Over the last century, endophytic fungi have gained tremendous attention due to their ability to produce novel bioactive compounds exhibiting varied biological properties and are, therefore, utilized for medicinal, pharmaceutical, and agricultural applications. Endophytic fungi reside within the plant tissues without showing any disease symptoms, thus supporting the physiological and ecological attributes of the host plant. Ground breaking lead compounds, such as paclitaxel and penicillin, produced by endophytic fungi have paved the way for exploring novel bioactive compounds for commercial usage. Despite this, limited research has been conducted in this valuable and unique niche area. These bioactive compounds belong to various structural groups, including alkaloids, peptides, steroids, terpenoids, phenols, quinones, phenols, and flavonoids. The current review focuses on the significance of endophytic fungi in producing novel bioactive compounds possessing a variety of biological properties that include antibacterial, antiviral, antifungal, antiprotozoal, antiparasitic, antioxidant, immunosuppressant, and anticancer functions. Taking into consideration the portal of this publication, special emphasis is placed on the antimicrobial and antiviral activities of metabolites produced by endophytes against human pathogens. It also highlights the importance of utilization of these compounds as potential treatment agents for serious life-threatening infectious diseases. This is supported by the fact that several findings have indicated that these bioactive compounds may significantly contribute towards the fight against resistant human and plant pathogens, thus motivating the need enhance the search for new, more efficacious and cost-effective antimicrobial drugs. In recent years, there has been a dramatic shift towards a more sustainable, eco-friendly, and natural way of living. Many researchers are of the opinion that there is an alarming increase in drug resistance worldwide and the problem is escalating tremendously, thus rendering the current antimicrobial agents ineffective ,2,3. AppIn developing countries, especially those in Africa, Asia, and Latin America, more than 80% of the population utilize medicinal plants to address their primary healthcare needs and wellness . With ovIn addition, the ratio of fungal species to vascular plants is approximately 1:6, which is estimated at 1.5 million fungal species . EndophyPenicillium chrysogenum, which was a milestone in the development of antibiotic drugs, as a “front-line” antibiotic which saved millions of lives; hence, it was referred to as “wonder drug” [Taxomyces andreanae for chemotherapy purposes [Bioactive compounds are primarily responsible for the beneficial characteristics displayed by endophytic fungi. History has shown that such metabolic compounds might aid in ground-breaking discoveries, e.g., the discovery of penicillin produced by er drug” . Anotherpurposes . These bpurposes . They hapurposes . New biotechnological advances concentrate on the search for and utilization of novel bioactive compounds extracted from endophytic fungi. Despite all of this, only a tiny portion of endophytic fungi have been isolated and investigated for their biological activities. In this current review, we focus in depth on the various biological properties demonstrated by endophytic fungi. We also identify novel bioactive compounds associated with the activities. Furthermore, we shed light on the activities against resistant pathogenic microorganisms. Unexplored niche areas such as these will progress the drug development process tremendously. Endophytic fungi are an abundant source of natural bioactive compounds which are novel, renewable, and low in toxicity and more efficacious, more potent, more affordable, safer, and less resistant than conventional antimicrobial agents. Hence, this will alleviate the massive burden on public healthcare systems and aid in the medical and pharmaceutical industries. Various biological activities, such as antibacterial, antifungal, immunosuppressant, antiviral, antiparasitic, antioxidant, anti-inflammatory, and anticancer, were exhibited by endophytic fungi with a wealth of bioactive metabolites. In this section, we investigate endophytic fungi as a source of countless bioactive compounds which might be beneficial for human health. Since the golden era of penicillin, antibiotics have become a staple conventional medicine and have reduced the fatality rate by saving millions of lives, as was proven in World War II . In the Aharwal and co-workers , recogniAlternaria alternate (AE1) was isolated from healthy and mature leaves of Azadirachta indica plants located in Santiniketan. The fungal extracts exhibited excellent antimicrobial activities against both Gram-positive and Gram-negative bacteria  with minimum inhibitory concentration (MIC) values of 300–400 μg/mL. The mode of action was determined as being cidal and further analysis demonstrated that cell lysis or leakage of cell membrane was occurring. Gas chromatography–mass spectrometry (GC-MS) analysis confirmed that the fungal extracts consist of several bioactive compounds [Penicillium sp. demonstrated strong effectiveness against Enterococcus faecalis with an MIC value of 62.50 mg/mL [Alternaria sp. were successfully isolated from native South African Pelargonium sidoides, and this is considered as a first report. These fungal metabolites inhibited several food-borne and food spoilage bacteria, including Bacillus cereus, Escherichia coli, and Enterococcus faecium, and E. gallinarum showed a 2–12-mm zone of inhibition [Meloidogyne incognita are persistent parasites in plants and contribute to about 5% of global crop losses [Piriformospora indica was capable of inhibiting the root-knot nematode parasite and, thus, enhanced plant growth [Recently, numerous scientists have demonstrated that endophytic fungi have a variety of beneficial, novel, and effective bioactive metabolites possessing antibacterial activity. Endophytic ompounds . The bio50 mg/mL . Linoleip losses , a biocot growth .Alternaria sp., isolated from the mangrove  plant located in China. The metabolites exhibited strong antibacterial properties against Enterococcus faecalis, Pseudomonas aeroginosa, and Staphylococcus epidermidis [Kjer et al. (2009) discovered two new secondary metabolites , and 2-methyloctanoic acid 6-oxo-2-propenyl-3,6-dihydro-2H-pyran-3-yl ester, (2) [Ding and colleagues (2019) also reported compounds 1 and 2 as novel isocoumarin derivatives  with a d32 μg/mL . Besidester, (2) . Figure The diversity of fungal species is currently estimated at 2.2 to 3.8 million worldwide, with soil fungal populations contributing a significant portion, and fungi are regarded as ubiquitous . This suCandida and Aspergillus sp. with 19 and 30 mm, respectively. Furthermore, in vivo studies conducted on mouse models showed moderate efficacy against candidiasis [Favolaschia calocera—which displayed antifungal effectiveness. The minimum inhibitory concentration (MIC) displayed by compound 1 was ≤ 9.37 and ≤ 18.75 μg/mL against Candida tenuis and Mucor plumbeus, respectively [Penicillium sp. (C7B) and Trichoderma sp. ) were effective against Escherichia coli, Staphylococcus aereus, and Vibrio alginolyticus, displaying clear-zone diameters of 17.91 ± 0.84 mm; 17.78 ± 0.83 mm; 17.66 ± 0.83 mm; 16.72 ± 1.15 mm, and 13.65 ± 0.27 mm, respectively [Research advances in modern antifungal agents rely highly on screening for novel bioactive compounds that have gained popularity in the drug development process and will aid in the fight against animal and human fungal pathogens. Peláez and co-workers (2000) established that new triterpene glycoside exhibited good antifungal inhibitory activities against didiasis . Recent ectively . Similarectively .Fusarium culmorum, Gibberella zeae, and Verticillium albo-atrum, exhibiting MIC activities of 7.2, 144.4, and 114.4 Μm respectively [“Pestalachlorides A” (C21H21Cl2NO5) and “B” (C20H18Cl2O5) are chlorinated benzophenone derivatives that inhibited plant pathogenic fungi, ectively . Well knHumicola sp. (JS-0112 strain) proved to be an ideal candidate for the development of novel fungicides by controlling plant pathogenic Sclerotinia homoeocarpa [12-C6” carbon skeleton isolated from endophytic Phomopsis sp. (CFS42). Moreover, the compounds inhibited activities against Bipolaris sorokinian, Alternaria alternat, Fursarium avenaceum, and Curvularia lunata. Therefore, this reiterates the untapped wealth of novel bioactive compounds produced by endophytic fungi [Endophytic oeocarpa . Currentic fungi .According to the World Health Organization (WHO), cancer is a group of diseases caused by the malignant growth of cells or tumor resulting from uncontrolled cell division . Cancer 47H51NO14 is formulated from ‘taxol”, the first anticancer drug to generate billions of dollars. The endophytic fungus (Taxomyces andreanae) isolated from the Pacific yew bark (Taxus brevifolia) produces the anticancer bioactive compound paclitaxel. A number of studies have also isolated taxol and its related compounds from a variety of endophytes, including Grammothele lineata [Aspegillus aculeatinus [Alternaria brassicicola [Acremonium, Colletotrichum, and Fusarium spp., from an ecologically altered Taxus baccata [Taxus plants is a complex process, means that an alternative will be to constantly assess taxol-producing endophytic fungi due to the ease and practicality of obtaining these valuable compounds. In order to understand the complexity of the processes or molecular mechanisms involved in the production of taxol, [Aspergillus aculeatinus Tax-6, indicating that genes involved in two pathways  and nonmevalonate (MEP)) need to be expressed. Despite the fact that the potential of endophytes to produce taxol varies, with Grammothele lineata producing 382.2 μg/L [Alternaria brassicicola producing 140.8 μg/L taxol [Acremonium, Colletotrichum, and Fusarium spp., which also produced up to 116.19 μg/L [A. aculeatinus BT-2 when compared to the wild-type A. aculeatinus Tax-6 strain [Pestalotiopsis microspore, Alternaria alternata, Periconia sp., Pithomyces sp., Chaetomella raphigera, Monochaetia sp., Seimatoantlerium nepalense, Botryodiplodia theobromae, Phyllosticta spinarum, Pestalotiopsis terminaliae, and Bartalinia robillardoides, have also been found to produce anticancer bioactive agents that chemically belong to taxols [The ground-breaking study resulting in the discovery of paclitaxel, also known as the “golden” compound, produced renewed hope in the search for novel anticancer agents, and the compound gained significant popularity because of its distinctive mode of action when compared to other anticancer agents. Paclitaxel obstructs the uncontrolled duplication of cancerous cells, thereby reducing their growth and spread. Paclitaxel with a chemical formula of C lineata , Aspegilsicicola , and Acr baccata . Taxol a baccata ,51,52,532.2 μg/L , Alternao taxols ,54. Seveo taxols .20H16N2O4), a bioactive alkaloid compound initially isolated from Camptotheca acuminata wood in China, also exhibited anticancer properties [Chaetomium globosum, which lives synergistically in the stem of healthy Imperata cylindrica [Chaetomium globosum, inhibited antitumor activities on the cancer cell line A549 [Chaetomium globosum from the Ginkgo biloba plant contained three novel compounds that include azaphilone alkaloids, chaetomugilides A–C, and chaetoviridin E, all of which displayed high cytotoxic activities against the human cancer cell line HePG2 [Xylaria, Phoma, Hypoxylon, and Chalara have also been reported to produce cytochalasins, which possess antitumor activities. In addition, three novel cytochalasins, namely cytochalasin H, cytochalasin J, and cytochalasin E, were also extracted from Rhinocladiella sp. isolated from Tripterygium wilfordii [22H22O8), a lignan-type bioactive compound which was originally isolated from Podophyllum peltatum L. in the 1880s, and its derivatives are well-known to possess a variety of biological properties and are used as cathartic, purgative, antiviral, vesicant, antibacterial, antihelminthic, and antitumor agents. Etoposide and teniposide are chemotherapeutic medications produced from podophyllotoxin, and they are currently available in the market. In addition, endophytes Fusarium oxysporum, Aspergillus fumigatus, Phialocephala fortinii, and Trametes hirsute, as well as some belonging to the genera Trichoderma, Penicillium, and Phomopsis, have also been reported to produce the compound podophyllotoxin, which exhibits anticancer activities [In addition to paclitaxel, camptothecin  wood located in China. Camptothecin (C20H16N2O4) is an alkaloid topoisomerase compound that displayed remarkable potent antineoplastic efficacy. Camptothecin and 10-hydroxycamptothecin were utilized in the development of the drugs topotecan and irinotecan, which are chemotherapeutic. Since then, precursor 9-methoxycamptothecin and 10-hydroxycamptothecin have been reported to exhibit powerful anticancer properties [The discovery of other lead compounds was intensified by the isolation of camptothecin from the endophytic fungi operties ,64. In aoperties .Aspergillus sp. (ZJ-68), associated with mangrove [Cladosporium species inhibited cell growth and induced apoptosis of human cancer cell lines HL-60, U937, and Jurkat [Another significant group of bioactive compounds are phenylpropanoids, which are naturally synthesized by plants. However, research has shown that endophytes also produce phenylpropanoids. These compounds belong to the largest group of secondary metabolites that possess an aromatic ring with a 3-carbon propene tail, thus resulting in a C6-C3 carbon skeleton. These compounds are from the amino acids phenylalanine and tyrosine . In cancmangrove . In anots sp. ZJ-, associas sp. ZJ-, associas sp. ZJ-, associad Jurkat . NonetheOxidation is a chemical reaction resulting in the loss of electrons from an atom, and this may produce free radicals. Naturally occurring free radicals are unstable molecules produced during chemical reactions such as digestion. These free radicals may participate in chain reactions which might potentially cause cell damage in the human body ,71. ThisFusarium oxysporum from the leaves of Otoba gracilipes exhibited antioxidant activity with as much as 51.5% of a scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH) after 5 min of reaction [Xylaria sp. were isolated from the medicinal plant Ginkgo biloba, and these compounds displayed antibacterial, antioxidant, anti-cardiovascular, anticancer, and antimicrobial properties. Phenolic and flavonoid compounds, among others, have been shown to possess very effective antioxidant properties [Alternaria alternata AE1 isolated from Azadirachta indica also revealed that it produced secondary metabolites that possess very effective antioxidant properties [50 value of 38.0 and 11.38 μg/mL, respectively [Pestalotiopsis microspore housed in Terminalia morobensis, which also displayed very effective antioxidant activities [Antioxidant agents are utilized to combat, prevent, and treat diseases that are linked with the presence of reactive oxygen species (ROS), and these agents have displayed very high efficacy against damage caused by ROS. ROS have been reported to boost the immune system by enabling cell signaling to occur. Various industries, such as the food, pharmaceutical, and agricultural sectors, use antioxidant compounds for beneficial purposes. Irrespective of the health concerns associated with oxidative stress, the search for safer, more efficacious, and cost-effective natural antioxidants is highly anticipated. Novel natural bioactive compounds have been reported to serve as a shelter against oxidative damage by preventing or reducing free radicals and reactive oxygen species. Various studies have demonstrated that molecules, including phenolic acids, phenylpropanoids, and flavonoids, lignin, melanin, and tannins, exhibited antioxidant activity ,75. Therreaction . A totaloperties . Recentloperties . DPPH frectively . Chemicaectively . In additivities .Fusarium oxysporum Dzf17 from Guazuma tomentosa displayed antioxidant properties [Phyllosticta sp. displayed significant antioxidant properties. When subjected to the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and DPPH radical assays, these compounds exhibited EC50 values of 580.02 and 2030.25 μg/mL, respectively [Polysaccharides produced by plants and microorganisms have been widely studied due to their natural antioxidants properties. Three polysaccharides, namely exopolysaccharide (EPS), water-extracted mycelial polysaccharide (WPS), and sodium hydroxide-extracted mycelial polysaccharide (SPS), produced by operties . In addiectively . Nerium oleander L (Apocynaceae) isolated from Trachelospermum jasminoides also displayed antioxidant potentials [Myrothecium roridum IFB-E008, IFB-E009, and IFB-E012. In addition, an investigation into their antimicrobial properties revealed that the compounds exhibited sufficient cytotoxicity based on their bioactive data [Novel trichothecene macrolides  producedtentials . Using cive data .Parasitic infections in humans are caused by protozoa, helminths, and ectoparasites that live on or in a host organism and make use of the host resources for their survival. Disease-causing parasites are known to contribute significantly to the rate of morbidity and mortality in humans worldwide, especially in developing countries that have a large proportion of vulnerable populations . This, tDiaporthe phaseolorum-92C (92C), an endophytic fungus that inhabits the roots of Combretum lanceolatum, displayed significant anti-parasitic activity against Trypanosoma cruzi by reducing up to 82% of the number of amastigotes and trypomastigotes. The bioactive molecule 18-des-hydroxy Cytochalasin H exhibited nematocidal activity and reduced the viability of promastigotes of Leishmania amazonenses with an IC50 of 9.2 μg/mL [Penicillium herquei strain BRS2A-AR was potent against Plasmodium falciparum 3D7, Trypanosoma brucei, Leishmani donovani, and Leishmania sp., with IC50 values higher than 100 μM, therefore displaying very excellent anti-parasitic activities [Alternaria alternata P1210 from the roots of the halophyte Salicornia sp. produced two new biosynthesized dimeric compounds belonging to the class alternariol, namely (±)-alternarlactones A and B. Preliminary results revealed that these compounds possessed anti-parasitic potentials [.2 μg/mL . Anothertivities . Alternatentials .Hyalodendriella sp. Ponipodef12 using spectroscopic chemical analysis. These compounds were capable of inhibiting the growth of fourth-instar larvae of Aedes aegypti [n = 152) endophytic fungi to determine their anti-plasmodial activity using a 96-well microtiter plate. The results showed that extracts from Fusarium sp. AMst1 (IC50 = 1.16–1.43 µg/mL), Trichoderma afroharzianum AMrb7 (IC50 = 1.71–2.31 µg/mL), and Penicillium tropicum AMb3 (IC50 = 1.90 µg/mL) possess significantly effective anti-plasmodial activities against Plasmodium falciparum strains [Phyllosticta capitalensis from Tibouchina granulosa (Desr.) Cogn. (Melastomataceae) inhibited Leishmania species and Trypanossoma cruzi. Based on high-resolution mass spectrum analysis (UHPLC-HRMS), 18 compounds were identified from P. capitalensis and crude extracts from the endophyte displayed growth inhibitory activities against Leishmania amazonensis, L. infantum, and Trypanosoma cruzi with IC50 values of 17.2, 82.0, and 50.13 μg/mL, respectively. Given that the diseases Leishmaniasis and Chagas are abandoned tropical diseases caused by protozoa and infect over 12 million individuals globally, these findings present a significant hope for mankind [18 and others (Mao and colleagues (2019) discovered two novel decalin/tetramic acid hybrid metabolites—hyalodendrins A and B, isolated from the endophytic fungus  aegypti . Bioassa strains . The fin mankind . Citrinid others  are amonMycosphaerella nawae ZJLQ129, isolated from Smilax china leaves [Penicillium sp. ZJ-SY2, which was associated with mangrove Sonneratia apetala leaves. These compounds exhibited excellent immunosuppressive properties, with IC50 values ranging from 5.9 to 9.3 μg/mL [Xylaria longipes HFG1018, obtained from the basidiomycete Fomitopsis betulinus, which is associated with rotting of wood, produced eighteen new nor-isopimarane diterpenes, xylarinorditerpenes A–R (1–18), some of which possessed immunosuppressive potential [Immunosuppressive drugs, also known as antirejection medications, are utilized to suppress, reduce, or prevent allograft rejection during organ transplant in patients . They tha leaves . Furthera leaves . In addi.3 μg/mL . Xylariaotential . Viruses are microorganisms that multiply only within living cells and are a leading cause of mortality and morbidity in humans globally. Current antiviral drugs and vaccines are crucial in combating life-threatening diseases in humans . In addiEmericella sp. (HK-ZJ) from the mangrove plant Aegiceras corniculatum. A bioassay using the cytopathic effect (CPE) test revealed that the fungal extracts displayed potency against influenza A viral (H1N1) [Nigrospora sphaerica (No.83-1-1-2), Alternaria alternata (No.58-8-4-1), and Phialophora sp. (No.96-1-8-1) exhibited some antiviral activity against herpes simplex virus (HSV). Extraction and identification of compounds revealed two novel heptaketides, (+)--altenuene (1a) and (−)--isoaltenuene, along with six recognizable compounds, (−)--altenuene, (+)--isoaltenuene, 5′-methoxy-6-methyl-biphenyl-3,4,3′-triol, alternariol (4), alternariol-9-methyl ether, and 4-hydroxyalternariol-9-methyl ether [Isoindolones compounds, namely emerimidines A and B, emeriphenolicins A and D, as well as other compounds, including aspernidines A and B, austin, austinol, dehydroaustin, and acetoxydehydroaustin, were discovered in the endophytic fungus l (H1N1) . Fungal yl ether .Pleospora tarda was responsible for the potent antiviral agents classified as alternariol and alternariol-(9)-methyl compounds [Phoma sp., isolated from the roots of Aconitum vilmorinianum [Nigrospora sp. YE3033 which resides in the plant Aconitum carmichaeli. A preliminary bioassay indicated that these compounds displayed strong antiviral activity against the influenza viral strain A/Puerto Rico/8/34 (H1N1) [Recently, endophytic fungi were isolated from medicinal plants of Egyptian origin, which displayed significant antiviral properties against herpes simplex (HSV-2) and vesicular stomatitis viruses (VSV) . It was ompounds . Lui et rinianum . The comrinianum . In addi4 (H1N1) . Penicillium sp. FKI-7127 led to the detection of the brefeldin A compound  NS3-NS4A protease, with an IC50 of 27.0 µg/mL. Structural characterization using MS and NMR spectral analysis of the metabolites from this endophyte showed the presence of two diketopiperazines  of 0.78 µg/mL [Tuberculosis (TB) is a life-threatening disease usually affecting the lungs and is caused by the bacteria h Africa . Infectih Africa ,113. Givh Africa . In addi concern  and, thu 3 and 4 , compoun78 µg/mL . Other cGlycyrrhiza glabra L. plant cultivated in the Kashmir Himalayas harbored a wide diversity of endophytic fungi that comprised, but were not limited to, Fusarium oxysporum strain (KT166447) and Colletotrichum gleosporoides strain (KT166445), which displayed strong inhibitory potentials against Mycobacterium tuberculosis (M. tb) strain H37Rv, with MIC values of 18.5 and 75 µg/mL, respectively [These were the first reports of such compounds being produced by a fungus . Glycyrrectively . In a reectively . Table 2Currently, we are losing the fight against ineffective, toxic, and expensive therapeutic antimicrobial drugs. However, endophytes provide a suitable alternative since they are a warehouse filled with novel bioactive compounds with endless possibilities of biological properties. Over the past few years, endophytic fungi have attracted tremendous attention in the drug development process as they are ubiquitous and abundantly availability. Numerous studies have reported novel, beneficial bioactive compounds exhibiting biological properties, such as antibacterial, antidiabetic, antifungal, anti-inflammatory, antiprotozoal, antituberculosis, insecticidal, immunomodulatory, antiviral, anticancer activities, anthelmintic, etc., that were successfully isolated from endophytic fungi. Despite this, limited research has been conducted on the valuable bioactive compounds from endophytic fungi. Research priorities need to shift towards biotechnological advances to accelerate the screening of new biomolecules for the treatment of numerous life-threatening diseases, thus safe-guarding human health. All things considered, an untapped wealth of novel bioactive compounds resides within endophytes, thus ensuring the discovery of new bioactive compounds for potential applications in the agricultural, food, medical, and pharmaceutical industries.


The transcription factor p63 is an essential regulator of vertebrate ectoderm development, including epidermis, limbs, and craniofacial tissues. Here, we have investigated the evolutionary conservation of p63 binding sites (BSs) between zebrafish and human. First, we have analyzed sequence conservation of p63 BSs by comparing ChIP-seq data from human keratinocytes and zebrafish embryos, observing a very poor conservation. Next, we compared the gene regulatory network orchestrated by p63 in both species and found a high overlap between them, suggesting a high degree of functional conservation during evolution despite sequence divergence and the large evolutionary distance. Finally, we used transgenic reporter assays in zebrafish embryos to functionally validate a set of equivalent p63 BSs from zebrafish and human located close to genes involved in epidermal development. Reporter expression was driven by human and zebrafish BSs to many common tissues related to p63 expression domains. Therefore, we conclude that the gene regulatory network controlled by p63 is highly conserved across vertebrates despite the fact that p63-bound regulatory elements show high divergence. Possible changes in TF binding are gains or losses of BSs or changes in the affinity of the site (Alterations in CRE sequences will affect es (BSs)  since ththe site , althougthe site .TP63 human gene are associated with several hereditary malformations showing ectrodactyly , ectodermal dysplasia, and orofacial cleft as their principal phenotypes , and the Andalusian government.2, 0.33 mM MgSO4, and 0.1% methylene blue). Wild-type strains for zebrafish were AB/Tübingen (AB/Tu) and were maintained and bred under standard conditions  reporter gene under the control of the gata2 minimal promoter  using primers from promoter .Zebrafish transgenic embryos were generated using the Tol2 method . One-celChIP-seq data of human keratinocytes  and ChIPFor gene assignment to ChIP-seq peaks, zebrafish coordinates were converted to Zv9/danRer7 genome using the Liftover tool of the UCSC Genome Browser . Both zeDe novo motif discovery analysis identified a p53-like binding sequence that was virtually the same in both species despite their high evolutionary divergence .P < 3.183e-33 and P < 7.590e-43, respectively)  . In othectively) . Howeverctively) . Altogetdlx3b/DLX3, grhl1/GRHL1, and myh9a/MYH9 zebrafish/human genes, and in the introns of st14a/ST14, lama5/LAMA5, and map2k1/MAP2K1. Then, these zebrafish and human sequences were cloned into an enhancer detection vector composed of a gata2 minimal promoter, an enhanced GFP reporter gene, and the strong midbrain enhancer z48 , and the pectoral fin bud , olfactory placodes, otic placodes and vesicle, pharyngeal arches, and pronephros .JT and JS-P conceived and designed the project. LG-F performed the experiments. LG-F, JS-P, and JT analyzed the data and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background and aims: Chronic disease, particularly inflammatory bowel disease (IBD), requires a multifaceted approach to managing patients, but it is apparent that primary care pharmacists are being underutilized. To demonstrate the benefits of pharmacist interventions in primary care, a systematic review was conducted of interventions in asthma and type 2 diabetes where pharmacists have a defined role in chronic disease management. We also explored potential opportunities for primary care pharmacists to deliver tailored care to patients with inflammatory bowel disease. Methods: The search strategy retrieved original research articles from seven databases; eligible articles were assessed for inclusion. Quality appraisal was performed independently by two reviewers. Results: Thirty-seven included studies were grouped into four categories of interventions: education/counseling (43%), medication management (34%), monitoring/follow-up (17%), and screening/risk prevention (6%). Education plus counseling was reported as the main intervention delivered by pharmacists. Three measurable outcomes were identified: clinical, humanistic , and economic. Clinical outcomes (63%) were reported more commonly than humanistic (26%) and economic (11%) outcomes. Pharmacist interventions led to statistically significant improvements in control of disease, severity, and medication adherence, as well as improvements in overall patient satisfaction, quality of life among patients with asthma and type 2 diabetes. Conclusion: As one of the most accessible sources of primary health care, pharmacists are well-placed to minimize the impact of chronic diseases on patients and communities. Evidence suggests there are opportunities for primary care pharmacists to play a more active role in the management of chronic diseases such as IBD. Chronic diseases, also referred to as non-communicable diseases (NCD), are the leading causes of mortality and disability globally . The WorInflammatory bowel disease (IBD) is a group of chronic diseases including Crohn’s disease (CD) and ulcerative colitis (UC) . IBD is This high level of morbidity associated with chronic diseases has a substantial impact on individuals, their families and carers, communities, healthcare professionals, and the health system. Effective chronic disease management requires a holistic approach that is patient-oriented with the aim of reducing premature mortality and morbidity through multidisciplinary collaboration between primary, secondary, and tertiary sectors ,9. Two cAlthough all chronic diseases require a similar approach to management, namely, to improve patients’ quality of life, prevent complications, and reduce the burden of disease, IBD management is primarily dealt with by gastroenterologists . Many ofTo date, there are no published studies that evaluate the impact of pharmacist interventions in the primary care management of IBD. However, considerable research is available for type 2 diabetes and asthma, two chronic diseases that are predominantly managed in primary care through a collaborative multidisciplinary approach by General Practitioners (GPs) and pharmacists. Therefore, the primary aim of this review was to examine interventions provided by pharmacists in the management of type 2 diabetes and asthma and demonstrate the benefits of their interventions through measurable clinical, humanistic, and economic outcomes. Secondly, drawing on these examples, we discuss potential opportunities for pharmacists in primary care to deliver tailored care to patients with IBD as a means of addressing the current gap in the literature and contributing to the management of this chronic disease.This systematic review follows the PRISMA (Preferred Reporting Items for Systematic Reviews) guidelines and is supported by the use of a PRISMA 2009 checklist  15]..15].A systematic search of articles reporting original research in seven bibliographic databases and of the grey literature was conducted to identify studies relating to pharmacist interventions in type 2 diabetes and asthma, namely, International Pharmaceutical Abstracts, MEDLINE, SCOPUS, CINAHL, EMBASE, and PsycINFO; the search of these databases was conducted between September 2018 through to November 2019. An additional search in the Cochrane database was carried out to identify relevant systematic reviews.Studies focusing only on secondary or tertiary care, review articles, non-original studies, notes, commentaries, and editorials were excluded. Inclusion criteria were articles published in English, which described the impact of pharmacist intervention in asthma or diabetes. No date restrictions were imposed. The ECHO model  was used for the classification of interventions as this represents a conceptual framework for effective clinical practice improvements, logically compelling and more scientifically rigorous than a unidimensional approach to outcome studies . OutcomeThe search strategy was finalized in consultation with a senior research librarian using the following three Medical Subject Headings (MeSH): disease (diabetes OR asthma OR chronic disease), setting/profession , and management  adj3 manag*) OR health education OR health promotion). See Manual searches of all published editorials, review articles, and reference lists from potentially relevant studies were conducted to identify additional studies. Grey literature was assessed and included websites of the Australian Journal of Pharmacy, Australian Pharmacist, and relevant professional/industry journals of countries published in other countries. Furthermore, a manual bibliographic search of conference abstracts arising from the systematic database search was performed. A word search of the internet was conducted using Google™ (word search: pharmacist intervention in asthma/diabetes) and the first 100 results were reviewed.Duplicate articles were removed after the title and abstract screening, which was completed by two independent reviewers (SP and KD). These reviewers also screened full-text articles to assess their eligibility based on the inclusion and exclusion criteria. Any disagreement between these reviewers was resolved via consensus and did not require a third reviewer.Due to the wide range of experimental study designs, a quality appraisal was performed using appropriate quality appraisal tools from the Joanna Briggs Institute at the University of South Australia, namely, the checklists for Randomised Controlled Trials , Quasi-E® spreadsheet and included bibliographic references , study design/methodology, interventions , outcomes measured , key findings (results and conclusion), level of evidence, and comments. The process of extracting data was undertaken by two reviewers (SP and KD). A meta-analysis was not feasible due to the heterogeneity of the studies.The study characteristics of the included articles were systematically recorded using a customized ExcelThere were 1734 articles selected for screening, of which, 1604 remained after duplications had been removed. After initial abstract screening for relevance, 170 full-text studies were assessed for eligibility. After screening and quality appraisal, 37 studies were included in this review. See Nineteen articles were deemed ‘high quality’ based on a quality score between 75% to 100%, of which seven were randomized controlled trials ,26,27,28n = 32) of the included studies were of an experimental design including randomized controlled trials  [n = 15) [n = 1) [n = 1) [n = 1) [n = 5) were observational ones with cohort study design [n = 11) [n = 6) [n = 4) [n = 4) [n = 1) [n = 2) [n = 1) [n = 1) [n = 2) [n = 1) [n = 1) [n = 1) [n = 1) [n = 1) [Most  ,45,46,47d trials %; n = 14[n = 15) ,51,52,55 [n = 1) , and tim [n = 1) . The remy design ,56,57,58d trials %; n = 14y design ,57 and 1y design ,55,56,58d trials %; n = 14[n = 11) ,47,52,54 [n = 6) ,56,58, C [n = 4) ,29,46,49 [n = 4) ,41,42,55 [n = 1) , Belgium [n = 2) ,28, Spai [n = 1) , Netherl [n = 2) ,51, Serb [n = 1) , Malta ( [n = 1) , Nigeria [n = 1) , Brazil  [n = 1)   [n = 14, diabetes n = 13) [n = 7, diabetes n = 6) [n = 2, diabetes n = 3) [n = 29) applied multiple interventions, although only two studies implemented all four components [As part of the inclusion criteria for this review, all interventions had to be performed by pharmacists within a primary care setting. Interventions included at least of one the following four key components: education plus counselling  ,56,57,58 n = 13) ,56,57,58s n = 6) ,50,51,55s n = 3) ,36,40,45mponents ,56,57,58mponents ,49,54 ormponents ,45,50,55n = 31) of the interventions occurred in a community pharmacy and 15% were conducted in medical practices (n = 2), primary health centers (n = 2), and pharmacy/ambulatory clinics (n = 2). Although type 2 diabetes interventions were performed in all of the primary care settings mentioned, asthma interventions only occurred in community pharmacies  [Type 2 diabetes and asthma showed a similar trend for all three measured outcomes . Overalln = 10 ,40,41,43 [n = 2) ,56; four [n = 2) ,36,46,47p-value of <0.001, with a change in the fasting blood glucose (BGL) of 4.1 mmol/L (95% CI and p = 0.007) [p < 0.001 (OR = 3.06), improved mean Asthma Control Questionnaire (ACQ) scores (p < 0.001), increased number of controlled asthma patients by 30.1% (p < 0.001), improved medication adherence by 40.3% (p < 0.001), and better inhaler technique . No significant changes were observed in the control group [p = 0.001; 95 % CI 0.34, 0.86 (OR 0.54), p = 0.009) [Key outcomes from the included studies were clinical (63%), followed by humanistic (26%) and economic (11%). In studies among patients with type 2 diabetes, just under half (48%) of the studies reported only clinical outcomes, while 58% reported two outcomes  and a quarter (25%) reported all three outcomes. Clinical outcomes included glycosylated hemoglobin (HbA1c) of ≤7%, fasting blood glucose levels (BGL), cardiovascular (CV) risk factors, blood pressure, and body mass index (BMI). As demonstrated in the RxING study, 51% of participants achieved target HbA1c of ≤7% at the end of the study with 95% CI and = 0.007)  that repol group . Humanis= 0.009) . The scope for pharmacist interventions continues to evolve and has certainly increased in chronic disease management in recent decades . The revInterventions have a key role in chronic disease management . The resEducation plus counseling are fundamental to optimizing care in chronic disease sufferers ,66. PatiChronic abdominal pain is a very common and debilitating symptom for IBD sufferers, with up to 70% of patients experiencing pain due to exacerbation of the disease ,73,74. PThis review has some limitations. Studies differed in their design, outcomes, and measurements, and this heterogeneity reduced our ability to make precise assessments or conduct a meta-analysis. Articles published in languages other than English were excluded, which meant that potentially relevant studies from developed as well as developing nations were not included. This review focused on two specific chronic diseases (type 2 diabetes and asthma) and may have overestimated or underestimated the impact of pharmacist interventions on these diseases. Although the extent of involvement of pharmacists may vary according to the country in which they practice, primary care pharmacists are well-positioned to play an important role in holistic approaches to chronic disease management including IBD.The available data suggest that primary care pharmacists can contribute to the services that are required to manage patients with chronic diseases, such as type 2 diabetes, asthma, and IBD. This requires innovative multidisciplinary models and collaboration between healthcare professionals in various health care settings. Considering the complex health care needs of IBD patients, primary care pharmacists are uniquely placed to complement existing care models and ultimately may help to improve patient outcomes. The introduction of pharmacist-delivered interventions requires integrated care models with defined accountabilities and communication/reporting pathways for all providers. It is evident that for pharmacist-delivered services to be valuable in the routine clinical setting careful integration and coordination are required.


Biomedical research aims to understand the molecular mechanisms causing human diseases and to develop curative therapies. So far, these goals have been achieved for a small fraction of diseases, limiting factors being the availability, validity, and use of experimental models. Niemann–Pick type C (NPC) is a prime example for a disease that lacks a curative therapy despite substantial breakthroughs. This rare, fatal, and autosomal-recessive disorder is caused by defects in NPC1 or NPC2. These ubiquitously expressed proteins help cholesterol exit from the endosomal–lysosomal system. The dysfunction of either causes an aberrant accumulation of lipids with patients presenting a large range of disease onset, neurovisceral symptoms, and life span. Here, we note general aspects of experimental models, we describe the line-up used for NPC-related research and therapy development, and we provide an outlook on future topics. The prime purpose of biomedical research is to understand the molecular underpinnings of human diseases enabling the development of curative therapies. Unfortunately, these goals have been reached merely for a minuscule fraction of diseases. The large majority of ailments—affecting from just a handful of patients to millions worldwide—awaits a treatment ,2,3. TheNPC1 (OMIM #257220) [NPC2 (OMIM #607625) [NPC is a prime example for a disease that lacks a curative therapy despite impressive breakthroughs within the last decades ,15,16,17#257220)  or NPC2 #607625) ,20. The #607625) ,24,25 to#607625) ,27,28. I#607625) ,30 toget#607625) ,32. How #607625) ,36,37,38#607625) ,42,43,44#607625) . The sec#607625) ,41,44. T#607625) ,48,49,50#607625) .N-butyldeoxynojirimycin), which decelerates disease progression in some patients [The diagnosis of NPC disease is complicated by the heterogeneous clinical presentation and therefore depends on laboratory tests. This includes the so-called filipin test, the detection of plasma biomarkers ,53,54,55patients ,60,61. Tpatients ,63.Understanding the somewhat mysterious links between cellular damage and the unpredictable outcome in patients, and the development of diagnostic tests and of efficient therapies require appropriate experimental approaches and models. In the following, we will make some general remarks, we will present currently available models for NPC research, and we will highlight crucial points.The use of experimental models in research has a long history. The first “publication” dates to the 17th century, when William Harvey described physiologic experiments with animals such as shrimp, eel, chick, and pigeon to understand blood circulation . For cenMus musculus) introduced by Flanagan [Rattus norvegicus) [Caenorhabditis elegans has been genetically modified to generate models of Parkinson’s [Drosophila melanogaster serves as disease model for different organs including the brain [Today, biomedical research on human diseases depends entirely on experimental models that range from single cells to non-human primates. Disease models may emerge from spontaneous changes. A famous example is the nude mouse  . Other pvegicus)  and incrvegicus) ,77, respvegicus) . Meanwhivegicus) . Mice arkinson’s , Alzheimkinson’s , PolyQ dkinson’s , and lyskinson’s . The fruhe brain ,85,86, khe brain , and panhe brain .Oryctolagus cuniculus) and rats do not show some symptoms of cystic fibrosis [Dasypus novemcinctus for research on leprosy [Trachemys scripta to study brain hypoxia and anoxia [Chinchilla lanigera to investigate hearing loss [The usefulness of a model depends on the specific question. Ideally, the model accurately recapitulates key aspects of the disease of interest, for example pathologic changes in cells or symptoms of patients. Furthermore, it should allow extrapolating results to the target organism. Interestingly, history teaches that extrapolability does not necessarily scale with evolutionary kinship: closer may not be better. For example, thalidomide and aspirin are well tolerated by mammalian species but not by pregnant women , and chifibrosis , and no fibrosis . On the  leprosy , the turd anoxia , and theing loss . Diurnaling loss .Disease models based on cultured cells have seen a remarkable renaissance due to the possibility of generating specific human cells from patient-derived induced pluripotent stem cells . A recenNPC1, which is mutated in 95% of patients. Only a few experimental models are available to study mutant NPC2. The presence of multiple isoforms in specific phyla suggests important and so far undiscovered functions of these proteins. Numerous experimental models are available to study NPC disease , probablNPC1 orthologues in plants  [NCR1; Saccharomyces cerevisiae) [NPC2 orthologues were not known [NPC2, which can replace the human version [NPC1 deficiency [NPC1 and NPC2 homologues in the sterol auxotroph pathogen Entamoeba histolytica revealed their contribution to cholesterol uptake  [Caenorhabditis elegans contains two homologues of mammalian NPC1 . Elimination of both forms stalls a specific phase of larval development, which is probably due to defects in the intracellular transport of cholesterol and the production of essential steroid hormones [The knock-out of haliana)  and yeasevisiae)  have beeot known , it was  version . Moreoveficiency ,110,111 ficiency . The kno Ehnpc2) . The genhormones ,114,115 hormones  and by shormones ,118.Npc1a, one of two NPC1 homologues from Drosophila melanogaster, causes larval lethality  are currently used to study NPC (Bos taurus) with progressive neurologic symptoms due to mutant NPC1, suggesting the possibility of a bovine model [Of all mammalian species serving biomedical research, only mice and cats  [spm allele, spmNpc1) [Npc1 knock-out. These mice present a relatively early onset of the disease, which is characterized by hepatomegaly, weight loss, disturbed motor coordination, tremor, and ataxia. The mice die prematurely between 11 and 13 weeks of age (pfNpc1), a double mutation (P202A/F203A) abolishes cholesterol binding by NPC1 and invalidates its function, but it leaves its level and localization unaffected [The first mouse strains to study NPC carried spontaneous mutations in Npc1Nih) ,129 and Npc1spm) ,132 in Bs of age . Cells saffected   [I1061TNPC1 (Npc1) [ImagineNpc1) or the c.1920delG mutation, generating a truncated protein (PioneerNpc1)  were predominantly female [Npc1 allele generated by the CRISPR-Cas technique (em1PavNpc1) [More common en years . Maue an1nmf164) . Praggas1I1061T) . This mo1I1061T) ,212. The1I1061T)  Table 11I1061T) . The str1I1061T .y female . The lat1em1Pav)  (Table 11em1Pav) ,214. In 1em1Pav) ,217,218.1em1Pav) ,234,235.1em1Pav) , life sp1em1Pav) ,134, and1em1Pav)  and to p1em1Pav) ,238 were1em1Pav)  as in ot1em1Pav) ,241,242 1em1Pav) ,244.Npc1 were based on the Cre/loxP technique (tm1.1AplNpc1) [Npc1 from Purkinje cells induces their degeneration but leaves the life span of mice unaffected [Npc1 in specific cell types has been accomplished using classic transgenic mice to target GFAP-expressing cells [Npc1 knock-out [Npc1 expression in the liver rescued liver disease, but it did not prevent progressive neurodegeneration and premature death [Several mouse models were established to study the relevance of NPC1 in specific cell types or tissues . Using mm1.1Apl) ,245,246 affected . A mouseaffected ,186. Theng cells , the indng cells , and thenock-out   were first reported by Lowenthal and collaborators [NPC1 causes an amino acid change (C955S) [NPC1 [NPC2 [NPC-like symptoms in a domestic cat  . Two casS) [NPC1  and NPC2C1 [NPC2 , indicatNPC1-encoding cDNA [Cultured cells are instrumental to uncover basic protein functions and molecular disease mechanisms and to test potential therapeutic approaches at the cellular level . The useing cDNA  and of sing cDNA , and theing cDNA .An alternative method to induce the cellular hallmark of NPC, an accumulation of unesterified cholesterol, relies on hydrophobic amines such as U18666A ,269,270.spmNpc1 mouse was generated using a spontaneous immortalization (3T3) protocol [NPC1 in a neuroblastoma cell line (SH-SY5Y) was achieved by stable transfection with short hairpin RNA [NPC1 or NPC2 were obtained by transduction with lentivirus and short hairpin RNAs [NPC1 in Escherichia coli has been used to study its transport function [NPC1 was stably down-regulated in a neuron-like Neuro-2a line that over-expresses a specific form of the amyloid precursor protein [spmNpc1 mouse [The first cell lines to study NPC disease were established from patient-derived blood lymphocytes, which were immortalized through transformation by the Epstein–Barr virus . A similprotocol ,275. Immprotocol . A mouseprotocol . A line protocol . Severalprotocol ,280,281.protocol ,283, mycprotocol , as wellprotocol ,286,287,protocol . CRISPR-protocol  or transprotocol ,291,292 protocol . The knorpin RNA . Immortapin RNAs ,296. Thefunction . In the  protein . Schwannpm mouse . Knock-dpm mouse . The firpm mouse .Cell line-based models matter to uncover basic molecular functions of NPC1  or NPC2 An alternative to cell lines are primary cultures, where cells are isolated from the organism and used after different periods of culture without immortalization. Cultured cells retain their in vivo properties to degrees that depend on the cell type and the culture conditions, namely the artificial exposure to chemically undefined serum ,309,310.Most NPC patients suffer from debilitating neurologic symptoms and therefore, it appears imperative to study the impact of dysfunctional NPC1 or NPC2 on cells in the brain . The firPredominant in vitro models of NPC research are the above-mentioned patient-derived skin fibroblasts, which are mitotic primary cells, but not cell lines unless they have been immortalized. Only very few differentiated cell types are studied in the field . Liver aNPC1 in human embryonic stem cells and differentiated these cells to neurons [The differentiation of specific cell types from embryonic or induced stem cells has become popular, because this technology allows studying cells from patients and producing them in large quantities. Consequently, the number of publications related to these models in the NPC field is increasing . A first neurons . These n neurons . These c neurons ,348,349. neurons . The ste neurons ,350,351.Experimental models are indispensable for the preclinical exploration of therapeutic approaches. In the NPC field, cell-based screens for targets and drugs used yeast , immortaN-butyldeoxynojirimycin inhibits glucosylceramide synthase [NPC1 mutant cats [Few studies have delivered an approved drug or treatments reaching clinical development. The disease-modifying synthase . Curioussynthase . This wasynthase , arguingant cats ,360, proA potential treatment is based on 2-hydroxypropyl-beta-cyclodextrin (CD) that chelates cholesterol and other components  Table 2Table 2. Npc1 mutant mice claimed that repeated intra-peritoneal injections of vorinostat, an HDAC inhibitor, together with polyetheylene–glycol and CD slow down neurologic disease progression, but some controls were missing [Histone deacetylases (HDACs) emerged as a possible therapeutic target for NPC from a genetic screen in yeast  and from missing . A subse missing . Repeate missing  probably missing . A compa missing  (Table 2Npc1 mutant mice that heat-shock proteins protect Purkinje cells from degeneration suggested these components as new drug targets in NPC [Evidence from s in NPC ,227. Thes in NPC ,227,388 s in NPC  (Table 2Within the last years, NPC1-deficient mice also helped to explore gene therapy for NPC . First sThe diversity and validity of experimental models and their pertinence to topics of interest are key to advance biomedical research. Over the last decades, the NPC field has developed a gang of models that matter as they revealed the origin of the disease, provided important insight in disease mechanisms, and helped to explore new diagnostic and therapeutic approaches. Moreover, these models are used extensively outside the NPC field to understand fundamental aspects of cholesterol homeostasis  in diffeThe publication record indicates a clear preference for NPC1, mice, and fibroblasts as gene, animal, and cell of choice, respectively. A few points should be considered with respect to future developments and advances. The focus on NPC1 is understandable given that most patients bear mutations in this gene. However, new models targeting NPC2 are of high interest, as they can help for example to discern NPC1- and NPC2-dependent genetic, epigenetic, and sex-dependent disease modifiers. The identification of modifiers remains a top priority in the field. The predominance of mouse models in NPC research is readily explained by the increasing ease of genetic manipulations and the relative cost efficacy. However, mice impose several limitations, notably with respect to their small size and their limited behavioral repertoire . TherefoThe focus on fibroblasts originates from their availability through skin biopsies, their ease of maintenance, and their long-standing use as a diagnostic tool. However, studies of patient-derived fibroblasts cannot inform about the outcome of NPC1 dysfunction in highly specialized postmitotic cells such as neurons. Therefore, it is imperative to elucidate how specific cells, namely the most vulnerable, react to defects in NPC1 and NPC2. This will require a combination of preparations allowing to study the same type of cells in vivo, ex vivo, and in vitro  as well Clearly, the establishment of new models requires substantial investments and bears risks, but ultimately, all that matters are the models: they are indispensable to expose molecular mechanisms underlying the disease and to develop efficient therapies.


SWEET11, which is the regulatory target of PthXo1, is activated to confer bacterial virulence and induce the leaf blight disease in rice. Furthermore, the three translocators also undergo translocation, but only XopN is highly translocated to suppress rice defense responses, suggesting that different components of a T3 translocon deploy distinct virulence mechanisms in addition to the common function in mediating bacterial effector translocation.Type III (T3) proteic effectors occupy most of the virulence determinants in eukaryote-pathogenic Gram-negative bacteria. During infection, bacteria may deploy a nanomachinery called translocon to deliver T3 effectors into host cells, wherein the effectors fulfill their pathological functions. T3 translocon is hypothetically assembled by bacterial translocators, which have been identified as one hydrophilic and two hydrophobic proteins in animal-pathogenic bacteria but remain unclear in plant pathogens. Now we characterize Hpa2, HrpF, and XopN proteins as concomitant T3 translocators in rice bacterial blight pathogen by analyzing pathological consequences of single, double, and triple gene knockout or genetic complementation. Based on these genetic analyses, Hpa2, HrpF, and XopN accordingly contribute to 46.9, 60.3, and 69.8% proportions of bacterial virulence on a susceptible rice variety. Virulence performances of Hpa2, HrpF, and XopN were attributed to their functions in essentially mediating from-bacteria-into-rice-cell translocation of PthXo1, the bacterial T3 effector characteristic of transcription factors targeting plant genes. On average, 61, 62, and 71% of PthXo1 translocation are provided correspondingly by Hpa2, HrpF, and XopN, while they cooperate to support PthXo1 translocation at a greater-than-95% extent. As a result, rice disease-susceptibility gene  Many pathogenicity-determinative proteic effectors of Gram-negative bacteria, which are plant , or animXanthomonas campestris pv. vesicatoria (Xcv), the bacterial spot pathogen of pepper , which causes bacterial speck in a variety of plant species , the pathogen that causes bacterial blight in rice, into the host plant cells  as nominated with the bacterial genus landmark  single genes back to the corresponding mutants resulted in the generation of genetically complementing strains Δhpa2/hpa2, ΔhrpF/hrpF, and ΔxopN/xopN  inferior to the WT stain in bacterial populations, whereas, genetic complementation restored these mutants to the WT in extents of multiplication in the medium , and subsequently complemented strains  in terms of the two criteria for virulence assessment. When determined at the 3rd day post-inoculation (dpi), the bacterial WT and complementing strains displayed regular multiplication in leaf tissues, gaining bacterial populations around 108 colony formation unit (cfu) per leaf, but the Δhpa2, ΔhrpF, and ΔxopN mutants incurred significant reductions in cfu/leaf values  shorter in leaves inoculated with the bacterial mutant Δhpa2, ΔhrpF, or ΔxopN in contrast to the WT and complementing strains , leaves were sampled and used in determinations of TAL effector translocation of the in planta Xoo populations  reduction in the cytoplasmic cAMP quantity in leaves inoculated with any of the Δhpa2ΔpthXo1/pthXo1-cya, ΔxopNΔpthXo1/pthXo1-cya, and ΔhrpFΔpthXo1/pthXo1-cya strains  or recombinant strains that concurrently contain pthxo1, hpa2, hrpF, and xopN (OsSWEET11 expression was significantly (P < 0.001) decreased in Nipponbare leaves inoculated with bacteria in which any of pthXo1, hpa2, hrpF, and xopN had been delete. Consistently, scoring leaf blight lesion size confirmed that recombinant Xoo strains that concurrently contain pthxo1, hpa2, hrpF, and xopN showed to be as aggressive as the WT strain in causing leaf blight symptoms, but the bacterial aggression incurred significant (P < 0.001) impairments by deleting any of these genes from the bacterial genome  quantitative reductions in leaves of the seedlings inoculated with the single mutant Δhpa2ΔpthXo1/pthXo1-cya, ΔhrpFΔpthXo1/pthXo1-cya, or ΔXopNΔpthXo1/pthXo1-cya compared with ΔpthXo1/pthXo1-cya  provided by ΔpthXo1/pthXo1-cya. Clearly, the concomitant presence of Hpa2, HrpF, and XopN is essential for the maximal translocation of PthoXo1, suggesting that the three translocators cooperate to mediate PthXol translocation from Xoo bacteria into rice cells.To elucidate whether Hpa2, HrpF, and XopN synergize in mediating PthXo1 translocation, we performed parallel experiments to compare the single, double, and triple gene-knockout PXO99hXo1-cya . FurtherhXo1-cya . On averXoo strains, including ΔhrcV/hpa2-cya, Δhpa2/hpa2-cya, ΔhrcV/xopN-cya, ΔxopN/xopN-cya, ΔhrcV/hrpF-cya, and ΔhrpF/hrpF-cya tested in comparison with PXO99A. By protein secretion analysis, we confirmed that Hap2-Cya, HrpF-Cya, and XopN-Cya were secreted by the bacterial T3 pathway with the requirement for HrcV  increased concentration (9.6 pmol cAMP per μg leaf protein), scored as 3.5-fold higher than Hpa1 and 4.6-fold higher than HrpF. By contrast, the three translocators were little translocated from the ΔhrcV/hpa2-cya, Δhpa2/hpa2-cya, and ΔhrcV/xopN-cya mutants generated by gene deletion and recombination under PXO99A background  decreased bacterial populations in leaves as determined at 3 dpi (P < 0.001) reduced lesion length of leaf blight as observed at 12 dpi  higher  higher . Based oOsPR1a and OsMPK6 genes, which are molecular markers of the ETI and PTI pathways, respectively,  and PTI  pathways . Therefoctively, . In the f OsPR1a  inset an  serve as semiredudant concomitant translocators of the TALE AvrPto  and were then reproduced and maintained in this lab. Seeds were germinated on filter papers immersed in sterile water in Petri dishes. After 3–5 days, the germinal seedlings were moved into 12-L pots (2–5 plants per pot) filled with a substrate containing peat, sand and vermiculite (1:1:1 v/v). Seeds were incubated and the plants were grown in environment-controlled chambers under 26 ± 1°C, 12-h light at 250 ± 50 μmol quanta/mEscherichia coli was grown at 37°C in Luria-Bertani broth (LB) or on LB agar (LA) plates with the appropriate antibiotics. Xoo strains were cultured at 28°C on nutrient broth (NB) or NA agar (NA) medium  medium . BacteriA genome were performed by using the unmarked deletion method  were confirmed by PCR amplification of hpa2, hrpF, and xopN, respectively. To create double and triple mutants, the recombinant vectors pK18sacB:Δhpa2, pK18sacB:ΔhrpF, and pK18sacB:ΔxopN were transferred correspondingly into one of the Δhpa2, ΔhrpF, and ΔxopN mutants or double mutants generated in the transformation process.Gene deletion from and backfill to the PXO99n method . The hrc our lab . To crea primers . Every P enzymes  and ligahpa2, hrpF, pthXo1, or xopN sequence in the pZWpthXo1 plasmid vectors , followed by brief centrifugation at 10,000 g for 10 min. The supernatant was analyzed as per the manufacturer’s instruction book to determine intracellular cAMP concentrations. Total proteins in each sample for normalization were quantified by using a BCA protein assay kit .The Cya reporter is an accurate and complete roster of effector proteins translocated by pathogenic bacteria into host cells, offering a highly sensitive and robust assay for monitoring the translocation of the effectors . An effeOsActin2 gene was used as a reference. RT-qPCR analyses always employed specific primers  and treated with DNase I (Invitrogen) to remove DNA. Northern blotting and RT-qPCR were performed as previously described . The con primers  and inclAll experiments were repeated at least three times with similar results as provided. Quantitative data were analyzed by using the commercial IBM SPSS19.0 software package  installeThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.XM and LZ designed and performed the experiments and wrote the manuscript. YL, XW, JB, and KL performed the experiments. SZ analyzed the data and commented on the manuscript. LC supervised the research and commented on the manuscript. HD conceived the research, designed the experiments, and wrote and finalized the manuscript.HD, LZ, and LC are the inventors on a provisional patent application 201911394862.6 that covers an Xoo T3 translocator. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mammalian Caco-2 and THP-1 cell lines served as reference controls to select insecticidal mycotoxins harmless to mammalian cells. All tested mycotoxins significantly reduced the in vitro proliferation of the Sf-9 cells and evoked morphological changes. Ten of the mycotoxins found to strongly inhibit Sf-9 proliferation also had moderate or no effect on Caco-2 cells. The THP-1 cells were highly resistant to the tested mycotoxins: doses 103 times higher were needed to affect viability and morphology (1 μg/ml for THP-1 versus 1 ng/ml for Sf-9 and Caco-2). Nine mycotoxins significantly decreased Sf-9 cell proliferation with minor effects on mammalian cells: cyclosporins B and D, cytochalasin E, gliotoxin, HC toxin, paxilline, penitrem A, stachybotrylactam and verruculogen. These may be good candidates for future biopesticide formulations.The economic losses and threats to human and animal health caused by insects and the pathogens transmitted by them require effective and environmentally-friendly methods of controlling them. One such group of natural biocontrol agents which may be used as biopesticides is that of the entomopathogenic fungi and their toxic secondary metabolites (mycotoxins). The present  Mycotoxins constitute a group of varied compounds produced naturally by fungi as secondary metabolites which pose a risk to human and animal health and can cause a variety of ill effects from allergic responses to immunosuppression and cancer. They are typically not essential to the growth and reproduction of the producing organism . Many myBeauveria bassiana, cytochalasin C produced by Metarhizium anisopliae, and cyclosporin H produced by Tolypocladium inflatum. A single mold species may produce many different mycotoxins, and several species may produce the same mycotoxin [in vivo studies using insects provide useful information on toxicity toward the target organism, they are time consuming and demand the use of high levels of tested compounds. In contrast, in vitro cytotoxicity tests are less expensive, more reproducible, and much faster. The aim of this study was to identify commercially-available mycotoxins that could act as promising candidates for further studies on potential insecticides. A testable hypothesis was to check whether the commercially-available mycotoxins could affect the morphology and in vitro proliferation of insect cells. The study evaluates the sensitivity of the Sf-9 cell line from fall armyworm, Spodoptera frugiperda, an important crop pest, toward 65 mycotoxins and compares the findings with those obtained from two human cell lines, Caco-2 and THP-1, which are commonly used as in vitro mammalian models.Entomopathogenic fungi are natural enemies of insects, and their role in the regulation of insect populations is relatively well described . In respycotoxin –5; in adycotoxin –12 for wycotoxin . Most myycotoxin , 15; howycotoxin , 17, whihttp://www.axxora.com). All mycotoxins were dissolved in 99.8% ethanol (POCH) for use in in vitro tests.The following mycotoxins on were administered to the Sf-9, Caco-2, and THP-1 cells cultures: 3-acetyldeoxynivalenol, aflatoxicol, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin M1, aflatoxin M2, alternariol, alternariol-9-methyl ether, α-amanitin, β-amanitin, γ-amanitin, antibiotic PF 1052, apicidin, beauvericin, brefeldin A, chaetocin, citreoviridin, citrinin, cyclopiazonic acid, cyclosporin A, cyclosporin B, cyclosporin C, cyclosporin D, cyclosporin H, cytochalasin A, cytochalasin B, cytochalasin C, cytochalasin D, cytochalasin E, deoxynivalenol, diacetoxyscirpenol, fumagillin, fumigaclavine A, fumonisin B1, fumonisin B2, fusarenon X, gliotoxin, HC toxin, HT-2-toxin, moniliformin, moniliformin sodium salt, mycophenolic acid, neosolaniol, ochratoxin A, ochratoxin B, patulin, paxilline, penitrem A, phomopsin A, roquefortine C, skyrin, stachybotrylactam, sterigmatocystin, strobilurin B, T2 tetraol, T2 toxin, T2 triol, tenuazonic acid, territrem B, verruculogen, wortmannin, zearalenone, and α-zearalanol. Mycotoxins were purchased via Axxora platform  was cultured in Gibco Grace’s Insect Medium (Thermo Fisher Scientific) supplemented with 10% Fetal Bovine Serum , 10 mg/ml gentamycin (Sigma Aldrich) and 250 μg/ml amphotericin B (Sigma Aldrich). To examine the effect of the mycotoxins, the cells were transferred to 24-well plates (Nest). The cell density was 1 x 106 cells/well in 400 μl. After 24-hour incubation at 27°C, the mycotoxins dissolved in ethanol were added to the cells. The final concentration of toxin in the well was 1 ng/ml. The dose was selected on the basis of pilot studies in which various concentrations (0.1–50 ng/ml) were tested on SF-9 cells (not shown). The cells were incubated for 24 hours in optimal conditions. After that time, the effect of the toxins on the cells was examined. Two negative controls were used: C1- untreated cells and C2—cells that received only ethanol (1% per well). Each control was performed in 20 independent replications. The effect of each mycotoxin on Sf-9 cell line was tested in 3 independent replications performed separately for proliferation assays and for microscopy.The Sf-9 cell line from 2 culture flasks (Nest) and incubated at 37°C in a humidified atmosphere with 5% CO2. The cell line was subcultured at 70–80% confluence. Briefly, the cells were detached with 0.25% trypsin / 0.02% sodium EDTA solution (Merck) and split 1: 3–1: 6 .A Caco-2 continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells were purchased in Sigma Aldrich. The cells were cultured in Dulbecco modified Eagle medium  supplemented with 10% Fetal Bovine Serum , 10 mg/ml gentamycin (Sigma Aldrich) and 250 μg/ml amphotericin B (Sigma Aldrich). The cells were grown in 25 cm5 cells/well and allowed to grow in 400 μl medium until confluence. Mycotoxins dissolved in ethanol were added to the cells at a final concentration of 1 ng/ml. The dose was selected on the basis of pilot studies in which various concentrations (0.1–50 ng/ml) were tested on Caco-2 cells (not shown). The cells were incubated for 24 hours under optimal conditions (37°C in a humidified atmosphere with 5% CO2). After that time, the effect of toxins on cells was examined. Two negative controls were used: C1- untreated cells, and C2—cells that received only ethanol (1% per well). Each control was performed in 20 independent replications. The effect of each mycotoxin on Caco-2 cell line was tested in 3 independent replications performed separately for proliferation assays and for microscopy.To examine the effects of the mycotoxins, the cells were seeded in 24-well plates (Nest) at a density of 1.2 × 105 per well in a 24-well plate (Nest), to a total of 400 μl medium and incubated at 37°C in a humidified atmosphere of 5% CO2 for 24 hours. After this time, the test mycotoxins diluted in ethanol were added to the cells.THP-1 cells (Sigma Aldrich), a pro-monocytic cell line, were cultured in RPMI 1640 (Merck) supplemented with 10% Fetal Bovine Serum , 10 mg/ml gentamycin (Sigma Aldrich) and 250 μg/ml amphotericin B (Sigma Aldrich). The cells were seeded at a density of 7 x 102). Following this, the effect of the toxins on the cells was examined. Two negative controls were used: C1- untreated cells, and C2—cells that received only ethanol (1% per well). Each control was performed in 20 independent replications. The effect of each mycotoxin on THP-1 cell line was tested in 3 independent replications performed separately for proliferation and viability assays as well as for microscopy.As the THP-1 cells displayed high resistance to mycotoxins administered at a concentration of 1 ng/ml during preliminary studies, four randomly-selected mycotoxins  were added at various concentrations to determine appropriate test concentrations. The mycotoxins, dissolved in ethanol, were added to the cells to give the following final concentrations per well: 1 ng/ml, 100 ng/ml, 200 ng/ml, 400 ng/ml, 500 ng/ml and 1 μg/ml. The results of this trial  indicateAfter 24h incubation of Sf-9, Caco-2, and THP-1 cells with tested mycotoxins, the ability of cells to proliferate were investigated and morphological changes were analyzed. In the THP-1 cultures, cell viability was additionally tested.Cell Counting Kit-8  was used to measure cell proliferation. This reagent based on WST-8  is reduced by the dehydrogenases in cells to give an orange colored product (formazan). The amount of the formazan in cells was directly proportional to the number of living cells. Briefly, 40 μl of CCK-8 reagent was added to each well containing cell cultures (test or control), the plates were then incubated for four hours under the described above optimal conditions for each cell type. Absorbance readings were taken at 450 nm using a Synergy HT Multi-Mode Microplate Reader (BioTek). The results are presented as a percentage of control (C2) findings, assumed to be 100%.Of the tested cells, only the THP-1 line is non-adherent. Therefore, it was possible to check the viability of these cells with propidium iodide . The final concentration of the reagent in the wells of culture plates was 10 μg/ml. After a 10-minute incubation period at room temperature, an Arthur cell counter (NanoEnTek) was used to calculate the number of cells. The results are presented as a percentage of dead cells. Similar attempts to detach adherent Sf-9 and Caco-2 cells, both mechanically and enzymatically, resulted in very high mortality, which prevented the measurement of cell viability in these cultures.Phase contrast microscope images of control and treated Sf-9, Caco-2, and THP-1 cells were taken at various time points (from five minutes up to 24 hours after treatment) using a Axio Vert.A1 fluorescence inverted microscope (Zeiss) equipped with Axio Cam ICc 5 camera (Zeiss) and Zen lite 2012 software (Zeiss).t test and Dunnett test for post hoc analysis. The Kolmogorov-Smirnov (K-S) test, skewness and kurtosis were used to check normality of controls. All data are shown as means ± SD, and values of p ≤ 0.05 were considered as significant.Statistical analysis was performed using STATISTICA 6.1 software (StatSoft Polska). Any statistical relationships were evaluated using the one-way ANOVA, in vitro proliferation of Sf-9 cells as compared with control cells (C2) which received an appropriate amount of solvent (ethanol). Regarding the Sf-9 cells the strongest effect  was observed in the case of 24-hour incubation with 1 ng/ml of 19 mycotoxins: 3-acetyldeoxynivalenol, aflatoxins B1 and B2, alternariol, alternariol-9-methyl ether, beauvericin, brefeldin A, cyclosporin D, cytochalasin E, fumonisin B2, gliotoxin, ochratoxin A, patulin, paxilline, penitrem A, stachybotrylactam, sterigmatocystin, T2 tetraol, and verruculogen. In contrast, the weakest effect (cell proliferation drop to 70–91% of that observed in control C2) was detected in the case of 13 mycotoxins: aflatoxicol, aflatoxin M1, γ-amanitin, antibiotic PF 1052, citrinin, cytochalasin D, fumagillin, HT-2-toxin, roquefortine C, T2 triol, wortmannin, zearalenone, and α-zearalanol.From the data presented in In the case of Caco-2 cells, no effect on cell proliferation was found after application of aflatoxin M2, alternariol (which exerted potent effect on Sf-9 cells), γ-ammanitin, cyclosporine B, cytochalasin A or diacetoxyscripenol. It is noteworthy that verruculogen, which inhibited the proliferation of Sf-9 cells to 16 ± 3%, had the opposite effect on Caco-2 (140 ± 12% of cell proliferation). The lowest values of Caco-2 cell proliferation were measured after the application of T2 tetraol (35 ± 3%), stachybotrylactam (37 ± 6%), cytochalasin B (39 ± 4%) or wortmannin (39 ± 2%). Application of these four mycotoxins to Sf-9 cells exerted either a similar effect, i.e. strong inhibition of cell proliferation , or slightly decreased proliferation . The following mycotoxins strongly decreased the proliferation of Sf-9 cells (drop to 13–30%) but displayed a moderate or no effect on the proliferation of Caco-2 cells: 3-acetyldeoxynivalenol, alternariol, alternariol-9-methyl ether, brefeldin A, cyclosporines B and C, ochratoxin A, paxilline, sterigmatocystin, and verruculogen  before significant effect on cell viability was observed. A significant decrease in THP-1 cell viability was observed only after the administration of the 0.5 and 1 μg/ml doses for four mycotoxins randomly selected from the 65 tested compounds: alternariol, deoxynivalenol, aflatoxin G2 and HC toxin .The enormous resistance of THP-1 cells to mycotoxins is evidenced by the fact that the increase in mortality after administration of T2-toxin was observed only at concentrations of 400 ng/ml and more, and the complete lack of cell mortality elevation after administration of aflatoxin M1, even at the concentration of 1 μg/ml .No effect on THP-1 cell proliferation was observed for 22 mycotoxins administered at such a high concentration: aflatoxins B2 and M2, antibiotic PF 1052, cyclopiazonic acid, cyclosporine B and H, cytochalasin B, fumonisin B1 and B2, fusarenon X, HT-2 toxin, moniliformin, mycophenolic acid, ochratoxin A, patulin, phomopsin A, strobilurin B, T2 triol, territrem B, verruculogen, zearalenone and α-zearalanol . THP-1 cElevated THP-1 cell mortality was induced by 33 mycotoxins , with thControl Caco-2 cells showed a typical confluent monolayer appearance i.e. an adherent cell culture in which all cells were in contact with other cells. Mitotic Caco-2 cells were observed in untreated cells (C1) only. In Caco-2 cells, mycotoxin treatments resulted in changes in the shape of the cells, loss of cellular junctions and the loss of cell adherence to the growth surface. Detached and disintegrated cells were seen after administration of aflatoxin B1, antibiotic PF 1052, HT-2 toxin and wortmannin. Treatment of Caco-2 cells with roquefortine C resulted in complete disintegration of the monolayer and very rapid cell fragmentation. The addition of zearalenone induced monolayer disintegration and the appearance of highly-vacuolized cells which displayed a range of shapes, including characteristic starfish-like cells. The most frequently-observed change was the formation of monolayer surface protrusions: convex structures similar to blisters. Such structures, of various shapes and sizes, were observed after the administration of aflatoxicol, 3- acetyldeoxynivalenol, aflatoxin G2, α-amanitin, antibiotic PF 1052, cyclosporin H, cytochalasin B, stachybotrylactam, T2 tetraol, and wortmannin.THP-1 cells were found to be much more resistant to mycotoxins than Sf-9 and Caco-2 cells, as a 1000-fold greater dose was typically required to elicit a response. Typical mycotoxin-induced changes in THP-1 cell morphology are presented in The damage caused to crops and stored food by harmful insects, as well as their transmission of pathogens causing serious diseases for humans, animals and plants require more effective control of pest populations. The insecticides currently used for this purpose are typically synthetic ones based on non-selective chemical compounds which accumulate in the environment and pose a serious threat to bio-diversity; growing pressure to minimize these deleterious effects has led to increased interest in the potential to harness the natural enemies of insects. Among various potential biocontrol agents, entomopathogenic fungi seem to be the most promising , 19.The fungal pathogenesis of insects typically follows a series of events comprising conidial adhesion to the insect host cuticle, followed by penetration of the cuticle via fungal enzymes, the impairment of host immune responses, dissemination within the host and transmission from the host , 20. ToxDrosophila melanogaster [Calliphora erythrocephala, Aedes aegypti, Lygus spp., Spodoptera frugiperda and Schizaphis graminum [Galleria mellonella larvae and to display toxicity against two different insect cell lines: Sf-9 from Spodoptera frugiperda and Tni High Five from Trichoplusia ni [Tolypocladium species, was toxic to insects upon injection [Leptinotarsa decemlineata [Helicoverpa zea and Spodoptera frugiperda: aflatoxin B1, ochratoxin A, sterigmatocistin, citrinin, cyclopiazonic acid, penicillic acid, deoxynivalenol, diacetoxyscripenol, dihydrodeoxynivalenol, dihydroxycalonectrin, hydroxycalonectrin, moniliformion, sambucinol, T2 toxin, zearalenone, chaetoglobosin C, cytochalasin H, paspaline, paxilline, penitrem A, roseotoxin B, and verruculogen [Drosophila melanogaster is susceptible to aflatoxin B1, the larvae of Bombyx mori, Heliotis zea and Spodoptera frugiperda are sensitive to both aflatoxin B1 and T2 toxin [Drosophila can tolerate the fungal poison α-amanitin in wild mushrooms and in artificial diet [in vitro using insect cell lines [Numerous mycotoxins, with various chemical structures, show strong insecticidal activity against insect pests. Four decades ago, the insecticidal activities of aflatoxin B1, rubratoxin B, patulin and diacetoxyscirpenol have been examined in contact tests against nogaster , as has graminum . Fungal lusia ni . In addinjection , leucinomlineata , and desmlineata . Anotheruculogen . The actT2 toxin . Interesial diet . The actial diet , 30. Duell lines .in vitro. Sixteen of the mycotoxins used in the present study against the Sf-9 cells, including beauvericin, deoxynivalenol, diacetoxyscripenol, fusarenon X, fumonisin B1, gliotoxin, moniliformin, ochratoxin A and zearalenone  and/or cation-selective channels in lipid membranes, which affects cell homeostasis and the uncoupling of oxidative phosphorylation [Most studies focus on the major feration , 41. At rophages . Nivalenrophages . Zearalerophages , 43. Fumrophages . Beauverrylation , 44. Therylation . The majrylation .in vivo and Ca2+/calmodulin dependent elongation factor 2 phosphorylation in vitro [in vivo [Our comparison of the impact of individual mycotoxins on the three examined cell lines has allowed the selection of several promising candidates for further evaluation as potential insecticides. The mycotoxins cyclosporin B and D, cytochalasin E, gliotoxin, HC toxin, paxilline, penitrem A, stachybotrylactam and verruculogen significantly decreased Sf-9 cell proliferation while displaying rather mild effects on mammalian cells. The best candidate for mycotoxin-based insecticide appears to be cyclosporin B, which is known to be toxic to mosquito larvae . In contin vitro . A similin vitro , as well[in vivo , HC toxi[in vivo , and pax[in vivo –53.leukemia THP-1 cells  indicated the presence of extensive genome rearrangements in the cell line and provided the first genomic evidence of a high degree of instability in chromosomes in lepidopteran cell lines [in vitro are not able to bypass the cuticle/peritrophic membrane, or that mycotoxins may not be processed by host enzymes in vitro or in vivo. Deleterious side effects, such as carcinogenic effects may also interfere with the response of the whole organism. Further experiments are necessary to analyze all these parameters. Using of another insect cell line and hemocytes (insect immunocompetent cells) will explain if the insecticidal effect is insect cell line and/or cell type specific.It is worth mentioning the limitations of research carried out on cell lines. The observed differences between the insect cell line and mammalian cell lines cannot be interpreted as true differences between insects and mammals and the characteristics of the cell lines might be not representative of those of the organism they originate from. Furthermore, while the genomes of wild insect populations are typically highly heterogeneous, the Sf-9 cell line  ovaries . A compall lines . For thiS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Regenerative capacity in vital organs is limited by fibrosis propensity. Idiopathic pulmonary fibrosis (IPF), a progressive lung disease linked with aging, is a classic example. In this study, we show that in flow cytometry, immunoblots (IB) and in lung sections, FLIP levels can be regulated, in vivo and in vitro, through SIRT1 activity inhibition by CMH (4-(4-Chloro-2-methylphenoxy)-N-hydroxybutanamide), a small molecule that, as we determined here by structural biology calculations, docked into its nonhistone substrate Ku70-binding site. Ku70 immunoprecipitations and immunoblots confirmed our theory that Ku70-deacetylation, Ku70/FLIP complex, myofibroblast resistance to apoptosis, cell survival, and lung fibrosis in bleomycin-treated mice, are reduced and regulated by CMH. Thus, small molecules associated with SIRT1-mediated regulation of Ku70 deacetylation, affecting FLIP stabilization in fibrotic-lung myofibroblasts, may be a useful strategy, enabling tissue regeneration. Current treatments of lung fibrosis are not only ineffective, but are even shown to increase mortality in IPF , except +-dependent deacetylases with diverse cellular localization and targeting [Sirtuins are a class of NADargeting . SIRT1, argeting ,12,13. Sargeting ,15, withargeting  and withargeting ,18,19,20argeting ,22 relevargeting ,22), sucargeting , can be argeting . In factargeting . On the argeting . In previous studies, we found that the Fas surface molecule is overexpressed on lung myofibroblasts from mice with bleomycin-induced fibrosis, and in humans with IPF . NeverthPreviously, SIRT1 was associated with deacetylation of histones and downregulation of FLIP mRNA levels. Increased FLIP expression was previously detected by us and others ,28 in fi® CCL-191™)-IPF-lung, and LL 24 (ATCC® CCL-151™)-normal lung fibroblast cell lines were used.Differentiated fibroblasts from IPF-lungs and controls from patient lung biopsies performed for tumor diagnosis were purchased from Carol Feghali-Bostwick . Informed consent was obtained under a protocol approved by the Institutional Review Board for Human Research at the Medical University of South Carolina. The samples were labeled and delivered to our laboratory with a code to anonymize them. LL 97A (AlMy)  were used. All experiments involving human cells conform to the relevant regulatory standards and were approved by the Institutional Review Board of the Hadassah-Hebrew University Medical Center . Mice were maintained under specific pathogen-free conditions with adherence to institutional guidelines for the care and use of laboratory animals.Oropharyngeal aspiration of BLM (OA-BLM) was performed as previously detailed . Lung fiMyofibroblast isolation and culture has been described by us in detail elsewhere ,27.Apoptosis was assessed with Annexin V affinity labeling, trypan blue exclusion, and caspase-3 cleavage in WB, as seen in the figures and described previously ,27,32.Performed as we previously described ,27.Standard WB and flow cytometry were performed as we described previously .Standard protocols have been described in detail previously .AutoDock VINA v.1.1.2 was used based on human SIRT1 crystal structure from the Protein Data Bank (PDB entry 4zzj). Both ligands and water molecules in 4zzj were removed using the Discovery Studio Visualizer 4.5. Hydrogen atoms were added using the MolProbity server . KollmanThe Kruskal–Wallis test was applied to compare variables measured at different time intervals or following different treatments. The Mann–Whitney test with the Bonferroni correction was used to test for statistical significance. Two-way ANOVA was used to assess time and treatment effects and interactions. All data are presented as mean ± standard error.L and c-FLIPS levels in MCF-7 cancer cells [Employing a high-throughput chemical screening strategy, a small molecule inhibitor of c-FLIP, 4-(4-chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH) (5809354) that downregulates c-FLIP mRNA expression has been identified ,37. CMH er cells .As determined in silico by AutoDock VINA v.1.1.2, CMH, the pro-apoptosis molecule that downregulates FLIP in cancer cells ,36,37 do3 (green) to only 101 (purple) . FLIP in normal lungs had decreased by a negligible amount. Indeed, following CMH exposure , IPF-lung myofibroblasts although keeping stable SIRT1 expression levels (not shown), they show an affected activity of SIRT1, as indicated by the increased acetylation of Ku70, a SIRT1 nonhistone substrate ,25,29, f(purple) G was detRNA-seq array analyses of IPF-lung fibroblasts were performed before (vehicle), and after CMH treatments (30 µM 72 h), followed by unsupervised clustering for differentially expressed genes in SIRT1-mediated signaling . Heat maE2F1–SIRT1 interplay bears some resemblance to that of the p53-SIRT1 axis and may be part of the mechanism controlling the fine balance between cell repair and apoptosis . Of note5 viable cells in control compared to only 0.3 × 105 cells following CMH. In fact, CMH-treated cells were 10 times more likely to undergo Fas-induced apoptosis (2.8 compared to 0.3), as determined by the OD of cleaved to uncleaved caspase-3 ratios , in the experimental model, and in fibroblasts from lungs of humans with IPF ,28. We tBLM-treated mice exposed to CMH vs. vehicle alone had indeed lower expression of FLIP, as well as of SIRT1, in IHC of lung tissue sections to levels detected in control saline-treated mice C. MoreovConcomitantly, compared to vehicle, CMH-treated mice reduced hematoxylin and eosin (H&E) and collagen-trichrome staining E, and coIn our recent study , we showCMH inhibited the binding of Ku70 to SIRT1 C, indicaCMH was docked in isolation and according to the predicted binding energy. It is not presented in relation to a drug of known activity but as an endogenous substrate (acetylated Lys) as a reference. The CMH was docked only in a catalytic site window and not as a blind dock potentially limiting the information about CMH binding to other sites on SIRT1. With this molecular modeling strategy, we simply demonstrate that CMH is capable of fitting into the SIRT1 binding site in a similar way as a substrate does. Further, NMR data to show co-crystallization of SIRT1 and CMH and mutational analyses would be of interest. A direct confirmation of interaction between CMH and the lysine binding site of SIRT1 may constitute a subject of further study, in particular with other proteins known to be deacetylated by SIRT1, such as NF-κB or FOXO1 . Other my/y chimeric mice, reduced FLIP levels, increased apoptosis cascades and decreased survival pathways in lung fibroblasts, which led to attenuation of fibrosis. Moreover, Ku70 acetylation was considerably augmented in the SIRTy/y mice. Thus, although FLIP was previously demonstrated to be regulated at the transcription level by other histone deacetylases as HDAC-3, 6 and 8 [We have recently found increased SIRT1 activity in fibrotic-lung myofibroblasts from IPF patients and BLM-treated mice . In supp 6 and 8 ,43 our fIn addition, besides having a negative feedback on itself, SIRT1 regulates several apoptosis cascades vs. survival signaling pathways, particularly by repression of p53 . CMH seeIPF lung myofibroblasts resist Fas-induced apoptosis . PreviouWe demonstrate that inhibition of SIRT1 is associated with increased Ku70 acetylation, FLIP destabilization, neutralizing resistance to apoptosis in human IPF-lung fibroblasts and enhances a variety of apoptosis cascades. This pathway may be used to modulate fibrosis evolution in the lungs of humans with IPF. Ku70 acetylation induced by CMH may provide new therapeutic approaches to combat IPF.The findings described in this article are protected in a provisional patent USP No. 62/897,332.