We used the network pharmacological analysis method to explore the mechanism of multicomponent, multitarget, and multiway actions of Xiao-Xu-Ming decoction (XXMD) for cerebral ischemic stroke (CIS), which provided a basis on the research of innovative drugs.  We used the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) to retrieve the active ingredients and targets of 12 herbs of XXMD; we used the Gene Expression Omnibus (GEO) database of the National Center for Biotechnology Information (NCBI) to screen for differentially expressed genes in CIS to obtain the disease targets of CIS and to intersect it with the action targets of XXMD, and then the target drug efficacy is obtained. We used Cytoscape 3.6 software to construct the drug-active ingredient-action target interaction network of XXMD to treat CIS and conduct protein-protein interaction (PPI) network and topology analysis. The action target Gene Ontology (GO) biological processes and metabolic pathways in Kyoto Encyclopedia of Genes and Genomes (KEGG) of XXMD to treat CIS were enrichment analyzed with R software. кB signaling pathway, Th17 cell differentiation pathway, HIF-1 signaling pathway, folic acid synthesis pathway, galactose metabolism, and fructose and mannose metabolism.  We screened out 226 active ingredients and 3646 action targets for XXMD. Among them, XXMD to treat CIS has 144 active ingredients, 12 targets, and proteins in the core network of PPI having STAT3, HIF1A, etc. Pathway enrichment analysis was based on the GO and KEGG biological processes involved in active oxygen metabolism, smooth muscle cell proliferation, cytokine production, angiogenesis, redox coenzyme metabolism, and oxidative stress. The main action processes are significantly associated with CIS signal pathways involved in microRNAs, ovarian steroid hormones, NF- This study initially clarified the main targets and pathways of XXMD in the treatment of CIS, which can lay the foundation for further research on its pharmacological effects. Cerebral ischemic stroke (CIS) refers to cerebral blood supply dysfunction, ischemia, and hypoxia due to various reasons, which leads to necrosis and softening of brain tissues and focal or complete neurological deficit symptoms. CIS includes cerebral thrombosis, cerebral embolism, and transient ischemic attacks. They have the characteristics of high morbidity, high disability, and high mortality, which seriously endanger human physical and mental health –3. HowevAs an emerging field of pharmacology, network pharmacology emphasizes the concept of “multicomponent, multitarget therapeutic network” and highlights the whole idea of TCM. It offers new methods for studying the multitarget mechanism of Chinese medicine in the treatment of CIS. In this paper, using network pharmacology methods, we analyzed the mechanism of XXMD prevention and treatment against CIS. First, we obtained the XXMD active ingredients by screening from the TCMSP database, and the target prediction of the effective ingredients was made. Second, based on the analysis of compound-target interaction by network pharmacology, the compound-target network and the compound-target-CIS network were established. Finally, the bioinformatics analysis method was used to clarify the multitarget and multiway action mechanism of XXMD in the prevention and treatment of CIS.http://tcmspw.com/tcmsp.php) in turn to search. Through screening, the two factors of oral bioavailability (OB) value ≥ 30% and drug-likeness (DL) value ≥ 0.18 were met concurrently, and 12 respective active ingredients were obtained. Then, the action targets of the active ingredients of 12 herbs of XXMD in the TCMSP were collected, the UniProt (https://www.uniprot.org/) database was used to get the official gene symbol of the whole corresponding target information, and then this part of the information for subsequent network pharmacology data analysis was used.A total of 12 herbs of XXMD including Saposhnikoviae Radix, Zingiberis Rhizoma Recens, Ephedrae Herba, Paeoniae Radix Alba, Chuanxiong Rhizoma, Ginseng Radix et Rhizoma, Armeniacae Semen Amarum, Cinnamomi Ramulus, Aconiti Lateralis Radix Preparata, Stephaniae Tetrandrae Radix, Scutellariae Radix, and Glycyrrhizae Radix et Rhizoma were typed in the TCMSP (https://www.ncbi.nlm.nih.gov/geo/). Barr et al. published the GSE16561 data set in 2010 [p < 0.05.The data of the study number GSE16561 gene expression profile chip come from the GEO database of the National Center for Biotechnology Information  and the metabolic pathway in the Kyoto Encyclopedia of Genes and Genomes (KEGG). From the two aspects of gene function and pathway analysis, the molecular mechanism of XXMD acting on CIS is clarified.With the help of the TCMSP database, we obtained 226 active ingredients of XXMD, which simultaneously meet the two screening factors of OB value ≥30% and DL value ≥0.18. Among them, there are 18 active ingredients for Saposhnikoviae Radix, 5 active ingredients for Zingiberis Rhizoma Recens, 23 active ingredients for Ephedrae Herba, 13 active ingredients for Paeoniae Radix Alba, 7 active ingredients for Chuanxiong Rhizoma, 22 active ingredients for Ginseng Radix et Rhizoma, 19 active ingredients for Armeniacae Semen Amarum, 7 active ingredients for Cinnamomi Ramulus, 21 active ingredients for Aconiti Lateralis Radix Preparata, 3 active ingredients for Stephaniae Tetrandrae Radix, 36 active ingredients for Scutellariae Radix, and 92 active ingredients for Glycyrrhizae Radix et Rhizoma. The herbs have 40 repeated ingredients, and 3646 targets of XXMD are obtained.The screening of differential genes for CIS was carried out using R software and software packages such as limma and pheatmap to analyze the data in GSE16561. According to the screening criteria, 527 differential genes were screened of which 282 were upregulated and 245 were downregulated. Select the first 30 differential genes, and use the R software pheatmap to draw a differentially expressed gene heatmap of CIS whole-blood samples and healthy whole-blood samples. The color of the heatmap reflects the level of gene expression: green represents decrease, red represents increase, and the brightness of the color represents the increase and decrease in degree of gene, as shown in Screening for differential genes in CIS, we obtained 527 CIS targets. The intersection of 3646 XXMD targets and CIS targets is taken to get 12 intersection targets. The drug-active ingredient-target interaction network of XXMD in the treatment of CIS is shown in We use the Cytoscape 3.6 software BisoGenet package to construct the PPI network of intersection targets obtained in step 2.3 . Select the input nodes and its neighbors to obtain the PPI network of XXMD in the treatment of CIS, and then use CytoNCA package to perform network topology analysis on the PPI network and filter proteins with a higher number of adjacent nodes by degree centrality (DC) (DC > 61); 763 nodes were obtained to create a subnetwork. Next, we filter the closely interacting proteins according to betweenness centrality (BC) (BC > 30) and obtained 47 nodes. The proteins in the core network of PPI are STAT3, HIF1A, etc., which may be important direct targets of XXMD in the treatment of CIS are shown in p < 0.05 and q < 0.05. The results show that, in terms of enrichment results of biological processes, the targets of XXMD in the treatment of CIS are mainly involved in the regulation of active oxygen metabolism, smooth muscle cell proliferation, cytokine production, angiogenesis, redox coenzyme metabolism, and oxidative stress and other processes. From the enrichment results of molecular functions, the effect of XXMD in the treatment of CIS is mainly manifested in the following aspects: NADP retinol dehydrogenase activity, alditol, NADP + 1-oxidoreductase activity, oxidoreductase activity, alcohol dehydrogenase (ADH) activity, protein phosphatase binding, nuclear hormone receptor binding, aldo-keto reductase (AKR) activity, histone acetyltransferase binding, tumor necrosis factor receptor binding, protein phosphatase 2A binding, phosphatase binding, hormone receptor binding, chemokine receptor binding, etc. According to the enrichment analysis results of cell components, there is no target of XXMD in the treatment of CIS , molecular function (MF), and cell component (CC)—were used. Among them, screening conditions were p < 0.05 and q < 0.05; the results are shown in Figures кB signaling pathway, Th17 cell differentiation pathway, HIF-1 signaling pathway, folate biosynthesis pathway, galactose metabolism, fructose and mannose metabolism, and so on, which are closely related to the signal pathways of CIS.We used R software and colorspace, stringi, DOSE, clusterProfiler, and enrichplot package for KEGG enrichment analysis of the targets of XXMD for CIS, and screening conditions were кB signaling pathway, Th17 cell differentiation pathway, HIF-1 signaling pathway, folate biosynthesis pathway, galactose metabolism, fructose and mannose metabolism, and so on, which are closely related to the signal pathways of CIS.Network pharmacology studies the interaction between drugs, targets, and diseases from the perspective of biomolecular networks. By abstracting the interaction between drugs, active ingredients, targets, and their related diseases into a network model, the topology parameters of each node in the network are obtained as the basis for evaluating the importance of the node in the whole network. In this study, with the help of the TCMSP, 226 active ingredients of XXMD were obtained with OB values and DL values as screening conditions. The results showed that the most active ingredient in XXMD was Glycyrrhizae Radix et Rhizoma, and the next were Scutellariae Radix, Ephedrae Herba, Ginseng Radix et Rhizoma, Aconiti Lateralis Radix Preparata, Armeniacae Semen Amarum, Saposhnikoviae Radix, Paeoniae Radix Alba, Cinnamomi Ramulus, Chuanxiong Rhizoma, Zingiberis Rhizoma Recens, and Stephaniae Tetrandrae Radix. Then, we discussed the potential biological mechanism of XXMD for CIS. We used the GEO database to obtain 527 CIS-related targets and obtained 12 intersection targets by taking the intersection with the XXMD targets, namely CD14, PTGS2, STAT1, CYP1B1, AKR1C3, AKR1B1, HMGCR, STAT3, MMP9, HIF1A, CD40LG, and HK2. Among them, PTGS2 is the common target of the most active ingredients of XXMD. Studies have found that XXMD can reduce blood-brain barrier (BBB) destruction and cerebral ischemia, the mechanism probably realized by inhibiting the expression of MMP-9 and so on . ThroughкB signaling pathway is one of the initiating factors of vascular endothelial cell, neuron, and glial cell injury. It participates in the inflammatory process after cerebral ischemia and promotes apoptosis after ischemia. Estrogen can inhibit NF-кB activation by upregulating receptors [α (HIF-1α) is a transcriptional regulator produced during hypoxia. It can regulate many hypoxia-related genes such as vascular endothelial growth factor, glucose transporter, erythropoietin, inducible nitric oxide synthase, B-cell lymphoma gene 2, caspase, adenovirus interfering protein 3, Ngb, heat shock protein 70, etc., and these genes increase ATP release by promoting anaerobic metabolism, promote the reconstruction of microcirculation and vasodilation, promote erythropoiesis, increase oxygen-carrying capacity, reduce nerve cell apoptosis, etc., and reduce brain tissue injury after CIS [Herpes virus infection causes the proliferation of vascular smooth muscle, makes endothelial cells produce adhesion receptors, and then accelerates the formation of thrombus, which is closely related to CIS . MicroRNeceptors . After ceceptors . Th17 ceeceptors . HypoxiaкB signaling pathway, herpes virus infection pathway, microRNAs, ovarian steroid hormones, Th17 cell differentiation pathway, folate biosynthesis pathway, galactose metabolism, and fructose and mannose metabolism pathway. This research studied the efficacy mechanism of XXMD from an integrity and systematic perspective, which provided basis for further understanding and using this decoction in the future. Certainly, only using network pharmacology technology to study the active ingredients and targets of XXMD, there are some limitations; for instance, the information in the databases inclines to some hot areas and the limited number of small-molecular compounds and drug targets, and the original information comes from different experimental conditions. Consequently, it is necessary to further confirm the results of this research with the help of experimental research in the future, especially the regulatory mechanism of XXMD and its components of the HIF-1 signaling pathway and NF-кB signaling pathway.To sum up, the potential biological mechanism of XXMD in treating CIS has the multicomponent, multitarget, and multiway characteristics, and STAT3 and HIF1A may be the important direct targets of XXMD in the treatment of CIS. It may be related to the HIF-1 signaling pathway, NF-


Cells have evolved highly intertwined kinase networks to finely tune cellular homeostasis to the environment. The network converging on the mechanistic target of rapamycin (MTOR) kinase constitutes a central hub that integrates metabolic signals and adapts cellular metabolism and functions to nutritional changes and stress. Feedforward and feedback loops, crosstalks and a plethora of modulators finely balance MTOR-driven anabolic and catabolic processes. This complexity renders it difficult — if not impossible — to intuitively decipher signaling dynamics and network topology. Over the last two decades, systems approaches have emerged as powerful tools to simulate signaling network dynamics and responses. In this review, we discuss the contribution of systems studies to the discovery of novel edges and modulators in the MTOR network in healthy cells and in disease. Over the last years, detailed molecular studies of signaling proteins have been increasingly complemented with systems approaches that allow us to understand the dynamic network-tuning arising for instance from interconnected feedback and feedforward loops ,2. Fundaernetics ,4 and inernetics , Tyson aernetics , are nowIn cell signaling research, systems models informed by time-series data are used to simulate the adaptation of a signaling network to multiple inputs or perturbations, including drug treatments. Such strategies serve for instance to dissect the convergence of known feedforward and feedback loops on a common effector to predict the outcome of a drug perturbation. Furthermore, novel network nodes (e.g. proteins) and connections (e.g. protein–protein interactions) can be postulated and the likelihood of alternative hypotheses can be compared in a quantitative manner. Simulations of signaling outputs arising from alternative network topologies can guide the experimentation to test those hypotheses. Hence, the classical iterative workflow of theoretical and experimental physics is now being translated to the life sciences, and theoretical and experimental biology and medicine work hand in hand.The tools and methodologies in theoretical biology are as diverse as in the experimental life sciences and they are constantly developing according to the specific biological problems that are being investigated. For instance, theoreticians develop new ways to deal with noisy data ,8 or nonin silico network modeling with experimental time-series data and validation provides a powerful means to understand the behavior of biological networks in a feasible time frame and work effort. Given the size of the field and multiplicity of problems and studies, we won't attempt a comprehensive overview. Instead, we will outline recent developments and applications focusing on the signaling networks converging on the metabolic master regulator MTOR. We discuss systems approaches of the last decade, which identified and experimentally validated novel edges in the MTOR network, focusing on ordinary differential equation (ODE)-based models constituting the majority of dynamic systems studies on MTOR [Modeling studies are performed based on prior data, and they generate hypotheses that are tested in subsequent experiments, which in turn can be incorporated into the models. Such iterative combination of  on MTOR .Cells are living systems, which constantly exchange information with their environment. Environmental inputs are translated into cellular signals that are transmitted through signaling networks to elicit responses that enable a cell to adapt to its environment. The serine/threonine protein kinase MTOR is at the centre of such a network which in response to metabolic signals promotes anabolism and inhibits catabolism . A complMTOR kinase resides in two distinct multiprotein complexes, termed mTOR complex 1 (mTORC1) and mTORC2  results in the recruitment and tyrosine phosphorylation of the insulin receptor substrate 1 (IRS1) ,43 Figu. IRS1 isin vitro and in vivo [mTORC1 activation by insulin is tightly balanced by several feedback loops. On the one hand, mTORC1 phosphorylates GRB10 (growth factor receptor-bound protein 10) ,65, whic in vivo   regulation by mTORC1 and mTORC2 in the context of insulin resistance in T2D ,70 Tabl; b,c. FOin silico network modeling with time series data to unravel signaling crosstalk. What is the biological importance of concomitant AMPK and mTORC1 activity? Dalle Pezze et al. [In response to amino acids, mTORC1 translocates to the surface of the lysosomes where it encounters its activator RHEB . Hence, e et al.  proposede et al.  suggestein silico analysis Smith and Shanley [Next to metabolic signals, MTOR responds to numerous stressors including nutritional, oxidative, endoplasmic reticulum, and hypoxic stress ,42. The  Shanley  suggeste Shanley  and idenSystems studies have uncovered new crosstalk and mechanisms in the MTOR network. Thus, they complement experimental approaches and open new avenues to hypothesis building and testing in metabolic signaling. Next to applications in basic research, systems approaches are currently also being developed for medical applications ,125. MajSystems modeling complements experimental biology for hypothesis building and testing in metabolic signaling.Systems approaches constitute powerful tools to decipher complex network topologies and signaling dynamics upstream and downstream of MTOR.Computational models of metabolic signaling hold promise for applications in systems medicine.


Human infertility affects ~15% of couples worldwide, and it is now recognized that in half of these cases, the causes of infertility can be traced to men . The speSpermatozoa are terminally differentiated cells produced in the testes during the hormone-regulated process of spermatogenesis. Two somatic cell types are critical to this process: Sertoli cells protect and support the germ cell development, whereas interstitial Leydig cells produce necessary intratesticular steroids  ,14. AfteA key feature of sperm capacitation is the production of ROS at very low and controlled levels by the spermatozoon. This essential phenomenon for the acquisition of the fertility ability was first reported in humans , bovine 2, is a key element in the antioxidant defence to protect sperm membranes and DNA from oxidative damage [Oxidative stress can promote detrimental changes during spermatogenesis, epididymal maturation, and sperm capacitation that can lead to infertility ,33,34,35e damage ,40. Oxide damage ,42,43,44e damage ,46,47. Te damage ), and fre damage ,50,51,52The decline of fertility and the increase of abnormalities in the semen of men that is observed over more than two decades is worrisome. However, we still do not have sufficient information to understand why this is happening ,54,55. TThis Special Issue on ROS and male fertility is composed of four original contributions that provide new data on the role of SOD  and PRDXMany groundbreaking studies have contributed to our understanding of the field of ROS in male fertility. It is undeniable that these reactive molecules play an essential role in both physiological and pathological mechanisms of the male reproductive system. Antioxidant therapy is a common clinical strategy to improve sperm quality and function in infertile men ,67. Howe


Philasterides dicentrarchi is a marine benthic microaerophilic scuticociliate and an opportunistic endoparasite that can infect and cause high mortalities in cultured turbot . In addition to a cytochrome pathway (CP), the ciliate can use a cyanide-insensitive respiratory pathway, which indicates the existence of an alternative oxidase (AOX) in the mitochondrion. Although AOX activity has been described in P. dicentrarchi, based on functional assay results, genetic evidence of the presence of AOX in the ciliate has not previously been reported. In this study, we conducted genomic and transcriptomic analysis of the ciliate and identified the AOX gene and its corresponding mRNA. The AOX gene  contains four exons and three introns that generate an open reading frame of 915 bp and a protein with a predicted molecular weight of 35.6 kDa. The amino acid (aa) sequence of the AOX includes an import signal peptide targeting the mitochondria and the protein is associated with the inner membrane of the mitochondria. Bioinformatic analysis predicted that the peptide is a homodimeric glycoprotein, although monomeric forms may also appear under native conditions, with EXXH motifs associated with the diiron active centers. The aa sequences of the AOX of different P. dicentrarchi isolates are highly conserved and phylogenetically closely related to AOXs of other ciliate species, especially scuticociliates. AOX expression increased significantly during infection in the host and after the addition of CP inhibitors. This confirms the important physiological roles of AOX in respiration under conditions of low levels of O2 and in protecting against oxidative stress generated during infection in the host. In plants, AOX is considered a ubiquitous enzyme; however, in algae3, fungi9, protozoa13 and several invertebrate species14 it is quite sporadic. AOX is involved in the mitochondrial respiratory chain and provides an alternative pathway for the direct transfer of electrons from ubiquinone to oxygen, thus forming water15. It facilitates mitochondrial respiratory activity and therefore the functioning of the Krebs cycle after inhibition of the cytochrome route due to the presence of nitric oxide, cyanide (both inhibitors of the complex IV) or antimycin A (complex III inhibitor)16. However, AOX reduces the energy efficiency of respiration as only 1/3 of the ATP is produced by pumping protons through complex I17. In plants, AOX participates in important biological processes, such as survival and adaptation to stress factors in biotic and abiotic environments, resistance to oxidative stress and maintenance of mitochondrial and cellular energy homeostasis19. In parasitic protists, it is particularly associated with metabolic adaptation to the host and pathogenicity22. AOX is present in the mitochondria of protists such as the amoebozoans Acanthamoeba castellani23, A. polyphaga24 and Dictyostelium discoideum25, Trypanosoma brucei26, T. congolensi and T. evansi27, Plasmodium falciparum28, Cryptosporidium parvum29 and Blastocystis hominis22. AOX has also been detected in the Euglenozoa Euglena gracilis30, in the ciliates Paramecium tetraurelia31, Tetrahymena pyriformis32 and Ichthyophthirius multifiliis33; and in the scuticociliate parasites Pseudocohnilembus persalinus34 and Philasterides dicentrachi13. In previous studies, we have demonstrated the presence of a cyanide-resistant alternative respiratory pathway (AP) in mitochondria of the scuticociliate parasite P. dicentrarchi, in addition to the conventional cytochrome pathway (CP)13. This provided physiological evidence for the presence of a cyanide-insensitive oxidase, which was recognised by antibodies raised against a synthetic peptide derived from a fully conserved C-terminal consensus motif from plant AOX isoforms13. In addition, AOX inhibitors such as salicylhydroxamic acid (SHAM) and 3,4,5-trihydroxybenzoic acid  significantly inhibit in vitro growth of the ciliate36.Alternative oxidase (AOX), also known as ubiquinol-oxidoreductase, is included within the diiron carboxylate superfamily. The enzyme is characterized by cyanide resistance and is associated with maintenance of metabolic homeostasis via mitochondrial electron transport in plants subjected to various stress conditionsP. dicentrarchi, this enzyme has not previously been identified or characterized in detail at the molecular level. In the present study, we characterized the P. dicentrarchi AOX gene and detected the AOX protein in the mitochondria. We also investigated the transcriptional modulation of this enzyme in vitro, in response to experimentally generated hypoxia, as well as in response to the presence of inhibitors of CP and AP mitochondrial respiration.Although there is clear physiological evidence for the existence of an AOX in Scophthalmus maximus). Juvenile fish (50 g) were obtained from a fish farm in Galicia (NW Spain). The fish were held in 200 L fibreglass tanks with a water recirculation system, at 16 °C with constant aeration and a photoperiod of 12L:12D, and were fed daily with commercial pellets . The fish were acclimatized to the aquarium conditions for 2 weeks before the start of the experiments.Turbot  of weight 25 g were supplied by the Central Animal Husbandry Unit of the University of Santiago de Compostela (Spain).All animal experiments and protocols were conducted in accordance with Spanish and European Legislation (R.D. 53/2013 and Council Directive 2010/63/EU) and approved by the Institutional Animal Care and Use Committee of the University of Santiago de Compostela (Spain). When required during all experiments, the animals were anaesthetized with isoflurane (mice) or 100 mg/L of tricaine methane sulphonate-MSS-222-(turbot) and finally euthanized by decapitation (mice) or by overdose of anaesthesia (turbot).P. dicentrarchi37 used (I1) was obtained from ascites of experimentally infected turbot 38. The isolate was grown axenically at 21 °C in complete L-15 medium39 under (a) normoxic conditions, in culture flasks provided with vented caps to allow aeration of the culture medium, or (b) under hypoxic conditions, by bubbling the culture flasks for 10 min with argon gas and tightly closing the flask with non-vented caps, after addition or removal of the ciliates13.The isolate of P. dicentrarchi genome and transcriptome, trophonts (107) were concentrated by centrifugation, frozen in liquid nitrogen and sent on dry ice to Future Genomic Technologies . For sequencing of the complete genome of the ciliate, a combination of short reading sequencing (Illumina technology) and long reading sequencing (Nanopore technology) was used (Oxford Nanopore Technologies). For de novo assembly of the parasite genome, the data sets were combined using the TULIP program v0.440. Transcript sequences from Illumina RNA- Seq data (fragments of approximately 100 bp), obtained by amplification by SBS, were assembled using Trinity software (v2.6.5)41, included within the Galaxy application . The assembled sequences were analyzed by homology, with Blastgo 5.0 software , and annotated. The sequences that encode proteins that are potentially related to the ciliate AOX were then selected from the Tetrahymena thermophila gene and protein sequences database by using the BLASTx tool Wiki TGD (https://www.ciliate.org/blast/blast_link.cgi).For analysis of the P. dicentrarchi AOX was obtained with an open reading frame (ORF) search tool , from the annotated data obtained by analysis of a RNA-Seq experiment. The codons of the original nucleotide sequence in P. dicentrarchi were optimized using the Integrate DNA Technologies (IDT) bioinformatics tool (https://eu.idtdna.com/CodonOpt), to produce a recombinant protein in the yeast Klyuveromyces lactis. The AOX gene nucleotide sequence was synthesized by Invitro GeneArt Gene Synthesis (ThermoFisher Scientific), amplified by PCR with the primers FaoxKl / RaoxKl: 5′-CGC CTC GAG AAA AGA ATG CAG TCC TTC GCC AGG AAA-3′/5′-ATA AGA ATG CGG CCG C TTA GTG GTG GTG ATG ATG GTG ATG ATG ATG ATG CTG CTC GGG GGA GTA AGG GTT 3′ and cloned in the integrative expression vector pKLAC2. The K. lactis Protein Expression kit  was used to express the recombinant protein in yeast according to the manufacturer’s instructions and the protein secretion strategy, as previously described42.The complete nucleotide sequence that encodes 42 and adsorbed for 30 min at room temperature in 200 µL of a 1% chitosan hydrogel adjuvant42 (CH) (w/v) in PBS buffer, pH 7.0. At intervals of 15 and 30 days after the first immunization, mice were reimmunized with the same dose of rAOX and adjuvant. Seven days after the second immunization, the mice were bled by decapitation and the blood thus obtained could clot overnight at 4° C. The serum was clarified by centrifugation at 2000 × g for 10 min, mixed 1: 1 (v/v) with glycerol, and stored at −20 °C until use.The anti-rAOX antiserum used in the immunoassays was obtained from CD-1 mice immunized intraperitoneally (i.p.) with 200 µg of rAOX. The antiserum was purified by immobilized metal affinity chromatography (IMAC) with Ni-Sepharose44—and treated under reducing conditions (after the addition of 0.02 M dithiothreitol-DTT-) or under non-reducing (without the addition of DTT), was performed on linear 12.5% polyacrylamide minigels in a Mini-Protean Tetra cell system , as previously described35. Once electrophoresis was completed, the gels were stained with a solution of GelCode Blue Stain Reagent (Thermo Scientific) according to the manufacturer's instructions.SDS-PAGE of the rAOX protein and a ciliate lysate (CL)—obtained from cultures maintained under normoxic or hypoxic conditions35. The samples were first incubated with serum from mice immunized with AOX (anti-rAOX serum) and then with a polyclonal peroxidase-conjugated rabbit anti-mouse antibody  at 1:800 dilution. The blots were stained by adding a chromogenic enzyme substrate solution consisting of 0.003% H2O2 and 0.06% 3,30-diaminobenzidine tetrahydrochloride with 0.03% NiCl2 .Samples separated by electrophoresis were also analyzed by Western blot, with a slightly modified version of a previously described protocol46. Briefly, ciliates were fixed in a solution of 4% formaldehyde in PBS, permeabilized in a solution containing 0.3% Triton X-100 in PBS and blocked with a solution of 1% BSA. The ciliates were then incubated with a mouse antiserum containing anti-rAOX antibodies diluted 1:100 in PBS and with a secondary polyclonal rabbit anti-mouse immunoglobulin conjugated with fluorescein isothiocyanate  and diluted 1:1,000. The ciliates were then visualized by fluorescence microscopy .For AOX immunolocalization in trophonts, an IIF immunoassay was performed, as previously described47. Briefly, the cultured ciliates were fixed in 2.5% (v/v) glutaraldehyde in 0.1 M cacodylate buffer at pH 7.2, post-fixed in 1% (w/v) OsO4, pre-stained in saturated aqueous uranyl acetate, dehydrated in acetone series and embedded in Spurr’s resin. Ultrathin sections were stained with 2.5% uranyl acetate in 50% ethanol and 0.5% of lead citrate in 4.5 mM NaOH and viewed in a Jeol JEM-1011 transmission electron microscope  at an accelerating voltage of 100 kV.For TEM analysis we followed the technique described by Paramá et al.35. Briefly, the ciliates were cultured at an initial concentration of 5 × 104 trophonts/mL in complete L-15 medium in culture flasks fitted with vented caps that allowed aeration of the culture medium (normoxic conditions) and in tightly closed bottles bubbled with argon (hypoxic conditions). To ensure hypoxic conditions, the L-15 medium was bubbled with argon for 10 min until gas saturation, to completely displace the dissolved oxygen in the medium. The ciliates were then added, and the bottles were hermetically sealed to prevent entry of oxygen. To determine growth of the cultures under conditions of normoxia and hypoxia, aliquots of 100 μL were collected daily for 6 days and the number of ciliates was quantified in a Neubauer counting chamber48. After collection of each sample to quantify the ciliates, the argon bubbling was repeated in all cases under the same conditions as described above to maintain the hypoxic conditions.To determine the growth of the ciliates under conditions of normoxia and hypoxia, we followed the protocol described by Mallo et al.35. Briefly, the total RNA extracted from 106 trophozoites/mL of P. dicentrarchi cultured under hypoxic/normoxic conditions, in the presence of 1 mM SHAM and 1 mM KCN , was isolated with a NucleoSpin RNA isolation kit (Macherey–Nagel) according to the manufacturer’s instructions. The quality, purity and concentration of RNA were quantified in a NanoDrop ND-1000 Spectrophotometer . Two μg of sample RNA, 1·25 μM random hexamer primers (Promega) and 200 U of Moloney murine leukemia virus reverse transcriptase  were used for cDNA synthesis (RT). Quantitative polymerase chain reaction (qPCR) analysis was performed with AOX gene-specific primers forward/reverse pair FqAOX/RqAOX (5′ TCG GAG ACT CCT TCG CTT AC-3′/5′-CCA TCC TTG GTC TCT TTC CA-3′) and P. dicentrarchi elongation factor 1-alpha gene (EF-1α) (GenBank accession KF952262) forward/reverse primer pair (FEF1A/REF1A: 5′-TCG CTC CTT CTT GCA TCG TT-3′/5′-TCT GGC TGG GTC GTT TTT GT-3′) as housekeeping gene. The reaction mixture (10 μL) used in the qPCR contained 5 μL of PowerUp SYBR Green Master Mix (Applied Biosystems), 300 nM of the primer pair, 1 μL of cDNA and RNase–DNase-free water. The qPCR was run at 95 °C for 5 min, followed by 40 cycles at 95 °C for 10 s and 60 °C for 30 s, ending with melting-curve analysis at 95 °C for 15 s, 55 °C for 15 s and 95 °C for 15 s. in a StepOnePlus Real Time System (Thermo Fisher Scientific). Finally, relative quantification of gene expression was determined by the 2−ΔΔct method49 with the software included in the equipment.The RT-qPCR technique was basically performed as previously described, with slight variations50. The transmembrane topology and location of signal peptide cleavage sites in amino acid (aa) sequences were predicted with the PHOBIUS program51. The N-terminal region of the protein that may contain a mitochondrial signal sequence and its cleavage site were analyzed with the MITOPROT II -v1.101 program52. The physicochemical parameters were predicted for a given protein with the The PROTPARAM tool53. The O-ß-GlcNAc attachment sites were predicted with the YINGOYANG54 and OGLCNACSCAN55 programs. For visualization of proteoforms and integration of the protein topology, the PROTTER bioinformatics tool was used56. Sub-cellular localization was predicted with the LOCTREE3 program57. The SWISS-MODEL protein server was used for modelling protein structures and complexes58. The aa sequences were aligned using the CLUSTAL OMEGA multiple sequence alignment program 59, and the phylogenetic signal/noise ratio was improved with the TRIMAI tool for automated alignment trimming60. The phylogenetic trees were constructed by the Maximum Likelihood (ML) method, with the JTT model61. Branch support was given with 1,000 bootstrap replicates62 in MEGAX software63. The Bayesian inference (BI) analysis was performed with MRBAYES 3.2.664.Functional analysis of proteins and classification into different families to predict the domains and important sites was carried out with INTERPRO softwareP < 0.05.The values shown in the text and figures are means ± SEM. One-way analysis of variance (ANOVA) was used for comparison of more than two samples, and the Tukey-Kramer test was used for pairwise comparisons. The Student’s t-test was used for comparison of two samples. In all cases, differences were considered significant at The complete sequences of the AOX gene and cDNA were deposited in the NCBI GenBank database, with accession numbers MN193569 and MH427340, respectively. The gene encoding the AOX has a total length of 1,107 bp and contains 4 exons and 3 introns, with an average intron size of around 60 bp   Fig. A,B. The 3+) Fig. C.Figure P. dicentrarchi strains  in order to estimate the degree of identity , we used bioinformatic tools to identify and predict the structure of the gene and the mRNA that encodes the AOX. The AOX of P. dicentrarchi is encoded by a single nuclear gene consisting of 4 exons and 3 introns. In many plant species, AOX enzymes are encoded by a multigene family68; however, in fungi and in some protozoa the genes encoding AOX are less diverse than in plants and, in many cases, this enzyme is encoded by a single nuclear gene69. The typical structure of the AOX gene is characterized by the presence of four exons separated by three gene-specific introns of different lengths71. After transcription and splicing, the AOX gene in P. dicentrarchi generated a mature mRNA that encodes a protein of an estimated molecular weight of 35,638.68 daltons, a size like the standard sizes of the AOXs of various organisms ranging between 32–36 kDa15. AOX is an integral interfacial membrane protein that interacts with a single leaflet of the lipid bilayer26. The first studies of the membrane localization of AOX in plant mitochondria showed that AOX has two transmembrane helices and a short loop located in the matrix73. However, later topological studies indicated that AOX is an integral monotopic membrane protein that embeds into a single face (inner) of the membrane76. There is some controversy regarding prediction of the transmembrane topology of the AOX in P. dicentrarchi. According to the Phobius program, AOX has two transmembrane helix regions, while other bioinformatic programs predict a single transmembrane helix region. In the first case, it is assumed that the C- and N-terminal domains of the protein will be located in the mitochondrial matrix; however, in the second model the N-terminal motif would be located outside the inner membrane and the C-terminal towards the mitochondrial matrix77. To resolve these differences, new computational methods are probably needed, as suggested by other authors78. In any case, until the crystal structure of AOX of P. dicentrarchi is available, it will not be possible to definitively clarify the topology of this enzyme.AOX is a terminal ubiquinol oxidase that participates in an alternative mitochondrial pathway that couples oxidation of ubiquinol to the reduction of O81. Although the AOX family contains highly conserved active sites, small differences have been observed in ligands associated with the active center in several AOXs22. P. dicentrarchi has the typical C-terminal E-X-X-H motif82, in which the conserved glutamate and histidine residues participate in the coordination of two iron atoms21.Analysis of AOX crystal structure indicates that it is a homodimer with a non-haem diiron carboxylate protein in which the metal atoms are ligated to four fully conserved glutamate and histidine residues83. On the other hand, Maximum Likelihood methods have revealed the existence of two main clades of AOX proteins: a clade containing AOXs of Ascomycota and Basidiomycota fungi, invertebrates, kinetoplastids and alveolate protozoa, and another clade that includes plants, algae and various fungal groups83. In our study, phylogenetic analysis by ML and BI methods also indicated the existence of two main clades of AOXs: and Sar (apicomplexans and kinetoplastids), and another including Metazoa, Fungi and Sar (Ciliophora). The present results also indicate that the AOX of P. dicentrarchi is closely related to AOXs of other ciliate species, especially the scuticociliate P. persalinus, but it is relatively distant from other Sar members as apicomplexans and kinetoplastids. In addition, although the enzyme sequence is highly conserved among strains of P. dicentrarchi, some differences could be used, together with sequences of other proteins, to carry out multigenic genotyping, to enable accurate, intraspecific characterization of the isolates of this scuticociliate84.Phylogenetic studies of AOX sequences indicate that Viridiplantae, Metazoan, Fungi and SAR form monophyletic clades75. Immunochemical and immunohistochemical analysis indicates that P. dicentrarchi have monomeric forms (of about 35 kDa) and homodimeric forms (of 70 kDa) associated with the mitochondrial inner membrane.Plant studies indicate that AOX is an integral homodimeric membrane protein in which the monomers have an estimated molecular mass of 40 kDa and a mitochondrial directed sequence that is cleaved to generate mature forms with a molecular mass of over 32 kDa85. Therefore, the results obtained indicate that P. dicentrarchi may show two AOX forms: one monomeric of 35 kDa and other dimeric of 70 kDa, due to the presence of a mixture of “reduced” and “oxidized” forms in the mitochondrial membrane.These results strongly suggest that a disulfide bond can link two monomers . This is what occurs in plants, where the status of this disulfide bond impacts in enzyme activity, thus when there is not disulfide bond between two monomers (reduced form), the AOX activity is strongly activated by allosteric effectors, while the oxidized form (disulfide bond between two monomers) has little activity, even with high concentrations of the allosteric effectors88. This hypothesis can also be extended to P. dicentrarchi, a benthic (like most scuticociliates) marine organism, which is also microaerophilic89.Phylogenetic analysis of AOX from different organisms has indicated the existence of a monophyletic clade among benthic marine animals and the presence of AOX in these species, which is probably related to a mechanism of adaptation to variations in oxygen concentration, hypoxic conditions and increased hydrogen sulphide concentration18. AOX has a lower affinity for O2 than cytochrome oxidase, possibly indicating higher expression/activity under conditions of low oxygen concentration91. It has also been suggested that AOX expression can be increased in plants under hypoxia to reduce the formation of superoxide (O2−), nitric oxide (NO) and peroxynitrite (ONOO-), as well as to reduce the toxic effect of H2O294. P. dicentrarchi is a microaerophilic microorganism capable of surviving in conditions of low oxygen concentrations13. Under these conditions, amount of AOX transcript and of protein increase significantly in the ciliate. Gene transcription and protein expression increase during the stationary phase of growth, during which O2 levels in the culture are low13. The low concentrations of O2 and the increased levels of reactive oxygen species (ROS) are also associated with infection in the host. In the first case, O2 levels during the endoparasitic phase are low, while ROS levels are high as a result of the innate immune response produced during infection96. Both the infection and the innate humoral immune response generated by the host induce a significant increase in AOX transcription in the ciliate.Expression of AOX has been reported to be modulated in response to hypoxia and anoxia or after reoxygenation98. Hypoxia, generated either by the removal of O2 from the culture medium or by the addition of cyanide, significantly induces the transcription and expression of AOX13. On the other hand, the addition of the AOX inhibitor, SHAM99, only increases the transcription of the enzyme under hypoxic conditions. The data of the increase in AOX transcription obtained in this study are closely related to the results obtained at the physiological level. Thus, the determination of the mitochondrial oxygen consumption shows that under hypoxia conditions and in the presence of inhibitors of the cytochrome pathway, the ciliate preferably uses the alternative pathway respiration13.Agents that inhibit the cytochrome bc1 significantly induce AOX transcriptionP. dicentrarchi. In this ciliate, the AOX occurs in the monomeric and dimeric forms and is associated with the inner membrane of the mitochondria. The amino acid sequence was found to be highly conserved among the different strains of this ciliate and is closely related to AOXs of ciliate species and, particularly, to the AOX of scuticociliates. Gene transcription increased significantly under conditions of hypoxia and after infection and addition of inhibitors of cytochrome pathway respiration. The molecular characterization of AOX, together with the functional evidence of its role in adapting to parasitism and in resistance to oxidative stress induced after infection, opens a promising route for developing structure-based antiparasitic drug design strategies through molecular docking.In conclusion, we have identified the gene and the mRNA that encode a protein belonging to the AOX family, not previously characterized at the molecular level in the scuticociliate Supplementary information


The human T cell leukemic/lymphotropic virus type 1 (HTLV-1), discovered several years ago, is the causative agent for a rapid progressive haematological malignancy, adult T cell leukemia (ATL), for debilitating neurological diseases and for a number of inflammatory based diseases. Although the heterogeneous features of the diseases caused by HTLV-1, a common topic concerning related therapeutic treatments relies on the use of antiretrovirals. This review will compare the different approaches and opinions in this matter, giving a concise overview of preclinical as well as clinical studies covering all the aspects of antiretrovirals in HTLV-1 infection. Studies will be grouped on the basis of the class of antiretroviral, putting together both pre-clinical and clinical results and generally following a chronological order. Analysis of the existing literature highlights that a number of preclinical studies clearly demonstrate that different classes of antiretrovirals, already utilized as anti-HIV agents, are actually capable to efficiently contrast HTLV-1 infection. Nevertheless, the results of most of the clinical studies are generally discouraging on the same point. In conclusion, the design of new antiretrovirals more specifically focused on HTLV-1 targets, and/or the establishment of early treatments with antiretrovirals could hopefully change the perspectives of diseases caused by HTLV-1.  The human T cell leukemic/lymphotropic virus type 1 (HTLV-1) . It has HTLV-1 is the etiological agent of a rapid progressive malignancy, Adult T cell leukemia (ATL), which develops in 5% of infected people, as well as a debilitating neurological disease, HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), which develops in 4% of HTLV-1 positive individuals. In addition, a number of inflammatory HTLV-1 associated diseases such as uveitis, Hashimoto’s thyroiditis and Graves’ disease, HTLV-1 associated pulmonary disease, infective dermatitis associated with HTLV-1, HTLV-1 associated inflammatory myositis, and HTLV-1 associated arthritis have been reported . AlthougZidovudine , a nucleoside analogue acting as competitive inhibitor of reverse transcriptase was the first effective antiretroviral used in HIV infection . The potAn HTLV-1 associated neuromyelopathy, originally named tropical spastic paraparesis (TSP), was first uncovered in the Caribbean area. A similar neurological disorder was then described in Japan and named as HTLV-1 associated myelopathy (HAM). The two diseases were then recognized to be identical and here we refer to this neurological disease, occurring in HTLV-1 infected patients, as HAM/TSP. First clinical trials using AZT in HTLV-1-infected HAM/TSP patients provided conflicting reports. An open study conducted for six months on five patients (1 g/day) affected by HAM/TSP observed no effect of AZT on the encephalomyelopathy and therefore no clinical benefit . ConversObviously, since HAM/TSP is caused by HTLV-1 infection, the assessment of the effect of antiretroviral therapy on viral load could highlight the possible influence of inhibition of reverse transcription on the clinical response. However, in vivo response to AZT was variable, and therefore, studies with other nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) were performed. In particular, it was reported in observational studies that treatment with lamivudine  for 24 weeks was able to reduce HTLV-1 viral load in five out of five patients with HAM/TSP, but with oscillation rising toward the baseline, accompanied by parallel variation in CTL precursor frequency and CD25 expression. These data pointed out an effect of therapy towards reverse transcription and the return to base line proviral DNA was interpreted as a failure in drug phosphorylation within the cell or emergence of viral variants with less susceptibility to 3TC inhibition . MoreoveOne alternative strategy for the use of NRTIs in HAM/TSP could be a combination treatment including compounds acting on different targets from viral replication. Such a possible alternative approach was the investigation of the effects of a combination treatment of NRTIs with the epigenetic regulator valproic acid (VPA). Asymptomatic baboon naturally infected with STLV-1, almost identical to HTLV-1 in humans, were treated with a combination of AZT plus VPA resulting in a reduction of proviral load by 5–12-fold in 50% of the animal tested at early stage of infection. The treatment with VPA was shown to be associated to a transient rise in the proviral load early in the first week of treatment while combination with AZT prevented the rebound of the viral load. Given that the decrease of viral load in VPA/AZT treatment was accompanied by an increase of CD8+ effector-cell cytotoxic activity, it was hypothesized that the decrease in viral load was owed to the reactivation of the immune response against the virus facilitated by AZT-mediated inhibition of viral transmission. On the other hand, the rebound of viral load following interruption of the combined treatment might be due to virus production by reservoirs not affected by the therapy, similar to what is observed in HIV infection. On the basis of the results obtained in STLV-1 infection, the combination treatment with VPA and AZT could be potentially transferred to HAM/TSP patients if treated at an early stage of the disease .Taken together, studies carried out until now on NRTIs in HAM/TSP provide conflicting results, but it seems that there is no opinion excluding their potential effect at least at some stage of the disease. Accordingly, attention has been recently paid to a novel generation of NRTIs anti-HIV drugs, i.e., the acyclic nucleoside phosphonates (ANP). In particular, prodrugs of the already mentioned TFV, also named as PMPA [9-R-(2-phosphonomethoxypropyl)adenine], were investigated in a new model of HTLV-1 infection in vitro using as recipient cells LTR luciferase reporter Jurkat T-cells . InteresThus, clinical studies concerning antiretrovirals in HTLV-1 induced neurological disorders provide, at the moment, insufficient evidence to support the use of antiretroviral therapy for the treatment of HAM/TSP as asserted in a recent systematic review and consensus-based recommendations . NeverthIn contrast to HAM/TSP, NRTIs were fruitfully used to treat ATL caused by HTLV-1 infection. This choice was due to the poor therapeutic options for treating ATL which was resistant to the classical cytotoxic chemotherapy. One of the first reports about the use of antiretrovirals was done by Gill et al. in 1995 when AZT was combined with interferon alpha (IFNa) . This coThe above reported studies highlighted the need to elucidate whether AZT+IFNa combination exerted a direct antiretroviral effect. Actually, an indirect antiretroviral effect by AZT+IFNa combination was recently hypothesized as the consequence of an impact towards dendritic cells or macrophages in which viral replication occurs and that provide growth factors and cytokines useful for the survival of ATL cells . InteresTransplantation has been recently recognized as a potentially efficacious HTLV-1 transmission route. The lack of routinely screening of organ donors for HTLV-1 makes difficult to precisely state that the transplant is actually responsible for ATL or HAM/TSP development in the recipient. Some scattered case reports were reported in the literature regarding HTLV-1 transmission through organ transplantation in France, Spain, and United States ,45,46,47Beside NRTIs, only two other classes of antiretroviral agents were translated from HIV experience to possible anti-HTLV-1 drugs. They belong to the classes of integrase and protease inhibitors. No study on anti-HIV non-nucleoside reverse transcriptase inhibitors in HTLV-1 infection has been reported.Several integrase strand transfer inhibitors (INSTIs) have been developed and its use has been introduced in HIV treatment . UnfortuRegarding protease inhibitors, only one recent evidence for the effect of an already used anti-HIV agent belonging to this class has been reported in . RitonavCollectively, the reported data suggest that antiretroviral therapy in HTLV-1 infection could provide benefit although there is not a common protocol to be suggested. The preclinical studies, have clearly demonstrated that antiretroviral therapies based on drugs translated by the HIV experience efficiently repress HTLV-1 infection, with exception of 3TC owe to a natural resistance of the virus. Nevertheless, the clinical results are still relatively discouraging. Different aspects could account for this discrepancy. The first, as reported, is the scarce reliability of known anti-HIV drugs capable to exert any activity towards the vertical spread of HTLV-1 through clonal expansion of already infected cells. Another related point that might be taken into consideration, based on 3TC experience, is that not all the drugs utilized in HIV infection should exert their optimal activity in HTLV-1 infection. Therefore, the still limited results of antiretrovirals in HTLV-1 might depend, at least in part, on the lack of compounds specifically designed and developed towards HTLV-1. Another aspect that should be taken into consideration is that HTLV-1 preferentially infects through cell-to-cell transmission rather than in a direct virus-to-cell way. Thus, in vitro experimental models might have some limitations owing to this fact. Nevertheless, recent results on new anti-HIV integrase inhibitors and on anti-HIV protease inhibitors in HTLV-1 infection could open new scenarios on usage of antiretrovirals in diseases caused by the virus. Relevant to the later, another aspect to be considered is that antiretroviral compounds could be also endowed with properties displaying activities that exert beneficial effects towards diseases caused by HTLV-1 independently on their antiviral activity and/or in addition to it . Finally


Homeostasis of the intestine is maintained by a delicate balance of signaling networks that regulate self-renewal and differentiation. In the past years, increasing evidence suggests that long non-coding RNAs (lncRNAs) are involved in the control of intestinal crypt turnover. Indeed, their deregulation can enable and drive malignant cell growth. Notably, lncRNAs have high tissue specificity, and therefore hold great potential for therapeutic intervention. Here, we address the function of lncRNAs in the intestine in physiological and pathological conditions and discuss promising interference systems to target oncogenic lncRNAs.Maintenance of the intestinal epithelium is dependent on the control of stem cell (SC) proliferation and differentiation. The fine regulation of these cellular processes requires a complex dynamic interplay between several signaling pathways, including Wnt, Notch, Hippo, EGF, Ephrin, and BMP/TGF-β. During the initiation and progression of colorectal cancer (CRC), key events, such as oncogenic mutations, influence these signaling pathways, and tilt the homeostatic balance towards proliferation and dedifferentiation. Therapeutic strategies to specifically target these deregulated signaling pathways are of particular interest. However, systemic blocking or activation of these pathways poses major risks for normal stem cell function and tissue homeostasis. Interestingly, long non-coding RNAs (lncRNAs) have recently emerged as potent regulators of key cellular processes often deregulated in cancer. Because of their exceptional tissue and tumor specificity, these regulatory RNAs represent attractive targets for cancer therapy. Here, we discuss how lncRNAs participate in the maintenance of intestinal homeostasis and how they can contribute to the deregulation of each signaling pathway in CRC. Finally, we describe currently available molecular tools to develop lncRNA-targeted cancer therapies. Intestinal tissue homeostasis is regulated by a variety of signaling pathways influencing cell proliferation and differentiation. Through collaborative or antagonistic activity, these pathways adjust cellular states along the crypt–villus axis. Indeed, the intestine is organized into a succession of self-renewing protrusions and invaginations respectively known as villi and crypts  that incAPC) gene, an essential factor for balancing Wnt activity, are found in approximately 80% of all colorectal carcinomas. Most of these mutations generate truncated versions of APC with reduced capacity to label β-catenin for degradation, resulting in the hyperactivation of Wnt signaling. Intracellular accumulation of β-catenin eventually leads to its nuclear translocation and consequent activation of the Wnt transcriptional program, thereby promoting proliferation and expansion of undifferentiated cells ,75HES1) Due to its role in the regulation of differentiation, Notch signaling is crucial for crypt homeostasis. While deletion of either Notch1 or Notch2 does not impair intestinal development, double deletion results in goblet cell hyperplasia ,77,78. CNotch signaling is known to play an important role in CRC. In stem cell-like cancer cells, Notch signaling is 10- to 30-fold more active than in differentiated CRC cells. Constitutive expression of active Notch-1 promotes EMT and supports the maintenance of stem-like features . FurtherDrosophila, is key in regulating intestinal homeostasis and regeneration. Its activation can be triggered by various extrinsic  and intrinsic  cues, as well as via the crosstalk with other signaling pathways, including the Wnt and Notch pathways  treatment . I. IDrosopreatment ,93. ThesMst1/2 in the intestinal epithelium of mice results in the formation of dysplasia and adenomas in the colon . F. F95]. FTaken together, these studies highlight the important roles of lncRNAs in the regulation of the Hippo pathway and underscore their conceivable use as therapeutic targets in CRC.Lrig1, a negative regulator of EGFR, show severe crypt expansions and enlarged intestines . . 100]. Itestines ,105. TheA large number of CRCs display mutations in components of the EGF pathway, among which the most frequent are activating mutations in KRAS, BRAF, and PIK3CA ,106. In HOXA6-HOXA5 locus and upregulated in advanced colon tumors, has been shown to enhance CRC growth in a xenograft mouse model. Although the mechanism remains to be clarified, HOXA5 short RNA mediates its action by modulating the expression of genes related to EGF signaling as well as by increasing the levels and phosphorylation of EGFR . . 133]. UIn CRC, TGF-β has long been known to increase metastasis formation . FurtherImportantly, in CRC, TGF-β and BMP signaling pathways can be influenced by deregulated lncRNAs f. For inAdditionally, the BMP transcriptional program utilizes lncRNAs to support tumor progression. For example, the lncRNA linc-POU3F3  was shown to support CRC cell survival, proliferation, and dissemination by influencing the expression of several genes, including caspase-3/9, cyclin D1, cyclin-dependent kinase 4 (CDK4), retinoblastoma (Rb), and SLUG . InteresInhibition of signaling pathways that are essential for CRC cells is key to counteract tumor growth and progression. For instance, therapeutic agents that can block Wnt signaling are of great interest, as hyperactivation of Wnt is a recurrent feature observed in a large proportion of CRCs. So far, most Wnt signaling inhibitors are directed against components acting upstream of APC , and are therefore unlikely to be effective in CRC, where APC is frequently mutated . On the C. elegans by Andrew Fire and Craig Mello . . 166]. TSeveral reports have demonstrated the potential of ASOs against lncRNAs in various mouse models. For example, silencing the lncGATA6 with intratumoral injection of ASOs in CRC patient-derived xenograft (PDX) models has been shown to significantly reduce tumor growth . SubcutaEmmanuelle Charpentier and Jennifer Doudna were awarded the 2020 Nobel prize in Chemistry for the discovery of CRISPR/cas9 gene editing system. Since their breakthrough publication in 2012 , the CRIHowever, CRISPRi- or Cas13-based therapeutic approaches face several challenges. One of the major limitations is the efficient delivery of a large cargo size (dCas9-KRAB or Cas13 and a guide RNA) to the cells of interest, using viral or nonviral vectors . Among vLncRNAs are increasingly recognized as critical regulators of numerous cell functions. In the intestine, lncRNAs modulate several signaling pathways, which are pivotal in maintaining tissue homeostasis. In contrast, their deregulation in diseases, such as cancer, can rewire these signaling cascades to enable malignant cells to proliferate and disseminate. Because of their high tissue (tumor) specificity, lncRNA-targeted therapy represents an exciting avenue to specifically disrupt important signaling pathways, such as the Wnt, Notch, and Hippo pathways, in tumor cells without harming their essential functions in normal tissues. Molecular tools, such as siRNAs, ASOs, and CRISPR technology, are now opening the race for the development of RNA-based therapeutics that target oncogenic lncRNAs.


Human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes adult T-cell leukemia/lymphoma (ATL), an aggressive form of T-cell malignancy. The relationship between HTLV-1 infection and cancer progression is controversial. HTLV-1 encodes oncogenic protein TAX1 and it is hypothesized that HTLV-1 infection is associated with breast cancer progression. In this study, we evaluated the relationship between HTLV-1 infection and clinicopathological factors in breast cancer patients. We retrospectively analyzed 610 patients with primary breast cancer who underwent surgical treatment without preoperative chemotherapy at Kagoshima University Hospital between January 2001 and January 2015. When patients with and without HTLV-1 infection were compared, no differences in clinicopathological factors were observed, except for age. Disease-free survival and overall survival rates did not differ between groups. HTLV-1–positive patients were significantly older than HTLV-1–negative patients. It was supposed to be due to the fact that the HTLV-1 infection rate is decreasing. Any effect of HTLV-1 infection on breast cancer progression appears to be negligibly small. Human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes an aggressive form of T-cell malignancy called adult T-cell leukemia/lymphoma (ATL) . The association between HTLV-1 infection and cancer occurrence is controversial. HTLV-1–infected patients are reported to be at significantly high risk of developing liver cancer and lymphomas other than ATL . However, a lower incidence of HTLV-1 in gastric cancer patients compared to the general population suggests that HTLV-1 infection reduces the risk of gastric cancer . To the best of our knowledge, no reports have demonstrated a significant difference in breast cancer incidence between HTLV-1–infected patients and uninfected patients. Tax1, a protein encoded by HTLV-1, has demonstrated oncogenic properties via the Ras-Raf-MEK-ERK signaling pathway in breast cancer cell lines . Thus, HTLV-1 infection has been hypothesized to be associated with breast cancer progression. In this study, we evaluated the relationship between HTLV-1 infection and clinicopathological factors in breast cancer patients.PatientsWe enrolled 686 patients with primary breast cancer who underwent curative surgical treatment without preoperative chemotherapy at Kagoshima University Hospital, Kagoshima, Japan between January 2001 and January 2015. During this period, HTLV-1 infection status was checked as part of the preoperative routine examination. Diagnostic method for HTLV-1 infection was chemiluminescence immunoassay against HTLV-1 antigen. Patients with recurrent breast cancer and those for whom HTLV-1 infection status was not checked were excluded. As a result, 610 patients were included in the present analysis. Clinicopathological information was collected retrospectively using medical records, and the seventh edition of the TNM classification was used for staging. Adjuvant therapy was selected for each patient based on the National Comprehensive Cancer Network guidelines. Statistical analysisDifferences in clinicopathological factors between groups were evaluated using analysis of variance for continuous variables and the Pearson chi-squared test for categorical variables. Disease-free survival (DFS) and overall survival (OS) were measured from the time of first surgery until the date of death or last follow-up. Survival curves were calculated using the Kaplan-Meier method, and statistical significance between groups was assessed using the log-rank test. Statistical analysis was performed using JMP Pro, version 12.1.0 for Mac OS . P values <0.05 were considered statistically significant.Patient characteristics Clinicopathological characteristics of enrolled patients are shown in Table 1. The mean age was 60.4 years, and 11% (n=66) of patients were HTLV-1–positive. No patient had ATL within the observation period. Immunohistochemical analysis was not performed in a small number of patients. Additionally, HER2 and Ki67 expression were not evaluated in some patients with ducal carcinoma in situ. The recurrence rate was 14% (n=85) among patients with stage 0-III disease, and 14% of patients (n=85) died during the observation period.Comparison of clinicopathological characteristics between patients with and without HTLV-1 infectionWhen patients with and without HTLV-1 infection were compared, no differences in clinicopathological factors were observed, except for age (Table 2); HTLV-1–positive patients were significantly older than HTLV-1–negative patients. Rates of recurrence and death were not different (Table 2), and DFS and OS did not differ between groups .HTLV-1 is a retrovirus that causes ATL, an aggressive form of T-cell malignancy . HTLV-1 is transmitted from mother to infant via breast milk and is known to be prevalent in several specific areas in the world, including Southwestern Japan . It was estimated that there were 10-20 millions HTLV-1 infected individuals in the world . In Japan, HTLV-1 infection has been extensively studied for many years, and infected individuals was estimated to be around 1.2 million . The geographic distribution of HTLV-1 carriers is quite uneven in Japan and the greatest prevalence is observed in Southwestern Japan . Our hospital located at Kagoshima in Southwestern Japan, and Kagoshima was reported to have highest HTLV-1 seropositive women rate in the world . So, we routinely checked HTLV-1 infection status for all preoperative patients for a period of time. In the present cohort, 11% of patients had HTLV-1 infection. Although this infection rate is relatively high, it is compatible with previously reported rates in our geographic region. To the best of our knowledge, no reports of a significant association between HTLV-1 infection and breast cancer occurrence have been published. We did not have access to data from healthy controls to compare with the patients from the present cohort, so we could not analyze the association between HTLV-1 infection and breast cancer incidence. Rather, in this study we aimed to evaluate the effect of HTLV-1 infection on clinicopathological factors following breast cancer diagnosis. No significant differences were observed between HTLV-1–positive and –negative patients, except for age: the age at diagnosis was significantly higher among HTLV-1–positive patients compared HTLV-1–negative patients. This may be due in part to the fact that the HTLV-1 infection rate is decreasing. Overall, Satake et al. (2012) reported that the number of HTLV-1 carriers has decreased 10% over the past 2 decades (1980s to 2000s), so the age distribution of carriers has trended toward older patients .HTLV-1 encodes Tax1, which was originally identified as a transcriptional activator that plays a central role in the immortalization of infected T-cells . Tax1 has been reported to promote cancer cell proliferation via the Ras-Raf-MEK-ERK signaling pathway . In addition, Tax1 sensitizes breast cancer cells to malignant transformation induced by environmental carcinogens through the inhibition of estrogen-induced ERα-mediated BRCA1 expression . Thus, we hypothesized that HTLV-1 infection is associated with breast cancer progression. However, no significant differences in tumor size, lymph node metastasis, stage, or other factors were observed between HTLV-1–positive and –negative patients. Furthermore, with respect to prognosis, no significant differences in recurrence or OS were observed. Any effect of HTLV-1 infection on breast cancer progression therefore appears to be negligibly small.This study was approved by the institutional review board of Kagoshima University Hospital. And for this type of study formal consent is not required.There is no financial relationships or other interests associated with this manuscript, which might be construed as constituting a conflict of interest.


ABL1 is an important gene in HAM/TSP. ABL1 is a known survival factor for T- and B-lymphocytes and is part of the fused gene (BCR-ABL) known to be responsible for chronic myelogenous leukemia (CML). ABL1 tyrosine kinase inhibitors (TKIs), including imatinib, nilotinib, and dasatinib, are used clinically for treating CML. To evaluate whether ABL1 is indeed important for HAM/TSP, we investigated the effect of TKIs on HTLV-1-infected cells. We developed a propidium monoazide-HTLV-1 viability quantitative PCR assay, which distinguishes DNA from live cells and dead cells. Using this method, we were able to measure the HTLV-1 proviral load (PVL) in live cells alone when peripheral blood mononuclear cells (PBMCs) from HAM/TSP cases were treated with TKIs. Treating the PBMCs with nilotinib or dasatinib induced significant reductions in PVL  in live cells. Furthermore, ABL1 siRNA transfection reduced cell viability in HTLV-1-infected cell lines, but not in uninfected cell lines. A retrospective survey based on our clinical records found a rare case of HAM/TSP who also suffered from CML. The patient showed an 84.2% PVL reduction after CML treatment with imatinib. We conclude that inhibiting the ABL1 tyrosine kinase specifically reduced the PVL in PBMCs from patients with HAM/TSP, suggesting that ABL1 is an important gene for the survival of HTLV-1-infected cells and that TKIs may be potential therapeutic agents for HAM/TSP.Human T-cell leukemia virus type 1 (HTLV-1) causes incurable adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Patients with HAM/TSP have increased levels of HTLV-1-infected cells compared with asymptomatic HTLV-1 carriers. However, the roles of cellular genes in HTLV-1-infected CD4+ T cells await discovery. We performed microarray analysis of CD4+ T cells from HAM/TSP patients and found that the  ABL1 as a gene frequently found in the signal transduction pathways in HTLV-1-infected CD4+ T cells. Therefore, ABL1 appears to be important in the pathogenesis of HAM/TSP. Inhibiting ABL1 with tyrosine kinase inhibitors (TKIs), which is used for chronic myelogenous leukemia (CML), reduced the proviral load (PVL) in vitro. We found a rare case of a patient with HAM/TSP and CML by our clinical records, who showed a decrease in PVL after TKI treatment for CML. Hence, TKIs are potential therapeutic agents for HAM/TSP.Human T-cell leukemia virus type 1 (HTLV-1) is integrated as a provirus in the genomic DNA mainly of CD4+ T cell population in the infected people. HTLV-1-infected CD4+ T cells are transmitted via breast milk, semen, and blood transfusions. HTLV-1 is endemic in Japan, the Middle East, Africa, Caribbean islands, and Central and South America. A small proportion of infected people develop adult T-cell leukemia, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and other diseases. HAM/TSP, a chronic neuroinflammatory disorder, is characterized by spastic paraparesis and urinary disturbance. HTLV-1-infected CD4+ T cells infiltrate the spinal cord and cause inflammation, which results in such neurological symptoms. We have identified the tyrosine kinase gene  Both viral genes have been investigated in relation to HTLV-1-related diseases propyl}-6-Phenylphenanthridinium)  for 5 min, then photo-crosslinked by exposure to halogen lamp. Each of the other half samples was not pretreated. After that, genomic DNA from all the samples was extracted. PMA is a membrane-impermeable nucleic acid-intercalating fluorescent dye and forms covalent bond by halogen lamp exposure. In PCR with PMA pretreatment, DNA from dead or dying cells is intercalated and is not amplified. PMA pretreatment was performed to quantify tax gene numbers only in the live cells.Cryopreserved PBMCs from six patients with HAM/TSP were thawed. The cell concentrations were counted by the trypan blue exclusion method. PBMCs at 95.0% viability or higher were adjusted to 1×10tax gene in the pX region of its genome [tax gene and β-actin  as previously described [tax amount)/((β-actin amount)/2)×104 (copies/104 cells)HTLV-1 carries the s genome . Using 2escribed . Real-titax and β-actin, and we have tentatively designated it as ‘PMA-HTLV-1-viability qPCR’. Amplification was performed in triplicate using DNA (20 ng) from the PMA pretreated samples. The cycle threshold was automatically defined by the thermal cycler. The HTLV-1 PVL in live PBMCs (PVLlive) is given as:A new technique, PMA viability PCR has been used to detect live microorganisms in the food and environmental sciences field –30. The Using ADP-Glo Kinase Assay Kit  and ABL1 Kinase Enzyme System  including Ablitide, a specific peptide substrate for ABL1 tyrosine kinase, ABL1 tyrosine kinase assay was performed according to the manufacturer’s protocol. For evaluating the effect of TKIs, cell lysate extracted after 48 h of incubation with TKIs  was applied for this assay in a 96-well plate.ABL1 mRNA in HTLV-1-infected and uninfected cell lines. ABL1 HSS100045 siRNA , mixture of Line 1 (5’-CCAACUACGACAAGUGGGAGAUGGA-3’) and Line 2 (5’-UCCAUCUCCCAUUUGUCGUAGUUGG-3’) target ABL1 mRNA variant α and β exon 4 sequence, very close region to the kinase domain of ABL1 tyrosine kinase. HTLV-1-infected cell lines  and uninfected cell lines (Molt-4 and CEM) were transfected with either of siRNA, RNAi Negative Control , or blank control using 1 μl of Lipofectamine RNAiMAX  per 600 μl of Opti-MEM I Reduced Serum Medium . Cells treated with 50 pmol/ml of siRNA were plated at a concentration of 1.67×106 cells/ml. After incubation for 48 h at 37°C in a CO2 incubator, cells were harvested, and cell viabilities were evaluated by the trypan blue exclusion method. RNA and cell lysate were also prepared from the cells. Transfection efficiency was assayed by RT-PCR. Ten nanograms of cDNA was applied for RT-PCR using primers for ABL1 mRNA variant β (forward: 5’-AAATGGACTGCACCCGAGAG-3’ and reverse: 5’-CTCATGGTCCAGAGGATCGC-3’) and ACTB mRNA (forward: 5’-ACACTGTGCCCATCTACGAC-3’ and reverse: 5’-TGGCCATCTCTTGCTCGAA-3’). The gel image was analyzed by Image J software.We performed siRNA experiment for All statistical analyses were performed using Statcel ver.4.0  and GraphPad Prism 6J .Through filtering and setting a fold change threshold of DEGs, we extracted 310 genes. By mapping to a Venn diagram, the DEGs were refined to the genes assigned to the area in the diagram where the up-regulated genes and the down-regulated genes belonging to only HAM/TSP. The refined genes were thought to be most important for HAM/TSP. We finally extracted significant 177 DEGs that were up-regulated only in HAM/TSP  and mappABL1 tyrosine kinase gene clearly stood out as being commonly included in 11 of the 12 pathways. However, its mRNA expression level was situated in the medium ranking of the up-regulated genes list  and ACs (P = 0.029) by Student’s t-test  showed a significant reduction of 17.5% and 21.0% after incubation with either dasatinib or nilotinib, respectively  as well as in uninfected cell lines (Molt-4 and CEM)  , and ABLand CEM) . In a ceand CEM) .i.e., able to walk more than 10 m with a cane and holding on to something) [i.e., walking without a cane but a handrail is necessary when climbing steps). Eight years later, he was diagnosed with CML because of leukocytosis, a Philadelphia-positive chromosome, and a low neutrophil alkaline phosphatase score. On admission, the OMDS was 6/13, the PVL was 2,844 copies/104 in PBMCs, and neopterin in the cerebral spinal fluid was 86.2 nmol/ml . He was administered imatinib (400 mg/day) for CML treatment for the first month, followed by 300 mg/day for 30 months. After imatinib treatment, he showed molecular genetics-based remission of the CML. Additionally, the OMDS improved from 6/13 to 5/13 . Likewise, the PVL decreased from 2,844 to 448 copies/104 PBMCs (an 84.2% reduction). Severe adverse effects by the imatinib treatment were not seen during the whole treatment period.As a retrospective survey based on our clinical records, we searched for HTLV-1-positive CML cases where the PVL was estimated before and after the treatment of the CML with a TKI. We found only one case who had both CML and HAM/TSP. The patient was a 52-year-old male diagnosed as having HAM/TSP. Osame’s Motor Disability Score (OMDS) was 6/13 for this patient (mething) . After pABL1 gene was involved in many of the relevant pathways for HAM/TSP specific genes extracted by the microarray data. The ABL1 expression level was relatively high in the CD4+ T cells from HAM/TSP patients and ranged in the list for the specific genes for HAM/TSP . When the ratio of dead cells was increased, the Ct value in the case with PMA treatment became high compared to that without PMA treatment, suggesting that the treatment made it possible to evaluate PVL in live cells more sensitive. By applying the method to clinical samples with TKI treatment, we could more accurately evaluate PVL in live cells than the conventional qPCR and found that the TKIs reduced PVL . Our data suggest that PMA-HTLV-1 viability qPCR can be expanded to an application that quantifies another target in the mixture of live and dead cells.QPCR for PVL quantitation is a standard method for estimating HTLV-1 provirus in genomic DNA from PBMC. However, when cells are dead during a short incubation with a drug, it was known that DNA extracted from the dead cells could be amplified by the qPCR . In our uced PVL . Of courABL1 mRNA induced cell death in HTLV-1-infected cell lines. We also found a HAM/TSP case who was complicated by CML. He showed an 84.2% reduction in PVL after imatinib treatment. These data suggest that inhibiting ABL1 tyrosine kinase by TKI can reduce HTLV-1 PVL in patients with HAM/TSP.We identified the ABL1 tyrosine kinase gene as being important in HAM/TSP by combining microarray and pathway analysis. The addition of the ABL1 tyrosine kinase inhibitors, nilotinib, and dasatinib, into the PBMC culture significantly reduced the PVL in live PBMCs by 21.0% and 17.5%, respectively. The knockdown of The Ethics Committee of Kagoshima University Hospital, Japan, approved this study. All human subjects were adults except one. We obtained written informed consent from all subjects. We explained to the child about blood sampling and obtained written informed consent from him with his parents. This report does not contain any individual data.S1 Table(DOCX)Click here for additional data file.S1 FigpX gene in triplicate and repeated twice. Error bars represent standard error of the mean.To evaluate whether the mixture of live and dead cells and PMA pretreatment affect HTLV-1 PVL measurement, we induced dead cells by digitonin treatment at 300 ng/ml for 2 h and made five different cell mixture  using HTLV-1-infected cell line HUT102. We divided these cell mixtures into two groups: one was pretreated with PMA (which enters only dead cells and fix the DNA), and the other was not. Genomic DNA was extracted and we performed quantitative PCR targeting HTLV-1 (TIF)Click here for additional data file.


Three, 7 and 14 days after the treatment, corneas were collected and stained with FITC-conjugated CD31 and LYVE-1 followed by Cy3-conjugated secondary antibody to quantify corneal blood and lymphatic vessels. Relative mRNA expression of VEGF in the cornea was quantified by using qPCR. FND cauterization as monotherapy significantly obliterated (lymph)angiogenesis at early time points; however, this treatment led to secondary corneal hem- and lymphangiogenesis associated with significant upregulation of pro(lymph)angiogenic VEGF-A, VEGF-C, VEGF-D and infiltration of macrophages. Combining FND cauterization with VEGF TrapR1R2 treatment prevented the undesired effect of the FND procedure alone and significantly better regressed corneal blood and lymphatic vessels at 1 week after the treatment compared to monotherapy and control group (p < 0.01).Fine needle diathermy (FND) is an effective method to destroy and regress pathologic corneal blood and lymphatic vessels. However, it is unknown whether FND itself causes a rebound corneal neovascularisation and whether that can be prevented by VEGF blockade. In female BALB/c mice, the suture-induced inflammatory corneal neovascularisation model was used to induce hem- and lymphangiogenesis. Thereafter, prevascularized mice were divided into 2 groups: the combination therapy group received FND cauterization and subsequent VEGF TrapR Various diseases, however, can result in corneal neovascularisation (CoNV), i.e. sprouting of new vessels from the limbal vascular arcade into the cornea3. The presence of blood and lymphatic vessels disrupts not only the”angiogenic privilege” but also the ‘’immune privilege” of the cornea6 and leads to a significant increase of graft rejection after subsequent corneal transplantation9.The cornea is one of the few human tissues devoid of blood vessels (BVs) and lymphatic vessels (LVs). Therefore, it is an ideal location to investigate the mechanism of pathological hemangiogenesis and lymphangiogenesis. The avascular status of the cornea, also termed”corneal (lymph)angiogenic privilege”, is maintained by the balance between proangiogenic factors and anti-angiogenic factors13. More recently, photodynamic therapy and UV light crosslinking were reported to significantly regress corneal vessels in mouse models15. Another approach is anti-vascular endothelial growth factor (VEGF) therapy which has been broadly used off-label to inhibit progressive corneal angiogenesis and lymphangiogenesis17. However, anti-VEGFs as a monotherapy seems to be less effective in regressing mature vessels due to the fact that these vessels depend less on angiogenic growth factors18.To regress pathological vessels prior to transplantation, different approaches have been used such as antiangiogenic argon laser or yellow dye laser and antisense oligonucleotide GS10120. The efficacy of this technique was documented by several studies in both clinical and animal settings25. We could recently show that the combination of FND and corticosteroids can regress both blood vessels and clinically invisible lymphatic vessels25. Based on clinical experience, many authors assume that FND in itself can induce rebound neovascularisation and should therefore be combined with anti-inflammatory eye drops or subconjunctival injection of anti-VEGF or with corneal angiography to lessen undesired effects of FND28.Fine needle diathermy (FND) is clinically used since 2000 to regress CoNV and is currently a promising clinical choice for managing mature pathologic corneal blood vessels1R2 and FND on dampening the undesired effect of monotherapy as well as on regressing mature blood and lymphatic vessels and prevention of their recurrence.However, so far there has been no formal evidence regarding expression level of angiogenic growth factor VEGF-A, VEGF-C and VEGF-D after FND treatment and FND induced rebound neovascularisation. Therefore, in this study, we investigated the potential additional angiogenic stimulus of FND procedure itself as a monotherapy and the effect of combined treatment of VEGF TrapR1R2 could prevent this rebound effect with a regression of blood vascularized (BV) area by 53% and lymphatic vessels (LV) by 65% compared to FND group (p < 0.0001) and 36% BV area and 55% LV area compared to control group (p < 0.01) at day 7  compared to the control groups at day 7 and 14 (p < 0.01). Furthermore, we could show that the additional treatment with VEGF TrapRTo further test the hypothesis of a VEGF driven rebound neovascularization effect of FND monotherapy, we investigated the effect of this treatment on the corneal expression of VEGF-A, VEGF-C and VEGF-D. Corneas treated with FND alone were harvested seven days after the treatment and analyzed via qPCR. The expression level of VEGF-A, VEGF-C, VEGF-D was significantly upregulated after FND monotherapy Fig. .Figure 329.Inflammatory cells, especially macrophages, are important sources of angiogenic and lymphangiogenic growth factors. Therefore, we also studied the infiltration of macrophages into the cornea after different treatments. The area covered by LYVE-1 (+) macrophages was significantly increased in the FND monotherapy group in comparison to the other groups Fig. . In addi30. To investigate the source of VEGF in the FND model, inducing the rebound of neovascularization, we performed corneal macrophage depletion as previously described31. Mice were treated with subconjunctival injections of 10 µl clodronate liposomes one day before FND treatment and every other day afterwards for one week. Control group was treated with subconjunctival injection of equal amounts of control liposomes with similar regime. Our results show that both blood and lymphatic vessels were significantly reduced at day 7 in the macrophage depleted group compared to the control group FND itself as a monotherapy is effective in significantly regressing both mature corneal blood and lymphatic vessels at early time points after treatment.(ii)FND monotherapy significantly increases the expression of VEGF-A, VEGF-C and VEGF-D - the important pro(lymph)angiogenic mediators – as well as macrophage infiltration into the cornea thereby inducing rebound revascularization.(iii)1R2 is a potential and effective treatment to regress mature corneal blood and lymphatic vessels and prevent early recurrence.The rebound effect of FND monotherapy can be prevented by VEGF-A blockade. The combination therapy of FND and VEGF TrapRThree important conclusions can be drawn from this study:et al.19 - has been clinically used to obliterate mature pathologic corneal blood vessels since then. Based on clinical experience, many authors assumed that FND monotherapy could induce micro trauma causing release of proangiogenic factors and should therefore be combined with anti-inflammatory eye drops or subconjunctival injection of anti-VEGF and with corneal angiography to reduce the undesired effect of FND26. Indeed, we recently showed that the combination of FND and corticosteroids can regress not only mature blood vessels but also clinically invisible lymphatic vessels and thereby promote subsequent high-risk corneal graft survival25. Nevertheless, there was no study so far formally testing whether FND treatment alone induces release of proangiogenic factors or causes rebound neovascularisation.Corneal neovascularisation, a sight-threatening condition, occurs in a wide variety of corneal pathologies. The regression of CoNV and especially of mature blood and lymphatic vessels is a challenge for ophthalmologists. The fine needle diathermy technique - first described by Pillai 30) thereby induces rebound neovascualrization of blood and lymphatic vessels at 7 and 14 days after the treatment compared to control group  significantly upregulates mRNA expression of VEGF-A, VEGF-C and VEGF-D –main pro(lymph)angiogenic factors - as well as the infiltration of macrophages (important source of (lymph)angiogenic growth factorsup Figs. . These r29. Oshima et al. also indicated that macrophage infiltration was markedly reduced in CCR2 KO mice and MCP-1 KO mice32. In fact, our findings showed that FND also causes an upregulation of MCP-1 and thereby resulting in the significant influx of macrophages after the treatment , a chemokine released from corneal epithelial cells and binding to chemokine receptor CCR2, subsequently recruits monocytes/macrophages to the cornea in the cauterization model33. This is due to the antiangiogenic function of corneal epithelium33. In our experiment, although the corneal epithelium was intact, the circular cauterization provoked rebound (lymph)angiogenesis. Conflicting results may be due to the different models studied and the different approach and intensity of FND applied. We here performed a circular perilimbal cauterization on prevascularized corneas instead of mild central cautery on nonvascularized corneas.It had been previously shown that mild cautery of the cornea without removal of the corneal epithelium fails to evoke a neovascular response whereas cautery with previous epithelial removal can cause an angiogenic response1R2 is a fusion protein containing the domain of VEGFR1 and VEGFR2 fused to the Fc domain of human IgG. Therefore, this trap can bind and block all forms of VEGF-A. Due to this effect, VEGF TrapR1R2 is an ideal candidate to dampen the upregulation of VEGF-A caused by FND alone. Indeed, this combination treatment could significantly regress blood and lymphatic vessesels at day 7 and day 14 in comparision with the FND monotherapy. These results are compareable to our previous study where FND was combined with corticosteroids25. Corticosteroids are clinically used to control inflammation and CoNV due to their anti-inflammatory and anti-angiogenic properties34. Many studies recently showed the additional antilymphangiogenic properties of corticosteroids37. Both dexamethason and anti-VEGF treatments delay cell migration, vessel dilation at the early phase and decrease the affected area of vascularization and the expression of VEGF41. However, long-term use of topical corticosteroids can result in severe side effects namely cataract, glaucoma, herpes simplex recurrence and superinfection. The long-term use of anti-VEGF seems to be more safe. However, they were reported to cause epithelial defects, corneal stromal thinning or neurotropic keratopathy. Clinically, Koenig20, Elbaz23 and Hussain24 combined FND with topical or subconjunctival injection of Bevacizumab to treat corneal neovascularisation. This combined treatment was reported to succesfully treat lipid keratopathy and CoNV in preparation of high risk keratoplasty without causing any serious side effects42. However, these studies lacked a control group and evidences why FND should be combined with anti-VEGFs42.VEGF TrapR1R2 mainly binds VEGF-A and inhibits progressive corneal (lymph)angiogenesis18. Our results show that FND combined with VEGF TrapR1R2 compensates for the limitation of the FND monotherapy and can effectively regress both mature corneal blood and clinically invisible lymphatic vessels in high-risk eyes with reduced risk of recurrence.Taken together, fine needle cautery is an option for treatment of mature pathological corneal neovascularisation. However, as a monotherapy FND results in significant upregulation of VEGF-A, VEGF-C and VEGF-D. VEGF TrapRAll experimental procedures were approved by Landesamt fuer Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Germany (license: AZ 84-02.04.2016.A055) and conformed to the Association for Research in Vision and Ophthalmology’s Statement for the Use of Animals in Ophthalmology and Vision Research. Female BALB/C mice, purchased from Charles River Laboratories, Sulzfeld, Germany (aged 6–8 weeks) were used in the mouse model of suture-induced corneal inflammation and neovascularization assay. Prior to surgery, a mixture of Ketanest (8 mg/kg) and Rompun (0.1 ml/kg) was intraperitoneally injected to deeply anesthetize the mice.43. Three 11–0 nylon sutures  were inserted intrastromally on the right eye with two stromal incursions extending over 120 degrees of corneal circumference each. The outer point of suture placement was chosen near the limbus and the inner suture point was placed near the center of cornea equidistant from the limbus to obtain standardized angiogenic responses. To achieve the peak of (lymph)angiogenesis, sutures were left in place for 14 days and were then removed. After taking out sutures, mice were separated into three groups (n = 10 each): combination treatment of FND and VEGF TrapR1R2; FND alone and control group complied with the protocols for each group mentioned below.The mouse model of suture-induced inflammatory neovascularisation was performed as previously described25. A stainless steel 3/8 circle round-bodied, single-armed needle was inserted intrastromally close to and parallel to the corneal limbus. Electrode needle connected to diathermy unit with lowest power in soft-coagulating mode was brought into contact with the shaft of the surgical needle. The cauterization was stopped when the vessels were coagulated with corneal blanching and/or corneal shrinkage. This procedure was repeated until reaching 360-degree circumferential cauterization.FND was performed as previously described under topical anesthesia with Conjuncain®EDO® 1R2  three drops (3 µl/drop) per day until the end of experiments. Mice in the control group did not obtain FND or VEGF TrapR1R2.After the cauterization, group treated with only FND were not received any further treatment, whereas, combination treatment group was additionally treated with topical VEGF TrapR1R2) on regression of both corneal blood and lymphatic vessels, mice were euthanized and corneas were harvested one and two weeks after the treatment. Whole-mount double-immunohistochemistry using CD31 and LYVE-1 was performed as described below.To investigate the angiogenic stimulus of FND procedure when solely using and the effect of combination treatment . Seven days after FND treatment, corneas were harvested and stained with CD31 for blood vessels and LYVE-1 for lymphatic vessels.To investigate the source of VEGF which induces the rebound of neovascularization, mice in treated group received FND therapy and subconjunctival injections of 10 µl clodronate liposomes  to deplete macrophages as previously described46. Excised corneas were rinsed in PBS and then fixed in acetone for 20 minutes. After washing in PBS three times, and blocking with 2% bovine serum albumin (BSA) in PBS, the corneas were stained overnight  with rabbit anti-mouse LYVE-1  for lymphatic vessels and with a FITC-conjugated rat anti-mouse CD31 antibody  for blood vessels. On the next day, a goat-anti-rabbit Cy3-conjugated secondary antibody  was added to detect LYVE-1. Finally, samples were mounted on slides using fluorescence mounting media (Sigma).The blood and lymphatic vessels in corneal wholemounts were double stained as described previously43.Double stained wholemount images were assembled automatically from nine to 12 images taken at x100 magnification with a fluorescence microscope . Afterwards, the areas covered with blood and lymphatic vessels, and the infiltration of LYVE-1 (+) macrophages at day three and seven were detected with an algorithm established in the image analyzing program Cell^F , as previously describedTotal RNA was isolated from the corneas using the RNeasy Micro Kit . As only the centre was analysed, corneas were excised without the limbus. Three corneas per group were pooled in one tube to obtain enough RNA. RNase-free DNase Set (Qiagen) was used to eliminate traces of genomic DNA. Subsequently, single strand cDNA (500 ng per group) was synthesized using the First Strand cDNA Synthesis Kit  according to manufacturers’ protocol. PCR reactions (25 µL) contained 50 ng of cDNA, 0.4 mmol/L of each forward and reverse primer, and master mix . The primers used for PCR were shown below Table . All reaStatistical analyses were performed with Prism 6 version 6.07 . Statistical significance between two groups was determined using Student’s t-test or ANOVA for multiple groups. Data reported as mean ± SD. Values with p < 0.05 was considered statistically significant. Graphs were drawn using Prism6.Supplementary information.


Higher-order functions have become a staple of modern programming languages. However, such values stymie concolic testers, as the SMT solvers at their hearts are inherently first-order.This paper lays a formal foundations for concolic testing higher-order functional programs. Three ideas enable our results: (i) our tester considers only program inputs in a canonical form; (ii) it collects novel constraints from the evaluation of the canonical inputs to search the space of inputs with partial help from an SMT solver and (iii) it collects constraints from canonical inputs even when they are arguments to concretized calls. We prove that (i) concolic evaluation is sound with respect to concrete evaluation; (ii) modulo concretization and SMT solver incompleteness, the search for a counter-example succeeds if a user program has a bug and (iii) this search amounts to directed evolution of inputs targeting hard-to-reach corners of the program.