Salmonella typhimurium is mucosa38. Therefore, mucosal immune induced by PhoP/PhoQ was used in this study, which presented an effective immune response and delivery ability in mice. However, additional preclinical and clinical experiments are required to examine the safety and effectiveness of phoP/phoQ and its mucosal immune pathway.Despite the promising anti-tumor effect of the co-expression vaccine on cervical cancer in mice model in this study, this delivery way still should be selected prudently in the future clinical trial. A previous phase I clinical trial showed that after injecting with high doses of the Salmonella typhimurium (VNP20009) through vein, the inability to fully colonize tumors in metastatic melanoma patients was different from that in rodent tumor modelsIn conclusion, a pcDNA3.1-HPV16-L1-siE6 plasmid carried by attenuated Salmonella phoP/phoQ was constructed for therapy of cervical cancer through a nasal mucosa vaccination way. As a DNA vaccine, it up-regulated the expression of HPV16-L1 in the xenograft, induced the production of HPV16-L1 antibody and immune cytokines in serum, and down-regulated the expression of E6/E7 gene in tumor tissues. The premise supported that phoP/phoQ-delivered co-expression plasmid is an attractive candidate for cervical cancer treatment and require an evaluation of its safety and immunogenicity in women volunteers.2 atmosphere.Human cervical cancer cells Siha and baby hamster kidney cells BHK were purchased from American Type Culture Collection . The cells were cultured in IMDM medium  supplemented with 10% fetal bovine serum (FBS)  and maintained at 37 °C in 5% COSix-week-old BalB/c female mice and BalB/c nu/nu female nude mice were purchased from the Institute of Laboratory Animals Science (China). All animals were housed at the Laboratory Animal Center of Jilin University, with freely access to food and water. The animal experiments were approved by the Institutional Animal Care and Use Committee of Jilin University (China). All animal care was in accordance with the recommendations in the ARRIVE guidelines and the Good Laboratory Practice Regulations.To construct pcDNA3.1-HPV16-L1, genomic DNA solution of cervical cancer tissues was prepared as templates. HPV16-L1 open reading fame was amplified using primers P1, 5′-CGAAGCTTGCCACCATGGCTCTTTGGCTGCCTAGTG-3′, and P2, 5′-CCGGTACCTTACAGCTTACGTTTTTTGCG-3′. The production of PCR was cloned into HindIII and KpnI sites of pcDNA3.1(+) plasmid to construct pcDNA3.1-HPV16-L1. The new plasmid was transformed into attenuated Salmonella phoP/phoQ or Ty21a strains by electroporation.The sequences of siRNA-E6A, siRNA-E6B and siRNA-E7 were shown in Table U6 promoter and siRNA-E6A fragment were amplified with primer P3, 5′-GCAGATCTTGCTTCGCGATGTACGGGCC-3′ and P4, 5′-GGTCGCGAGGGCTATGAACTAATGACCC-3′, and PCR production was cloned into BglII and NruI sites of pcDNA3.1-HPV16-L1 to generate pcDNA3.1-HPV16-L1-siE6 plasmid.BHK cells were transfected with pcDNA3.1-HPV16-L1, and Siha cells were transfected with various pGC-siRNA plasmids using the Lipofectamine 2000 reagent .5 cells/well and transfected with pcDNA3.1-HPV16-L1 for 72 h. Standard ICC procedures were carried out according to the protocol of manufactory . Rabbit anti-HPV16-L1  was used as the primary antibodies and biotinylated goat anti-rabbit IgG was used as the secondary antibody.BHK cells were seeded to 24-well plates with cover slips on the bottom at 2 × 105 cells/well and transfected with pGC-siRNA plasmids for 48 h, standard immunofluorescence procedures were carried out as our previous study39. Rabbit anti-HPV16-E6/E7  antibodies were used as the primary antibody. Goat anti-Rabbit IgG (H + L) conjugated with Alexa Fluor Plus 594 (Thermo Fisher) was used as the secondary antibody. The cover slips were analyzed using a fluorescence microscope . Blue represented the nucleus stained with Hochest and red represented HPV16-E6 or HPV16-E7 protein.Siha cells were seeded to 24-well plate at 2 × 1040. Sections were finally counter stained with hematoxylin.After transfection for 48 h, the cell apoptosis rates were assessed with terminal dUTP nick-end labeling (TUNEL) assay according to manufactory 48 h after transfection with the pGC-siRNA plasmids, Siha cells were fixed with 70% cold ethanol at 4 °C for 12 h and stained with 1.5 mL (0.5 mg/L) propidium iodide (PI) solution. The flow cytometer  was used for single parameter analysis. The apoptosis peak was presented as the hypodiploid peak in front of G1 peak. The cell cycles were calculated as well.41. Simply, Siha cells were seeded into 96-well plates at 2 × 103 cells/well. After being transfected with the pGC-siRNA plasmids, MTT  solution  was added into each well. The absorbance was detected at 490 nm using a Microplate Reader (Japan).MTT assay was performed as described previously17. Rabbit anti-HPV16-L1, anti-HPV16-E6, anti-HPV16-E7 , Rabbit anti-P53, anti-Bax, anti-Caspase9, anti-Caspase3, anti-cleaved-Caspase3 and anti-β-actin  were used as the primary antibodies. After being incubated with secondary-HRP-antibodies , the protein levels were detected with BCIP/NBT Alkaline Phosphatase Color Development Kit  and the densities of the specific bands were quantified with an imaging densitometer .Western blot was performed as described in previous studyTotal RNA was extracted from the cells or tumor tissues using Trizol X-100 Reagent . 5 μg of total RNA was used to reversely transcribed to cDNA using a commercially available RT-PCR kit . The primers were shown in Table Female BalB/c mice were randomly divided into 5 groups (n = 6) and intranasally treated with the following dilution at the volume of 10 μL: (a) PBS, (b) phoP/phoQ (pcDNA3.1), (c) Ty21a (pcDNA3.1), (d) phoP/phoQ (pcDNA3.1-HPV16-L1), (e) Ty21a (pcDNA3.1-HPV16-L1). The treatment was performed on day 0, 7, and 21. Mice were performed euthanasia followed by AVMA guidelines on day 28. The serum and genital secretions were collected for ELISA assay.Another group of Female BalB/c mice were randomly divided into 7 groups (n = 6) and intranasally treated with 10 μL phoP/phoQ (pcDNA3.1-HPV16-L1). The treatment was performed only on day 0. Mice were performed euthanasia followed by AVMA guidelines on day 0, 5, 10, 15, 20, 25, and 30.6 Siha cells. When the tumors grown to a diameter of 3 mm, the mice were randomly split into 3 groups (n = 5), injected PBS, pGCsi-scramble and pGCsi-E6A (20 μg) respectively into tumor, and performed with electro-transfection twice. The treatment was repeated on day 15. The diameter of tumor was measured weekly. Mice were sacrificed on week 8 and the tumors were collected.Female BalB/c nu/nu nude mice were subcutaneously injected with 5 × 10Other subcutaneous xenograft models were randomly split into 5 groups (n = 5), and intranasally administrated with the following dilution at the volume of 10 μL: (a) Mock (PBS), (b) Scramble (phoP/phoQ-pcDNA3.1), (c) siE6 (phoP/phoQ -pGCsi-E6A), (d) HPV16-L1 (phoP/phoQ -pcDNA3.1-HPV16-L1), (e) siE6 + L1 (phoP/phoQ -pcDNA3.1-HPV16-L1-siE6). The diameter of tumors was measured weekly. Mice were sacrificed on week 8 and the tumors and serum were collected.41. The images were subsequently acquired using a light microscope with 200× magnification.Hematoxylin and eosin (H&E) stained was performed as described previously17. Histological slices from tumors were prepared for HPV16-L1, HPV16-E6 and HPV16-E7 IHC staining (Immunohistochemistry was performed as described previouslystaining .Anti-HPV16-L1 antibody was detected by an enzyme-linked immunosorbent assay (ELISA). The DuoSet ELISA Ancillary Reagent Kit  was purchased from R&D company. The clear 96-well microplate was coated with 100 μL of purify HPV16-L1 protein at a concentration of 10 μg/mL. Seal the plate and incubate overnight at 4 °C. Then, the plate was washed with the wash buffer . After blocking the plates with 1% BSA diluting in PBST, 50 μL of diluted serum (10 μL of each sample was diluted in 40 μL blocking buffer) was added to each well. The plates were incubated for 45 min at 37 °C. After washing four times, 50 μL of biotin-labeled goat anti-human IgG  was added for 30 min at 37 °C. After washing four times, 50 μL of the working dilution of streptavidin-HRP was added to each well for 30 min at 37 °C. After washing 4 times, each well was added with color reagent A and color reagent B. After 15 min of incubation in the dark at 37 °C, the reaction was stopped by the addition of 50 μL of stop solution to each well. The absorbance at 450 nm were detected with a Microplate Reader . IL-2 and IFN-γ in serum and genital secretions were detected by ELISA kits according to the manufacturer’s instructions .Data was analyzed with the statistical software SPSS 17.0 and the results expressed as means ± SD. Statistical analysis were made for multiple comparisons using one-factor analysis of variance (ANOVA). P < 0.05 was considered as statistical differences.Supplementary Information.


Progressive fibrosing interstitial lung disease (PF-ILD) is a progressive phenotype of fibrosing ILDs with varying definitions and elusive clinical characteristics. We aimed to clarify the clinical features and prognosis of PF-ILD cases based on the deterioration of pulmonary function.Altogether, 91 consecutive ILD patients who underwent at least 2 pulmonary function tests (PFTs) with an interval of at least 24 months, as the screening period, between January 2009 and December 2015 were retrospectively reviewed. The deterioration of forced vital capacity (FVC) and diffusing capacity of the lung for carbon monoxide (DLco) was calculated based on PFT data and screening period. The definition of PF-ILD was1.relative decline of 10% or more in FVC per 24 months or2.relative decline in FVC of 5% or more with decline in DLco of 15% or more per 24 months.Medical records of 34 patients with idiopathic pulmonary fibrosis (IPF), 11 patients with non-IPF, PF-ILD, and 46 patients with non-IPF, non-PF-ILD were retrospectively analyzed. Patient characteristics, pharmacologic or non-pharmacologic treatment status, and prognosis were compared between the IPF and non-IPF groups and between the non-IPF, PF-ILD and non-IPF, non-PF-ILD groups.Eleven patients (19.3%) showed a progressive phenotype in the non-IPF group. The pulmonary function data at the first PFT were worse in non-IPF, PF-ILD patients than in non-IPF, non-PF-ILD patients. There were no differences in the proportion of patients who were observed without pharmacologic treatment or of those receiving pharmacologic treatment between the non-IPF, PF-ILD and non-IPF, non-PF-ILD groups. Low %FVC at the first PFT and the usual interstitial pneumonia-like fibrotic pattern on high-resolution computed tomography were risk factors for PF-ILD in the non-IPF group. The mortality in the non-IPF, PF-ILD group was significantly worse than that of the non-IPF, non-PF-ILD group and was as poor as that of the IPF group. Multivariate logistic analysis showed that aging and low %DLco at the first PFT were risk factors for mortality within the non-IPF group.The prognosis of non-IPF, PF-ILD patients was as poor as that of IPF patients. Non-IPF, PF-ILD patients require more intensive treatment before disease progression. Recently, these fibrosing ILDs were grouped under the term “progressive fibrosing ILDs” (PF-ILDs).Idiopathic pulmonary fibrosis (IPF), the most common form of idiopathic interstitial pneumonia (IIPs), is a chronic and progressive fibrosing interstitial lung disease (ILD) of unknown etiology. In most clinical trials or observational studies, the rate of decline in forced vital capacity (FVC) was used to assess disease progression in ILD patients.–16 Furthermore, gas exchange parameters, including the diffusing capacity of the lung for carbon monoxide (DLco), worsening of symptoms, exercise capacity, deterioration in health-related quality of life, the extent of lung fibrosis on high-resolution computed tomography (HRCT), or the initiation of supplemental oxygen therapy, were used to assess disease progression.The definitions of disease progression of ILD vary across studies. non-IPF, PF-ILD was defined as a relative decline in FVC, worsening of symptoms, or increased extent of fibrotic changes on chest HRCT within 24 months. Nintedanib demonstrated slowing disease progression in non-IPF, PF-ILD in INBUILD trials, and now, nintedanib is one of the essential treatment options in ILDs with a progressive phenotype. However, the clinical features of non-IPF, PF-ILD in a real-world setting remain unknown.In INBUILD trials,In this study, we retrospectively analyzed the clinical features of non-IPF, PF-ILD patients based on the deterioration of their pulmonary function in the past 24 months in a real-world setting.22.1This study was approved by the Ethics Committee of the Shinshu University School of Medicine  and was performed in accordance with the Declaration of Helsinki and its subsequent amendments. The need for obtaining patient written informed consent was waived, owing to the retrospective nature of this study.This retrospective, single-center study reviewed the medical records of ILD patients, including those with IIPs, CTD-ILDs, and other related ILDs, such as drug-induced lung disease, who underwent at least 2 pulmonary function tests (PFTs) with an interval of at least 24 months, as the screening period, at the Shinshu University Hospital between January 2009 and December 2015. For patients who underwent multiple PFTs during the same period, the PFT performed closest to January 2009 was defined as the first PFT, and that performed at 24 months after the first PFT was defined as the second  were diagnosed as having ANCA-associated ILDs.,24 Patients with sarcoidosis and drug-induced pneumonitis were diagnosed according to the respective criteria.,26 In this study, PF-ILD was defined only by the deterioration of pulmonary function, in consideration of data loss such as subjective symptoms or deterioration of fibrosis on HRCT. In particular, PF-ILD was defined as theThe clinical, radiological, and pathological findings and disease behaviors were reviewed, and ILD diagnoses were made in a multidisciplinary discussion. IPF was diagnosed according to the 2018 Clinical Practice Guideline.1.relative decline of 10% or more in FVC per 24 months and2.the relative decline in FVC of 5% or more with decline in DLco of 15% or more per 24 months, with reference to recently suggested definitions.A flow diagram showing patient selection is presented in Figure 2.2 When the HRCT findings met criteria A, B, and C as described below, or criteria A and C or B and C, the HRCT pattern was defined as a UIP-like fibrotic pattern. These HRCT criteria were as follows: A) definite honeycomb lung destruction with basal and peripheral predominance; B) presence of reticular abnormality and traction bronchiectasis consistent with fibrosis, with basal and peripheral predominance; and C) absence of atypical features, specifically nodules and consolidation. Ground glass opacity, if present, was less extensive than the reticular opacity pattern.Two radiologists (S.K. and M.M.) independently reviewed the chest HRCT findings during the first PFT and determined whether the patient showed a usual interstitial pneumonia (UIP)-like fibrotic pattern or a non-UIP pattern without referring to clinical information. In case of disagreement on HRCT findings between the 2 reviewers, they discussed and resolved the case by mutual consensus. The UIP-like fibrotic pattern was defined according to the radiological criteria in the INPULSIS trials.2.3,29 The composite physiologic index (CPI),,31 representing a combination of pulmonary ventilation and diffusing capacity, was also calculated based on the pulmonary function data.In this study, patients underwent PFTs, including spirometry and measurements of the DLco, functional residual capacity, total lung capacity (TLC), and residual volume, using a pulmonary function testing system , as previously reported.Relative decline in FVC and DLco per 24 months was calculated based on PFT data and screening period. For example, when the percent predicted FVC (%FVC) at the first PFT was 80% and that at the second PFT 30 months later was 72%, the relative decline in FVC per 30 months was calculated as 10%, and the relative decline in FVC per 24 months was calculated as 8% . Statistical significance was established at P values of <.05.Continuous data are presented as median and range, and categorical data are presented as the number in each group. While the Mann–Whitney 33.1P = .04). The respective pulmonary function data and CPI at the first PFT in the IPF and non-IPF groups did not differ significantly, except for percent predicted TLC (%TLC). The %TLC and %DLco at the second PFT were poorer in the IPF group than in the non-IPF group . CPI at the second PFT was also poorer in the IPF group than in the non-IPF group (P < .01). Relative decline in FVC ≥10% per 24 months was observed in 6 (17.6%) patients in the IPF group and 9 (15.8%) patients in the non-IPF group. Relative decline in DLco ≥15% per 24 months was observed in 17 (50.0%) patients in the IPF group and 11 (19.3%) patients in the non-IPF group.The clinical characteristics of the IPF and non-IPF patients are described in Table 3.2P = .03). In the non-IPF group, 2 of the 7 patients (28.6%) with idiopathic non-specific interstitial pneumonia (iNSIP), 2 of the 16 patients (12.5%) with unclassifiable IIPs, and 3 of the 22 patients (13.3%) with CTD-ILD were diagnosed with non-IPF, PF-ILD. The %FVC and %TLC at the first PFT in the non-IPF, PF-ILD group were poorer than those of the non-IPF, non-PF-ILD group. Additionally, pulmonary function data and CPI at the second PFT in the non-IPF, PF-ILD group were also poorer than those of the non-IPF, non-PF-ILD group.Patients with non-IPF were further divided into the non-IPF, PF-ILD and non-IPF, non-PF-ILD groups. Eleven of 57 (19.3%) patients with non-IPF showed a progressive phenotype (9 patients showed the relative decline of 10% or more and 2 patients showed the relative decline in FVC of 5% or more with decline in DLco of 15% or more per 24 months). Clinical characteristics of the non-IPF, PF-ILD and non-IPF, non-PF-ILD patients are described in Table P = .02 and P = .03, respectively). However, in the multivariate analysis, these were not significant risk factors for PF-ILD in the non-IPF group.We examined the risk factors for PF-ILD in 57 non-IPF patients Table . The log3.3Treatment status throughout the study period for each disease of the IPF and non-IPF groups are shown in Table 3.4P = .02). Similarly, the non-IPF, PF-ILD group had a significantly worse survival than the non-IPF, non-PF-ILD group (P = .01). A similar trend was demonstrated in the survival analysis between the IPF and non-IPF, PF-ILD groups.The Kaplan–Meier curves of overall survival in each disease group from the first PFT are shown in Figure P < .01, P = .02, and P = .03, respectively). In a multivariate analysis, aging and low %DLco at the first PFT were independent risk factors for mortality .We identified the risk factors for mortality in 57 non-IPF patients by performing a Cox proportional hazards regression analysis Table . The uni4This study showed 3 main findings regarding the clinical characteristics of patients with non-IPF, PF-ILD. First, the pulmonary function data at the first PFT were worse in the non-IPF, PF-ILD than in the non-IPF, non-PF-ILD group. Second, approximately half of patients with non-IPF, PF-ILD underwent observation without pharmacologic treatment, despite the deterioration of their pulmonary function. Last, the prognosis of the non-IPF, PF-ILD group was significantly worse than that of the non-IPF, non-PF-ILD group and was as poor as that of the IPF group. unclassifiable ILD, CTD-ILDs, including rheumatoid arthritis-associated ILD, systemic sclerosis-associated ILD, chronic hypersensitivity pneumonitis, and exposure-related disease.,13 The definition of PF-ILD varies across studies; therefore, there is no consensus on the definition of disease progression in patients with ILDs. Nevertheless, FVC decline has been used in some trials for IPF patients,,36 and is a well-established predictor of mortality. A recent phase III study, the INBUILD trial, demonstrated slower disease progression in non-IPF, PF-ILD patients with nintedanib treatment. This trial recruited non-IPF patients showing diffuse fibrosing lung disease with >10% extent of fibrosis on HRCT scans who met the criteria for disease progression at 24 months before screening, based on a relative decline of 10% or more in FVC, relative decline in FVC of 5% or more along with the increased extent of fibrosis on HRCT, relative decline in FVC of 5% or more along with worsening of respiratory symptoms or worsened respiratory symptoms and increased extent of fibrosis on HRCT. Recently, a definition of PF-ILD was proposed by adding the criteria of relative decline in FVC of 5% or more with a decline in DLco of 15% or more per 24 months to the INBUILD criteria. In this study, we defined PF-ILD according to the deterioration of the pulmonary function in the past 24 months.The term “PF-ILD” describes chronic fibrosing ILDs and mostly includes IPF, iNSIP, with varied diagnostic concordance across multidisciplinary teams. In this study, chronic hypersensitivity pneumonitis might have been diagnosed as IPF or unclassifiable IIPs in our multidisciplinary discussion.In our study, 11 of 57 patients with non-IPF were diagnosed with ILD with a progressive phenotype (PF-ILD). Here, PF-ILDs included iNSIP, unclassifiable IIPs, pleuroparenchymal fibroelastosis, CTD-ILDs, and ANCA-related ILDs. Chronic hypersensitivity pneumonitis was not identified in this study; however, some cases of chronic hypersensitivity pneumonitis show a progressive phenotype. The diagnosis of chronic hypersensitivity pneumonitis is challenging, meaning, these patients can now be treated with antifibrotic agents. To avoid FVC decline, early treatment is essential.First, in the present study, the pulmonary function at the first PFT was worse in the non-IPF, PF-ILD group than in the non-IPF, non-PF-ILD group. Low %FVC may be a risk factor for PF-ILD in the non-IPF group in addition to the UIP-like fibrotic pattern on HRCT. Thus, the pulmonologist should administer additional treatment before disease progression. Nintedanib has been shown to slow the FVC decline in non-IPF, PF-ILD patients;Second, 6 of 11 patients (54.5%) in the non-IPF, PF-ILD group were observed without pharmacologic treatment, despite showing the deterioration of their pulmonary function. In these cases, there is a possibility that pharmacologic treatment was not administered due to concerns for adverse events of immunomodulation treatment. In addition, these patients may have only been observed because there was no effective treatment option so far. Nintedanib might be expected to be effective in these cases.,6 which could be due to the difference in the inclusion criteria. The eligible patients in this study underwent PFT including DLco measurements twice or more for at least 24 months apart. Since patients with low FVC cannot undergo DLco assessments, ILD patients with rapid disease progression were not eligible for this study. Although the criteria were similar in the non-IPF group, IPF patients with rapid disease progression, that is, a poor prognosis, were not included in this study.Third, the prognosis of patients with non-IPF, PF-ILD was as poor as that of IPF patients. Aging, low %DLco, and PF-ILD were risk factors for mortality in the non-IPF group. The prognosis of IPF patients in this study was better than that previously reported, They reported that 168 of 617 (27.2%) patients with non-IPF, ILD had progressive fibrosing phenotypes. This result was higher than our study; however, the definition of progressive phenotypes used in their study was based on INBUILD trial. Differences in the definition of progressive phenotypes might therefore have contributed to the difference in the results. Third, this was a retrospective study; thus, whether the management and assessment for disease behavior were proper or not was uncertain. A prospective study design with well-defined endpoints and a larger sample size is therefore advocated. Despite these limitations, we demonstrated the clinical characteristics of non-IPF, PF-ILD patients and showed that their prognosis was as poor as that of IPF patients.Our study had some limitations. First, several patients with rapid disease progression who could not undergo follow-up PFTs were not included in this study; therefore, their prognosis might have been better than the actual prognosis of the IPF and non-IPF, PF-ILD groups. Second, this was a single-center study with a small sample size. Recently, Nasser et al reported the clinical characteristics of patients with PF-ILD other than IPF in a real-world setting.In conclusion, the prognosis of patients with non-IPF, PF-ILD was as poor as that of IPF patients. Low %FVC may be a risk factor for PF-ILD in the non-IPF group. Non-IPF, PF-ILD patients require more intensive treatment before disease progression, and nintedanib might be an option.www.editage.jp) for English language editing.We would like to thank Editage (Conceptualization: Hiroshi Yamamoto, Masayuki Hanaoka.Data curation: Masamichi Komatsu, Hiroshi Yamamoto, Yoshiaki Kitaguchi, Satoshi Kawakami, Mina Matsushita, Takeshi Uehara, Yosuke Wada, Takumi Kinjo, Takashi Ichiyama, Kazuhisa Urushihata, Atsuhito Ushiki, Masanori Yasuo.Formal analysis: Masamichi Komatsu.Funding acquisition: Masamichi Komatsu.Investigation: Masamichi Komatsu.Methodology: Masamichi Komatsu.Project administration: Hiroshi Yamamoto.Supervision: Hiroshi Yamamoto, Masayuki Hanaoka.Validation: Masamichi Komatsu.Visualization: Masamichi Komatsu.Writing – original draft: Masamichi Komatsu.Writing – review & editing: Hiroshi Yamamoto, Masayuki Hanaoka.


This experimental study investigates the mechanical properties of polymer matrix composites containing nanofiller developed by fused deposition modelling (FDM). A novel polymer nanocomposite was developed by amalgamating polycarbonate-acrylonitrile butadiene styrene (PC-ABS) by blending with graphene nanoparticles in the following proportions: 0.2, 0.4, 0.6, and 0.8 wt %. The composite filaments were developed using a twin-screw extrusion method. The mechanical properties such as tensile strength, low-velocity impact strength, and surface roughness of pure PC-ABS and PC-ABS + graphene were compared. It was observed that with the addition of graphene, tensile strength and impact strength improved, and a reduction in surface roughness was observed along the build direction. These properties were analyzed to understand the dispersion of graphene in the PC-ABS matrix and its effects on the parameters of the study. With the 0.8 wt % addition of graphene to PC-ABS, the tensile strength increased by 57%, and the impact resistance increased by 87%. A reduction in surface roughness was noted for every incremental addition of graphene to PC-ABS. The highest decrement was seen for the 0.8 wt % addition of graphene reinforcement that amounted to 40% compared to PC-ABS. Thermoplastics and thermoset plastics are two categories of polymer materials that are used based on application. Thermoplastics polymers are the most abundantly available materials that are used in the FDM process due to lower cost and lower melting temperature . The mosThree-dimensional printing, also known as additive manufacturing (AM), is gaining popularity among industrialists and researchers. According to Ford et al., fused deposition modeling (FDM), a significant shareholder of AM in the current market, uses polymer in the form of filament as a raw material to develop either prototype or functional models. Any complex model can be quickly created using FDM. The printed model is created in any CAD format and then converted to STL file format, which is then sliced and fed into the 3D printer. The option of using AM opens up a wide range of possibilities for improving design properties and printing speed . Keshava2 compared to pure ABS; however, tensile strength decreased for jute fiber and TPE [Melenka et al. conducted an experimental study with a polymer matrix as ABS and a reinforcement of Kevlar fibers. To test the tensile properties, the composite was created with different volume percents of 4.04 percent, 8.08 percent, and 10.1 percent. It was discovered that increasing the filler content increased the Young’s modulus and tensile strength . Perez e and TPE . Vijay e and TPE .2 could improve thermal stability, and Weng Z. et al. [Brennan et al. investigated the improvement of mechanical properties for low loading of graphene oxide with carboxyl and hydroxyl functional groups. The addition of nanofiller material would significantly improve the required properties for FDM-printed parts. The improvement in the multifunctional property was observed for the addition of less than 0.1 wt percent of GO . Lin et . et al.  investigVijay T. et al. recently investigated the filament characterization of a PC-ABS filament reinforced with graphene nanofiller. The filler was added in increments of 0.2, 0.4, 0.6, and 0.8 weight percent. The SEM and elemental mapping provided useful information about the dispersion of filler content in filaments produced by the twin-screw extrusion process. The presence of filler had no effect on the diameter of the extrusion wire . The devCertain polymers have distinct properties and serve as a tough material. Polycarbonate (PC) is one such polymer with a high toughness and resistance value. It can behave as a brittle material under certain strain conditions. The addition of a limited amount of ABS to PC resulted in balancing certain properties and improvements in mechanical stability and economic value . PolymerThe extraction of PC-ABS in the form of filament, which is required for FDM, could be performed in a smooth flow while retaining the desired properties. To create the composite, graphene, a novel material with extremely high mechanical and thermal properties, is used as nanofiller. In the current study, graphene is used as a filler material in the PC-ABS matrix at 0.2, 0.4, 0.6, and 0.8 wt percent. Physical and mechanical properties of the developed polymer composite are tested.The following PC and ABS in the form of pellets were mixed in the ratio 70:30. The parts to be fabricated by polymers were obtained in the form of pellets. As shown in The addition of graphene has exhibited proper amalgamation with matrix material, leading to even surface diameter of the filament. The macroanalysis showed no cracks or flaws in the developed filament, and microanalysis did not exhibit any visible defects. The addition of graphene reinforcement to the matrix showed the diameter readings to be similar to that of pure PC-ABS without any effect on surface smoothness.JSM 7100F Jeol model field emission scanning electron microscope was used for microstructure analysis of graphene. Scanning electron microscope  was used for SEM and EDAX studies.Philips X’Pert Pro X-ray diffractometer was used for taking XRD patterns on developed composites. This ray is diffracted from the specimen and is recorded in the acquisition software.TM Raman system, Bangalore, India. First, a 785 nm wavelength laser with a power of 5–300 mW was implemented in excitation of the sample. A 100 micron laser spot size was noted. The system comprises TE cooled, efficient CCD detector arrays which are cooled at −20 °C. A vial holder is used to place the sample. High sensitivity, resolution, and stability of the machine are met with utilization of USP monograph 1120. Rayleigh scatters are filtered by deep blocking laser, which helps isolate Raman scatter for valuable molecular analysis. The Raman spectra were recorded with RSIQ software. The resolution of the spectra is ~6 cm−1. The accumulation time is 5 s.The Raman spectrum was recorded with Peak SeekerPro3 enclosed chamber was the build volume of the printer. Optimal parameters were chosen for the development of models as follows: infill density of 100%, layer thickness of 0.1 mm, shell thickness of 0.4 mm, top and bottom layer thickness of 1.2 mm, speed of 5 mm/s, the orientation of 45°, the temperature of the nozzle for PC-ABS fabrication maintained at 240 °C and varied for other proportions, and bed temperature maintained at 80 °C. The 3D printer used for printing is shown in FDM follows a process of developing the models by the addition of material layer-wise. FDM is one of the most widely used 3D manufacturing techniques used for the development of polymer parts. The 3D model is developed through a CAD file. Any complex part can be easily developed through this process . A pramaSurface roughness (Ra) was measured for the FDM parts using Miyu surface roughness tester (M35:2010), Bangalore, India. The specimen of 10 mm × 10 mm × 20 mm was developed for measurement of surface roughness Ra, through which three trials were conducted at various lengths of the surface as shown in ASTM D638 standard procedure was implemented for conduction of tensile test , make ofFDM parts’ ability to hold the load with the increase in filler content was analyzed using Izod impact test following a procedure of ASTM D4812 . The tes−1. The D band indicates the presence of disorder either in vacancies, grain boundaries, and carbon content’s amorphous nature. Johra et al. [The obtained XRD pattern for graphene is represented in a et al.  obtainedThe studies of the dispersion of nanofiller have stated that graphene dispersion occurs in the styrene-acryolnitrile phase and not in the polybutadiene phase of ABS . Gao et d spacing of 3.36 Ᾰ. Thus, the presence of peaks proves the existence of graphene in the PC-ABS matrix, with multilayer graphene having a specified d spacing. The FESEM image shown above in The first layer of filament that is laid on the heating bed from the nozzle forms a binding zone between the adjacent filament surfaces that are laid one above the other; gradually, the diffusion occurs because of variation in temperature between the adjacent layers and also between the layer and the bed, the rapid cooling of the layers leading in enhancing the bonding between the layers ,29. HoweThe roughness (Ra) average values are used to compare the roughness with different specimens and are plotted in the graph as shown in Compared to pure PC-ABS addition of 0.2 wt % of graphene, it has a decrement of 25.8% in surface roughness. Similarly, the decrement trend is followed with addition of reinforcement. The addition of 0.4 wt % graphene PC-ABS saw a reduction of 11.1% in comparison to 0.2 wt % graphene addition. Similarly, a reduction of 12.5% was noted for 0.6 wt % addition of graphene compared to 0.4 wt % addition. The highest decrement is seen for the 0.8 wt % addition of graphene reinforcement that amounts to 40% compared to PC-ABS. The calculation of error bars was performed by considering the standard deviation and dividing it by square root of the overall measurements that constitute the mean.ASTM D638 testing method was followed to determine the ultimate tensile strength of the fabricated part. The graph in The increased tensile strength with graphene addition is attributed to the massive intermolecular linkages and reinforcement alignment . This prSeyeon et al. developed a polymer nanocomposite using ABS as a matrix and iron and copper as filler material. The tensile strength analysis showed that the specimens printed with 3D printing technology would have better tensile properties with increased fill density. An impressive result was revealed such that as the increment in metal filler increases above a specific value, the decrement in tensile strength is observed .The impact strength of pure PC-ABS had a value of nearly 1 J, which increased with the incremental addition of graphene reinforcement; 0.4 wt % addition of graphene gave an incremental value of nearly 4 J, an increase of 75% with impact resistance. The highest value of 87% was observed for the addition of 0.8 wt % of graphene. Suarez et al. have stated that the addition of PC to ABS increased the developed specimens’ impact strength . An incrThe incorporation of graphene into the matrix tends to inhibit deformation and ductile mobility of polymer molecules while also absorbing energy during crack propagation. Fewer flaws in composites result from fewer void spaces, which improve composite impact strength. The following conclusion has been derived from the experimental study carried out in determining the mechanical properties of polymer nanocomposites developed by FDM:Fixed filament of required diameter was successfully carried out for PC-ABS + graphene filament by compounding and twin-screw extrusion.The PC-ABS and composite specimens have been tested perpendicular to printing, as this direction offers the highest mechanical properties in the 3D printing process.Microstructure studies indicate proper dispersion of graphene in the PC-ABS matrix and elemental mapping suggests no formation of aggregates.Reduction in surface roughness was noted for every incremental addition of graphene to PC-ABS. A 0.2 wt % addition of graphene showed a decrement of 25.8% in comparison to PC-ABS. The highest decrement was seen for the 0.8 wt % addition of graphene reinforcement that amounted to 40% compared to PC-ABS.The increment in tensile strength is in linear trend with the addition of graphene. With a maximum addition of 0.8 wt % graphene in this experiment, the tensile strength increased by 57% compared to pure PC-ABS. A 34.32% increase in yield strength has been noted for PC-ABS + 0.8 wt % when compared to pure PC-ABS. Percentage elongation was reduced with every increment of graphene added to the PC-ABS matrix.An increase in resistance to breaking energy with the addition of graphene has been observed.Graphene nanosheets addition has significantly improved the physical and mechanical properties of FDM parts.


Saccharomyces cerevisiae. Markedly, Fkh1 impinges chromatin architecture by recruiting large regulatory complexes. Implication of Fkh1 with transcriptional corepressor complexes remains largely unexplored. In this work we show that Fkh1 directly recruits corepressors Sin3 and Tup1 (but not Cyc8), providing evidence for its influence on epigenetic regulation. We also identified the specific domain of Fkh1 mediating Sin3 recruitment and substantiated that amino acids 51–125 of Fkh1 bind PAH2 of Sin3. Importantly, this part of Fkh1 overlaps with its Forkhead-associated domain (FHA). To analyse this domain in more detail, selected amino acids were replaced by alanine, revealing that hydrophobic amino acids L74 and I78 are important for Fkh1-Sin3 binding. In addition, we could prove Fkh1 recruitment to promoters of cell cycle genes CLB2 and SWI5. Notably, Sin3 is also recruited to these promoters but only in the presence of functional Fkh1. Our results disclose that recruitment of Sin3 to Fkh1 requires precisely positioned Fkh1/Sin3 binding sites which provide an extended view on the genetic control of cell cycle genes CLB2 and SWI5 and the mechanism of transcriptional repression by modulation of chromatin architecture at the G2/M transition.Transcription factors are inextricably linked with histone deacetylases leading to compact chromatin. The Forkhead transcription factor Fkh1 is mainly a negative transcriptional regulator which affects cell cycle control, silencing of mating-type cassettes and induction of pseudohyphal growth in the yeast The online version contains supplementary material available at 10.1007/s00294-021-01158-3. S. cerevisiae encodes four Fox proteins among which Fkh1 and Fkh2 (Forkhead homolog) are important regulators of the CLB2 cluster of cell cycle-regulated genes, responsible for the transition of the G2/M boundary (Jorgensen and Tyers ForkHead Associated (FHA) domain which is required for protein–protein-interactions with an emphasis on recognition of phosphopeptides . Complete genotypes of all used strains are available in the supplementary file, Table For ChIP analyses, derivatives of Escherichia coli expression plasmids  encoding various glutathione S-transferase (GST) fusions were constructed. Length variants of coding regions of the FKH1 gene were amplified by PCR and fused behind GST. Similarly, HA-tagged length variants of Sin3 representing PAH domains, Cyc8 and Tup1 were expressed in yeast using plasmid p426-MET25HA  has been described , pFK77  and pRAR110  derived from pASK-IBA5  were used. To confirm authenticity of gene fragments obtained by PCR, GST fusions which encode minimal length variants of Fkh1 were verified by DNA sequencing . Plasmid names and fused sequences are mentioned in legends of figures and are described in detail in supplementary file Table FKH1 gene (Δfkh1::LEU2). To construct this plasmid, flanking sequences upstream and downstream of the respective coding region were amplified by PCR and inserted on both sides of LEU2 selection marker, allowing total deletion of the FKH1 reading frame.To perform interaction assays, E. coli strain BL21 (Stratagene/Agilent). The tac promoter controlling GST fusion genes was induced with 1 mM IPTG. Similarly, tetR-dependent gene expression was activated by 0.2 mg/l anhydrotetracycline. Derepression of MET25-dependent gene fusions was achieved by cultivating yeast transformants in the absence of methionine. GST fusion proteins synthesized in E. coli were released by sonication, immobilized on glutathione (GSH) sepharose and subsequently incubated with yeast or bacterial total protein extracts containing HA fusions. To avoid unspecific interactions, protein extracts were pre-cleared by treatment with GSH Sepharose beads prior to incubation with GST fusions. After the release of GST fusions with free GSH (10 mM), eluates were separated by SDS-PAGE and proteins transferred to a filter. Following incubation with anti-HA-peroxidase conjugate, HA fusion proteins were detected with POD chemiluminescent substrate .GST- and HA-tagged proteins used for interaction assays by affinity chromatography were synthesized by MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4Δ gal80Δ UASGAL2-ADE2 LYS2::UASGAL1-HIS3 met2::UASGAL7-lacZ; James et al. BDTRP1) while Fkh1 domains  were inserted in pGAD-C1 (2 µm GAL4ADLEU2). Double-transformed strains containing both types of fusion plasmids were first selected on a medium lacking leucine and tryptophan (-L-T) and subsequently transferred to a medium devoid of adenine (-L-T-A).To perform two-hybrid assays, strain PJ69-4A was used (fkh1 (L74A and I78A) and the absence of any other change in the plasmids obtained .To alter selected residues in the coding region of Fkh1, the QuikChange site-directed mutagenesis kit of Stratagene was used. To obtain mutations within the FHA domain, plasmid pRAR73 containing the Fkh1 coding region was used. To replace selected residues against alanine, we used pairs of mutagenic primers introducing a GCA codon instead of the natural codon, flanked by 15–19 nucleotides on both sides. DNA sequencing was used to confirm the presence of the desired mutant alleles of FKH1 was modified such that it expressed a His-tagged Fkh1 without alteration of gene copy number or control region. Tagging was performed by the transformation of strain C13-ABY.S86 with a gene-specific modification fragment and selection for resistance against geneticin. The modification fragment was amplified by PCR, using gene-specific primers and plasmid pU6H3HA as a template , FKH11 (SIN3-HIS6-HA3-kanMX) and its isogenic fkh1 derivative RAY5 (Δfkh1 SIN3-HIS6-HA3-kanMX) grew until mid-log phase and were treated with formaldehyde for 15 min.Chromatin immunoprecipitation (ChIP) analysis followed the procedure described by Cobb and Van Attikum . Chromosg to remove insoluble material and incubated for at least 4 h with His-Tag Dynabeads® . After elution of affinity-purified proteins and bound DNA with a buffer containing 300 mM imidazole, cross-linking was reversed by heating to 65 °C overnight. DNA was recovered and analyzed by PCR (29 amplification cycles), using specific primers for promoters of CLB2 (− 880/− 580), SWI5 (− 420/− 170) or ACT1 .The crosslinking reaction was subsequently quenched for 5 min by the addition of glycine to a final concentration of 125 mM. After lysis, cells were sonicated five times for 30 s to shear chromatin, using a Bandelin Sonoplus UW 70 microtip (35% power). After sonication, lysates were centrifuged for 10 min at 16,000 Based on the methods of homology, the secondary structure of Fkh1 protein was predicted using an accurate web-based predicting tool Phyre2 , selecting on SCD -Ura -Leu medium. Transformants were cultured in double-selective medium at 30 °C until mid-logarithmic growth phase and then specific β-galactosidase activities were determined in the crude extract. The empty repressor test plasmid pRT-lexA was used as a control.Effector plasmid encoding Fkh1 (full length) fused with the DNA binding domain of lexA (supplementary Table 1b) was transformed into two strains containing integrated reporter genes -Fkh1 fusion protein  was synthesized in E. coli and subsequently bound to glutathione (GSH) sepharose. A protein extract from S. cerevisiae transformants containing epitope-tagged Sin3 (full length) was added to this affinity matrix. After intensive washing with increasing stringency, specifically bound protein was eluted by the addition of free GSH. As is shown in Fig. 3-Sin3 could be bound by GST-Fkh1 under stringent conditions but not by GST. Thus, Fkh1 may execute its function by recruiting the general corepressor Sin3.A high-throughput characterization of yeast protein complexes by mass spectrometry provided evidence for a physical interaction of Fkh1 with corepressor Sin3  can bind to PAH1 and PAH2 of Sin3. To inspect the minimal core domain of Fkh1 in charge of Sin3 recruitment, we tested shorter Fkh1 length variants which revealed that aa 81–160 of Fkh1 are able to mediate Sin3 binding. More precise, 75 amino acids at the N-terminus  turned out as the domain which is sufficient to interact with PAH1 and PAH2 of Sin3.As is apparent from Fig. Vice versa, the physical map of interacting domains within Sin3  and PAH2 (aa 301–888) were fused with the DNA-binding domain (BD) of Gal4. Length variants of Fkh1 which in vitro showed Sin3 binding were fused with the Gal4 transcriptional activation domain (AD). In vivo Sin3-Fkh1 interactions should reconstitute a functional Gal4 activator being able to stimulate expression of the 51–125 minimal binding domain which has displayed in vitro interaction with PAH2 of Sin3 is able to show in vivo binding as well, proving the interaction between Fkh1 core domain (aa 51–125) and PAH2 of Sin3.As is apparent from the results summarized in Table In contrast to in vitro results, BD fusions of Sin3  in combination with AD fusions of Fkh1 (aa 81–160 and aa 51–125) were unable to mediate growth on medium lacking adenine. Presumably, formation of functional interaction domains in vivo is prevented with certain length variants due to failure of correct protein folding. In summary, use of the in vivo two-hybrid system confirmed that PAH2 domain of Sin3 exclusively interacts with Fkh1.Based on the methods of homology, the secondary structure of the full length of Fkh1 protein was predicted to consist of 15 β-sheets and 7 α-helix structures Fig. a. The FkAs aforementioned, the N-terminus of Fkh1 residues 51–125) with its FHA domain is able to bind PAH2 of Sin3 in vitro and in vivo. Within the minimal identified domain of Fkh1, a short pattern of alternating hydrophobic amino acids which extends from aa 67–80 could be identified. To scrutinize the significance of these residues for interaction with Sin3 (and consequently for regulated expression of Fkh1 target genes), we implemented a site-directed mutagenesis at selected positions leading to the replacement of large hydrophobic amino acids to alanine [single mutations L74A and I78A; pointed out with arrows  were completely defective for interaction with Sin3. In vivo binding of Fkh1 missense variants to Sin3 was again investigated by the two-hybrid system.The impact of alanine substitutions in Fkh1 domain aa 51–125) on binding to Sin3 was subsequently investigated in vitro (by GST pull-down) and in vivo (two-hybrid system). To assay for in vitro interaction of Fkh1 missense variants with Sin3, GST-Fkh1 fusions (comprising aa 51–125) with mutational variants L74A and I78A were synthesized in 1–125 on 51–125 fusion used above together with its mutational variants L74A and I78A were co-transformed into strain PJ69-4A, containing a plasmid encoding BD-Sin3301–888 (comprising PAH2). In contrast to the wild-type minimal binding domain (Fkh151–125), Fkh1 variants L74A and I78A were unable to activate the GAL2-ADE2 reporter gene, resulting in the failure of transformants to grow in the absence of adenine  was employed to monitor directly the occupancy of particular promoters by both regulatory proteins. A substantial body of data points to the significant role of Fkh1 in the regulation of the cell cycle  during the G2/M phase of the mitotic cell cycle .Fkh1 regulates the expression of the CLB2 and SWI5 are key players in cell cycle regulation  and then Sin3 recruitment to target genes was investigated again. As is apparent from Fig. ∆fkh1 mutant strain although Sin3 was detected in the presence of an intact copy of Fkh1. Thus, Sin3 corepressor can be efficiently recruited to gene promoters when the Fkh1 protein is present.A strain encoding an epitope-tagged variant of Sin3  has been previously described  together with the Fkh1 coding region was constructed. Plasmid pRAR28 (lexABD-FKH1) together with the empty lexA plasmid pRT-lexA (negative control) was transformed into two strains containing integrated reporter genes . As shown in Fig. Although we here definitely confirmed the direct interaction between Fkh1 and Sin3, the in vivo significance of this result still remains open. According to previous findings, the assumption that Fkh1 may act as a repressor is obvious . However, a corresponding experiment with GST-Fkh1 and E. coli extract containing HA-tagged Tup1 revealed a direct interaction between Fkh1 and Tup1 , indicating that the repressor function of Fkh1 may be redundantly executed by two separate corepressor complexes.Previous studies have shown that repressor proteins may utilize more than a single corepressor complex to negatively affect transcription  was initially identified as a surrogate for SIR1 (silent information regulator one) gene as it recovered mating ability in a sir1 mutant . There was an initial study that highlighted Sin3 as an associated protein to Fkh1 through high-throughput mass spectrometric protein complex identification  and Ndd1 is dephosphorylated, Fkh proteins may instead recruit Sin3 which allows promoter access for various HDACs being associated with Sin3  domain. While the DNA binding domain of Fkh1 turned out as inessential for Sin3 corepressor recruitment, we could demonstrate that aa 51–125 precisely covering its FHA domain are sufficient to bind PAH1 and PAH2 of Sin3 in vitro. Moreover, in vivo experiments indicated the inability of Fkh1 aa 81–160 and aa 51–125 length variants to bind PAH1 domain of Sin3 which suggests that PAH2 could be the genuine docking site playing role in stabilization of Fkh1/Sin3 interaction. Importantly, the FHA domain (at least of Fkh2) has been also described as a binding site for phosphorylated Ndd1 (dependent on protein kinases Cdk-Clb and Cdc5) which is indispensable for transcriptional activation of Op-CYC1-lacZ reporter gene 4.6 fold in asynchronously growing cells. Similarly, Cti6 and Ume6 were able to repress transcription by factors of 5.6 and 7.0, respectively , indicating that Sin3 recruitment is Fkh1-dependent. Previously, Veis et al. . Future studies should investigate whether Fkh1 (and/or Fkh2) directly interact with Isw1 and Sir2 under both repression and derepression conditions. As a conclusion, the findings of previous work and the results described here are summarized by the scenarios depicted in Fig. To strengthen the in vivo significance of our results we also investigated chromatin occupancy of Fkh1 and Sin3 by ChIP. Although asynchronously growing cells were used, an association of Fkh1 and Sin3 with cell cycle-regulated promoters s et al.  reportedf et al.  could inSupplementary file1 (DOCX 5485 KB)Below is the link to the electronic supplementary material.