Autophagosome fusion with vacuoles requires a conserved fusion machinery, though the topology remained unclear. Two papers in this issue, Bas et al. and Gao et al., uncover Ykt6 as the required autophagosomal SNARE. Autophagy mediates the bulk degradation of cytoplasmic material, particularly during starvation. Upon the induction of autophagy, autophagosomes form a sealed membrane around cargo, fuse with a lytic compartment, and release the cargo for degradation. The mechanism of autophagosome–vacuole fusion is poorly understood, although factors that mediate other cellular fusion events have been implicated. In this study, we developed an in vitro reconstitution assay that enables systematic discovery and dissection of the players involved in autophagosome–vacuole fusion. We found that this process requires the Atg14–Vps34 complex to generate PI3P and thus recruit the Ypt7 module to autophagosomes. The HOPS-tethering complex, recruited by Ypt7, is required to prepare SNARE proteins for fusion. Furthermore, we discovered that fusion requires the R-SNARE Ykt6 on the autophagosome, together with the Q-SNAREs Vam3, Vam7, and Vti1 on the vacuole. These findings shed new light on the mechanism of autophagosome–vacuole fusion and reveal that the R-SNARE Ykt6 is required for this process. Macroautophagy, hereafter referred to as autophagy, is an intracellular degradation and recycling pathway that is highly conserved among eukaryotes. During autophagy, cellular material is engulfed within a newly formed double membrane vesicle, the autophagosome. Once complete, the outer autophagosomal membrane fuses with a lytic compartment, that is, the lysosome in mammals or the vacuole in yeast and plants. Fusion releases the inner vesicle, the so-called autophagic body, for degradation in the lumen of the lytic compartment.Drosophila melanogaster and yeast (Over 40 autophagy-related (Atg) proteins are known to act in the yeast autophagy pathway, and many functional homologues of these players have been identified in higher eukaryotes . The cornd yeast . Activatnd yeast , which ind yeast . SNARE pnd yeast . WhetherSNAREs are membrane proteins that have canonical heptad repeat domains (SNARE motifs) with a central argininyl (R) or glutaminyl (Q) residue and therefore are termed R- or Q-SNAREs, respectively. SNARE complexes are composed of four-helix bundles consisting of three Q-SNAREs and one R-SNARE . SNAREs SNARE proteins also act in earlier steps in autophagy such as autophagosome formation and closure. The SNAREs required for yeast autophagosome formation are Sso1, Sso2, Sec9, Tlg2, Sec22, and Ykt6 . MutantsTechnical limitations have obscured insight into the mechanism of autophagosome–vacuole fusion. For instance, disrupting membrane fusion factors in intact cells may not only affect autophagy directly but will affect vesicular trafficking in general, with potential indirect effects on autophagy. Also, if a fusion factor is required in early steps of autophagosome formation, this requirement will mask its roles in late stages of the pathway such as fusion to the vacuole. As a result, the factors that are required for autophagosome-to-vacuole fusion, where they localize and act, and how they are recruited have remained a mystery.We have overcome this critical barrier by developing a new approach that recapitulates autophagosome–vacuole fusion in vitro using isolated yeast vacuoles, an autophagosome-enriched fraction, and a cytosolic extract. This system allows us to uncover the players in autophagosome–vacuole fusion and to dissect the molecular steps of autophagosome–vacuole fusion by determining whether a player acts on the autophagosome, on the vacuole, or in the cytosol.Saccharomyces cerevisiae to test in an in vitro fusion assay . We further separated the pellet on a 0–4–8–15% Ficoll step gradient, isolating vacuoles at the 0–4% Ficoll gradient interface as previously described , and Atg1  in the lumen of the vacuoles ; Atg11 and Atg19 (Cvt pathway); Atg9 and Atg18 (Atg9 and Atg2-18 system); Atg14 (PI3-kinase complex I); and Atg8. To screen for the involvement of these proteins, we prepared cytosolic fractions from multiple knockout strains. We found that vacuoles .vps34Δ cells would support fusion. We found that fusion was strongly diminished with cytosolic fractions lacking Vps34  variant of the HOPS component Vps11  but not at the permissive temperature  and spheroplasted by lyticase (90 U/ml) treatment at 30°C for 30 min. Spheroplast formation was monitored by diluting the spheroplasts 1:100 in SP buffer or in H2O. Efficient spheroplasting causes a 10-fold drop at OD600 in the H2O sample. Spheroplasts were collected by centrifugation at 1,500 g for 10 min at 4°C, resuspended to 20 OD/ml in PS200 buffer , and lysed on ice by passing through a 22G needle 30 times. The lysate was cleared from unbroken cells by 500 g centrifugations at 4°C until no pellet was observed, and the cleared lysate was then spun at 20,000 g for 30 min at 4°C. The resulting pellet fraction was washed with PS200 buffer and resuspended to 10,000 OD/ml in PS200 buffer and used as autophagosome-enriched fraction.Yeast cells were grown in YPD to mid–log phase, followed by starvation for 16 h in SD-N. The cells were then harvested and incubated with 50 OD/ml DTT buffer  for 15 min at 30°C, spun at 3,000 g for 5 min, resuspended to 50 OD/ml in SP buffer, and spheroplasted by lyticase (60 U/ml) treatment at 30°C for 30 min. Spheroplast formation was monitored by a drop in OD600 as described above. Spheroplasts were then pelleted at 1,500 g for 10 min at 4°C, resuspended to 20 OD/ml in PS200 buffer, and lysed on ice by passing through a 22G needle 30 times. The lysate was cleared by 500 g centrifugations at 4°C until no pellet was observed, and the cleared lysate was then spun at 6,000 g for 30 min at 4°C. The resulting pellet fraction was washed with PS200 Buffer and resuspended to 2,500 OD/ml in PS200 buffer and used as crude vacuole fraction.Yeast cells were grown in YPD to mid–log phase, harvested, and resuspended to 50 OD/ml in DTT buffer. After incubation at 30°C for 15 min, they were spun at 3,000 g pellet was resuspended in 3 ml 15% Ficoll buffer . The resuspended crude vacuoles were transferred into a SW40 centrifugation tube (Beckman Coulter) and overlaid with 3 ml of 8% Ficoll, 3 ml of 4% Ficoll, and 1 ml 0% Ficoll . The step gradient was spun at 180,000 g at 4°C for 90 min, and the vacuoles were collected from the 0–4% interphase.To obtain a pure vacuole fraction, crude vacuoles were prepared as described above from 1,000 OD cells, and the 6,000 2) in reaction buffer . After 120 min at 30°C the reactions were transferred to ice, and 10 µl of the reaction was mixed with 10 µl of 1% low melting agarose dissolved in reaction buffer (preheated to 45°C), immediately transferred onto glass slides, and covered with a coverslip. The slides were incubated at RT for 5 min for the agarose to solidify before visualization.Standard fusion reactions (20 µl) contained 25 OD equivalents of crude vacuoles, 50 OD equivalents of the autophagosome-enriched fraction, 3 mg/ml cytosol, and an ATP regeneration system Protease protection experiments were performed as previously described . In brieg for 5 min). Pellets were washed in 1 ml distilled H2O, followed by centrifugation at 4°C and resuspension of the pellet in 2 ml ice-cold 0.85% NaCl containing 1 mM PMSF. After another centrifugation step, pellets were resuspended in 16 µl/OD unit lysis buffer . Cells were lysed by bead beating, and extracts were cleared by centrifugation at 16,000 g for 5 min at 4°C. Protein concentration of the supernatant was adjusted to 50 µg in 100 µl lysis buffer. Samples were prepared in duplicates for enzymatic reactions and control reactions. 400 µl reaction buffer  containing 6.25 mM α-naphthylphosphate  with an UPlanSApo 100×/1.4 oil objective, a CoolSNAP HQ2 camera, and an InsightSSI solid state illumination unit as time-lapse images of 0.6-s intervals with SoftWoRx software , mouse monoclonal anti-Pgk1 antibody (Invitrogen), mouse monoclonal anti-Vph1 (Abcam), mouse monoclonal anti-Pho8 (Abcam), and polyclonal rabbit anti-Ape1 antibody . Polyclovam3ts mutant  via BamHI-SalI into pRS315. pLB52 was generated by subcloning vps11-1ts into pRS315 from a plasmid obtained from W. Wickner .Plasmids are listed in Table S2 . pLB42 wt test, apart from Fig. S1 C, in which the two-tailed Student’s t test was used. P values are listed in Table S3.P values were calculated using the two-tailed Welch’s Fig. S1 shows the fusion assay and selective autophagy experiments. Video 1 shows a time lapse of an autophagosome fused with a vacuole, and Video 2 shows a time lapse of a fusion reaction without cytosol. Table S1 describes the yeast strains, Table S2 describes the plasmids used in this study, and Table S3 lists the P values.


In ER negative breast cancer the MGMT promoter is frequently methylated. In ER positive breast cancer MGMT is upregulated and modulates ER function. Here, we evaluate MGMT’s role in control of other clinically relevant targets involved in cell cycle regulation during breast cancer oncogenesis. We show that O6-benzylguanine (BG), an MGMT inhibitor decreases CDC2, CDC20, TOP2A, AURKB, KIF20A, cyclin B2, A2, D1, ERα and survivin and induces c-PARP and p21 and sensitizes ER positive breast cancer to temozolomide (TMZ). Further, siRNA inhibition of MGMT inhibits CDC2, TOP2A, AURKB, KIF20A, Cyclin B2, A2 and survivin and induces p21. Combination of BG+TMZ decreases CDC2, CDC20, TOP2A, AURKB, KIF20A, Cyclin A2, B2, D1, ERα and survivin. Temozolomide alone inhibits MGMT expression in a dose and time dependent manner and increases p21 and cytochrome c. Temozolomide inhibits transcription of TOP2A, AURKB, KIF20A and does not have any effect on CDC2 and CDC20 and induces p21. BG+/-TMZ inhibits breast cancer growth. In our orthotopic ER positive breast cancer xenografts, BG+/-TMZ decreases ki-67, CDC2, CDC20, TOP2A, AURKB and induces p21 expression. In the same model, BG+TMZ combination inhibits breast tumor growth in vivo compared to single agent (TMZ or BG) or control. Our results show that MGMT inhibition is relevant for inhibition of multiple downstream targets involved in tumorigenesis. We also show that MGMT inhibition increases ER positive breast cancer sensitivity to alkylator based chemotherapy.The DNA damage repair enzyme, O MGMT, a DNA repair protein, mediates resistance to alkylating agents. Increased MGMT expression level is exclusively a tumor phenotype trait as MGMT levels in normal tissues do not vary between patients with benign or malignant disease . In breaMGMT has been reported to play a role in integrating DNA damage/repair related signals with replication and cell cycle progression , 9. In t6 benzylguanine (BG) inhibits MGMT and potentiates the cytotoxicity of alkylating agents  in deionized Ho plugin . Resultst test, assuming equal variance, and each p value was calculated based on two-tailed test. A p value of <0.05 was considered statistically significant. P values are reported using a star system as follows: *p<0.05; **p<0.005 and ***p<0.0005.Experiments were performed in triplicates and data presented as mean ± SD. Statistical analysis was done using Student’s


HCMV is an important human pathogen that establishes lifelong latent infection in myeloid progenitor cells and reactivates frequently to cause significant disease in immunocompromised people. Our observation that viral gene expression is first turned on and then turned off to establish latency suggests that there is a host defense, which may be myeloid cell specific, responsible for transcriptional silencing of viral gene expression. Our observation that TNF-α induces reactivation independently of differentiation provides insight into molecular mechanisms that control reactivation. wt-GFP, flow sorted for green fluorescent protein-positive (GFP+) cells, and cultured for various times to monitor establishment of latency, as judged by repression of viral gene expression (RNA/DNA ratio) and loss of virus production. We found that, in the vast majority of cells, latency was established posttranscriptionally in the GFP+ infected cells: transcription was initially turned on and then turned off. We also found that some of the GFP− cells were infected, suggesting that latency might be established in these cells at the outset of infection. We were not able to test this hypothesis because some GFP− cells expressed lytic genes and thus it was not possible to separate them from GFP− quiescent cells. In addition, we found that the pattern of expression of lytic genes that have been associated with latency, including UL138, US28, and RNA2.7, was the same as that of other lytic genes, indicating that there was no preferential expression of these genes once latency was established. We confirmed previous studies showing that tumor necrosis factor alpha (TNF-α) induced reactivation of infectious virus, and by analyzing expression of the progenitor cell marker CD34 as well as myeloid cell differentiation markers in IE+ cells after treatment with TNF-α, we showed that TNF-α induced transcriptional reactivation of IE gene expression independently of differentiation. TNF-α-mediated reactivation in Kasumi-3 cells was correlated with activation of NF-κB, KAP-1, and ATM.We used the Kasumi-3 model to study human cytomegalovirus (HCMV) latency and reactivation in myeloid progenitor cells. Kasumi-3 cells were infected with HCMV strain TB40/E Human cytomegalovirus (HCMV) is a ubiquitous human pathogen of the betaherpesvirus family. Infection is transmitted through contact with body fluids, including saliva, urine, blood, and genital secretions. Hematogenous spread of the virus leads to a systemic infection, with multiple cell types infected in many organs. In immunocompetent hosts, primary infection is typically subclinical and is resolved by activation of innate and adaptive immunity. However, latently infected cells, which carry viral DNA but do not produce infectious virus, are able to escape immune surveillance and can persist for the life of the host.+/R− combination; specific immunosuppression protocols; and inflammatory conditions associated with high tumor necrosis factor alpha (TNF-α) secretion, including allograft rejection and sepsis  in the bone marrow are sites of latency in vivo –10. Expein vivo ––16. Celln vivo ––, 17–26.  ––17–26–. Differe ––17–26–, 29–32. + HPCs; Kasumi-3 cells, a CD34+ myeloblastic cell line derived from a patient with acute myeloid leukemia (AML)  , which iia (AML) , 36, 37;ar cells , 36–43. in vivo occurs in the context of infection and inflammation  promoter-driven green fluorescent protein (GFP) reporter, as well as viral genes at early times postinfection , early , and late (UL32) phases of the viral life cycle. Expression of these genes peaked at 4 dpi and then fell throughout the course of infection. Viral DNA copy number also increased following infection, peaked 4 to 8 dpi, and then decreased significantly  and E. T10.1128/mBio.01560-18.2FIG S1 FIG S1, PDF file, 0.9 MB.Analysis of viral RNA and DNA over the course of infection in Kasumi-3 cells. RNAs and DNAs from representative immediate early, early, and late genes were analyzed by reverse transcription-qPCR and qPCR, respectively, at various times postinfection as described in Copyright © 2018 Forte et al.2018Forte et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the + cells, some DNA was detectable in the GFP− population  analysis revealed that, although the vast majority of the DNA was present in the GFPpulation . We thenpulation . Expresshis time . However Relative mRNA expression and DNA amount of UL122, UL54, and UL32 analyzed by reverse transcription-qPCR and qPCR, respectively, at the indicated times postinfection and expressed relative to day 1. Statistical significance was calculated by a one-way analysis of variance with Dunnett’s multiple-comparison test (n = 3). The error bars represent the standard errors of the means, and the asterisks indicate P values  calculated by comparison to the peak at day 4. Download FIG S2, PDF file, 0.8 MB.Kinetic analysis of HCMV DNA and RNA expression in GFPCopyright © 2018 Forte et al.2018Forte et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the − population containing quiescent viral DNA, we resorted these cells at 4 dpi into GFP-positive and -negative cells for analysis of viral DNA and RNA  th+ cells over the course of infection in Kasumi-3 cells. RNA2.7 is expressed at very high levels during lytic infection of fibroblasts  , 51–53. 51–roblasts  and at eroblasts . UL138 aroblasts . When weroblasts . The RNAroblasts . Thus, twt-GFP virus at a multiplicity of infection (MOI) of 1 to 2, flow sorted for GFP+ cells, and cultured for 14 days to establish latency. The cells were then divided into different pools and incubated for a further 3 days in the presence of 5 ng/ml TNF-α or left untreated. Relative to untreated controls, TNF-α induced statistically significant increases in viral DNA copy, RNA expression, and the RNA/DNA ratio, as well as production of infectious virus  and latently infected (B) cells for the expression of the hematopoietic progenitor marker CD34 and markers of myeloid differentiation . Download Copyright © 2018 Forte et al.2018Forte et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Binding of TNF-α to the TNFR1 receptor activates signaling cascades, which lead to activation of transcription factors that control activation of the HCMV major immediate early promoter (MIEP), including canonical NF-κB (p65/p50) –59. Beca–Previous studies showed that TNF-α induces activation of ATM  and that+ at 1 dpi. From 4 to 8 dpi, expression of all classes of viral genes was activated in this population, viral DNA was amplified, and infectious virus was produced. At later times, viral RNA expression, DNA copy number, and the RNA/DNA ratio decreased. Some lytically infected cells die after infection, and the drop in RNA/DNA ratio may be due in part to loss of these cells. However, we observed a significant decrease in RNA/DNA ratio between live GFP+ cells isolated at 1 dpi, prior to robust viral replication, and those isolated at 17 dpi, when the cells maintain viral DNA but expression of viral genes is nearly undetectable. This difference is due at least in part to transcriptional repression of viral genes during the transition from a lytic to latent infection.HCMV establishes latency in myeloid progenitor cells through heterochromatinization and repression of viral gene expression, but the timing and molecular mechanisms that control this process are not well understood. While some studies have shown that viral gene expression is silenced immediately after infection , 46, oth+ HPCs, previous studies have also shown activation of viral gene expression in HPCs prior to establishment of latency  showed that LPS-induced reactivation of latent HCMV required differentiation to a dendritic cell phenotype , 52, 78.+ hematopoietic progenitor cells, viral genomes are bound to KAP-1 and that KAP-1 is required to establish latency through heterochromatinization and transcriptional silencing  . ATM is 81–wt-GFP . Fluorescently conjugated monoclonal antibodies used in this study are listed in wt-GFP  viral st10.1128/mBio.01560-18.1TEXT S1 TEXT S1, PDF file, 0.1 MB.Supplemental methods. Download Copyright © 2018 Forte et al.2018Forte et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the wt-GFP (+ and GFP− cells were sorted on a FACSAria-6 laser cell sorter (BD Biosciences). Infected cells were cultured for 13 days to establish latency, with medium changes every 2 to 3 days. For reactivation studies, cells were treated with 5 ng/ml TNF-α at 14 dpi for 3 days at 37°C. A 50% tissue culture infective dose (TCID50) assay was used to determine the infectious titer of infected cell supernatants.Kasumi-3 cells were infected with HCMV strain TB40/Ewt-GFP  (MOI of wt-GFP . GFP+ anDNA was isolated with the Arcturus Pico pure DNA extraction kit (Thermo Fisher Scientific) as recommended by the manufacturer. RNA was purified with the Direct-zol RNA miniprep kit (Zymo Research) with a DNase digestion step according to the manufacturer’s instructions.CT−ΔΔ) method, using predesigned assays (Life Technologies) for RNase P  or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively, as the normalization controls. This method was validated by determining that the efficiencies of the target and reference genes were approximately equal  for each dilution were calculated and plotted against the log nanograms of DNA. Download FIG S5, PDF file, 0.1 MB.Analysis of the efficiencies of amplification of viral genes versus RNase P. Viral genes and the cellular gene RNase P were amplified in samples prepared from serial dilutions of DNA isolated from lytically infected MRC-5 fibroblasts. The ΔCopyright © 2018 Forte et al.2018Forte et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01560-18.7FIG S6 CT values  for each dilution were calculated and plotted against the log nanograms of cDNA. Download FIG S6, PDF file, 0.1 MB.Analysis of the efficiencies of amplification of viral RNAs versus GAPDH. Viral RNAs and cellular GAPDH RNA were amplified in samples prepared from serial dilutions of cDNA prepared from RNA isolated from lytically infected MRC-5 fibroblasts. The ΔCopyright © 2018 Forte et al.2018Forte et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01560-18.8FIG S7 CT values ± standard deviation for GAPDH at various times after infection. n = 4. Download FIG S7, PDF file, 0.1 MB.Validation of GAPDH as a normalization control in HCMV-infected Kasumi-3 cells. Data show average Copyright © 2018 Forte et al.2018Forte et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Cells were stained with antibodies specific for CD34, CD64, CD14, CD15, CD11c, CD1c, and IE1/2 (CH160)  as descrKasumi-3 cells were treated or not treated with 5 ng/ml TNF for 30 min at 37°C, fixed, permeabilized, and washed according to previously described protocols , 88. Cel10.1128/mBio.01560-18.9FIG S8 FIG S8, PDF file, 0.9 MB.Antibody staining validation. (A) Representative flow cytometric analysis of HeLa cells, untreated (red) or treated with human TNF-α (20 ng/ml) and calyculin A (100 nM) for 15 min (blue), using phospho-NF-κB p65 (Ser536) rabbit monoclonal antibody and total NF-κB p65. (B and C) Representative flow cytometric analysis of HCT116 treated with 200 nM newborn calf serum (NCS), using phospho-ATM (S1981), phospho-KAP-1 (S824) monoclonal antibody, ATM, and total KAP-1 monoclonal antibody (blue) compared to untreated control cells (red). Download Copyright © 2018 Forte et al.2018Forte et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


Existing photoresists for 3D laser lithography that can be removed after development in a subtractive manner typically suffer from harsh cleavage conditions. Here, we report chemoselectively cleavable photoresists for 3D laser lithography based on silane crosslinkers, allowing the targeted degradation of 3D printed microstructures under mild conditions. Three bifunctional silane crosslinkers carrying various substitutions on the silicon atom are synthesized. The photoresists are prepared by mixing these silane crosslinkers with pentaerythritol triacrylate and a two-photon photoinitiator. The presence of pentaerythritol triacrylate significantly enhances the direct laser written structures with regard to resolution, while the microstructures remain cleavable. For the targeted cleavage of the fabricated 3D microstructures, simply a methanol solution including inorganic salts is required, highlighting the mild cleavage conditions. Critically, the photoresists can be cleaved selectively, which enables the sequential degradation of direct laser written structures and allows for subtractive manufacturing at the micro- and nanoscale. Subtractive manufacturing of microstructures is important for many applications, yet photoresists for 3D laser lithography allow only removal after development under harsh cleavage conditions. Here, the authors introduce a set of chemoselective cleavable photoresists allowing the orthogonal cleavage of microstructures under mild conditions. This light-based micro- and nanoscale printing method is based on a multi-photon polymerization of a photoresist employing a femtosecond laser, in which two  photons are absorbed simultaneously. Due to the nonlinearity of the multi-photon process, the chemical reaction occurs exclusively within the focal spot of the laser and allows for locally defined crosslinking. Thus, DLW is capable of fabricating complex 3D structures on the submicron length scale. This high resolution—in some instances sub-diffraction resolution by exploiting STED principles8—is particularly attractive for applications where sophisticated structures with high precision are required, including optical metamaterials, biomedicine, microfluidics, and microelectronic12. To date, there exists no other 3D manufacturing approach on the submicron length scale that would be even remotely as versatile.The direct structuring of soft matter in three dimensions via direct laser writing (DLW), also referred to as 3D laser lithography or 3D laser printing, has revolutionized the field of micro-optics4. Classically, 3D microstructures obtained via DLW are irreversibly crosslinked into a permanent form. For many applications, however, it is important that the 3D structure consists of a material, which is removable or replaceable at a later stage. Such an approach, which has also been termed subtractive manufacturing, is particularly interesting for applications where natural damage results in a short lifetime and material parts need to be replaced. Furthermore, direct laser written 3D structures have been used as templates for complex yet well-controlled architectures of inorganic materials or for the design of flying features in specific structures13. However, existing resists suffer from incomplete template removal and distortion of cavities due to harsh cleavage conditions. Thus, progress in these research areas has been limited by the provision of appropriate degradable photoresists.An important research field for current and future applications of DLW is to extend the range of available functional photoresists14. The next frontier in cleavable photoresist design is the ability of the resulting structures to be removed by orthogonal chemical triggers, allowing the targeted removal of specific material elements within a structure. Ideally, the number of selectively removable parts in a 3D structure should be as large as possible enabling the fabrication of previously unknown classes of multifunctional and complex materials. Here, we introduce a class composed of three such photoresists capable of 3D laser lithography, based on chemospecifically addressable silane linkers. We take inspiration from linkers employed in the realm of degradable biomaterials15, and demonstrate that well-defined 3D microstructures become accessible. Critically, we show that completely selective cleavage can be achieved, with the individual cleavage conditions not affecting the fabricated structures written with disparate material properties. As a proof of principle, we report the controlled degradation of three individual microstructures on the same substrate in a sequential manner.It is noteworthy that the availability of photoresists for 3D laser lithography that can be cleaved on demand is still in its infancy and only recently we have introduced a first photoresist leading to microstructures that can be degraded upon a defined chemical trigger17. The stability of silyl ethers, i.e., the ease of cleavage, depends on the substituent on the silicon atom. In general, sterically bulky substituents on the silicon atom decrease the rate of cleavage. As a result, triethylsilyl ethers are more stable than trimethylsilyl ethers, yet less stable than triisopropylsilyl ethers towards acid or base hydrolysis. This difference in silyl ether stability allows selective deprotection of individual hydroxyl groups in the same molecule, which is commonly employed in the organic synthesis of natural products. In analogy to established strategies for degradable soft materials, we prepared crosslinkers with labile linkages based on silyl ethers that undergo cleavage under mild yet specific conditions. Notably, we have synthesized acrylamide-based crosslinkers to introduce additional physical crosslinks, i.e., hydrogen bonds. It has been shown that networks composed of amide bonds exhibit much higher stiffness and stability compared to their ester analogs19. This aspect is particularly beneficial for true 3D nano- and microstructures having freely suspended segments. In addition to physical crosslinks, the degradation of DLW structures benefits from the higher hydrophilic character of amide bonds20. It was observed that degradation of DLW structures is slow due to the high crosslinking density14. Increasing the hydrophilicity allows for greater swelling of written structures in polar solvents as here employed, associated with greater accessibility of the cleavable bonds. Figure 1H and 13C nuclear magnetic resonance (NMR) spectroscopy . PETA is a commonly used multi-functional monomer for DLW22. Figure 24. Because all polymer lines were fabricated with the same writing conditions, we assume that an increase in the number of polymerizable groups per molecule translates to a higher crosslinking density of 3D structures allowing for thinner lines. In order to evaluate whether this assumption is correct, we added PETA to the photoresist to increase the crosslinking density. It is important to note that we kept the PETA amount at 2.5 mol% to retain the cleavable properties of the direct laser written structure. We observed that when the PETA concentration is above 2.5 mol%, non-cleavable 3D structures were fabricated -1-[4-(morpholinyl) phenyl)]-1-butanone) and employed for the fabrication of line patterns to determine the minimum feature size . It is advantageous for a DLW photoresist to support a high spatial resolution and small feature sizes. All experimental DLW was carried out with a commercially available 3D DLW lithography system  using an oil-immersion configuration. Line patterns were fabricated with a writing speed of 50 μm s4. All woodpiles were fabricated at identical writing conditions as for the line pattern. Figure a = 800 nm was fabricated. This woodpile showed a moderate level of shrinkage  a saturated solution of K2CO3 in MeOH at room temperature (RT), and (3) a saturated solution of KF in MeOH at RT. The cleavage of the 3D microstructures was visualized by scanning electron microscopy (SEM) and optical microscopy  a saturated solution of NaHCO a satura27. First, time-lapse images using optical microscopy were taken at different times in order to monitor the degradation process .Once the unique selectively of our cleavable photoresists was proven, we fabricated MSEA-, ESEA-, ISEA-, and PETA-based model structures on a single glass substrate, and degraded them in a consecutive manner as shown in Fig. 3, K2CO3, and KF, in methanol. The degradation process was visualized by scanning electron and optical microscopy, showing rapid and complete removal independent of the used photoresist. Our degradation studies demonstrate the unique chemospecificity of our pioneered cleavable photoresist. MSEA-based structures can be cleaved, while ESEA- and ISEA-based structures remain intact. Furthermore, ESEA- and ISEA-based structures are orthogonal to each other. Both photoresists can be selectively cleaved one over another, and vice versa. In order to emphasize the chemospecificity of our set of photoresists, we degraded MSEA-, ESEA-, and ISEA-based 3D microstructures on a single glass substrate in three consecutive steps. Critically, our reference 3D microstructure made of PETA remained unaffected during these cleavage steps highlighting the mild cleavage conditions. Due to the mild, efficient and selective nature of the cleavage process, we submit that the class of photoresists presented here hold large potential and will allow for the fabrication of a variety of complex and multifunctional 3D nano- and microstructures that are presently inaccessible using current state of the art photoresists and/or subtractive manufacturing methodologies. In addition, we anticipate the fabrication of multi-materials using our photoresist system can be expanded to other 3D printing technologies, including stereolithography and digital micromirror device-based projection printing.In summary, we have introduced a class of cleavable photoresists for 3D laser lithography based on labile silane crosslinkers, allowing the targeted and subsequent degradation of three different direct laser written 3D microstructures. Specifically, three silane crosslinkers with methyl, ethyl, or isopropyl as substituents on the silicon atom were prepared using a facile and rapid one-step synthesis. We observed that addition of 2.5 mol% PETA significantly enhanced the direct laser written structures with regard to resolution, while the 3D microstructures remained cleavable. For the cleavage of the 3D microstructures, we only employed inorganic salts, i.e., NaHCOd6 , ethyl acetate , Irgacure 369 (Ciba Inc.), hydroxyethyl acrylamide , pentaerythritol triacrylate , sodium bicarbonate (Sigma-Aldrich), 3-(trimethoxysilyl)propyl methacrylate , potassium fluoride (Sigma-Aldrich), potassium carbonate (Sigma-Aldrich), triethylamine , MeOH .All chemicals were used without further purification. Dichlorodimethylsilane , dichloromethane , diethyldichlorosilane , diisopropyldichlorosilane , dimethylformamide , dimethylsufoxide-1H NMR) spectra were recorded on a Bruker AM 400 (400 MHz) spectrometer. Chemical shifts are expressed in parts per million (ppm) and calibrated on characteristic solvent signals as internal standards. Carbon nuclear magnetic resonance (13C NMR) spectra were recorded on a Bruker AM 400 (101 MHz) spectrometer.Proton nuclear magnetic resonance  mass spectrometer  equipped with a HESI II probe. Calibration of the instrument was carried out in the m zSEM images were recorded using a Zeiss Supra 55VP. All samples were sputter coated with a 10 nm gold layer.Time-lapse microscopy was carried out on a Zeiss Axiophot microscope.1H NMR, 13C NMR, and ESI MS) for all molecules see Supplementary Figs. For the characterization data (3) (13 mL) and 40 mL dichloromethane (DCM) were added. After stirring for 10 min, dichlorodimethylsilane  in 7 mL DCM was added dropwise and allowed to stir overnight for 12 h at ambient temperature. DCM was removed under reduced pressure and the crude product was purified by column chromatography . After the residual solvent was removed under reduced pressure, MSEA was obtained as a colorless liquid. Yield: 1.2 g (54%).To a 100 mL Schlenk round-bottom flask equipped with a magnetic stirrer hydroxyethyl acrylamide (HEA) , triethylamine : δ (ppm) = 0.07 , 3.24 , 3.65 , 5.58 , 6.07 , 6.24 , 8.15 13C NMR : δ (ppm) = −3.18 (CH3), 40.94 (CH2), 60.59 (CH2), 125.06 (CH2), 131.68 (CH), 164.73 (CO)−1) calculated for C12H22N2O4Si, [M]+ = 286.1343, [M + Na]+ = 309.1241; found [M + Na]+ = 309.1241MS (m z3 (9.5 mL) and 40 mL DCM were added. After stirring for 10 min, dichlorodiethylsilane  in 7 mL DCM was added dropwise and allowed to stir overnight for 12 h at ambient temperature. DCM was removed under reduced pressure and the crude product was purified by column chromatography . After the residual solvent was removed under reduced pressure, ESEA was obtained as a colorless oil. Yield: 3.27 g (62%).To a 100 mL Schlenk round-bottom flask equipped with a magnetic stirrer HEA , NEt1H NMR : δ (ppm) = 0.56 , 0.9 , 3.25 , 3.67 , 5.57 , 6.07 , 6.24 , 8.14 13C NMR : δ (ppm) = 3.21 (CH3), 6.28 (CH2), 41.03 (CH2), 60.69 (CH2), 125.07 (CH2), 131.68 (CH), 164.76 (CO)-1) calculated for C12H22N2O4Si, [M]+ = 314.1656, [M + Na]+ = 337.1554; found [M + Na]+ = 337.1449MS (m z3 (9 mL) and 50 mL DCM were added. After stirring for 10 min, dichlorodiisopropylsilane  in 7 mL DCM was added dropwise and allowed to stir overnight for 12 h at ambient temperature. DCM was removed by rotary evaporation under reduced pressure and the product was isolated by column chromatography . After the residual solvent was removed under reduced pressure, ISEA was obtained as a colorless oil. Yield: 1.8 g (65%).To a 100 mL Schlenk round-bottom flask equipped with a magnetic stirrer HEA , NEt1H NMR : δ (ppm) = 0.98 , 3.27 , 3.72 , 5.57 , 6.07 , 6.24 , 8.13 13C NMR : δ (ppm) = 11.36 (CH), 17.11 (CH3), 41.06 (CH2), 61.02 (CH2), 125.04 (CH2), 131.68 (CH), 164.79 (CO)−1) calculated for C12H22N2O4Si, [M]+ = 342.1969, [M + Na]+ = 365.1867; found [M + Na]+ = 365.1864MS propyl methacrylate in toluene (0.5 mM). Subsequently, the substrates were ultrasonicated in toluene (5 min) and acetone (5 min) to remove physisorbed silane.−1 and a laser power of 5 mW. 3D microstructures were fabricated with writing speeds ranging from 1 mm s−1 to 10 mm s−1 and a laser power close to the damage threshold. For additional information about the writing conditions see Supplementary Figs. The commercially available DLW setup Photonic Professional GT  was used. The instrument was equipped with a high-numerical aperture  oil immersion objective lens. A mode-locked and frequency-doubled Er-doped fiber laser was employed, emitting femtosecond pulses at 780 nm center wavelength. All employed glass substrates were silanized. Line patterns and woodpiles were fabricated with a writing speed of 50 μm sData supporting the findings of this study are available within the article and its Supplementary Information file, and from the corresponding authors upon reasonable request.Supplementary InformationPeer Review File


Oxidative stress was evaluated by reduced glutathione content, reactive oxygen species formation, mitochondrial dysfunction, and gene expression of heme oxygenase-1b and mitochondrial superoxide dismutase; organ injury, iron accumulation, and autophagy were measured by microscopical, cytofluorimetric analyses, and inductively coupled plasma‒optical mission Spectrometer (ICP-OES). Our results showed that FAC results in a significant increase of tissue iron accumulation, oxidative stress, and autophagy and such detrimental effects were reversed by ALA treatment. In conclusion, ALA possesses excellent iron chelating properties that may be exploited in a clinical setting for organ preservation, as well as exhibiting a good safety profile and low cost for the national health system.Iron toxicity is associated with organ injury and has been reported in various clinical conditions, such as hemochromatosis, thalassemia major, and myelodysplastic syndromes. Therefore, iron chelation therapy represents a pivotal therapy for these patients during their lifetime. The aim of the present study was to assess the iron chelating properties of α-lipoic acid (ALA) and how such an effect impacts on iron overload mediated toxicity. Human mesenchymal stem cells (HS-5) and animals (zebrafish,  Furthermore, the authors showed that ALA also resulted in a significant increase of GSH content through the activation of the same pathway. Consistently with these observations, our results showed that iron overload following FAC treatment resulted in a significant increase in GSH content and that the concomitant treatment with ALA further increased such content when compared to FAC alone. Studies show ALA and DHLA may enhance cellular antioxidant defenses by a number of different mechanisms, including a direct antioxidant effect, and indirectly by augmenting the cellular GSH pool by increasing the expression of γ-glutamylcysteine ligase, the rate-controlling enzyme for GSH synthesis, Nrf2 activation [Previous studies suggested that the accumulation of mitochondrial iron contributes to the decay of mitochondria and decreases life-sustaining functions, such as adenosine triphosphate (ATP) production, intracellular Capoptosis ,24. Our poptosis . Previoupoptosis ,25,26. Ith Fe II ,31,32,33th Fe II . Our resth Fe II  and its th Fe II  showed ttivation . In this® CRL-11882™), was maintained in Dulbecco’s Modified Eagle Medium-high glucose , supplemented with 10% fetal bovine serum (FBS) , 10,000 U/mL penicillin, and 10 mg/mL streptomycin. Cells were grown to confluence and used from passages 4 through 7. Iron overload was obtained by ferric ammonium citrate  (120 µg/mL for 24 h). In a separate set of experiments, cells were pretreated (2 h) with 20 µg/mL of ALA to evaluate its antioxidant and chelating properties.Human mesenchymal stromal cell line, HS-5  was performed according to the manufacturer’s instructions. Briefly, tissue sections were passaged in distilled water and stained with Perls staining  for 20 min. Sections were then rinsed in distilled water, dehydrated in ascending alcohols, cleared in xylene, and finally mounted for microscopic analysis.To determine the intracellular ROS generation (mainly superoxide), cells were stained with 5 mM dihydroethidium  in PBS for 30 min at 37 °C. Fluorescence  was determined by fluorescence-activated cell sorting  . N-acetyλ = 412 nm . Measurements were performed in triplicate.Intracellular content of reduced glutathione (GSH) was measured using a spectrophotometric assay based on the reaction of thiol groups with 2,2-dithio-bis-nitrobenzoic acid (DTNB) at Cells were grown directly on coverslips before immunofluorescence. After washing with phosphate-buffered saline (PBS), cells were fixed in 4% paraformaldehyde  for 20 min at room temperature. After fixation, cells were washed three times in PBS for 5 min and blocked in Odyssey Blocking Buffer for 1 h at room temperature. Subsequently, the cells were incubated with primary antibody against HO-1  at a dilution of 1:200 and LC3IIb  and TUFM  at a dilution of 1:200, overnight at 4 °C. Next day, cells were washed three times in PBS for 5 min and incubated with secondary antibodies: FITC  at a dilution of 1:200, and FITC  at a dilution of 1:200 and PE-cojugated  for 1 h at room temperature. All antibodies were diluted in Odyssey Blocking Buffer. The slides were mounted with medium containing DAPI  to visualize nuclei. The fluorescent images were obtained using a Zeiss Axio Imager Z1 Microscope with Apotome 2 system .Autophagic cells and formation of acidic vesicular organelles were quantified by FACS following acridine orange staining. Briefly, 20 µg/mL solution of acridine orange in appropriate buffer were added to100 µL of cell suspension and incubated at room temperature for 20 min. Finally, the appropriate isotopic control was also included and labeled cells were acquired using a Beckman Coulter FC-500 flow cytometer ,34.5 cells/mL per plate in a 6-well plate and incubated for 24 h. Cellular mitochondrial membrane potential was assayed using the Muse MitoPotential Kit according to the user’s guide. A total of 1 × 105 cells were collected by centrifugation  and washed with PBS. The supernatant was then removed and the cell pellets were stained with the Muse MitoPotential Kit  for 25 min at 37 °C. The data was analyzed using the Muse™ Cell Analyzer Assay.Cells were seeded at 1 × 10TM 8% Bis- Tris Plus Invitrogen  followed by electrotransfer to nitrocellulose membrane iBlot® Gel Transfer Stacks Nitrocellulose Regular  using iBLOT Invitrogen transfer . Subsequently, the membrane was blocked in Odyssey Blocking Buffer  for 1 h at room temperature. After blocking, the membrane was washed three times in PBS for 5 min and incubated with primary antibodies against HO-1 (1:1000)  and β-actin (1:1000)  overnight at 4 °C. The following day, membranes were washed three times in PBS for 5 min and incubated with Infrared anti-mouse IRDye800CW (1:5000) and anti-rabbit IRDye700CW secondary antibodies (1:5000) in PBS/0.5% Tween-20 for 1 h at room temperature. All antibodies were diluted in Odyssey Blocking Buffer. The blots were visualized using an Odyssey Infrared Imaging Scanner  and protein levels were quantified by densitometric analysis of antibody responses. Data were normalized to the total protein levels of β-actin.Briefly, for western blot analysis, 50 µg of protein was loaded onto a 12% polyacrylamide gel Bolt n = 10, for each group) were used for this study. Fishes were tested to be free from Pseudoloma neurophilia, Pseudocapillaria tomentosa, Mycobacterium spp., and Edwardsiella ictalurias determined by twice-yearly sentinel monitoring. Fishes were housed at a density of 5 fishes per tank in mixed-sex groups in 2.5 L tanks on a recirculating system in 28 °C water in a room with a 14:10 h light:dark cycle. System water was carbon-filtered municipal tap water, filtered through a 20 µm pleated particulate filter, and exposed to 40 W UV light [Adult wildtype AB zebrafish (UV light . Standarn = 10) was randomly assigned to a different treatment condition. FAC 120 µg/mL, ALA 20 µg/mL, and DFO (deferoxamine) 131 µg/mL , the chemical agents tested were introduced into a static 2 L tank filled with system water obtained from the main recirculating system. A behavioral control group of untreated fish housed under the same conditions as the experimental groups was tested in parallel. Experimental fish were monitored up to 48 h.Each experimental group  was used for real-time RT-PCR experiments in a FAST-HT 7900 Real Time PCR System (Applied Biosystem) under the following conditions: 95 °C for 3 min, 95 °C for 15 s/60 °C for 30 s (40 cycles). To ensure that only a single product was amplified, all real-time RT-PCR experiments were followed with a melt-curve analysis. For all experiments, GAPDH RNA was used as a ‘housekeeping’ gene for normalization. GAPDH RNA levels were not affected by any pro-oxidant treatments. The following sense and antisense primers (5′→3′) were used: Zebrafish HMOX1b sense: 5′-GCAGTGATCTGTCTGAACAG-3′, antisense 5′-GCTTGTACTGTGTTTGTGTG-3′; zebrafish mtSOD sense: 5′-ATGGCTTTAACATATCCGG-3′, antisense: 5′-TTCAGGGCTCAGGCTGG-3′; zebrafish FPN1: 5′-GGCCAGCACAGCTATGTC-3′ antisense: 5′-GCCAGAATGTTGGTCAAC-3′.RNA was extracted from dissected liver, intestine, gills, and heart by using TRIzol (Invitrogen). RNA was measured, and 1 µg of RNA with a 260/280 ratio>1.8 was used for reverse transcription by using a high-capacity cDNA kit (Applied Biosystem). PerfeCTaZebrafish intestinal mucosa tissues were collected and fixed in 10% buffered-formaldehyde; after an overnight wash, specimens were dehydrated in graded ethanol and paraffin-embedded, preserving their anatomical orientation. Three to four micrometer thick sections were obtained according to routine procedures, mounted on sialane-coated slides and air-dried. Slides were dewaxed in xylene, hydrated using graded ethanol, and stained for histological studies (Hematoxylin and Eosin and Perls staining).® for trace analysis (Carlo Erba). After digestion, ultra-pure water (Merck) was added to the samples up to a volume of 2 mL and iron (Fe) was quantified with an Inductively Coupled Plasma‒Optical Emission Spectrometer . Standards for the instrument calibration were prepared on the basis of mono-element certified reference solution ICP Standard (Merck) in the same acid matrix of the samples as well as the calibration blank. The method detection limits (MDL) estimated with 10 blanks was 5.4 µg/L, calculated according to the following equation: MDL= One-tailed student’s t-test  × Sr. A laboratory-fortified matrix (LFM) was determined as quality control and a recovery rate of 111% was obtained.The samples (cellular pellet or homogenate) were digested overnight with 200 µL of Nitric Acid 65%, Suprapurp < 0.05.Results are expressed as the means ± standard deviation (SD) of at least three independent experiments. Statistical analysis was carried out by one-way analysis of variance using the GraphPad Prism 4.0 software . Differences were considered significant at In conclusion, ALA may represent a valuable tool to be used in iron overload conditions because of its pleiotropic mechanisms of action, impacting on various, important pathophysiological mechanisms involved in cellular dysfunction and organ injury.


Antrodia cinnamomea, a well-known traditional medicine used in Taiwan, is a potent anticancer drug for colorectal cancer, but the upstream molecular mechanism of its anticancer effects remains unclear. In this study, A. cinnamomea extracts showed cytotoxicity in HCT116, HT29, SW480, Caco-2 and, Colo205 colorectal cancer cells. Whole-genome expression profiling of A. cinnamomea extracts in HCT116 cells was performed. A. cinnamomea extracts upregulated the expression of the endoplasmic reticulum stress marker CHOP and its downstream gene TRB3. Moreover, dephosphorylation of Akt and mTOR as well as autophagic cell death were observed. Gene expression and autophagic cell death were reversed by the knockdown of CHOP and TRB3. Autophagy inhibition but not apoptosis inhibition reversed A. cinnamomea-induced cell death. Finally, we demonstrated that A. cinnamomea extracts significantly suppressed HCT116 tumour growth in nude mice. Our findings suggest that autophagic cell death via the CHOP/TRB3/Akt/mTOR pathway may represent a new mechanism of anti-colorectal cancer action by A. cinnamomea. A. cinnamomea is a new CHOP activator and potential drug that can be used in colorectal cancer treatment. Although there are many treatment strategies for colorectal cancer, such as chemotherapy, surgery, radiation therapy, targeted therapy, and immunotherapy, nearly 0.7 million people are estimated to have died from colorectal cancer in 2012 worldwide1. For this reason, finding new drugs against colorectal cancer is urgent.Colorectal cancer is one of the most common cancers (an estimated 1.36 million new cases occurred in 2012) worldwide3. Paclitaxel is one of the most well-known natural products in cancer treatment. In addition, previous studies indicated that many natural products, such as curcumin, epigallocatechin gallate, and shikonin, are potent drug candidates for cancer treatment5.Over the past decades, natural-source cancer drugs have sufficiently served to combat cancer, and over 60% of the anticancer agents approved since 1940 that are available for use can be traced to a natural productAntrodia cinnamomea , famed as “the ruby of the forest” in Taiwan, is a rare mushroom that grows only on the native Taiwanese tree Cinnamomum kanehirai6. A. cinnamomea has been used in traditional medicine for hundreds of years to treat discomforts caused by alcohol consumption, exhaustion, diarrhoea, abdominal pain, hypertension and cancer8. Several researchers have reported on the different biological activities of A. cinnamomea, which include anticancer activities against liver cancer9, colorectal cancer10, lung cancer11, breast cancer12, and leukaemia13; anti-inflammatory and immunomodulatory effects14; hepatoprotective activities15; and antioxidant activities16.A. cinnamomea showed potent anticancer activities in vitro18 and in vivo20. Treatment with A. cinnamomea extracts alone or combination with amphotericin B induced cell cycle arrest in HT29 human colorectal cancer cells20. Treatment with SY-1, a compound purified from A. cinnamomea, induced G0/G1 cell cycle arrest in Colo205 human colorectal cancer cells19. Treatment with A. cinnamomea also caused HT29 and Colo205 cells to undergo apoptotic cell death17. Furthermore, antroquinonol, a derivative of A. cinnamomea, suppresses colon cancer stem cell-like properties via targeting the PI3K/AKT/β-catenin signalling pathway18.In colorectal cancer, A. cinnamomea in colorectal cancer, more evidence of the pharmacological mechanisms at the molecular level remains necessary for better understanding. Microarray technology and the associated bioinformatic tools have become widely used methods to investigate the molecular mechanisms of traditional Chinese medicines22. According to microarray gene expression profiles, Si-Wu-Tang, a traditional Chinese medicinal formula utilized for menstrual discomfort relief, was identified as a Nrf2 activator and suggested to be used as a nontoxic chemopreventive agent23. Gene expression profiles indicated that VI-28, a traditional Chinese medicinal formula originally designed to be an anti-aging health product, was shown to regulate innate and adaptive immune gene expression24. Microarray analysis results showed that a new immunomodulatory protein, ACA, purified from A. cinnamomea exhibited TLR2-dependent NF-KB activation in murine macrophages25. We presume that whole-genome expression profiling can provide deep insights into the molecular mechanisms mediating the anticancer activity of A. cinnamomea in colorectal cancer.Although the expression of several genes has been shown to mediate the effects of A. cinnamomea can help fight against colorectal cancer and identify the molecular mechanisms underlying its anticancer activity. First, we evaluated the antitumour activity of A. cinnamomea in five colorectal cancer cell lines. Then, next-generation sequencing (NGS) was used to analyse gene expression changes after A. cinnamomea treatment. Finally, we examined the expression of genes identified using whole-genome expression profiling and confirmed the molecular mechanisms underlying the anticancer effects of A. cinnamomea in colorectal cancer.The aims of this work were to examine whether A. cinnamomea fruiting bodies used in this study are shown in Fig. A. cinnamomea extracts AC, ACF1, ACF2, and ACF3 were obtained -5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) assay was performed to evaluate its cytotoxic role on HCT116, HT29, SW480, Caco-2 and Colo205 human colorectal cancer cells. As shown in Fig. 50 values of 33.21 ± 13.25, 49.28 ± 34.23, 98.53 ± 10.63, 84.89 ± 7.94, and 55.28 ± 10.53 μg/ml, respectively. Direct trypan blue exclusion cell counts were performed to confirm the results of the MTS assay. The cell count results for HCT116 cells treated with A. cinnamomea extracts were consistent with the IC50 values determined by the MTS assay . The 15 top differentially expressed genes are listed in Table 27. The genes listed in Table 31. After ACF2 treatment, NGS analysis showed no statistically significant difference in these genes except for tribbles pseudokinase 3 , which was increased by 6.48-fold after ACF2 treatment  induction33. However, as shown in Table 34, and negative regulation of the Akt-mTOR axis activates Ulk1. After the release of ULK1 from inactive mTOR, autophagosome formation is induced29. HCT116 cells were treated with increasing concentrations of ACF2 for 24 h, and phospho-Akt, total Akt, phospho-mTOR, and total mTOR were detected by Western blot analysis. As shown in Fig. 35. We evaluated the autophagic process via detecting LC3 conversion by Western blot analysis. As shown in Fig. To investigate the anticancer mechanism of ACF2, we analysed molecules downstream of CHOP and TRB3 at the protein level. TRB3 is involved downstream of the CHOP-mediated apoptosis pathwayTo verify our hypothesis that the anticancer mechanism of ACF2 in colorectal cancer is due to CHOP/TRB3/Akt/mTOR-mediated autophagy, CHOP and TRB3 small interference RNA (siRNA) were employed to suppress CHOP and TRB3 expression in HCT116 cells. HCT116 cells were transfected with siRNA targeting human CHOP or TRB3 or control siRNA. After 48 h of stimulation, HCT116 cells were treated with 75 μg/mL ACF2 for 24 h. Compared with non-transfected and control siRNA-transfected cells, CHOP siRNA clearly decreased ACF2-induced CHOP protein overexpression Fig. . TRB3 si29, we next investigated the role of autophagy in the anticancer mechanism of ACF2. The MTS assay was used to evaluate the viability of HCT116 cells. As shown in Fig. 36. 5-FU obviously increased the protein expression levels of cleaved caspase3. Cleaved caspase3 was not observed after ACF2 treatment  for 15 days. As shown in Fig. p < 0.005). On the sixth day after treatment, the tumour volume in the 200 mg/kg ACF2 treatment group was significantly decreased compared with that in the control group (p < 0.05). However, after 9, 12 and 15 days of administration, the tumour volume in the 200 mg/kg ACF2 treatment group increased, and there was no statistically significant difference compared with the control group. Changes in tumour volume in the 100 mg/kg ACF2 treatment group were similar to those in the control group, with the two groups presenting no significant difference at any time point. As shown in Fig. p < 0.005). The body weight of each mouse was observed during the experiments. Importantly, there were no significant decreases in body weight in the ACF2 treatment groups at any of the dosages , was originally identified in response to UV-induced DNA damage31. CHOP has been shown to trigger apoptotic cell death in lung, colorectal, prostate, and ovarian cancer44. On the other hand, CHOP also induces the transcriptional upregulation of many autophagic genes, such as SQSTM1, NBR1, and members of the ATG family27. In our study, the expression of CHOP and its downstream target TRB3 were upregulated by A. cinnamomea. TRB3 can promote apoptosis via Akt dephosphorylation, followed by BBC3 induction33. TRB3 can also promote autophagy via Akt and mTOR dephosphorylation29. Akt and mTOR dephosphorylation were observed after A. cinnamomea treatment, but the mRNA expression of BBC3 was not increased. Conversion of the autophagy marker LC3 was increased by A. cinnamomea treatment 53. Previous studies showed that CXCL8 promoted cell proliferation via the activation of Akt and inhibited apoptosis in colorectal cancer56. In response to the chemotherapy drug 5-fluorouracil, HCT116 cells upregulated the expression of CXCL8, CXCR1, and CXCR2; CXCR1 and CXCR2 are receptors of CXCL857. In our study, CXCL1, CXCL2, CXCL3, and CXCL8 were upregulated after A. cinnamomea treatment. However, our results indicated that A. cinnamomea has anticancer effects both in vitro and in vivo  were soaked in 3000 mL of ethanol for 120 h. The samples were extracted three times using filter paper . The combined filtrates collected from the three extractions were evaporated to dryness under vacuum. The ethanol extract  from the powdered fruiting bodies of A. cinnamomea (100 g) was applied to a Diaion HP-20 column (460 g) and washed with 50% ethanol, 95% ethanol, and ethyl acetate to yield 3 fractions: ACF1 , ACF2 , and ACF3 . The scheme depicting the methodology is shown in Fig. mea Fig.  was cult2 in air and passaged every 2 days to obtain exponential growth. All five cell lines were maintained in culture no more than 15 passages. All culture reagents were sourced from Gibco .Five human colorectal cancer cell lines  were obtained from the Bioresource Collection and Research Center . Dulbecco’s Modified Eagle’s Medium containing 10% foetal bovine serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin served as the cell culture medium throughout all experiments. The cells were maintained at 37 °C in a humidified atmosphere of 5% CO4 cells per well and treated with increasing concentrations of AC, ACF1, ACF2 or ACF3 for 48 h. The cytotoxic effects of AC, ACF1, ACF2, and ACF3 on colorectal cancer cells were assessed by the MTS assay . The absorbance was measured at 490 nm with a microplate reader. The IC50 values were calculated using GraphPad Prism 5.HCT116, HT29, SW480, Caco-2, and Colo205 cells were seeded in 96-well plates at a density of 5 × 10ACF2 was further separated using a Thermo HPLC system  with an Eclipse XDB-C18 analytical column . Gradient elution was performed with mobile phases A (0.1% formic acid aqueous solution) and B (acetonitrile). The gradient elution profile was as follows: 0–3 minutes, 70% A and 30% B to 60% A and 40% B (linear gradient); 3–15 minutes, 60% A and 40% B to 42% A and 58% B (linear gradient); 15–21 minutes, 42% A and 58% B (isocratic); 21–26 minutes, 42% A and 58% B to 35% A and 65% B (linear gradient); 26–35 minutes, 35% A and 65% B to 0% A and 100% B (linear gradient); and 35–50 minutes, 0% A and 100% B (isocratic). The flow rate was 0.8 mL/min, and the wavelength of the photodiode array detector was 254 nm.6 cells per plate and treated with 50 μg/ml ACF2 for 24 h. Total RNA was extracted using the RNeasy Mini Kit .HCT116 cells were seeded in 10 cm plates at a density of 2 × 1058. A total of 20 million reads were randomly selected for quality control with Bowtie 2 and mapped to Genome Reference Consortium Human Build 37/hg1959. Differential expression was analysed between different subgroups using Bioconductor EBseq. (version 1.1.6)60. A false discovery rate of 0.05 was applied to the resulting P values to correct for multiple hypothesis testing. T-tests were employed to screen genes differentially expressed between control and ACF2-treated cells, with a threshold of P < 0.05 and a posterior probability fold change >2.An RNA sequencing library was generated using the TruSeq Stranded mRNA Library Prep Kit according to the user’s instruction manual . Briefly, the poly-A containing mRNA was isolated from total RNA using poly-T oligo-attached magnetic beads and fragmented and primed for cDNA synthesis. After double-strand cDNA synthesis, adenylation of the 3′-end, and multiple indexing adapter ligation, DNA purification with magnetic beads and PCR selective amplification were performed. Next, the amplified library was purified, quantified, and applied for template preparation. The NextSeq 500 platform was utilized to generate 75-bp single-end sequencing reads. The initial sequence reads were generated by the Genome Analyser , and the expression values were calculated using RNA-Seq by Expectation Maximization (RSEM) for each gene , Homo.sapiens.hg19.gtf) in individual samples61, TRB3 , and GAPDH 62. Reactions were performed using the CFX Connect Real-Time PCR Detection System  with the following reaction programme: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The relative changes in gene expression were calculated by the 2−ΔΔCT method, and GAPDH served as the internal control.HCT116 cells were treated with 25, 50, and 75 μg/ml ACF2 for 24 h. Total RNA was isolated using the RNeasy Mini Kit . cDNA was synthesized from the RNA using the SuperScript III First-Strand Synthesis System  according to the manufacturer’s instructions. Quantitative real-time PCR was performed using iQ SYBR Green Supermix  with the following specific primers: CHOP HCT116 cells were treated with 25, 50, 75, 100, and 125 μg/ml ACF2 for 24 h. Total cellular protein was extracted using M-PER™ mammalian protein extraction reagent  and then quantified using the Bradford protein assay . Denatured protein was loaded onto 10–15% SDS polyacrylamide gels. Following electrophoresis, the proteins on the gel were transferred onto polyvinylidene difluoride membranes using the TE22 mighty small transfer tank . The membranes were blocked with 5% skim milk at room temperature for 30 minutes, followed by incubation at 4 °C overnight with the following primary antibodies: CHOP, TRB3, caspase3, actin , total and phosphorylated Akt (ser473), total and phosphorylated mTOR (ser2448), and LC3 . The membranes were then washed three times for 10 minutes with PBS-Tween buffer and incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies  at room temperature for 1 h. All antibodies were diluted 1:2,000. The antibody binding proteins were detected using ECL chemiluminescence substrates and then captured on the Amersham Imager 600 . The membranes were reprobed for actin protein expression to show that similar amounts of protein were loaded in each lane.32 were synthesized by Invitrogen. HCT116 cells were transfected with siRNA using Lipofectamine RNAiMAX  according to the manufacturer’s protocol.For siRNA inhibition, double-stranded RNA duplexes targeting human CHOP (5′- GCC UGG UAU GAG GAC CUG C-3′) or human TRB3 (5′-CGA GCU CGA AGU GGG CCC C-3′) and control siRNA (5′-GCG CGC UUU GUA GGA UUC G-3′)nu/nu mice were obtained from the National Laboratory Animal Center  and housed in clean specific pathogen-free (SPF) rooms. The mice were inoculated with 3 × 106 HCT116 cells suspended in PBS subcutaneously into the right flanks. Twenty-four mice were randomly divided into four groups (6 mice in each group): a control group  and 100, 200, and 400 mg/kg ACF2 treatment groups. ACF2 treatment was started on the seventh day after tumour inoculation by daily oral gavage for 15 days. The body weights of the mice were recorded every three days. Tumour volume was measured every three days using digital callipers and calculated as length × (width)2/2. The experimental protocol was approved by the Institutional Animal Care and Use Committee at National Taiwan University. All methods were carried out in accordance with relevant guidelines and regulations.Six-week-old male nudep < 0.05, **p < 0.005 and ***p < 0.0005.All experiments were performed in triplicate, and data are presented as the mean ± standard deviation. Statistical analysis was performed by the ANOVA and LSD tests using SPSS 12.0 software . P values of statistical significance are represented as *Supplementary fileDataset 1


Rabbit adipose mesenchymal stem cells were used for the purpose of studying acquisition of the chondrogenic phenotype over time at 1, 14 and 28 days after in vitro incubation with differentiation media, using nano-liquid chromatography electrospray ionization tandem mass spectrometry analysis. This was part of a preliminary study of the behavior of differentiated adipose stem cells for use in a rabbit model of laryngoplasty.The data comprise .MGF, .RAW, .MZID, and .XLSX, lists of peaks, peptides and proteins identified by nano-flow liquid chromatography electrospray ionization tandem mass spectrometry analysis upon incubation with non-differentiating (ND) or chondrogenic differentiating (CHD) media (ProteomeXchange ID PXD010236). XLSX files contain the following information: day 1 CT , day 14 ND (N = 3106 proteins), day 28 ND (N = 3116 proteins), day 14 CHD (N = 2901 proteins), and day 28 CHD (N = 2876 proteins). Proteins are characterized with respect to their − 10lgP value, percent coverage, number of total as well as unique peptides after trypsin digestion, derivatization method , average mass, and include a full description. Adipose mesenchymal stem cells (ADSCs) are fast gaining popularity for various regenerative medicine applications due to their pluripotency as well as relative use of collection and processing. This has called for increased scrutiny and standardization of protocols involved in tissue collection, differentiation and analysis . While aFive lists of proteins were generated by nLC–MS/MS analysis of rabbit ADSC incubated with non-differentiating (ND) or chondrogenic differentiating (CHD) media for 1, 14, or 28 days. List 1: day 1 (N = 3499 proteins); List 2: ND media, day 14 (N = 3106 proteins); List 3: ND media, day 28 (N = 3116 proteins); List 4: CHD media, day 14 (N = 2901 proteins); and List 5: CHD media, day 28 (N = 2876 proteins). Proteins are displayed in rows and descriptors in columns, as follows: (1) Accession Number, (2) Protein Group, (3) Protein ID, (4) − 10 logP value, (5) % Coverage, (6) No. of Total Peptides, (7) No. of Unique Peptides, (8) Derivatization Method, (9) Average Mass, and (10) Description. A Protein Group is a list of proteins sharing a significant number of identical matched peptides. − 10 logP expresses the confidence of making a protein call; high values reflect high confidence [5 cells with 50 µL of collagen followed by sedimentation at 500 g for 5 min and drop-wise addition of StemPro Chondrogenic differentiation media . This was supplemented with 5 ng/mL of TGF-β2 . Non-differentiating media consisted of the Basal Medium alone. All media was replenished every 2–3 days. Chondrogenic differentiation was confirmed by Alcian blue staining and sulfated glycosaminoglycan (s-GAG) level analysis with DNA content correction using PicoGreen . Parallel cultures in adipogenic and osteogenic differentiation media  were performed to confirm the presence of pluripotent stem cells in our samples. These data were published elsewhere [Chondrogenic differentiation was performed as previously described . BrieflyAfter 1, 14, or 28 days the cell pellets were washed ten times with PBS, fixed in 4% paraformaldehyde and processed for nLc–MS/MS analysis as previously reported . Only prThe data presented here describe acquisition of the chondrogenic phenotype by rabbit ADSCs. The data are useful for the appreciation of time-dependent cellular changes in ADSCs at 14 and 28 days of incubation with chondrogenic differentiation media. The data are also useful for a comparison across species, for example with mouse or human ADSCs, aimed at identifying patterns of similar or differential expression of proteins implicate in acquisition of the chondrogenic phenotype. The main limitations of the data are the lack of replicate measurements and time points beyond day 28. Furthermore, because of the lack of a finer temporal resolution, transient, or intermediary events are not properly captured.


Results confirmed chemical structures of two acetogenins as present in Avosafe® (AcO-avocadyne-(0) and AcO-avocadiene B-(3)), and together with seven other previously known compounds, quantified 94.74 ± 5.77% w/w of its solids as acetogenins. Safety evaluations indicated that Avosafe® was non-mutagenic and had an acute median lethal oral dose (LD50) to rats higher than the maximum concentration tested (>2000 mg·kg−1), with no signs of macroscopic abnormalities in organs. Mean body weight and hematological and biochemical parameters were normal after 14 days of a single oral dose of 2000 mg·kg−1. The results advance scientific information on the safety of avocado seed acetogenins and also generate new knowledge on profiles and concentrations of individual acetogenins found in avocado tissues  and in Avosafe®.Acetogenins are bioactive fatty acid derivatives found in avocado tissues. Their efficacy as antimicrobials has been documented and initiated interest to use them as replacements of synthetic food additives. The present work focused on evaluation of multiple analytical methodologies for detection and quantification of organic solids present in a food-grade acetogenin-enriched extract (Avosafe Persea (family: Lauraceae) +), its corresponding sodium adduct at m/z 349 ([M + Na]+), and a dimer with sodium adduct at m/z 675 ([2M + Na]+), suggesting that they might be isomers. Moreover, both compounds displayed the characteristic ion pattern of acetogenins [m/z 309 ([M − H2O]+), 291 ([M − 2H2O]+), 249 ([M + H − H2O − CH3COOH]+), and 267 ([M + H − CH3COOH]+), which corresponded to losses of 18, 36, 78, or 60 amu from molecular ion at m/z 327 ([M + H]+), respectively  + = m/z 249). As shown in 0 produced a rich pattern of peaks separated by 14 amu (from m/z 66 to 192), indicative of consecutive losses of methylene groups (CH2), which has been reported to be typical for the fragmentation of saturated alkyl side chains of annonaceous acetogenins . Th. Th50 vatoxicity . Hence,  14 days , no deat 14 days ), whereahemicals , which m−1 bw of Avosafe® and none of the treated rats died nor showed signs of toxicity after 15 days of observation. Three rats were used in the subsequent evaluations and were administered with 2000 mg·kg−1 of Avosafe®, and their clinical signs, hematology, serum biochemistry, and body weight were within normal levels and comparable to the control group, which ingested only the PG vehicle  detected no abnormalities  and Persenone A (6)) [Our results from the three different safety studies indicated that the acetogenin-enriched extract did not demonstrate signs of toxicity. Comparison of our results with prior scientific reports proved to be a difficult task, since, to the best of knowledge, only four studies have assessed biological effects of purified acetogenins in mammals (including Persin (e A (6)) ,28,56,57e A (6)) .7)  and the authors observed necrosis of the secretory mammary gland (2 to 5 days after exposure) [−1, other tissues were affected  [7) in the leaves [7) concentrations were not measured in the study that treated lactating goats and observed negative effects [7) administered in those experiments based on the quantification of Persin (7) in avocado leaves conducted by Carman and Handley (1990) [7) concentrations in avocado leaves [7) levels contained in the Guatemalan avocado leaves ingested by lactating goats [−1 bw. Even though there are some discrepancies on the horticultural race of the ’Hass’ avocado cultivar used in the present work  levels measured in the present study (7) was 55 mg·kg−1 bw. Consistently, the presumed Persin (7) exposure in the goat study (38–95 or 55 mg of Persin (7) kg−1 bw) resulted considerably higher values (3–14 times) than the dose that caused necrosis of mammary gland in mouse [7) kg−1 bw in goat (average bw of 17 kg [7) level in leaves of the Mexican varieties were up to 5-times lower than the present in Guatemalan varieties, which may partially explain the absence of detrimental effects observed when avocado leaves from the Mexican varieties were tested in the lactating goat model [In prior publications, two similar studies were conducted using lactating mice models treated with single oral doses of Persin (xposure) ,56; at dcardium) . The samcardium) , resultse leaves . Interese leaves . Persin  effects , therefoy (1990)  and concy (1990) ,59 for to leaves , we esting goats  ranged batemalan  or hybrint study , the hypin mouse ,56, whicof 17 kg ), considof 17 kg ,59. Moreat model .7) and Persenone A (6), on d-galactosamine-induced liver injury. In their studies, the purified compounds were directly administered into rats’ stomachs using a catheter (as a single dose of 100 mg·kg−1 of bw), and then 4 h later, d-galactosamine was injected intraperitoneally. Activities of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured after 22 h. Apparently, all the compounds exhibited strong liver injury suppressing activities, as reduced plasma ALT and AST activities were observed [6) (at dose of 25 mg·kg−1 bw) was administered intraperitoneally to mice, resulting in higher blood clotting times and antithrombotic activities after 24 h of exposure [Differing from the above-mentioned reports, Kawagishi and others (2001)  describeobserved . Anotherexposure .7) at 60–100 mg·kg−1 bw, in mice [−1 bw in rat for Persin (7) and Persenone A (6) [−1 bw) were 1.6–3-times lower that doses at which therapeutic effects were reported, without signs of toxicity. Data from our acute oral toxicity experiments  of Avosafe®  and Persenone A (6), respectively. In the present work Persin (7) was not tested in a pure form, however it was present in Avosafe® at concentrations that were 4–8.7-times higher than the levels previously reported to be harmful [50 in rats reported in literature for sodium nitrite was 77–130 mg·kg−1 bw [As discussed, prior literature has reported contradictory results regarding the safety of acetogenins in vivo. In summary, toxic effects were observed after the administration of purified acetogenins, particularly Persin  ,57,61. Ane A (6) , we obseeriments  estimateAvosafe® , the obs harmful ,56. Acet50 determination), which have been reviewed by international regulations and guidelines established by the Organization for Economic Cooperation and Development (OECD), the EC Commission, and government agencies in the United States (EPA and FDA) for the testing of chemicals [The current work presented various strengths in its experimental design. One advantage being that a high purity food-grade avocado seed extract of a known chemical profile (94.74% acetogenins) was studied, which was characterized and quantified using improvements in analytical and detection methodologies described in the present work. Other strengths also being the use of standardized procedures for the AMES test and for the acute oral toxicity to the rat assays  were acquired from DEQ . HPLC grade solvents  were purchased from Fisher Scientific . Analytic standards of acetogenins were purified from avocado seed in our laboratory (>97 wt% purity), as described by Rodríguez-Sánchez and others (2013b). Compounds: AcO-avocadyne (0), 1-acetoxy-2,4-dihydroxy-heptadec-16-yne; AcO-avocadenyne (1), 1-acetoxy-2,4-dihydroxy-heptadec-12-en-16-yne; AcO-avocadene (2), -1-acetoxy-2,4-dihydroxy-n-heptadeca-16-ene; AcO-avocadiene B (3), 1-acetoxy-2,4-dihydroxy-heptadeca-12,16-diene; Persenone C (5), -1-acetoxy-2-hydroxy-4-oxononadeca-5,16-diene; Persenone A (6), -1-acetoxy-2-hydroxy-4-oxoheneicosa-5,12,15-triene; Persin (7), 2R,12Z,15Z)-1-acetoxy-2-hydroxy-4-oxoheneicosa-12,15-diene; and Persenone B (8), (5E)-1-acetoxy-2-hydroxy-4-oxononadeca-5-ene. Numbers 0–8 next to compounds chemical names were assigned based on their chromatographic elution order . Food-grade propylene glycol (PG) and lysozyme were purchased from Sigma Aldrich .Reagent grade solvents  in a stage of commercial ripeness of the State of Michoacán, Mexico, was used and pulp and seed were manually separated. Both mature and young ‘Hass’ avocado leaves were collected from mature avocado trees growing at the Tecnologico de Monterrey Plant Nursery, Monterrey, NL, México. Three-stage Acetogenin extraction from avocado seed, pulp, and leaf was carried out as described by Rodríguez-López and collaborators (2015) [−1), and filtered using a 0.45 mm PTFE filter for HPLC injection. Whereas Avosafe®, already an acetogenin-enriched extract, was directly dissolved in HPLC-grade isopropanol and adjusted at a concentration of 1 mg·mL−1, as previously described.Avocado  and methanol 100% (B). Solvents were pumped at 1 mL·min−1 using gradients of: 0−15 min, 80−95% B; 15−25 min, 95−100% B; and 25−30 min, 100–80% B, followed by 10 min re-equilibration. The column used was a Synergy Hydro RP C18  kept at 35 °C. The following detector signals were considered for each compound: ELSD for compound 0, PDA at 220 nm for compounds 1 to 6, PDA at 208 nm for compounds 7 and 8, while the rest of putative acetogenins  , with so015 , wittogenins  were qua1, 2, and 4 to 8 in 0 and 3 in −1 using gradient of: 0–5 min, 80–84% B linear; 5–10 min, 84–88% B linear; 10–10.5 min, 88–100% B linear; 10.5–12 min, 100% B isocratic, followed by 5 min of re-equilibration. The measurements were performed in the positive ion mode (ESI+) controlled by the MassLynx™ software . Parameters of chromatogram and spectra acquisition (ESI-MS) included capillary, sample cone, and extraction cone voltages of 2.5 kV, 40 V, and 3 V, respectively; desolvation temperature of 40 °C; source temperature of 120 °C; cone gas (N2) flow of 50 L·h−1; desolvation gas (N2) flow of 250 L·h−1; and scan range of m/z 200–800. For ESI-MS/MS (positive ion mode), the sample was introduced into a Quattro Premier XE Micromass  triple quadrupole mass spectrometer via a syringe pump at a flow rate of 20 µL·min−1; drying gas (N2) flow rate was set at 200 L·h−1 at 40 °C; capillary, cone and extractor voltages were 4 kV, 4 V, and 5 V, respectively; and source temperature was set at 120 °C. The collision energy was set to 10 or 20 V, a spread of ±1 V, and collision gas (Ar) at 7.46 × 10−4 mbar with a flow rate of 0.35 mL·min−1.Elucidation of the chemical identity of compounds ® mutagenicity was assessed by in vitro Bacterial Reverse Mutation test (AMES test). The evaluation was conducted under contract by Envigo Research Limited  in full compliance with international regulations and guidelines established by Organization for Economic Co-operation and Development (OECD), EC Commission, and U.S. organizations (EPA and FDA) the for testing of chemicals [Salmonella typhimurium  and a tryptophan-dependent mutant strain of Escherichia coli  were tested in triplicate at the seven doses  of Avosafe®. The highest concentration of Avosafe® (5000 µg·plate−1) tested in this study was the standard limit concentration recommended in the regulatory guidelines for this assay [v/v for test 1 and 20% v/v for test 2), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM), and NADH (4 mM) in water). Positive controls with and without S9 activator  four rats of the same sex for the main study. The animals were allowed free access to a standard rodent diet (Harlan Teklad 2014C Diet), except for overnight prior to and approximately four hours after dosing. This diet contained no added antibiotic or other chemotherapeutic or prophylactic agents. Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes. The animals were allowed to acclimatize to the conditions described below for at least 5 days before treatment. The acute oral toxicity study was carried out according to guidelines established by the OECD (No. 420) [An acute oral toxicity test in rats was performed under contract by Envigo Research Limited , with the care and use of experimental animals being in compliance with the United Kingdom Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 (the Act) . HealthyNo. 420) , Japan [No. 420) , and theNo. 420) . The app®, formulated in PG (at 30 or 200 mg of Avosafe® mL−1) for administration at a volume of 10 mL·kg−1 bw, corresponding to dose level of 300 or 2000 mg·kg−1 bodyweight (bw), respectively. For the main study, based on the results of the sighting investigations, 4 new animals were administered 2000 mg·kg−1 bw.In the sighting study, two fasted female rats were given a single oral gavage dose of AvosafeAll animals were observed for 14 days after dosing, recording clinical condition and bw. All animals were euthanized on day 15 by carbon dioxide asphyxiation. All animals were subjected to a macroscopic examination, which consisted of opening the cranial, thoracic, and abdominal cavities. The macroscopic appearance of the brain, caecum, duodenum, heart, kidneys, small and large intestine, liver, lungs and bronchi, spleen, stomach, subcutaneous tissue, and urinary bladder was recorded.®: WIST albino rats weighing 200–250 g were obtained from Scientific Services Tetrarium. Animals were distributed in two groups  based on their bw using a balanced design. Animals were housed in groups at constant temperature (21–24 °C) and light–dark cycles of 12 h and allowed to acclimatize for 5 days before treatment. Food and water were provided ad libitum, except for overnight prior to and approximately four hours after dosing, as well as overnight before scheduled necropsy. Avosafe® formulated in PG (vehicle) was administered to each rat (2000 mg of Avosafe® kg−1 bw) by oral gavage using a plastic syringe and plastic catheter. Control animals only received the vehicle (2000 mg of PG kg−1 bw). All animals were observed throughout the experimental period (14 days after dosing). The study was performed at Tecnologico de Monterrey  after approval of the Institutional Committee for the Right Use and Care of Laboratory Animals . Procedures were conducted following guidelines established in the Mexican National Protection Laws on Animal Protection and the General Health Law Related to Health Research (NOM-062-Z00-1999). Nulliparous and non-pregnant female RccHanThe clinical conditions of treated animals were monitored over 14 days after dosing, at hourly intervals during the first 6 h of day 1 and daily on subsequent days. Recorded clinical condition included changes in skin and fur appearance, behavioral patterns , urine, and fluid secretion and excretion. The body weight (bw) of each animal was recorded prior to dosing (day 1) and subsequently on day 8 and 15, and bw changes from days 1–8 and from days 8–15 were calculated for each animal group.On day 14, overnight fasted animals were anesthetized with sodium pentobarbital before blood was collected from the tail vein into tubes containing either EDTA (for hematology parameters) or lithium heparin . Hematologic analysis was carried out . For biochemical analysis, we determined levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatine phosphokinase, total bilirubin, glucose, total cholesterol, triglycerides, total protein, albumin, albumin/globulin ratio, blood urea nitrogen, creatinine, inorganic phosphorus, calcium ions, sodium ions, potassium ions, and chloride ions. For both studies, an automated CELL-DYN Ruby Hematological Analyzer  was employed to carry the blood samples analysis.Animals were euthanized on day 14 by an overdose of sodium pentobarbital. The macroscopic appearance and weight of heart, brain, liver, and pair of kidneys were recorded. Ratios of organ weight relative to body weight were calculated (grams per 100 g of bw). Organs were fixed in formalin, dehydrated, and embedded in paraffin for further staining with hematoxylin and eosin pathology score. Organs for which there were no abnormalities in macroscopic appearance or weight relative to body weight were retained in fixative and not analyzed histopathologically.p < 0.05. Grouping by Tukey’s Honestly Significant Difference (HSD) was performed (α < 0.05). Differences between the vehicle control and the treated group in in vivo experiments were estimated by LSMean Dunnett’s test (p < 0.05). Statistical analysis was performed using JMP® software version 13.0.0 .Results were expressed as means ± standard deviation. Statistically significant differences among groups were analyzed employing one-way analysis of variance (ANOVA). Differences were considered significant at a level of ®, and together with other seven major peaks quantified 94.74 ± 5.77% w/w of its total solids. Eleven other small chromatographic peaks exhibiting the characteristic ion pattern of acetogenins were also identified as present in the enriched extract and individually contributed concentrations that ranged between 0.34 to 1.41% to the Avosafe® total solids .A multiple detection strategy that used HPLC-PDA/ELSD, HPLC-ESI-TOF-MS, and ESI-MS/MS was developed for identification and quantification of additional acetogenins present in avocado tissues  and in an enriched food-grade extract obtained from avocado seed. The use of low-energy CID aided the assignment of the location of unsaturations in acetogenins, resulting in the confirmation of the chemical structures of two molecules (AcO-avocadyne (0) and AcO-avocadiene B (3)), as present in Avosafe7) concentrations . Safety evaluations indicated that Avosafe® was non-mutagenic, had an acute median lethal oral dose (LD50) for rats estimated to be higher than the maximum concentration tested (>2000 mg·kg−1), and showed no signs of macroscopic abnormalities in organs. Mean body weight, hematological, and biochemical parameters were also normal after 14 days of administration of the single oral dose of 2000 mg·kg−1. More tests are desirable to further characterize the safety of acetogenins as food additives; however, the present work advanced scientific knowledge on this family of lipid derivatives contained in the widely consumed avocado fruit.Quantification of individual acetogenins found in avocado tissues indicated that seed contained the highest total concentrations (2.31 ± 0.29% dw). Seed profiles and concentrations of individual acetogenins were different when compared with those of pulp and leaves, the latter featuring the highest Persin (Some of the potential commercial uses of acetogenins in the food, pharmaceutical, and personal care industries are protected under patent applications WO/2012/042404 and US 20180103671A1.