Curvularia is a Pleosporalean monophyletic genus with a great diversity of species, including relevant phytopathogenic, animal and human pathogenic fungi. However, their microscopic identification is difficult due to overlapping morphological features amongst species. In recent years, multi-locus sequence analysis using the ITS region of the rDNA and fragments of the genes gapdh and tef1 revealed numerous cryptic species, especially in isolates that commonly produced 3-septate conidia. Therefore, based on sequence analysis of the above-mentioned DNA barcodes recommended for species delineation in Curvularia, we propose three novel species, C.paraverruculosa, C.suttoniae and C.vietnamensis, isolated from soil, human clinical specimens and plant material, respectively, collected in different countries. These new species are morphologically characterised and illustrated in the present study. Curvulariaparaverruculosa differs from its counterparts, C.americana and C.verruculosa, mainly by its narrower conidia. Curvulariasuttoniae and C.vietnamensis are closely related to C.petersonii, but the former two have larger conidia. CurvulariaC.lunata (Wakker) Boedijn, belongs in Pleosporaceae, Pleosporales  region of the rDNA and the protein-coding loci glyceraldehyde-3-phosphate dehydrogenase (gapdh) and translation elongation factor 1-a (tef1)  . Nearly nclature .Curvularia species are proposed here, isolated from human clinical specimens in the USA, soil in Mexico and seed and plant debris in Vietnam and Indonesia, respectively.Based on a polyphasic approach, combining morphological and phylogenetic analyses, three novel Curvularia isolates, maintained in the fungal collection of the Medical School of the Rovira i Virgili University , were included in the study. Two of these  were isolated from human specimens in the USA by Deana A. Sutton of the Fungus Testing Laboratory at the University of Texas Health Sciences Center  and the other three  were isolated from environmental samples; the first from sorghum seeds collected in Indonesia, the second from soil collected in the Mexican region of Michoacán and the third from unidentified plant material collected in the north-east of Vietnam.Five unidentified PDA; Pronadisa, Madrid, Spain) for 7 to 10 days at 25 °C in darkness and following the protocol of ITS barcode, including the 5.8S gene and the genes gapdh and tef1 were analysed following ITS region  using the Basic Local Alignment Search Tool (BLASTn) for their molecular identification. To establish the phylogenetic position of unidentified isolates with respect to the most accepted species in Curvularia, we carried out individual (data not shown) and combined alignments of the three loci complemented by all available sequences of the ex-type and reference strains of Curvularia species retrieved from NCBI  software v.6.0. (ML) and Bayesian Inference (BI) approaches under RAxML-HPC2 on XSEDE v.8.2.12  in CIPRES Science gateway portal ; the combined analysis of these three phylogenetic markers was tested through Incongruence Length Difference (ILD) implemented in the Winclada programme  ≥ 70% were considered significant.For the rogramme . ML bootBI phylogenetic analysis, the best nucleotide substitution model was determined using jModelTest , for gapdh General Time Reversible with gamma distribution (GTR+G) and for tef1 General Time Reversible with invariant sites (GTR+I). The parameter settings used were two simultaneous runs of 5M generations, four Markov chains, sampled every 1000 generations. The 50% majority-rule consensus tree and posterior probability values were calculated after discarding the first 25% of the samples. A posterior probability (pp) value of ≥ 0.95 was considered significant.For the odelTest . For thehttp://treebase.org).Sequence data generated in the present study were deposited in GenBank Table  and the PDA, oatmeal agar  and potato carrot agar , after 7 days at 25 °C in darkness. Colours of the colonies in descriptions were based on Kornerup & Wanscher (1978). Cardinal temperatures for growth were obtained on PDA after 7 days in darkness.Macroscopic characterisation of the colonies was made on Microscopic features were studied from the specimens mounted in Shear’s solution growing on the same media . At leasCBS, The Netherlands).Nomenclatural novelties and descriptions were deposited in MycoBank . Ex-typeBLASTn results with gapdh sequences showed that the isolate FMR 17656 was ≤ 97.6%, similar to C.verruculosaCGMCC 28792; FMR 11956 and FMR 17659 showed a similarity of 93.31% and 93.6%, respectively, with C.spiciferaCBS 198.31; and isolates FMR 10992 and FMR 11690 both exhibited a similarity of 94.7% with the ex-type strain of C.petersonii (BRIP 14642). Sequence similarity with this marker between FMR 11956/17659 and FMR 10992/11690 was 97%. These values suggested that the unidentified isolates represented putative new species for the genus, which were then confirmed by multi-locus sequence analysis of ITS, gapdh and tef1 barcodes. The combined analysis included 128 sequences representing 126 taxa in the genus Curvularia and these were rooted with Bipolarismaydis (CBS 136.29) and B.saccharicola (CBS 155.26) , including 546 variable sites  and 445 phylogenetically informative . The unidentified isolates were allocated to three single lineages in the same clade (74/0.99) together with sequences of the ex-type strains of C.americana (UTHSC 08-3414), C.petersonii (BRIP 14642) and C.verruculosa (CBS 150.63), but with enough distance to be considered distinct species. The two clinical isolates (FMR 10992 and FMR 11690) formed a fully-supported clade closely related to isolates FMR 11956 and FMR 17659, which were collected in Indonesia and Vietnam, respectively and to C.petersonii. The fifth isolate (FMR 17656) was related to C.verruculosa and C.americana, but formed an independent and distant branch from the previously-mentioned species.Curvularia isolates , with 298 variable sites  and 240 being phylogenetically informative . The phylogenetic analyses show that these isolates indeed represent three new species, which are described and illustrated in the Taxonomy section. The species can be morphologically differentiated mainly by features of their conidia .Mexico, Michoacán, Villa Jiménez, from soil, Sept 2016, PDA at 25 °C). Mycelium composed of branched, septate, subhyaline to pale brown, thin- and smooth-walled hyphae, 2–4 μm wide. Conidiophores semi- to macronematous, mononematous, septate, straight or flexuous, geniculate at upper part, unbranched or slightly branched, smooth-walled, yellowish-brown to brown, 19–85(–145) × 3–6 μm (av. (±SD) 49.6 (±43.8) × 4.6 (±0.69)). Conidiogenous cells terminal or intercalary, polytretic, proliferating sympodially, yellowish-brown, with darkened scars, subcylindrical, 4–6 μm wide. Conidia 3(–4)-septate, mostly curved at the third cell from base which is usually larger than the others, sometimes apically bifurcate, verruculose to verrucose, apical and basal cells subhyaline to pale brown, middle cells brown, 11–37 × 8–12 μm (av. (±SD) 24 (±18.38) × 9.58 (±1.66)); hila slightly protuberant, thickened and darkened. Sexual morph not observed., final edge whitish, velvety, flat, margin regular and fimbriate; reverse dark green (30F8). On PCA and OA, reaching 58–60 mm diam., dark green (30F8), final edge whitish, slightly floccose, flat, margin regular and fimbriate; reverse dark green (30F8). Sporulation was abundant on the three media.(7 d at 25 °C). Optimum 30 °C, maximum 37 °C, minimum 15 °C.Mexico.Curvulariaparaverruculosa is allocated phylogenetically to a strongly-supported clade (100/1) with C.verruculosa and C.americana   and smooth-walled with a slightly verruculose basal cell  . Furthersal cell . In addiTaxon classificationFungiPleosporalesPleosporaceaeIturrieta-González, Wiederhold, Gené & Dania Garcíasp. nov.D6763586-FEDC-5E6F-9484-1D26368AE9FD833025Named in honour of the American mycologist Deanna A. Sutton for her contribution to the body knowledge of microfungi.D.A. Sutton .USA, Texas, from a human leg wound, 2009, PDA at 25 °C). Mycelium consisting of branched, septate, pale brown, smooth-walled to verruculose hyphae, 1–4 µm wide. Conidiophores mononematous, semi- to macronematous, erect to slightly flexuous, geniculate at the apex, unbranched or branched, smooth-walled to verruculose, pale brown, 43–103 × 3–5 µm (av. (±SD) 80 (±32.35) × 3.7 (±0.67)). Conidiogenous cells terminal, subterminal or intercalary, polytretic, proliferating sympodially, pale brown, darkened scars, subcylindrical to slightly swollen, 3–5 µm wide. Conidia (2–)3-septate, straight or curved, with the third cell often larger than the rest, apical and middle cells smooth-walled, basal cell verruculose, pale brown to brown, apical and basal cells paler than the middle cells, 8–22 × 5–9 µm (av. (±SD) 15 (±9.89) × 6.88 (±1.18)); hila protuberant, thickened and darkened. Sexual morph not observed., velvety, flat, margin slightly irregular and fimbriate; reverse black to brownish-orange (5C4); soluble pigment brown (6E6) present in cultures between 30–37 °C. On PCA, reaching 67 mm diam., olive grey (3D2), slightly floccose at the centre, flat, margin regular and whitish; reverse olive grey (3D2), whitish towards periphery. On OA, reaching 64 mm diam., olive grey (3F2), slightly floccose at the centre, flat, margin regular and whitish; reverse olive grey (3F2). Scarce sporulation on the three media.(7 d at 25 °C). Colonies on Optimum 25–30 °C, maximum 37 °C, minimum 5 °C.USA.D.A. Sutton .USA, South Carolina, from human sphenoid sinus, 2008, Curvulariasuttoniae is included in a well-supported clade with C.petersonii and C.vietnamensis, the latter also described here. Although the three species are clearly differentiated phylogenetically   than those of C.suttoniae (8–22 × 5–9 µm). In addition to these morphological features, gapdh sequences easily distinguish the two latter species.lly Fig. , they caµm wide)  and, in Taxon classificationFungiPleosporalesPleosporaceaeIturrieta-González, Gené & Dania Garcíasp. nov.1C8CCD40-8C4C-5E77-A738-3C8970080434833027Name refers to the country where the species was collected.J. Guarro .Vietnam, north-east region, on an unidentified dead leaf, Aug 2011, PDA at 25 °C). Mycelium composed of branched, septate, subhyaline to pale brown, thin and smooth-walled to verruculose hyphae, 2–4 μm wide. Conidiophores macronematous, mononematous, septate, straight or flexuous, sometimes slightly geniculate at upper part, unbranched to slightly branched, smooth to verruculose, pale brown to brown, 11–136(–194) × 3–6 μm (av. (±SD) 92.2 (±72.86) × 4.21 (±0.85)). Conidiogenous cells terminal or intercalary, mono- or polytretic, proliferating sympodially, pale brown, with darkened scars, subcylindrical to swollen, 3–7 μm wide. Conidia (1–)3(–4)-septate, curved, with the third cell from base unequally enlarged, some apically bifurcate, smooth-walled, apical and basal cells pale brown, middle cells brown, 15–28 × 5–12 μm (av. (±SD) 21.38 (± 3.44) × 9.34 (±1.83)); hila slightly protuberant, thickened and darkened. Sexual morph not observed., final edge white, umbonate, densely floccose, margin regular; reverse grey (29F1), final edge pale grey (1B2). On PCA, reaching 58 mm diam., olive grey to grey (3F2/3B1), slightly floccose at the centre, margin regular, final edge whitish; reverse olive grey to grey (3F2/3B1). On OA, reaching 74 mm diam., olive (2F3) slightly floccose at the centre, margin regular, flat; reverse olive to greenish-grey (2F3/1C2). Sporulation abundant mainly on PCA and OA.(7 d at 25 °C). Optimum 30 °C, maximum 37 °C, minimum 15 °C.Indonesia and Vietnam.Sorghum seed, 1948, J. van der Vecht (CBS 188.48 = FMR 11956).Indonesia, from C.suttoniae described above.See Curvularia is currently a well-delineated genus on the basis of molecular data  has been used to study the species diversity in Curvularia and phylogentic relationships with other similar genera  than C.suttoniae (8–22 µm) and C. vietnamensis (15–28 µm) and it differs from C.paraverruculosa by its smooth-walled conidia.The Thailand , is alsoCurvularia identification, two species were recently characterised exclusively, based on morphological data and host association, i.e. C.tremae on living leaves of Tremaorientalis (martyniicolaC  on Martyniaannua (Curvulariatremae produces up to 4-septate and larger conidia (average length 152.21 μm and 67.75 μm wide at the broadest part) than those described here. Despite the conidia being mostly 3-sepetate, as in our species, C.martyniicola differs by having longer conidiophores (95–200 μm) than those of C.paraverruculosa (19–85(–145) μm) and C.suttoniae (43–103 μm) and by larger conidia (25–45 × 10–15 μm) than those observed in C.vietnamensis (15–28 × 5–12 μm).Despite the fact that DNA sequence analysis is currently mandatory for ientalis  and C maiaannua , both fr


Bacillus licheniformis WX-02. Firstly, the expression and secretion of PgdS were regulated through engineering its expression elements , which generated γ-PGAs with molecular weights ranging from 6.82 × 104 to 1.78 × 106 Da. Subsequently, through combination of promoters with signal peptides, the production of γ-PGAs with a specific molecular weight could be efficiently obtained. Interestingly, the γ-PGA yield increased with the reduced molecular weight in flask cultures . Finally, in batch fermentation, the highest yield of γ-PGA with a weight-average molecular weight of 7.80 × 104 Da reached 39.13 g/L under glutamate-free medium. Collectively, we developed an efficient strategy for one-step production of γ-PGAs with specific molecular weights, which have potential application for industrial production of desirable γ-PGAs.Poly-γ-glutamic acid (γ-PGA) is an emerging biopolymer with various applications and γ-PGAs with different molecular weights exhibit distinctive properties. However, studies on the controllable molecular weights of biopolymers are limited. The purpose of this study is to achieve production of γ-PGAs with a wide range of molecular weights through manipulating the expression of γ-PGA depolymerase (PgdS) in  Bacillus species  (4 to over 2.0 × 106 Da (5 Da) can efficiently remove basic dyes from solution (4–2.7 × 105 Da) can be used as the drug carrier and tissue engineering nanocomposite in the biomedical industry (Poly-γ-glutamic acid (γ-PGA), an anionic polymer composed of repeated D- and L-glutamic acid units via γ-amide linkages, is mainly produced by faciens) . Due to faciens) . Notably× 106 Da . The γ-P× 106 Da . For exa× 106 Da . The medsolution . Low-Mw industry . Thus, tPreviously, efforts have been made to change the Mws of γ-PGAs through changing fermentation conditions . Also, s4–8.5 × 106 Da) were achieved in B. subtilis via expression of different γ-PGA synthetases -ori were used to construct the expression and deletion vectors, respectively. All strains were generally cultivated at 37°C in Luria Bertani (LB) medium. When necessary, antibiotics  were supplemented into the media.All strains and plasmids employed in this work were listed in B. licheniformis cells were precultured in LB medium and incubated at 37°C for 12 h. For γ-PGA fermentation, 250 mL flasks containing 50 mL medium  were inoculated with 3% (v/v) seed cultures and cultivated at 37°C.For the seed cultures, B. licheniformis cells grow in 1-L bioreactor  with 0.60 L of media for 10 h. The aeration rate was 1.0 vvm, and the stirring speed was 300 rpm. The volume fractions of exhausted O2 and CO2 were measured by exhaust gas analyzer, and the volumetric mass transfer coefficient were calculated by the exhaust gas analysis system   .pgdS was further verified by diagnostic PCR and DNA sequencing.Batch fermentation was carried out in the 3-L bioreactor  containing 1.8 L media with an aeration rate of 1.0 vvm. The agitation speed was starting to set at 300 rpm and increased to 600 rpm after 12 h. Samples were collected periodically to analyze the cell growth, glucose, and γ-PGA concentrations. ΔB. subtilis 168, signal peptide of levansucrase SacB (SPsacB), gene pgdS  and amyL terminator of B. licheniformis WX-02 were amplified and fused by overlapping PCR to obtain the expression cassettes  was described. Briefly, P43 promoter of assettes . The expg, 20 min). Enzyme activity was measured as described by 6 Da γ-PGA substrate, 2 μM dithiothreitol and 10 μL of crude enzyme solution, and was incubated at 37°C for 4 h. After stopped, 100 μL of reaction solution was withdrawn and mixed with 100 μL of 0.2 M borate buffer (pH 8.5) and 20 μL of 10 mmol/L fluorodinitrobenzene . The hydrolysis of FDNB-modified fragments and the assay of dinitrophenyl glutamate monomers (DNPGlu) were performed as described previously . The precipitate was washed with absolute alcohol, and re-dissolved in a 100 uL solution which containing 2 mol/L thiourea and 8 mol/L urea. Protein concentrations were quantitated by Bradford assay  was used as a protein standard. Briefly, 1 mL fermentation broth was centrifuged at 10000 × rd assay  and BSA rd assay . The gelrd assay , and the®II Q RT SuperMix for qPCR (+gDNA wiper)  was employed for cDNA synthesis. The real-time PCR was performed using iTaqTM Universal SYBR® Green Supermix . The experiments were performed in three replicates, and 16S rRNA was used as the reference gene  was measured using the 752N spectrophotometer . The glucose concentration was determined by SBA-40C bioanalyzer .The cell density  among the SP12–SP18 strains, except the control strain SP01 and SP11 (carrying cassette with the combination of P43-SPsacC). Among these strains, the secretion efficiency ratios were SPsacB > SPyvpA > SPpgdS > SPaprE > SPggt > SPbprA > SPvpr > SPsacC (4 to (1.71 ± 0.08) × 106 Da, compared with the control strain (1.99 ± 0.11) × 106 Da. Interestingly, cell growth showed no significant differences between the recombinant strains and control strain SP01. Therefore, our results indicated that γ-PGA with a wide range of Mws (6.82 × 104–1.71 × 106 Da) could be achieved via modulating the secretory capability of PgdS.In our previous study, we have construct SP library in reporter . We choornatants  showed a> SPsacC . As indi> SPsacC . The MwspgdS on Mws by using promoters with different strength. Four promoters P43, PpgdS, PbacA, and PbprA were applied to drive the transcription of pgdS. The four recombinant vectors harboring the signal peptide BprA were constructed and transformed into the WX-02ΔpgdS, generated four recombinant strains SP13, SP23, SP33, and SP43, respectively   . The exp < 0.05) , and the < 0.05) , the res5, (9.55 ± 0.46) × 105, (14.41 ± 0.67) × 105, and (17.80 ± 0.87) × 105 Da, respectively (5 Da). However, the cell growth of these four recombinant strains were approximately the same as that of SP01. Collectively, our results showed that an apparent reduction of Mws along with the enhanced promoter strength.After 36 h cultivation, we measured the Mws and yields of γ-PGA produced by these recombinant strains. The Mws of the polymer produced by SP13, SP23, SP33, and SP43 were (7.78 ± 0.23) × 10ectively , which dpgdS. The transformed strains were designated as B. licheniformis SP21, SP25, SP28, SP31, SP35, SP38, SP41, SP45, and SP48, respectively.In order to further boost the performance, the promoter and signal peptide were combined to regulate PgdS expression. Three promoters  and signal peptides  were selected and combined to form nine expression cassettes . Every e4 to (1.91 ± 0.08) × 106 Da. To assess the relationship between PgdS expression and γ-PGA Mws, the Pearson correlation coefficients were calculated (P < 0.01) (As shown in lculated . A corre < 0.01) . Taken tIn this study, we achieved the production of low-, medium-, and high-Mw γ-PGA in the same microbial chassis. The effect of PgdS expression levels on γ-PGA yield was further studied . We founB. licheniformis SP01 , SP12 , SP14 , and SP18  were compared in 1-L fermenter , which is proportional to the volumetric mass transfer coefficient and substrate utilization. The OTR plays vital roles in the growth of strains and the production of target metabolites . We hypoermenter . All rec strains . These r4–1.99 × 106 Da) was developed by regulating PgdS expression. Therefore, in order to further explored the applicability of this system in large-scale γ-PGA production, the engineered strains SP01, SP12, SP14, and SP18 were carried out in a 3-L bioreactor, respectively  has been characterized from various strains  and usedlP tool1 . The eff99 FU/mL . In this99 FU/mL . Besides99 FU/mL . In thispeptides , which ipeptides .pgdS transcriptional levels on γ-PGA Mws using promoters with different strengths . P43 is considered as a strong promoter for protein expression  × 106 Da]. Based on these results, regulating PgdS expression was an efficient and simple method to obtain γ-PGAs with specific Mws. Compared to other methods, we could achieve broader range of Mws of γ-PGAs, which is beneficial to broaden the industrial application of γ-PGAs. Previous studies have mainly focused on increasing the production of high-Mw γ-PGAs (n = 9). We will achieve precise control of Mws via the titratable regulation of PgdS expression using the further development of tools.In order to further evaluate the effect of regulated PgdS expression on the Mws of γ-PGAs, we explored various levels of PgdS expression by combining promoters and signal peptides. Through this strategy, the combined strains provided γ-PGA with a wide range of Mws [(7.74 ± 0.80) × 10w γ-PGAs , limitinpgdS gene produced higher γ-PGA yields compared with the control strain SP01, which confirmed that overexpression of PgdS is an efficient approach to enhance γ-PGA production. Due to the high viscosity of high-Mw γ-PGAs, it severely decreases oxygen transfer in the fermentation process, resulting in inhibition of cell growth and limit of γ-PGA production  without additional glutamic acid using the recombinant strains in 3-L fermenter. The results from this work and related studies were summarized in roducers . MoreoveB. licheniformis for the first time. The ability to produce γ-PGA with low, medium, and high Mws ranging between 6.82 × 104 Da and 1.99 × 106 Da was demonstrated by manipulating the promoter and signal peptide independently and in combination. The maximum production of γ-PGA  reached 39.13 g/L from glutamate-free medium in batch fermentation. Our study presented a potential method for commercial production of specific-molecular-weight γ-PGA, and this strategy could also be used to produce other biopolymers by precisely controlling corresponding depolymerase expression.In conclusion, we developed an efficient system for one-step production of specific Mws γ-PGA through regulating the PgdS expression in All datasets generated for this study are included in the article/SC and FS designed and supervised the study. HW, DW, YX, JD, and JC performed the experiments. DW and HW analyzed the data and wrote the manuscript. YZ, QW, DC, SC, and FS revised the manuscript. All authors read and approved the final 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.


Myzus persicae and antiproliferative activity against four selected cancer cell lines are reported. Products were obtained in good yields (19–96%) and their structures were fully characterized by spectroscopic data . Four synthesized δ-halo-γ-lactones (4–7) are new and have not been previously described in the literature. In the choice test phytol (1) appeared deterrent to M. persicae, whereas modifications of its structure did not cause the avoidance of the treated leaves by the aphids. In contrast, aphids were attracted to the leaves treated with the new trans-δ-chloro-γ-lactone (6). Electrical Penetration Graph (EPG) technique applied to explore the aphid probing and feeding activity revealed that neither phytol nor lactone 6 affected aphid probing and the consumption of phloem sap, which means that both phytol and the lactone 6 might have acted as postingestive modifiers of aphid behavior. The results of in vitro antitumor assays showed that obtained phytol derivatives exhibit cytotoxic activity against studied cancer cell lines . Halolactones 4–6 were identified as the compounds, which arrest cell cycle of leukemia cells mainly in G2/M and S phases.The synthesis of phytol-derived γ-butyrolactones as well as their evaluation for deterrent activity towards peach-potato aphid  This type of molecules is produced by algae, sponges, fungi and liverworts4. Some of them have been also isolated from Streptomyces and Hortonia species6. γ-Butyrolactones determine the aroma and taste of fruits, vegetables and cheese10 and are valuable fragrance compounds of high-quality alcohol drinks such as wine, sherry, and whisky13. Among biological activities that γ-lactones show, to the fore comes extensively described in the literature their significant role which they play in the world of insects and the role of anticancer agents. This type of molecules is responsible for effective sexual signaling as sex attractant pheromones between different species of insect14, exhibits good antifeedant activity against insects15 and is also well-known as strong antitumor, antibiotic, antifungal and antiviral agents16.Mono-, di- and trisubstituted γ-butyrolactones are crucial structural elements of many biologically active natural and synthetic compounds. It has been estimated that almost 10% of natural products contain in their structure a γ-butyrolactone ring22, for which it has been observed that their strong deterrent activity is mostly related to presence in their structure bromine or chlorine atom. For example, δ-halo-γ-lactones have been demonstrated as the most active lactone derivatives of damascone with strong antifeedant activity able to induce significant changes in the peach-potato aphid Myzus persicae behavior23. While β-damascone appeared a behaviorally inactive compound, the transformation of β-damascone into δ-bromo-γ-lactone caused frequent interruptions in the probing activity in peripheral plant tissues and a reduction in the ingestion of phloem sap20. Likewise, in the case of lactones with p-menthane system, the halolactones derived from piperitone had a negative effect on probing, feeding, and settling behavior of M. persicae, which was in contrast to the natural compound that showed weak attractant properties24. In some cases, like for racemic δ-halo-γ-lactone synthesized from farnesol it was shown that γ-butyrolactones are also able to inhibit the growth of cancer cell lines, A549 (human lung adenocarcinoma) and HL-60 (human promyelocytic leukemia)23.Especially valuable γ-butyrolactones are those which structure is based on the isoprenoid skeleton. These lactones isolated from plants constitute one of the most numerous groups of antifeedants. However, their practical application is economically unjustified in the context of their low concentration in natural sources as well as high costs and difficulties with their isolation. From practical point of view more accessible are their synthetic analogs, like previously reported lactones synthesized from pulegone, limonene, myrtenol, pinene, damascone or farnesol1). Our interest in phytol (1) was inspired by its easy availability and reports on its broad spectrum of biological activity. PYT is an acyclic monounsaturated diterpene alcohol widely distributed in the plant world but can be also found in algae26, in bacteria28, in gut of the ruminant’s animals and their dairy products29. Biosynthesis of PYT goes mainly on the mevalonic acid (MVA) pathway30 or 2-C-methylerythritol-4-phosphate (MEP) pathway28. Phytol (1) plays an important role in the plant world being a structural element of chlorophyll responsible for its anchoring to the thylakoid membrane of chloroplast31. It is a cost-effective chemical widely used in the cosmetics industry as a fragrance component and well known as a non-mutagenic food additive. The world use of PYT reaches 0.1–1 metric ton per year and it’s a non-toxic effect for the mammalian was evaluated in the studies on albino mice and rats. The calculated lethal doses (LD50) of PYT for them are above 5 g and 10 g/kg, respectively32.Therefore, looking among isoprenoids for a biologically active natural carbon skeleton that could be the basis structure for developing its active lactone derivatives which can be useful as a new type of insecticides and cytostatic molecules we selected phytol  (PYT)  exhibits cytotoxic potential against certain cancer cell lines: leukemia (MV4-11 and HeLa), breast (MCF-7), prostate (PC-3) and lungs (A-549)39 and is the substance promising for the treatment of cancer.PYT is known to exhibit insecticidal activities against 1) and evaluate them as the antifeedant and anticancer agents. We synthesized a series of novel PYT derivatives and evaluated their feeding-deterrent activity against the peach-potato aphid Myzus persicae (Sulz.) (Hemiptera: Aphididae) and antiproliferative activity against four selected cancer cell lines. The influence of novel PYT derivatives on aphid activities was assessed by monitoring the settling behavior of freely moving aphids in choice situation. We also monitored the individual phases of aphid probing and feeding in the no-choice situation using the Electrical Penetration Graph (EPG) technique which visualizes the movements of aphid mouthparts within individual plant tissues. The antiproliferative activity of synthesized phytol derivatives was evaluated against selected cancer cell lines  whereas their molecular mechanism of action including of cell cycle, cell death evaluation and their influence on the activity of caspase 3/7 were studied towards human biphenotypic B myelomonocytic leukemia cell line MV4-11.The low and well-characterized toxicity of PYT and its high tolerance by mammals makes this compound a strong candidate for the development of new and environmentally friendly insecticides and cytostatic agents. Therefore, the aim of this work was to obtain more biologically active halolactone derivatives of phytol (cis/trans (35%:65%) isomers of phytol (1) (PYT) (97% purity), N-bromosuccinimide  and N-chlorosuccinimide  were purchased from Sigma-Aldrich Chemical Co. , while trimethylortoacetate was purchased from Fluka. Analytical grade acetic acid, sodium hydrogen carbonate, acetone, hexane, diethyl ether, tetrahydrofuran (THF), anhydrous magnesium sulfate, sodium chloride were purchased from Chempur (Poland).Racemic mixture of 4)2 and 2% H3[P(Mo3O10)4] (2 g) in 10% H2SO4, followed by heating to 120–200 °C.Analytical Thin Layer Chromatography (TLC) was carried out on silica gel coated aluminium plates  with a mixture of hexane, acetone and diethyl ether in various ratios as the developing systems. Compounds were visualized by spraying the plates with solution of 1% Ce using a mixture of hexane, acetone, and diethyl ether (in various ratios) as eluents.Gas chromatography (GC) analysis was carried out on an Agilent Technologies 6890 N Network GC instrument  equipped with autosampler, split injection (20:1) and FID detector using a DB-5HT column   with hydrogen as the carrier gas. Products of the chemical reactions were analysed using the following temperature programme: injector 250 °C, detector (FID) 250 °C, initial column temperature: 100 °C, 100–300 °C (rate 30 °C/min), final column temperature 300 °C (hold 2 min).1H NMR, 13C NMR, DEPT 135, HSQC, 1H–1H COSY and NOESY were recorded in CDCl3 solutions with signals of residual solvent (δH = 7.26 δC = 77) on a Brüker Avance II 600 MHz  spectrometer.Nuclear magnetic resonance spectra High-resolution mass spectra (HRMS) were recorded using electron spray ionization (ESI) technique on spectrometer Waters ESI-Q-TOF Premier XE .2 and acid 3 has been illustrated in detail in our previous work39, and so the synthesis method would not be listed here.The preparation of ester 3 (7.8 mmol) in THF (30 mL) the N-bromosuccinimide (7.8 mmol) or N-chlorosuccinimide (7.8 mmol) was added. The mixture was stirred at room temperature for 48–96 h. When the substrate reacted completely  the mixture was diluted with diethyl ether and washed with saturated NaHCO3 solution and brine. Organic layer of ether extract was separated and dired over anhydrous magnesium sulfate and evaporated on a rotary evaporator. New δ-halogeno-γ-lactones (4–7) were separated by silica gel column using for elution hexane/diethyl eter in gradient system. Bromo- and chlorolactonization afforded products with the following physical and spectral data presented below:To a solution of acid 1H NMR : δ 0.85 2–12′), 1.05–1.55 , 1.24 , 2.30 and 2.60 , 3.47 , 3.55 , 4.39 ; 13C NMR : δ 19.61, 19.69 (CH3)2–12′), 22.65, 22.75 , 24.50 (CH3-4), 29.21 (CH2-Br), 41.49 (CH2-3), 42.86 (C-4), 22.00, 24.47, 24.82, 34.08, 37.28, 37.41, 37.61, 37.70, 39.38 , 28.00, 32.69, 32.80 , 87.66 (H-5), 174.92 (C-2); HRMS (ESI): m/z calcd. for C22H41BrO2 [M + Na]+ 439.2188; found 439.2182.(25% yield); 1H NMR : δ 0.85 2–12′), 1.04–1.55 , 1.08 , 2.38 and 2.49 , 3.48 , 4.41 ; 13C NMR : δ 18.96 (CH3-4), 19.69, 19.76 (CH3)2–12′), 22.65, 22.75 , 29.17 (CH2-Br), 42.70 (CH2-3), 43.09 (C-4), 22.20, 24.46, 24.82, 37.28, 37.39, 37.41, 37.49, 39.38, 39.96 , 28.00, 30.95, 32.72 , 86.46 (H-5), 174.76 (C-2); HRMS (ESI): m/z calcd. for C22H41BrO2 [M + Na]+ 439.2188; found 439.2183.(46% yield); 1H NMR : δ 0.84 2–12′), 1.03–1.54 , 1.23 , 2.23 and 2.60 , 3.67 , 3.73 , 4.33 ; 13C NMR : δ 19.61, 19.67 (CH3)2–12′), 22.65, 22.75 , 24.67 (CH3-4), 42.30 (CH2-Cl), 41.48 (CH2-3), 42.38 (C-4), 22.09, 24.46, 24.82, 34.21, 37.29, 37.39, 37.61, 37.70, 39.38 , 28.00, 32.70, 32.80 , 87.45 (H-5), 175.21 (C-2); HRMS (ESI): m/z calcd. for C22H41ClO2 [M + Na]+ 395.2693; found 395.2698.(21% yield); 1H NMR : δ 0.85 2–12′), 1.04–1.56 , 1.06 , 2.38 and 2.47 , 3.67 , 4.35 ; 13C NMR : δ 19.06 (CH3-4), 19.69, 19.76 (CH3)2–12′), 22.65, 22.74 , 42.52 (CH2-Cl), 42.39 (CH2-3), 42.54 (C-4), 22.14, 24.46, 24.83, 37.28, 37.37, 37.41, 37.49, 39.38, 40.16 , 28.00, 32.64, 32.80 , 86.24 (H-5), 175.04 (C-2); HRMS (ESI): m/z calcd. for C22H41ClO2 [M + Na]+ 395.2693; found 395.2697.(39% yield); Myzus persicae (Sulzer) and the Chinese cabbage Brassica rapa subsp. pekinensis (Lour.) Hanelt were reared in laboratory at 20 °C, 65% r.h., and L16:8D photoperiod. One to seven days old apterous females of M. persicae and 3-week old plants with 4–5 fully developed leaves were used for experiments. M. persicae were obtained from the laboratory culture maintained at the Department of Botany and Ecology for many generations since 2000. All experiments were carried out under the same conditions of temperature, relative humidity, and photoperiod. The bioassays were started at 10–11.a.m. Each compound was dissolved in 70% ethanol to obtain the recommended 0.1% solution40. All compounds were applied on the adaxial and abaxial leaf surfaces by immersing a leaf in the ethanolic solution of a given compound for 30 s.20. Control leaves of similar size were immersed in 70% ethanol that was used as a solvent for the studied compounds. Experiments were performed 1 h after the compounds application to allow the evaporation of the solvent. Every plant and aphid were used only once.The peach potato aphids 41. In the present study, aphids were given free choice between control and treated excised leaves that were placed in a Petri dish. Aphids were placed in the dish equidistance from treated and untreated leaves, so that aphids could choose between treated  and control leaves . Aphids that settled, i.e. they did not move, and the position of their antennae indicated feeding, on each leaf were counted at 1 h, 2 h, and 24 h intervals after access to the leaf. Each experiment was replicated 8 times . Aphids that were moving or not on any of the leaves were not counted.This bioassay allows the study of aphid host preferences under semi-natural conditionsM. persicae was monitored using the technique of electronic registration of aphid probing in plant tissues, known as EPG , that is frequently employed in insect–plant relationship studies considering insects with sucking-piercing mouthparts44. In this experimental set-up, aphid and plant are connected to electrodes and thus made parts of an electric circuit, which is completed when the aphid inserts its stylets into the plant. Weak voltage is supplied in the circuit, and all changing electric properties are recorded as EPG waveforms that can be correlated with aphid activities and stylet position in plant tissues46. The parameters describing aphid behaviour during probing and feeding, such as total time of probing, proportion of phloem patterns E1 and E2, number of probes, etc., are good indicators of plant suitability or interference of probing by chemical or physical factors in individual plant tissues46. In the present study, aphids were attached to a golden wire electrode with conductive silver paint (epgsystems. eu) and starved for 1 h prior to the experiment. Probing behaviour of 12 apterous females/studied compound and control was monitored for 8 h continuously with Giga-4 and Giga-8 DC EPG with 1 GΩ of input resistance recording equipment . Each aphid was given access to a freshly prepared plant and each aphid/plant combination was used only once. Various behavioural phases were labelled manually using the Stylet + software (www.epgsystems.eu). The following aphid behaviours were distinguished: no penetration (waveform 'np' – aphid stylets outside the plant), pathway phase—penetration of non-phloem tissues (waveforms 'ABC'), phloem phase , and xylem phase . Waveform ‘G’ occurred rarely irrespective of the treatment. Therefore, in all calculations the xylem phase was added to the pathway phase and termed as probing in non-phloem tissues. The E1/E2 transition patterns were included in E2. Waveform patterns that were not terminated before the end of the experimental period (8 h) were not excluded from the calculations. The parameters derived from EPG recordings were analyzed according to their frequency and duration in configuration related to activities in peripheral and vascular tissues.Aphid probing and the phloem sap uptake by p < 0.05), the compound was described as having attractant properties. If aphids settled mainly on the control leaf (p < 0.05), the compound tested in the respective choice-test was stated a deterrent. From the data thus obtained the relative index of deterrence (DI) was calculated: DI = (C − T/C + T) where C was the number of aphids settled on control leaf, T was the number of aphids settled on the leaf treated with the tested compound. The value of DI ranged between 1  and − 1 41.The data of the choice-test were analyzed using Student’s t-test (STATISTICA 13.1. package). If aphids showed clear preference for the leaf treated with the tested compound  STATISTICA , version 12, 014 STATI47.Human biphenotypic B myelomonocytic leukemia MV4-11, human colon cancer LoVo cell line and normal mouse fibroblast BALB/3T3 cells were obtained from American Type Culture Collection , human lung carcinoma A549 cells were obtained from European Collection of Authenticated Cell Cultures (UK). All the cell lines are being maintained at the Hirszfeld Institute of Immunology and Experimental Therapy, PAS, Wroclaw, Poland and were cultured according the procedure described before48. MV4-11 cells were cultured in RPMI 1640 medium  with 1.0 mM sodium pyruvate, and 10% fetal bovine serum (FBS) . A549, LoVo and LoVo/DX cells were cultured in RPMI 1640 + Opti-MEM (1:1)  supplemented with 5% fetal bovine serum, 1.0 mM sodium pyruvate (LoVo and LoVo/DX cells)  and 0.1 μg/mL doxorubicin chloride (Accord) (only LoVo/DX cells). BALB/3T3 cells in Dulbecco medium  supplemented with 10% fetal bovine serum . All culture media were supplemented with 2 mM l-glutamine , 100 units/mL penicillin, and 100 µg/mL streptomycin . All cell lines were grown at 37 °C with 5% CO2 humidified atmosphere.Cell lines were cultured according to the protocol described before4 or 0.5 × 104 (A549) cells per well. The assay was performed after 72 h of exposure to 625, 125, 25 and 5 μM of the tested agents. The in vitro cytotoxic effect of all agents was examined using the MTT (MV4-11) or SRB assay, described previously47. The results were calculated as an IC50 (inhibitory concentration 50%) the concentration of tested agent, which is cytotoxic for 50% of the cancer cells. IC values were calculated for each experiment separately and mean values ± SD are presented in Tables The solutions of the compounds (50 mM) were prepared by dissolving the substances in DMSO . Then the tested compounds were diluted in culture medium to reach the final concentrations of 625, 125, 25 and 5 μM. Before adding of the tested compounds (24 h prior), the cells were plated in 96-well plates  at a density of 1 × 1048. The MV4-11 cells were seeded at the density of 1 × 105 cells/well of culture medium on 24-well plates  to the final volume of 2 mL and were exposed to the test compounds at concentrations 75 μM for 72 h. After incubation, the cells were collected and 1 × 106 of cells were washed twice in cold PBS and fixed for 24 h in 70% ethanol at − 20 °C. Then the cells were washed twice in PBS and incubated with RNAse  at 37 °C for 1 h. The cells were stained for 30 min. with propidium iodide  at 4 °C and the cellular DNA content was analyzed by flow cytometry using BD LSRFortessa cytometer . Compounds at each concentration were tested at least three times independently. Obtained results were analyzed using Flowing software 2 .Cell cycle analysis was carried out according to the procedure previously described48. The MV4-11 cells were seeded at the density of 1 × 105 cells/mL of culture medium on 24-well plates  to the final volume of 2 mL. The cells were exposed to the test compounds at concentrations 75 μM or campthothecin (0.05 μg/mL) as a positive control, for 72 h. After 72 h of the incubation, the cells were collected and centrifuged . Cells were suspended in 50 µL of ice-cold lysis buffer  sucrose, 150 mM NaCl, 2 mM EDTA, 1% (v/v) Triton X-100, pH 7.3, IIET, Poland) and incubated 30 min. at 4 °C. After the incubation, 40 µL of each sample was transferred to a white, 96-well plate  containing 160 µL of the reaction buffer   with 9 µM Ac-DEVD-ACC fluorogenic substrate . The fluorescence increase correlated with the caspase-3/7 level was continuously recorded at 37 °C for 120 min using a Biotek Synergy H4 . Compounds were tested in duplicates in single experiment and each experiment was repeated at least three times independently. Results were normalized to the number of cells in each well and are reported as mean relative caspase-3/7 activity compared to untreated control sample ± SD.Caspase-3/7 activity determination was performed according to the protocol previously describedp Values lower than 0.05 were considered as statistically significant.Statistical analysis was performed in Statsoft Statistica 10. All datasets were analyzed using t-test. cis/trans (35% : 65%) isomers of phytol (1) (PYT) (97% purity). This natural allylic alcohol (1) was subjected in the first step of the synthesis to the Johanson-Claisen rearrangement using triethylorthoacetate in the presence of a catalytic amount of propionic acid. The reaction afforded known ethyl 3,7,11,15-tetramethyl- 3-vinylhexadecanoate (2) in 86% yield, which was subsequently hydrolyzed in ethanolic KOH solution to the corresponding γ,δ-unsaturated acid (3) in 96% yield39. The acid 3 was next the substrate in the halolactonization reactions, which were carried out in tetrahydrofuran with N-bromosuccinimide or N-chlorosuccinimide, respectively and afforded four novel δ-halo-γ-lactones (4–7).The synthesis of phytol-derived γ-butyrolactones is outlined in Fig. 4, 5) in a ratio of 28% : 72% according to GC (after 2 days). We separated them using column chromatography and their structures were established based on spectroscopic methods. The products of cyclization were trans-δ-bromo-γ-lactone (4)  and cis-δ-bromo-γ-lactone (5) . The lactones were obtained in 25 and 46% yield, respectively. The reaction of γ,δ-unsaturated acid (3) with N-chlorosuccinimide (NCS) was carried out to obtain chlorolactones . Using chloride as an electrophilic agent, we observed a similar situation as in the process of bromolactonization, but this time the reaction proceeded longer, 4 days. Two new δ-chloro-γ-lactones (6) and (7) were formed as products of cyclization. According to GC, the reaction mixture consisted 31% of trans-δ-chloro-γ-lactone (6) and 69% of cis-δ-chloro-γ-lactone (7), which were obtained in 21 and 39% yield, respectively. The structures of halolactones 4–7 were confirmed by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. Correlation spectroscopy (COSY), heteronuclear single quantum correlation spectroscopy (HSQC), nuclear overhauser effect spectroscopy (NOESY) and mass spectra (HRMS) were also applied for this purpose . A considerable difference can be seen in the analysis of the position of the signal from the carbon atom C-6 in the 13C NMR spectra of bromo- and chlorolactones. The signals from C-6 carbon atom on bromolactones spectra are present at 29.21 and 29.17 ppm, respectively and at 42.30 and 42.52 ppm in the case of chlorolactones.5 and 7 correlation signals confirmed the coupling of protons of halogenomethyl group with protons methyl group at C-4, indicating their close location in space (cis to each other). These correlations were not observed in the spectra of 4 and 5 made in NOESY technique. Formation of higher amounts of trans-lactones 5 and 7 where in their structure is trans location of halogenomethyl substituent and isoprenoid chain at C-4 can be explained by the preferential formation of bromonium/chloronium ion from the less shadowed side of double bound it means from the side of the methyl group, not a isoprenoid substituent. Carboxylate ion, according to the mechanism of halolactonization, formed a lactone ring, reacting from the side opposite to the bromine/chlorine atom.The spatial orientation of the halogenomethyl groups of synthesized bromo- and chlorolactones relative to the isoprenoid chain at carbon atom C-4 has been confirmed by spin–spin coupling through the space on the spectrum made in NOESY technique. Observed in the spectra of lactone 49. Therefore, the number of aphids that settle and feed on a given substrate is a good indicator of its suitability. Exogenously applied substances may alter plant suitability to aphids50. In the present work, we determined the settling success of the peach potato aphid after the application of phytol (1) and its newly obtained derivatives (2–7). The settling preferences of aphids depended on the compound and the time after the exposure of plants to the compound applied. The application of phytol (1) caused an initial acceptance of the treated leaves by M. persicae but from the second hour after application onwards, aphids tended to prefer the untreated to the treated leaves  to M. persicae. Such switches in aphid responses in the course of time have previously been reported for other substances, such as piperitone-derived halolactones and hydroxylactones51. In contrast, aphids preferred the leaves treated with the new trans-δ-chloro-γ-lactone 6, which indicates the attractant properties of this lactone to M. persicae. The aphid responses to phytol and lactone 6 were the strongest and statistically significant 24 h after exposure. The phytol derivatives 2–5 and 7 did not affect aphid settling behavior significantly. However, aphids showed a detectable tendency to avoid the leaves treated with the new γ,δ-unsaturated acid 3 and two new δ-bromo-γ-lactones 4 and 5 and prefer the leaves treated with the new cis-δ-chloro-γ-lactone 7 , probing in non-phloem tissues (35%), and probing in phloem tissues (55%). Sap ingestion occupied 98% of the phloem phase. Aphid probing activities were divided into 24.2 (± 4.8) probes on average and these probes were approximately 0.7 (± 0.3) hours long. M. persicae needed approximately 2.1 (± 0.4) hours and 14.1 (± 3.7) probes to reach phloem vessels and commence sap ingestion. 8.8 (± 2.5) of the probes before the first phloem phase were short eipdermal probes. The first contact with sieve elements (the first phloem phase) was 1.8 (± 0.7) hours long , ingestive , and postingestive (long-term effects involving various aspects of digestion and absorption of food)2–7 in the in vitro model have been evaluated in MV4-11 (human biphenotypic B myelomonocytic leukemia) cell line using 3--2,5-diphenyl tetrazolium bromide (MTT) assay and A549 (human lung adenocarcinoma), LoVo (colon cancer) and LoVo/DX (doxorubicin resistant subline) cell lines using sulforhodamine B (SRB) assay. The toxicity of these compounds was also tested towards the normal mouse fibroblasts BALB/3T3. Cisplatin and doxorubicin were used as the positive controls and the results expressed as half-maximal inhibitory concentration (IC50) values are summarized in Table The cytotoxic activities of the target compounds 1). According to the values summarized in Table 1–7). The phytol derivatives 2–7 were active towards leukemia at concentration range of 39.28–110.5 μM. In this group the highest activity was reported for 3,7,11,15-tetramethyl- 3-vinylhexadecanoic acid (3) and for trans-δ-bromo-γ-lactone (4) with IC50 = 39.28 and 46.9 μM, respectively. Considering the effect of phytol derivatives on the normal cells the potential application exhibit mainly δ-halo-γ-lactones (4–6) for which strong selectivity action towards cancer cells was observed. The obtained results of the antiproliferative effect of lactones 4–7 proved some correlation between their structures and cytotoxicity. While the differences in the activity of individual lactones containing bromine or chlorine atoms are almost not noticeable, in the case of orientation of the halogenomethyl group at carbon atom C-5 are clearly visible. trans-δ-Bromo-γ-lactone (4) and trans-δ-chloro-γ-lactone (6) exhibit higher activity than their cis analogues 5 and 7.Cell viability analysis indicates that synthesized derivatives show more pronounced cytotoxic activity than phytol (3 at the concentration 49.5 μM which was slightly lower than this appointed for phytol (1). The proliferation of colon cancer LoVo cells was also inhibited only by acid 3 and the established IC50 value 59.5 μM was 4 times lower than this observed for phytol (1) (IC50 255.2 μM).In contrast to the cytotoxicity of phytol-derived products against MV4-11 cells the proliferation of human lung adenocarcinoma (A549) was inhibited only by acid 1) and compounds 3 and 6 were able to overcome drug resistance (IR < 2).To evaluate agents activity against the MDR (multidrug resistance) phenotype cancer cell line LoVo together with its drug resistant subline LoVo/DX were tested and indexes of resistance (IR) were calculated. The IR value indicates how many times more a resistant subline is chemoresistant relative to its parental cell line. The data presented in Table 50) of each compound against each cancerous cell line with that of the normal fibroblasts BALB/3T3 (Table 4–6) against MV4-11 cells were much higher than for cisplatin. Phytol (1) also demonstrated lower selectivity towards cancer cells than its synthesized derivatives 3–7. The 3,7,11,15-tetramethyl- 3-vinylhexadecanoic acid (3) had efficacy against all tested tumor cells greater than toxicity against normal cells.Many chemotherapeutics are toxic not only against malignant cells but also normal ones. Therefore, a very serious problem is the degree of toxicity of drugs depending on the doses required to achieve response in clinical trials. Selectivity of the cytotoxic activity of the phytol derivatives was determined by comparing the cytotoxic activity . This result is with agreement with result of analysis of caspase 3 activity , whereas ester 2 and trans-δ-chloro-γ-lactone (6) increased activity of caspase about 1.5-times -Val-Asp-7-amino-4-carbamoylmethylcoumarin (Ac-DEVD-ACC). At the concentration of 75 μM the acid -Val-Asp-In summary, a series of phytol-derived compounds were synthesized in good yields and their aphid behavior modifying activity and cytotoxic potency were evaluated.trans-δ-chloro-γ-lactone 6. Both phytol and the chlorolactone 6 acted as postingestive modifiers of aphid behavior. The phytol derivatives 2–5, and 7 did not affect the feeding activity or settling behavior of M. persicae.The observation of freely moving aphids and the electronic monitoring of aphid probing in response to phytol and phytol-derived lactones demonstrated the deterrent potential of phytol and the attractant potential of the new 4–6 were identified as the compounds, which arrest cell cycle of leukemia cells mainly in G2/M and S phases. We have found that the antiproliferative activity of individual lactones depends mainly on the orientation of halogenomethyl group at carbon atom C-5, whereas we did not observe bigger differences in the activity of bromo- and chlorolactones. However, further laboratory research in the area of the synthesis of more derivatives of phytol and broaden studies on their molecular mechanism of action are needed to be able to analyze the structure activity relationships. In the aspect of obtained results so far, the important fact is that calculated selectivity factors of almost all phytol derivatives were higher than those of cisplatin, what makes those products promising for further biological investigations. Intermediate acid 3 derived from phytol effectively inhibits the growth of all four types of tested cancer cells and is pro-apoptotic agent of leukemia cells that acts by activation of caspase-3/7 on the level comparable with camptothecin.Phytol derivatives were also evaluated towards four human cancer cell lines  and normal mice fibroblasts (BALB/3T3). Obtained results confirmed that introduction of carboxy group and lactone ring into the structure of phytol increase its anticancer potential. Halolactones Supplementary Information


Autophagy is a cellular homeostatic pathway with functions ranging from cytoplasmic protein turnover to immune defense. Therapeutic modulation of autophagy has been demonstrated to positively impact the outcome of autophagy-dysregulated diseases such as cancer or microbial infections. However, currently available agents lack specificity, and new candidates for drug development or potential cellular targets need to be identified. Here, we present an improved method to robustly detect changes in autophagy in a high-throughput manner on a single cell level, allowing effective screening. This method quantifies eGFP-LC3B positive vesicles to accurately monitor autophagy. We have significantly streamlined the protocol and optimized it for rapid quantification of large numbers of cells in little time, while retaining accuracy and sensitivity. Z scores up to 0.91 without a loss of sensitivity demonstrate the robustness and aptness of this approach. Three exemplary applications outline the value of our protocols and cell lines: (I) Examining autophagy modulating compounds on four different cell types. (II) Monitoring of autophagy upon infection with e.g. measles or influenza A virus. (III) CRISPR/Cas9 screening for autophagy modulating factors in T cells. In summary, we offer ready-to-use protocols to generate sensitive autophagy reporter cells and quantify autophagy in high-throughput assays. Basal levels of autophagy are required to maintain homeostasis of eukaryotic cells, mediating continuous turnover of organelles and proteins. Autophagy is active in almost all cells of the body. Upon induction of autophagy, cytoplasmic cargo is engulfed by double-layered membranes, which upon elongation and closure form so-called autophagosomes, eventually fusing with lysosomes. Their contents along with the inner membrane are degraded at a low pH by lysosomal peptidases and hydrolases. This process is either specifically (selective autophagy) targeting compounds recognized by so-called autophagy receptors like SQSTM1 (p62) or leads to bulk degradation of parts of the cytoplasm4. Specific autophagy targets are usually earmarked for destruction by ubiquitin chains. Overall, autophagy primarily has a pro-survival, cytoprotective role5. Diverse cellular signaling pathways are regulated by autophagy, including the turnover of key signaling components6. Furthermore, cellular proteins processed by autophagy may lead e.g. to the release of anti-microbial peptides7. In addition, autophagy targets foreign compounds such as viral proteins in the cytoplasm (Xenophagy). Thus, it acts as an innate immune defense pathway10 and eventually provides peptides for display on major histocompatibility complex (MHC) molecules on antigen-presenting cells11.Macroautophagy  is a catabolic cellular process13. Diseases like cancer and neuropathies are characterized by dysregulation of autophagy15. Hereditary diseases like Crohn’s disease are linked to mutations in e.g. autophagy-related protein 16L (ATG16L), a protein necessary for autophagy induction16. Moreover, autophagy is involved in the immune defense system targeting intracellular viruses, parasites, or bacteria and impaired autophagy may be detrimental during microbial infections12. Therefore, there is a growing need to understand the molecular basis of autophagy in human diseases and upon microbial challenge. Importantly, exogenous modulation of autophagy could be key to novel therapies. Indeed, cancer therapy is already successfully aided by drugs that, depending on the type and stage of cancer, induce or block autophagic flux, such as Rapamycin and Chloroquine18. However, the currently available drugs lack specificity. Therefore, novel compounds that modulate autophagy in a more specific manner need to be identified19. To discover novel regulators of autophagy (both cellular and exogenous), robust, rapid, and cost-effective experimental approaches are necessary. Unfortunately, activation of autophagy does not result in transcriptional activation, so classical reporter-gene approaches are not applicable.Recent studies revealed that aberrant autophagy impacts several disorders20. ATG8-family members are modified in two steps. First, proteolytic cleavage occurs, driven by ATG4, that exposes the C-terminal glycine. In the second step, phosphatidylethanolamine (PE) is covalently attached to the final glycine of ATG8 proteins in a ubiquitin-like conjugation catalyzed by the ATG5-12/16L complex22. The human genome encodes several ATG8 homologs , with Microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B or short LC3B) being the most prominent member24. The conversion of LC3B-I to LC3B-II has long been recognized as a reliable marker for autophagosome quantification24. For example, conversion of LC3B can be monitored by western blotting as LC3B-I and II migrate at different sizes, with the more hydrophobic LC3B-II traversing faster through a gel. Furthermore, the recruitment of LC3B to autophagosomes can be observed using microscopy as puncta formation24. However, a lot of classical methods to study autophagy suffer from human bias, especially the manual counting of enhanced green fluorescence protein (eGFP)-LC3B puncta, high variability, and labor-intensive experimental procedures and are thus not apt for high-throughput applications25. Recent years have developed robust systems to study autophagy, that have already been successfully adapted for screening approaches, identifying novel parts of the autophagic machinery, regulation as well as drugs that modulate autophagy32. Eng et al. (2010) reported an easy and rapid method to quantify autophagosomes using flow cytometry, based on the quantification of lipidated, autophagosome-bound LC3B-II. To isolate the membrane-bound eGFP-LC3B-II, soluble, cytoplasmic eGFP-LC3B-I is removed from cells, using mild permeabilization and subsequent washing that retains the membrane(= autophagosome) associated eGFP-LC3B-II inside the cells33.One major feature of active autophagy is the presence of processed ubiquitin-like ATG8-family members that decorate the membrane of autophagosomesWe adopted this system for high-throughput experiments and present here a detailed protocol for robust, rapid, sensitive, and cost-effective measurement of autophagy using a simple flow-cytometry based readout. In addition, we provide details on the construction, selection, and cultivation of clonal autophagy reporter cell lines to assess cell-type specific differences in autophagy. In our system, the reporter construct is stably integrated into the genome of the target cell and no additional selection to maintain stable eGFP-LC3B expression is required. We generated and thoroughly characterized eGFP-LC3B expressing autophagy reporter cell lines based on HEK293T, HeLa, Jurkat, and THP-1 cells. These reporter cell lines allow quantification of autophagy with high sensitivity and are suitable for high-throughput approaches. We further provide examples and experimental conditions on how to use these eGFP-LC3B expressing cell lines for rapid flow cytometry-based quantification of autophagy to discover novel drug candidates, analyze time-dependent modulation of autophagy by viral infections, and identify key factors in autophagy by overexpression or knock out CRISPR/Cas9 analysis.All cell lines  were purchased from American type culture collection . HEK293T and HeLa cells were cultivated in Dulbecco’s Modified Eagle Medium  supplemented with 10% (v/v) fetal bovine serum , 100 U/ml penicillin (PAN-Biotech), 100 µg/ml streptomycin (PAN-Biotech) and 2 mM L-glutamine (PAN-Biotech) . Jurkat and THP-1 cells were cultivated in Roswell Park Memorial Institute  1640 medium supplemented with 10% (v/v) FBS (Gibco), 100 U/ml penicillin (PAN-Biotech), 100 µg/ml streptomycin (PAN-Biotech) and 2 mM L-glutamine (PAN-Biotech). Cells were tested for mycoplasma contamination by polymerase chain reaction (PCR) test and used if negative. Measles virus  was a generous gift from K.-K. Conzelmann . Influenza A virus (strain PR8/34 H1N1) and encephalomyocarditis virus  were purchased from ATCC .34. The open reading frame (ORF) of TRIM32 was subcloned into the pIRES_FLAG vector using Gibson assembly (New England Biolabs). The insert was amplified by PCR  with primers 1 and 2 , and the vector linearized with NotI and MluI restriction enzymes (New England Biolabs). pBabe-mCherry-eGFP-LC3B and pCSC-SP-PW-GFP (aka pBOB-eGFP) were kindly provided by Jayanta Debnath  and Inder Verma , respectively. pMDLg, RSV-Rev, and pMD.G together with the generated pBOB construct were used to rescue 3rd generation lentiviruses as previously described35. pSicoR-CRISPR-PuroR was kindly provided by Robert-Jan Lebbink36. ATG5 and non-targeting single guide RNA coding sequences were inserted by cutting the backbone with BsmBI and subsequent Gibson assembly (NEB) with oligonucleotides  to create pSicoR-CRISPR-PuroR-NT, pSicoR-CRISPR-PuroR-ATG5-1, and pSicoR-CRISPR-PuroR-ATG5-2. For all transfections, Polyethylenimine  or Lipofectamine RNAiMax (Invitrogen) was used as detailed in the protocol.pEGFP-N1_hTRIM32 was a gift from Martin Dorf HeLa eGFP-LC3B reporter cells were grown in 24-well plates and 18 h post-seeding, the cells were transfected with siRNA (Horizon Discovery) either targeting autophagy-related genes (ATG) or non-targeting as a negative control . The transfection was done using Lipofectamine RNAiMAX (Invitrogen) following the manufacturer’s instructions. 72 h post transfection, Chloroquine  was added to one set of wells, transfected with non-targeting control siRNA. Subsequently, the cells were harvested, treated with saponin, fixed using PFA, and analyzed in flow cytometry.35 were generated as described in the detailed protocol 1.2. The pBOB-eGFP-LC3B plasmid was exchanged for the respective pSicoR-CRISPR-PuroR CRISPR/Cas936 constructs harboring ATG5 targeting sgRNAs or non-targeting (NT) sgRNAs 37. Three days post-transduction, the medium of the transduced HeLa eGFP-LC3B cells was changed to DMEM + 3 containing 0.75 µg/ml puromycin for selection. The selected cells were grown to confluency and differences in autophagic flux between the pools were analyzed by flow cytometry.Third generation lentiviral particles10) by vortexing at maximum speed for 30 s. Cell debris was pelleted by centrifugation , and the cleared supernatants (SN) stored for analysis at -20 °C.Whole-cell lysates were prepared by collecting cells in Phosphate-Buffered Saline . The cell pellet  was lysed in transmembrane lysis buffer -1-piperazineethanesulfonic acid (HEPES) pH 7.4, 1% Triton X-100, 5 mM ethylenediaminetetraacetic acid (EDTA)Whole-cell lysates were mixed with 6xSDS-PAGE loading buffer  Glycerol, 6% (w/v) SDS, 0.3% (w/v) Orange G, 15% (v/v) 2-mercaptoethanol) and heated to 95 °C for 5 min. Samples were loaded on a precast 10% NuPAGE Bis–Tris gel (Invitrogen) and run for 2 h at 80 V in 1 × MES SDS running buffer (Invitrogen). Subsequently, the gel was blotted onto methanol activated PVDF-Membrane  using 1xSemi dry blot transfer buffer (Alfa Aesar) for 30 min at 30 V. Afterwards, the membrane was blocked using 0.5% (w/v) casein (Thermo Scientific) for at least 1 h at room temperature (RT), then incubated with primary antibodies, diluted in 0.05% (w/v) casein (Thermo Scientific) for 1 h at RT or overnight at 4 °C. The membrane was extensively washed with PBS-T (PBS containing 0.2% (v/v) TWEEN-20, Sigma-Aldrich). The respective secondary antibodies , diluted 1:20,000 in 0.05% (w/v) casein (Thermo Scientific), were incubated with the membrane for 30 min at RT, followed again by extensive washing with PBS-T for 30 min. The blot was developed on an Odyssey 9120 (Li-Cor) infrared imager. The following antibodies were used: anti-LC3B , anti-GFP , anti-p62  and anti-β-actin .2O 25% (v/v), 0.2 M Tris HCl pH 8.5 50% , DABCO 2.5% 38) to co-stain nuclei. The suspension cells were pelleted , suspended in the same mounting medium, and mounted between coverslips and microscope slides (VWR). All laser scanning images were acquired on a Zeiss LSM 710 confocal microscope. The total area of cytoplasmic eGFP-LC3B puncta in HeLa, HEK293T, Jurkat and THP-1 cells was determined using a custom ImageJ macro for ≥ 30 randomly selected cells.HeLa or HEK293T eGFP-LC3B reporter cells were grown on coverslips (VWR) in 24-well plates and treated as indicated. THP-1 or Jurkat eGFP-LC3B reporter cells were grown in 24-well plates and treated as indicated. Cells were fixed with 4% (w/v) paraformaldehyde  for 20 min at RT, permeabilized with 0.5% (v/v) Triton-X-100 (Sigma-Aldrich) in PBS and then blocked with 5% (v/v) fetal bovine serum (Gibco) in PBS for 1 h at RT. Adherent cells were mounted on microscope slides (VWR) in 4′,6-diamidino-2-phenylindole -containing Mowiol mounting medium , glycerol 25% , HPlease see the detailed protocol for the exact experimental procedures. Single cells to create clonal cell lines were sorted into 96-wells using BD FACSAria III. To measure eGFP or allophycocyanin (APC) fluorescence, a BD FACSCanto II or Beckman-Coulter CytoFLEX with attached high-throughput samplers were used. Voltage/gain for forward and sideward scatter were set to such values that allowed clear separation of the cell population from debris. For all co-staining, we chose APC as the fluorophore to avoid compensation with the eGFP signal. eGFP mean fluorescence intensity (MFI) in mock cells was set to above 1,000 to allow for linear monitoring of changes in both directions (less/more autophagosomes) using voltage/gain settings. A minimum of 10,000 intact single cells were measured per sample. Raw fluorescence-activated cell sorting (FACS) data were analyzed using FlowJo 10. Intact cells were gated in FSC-A vs SSC-A and single cells gated in FSC-H vs FSC-A. Stringent gating strategies to exclude debris or dead cells that exhibit autofluorescence were applied. eGFP-MFI of all samples was calculated and eGFP-MFI of the mock control subtracted (= baseline autophagic flux) from the other samples to depict the eGFP-MFI shift (= shift of autophagic flux).sgRNA PCR amplicons were mixed with 6 × loading dye  and loaded on a 1% agarose gel  in 1 × TAE buffer (Carl Roth) next to a 1 kb Plus DNA ladder (Thermo Scientific). The samples were run for 25 min at 140 V, stained using ethidium bromide (AppliChem GmbH), and visualized in a Bio-Rad Gel Doc XR + .40. Default settings were used to process the raw reads. Combined from three independent experiments, 6.4 million reads were obtained for the low sample and 4.2 million for the input sample. To align the reads to the GeCKO 2.0 gRNA table, quantify, and normalize them, the MAGeCK suite was used41. The raw sequencing data were deposited in GEO (ID: GSE147488).Sequencing was performed on the Illumina Genome Analyzer in the GeneCenter sequencing facility (LAFUGA). Obtained sequences were processed with the Trim Galore! toolkit to remove adapter sequences and reads with PHRED scores below 30 as previously describedP-values ≤ 0.05 were considered statistically significant. A p-value below 0.05 is annotated with *, p-values between 0.05 and 0.01 with **, and p-values below 0.001 with ***. To evaluate the quality of the method for high-throughput screening, we calculated the Z-factor as described previously42. A Z-factor above 0 indicates a method suitable for high-throughput approaches, 1 represents the maximum quality reachable.Data were collected at least in triplicates for all flow cytometry-based approaches and with at least 30 replicates for fluorescence confocal microscopy-based approaches. For the relative comparison of changes in autophagic flux, statistical differences were assessed by one-way ANOVA or unpaired t-test as indicated. Results were graphed by Graph Pad Prism 8, and are displayed as means ± standard error of the mean (SEM) with individual values shown. A detailed list of materials is provided in Table 1.11.1.1.Set up a PCR reaction to amplify the eGFP-LC3B fragment using Primers 3 & 4  and pBabe-mCherry-eGFP-LC3B as a template according to Tables 1.1.2.Digest the amplified insert  with BamHI and PmeI (both New England Biolabs), purify it on an agarose gel, and recover the DNA  to create the insert for ligation.1.1.3Digest the pBob-eGFP vector using BamHI and PmeI, purify on an agarose gel and recover the DNA  to create the backbone for ligation.1.1.4.Ligate the insert and backbone  to create the pBob-eGFP-LC3B vector.Optional: An internal ribosomal entry site (IRES) element can be inserted after the eGFP-LC3B fusion ORF to allow the expression of an antibiotic resistance protein. To avoid selection using antibiotics and stressing the autophagy reporter cells, we decided to omit this.Amplify a fused eGFP-LC3B ORF from the pBabe-mCherry-eGFP-LC3B template and insert it into the pBob-eGFP vector, replacing the existing eGFP ORF:1.2.35 for transduction of target cells: Transfect HEK293T cells in 6-well plates (about 70% confluency) using PEI .1.2.1.Mix A: Opti-MEM  and PEI (2 µl PEI per µg DNA).1.2.2.Incubate Mix A at RT for 5 min.1.2.3.Mix B: Opti-MEM  and plasmid DNA (for the amounts see Table 1.2.4.Combine Mix A and Mix B and incubate at RT for 20 min.1.2.5.Add the transfection mix dropwise onto the cells without disturbing the monolayer.1.2.6.Change the medium to DMEM + 3 (2.5% FBS) 6 h post-transfection.1.2.7.24 h post-transfection, the cells should exhibit bright green fluorescence. Repeat if no fluorescence is observed.1.2.8.Harvest the SN 48 h post transfection and clear it by centrifugation  and filtering .Generate third generation lentiviral particles (VSV-G pseudotyped)1.3.5 cells of the selected target cell line  with 100 µl of the SN harvested in step 1.2.8.Transduce 5 × 101.4.Two days post-transduction, detach the cells using Trypsin , pellet at 300 g, 3 min, 4 °C.1.5.6 cells per ml in a 15 ml Falcon tube. Filter through round-bottom tubes with a cell strainer cap . Sort samples on a FACS sorter e.g. BD FACSAria III, using a 100-micron ceramic nozzle. The flow rate was kept below 2, and the purity setting was set to a single cell. Sort one cell per 96-well F-bottom well.Remove the SN and replace it with fresh medium without FBS to a concentration of 5–10 × 10Important: The nozzle size needs to be larger than the cell passing through it. Avoid crushing or stressing the cells too much.1.6.Provide single cells with medium supplemented with 20% FBS according to the cell type. Select eGFP-LC3B cells, with moderate eGFP MFIs Fig. B.Figure Important: Some cell lines do not grow efficiently from single clones. Avoid isolating cell lines expressing too much or no eGFP-LC3B.1.7.Approximately 3–6 weeks post sorting, clones can be transferred from half-confluent 96-wells directly into 12-well plates. Avoid overgrowing in 96-wells or at any step.1.8.eGFP-LC3B expression and fluorescence can be monitored using a fluorescence microscope and clones with little to no detectable fluorescence are discarded. Clones with clearly visible aggregations of eGFP-LC3B in the cytoplasm are also discarded.1.9.Grow clones up to confluent T25 flasks and then proceed to verify the clones.1.10.1.10.1.Generate whole-cell lysates of the cell line (as described in the short Methods section), and immunoblot with anti-GFP antibody and anti-LC3B antibody. An additional band at around 50 kDa, representing the fusion protein, should appear in the stable cell lines compared to their parental cell lines.Note: In some cell lines an additional band at the height of eGFP (ca. 27 kDa) may appear due to the processing of eGFP-LC3B. We have not observed any negative impact of this additional eGFP band on the sensitivity and responsiveness of the respective cell lines. Cell lines with further degradation products should be discarded.1.10.2.Fix the cells using 4% PFA on glass slides for confocal microscopy (as described in the short methods section). Take confocal images and analyze the number of autophagosomes upon treatment with positive controls such as Rapamycin (1 µM) or Chloroquine (10 µM) for 4 h. The number of autophagosomes (= eGFP-LC3B) puncta per cell should significantly increase compared to mock-treated cells.1.10.3.Follow the protocol for high-throughput autophagy quantification to verify that the cell line is suitable for the approach and contains enough eGFP-LC3B for cytoplasmic washout and subsequent analysis. Use treatment with Rapamycin (1 µM) or Chloroquine (10 µM) for 4 h and compare to cells treated with the carrier (e.g. Dimethyl sulfoxide (DMSO) or water).Important: The level of expressed eGFP-LC3B impacts the sensitivity of the assay. Generally, we have found that a medium amount of eGFP-LC3B seems to be optimal. Too low eGFP-LC3B expression causes a loss of detectable eGFP-LC3B fluorescence after washout. Too high eGFP-LC3B causes a high baseline of autophagosome associated eGFP-LC3B, as well as occasionally aggregates observed in confocal microscopy.Produce frozen stocks as early as possible to preserve the generated cell line at a passage number as low as possible.Confirm whether the cell clones contain the reporter:For the construction of the cell lines use low passage numbers. For accurate autophagy measurements, make sure that the cells never overgrew, even during the construction of the cell lines, discard any flasks which are too dense. Cell lines should be discarded once the passage number is higher than 20 (assuming a biweekly split-cycle).A detailed list of materials is provided in Table 2.1.Grow cells in either F-bottom (adherent cell types) or V-bottom (suspension cell types) 96-well plates. Seeding of 50,000 cells per well for adherent cells and 100,000 cells per well for non-adherent cells is recommended, 18 h before a 4-h assay.Important: At the time of harvest, the cells should be approximately confluent, and kept in fully supplemented medium which is still at optimal pH. Adjust the cell number/density at the time of seeding to meet these criteria. Do not change medium/nutrient conditions before treatment/harvesting. Any stress on the cells impacts autophagy and thus may bias the assay. Consider appropriate negative and positive controls. Established drugs like Rapamycin, Chloroquine, or Bafilomycin A1 are recommended as controls. Negative controls should be treated the same way as the samples, e.g. add the carrier for a drug in the same concentration to the mock SN. Screening results have to be confirmed using orthogonal methods for assessing autophagy levels.2.2.For adherent cells: discard the SN without disturbing the monolayer.for suspension cells: Spin down the cells at 500 g, 3 min, 4 °C, discard the SN.2.3.Wash the treated (or transfected or infected) cells with 100 µl 1xPBS (Gibco).Important: Proceed with the harvesting as quickly as possible until the cells are in saponin-containing PBS (step 2.6)!2.4.2.4.1.Discard the SN without disturbing the monolayer.2.4.2.Detach cells with 50 µl Trypsin , incubate for 5 min at 37 °C.2.4.3.Transfer cells to a V-bottom 96-well plate.2.4.4.Add 50 µl DMEM + 3 to the original F-bottom 96-well plate, transfer the medium to the same V-bottom 96-well plate as in the previous step.Only for adherent cells: Transfer cells into V-bottom 96-well plate.2.5.Spin down the cells at 500 g, 3 min, 4 °C, discard the SN.2.6.Add 100 µl 0.05% saponin in 1xPBS.2.7.Resuspend the cells in the plate by shaking or pipetting up and down.2.8.crucial!) at 4 °C.Incubate for at least 20 min  with 100 µl 1xPBS.Wash cells at least twice .2.12.2.3.Incubate for 20 min at RT in the dark.2.12.2.4.Spin down at 500 g, 3 min, 4 °C, discard SN.2.12.2.5.Add 100 µl 1xPBS (Gibco).Important: Fixed cells can be stored in the fridge (4 °C) for analysis up to a week. But fluorescence of the cells wanes over time. Analyze as early as possible.Paraformaldehyde (PFA) fixation:2.12.3.2.12.3.1.Spin down 500 g, 3 min, 4 °C, discard SN.2.12.3.2.Add 100 µl -20 °C cold 100% methanol (MeOH) (Merck) per well.2.12.3.3.Incubate 20 min at -20 °C.2.12.3.4.Spin down 500 g, 3 min, 4 °C, discard SN.Important: Do not let the MeOH on the cells warm up to RT!2.12.3.5.Add 100 µl ice-cold 1xPBS.2.12.3.6.Spin down 500 g, 3 min, 4 °C, discard SN.Important: Get rid of traces of MeOH for downstream analyses.2.12.3.7.Add 100 µl 1xPBS.Important: Fixed cells can be stored in the fridge (4 °C) for analysis up to a week.But fluorescence of the cells wanes over time. Analyze as early as possible.Methanol fixation:Use one of three different subsequent treatments .A detailed list of materials is provided in Table Important: All hits in screening approaches have to be verified using orthogonal methods to assess autophagy (for a comprehensive overview of available methods see24).3.1.3.1.1.Seed eGFP-LC3B reporter cells in 96-well plates, 50,000 cells per well in F-bottom plates for adherent cells, and 100,000 cells per well in V-bottom plates for non-adherent cells. The total amount of medium should be 90 µl fully supplemented medium (e.g. DMEM + 3) per well.3.1.2.2, 90% humidity. Seed non-adherent cells directly before applying the treatment.Grow adherent cells overnight at 37 °C, 5% COImportant: Do not seed cells from overgrown flasks or nutrient-deprived cell stocks.3.1.3.Add 10 µl of treatment solution to the cells. Several examples of drug treatment were used to verify the generated cell lines. As a control, use the same amount of carrier liquid (e.g. water or DMSO).Important: Do not exchange the medium before treatment. Do not vary the volume between treatments and do not add additional medium, FBS, or salt (beyond 150 mM). Any stress may cause changes in autophagic flux and obscure effects of drugs, thus treat your cells carefully.3.1.3.1.43, was used in a range of 2 µM to 15 nM , which induces autophagy by inhibiting the mTORC1 complex3.1.3.2.46, was used in a range of 20 µM to 150 nM , which blocks the turnover of autophagosomes3.1.3.3.48, was used in a range of 625 nM to 5 nM , which blocks the turnover of autophagosomes3.1.3.4.Amino acids (Sigma) dissolved in water were used at a final concentration of 1 mg/ml. Fig. E.For our exemplary experiments Fig.  we used:3.1.4.Harvest and analyze the cells 4 h post-treatment as described in the basic protocol.Optionally: Cells can be treated longer or shorter, we have observed that a time frame between 2 and 4 h is optimal for most drug-based applications. However, drugs that are expected to block autophagic flux may be kept on the cells for longer to measure the accumulation of vesicles from basal autophagy. Please note that especially blocking autophagy decreases the viability of the cells after 4–6 h.High-throughput screening for novel autophagy modulating drugs.3.2.3.2.1.Seed eGFP-LC3B reporter cells in 96-well plates, 50,000 cells per well in F-bottom plates for adherent cells, and 100,000 cells per well in V-bottom plates for non-adherent cells. Seed cells in 90 µl fully supplemented medium (e.g. DMEM + 3) or infection medium per well.Important: Control medium changes with parallel medium changes in mock-infected wells.3.2.2.Infect cells by addition of 10 µl virus-containing solution (or mock carrier) with an appropriate MOI at different time points, e.g. as indicated in Fig. Important: Add the same type/amount of fluid (lacking virus) used for infection to the medium of the mock wells. E.g. if the virus was produced in Vero cells, add an equal amount SN from non-infected Vero cells to the mock wells. Alternatively, use the buffer the virus is stored in, e.g. after purification.3.2.3.Harvest cells and analyze as described in the basic protocol.Important: Discard virus-containing SN according to your biosafety protocols. Keep in mind that some viruses like EMCV are not inactivated by saponin-treatment.Manipulation of autophagy by viruses.3.3.3.3.1.Seed 30,000 eGFP-LC3B HEK293T reporter cells in F-bottom 96-well plates in 100 µl DMEM + 3 per well.3.3.2.3.3.2.1.Mix A: Mix Opti-MEM (10 µl per well of the 96-well plate) and PEI (2 µl PEI per µg DNA) and incubate for 5 min at RT.3.3.2.2.Mix B: Mix Opti-MEM (10 µl per well of the 96-well plate) and DNA (0.15 µg per well of the 96-well) and add the same volume of Mix A.3.3.2.3.Incubate the transfection mix for 20 min at RT.3.3.2.4.Add the transfection mix dropwise onto the cells without disturbing the monolayer.Important: Transfection induces autophagy. We have tested a few transfection reagents and found that PEI and calcium phosphate transfection induce the lowest amount of autophagy as opposed to commercially available transfection reagents. We generally use PEI, as it results in higher transfection rates/expression rates than calcium phosphate transfection. Thus, pre-test your transfection method. Alternatively, transduction can be used, however, gene expression is lower and an autophagic response is still induced albeit to a lesser degree.Transfect vectors coding the proteins of interest 18 h post-seeding and the empty vector control using PEI and a maximum of 0.15 µg of DNA per well.3.3.3.Change medium to DMEM + 3 4–6 h post-transfection.3.3.4.48 h post-transfection, cells are harvested and analyzed as described in the basic protocol.Identification of novel autophagy regulating factors using overexpression3.4.Identification of novel autophagy regulating factors using CRISPR/Cas9.Important: This procedure was modified from Joung et al49. Please consult the original publication for a comprehensive protocol. Create/Amplify/Determine the titer of the Human CRISPR Knockout Pooled Library (GeCKO v2)37 according to49.3.4.1.9 autophagy reporter cells (e.g. Jurkat eGFP-LC3B) with an MOI of 0.3.Transduce 1 × 103.4.2.24 h post-infection, select with appropriate concentrations of puromycin .3.4.3.Add fresh, fully supplemented medium to the cells every two days.Important: Do not let the cells starve or overgrow.3.4.4.10 days post-infection, harvest the cells by pelleting .3.4.5.10 and 1 × 1011.Determine the initial cell number for the protocol. It should range between 1 × 103.4.6.8 cells from the ‘sample’ as ‘input’ for the CRISPR screen.Take 1 × 103.4.7.Process the ‘sample’ and ‘input’ with saponin according to the basic protocol.3.4.8.!) according to the basic protocol.Fix cells in MeOH .3.4.10.7 cells for each fraction. Discard the medium autophagy inducing cells.Sort the ‘sample’ cells using a FACS sorter into high, medium, and low autophagosome content cells see Fig. D. Sort aImportant: We usually get about 8–10% of the total cell number for the high eGFP-LC3B fluorescence cells and 8–10% of the total cell number for the low eGFP-LC3B fluorescence cells. In cells transduced with non-targeting CRISPR vector, less than 1% of the total cell number should fall into the low or high category. Sorting of the cells takes a few hours (~ 8–10) depending on the flow rate. Adjust the flow rate and the concentration of the treated sample to the highest possible flow rates.3.4.11.Pellet the sorted and input cells .3.4.12.Use the Zymo Research Quick-gDNA MidiPrep Kit to extract the genomic DNA from the cells (according to the manufacturer's protocol).3.4.13.Elute the genomic DNA in 200 µl Pyrogen/RNase/DNase free water and measure its concentration.Important: The concentration may be very low (< 10 ng/µl) depending on the number of sorted cells. The DNA may be concentrated using an 80% isopropanol precipitation.3.4.14.Use PCR to amplify the genomically integrated sgRNAs, use individual barcodes for ‘input’, ‘sample-high’, and ‘sample-low’.Set up 10 PCR reactions according to Tables 3.4.15.A single band at 260–270 bp should appear on an agarose gel.3.4.16.Combine 2 PCR reactions each for one NEB Monarch PCR & DNA Cleanup Kit (5 µg) column according to the manufacturer’s instructions (e.g. 5 purifications per sample).3.4.17.Elute in 10 µl Pyrogen/RNase/DNase free water.3.4.18.Pool the extracted DNA to have three samples: ‘input’, ‘high’, and ‘low’. Proceed with next-generation-sequencing.33. To this end, we constructed autophagy reporter cell lines stably expressing eGFP-LC3B from a genomically integrated, CMV-promoter controlled, expression cassette. Third generation lentiviral particles harboring the expression cassette were generated and target cell lines transduced. The cell lines include cell lines (HeLa) classically used for autophagy research, easy-to-transfect cell lines (HEK293T) and cells of the immune system like Monocyte-like and T cells (THP-1 and Jurkat)  Fig. A. Finalllines HEK93T and clines HEK93T and clines HEK93T and clines HEK93T and cmes Fig. B. Taken 33. As determined by flow cytometry, the cells decreased in size and granularity .Drug screenings, as exemplified for the known autophagy manipulating drugs Rapamycin, Chloroquine, and Bafilomycin A1, can be readily performed Fig. A–D using55, measles virus (MeV) infection induced high numbers of autophagosomes at late time points 57. In monocyte-like cells, both IAV and MeV infection induced autophagy at early time points . These methods include visualization of autophagosomes by electron microscopy, monitoring of degradation of targets of autophagy such as p62 using western blotting, processing of endogenous LC3B, and visualization of LC3B puncta using eGFP-LC3B and confocal fluorescence microscopy. Advanced imaging methods using automated image processing reduce the manual labor required for confocal image acquisition and analysis50. Several currently used methods to quantify autophagy rely on monitoring a hallmark of autophagy induction, the lipidation and translocation of (eGFP-LC3B) to autophagosomal membranes. However, most of these methods are not applicable for high-throughput approaches. Here, we describe a detailed protocol for an easily accessible method to robustly and rapidly quantify autophagosomes for high-throughput applications based on flow cytometry-mediated quantification of membrane (= autophagosome)-bound eGFP-LC3B. Compared to classical methods for monitoring autophagy, like western blotting, this system is less labor-intensive, faster, and allows the quantification of large numbers of cells  at once, allowing extraction of robust means of autophagy levels but also visualization of the heterogeneity of cell systems. Therefore, this system is well fitted for approaches that measure the mean induction of autophagy by e.g. drugs or peptides, but also approaches relying on single-cell autophagy levels like CRISPR/Cas9 screens . Complementary assays to monitor autophagy, that do not rely on LC3B are mandatory to properly assess the status of autophagy in a cell. For example, the cellular levels of SQSTM1/p62 are decreased upon induction of autophagic flux but p62 accumulates if autophagy is blocked63. Monitoring p62 levels will provide further hints whether a compound/molecule induces or blocks autophagy62. Alternatively, other cellular proteins targeted by autophagy like NBR164 or cGAS65 can be used as indicators of autophagic degradation. Autophagy-like cellular processes such as LC3-associated phagocytosis66 or viruses may use LC3B and redirect it to membranes, thus giving rise to false-positive signals in our flow cytometry assay and other methods that measure autophagy using LC3B processing or localization66. In general, for the majority of non-canonical autophagic processes, degradation of p62 is not observed. Furthermore, the activity of the compound/factor should be dependent on multiple essential factors of the core machinery of autophagy such as ATG5, ATG4, and ATG16L. For example, while LC3B associated phagocytosis is dependent on most core machinery factors, it is independent of e.g. ATG14L, which is required for canonical autophagy66. Finally, visual analysis of double-layered membrane structures by electron microscopy will support the notion of whether a compound/regulatory factor modulates classical autophagy68.One major issue of all systems relying on the processing of LC3B is that both de novo induction of autophagy and blockage of autophagic flux increases the amount of autophagosomes (or processed LC3B) at a given time point. Thus, a rigorous secondary assessment of hits obtained in the primary screen has to take place  allow rapid quantification of autophagy without cytoplasmic washout26. Using a reporter construct expressing a fusion protein of GFP, LC3, RFP, and a non-cleavable variant of LC3B, LC3BΔG, this system allows, similarly to the double-labeled LC3B, monitoring of autophagy based on the ratio between GFP and RFP fluorescence. Upon autophagy induction, the protease ATG4 is activated and cleaves the fusion protein into GFP-LC3and RFP-LC3BΔG. Eventually, only GFP-LC3B can be incorporated into autophagosomes and is degraded. Thus, upon induction of autophagy, the ratio between GFP and RFP fluorescence decreases. This system has allowed the identification of several novel autophagy modulators26. However, recombination between the two LC3B ORFs, especially during lentiviral driven applications like CRISPR screens, may render the system inactive.Recently, another sophisticated approach to quantify autophagic flux using flow cytometry was described29, the ufmylation pathway28, or the acetyltransferase EP30027. However, upon induction of autophagy, transcriptional upregulation of p62 has been observed in some instances. Furthermore, longer assay times are needed to allow the degradation to proceed enough to detect decreased levels of p62.Instead of assessing the number of autophagosomes to quantify autophagy, the consequences of the induction of autophagic flux can also be monitored by examining p62 levels. Based on this approach several genetic screens have identified novel factors involved in autophagy regulation, such as 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase73. However, as acidification of lysosomes is not a process exclusive to autophagy induction, such approaches may lead to high false positive/false negative rates. Thus, systems based on lysosomal dyes are rarely used to monitor autophagy.Lysosomal dyes like lysotracker may be used to stain lysosomes or autophagolysosomes and even to monitor changes in pHTaken together, using eGFP-LC3B as a reporter for quantification of autophagy is currently still the most established and advantageous system for quantifying autophagy. It avoids convoluted reporter systems and thus directly quantifies autophagosomes. Our method applies this established tool for high-throughput approaches.Accurate and robust quantification of autophagosomes by measuring the mean fluorescence intensity of membrane-bound eGFP-LC3B is a powerful high-throughput tool to study autophagy. Our protocol is designed to easily approach e.g. drug discovery, key factor identification, pathway analysis, and virus/host response monitoring. The setup is easy to adopt and provides a robust, flexible, and rapid readout. However, screening results have to be confirmed using orthogonal methods for assessing autophagy levels. We strongly believe our high-throughput approaches may pave the way for the discovery of novel compounds modulating autophagy and provide an immediately accessible and thoroughly tested system for labs to test their compounds/proteins/viruses for autophagy modulation.Supplementary information.