OPE12− displayed selectivity for PHFs in fluorimetry assays and strong staining of neurofibrillary tangles (NFTs) in mouse and human brain tissue sections, while OPE24+ stained both NFTs and Aβ plaques. Both OPEs stained the brain sections with limited background or non-specific staining. This novel family of sensors outperformed the gold-standard dye Thioflavin T in sensing capacities and co-stained with conventional phosphorylated tau (AT180) and Aβ (4G8) antibodies. As the OPEs readily bind protein amyloids in vitro and ex vivo, they are selective and rapid tools for identifying proteopathic inclusions relevant to AD. Such OPEs can be useful in understanding pathogenesis and in creating in vivo diagnostically relevant detection tools for neurodegenerative diseases.The identification of protein aggregates as biomarkers for neurodegeneration is an area of interest for disease diagnosis and treatment development. In this work, we present novel super luminescent conjugated polyelectrolyte molecules as ex vivo sensors for tau-paired helical filaments (PHFs) and amyloid-β (Aβ) plaques. We evaluated the use of two oligo-p-phenylene ethynylenes (OPEs), anionic OPE Proteinaceous deposits composed of β-sheet rich fibrils are pathological hallmarks of a range of neurodegenerative disorders, including Alzheimer’s (AD) and Parkinson’s diseases (PD). Following DNA translation, proteins normally fold into their functional conformations driven by thermodynamics and with the help of chaperone proteins. Many proteins undergo a poorly understood misfolding and aggregation process to form toxic oligomers and then β-sheet rich fibrils, which eventually deposit into large inclusion bodies ,2,3,4. SMAPT gene [The Aβ peptide varies in length from 36–42 amino acids and is cleaved from the amyloid precursor protein by β-secretase followed by γ-secretase. Aβ’s function in the brain is still unclear, but the excessive presence of Aβ or hyperphosphorylated/aggregated tau could be a metabolic by-product with gain-of-toxic function effects or an antimicrobial peptide/modifications evolutionarily derived for innate immunity . Aβ can APT gene , which iAPT gene ,9,10.While many factors may contribute to AD etiology ,5, inclu18F-florbetapir, 18F-florbetaben, and 18F-flutemetamol [18F-T807  showed promise as a cortical tau tracer in AD patients but not in non-AD tauopathies. [The detection of amyloid protein fibrils has traditionally been carried out using Thioflavin T (ThT) and Congo Red stains ,22,23. Temetamol ,28,29. Temetamol ,32,33,34pathies.  As NFT bpathies. ,37,38, tp-phenylene ethynylenes (OPEs) ranging in size and photophysical properties for their specificity and affinity towards fibrillar aggregates of two model amyloid proteins, insulin and lysozyme [The development of protein aggregate sensors includes the synthesis and characterization of various classes of compounds based on their biophysical properties. Luminescent conjugated oligo-and polythiophenes  have been introduced for broad amyloid identification ,12,13,39lysozyme , and dislysozyme . OPEs polysozyme . In addilysozyme . Our stulysozyme . OPEs anlysozyme . All thelysozyme ,15,16,21lysozyme  and Aβ40lysozyme  at multilysozyme . TherefoAll experimental protocols involving animals were performed in accordance with US National Institutes of Health guidelines on animal care and were approved  by the University of New Mexico Institutional Animal Care and Use Committee. Animals of both sexes were used in this study.12− and OPE24+ were synthesized as previously reported [370nm = 3.92 × 104 M–1 cm–1 for OPE12− and ε370nm = 8.29 × 104 M−1 cm−1 for OPE24+. ThT was purchased from Thermo Fisher Scientific . To prepare the ThT stock solution, ThT powder was solubilized in 70% ethanol in water at 0.5% w/v (1.56 mM). All dye solutions were covered with aluminum foil and stored in the dark at 4 °C until use.OPEreported  and soluw/v% sucrose at pH 7.4). After sonication and centrifugation at 22,000× g for 30 min at 4 °C, the supernatant was adjusted to 1 w/v% N-laurylsarcosine and 1% β-mercaptoethanol and incubated for 2 h at 37 °C with agitation. Next, the solution was centrifuged at 150,000× g for 35 min. The pellet containing the Sarkosyl-insoluble fraction was washed multiple times in 1% buffer H. Then, the pellet was resuspended in Buffer H with 1 w/v% CHAPS and 1 v/v% β-mercaptoethanol and filtered with a 0.45 µm cellulose acetate syringe filter. This filtered solution was centrifuged at 35,000× g rpm for 1 h at room temperature. The pellet was then collected and resuspended in buffer H with 1 v/v% β-mercaptoethanol and then purified by layering over a sucrose gradient and centrifuging at 35,000× g rpm for 2 h in a Beckman SW 41 Ti rotor at 4 °C. The sucrose gradient was composed of 6 mL of 50% sucrose and 4 mL of 35% sucrose in buffer . Following centrifugation, PHFs were collected from the 35–50% sucrose layer interface with a syringe. Further PHF purification was performed using chloroform/methanol precipitation. First, a 4:1 methanol to protein (v/v) solution was prepared and vortexed, then a volume of chloroform was added. The sample was vortexed and then centrifuged at 10,000× g for 5 min. The top methanol layer was removed, four more volumes of methanol were added, and the solution was vortexed and centrifuged again at 10,000× g for 15 min. The supernatant was removed. The remaining pellet containing PHFs was air-dried and resuspended with cell media. Protein concentration was determined using Nanodrop .The recombinant tau-441 (2N4R-Tau) monomers were generously provided by Dr. Rakez Kayed, and prepared as previously described . A proteWestern blot assay was carried out to confirm that the fraction isolated from the extraction comprised of the tau protein. Sample separation was performed via a 4–12% Bis-Tris Novex NuPage gel from Invitrogen, then transferred to a polyvinylidene difluoride (PDVF) membrane. The membrane was blocked with 5% milk and then incubated with Tau12 antibody from Millipore at 1:10,000 dilution overnight, followed by goat-anti mouse secondary antibody from Jackson ImmunoResearch. For visualization, the membranes were developed using the ECL reagent from Thermo Fisher Scientific with a 5-min exposure. The immunoreactive bands were quantified using Image J ,47.The Sarkosyl insoluble pellets were imaged via transmission electron microscopy (TEM) as previously described . Briefly®, Manassas, VA, USA) cells were grown in Dulbecco’s Minimum Essential Medium  supplemented with 10% Fetal Bovine Serum (FBS) and 100X Antibiotic-Antimycotic (Thermo Fisher Scientific). The N2a cells were passaged and incubated at 37 °C with 5% CO2. For the toxicity assay, the cells were plated on 96-well plates at 32,000 cells/well and incubated over night for cell attachment. To add the dyes to the cells, the cell media was first replaced with 10% FBS in DMEM with OPE or ThT at concentrations used for staining. Cell media solution containing 10% ethanol was used as a negative control. All samples were incubated for 24 h. To measure cell viability, cell media was replaced with 10% FBS in DMEM and MTT . The plates were incubated for 4 h before taking absorbance readings at 490 nm using a SpectraMax M2e plate reader spectrophotometer. Five samples were prepared and measured for each experimental condition, and three experiments were run for all conditions. Means and standard error of means (SEMs) of cell viability for each condition were calculated and reported.Neuroblastoma  monomers and brain-extracted PHFs was evaluated by measuring the fluorescence of the OPEs (1.6 μM) when mixed with each protein sample (6.67 μg/mL) using a PTI QuantaMaster 40 spectrofluorometer. OPEv/v% glycerol, 20% 0.4 M Sorenson’s buffer) prior to sectioning. A day before sectioning, the brains were transferred to a cryostorage solution . To section, the brains were first placed on a flat surface made of frozen 30% sucrose solution and then flash-frozen using dry ice and ethanol. The frozen brains were then placed on the stage of a Leica Sliding Knife Microtome 2010R  and cut into 30 μm thick sagittal sections. Brain sections were placed into cryostorage solutions and kept at −20 °C until immunohistochemical or fluorescence staining was performed. The sections were washed three times with PBS, placed on a shaker, and blocked with a standard blocking solution ) for one hour prior to staining.Tissue from multiple transgenic 9-month-old rTg4510, 9-month-old non-transgenic C57BL/6J mice, and 6-month-old non-transgenic C57BL/6J mice were obtained. Following anaesthetization, mice were transcardially perfused with PBS at pH 7.4. The brains were removed, and the left hemispheres were taken for immunochemical assays. After fixing by immersion in 4% paraformaldehyde (PFA), the brains were then placed into a cryoprotection solution (20 w/v% in 70% ethanol) for five minutes at room temperature with rotation, followed by a 1-min wash in 70% ethanol and five 5-min washes with 1xPBS. To stain the brain sections with ThT, the dye was solubilized in 50% ethanol to a final concentration of 0.05 w/v%. Sections were incubated with ThT for 8 min in the dark, followed by two 10-s 80% ethanol washes and then 3 water washes. Following all staining treatments, the sections were mounted using VECTASHIELD HardSet Antifade Mounting Medium without DAPI  and hardened for at least 24 h at 4 °C before imaging.Prior to staining, brain sections were washed in 1xPBS pH 7.4 to remove residual cryostorage solution, then blocked with 5% normal goat serum in 1xPBS-0.4% Triton X for one hour at room temperature with rotation. To stain the brain sections with OPEs, each OPE was diluted from the stock solutions to 5 μM or 0.5 μM using 1xPB at pH 7.2. The brain sections were then immediately immersed and incubated in a staining solution for an hour at room temperature with rotation; samples were covered with foil to reduce light exposure at all times. The sections were then washed 3 times at 5 min each with PBS to remove excess dye. OPE-stained sections were then stained with 1% Sudan Black B , AT180 and 4G8 were used at a 1:500 dilution, and AF555 was used at a 1:1000 dilution. Sections were incubated with AT180 or 4G8 overnight at 4 °C; secondary antibody incubation was done for one hour at room temperature in the dark. Following incubation, the excess dye was removed by PBS washes. To block lipofuscin auto fluorescence signals, the sections were treated with 1% Sudan Black B  for 5 min followed by a 1-min rinse with 70% ethanol, then five 5-min rinses with PBS. Prior to staining, the sections were washed with PBS to remove any residual cryostorage solution and then blocked with 5% normal goat serum in 1xPBS-0.4% Triton X for 1 h at room temperature. Following all staining treatments, the sections were mounted using VECTASHIELD HardSet Antifade Mounting Medium without DAPI and hardened at 4 °C for at least 24 h before imaging.w/v% ThT or 500x diluted 4G8 anti-Aβ antibodies. In these animals, we visualized the pathology in the hippocampus, which is affected in AD [For rat sections, we used brain sections from 10-month-old transgenic TgF344-AD and non-Tg Fisher-344 rats. The 30 μm brain sections were prepared using on the same protocol as described previously. Free-floating sections were incubated with 5 μM OPE, 0.05 ed in AD .12− and OPE24+ stained samples were excited using the 405 nm laser line, with emission collected at 420–600 nm or 440–650 nm, respectively. ThT-stained samples were excited using the 458 nm argon line, with their emission collected from 460–650 nm. Sections co-stained with anti-phospho tau antibodies (AT180) or Aβ-specific (4G8) and Alexa Fluor 555 were excited using a tunable and pulsed white light laser (470–670 nm), and emissions were collected from 560–700 nm. All sections were initially imaged using a 20x objective with immersion oil to identify anatomy and then by the 63x objective to collect images. Emission intensities were also taken at 10–20 nm intervals to generate emission spectra. Ex vivo OPE-stained brain tissue images shown in Images were taken using a Leica TCS SP8 confocal microscope equipped with 405 nm diode laser line, white light laser, multi-argon lines, and Hybrid Spectral Detectors. OPE12− and AT180 staining of NFTs, single-stained images from wildtype and transgenic mouse brains in the cortex and hippocampus were analyzed. For each analysis, 3 images were taken per sagittal section, 3 sections were taken from each animal, and 3 animals were used. Thus, a total of 27 images were analyzed for each staining condition. All images were converted to 8-bit, and a threshold was applied to subtract the background signal or remaining lipofuscin fluorescence. To obtain positive staining area in each image, Fiji’s measure tool [2 image area. Average percent positive area values and their associated standard errors of the mean (SEM) were reported.To quantify OPEure tool ,50 was u2) from the hippocampus of single-stained wildtype and transgenic rats were analyzed. For each analysis, the number of plaques was counted in three areas within the hippocampus, 3 sections were taken from each rat, and 3 rats were used. In single-stained sections (4G8 or OPE24+), the plaques were easily visualized. Thus, they were counted without specific signals or size cut-offs. A plaque was counted when the staining pattern and size from the 4G8 antibody and OPE24+ dye corresponded to each other.A similar approach was used to quantify 4G8 and OPE staining of Aβ plaques in rat sections. Images -based oligomers (OPEs) and polymers (CPEs) for the detection of fibrillar aggregates of two model amyloid proteins, insulin, and lysozyme . These tc OPE12− , bound tce yield ,41. In t24+ and anionic OPE12− towards the detection of brain-derived tau PHFs. The PHFs were extracted and isolated from rTg4510 transgenic mice and post-mortem brain tissues from human FTD-tauopathy using a Sarkosyl-insoluble extraction protocol [First, we tested the efficacy and selectivity of cationic OPEprotocol , showed We have previously shown that when solubilized in an aqueous buffer, the OPE sensors are quenched and undergo a non-radiative, water-mediated decay process upon excitation by photon absorption . When bo12− alone is weakly fluorescent, displaying a small peak at around 440 nm ADF=Iectively , indicat24+ similarly increased in the presence of PHFs compared to the quenched sensor alone  [w/v was used . The rTg4510 mouse model expresses human FTDP-17) . rTg4510FTDP-17) ,57,58. BFTDP-17) , where 0was used . 9-monthwas used .12− and OPE24+ detected NFTs in transgenic mice brain sections at 5 and 0.5 μM dye concentrations. OPE12− stained NFTs in the cortex with minimal background fluorescence  on tau is one of the well-established neuropathological markers of tau pathology [12− then further processed for AT180 binding and the staining of corresponding anti-mouse secondary antibody conjugated to Alexa Fluor 555 fluorophore (AF555). Each fluorophore was excited separately, and images were combined to visualize for overlap  with Swedish mutant human APPSW and presenilin (PS1ΔE9 mutations) and is in Fisher 344 genetic background [In previous studies, we found OPEs detected Aβckground ,63. Thisckground ,65. Whilckground . The TgFckground ,63. Ratsckground . The patckground . Spatialckground . Howeverckground .12− had no apparent selective staining of Aβ plaques or any pathology in TgF344-AD rat brain sections, except that it showed some non-specific staining -2,5-diphenyltetrazolium bromide (MTT) cell metabolism assay . Cells wried out . All meama cells .12− selectively stained tau NFTs in mouse and human tissue at 5 µM. The cationic OPE24+ at 5 µM showed some non-specific binding in all brain sections, but at a lower concentration of 0.5 µM, this problem was resolved and the sensor displayed selectivity for staining tau NFTs. OPE12− staining co-localized with the binding of the phosphorylated tau antibody AT180 and ThT in mouse and human sections, confirming that the OPE-stained tau NFTs. Furthermore, quantitative analysis showed that OPE12− is better at staining NFTs than AT180. In the rat AD model, OPE24+ displayed selectivity for Aβ plaques at 5 µM with minimal background fluorescence in the Fisher-344 wildtype sections. OPE24+ co-localized with Aβ specific antibody 4G8 on the same plaques but stained the core, while the antibody stained the more diffuse morphology. In the rat sections, we did not observe consistent OPE12− staining of plaques. The rat brain sections did not have tau pathology for the OPEs to detect; therefore, we did not see NFT staining in rats. Lastly, we evaluated the OPEs’ toxicity toward N2a cells using the MTT assay. Neither OPE caused a significant loss of cell viability at concentrations up to 10 µM. In comparison, ThT at a tissue staining concentration of 1.56 mM caused a complete loss of viability of the N2a cells. Our results thus show that OPEs are effective ex vivo markers for the selective staining of tau NFTs and Aβ amyloid plaques at concentrations orders of magnitude lower than ThT. This work paves the way for developing these novel sensors for in vivo diagnosis and tracking of disease progression in numerous neurodegenerative diseases. The versatility of the OPEs opens the door not only for the detection of neurodegeneration but also for the detection of general amyloidogenic disorders.As our population ages, there is an increased need to develop diagnostically relevant tools to aid in the detection and treatment of neurodegenerative diseases such as AD. In previous work, we found that small OPEs selectively bind to and detect fibrillar and pre-fibrillar aggregates of amyloid proteins in vitro. In this study, we evaluated and characterized the capacity of two OPEs as ex vivo amyloid sensors. Brain tissues from wildtype  and transgenic AD  mice and rats along with human brain tissue from non-demented healthy controls and FTD were incubated with two OPEs and characterized for staining patterns. The anionic OPE


N-(2-morpholinoethyl)acetamide-4-morpholine-1,8-naphthimide (MMN), was designed and synthesized through an amidation reaction. MMN was fabricated as a multifunctional fluorescent probe for monitoring pH and isoxaflutole. MMN exhibited excellent stability in MeCN/H2O , with an obvious “off–on” fluorescence response toward pH changes due to intramolecular charge transfer (ICT), where the linear response ranges of MMN in the weakly acidic system were from 4.2 to 5.0 and from 5.0 to 6.0 with apparent paK = 4.62 ± 0.02 and 5.43 ± 0.02. Based on morpholine as the lysosome targetable unit, MMN could selectively locate lysosomes in live cells. MMN also successfully detected the presence of H+ in test papers. Finally, MMN could specifically recognize isoxaflutole at a detection limit of 0.88 μM. A possible sensing mechanism was identified based on density function theory calculations. These results indicate that MMN could be a superior potential chemosensor for detecting pH and isoxaflutole selectively and sensitively and could be used in real sample detection.A new chemosensor, namely  IntraceIn the past few years, various techniques have been used for pH detection such as the colorimetric method , electroPesticides refer to chemical agents used in agriculture to control pests and to regulate plant growth. Pesticides are widely used in agriculture, forestry and animal husbandry; in environmental and family hygiene, pest control and epidemic prevention; and in industrial products to prevent mildew and moths . IsoxaflCurrent reports on the detection methods for isoxaflutole residue mainly involve liquid chromatography and liquid chromatography-tandem mass spectrometry ,19. EvenN-(2-morpholinoethyl)acetamide-4-morpholine-1,8-naphthimide (MMN), was synthesized and characterized. MMN was synthesized by introducing the morpholine unit  represents molar concentration. The binding constant (K) was calculated as 3.7 × 105 M−1  calculations exhibited via the electron density or energy level between isoxaflutole and MMN. As shown in 3 electron-withdrawing group and mesotrione with the -NO2 electron-withdrawing group were relatively higher than those of other pesticides. Meanwhile, their LUMO energy levels were between the HOMO and LUMO energy levels of MMN, which could cause the transition of electrons from the LUMO orbital of MMN to the LUMO orbitals of isoxaflutole and mesotrione in the excited state  +) 453.2060, found 453.2144.The compound rocedure ,41,42. CMMN was prepared in CH3CN/H2O . The 1.00 × 10−2 M stock solutions of metal cations  were prepared with deionized water. The 1.00 × 10−2 M solutions of anions  were prepared by tetrabutylammonium (TBA) salts and sodium salts with deionized water. The ionic salts were dissolved in distilled water to prepare the stock solutions for the cations and anions (1.00 × 10−2 M). The stock pesticide solutions (1.00 × 10−2 M) were prepared in DMSO due to the poor solubility of some pesticides using cypermethrin, cyhalothrin, oxyfluorfen, mesotrione, cyhalofop-butyl, teflubenzuron, NTBC, sulcotrione, tembotrion, isoxaflutole, pyrazoxyfen, glyphosate, clethodim, fluazuron, flusilazole, fluorobenzene and benoxacor. For different pH solutions, 1.00 × 10−1 M HCl or NaOH was added to 10 mL of 1.00 × 10−5 M MMN solution and the apparent pH value was determined with a pH meter. To investigate the anti-interference of MMN, 50 μL aliquots of 1.00 × 10−2 M different cation and anion solutions were used as the background to measure the fluorescence spectra at two representative apparent pH values (4.2 and 6.0). Different instruments were used for fluorescence spectroscopic detection of pH and isoxaflutole. The slit widths of excitation and emission were set to 10 nm, and the excitation wavelength was set to 415 nm in the fluorescence spectral experiment.The stock solution of 2 atmosphere. One day before the fluorescence imaging experiment, the cells were placed on petri dishes (NESTC) at the bottom of 35 mm cover slides. The cells were incubated with Lyso-Tracker Red (70 nM) for 30 min, washed with PBS 3 times, fixed with 4% paraformaldehyde for 1 h, washed with PBS 3 times, washed with a solvent 3 times, stained overnight with MMN (100 nM), washed with a solvent 3 times, sealed and observed under a confocal microscope.Human stromal cell line (HSC) cells were purchased from the Chinese Academy of Sciences and cultured using the medium DMEM, which contains 15% fetal bovine serum, 100 µg/mL penicillin and 100 g/mL streptomycin at 37 °C within a humidified 5% COThe Perdew–Burke–Ernzerh (PBE) method of Generalized Gradient Approximation (GGA) in the Dmol3 module was used to calculate the exchange correlation energy using a DNP+ basis set calculation and dispersion correction with Grimme ,44,45.MMN). Because of the H+-induced intramolecular charge transfer (ICT) effect, MMN displayed significant pH sensitivity with apparent paK values of 4.62 ± 0.02 and 5.43 ± 0.02 as well as good linear apparent pH responses ranging from 4.2 to 5.0 and from 5.0 to 6.0, which were suitable for selectively detecting acidic lysosomes. MMN shows potential for the rapid detection of pH in test papers. Furthermore, MMN could quickly and easily detect isoxaflutole with an extremely low detection limit. Density function theory calculations revealed MMN’s potential underlying the sensing mechanism.In summary, we designed and developed a multifunctional naphthimide-based fluorescent probe (


Using a “synthetic biological” approach to assay the growth of the present-day bacteria engineered to resemble ancient ones, we show that it is possible to explain the emergence of bacterial CCMs if atmospheric CO2 was once much higher than today, consistent with geochemical proxies. Taken together, our results delineated an unexpected “CO2-catalyzed” pathway for the evolution of bacterial CCMs, whose multiple emergence has been challenging to understand.The emergence of biological novelty is often coupled to the evolution of Earth’s chemical environment. Here, we studied how the evolution of a bacterial CO 2-concentrating mechanisms (CCMs) to grow in today’s atmosphere (0.04% CO2). These complex physiological adaptations require ≈15 genes to produce two types of protein complexes: inorganic carbon (Ci) transporters and 100+ nm carboxysome compartments that encapsulate rubisco with a carbonic anhydrase (CA) enzyme. Mutations disrupting any of these genes prohibit growth in ambient air. If any plausible ancestral form—i.e., lacking a single gene—cannot grow, how did the CCM evolve? Here, we test the hypothesis that evolution of the bacterial CCM was “catalyzed” by historically high CO2 levels that decreased over geologic time. Using an E. coli reconstitution of a bacterial CCM, we constructed strains lacking one or more CCM components and evaluated their growth across CO2 concentrations. We expected these experiments to demonstrate the importance of the carboxysome. Instead, we found that partial CCMs expressing CA or Ci uptake genes grew better than controls in intermediate CO2 levels (≈1%) and observed similar phenotypes in two autotrophic bacteria, Halothiobacillus neapolitanus and Cupriavidus necator. To understand how CA and Ci uptake improve growth, we model autotrophy as colimited by CO2 and HCO3−, as both are required to produce biomass. Our experiments and model delineated a viable trajectory for CCM evolution where decreasing atmospheric CO2 induces an HCO3− deficiency that is alleviated by acquisition of CA or Ci uptake, thereby enabling the emergence of a modern CCM. This work underscores the importance of considering physiology and environmental context when studying the evolution of biological complexity.Cyanobacteria rely on CO Rubisco, the carboxylating enzyme of that pathway, is often considered inefficient due to relatively slow carboxylation kinetics  and depletion of atmospheric CO2 to the present-day levels of a few hundred parts per million . Historical CO2 levels are challenging to estimate, but are thought to have been substantially higher than today, perhaps as high as 0.1–1 bar early in Earth history , which help meet the challenge of fixing carbon in a low CO2 atmosphere. CCMs concentrate CO2 near rubisco and are found in several varieties in all Cyanobacteria, some Proteobacteria, as well as many eukaryotic algae and diverse plants  that HCO3- can enter the carboxysome diffusively, where it undergoes rapid CA-catalyzed dehydration  and tested their growth across a range of COall CCMs  and ask trations C. We foctrations , Ci trantrations , and homtrations , 25 are H. neapolitanus, in five CO2 partial pressures , supporting the view that CCM components play an important physiological role even in relatively high environmental CO2 concentrations.One approach to constructing contemporary analogs of CCM ancestors is to remove CCM genes from a native host. If CCM components were acquired sequentially, some single-gene knockouts would be analogous to recent ancestors, e.g., those lacking a complete carboxysome shell . We testressures , 27. As E. coli strain called CCMB1  in all metabolisms, and comment on how these results can inform our entwined understandings of bacterial physiology, CCM evolution, and the CO2 content of Earth’s ancient atmosphere.Removing single CCM genes from a native host can only produce analogs of recent ancestors, however. We recently constructed a functional α-carboxysome CCM in an engineered ed CCMB1 . This stpartment , but insH. neapolitanus contains all the genes necessary for a functional CCM  and ambient air (≈0.04%) via high-throughput sequencing , low , moderate , high , and very high . Replicate experiments were strongly correlated , implying a high degree of reproducibility. We, therefore, proceeded to ask whether H. neapolitanus CCM genes contribute to growth in a range of CO2 concentrations.To mimic the changes in atmospheric CO2 pressures, with genes grouped by their documented roles in the CCM. Each point in 2, as indicated by large growth defects in disruptive mutants  even in the absence of a complete and functional CCM. We considered testing this hypothesis in H. neapolitanus directly by constructing “ancestral-like” CCMs lacking carboxysome shell genes or Ci transporters. However, genetic manipulation of H. neapolitanus is cumbersome, and the native host’s regulatory network could complicate interpretation—a concern highlighted by three DNA-binding proteins that emerged in our screen as likely regulators of the CCM . We, therefore, decided to construct and test analogs of CCM ancestors in a nonnative host, namely E. coli.In high COe 5–10%, B. As sucE. coli strain, CCMB1, that depends on rubisco carboxylation for growth in minimal medium. This strain requires elevated CO2 for rubisco-dependent growth, but expressing the H. neapolitanus CCM from two plasmids enabled growth in ambient air , growth improved substantially in intermediate CO2 levels (0.5% and 1.5%). These data indicated that a carboxysomal CA plays a pivotal role in 0.5% and 1.5% CO2 , or both. This was achieved by cloning both Dab2 and Can into a dual expression vector and making targeted active site mutants  to isolate each activity. These vectors were cotransformed into CCMB1 with a constitutive version of p1A (p1Ac) so that rubisco expression would not be affected by induction of Dab2 or Can (Methods).Our observation that expression of the carboxysomal CA improves rubisco-dependent growth on its own  motivated us to test the effects of CA and Ci transport independently of carboxysome expression. We designed plasmids that express the nsporter , E. coli2 . Similar to the reference strain, a double-negative control strain expressing inactivated versions of both Dab2 and Can (“+Dab−+CA−”) grew poorly or not at all in 0.5% and 1.5% CO2, implying that the observed growth improvements were due to activity and not a side effect of heterologous gene expression.Expressing active Can and Dab2, whether alone or together, improved growth substantially in 1.5% COE. coli. We found the effects of Dab2 expression especially perplexing because Ci uptake is expected to generate intracellular HCO3− . To confirm that these results are not a side effect of working in an engineered E. coli strain, but rather a genuine feature of autotrophy, we pursued experiments in a natively autotrophic proteobacterium, C. necator.It was not immediately obvious to us how CA and Ci uptake activities improve rubisco-dependent growth of CCMB1  HCO3− 11, 20, 33 O3− . Consistent with our experiments in E. coli, this growth defect was complemented by heterologous expression of a human CA (“ΔCA+CA”) or a DAB-type Ci uptake system (“ΔCA+DAB”). Moreover, as in E. coli, coexpression of Ci uptake with native CAs was not deleterious (“WT+DAB”). Rather, this strain grew to higher densities than wild type in 1.5% and 5% CO2 .We found that expression of a CA, Ci transporter, or both improved rubisco-dependent growth of 1-1 cm/s , 45, 46.ynthesis . Here, w2 concentration relative to the reference strain expressing only rubisco. This could arise if rubisco activity depletes intracellular CO2 (Cin) significantly below its extracellular level (Cout) in the reference strain. In this setting, one might assume that CA activity accelerates the equilibration of CO2 across the membrane, thereby increasing Cin and carboxylation flux. However, this naive model cannot explain the growth benefits associated with expressing Ci uptake systems, which provide HCO3− and not CO2 , the maximum rubisco active site concentration is ≈1 mM. In this naive model, Cin is set by the balance of passive uptake through the membrane, with effective permeability α = PC × SA/V ≈ 104 s−1, and fixation by rubisco, with an effective rate constant of γ = [rubisco] × kcat/KM < 103 s−1 (neglecting inhibition by O2). These values give a steady-state Cin of α Cout / (α + γ) > 0.9 Cout. That is, even an extreme level of rubisco activity cannot draw Cin beneath 90% of Cout. In such conditions, rubisco would fix ≈1010 CO2/hour, supporting a 1-2 h doubling time . So, although the CO2 concentration is low in aqueous environments equilibrated with the present-day atmosphere (≈10–20 μM), passive CO2 uptake is expected to be very fast, giving intracellular CO2 concentrations nearly equal to extracellular ones and supporting substantial carboxylation flux. As Cin ≈ Cout, CA activity cannot substantially affect Cin or carboxylation flux in this setting.In a bacterium, rubisco might make up 20% of soluble protein at the very most . This am3−  and is therefore designed for coarse, order-of-magnitude comparisons with data, not direct quantitative correspondence.We instead argue that the effects of expressing CA or Ci transport can be explained by the ubiquitous dependence of growth on HCO3− –52. It h3−  be depleted beneath 50% of the extracellular level (Cout). Furthermore, when Cin ≈ Cout, neither CA activity nor Ci uptake meaningfully increased the modeled rubisco carboxylation flux . When the HCO3− dependence of biomass precursor production was included in our model, however, it became possible to rationalize the growth improvements associated with heterologous expression of a CA or Ci transporter, as both of these activities can supply HCO3- needed for anabolic carboxylations  produced faster growth, but the growth rate was insensitive to CA activity because slow CO2 hydration already provided sufficient HCO3− for the anabolic carboxylations involved in lipid, nucleotide, and amino acid biosynthesis . When rubisco activity was set to higher values  for anabolic carboxylations. In bicarboxylation-limited conditions, additional HCO3− could be supplied by CA, Ci uptake, or both . However, these activities are not equivalent, as we discuss below.We first used our model to confirm that rubisco should not be expected to deplete intracellular COylations A. When tylations B, growthylations B or the L,totJ = L,CJ + L,HJ) to biomass production by rubisco- and HCO3−-dependent carboxylases  greatly exceeded the HCO3− leakage flux  in most modeled conditions. Low Ci leakage is desirable; when L,totJ ≈ 0, the flux of Ci pumped into the cell is balanced by fixation of CO2 and HCO3− such that no energy is “wasted” on Ci uptake. When CA (δ) and Ci uptake (χ) activities were low, the model predicted slow growth and net diffusive Ci uptake  to balance carboxylation . Increasing Ci uptake (χ) could cause L,totJ to change sign from negative (connoting passive uptake) to positive (connoting leakage) passing through L,totJ = 0. If CA was also expressed—i.e., if δ was increased above a baseline value—L,totJ ≈ 0 could be achieved at lower Ci uptake activities due to CA-catalyzed dehydration of imported HCO3− producing CO2 used by rubisco. In other words, CA activity lowers the Ci uptake rate and, consequently, energy expenditure required to achieve the same rate of biomass production . When both activities were high, the model predicted substantial leakage, with L,totJ / BJ ≈ 100 in extreme cases. Such extreme levels of futile cycling are likely incompatible with growth. If Ci uptake was low, in contrast, CA activity could be increased arbitrarily without incurring leakage because CAs do not pump Ci into the cell . As HCO3− is supplied sufficiently by Ci uptake in this case, the colimitation model predicted that increased rubisco fluxes would translate into faster growth. For this effect to arise, however, the membrane permeability to CO2 would have to be 100–1,000 times lower than the measured  to support growth as atmospheric CO2 levels decreased . In CCMB1, we found that the coexpression of CA and Ci transport supported growth in low CO2 environments  only when all genetic components are intact  that CO2 ≈ 1% (25 PAL) improves the growth of organisms harboring partial CCMs , while H. neapolitanus uses O2 as the preferred terminal electron acceptor during chemoautotrophic growth  and whole cell physiology (the requirement for HCO3−) on the evolution of CO2 fixation. That is, neither rubisco nor the CCM should be considered in isolation, but rather in the context of a metabolism that demands both CO2 and HCO3− for growth. We hope that future research advances the synthetic approach to studying evolution and fully expect that this approach will enrich our understanding of biological processes that have shaped the evolution of biogeochemical cycles on Earth.It is intrinsically difficult to answer questions about Earth’s deep biological history; addressing such questions will surely require cooperation between scientific disciplines. Here, we took a “synthetic biological” approach to study the molecular evolution of bacterial COain ways , 66, 70.SI Appendix, Tables S4 and S5. The rubisco-dependent E. coli strain CCMB1 was derived from E. coli BW25113 and has the genotype BW25113 ΔrpiAB Δedd ΔcynT Δcan, as documented in ref. C. necator H16, we first knocked out the hdsR homolog A0006 as removal of this restriction enzyme increases electroporation efficiency  with 10 μg/ml kanamycin. These precultures were grown to an OD ≈ 0.07 (600 nm) in 5% CO2. The library was subsequently back diluted 1:64 and grown in various CO2 concentrations  on a platform shaker  in a Percival Intellus Incubator configured to mix pure CO2 with laboratory air to reach the desired CO2 partial pressure; 20 ml of preculture was pelleted by centrifugation  and saved as a T0 reference. Upon reaching 6.5–7.5 doublings, 50 ml of culture was spun down and gDNA was extracted for barcode PCRs as described in ref. 2 of the ratio of barcode abundance in the experimental condition over abundance in the T0. Gene-level fitness values were then calculated considering all transposon insertions expected to disrupt an individual gene. Each CO2 concentration was tested in biological duplicate except for 5% CO2, which was assayed in biological quadruplicate.Competitive fitness assays were performed following . The barE. coli strains were grown at 37°C. 60 μg/ml kanamycin and 25 μg/ml chloramphenicol were used for selection during routine cloning and propagation. For strains carrying two selectable markers, antibiotics were used at half concentration . All CCMB1 cultures were grown in 10% CO2 unless otherwise specified. When rubisco-independent growth was desired, CCMB1 was propagated in rich LB media. CCMB1 was cultured in a rubisco-dependent manner in M9 minimal media (pH 7.0) supplemented with trace elements and 0.4% glycerol (v/v) as described in ref. 2 with laboratory air to reach a defined CO2 partial pressure. For the data in can CA. 100 nM aTc was supplied to induce DAB2 and 1 mM IPTG for can.2 as a negative control; the remaining 4 ml was incubated in 10% CO2. CCMB1 strains carrying active site mutants of rubisco (cbbL K194M) were precultured in LB with the same antibiotic concentrations. Once cultures reached saturation, they were centrifuged at 4000 g for 8 min. Pellets were washed in 10 ml of M9 with no carbon source (M9 NoC) and resuspended in 5 ml M9 NoC. Optical density was measured in fivefold dilution at 600 nm , and cultures were normalized to 0.5 OD units. 96-well plates were inoculated by adding 2.5 μl of OD-normalized preculture to 247.5 μl M9 glycerol supplemented with appropriate antibiotics and induction to a starting density of 0.005 OD600. Kanamycin was omitted as the plasmid carrying kanamycin resistance also expresses rubisco, which is required for growth in glycerol media (2 concentration and measure the culture density (OD600) every 30 min. The humidity cassette was replenished after 48 h. All experiments were performed in biological quadruplicate and technical triplicate.Precultures were inoculated into 5 ml of M9 glycerol with 30 μg/ml kanamycin, 12.5 μg/ml chloramphenicol, and appropriate induction. 1 ml of each culture was transferred to a separate tube, which was incubated in ambient COC. necator strains were grown in ambient CO2 unless otherwise specified, and 200 μg/ml kanamycin was added to select for plasmid retention. When rubisco-independent heterotrophic growth was desired, C. necator was cultured in LB at 30°C. For autotrophic growth experiments, strains were precultured in 5 mL of LB media in 10% CO2 in 25 mL tubes with 20 mm butyl stoppers sealed by aluminum crimping. Precultures were incubated for 2 d at 30°C with 200 rpm shaking, washed three times in Cupriavidus minimal media (pH 6.7), and inoculated to an OD of 0.1 (550 nm) in minimal media in a 165-mL flask with a 20-mm butyl stopper sealed by aluminum crimping. Cupriavidus minimal growth media contained 3.24 mM MgSO4, 0.42 mM CaCl2, 33.5 mM NaH2PO4, 32.25 mM Na2HPO4, 2.6 mM K2SO4, 1 mM NaOH, 1.87 mM NH4Cl, and 1 mL/L of a 1000× trace mineral solution containing 480 mg/L CuSO4·5H2O, 2.4 g/L ZnSO4·7H2O, 2.4 g/L MnSO4·H2O, and 15 g/L FeSO4·7H2O (pH 6.7). The flasks were evacuated, and the headspace was filled with 60% H2, 10% O2, and indicated concentrations of CO2. The balance was air. Cells were grown for 48 h at 30°C with 200 rpm shaking, and OD550 values were taken using a Molecular Devices SpectraMax M2 spectrophotometer.Appendix 01 (PDF)Click here for additional data file.Dataset S01 (XLSX)Click here for additional data file.


Piperacillin-Tazobactam (PIP/TAZ) is a combination of a β-lactam antibiotic with a β-lactamase inhibitor, used to treat moderate to severe infections due to its broad-spectrum antibacterial activity. Its bactericidal effect is time-dependent. Therefore, the free-drug concentration should remain above the minimum inhibitory concentration during at least 50% of the dosing interval. Patients with severe infections develop pathophysiological changes that alter drugs pharmacokinetics (PK), leading to only 30% of probability of target attainment in clinical setting.A prospective observational study was performed in patients with severe infections from Hospital Central “Dr. Ignacio Morones Prieto”. The protocol was approved by the Research and Ethics Committee (register 05-20) and patients signed written informed consent. Samples were collected at steady-state and plasma concentrations were quantified by liquid chromatography coupled to mass spectrometry. Data were analyzed by a population approach using NONMEM® software.2. According to the Akaike information criterion and visual inspection, a one-compartment open model was chosen to describe the concentration vs time data (n=156) for both drugs. Typical values (relative standard error) of PK parameters obtained were Clearance [CLPIP (L/h)] = 8.79 (12%) and Volume of distribution [VPIP (L)] = 17.6 (13%); and CLTAZ (L/h) = 12.6 (14%), VTAZ (L) = 32.8 (13%). Interindividual variability (IIV) of each parameter was modeled by exponential error and reported as coefficient of variation as follows: 75.3% and 88.7% for CL; 67.2% and 68.8% for V of PIP and TAZ, respectively. Finally, residual error was modeled as additive and presented a standard deviation (SD) of 7.28 µg/mL for PIP, and for TAZ was modeled as a combined with a SD of 0.22 µg/mL and a coefficient of variation of 17.32%.A total of 52 patients were included  with a mean age of 46 ± 17 years and a body mass index of 25 ± 5 Kg/mIndividualization and optimization of β-lactam dosing are essential in drugs with wide IIV as PIP/TAZ; hence, development of a population PK model will provide a valuable aid in explaining and quantifying some of this variability to allow a priori predictions to design initial regimens to reach pharmacotherapeutic targets.All Authors: No reported disclosures.


Golgi membrane protein 1 (GOLM1) is a Golgi-resident type 2 transmembrane protein known to be overexpressed in several cancers, including hepatocellular carcinoma (HCC), as well as in viral infections. However, the role of GOLM1 in lipid metabolism remains enigmatic. In this study, we employed siRNA-mediated GOLM1 depletion in Huh-7 HCC cells to study the role of GOLM1 in lipid metabolism. Mass spectrometric lipidomic analysis in GOLM1 knockdown cells showed an aberrant accumulation of sphingolipids, such as ceramides, hexosylceramides, dihexosylceramides, sphinganine, sphingosine, and ceramide phosphate, along with cholesteryl esters. Furthermore, we observed a reduction in phosphatidylethanolamines and lysophosphatidylethanolamines. In addition, Seahorse extracellular flux analysis indicated a reduction in mitochondrial oxygen consumption rate upon GOLM1 depletion. Finally, alterations in Golgi structure and distribution were observed both by electron microscopy imaging and immunofluorescence microscopy analysis. Importantly, we found that GOLM1 depletion also affected cell proliferation and cell cycle progression in Huh-7 HCC cells. The Golgi structural defects induced by GOLM1 reduction might potentially affect the trafficking of proteins and lipids leading to distorted intracellular lipid homeostasis, which may result in organelle dysfunction and altered cell growth. In conclusion, we demonstrate that GOLM1 depletion affects sphingolipid metabolism, mitochondrial function, Golgi structure, and proliferation of HCC cells. Golm1 is not expressed in healthy rat hepatocytes; however, its expression increases during malignant transformation  (+ T cells and activating endoplasmic reticulum stress in tumor-associated macrophages (Golgi membrane protein 1 (GOLM1), a Golgi type 2 transmembrane protein also known as Golgi phosphoprotein 73 (GP73) or Golgi phosphoprotein 2 (GOLPH2), is expressed primarily in epithelial cells, and it is overexpressed upon viral infections including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Wilson disease, and cancers . GOLM1 eormation  and contormation . In addis (NASH) . GOLM1 es (NASH) . GOLM1 arophages , 12. Morrophages . FurtherHuman HCC is a complex and aggressive form of liver cancer with a high mortality rate, its incidence being four times higher in men than women , 15, 16.In this study, the effect of GOLM1 depletion on the lipid profile and metabolism was characterized in HCC cells. In addition, the changes in Golgi morphology, apoptosis, mitochondrial function, and proliferation in GOLM1-depleted cells were analyzed.Huh-7 and HepG2 HCC cell lines were cultured in Eagle's minimal essential medium (MEM), GlutaMAX™ Supplement , and MEM AQ™  containing 10% FBS , 100 U/ml penicillin, and 100 μg/ml streptomycin. Huh-7 cells were reverse transfected with 100 or 150 nM negative control siRNA  or GOLM1 siRNA  using Lipofectamine™ RNAiMAX Transfection Reagent  according to the manufacturer's protocol. Depending on the experiments, the cells were transfected for 24–72 h and used for downstream analyses.https://www.ncbi.nlm.nih.gov/geo/). The GSE62232 dataset included 22 tumors from alcohol-related etiology, 21 tumors with HBV or HCV as the etiology, and 10 HCC adjacent samples for examining viruses and alcohol-induced HCC (Two microarray datasets (GSE62232 and GSE164760) of HCC cohorts with different etiologies were downloaded from the Gene Expression Omnibus database  for 20 min, followed by three washes with ice-cold PBS. Cells were then permeabilized using 0.1% Triton X-100 for 5 min. The fixed and permeabilized cells were blocked for 60 min using 10% FBS (in PBS) at room temperature. After blocking, the cells were incubated at 4°C overnight with the primary antibody, anti-GOLM1 , and anti-Golgin subfamily A member 2 . The cells were washed three times with PBS for 5 min and probed with the respective secondary antibody  for 60 min at 37°C, followed by three washes with PBS. The cells were mounted with 50 mg/ml 1,4-diazabicyclo[2.2.2]octane , Mowiol  containing 5 μg/ml 4′,6-diamidino-2-phenylindole, dihydrochloride . Fluorescence was observed with a 63× oil objective in ZEISS LSM 880 with Airyscan at HiLIFE Biomedicum Imaging Unit, University of Helsinki. ImageJ (FIJI) software was used to quantify the cell number, Golgi stack distribution, and the length of the Golgi stack from the nucleus.GOLM1 protein expression was analyzed by Western blotting. After 72 h of silencing, control and GOLM1-silenced cells were lysed with RIPA buffer , and equal amount of protein was resolved on 10% or 12% SDS-polyacrylamide gels , followed by transferring onto PVDF membrane using Bio-Rad Transblot system. The membrane was blocked to eliminate nonspecific antibody binding using 5% milk in TBS with 0.1% Tween for 60 min and probed with anti-GOLM1 , anti-ORMDL3 , and corresponding HRP-conjugated secondary antibodies. Signals were developed with Pierce™ ECL Western Blotting Substrate  or Clarity™ Western ECL Substrate  and captured using ChemiDoc™ Touch Gel Imaging System . Protein expression was quantified using Image Lab™ Software (Bio-Rad) and is normalized to the total protein intensity of the blot lane.Total RNA from Huh-7 cells was extracted using PureLink ™ RNA Mini Kit  according to the manufacturer’s instruction, and RNA was reverse-transcribed into complementary DNA using SuperScript® VILO™ synthesis Kit . The mRNA expression of genes was analyzed by quantitative PCR (qPCR) using gene-specific primers and LightCycler® 480 SYBR Green  master mix. The reactions were carried out using the LightCycler 480 II Real-Time PCR system . The expression was calculated using the CT values and was normalized to the housekeeping gene expression .(−ΔCT) was calculated for both nuclear and mitochondrial gene measurements; further, mitochondrial measurements were normalized to genomic measurements. FABP1 and RPLPO were used as nuclear gene references. Primer sequences used are provided in the Mitochondrial DNA amount was measured using qPCR, and total DNA was isolated from GOLM1-depleted and control cells using genomic DNA isolation kit according to the manufacturer’s protocol . qPCR was performed using mitochondrial and nuclear gene–specific primers. 2GOLM1 knockdown and control Huh-7 samples were subjected to quantitative lipid MS. Cell homogenates were extracted according to the method of Bligh and Dyer  in the pm/z 184 was used for phosphatidylcholine, SM, and lysophosphatidylcholine. The following neutral losses were applied: phosphatidylethanolamine (PE) and lysophosphatidylethanolamine (LPE) 141, phosphatidylserine 185, phosphatidylglycerol 189, and phosphatidylinositol 277  was performed in positive ion mode using the analytical setup and strategy described previously . A fragmitol 277 . PE-baseitol 277 . Sphingoet al. (m/z 200). Multiplexed acquisition (MSX) was used for the [M + NH4]+ of free cholesterol (m/z 404.39) and D7-cholesterol (m/z 411.43)  . Data pr , sphingoid bases, and ceramide phosphate (CerP) .The extracted data were exported to Microsoft Excel 2016 and further processed by self-programmed macros including type II and type I correction as described. Lipid species were annotated according to the latest proposal for shorthand notation of lipid structures that are derived from MS . For QQQGOLM1-silenced and control cells were scraped in 2% sodium chloride 72 h after the transfection, and lipids were Bligh and Dyer  extracte3H] acetic acid . The cells were washed twice with PBS and then scraped in 2% sodium chloride. TLC was performed after isolating the total lipids according to the extraction protocol by Bligh and Dyer  scintillation liquid, and the radioactivity was measured using Wallac 1410 liquid scintillation counter. The results were normalized to total cell protein.GOLM1-silenced and control cells were grown in 6-well plates. At 72 h after transfection, the cells were labeled for 4 h in FBS-containing MEM with 20 μCi/well  thymidine  was added and incubated for 4 h. The cells were washed three times with cold PBS and incubated with 5% trichloroacetic acid for 10 min, followed by 0.1 M NaOH treatment. After 10 min of incubation, the cells were scratched, and 3 ml of scintillation liquid (OptiPhase Hisafe 3) was added to the lysates. The radioactivity was measured using a Wallac 1410 liquid scintillation counter.Reverse-transfected Huh-7 cells were seeded on a 6-well plate (1 × 103 cells per well) were seeded in opaque wall clear bottom 96-well plate . Post 24 h of transfection, 2× detection reagent from RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay Kit  was added on the cells. Luminescence (relative light unit) measures were taken at different time points in an EnSpire Multimode plate reader (PerkinElmer).Reverse-transfected Huh-7 cells (10 × 105 cells per well) were cultured for 72 h in a 6-well plate. The cells were then trypsinized and centrifuged. The cells were washed for three times with PBS, and the pellet was suspended in 500 μl of propidium iodide solution  and incubated for 15 min at room temperature. The samples were then run with BD Accuri C6 flow cytometer with FL2 detector, and the results were analyzed with FlowJo_v10.8.0 software (BD Bioscience) using the univariate Dean-Jett-Fox cell cycle model. The flow cytometry analysis was performed at the HiLIFE Biomedicum Flow Cytometry Unit, University of Helsinki.Reverse-transfected Huh-7 cells  or R 4.0.3 was used for the statistical analyses.The Mann-Whitney GOLM1 in HCC with different etiologies was analyzed using the data from the publicly available datasets GSE62232 and GSE164760. GOLM1 mRNA expression was elevated in HCC related with hepatitis viruses, high alcohol consumption, and NASH , sphingosine , sphinganine , and CerP along with CE was observed in the GOLM1-depleted cells as compared with controls, whereas a significant reduction was seen in membrane phospholipids such as LPE and PE A. Indivi3H] acetic acid. There was no increase in the synthesis of CE or free cholesterol, rather a tendency of decrease in cholesterol and a significant decrease in CE synthesis was observed  were decreased , and lactosylceramide (LacCer) in liver cells are known to adversely affect the mitochondrial function , 40. MorFurthermore, a decrease in mitochondrial DNA content  was observed in GOLM1 knockdown cells as compared with controls B.Since GOLM1 is a Golgi localized protein and an aberrant increase in HexCer was observed in GOLM1 knockdown cells, further efforts were made to study whether GOLM1 silencing affects Golgi structure and morphology. Control and GOLM1-silenced cells were stained for GM130, and its distribution was measured. Scattering of the Golgi stacks was observed in GOLM1-depleted cells A. In GOL3H] thymidine labeling, 22.5% reduction was observed at 72 h of GOLM1 silencing -2,5-diphenyl tetrazolium bromide proliferation assay A. Howeveilencing B. Furtheilencing C. Furtheilencing D. Cell cilencing E.Fig. 8SSPTLC2), serine palmitoyltransferase, long chain base subunit 2 (SPTLC3), serine palmitoyltransferase small subunit B (SPTSSB), delta 4-desaturase, sphingolipid 2 (DEGS2), and glycolipid transfer protein (GLTP) were observed (ORMDL1) and ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3), negative regulators of ceramide synthesis, was seen (VPS53 (vacuolar protein sorting 53) and VPS54, GARP (Golgi-associated retrograde protein) complex components were mildly but significantly downregulated (ACBD3 mRNA expression was observed in GOLM1-silenced cells (GRASP55) encoding another Golgi protein involved in GSL metabolism , and globotriaosylceramide (GB3) , rather an increase was noted suggesting that GlcCer transport to the trans Golgi network by FAPP2 may not be altered in these cells. Taken together, these data suggest that GOLM1 potentially plays a role in GSL metabolism similar to the other GOLPHs, possibly by regulating the intra-Golgi transport. The GARP complexes are involved in the retrograde transport from endosomes to Golgi. Deletion of a GARP complex component VPS53 caused an increase in long-chain bases like sphingosine, sphinganine, Cer, and HexCer. In addition, VPS53 and VPS54 mutants exhibited growth defects in yeast , GRASP55, and pleckstrin homology domain containing A8 (PLEKHA8-FAPP2), are known to affect sphingolipid and GSL metabolism , 52, 53.de (GB3) . A mild in yeast . VPS53 sin yeast , contribIn addition to alterations in sphingolipids and CEs, GOLM1 knockdown cells also displayed a reduction in PE and LPE. This might be due to a defect in mitochondrial function in these cells . On the A tight connection exists between Golgi morphology and sphingolipid metabolism. The Golgi resident proteins, GRASPs, are shown to maintain Golgi structure and function . ImportaIn conclusion, GOLM1 depletion in hepatocarcinoma cells resulted in aberrant accumulation of sphingolipids and CEs, possibly because of a defect in Golgi structure and function in retrograde trafficking. These changes in lipids were associated with reduced mitochondrial function and cell proliferation indicating putative future value of GOLM1 as a therapy target for cancers.All data used in this study are presented as main figures or supplemental figures and tables.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Vaccinium vitis-idaea L. (lingonberry) leaves and fruits and the evaluation of their cytotoxic potential. Qualitative and quantitative characterization of obtained extracts and triterpenoid fractions was performed using HPLC-PDA method, followed by complementary analysis by GC-MS. For each fraction, cytotoxic activities towards the human colon adenocarcinoma cell line (HT-29), malignant melanoma cell line (IGR39), clear renal carcinoma cell line (CaKi-1), and normal endothelial cells (EC) were determined using MTT assay. Furthermore, the effect of the most promising samples on cancer spheroid growth and viability was examined. This study allowed us to confirm that particular triterpenoid mixtures from lingonberry waxes may possess stronger cytotoxic activities than crude unpurified extracts. Fractions containing triterpenoid acids plus fernenol, complexes of oleanolic:ursolic acids, and erythrodiol:uvaol were found to be the most potent therapeutic candidates in the management of cancer diseases. The specificity of cuticular wax extracts of lingonberry leaves and fruits, leading to different purity and anticancer potential of obtained counterpart fractions, was also enclosed. These findings contribute to the profitable utilization of lingonberry cuticular waxes and provide considerable insights into the anticancer effects of particular triterpenoids and pharmacological interactions.Fruit and leaf cuticular waxes are valuable source materials for the isolation of triterpenoids that can be applied as natural antioxidants and anticancer agents. The present study aimed at the semi-preparative fractionation of triterpenoids from cuticular wax extracts of  Natural products constitute a considerable part of newly proposed drugs. Nowadays, Hippocrates’s famous quote “Let food be your medicine” has turned to plant constituents that could be used in the development of new therapeutic agents for the management and prevention of different diseases, including cancer. Phytochemical research tends to focus on finding novel anticancer phytopharmaceuticals to fulfill current needs ,2,3. AntTriterpenoids, distributed widely in higher plants, represent an important and abundant group of specialized plant metabolites with structural diversity in carbon skeletons and functional groups . These cVaccinium vitis-idaea L.) plant, a perennial evergreen Ericaceous dwarf shrub with edible fruits, is included in a healthy diet of the Northern Hemisphere due to its high nutritional value and richness of bioactive compounds. Data suggest that lingonberry raw materials are underutilized natural resources for developing medicines or functional food [Vaccinium berries are particularly distinguished by the abundance of triterpenoids as the main components of cuticular waxes. Although the triterpenoid profile of lingonberry fruit cuticles has been reported earlier [Lingonberry , malignant melanoma (IGR39), clear renal carcinoma (CaKi-1) cancer 2D- and 3D-culture models and normal endothelial cells (EC) using MTT and spheroid growth and viability assays. The extraction and fractionation technologies we employ can reduce the amount of waste and utilize other bioactive compounds as phenolics, which can be further isolated from the lingonberry matrix after extraction of triterpenoids from the cuticle. To the extent of our knowledge, this is the first comprehensive report on fractionating different groups of triterpenoids from lingonberry cuticular waxes to identify potential anticancer agents.trans-cinnamic, p-coumaric, sinapic, ferulic, maslinic and oleanolic acids, erythrodiol, α-amyrin, β-amyrin, β-sitosterol, taraxasterol, lanosterol, and lupeol were obtained from Sigma-Aldrich; betulinic and corosolic acids, betulin, uvaol, and friedelin were obtained from Extrasynthese ; and ursolic acid was purchased from Carl Roth . Helium used in GC-MS was purchased from Gazchema (Lithuania) and N,O-Bis(trimethylsilyl) trifluoro acetamide, Trimethylchlorosilane (BSTFA-TMCS) was acquired from Sigma-Aldrich.Analytical-grade solvents—namely chloroform, dimethylformamide (DMF), dimethyl sulfoxide (DMSO) methanol, and acetonitrile—used for extraction, sample preparation, and chromatography were purchased from Sigma-Aldrich , trifluoracetic acid—from Merck , and purified deionized water was produced using Milli-Q system . Authentic standards of benzoic, TM Express reagent, phosphate-buffered saline (PBS), and penicillin/streptomycin solution (100×) were acquired from Gibco .Chemicals for anticancer activity assays—namely, 3--2,5-diphenyltetrazolium bromide (MTT) and crystal violet—were obtained from Sigma-Aldrich. Cell proliferation reagent WST-1 was from Roche Diagnostics . The plasticware for cell cultures was purchased from Corning  and Techno Plastic Products . NanoShuttle magnetic nanoparticles were obtained from Nano3D Biosciences Inc. . Fetal bovine serum (FBS), Dulbecco’s modified Eagle high glucose medium (DMEM Glutamax), TrypLE2 in a humidified atmosphere and were grown to 70% confluence. During the passage, cells were trypsinized with 0.125% TrypLE™ Express solution and used till passage 20.Human colon adenocarcinoma cell line (HT-29), human malignant melanoma cell line (IGR39), human clear renal carcinoma cell line (CaKi-1), and human endothelial cells (EC) CRL-1730 were obtained from the American Type Culture Collection . The cell culture medium consisted of DMEM Glutamax medium supplemented with 10% FBS and 1% antibiotics. Cells were kept at 37 °C with 5% COLingonberry raw materials were collected in September 2019 from the wild at different natural sites in Lithuania ) and then pooled prior to extraction to obtain a homogeneous mixture of leaf and fruit samples, which were frozen (−20 °C) until analyses were performed. Lingonberry raw materials were identified according to morphological characteristics by Gabriele Vilkickyte and Lina Raudone. The voucher specimen was deposited in the herbarium of Vilnius University Siauliai Academy Botanical Garden.To obtain lingonberry cuticular waxes, sample sets of entire leaves and fruits (10–20 g) were immersed in an adequate amount of chloroform to fully cover the sample (the sample/solvent approximate ratio of 1:8 was used for leaves and 1:2 for fruits) at room temperature and gently stirred for 30 s. As reported earlier ,29,30, tFractionation of triterpenoids from crude cuticular wax extracts was conducted on a semi-preparative HPLC-PDA system  using an ACE C18 semi-preparative column . The crude cuticular wax extracts were reconstituted with methanol and then filtered through a 0.45 µm membrane before injection into the system using an injection volume of 0.5 mL. Gradient elution at a constant flow rate (4 mL/min) was carried out with a total run time of 85 min as follows: starting from an isocratic run at 100% solvent A for 33 min, then increasing to 100% solvent B over 10 min and maintaining isocratic at 100% solvent B to the end of the run. Equilibration time was at least 15 min between runs. Solvent A was acetonitrile and water  and solvent B was methanol and acetonitrile . Fraction collection was based on time, monitoring the chromatograms at 205 nm and allowing the collection of known and unknown components to generate many different fractions with no more than two major peaks in each for the screening of anticancer agents. The fractionation scheme used in the present study is presented in Triterpenoid composition and purity of obtained fractions were analyzed using Waters e2695 Alliance HPLC system coupled with a PDA detector and validated method as reported previously . ChromatAll samples were additionally screened for the residuals of phenolic compounds using the same HPLC-PDA system and ACE Super C18 column , exactly as described earlier . A gradi2 stream. Dried residues were reconstituted with 100 µL of DMF, silylated by adding 100 µL of BSTFA-TMCS (99:1), and held at 90 °C in a glycerol bath for 2 h. After derivatization, the resultant solutions were injected ) with a Shimadzu autoinjector AOC-5000  for triterpenoid qualitative analysis using a robotic autosampler and a split/splitless injection port.To confirm the identity of triterpenoids in obtained fractions, additional GC-MS analysis was performed according to Caligiani et al.  and Sáncm/z, and a scan time of 0.2 s., full-scan (m/z 29–700). Identification of triterpenoids was achieved by comparing their mass spectra with literature and library data from LabSolution GC/MS (version 5.71) (Shimadzu Corporation) and by comparison of their retention times and corresponding mass spectra with those of reference standards, where available.A Shimadzu GC/MS-QP2010nc Ultra chromatography system coupled to an Electron Ionization ion source and a single quadrupole MS  and Rxi-5ms column  were used. The oven temperature was programmed at 200 °C for 2 min increased to 300 °C at the rate of 4 °C/min and then increased to 320 °C at the rate of 10 °C/min and held for 6 min. The total run time was 35 min. Helium was used as the carrier gas at a flow rate of 1.07 mL/min. The detector ion source and interface temperatures were 200 °C and 280 °C, respectively. Mass spectra were acquired at an ionization voltage of 70 eV, a scan rate of 2500 50 values that represent the concentration, which is effective in reducing cell viability by 50%, were calculated using the Hill equation as described earlier [The effect of lingonberry cuticular wax extracts and obtained fractions on cancer and endothelial cell viability was determined using an MTT assay, according to Grigalius and Petrikaite . HT-29,  earlier .y=Emax+y—response measure at dose D;E0 and Emax—top and bottom asymptotes of the response;50—concentration at half-maximal effect;ECHS—slope parameter analogous to the Hill coefficient.2 for 8 h. After magnetization, cells were trypsinized, centrifuged, and seeded into ultralow attachment 96-well plates in a volume of 100 µL (1500 cancer and 1500 human fibroblast cells/well). The magnetic drive  was used for the plate incubation at 37 °C in a humidified atmosphere containing 5% CO2 for 2 days to form spheroids. Then photos of spheroids were taken every 48 h using a phase-contrast Olympus IX73 inverted microscope . The medium was replaced by the fresh one, containing 100% of solutions of the most promising samples  at concentrations 5 and 25 μg/mL, every 48 h. ImageJ software  was used to evaluate the effect of tested lingonberry samples on reducing the size of cancer spheroids, while the WST-1 assay was used to determine the change of cell viability in spheroids. Briefly, on the last day of the experiment, the medium of spheroids was replaced by the fresh one containing 10% of WST-1 and incubated for 6 h. Then 50 µL of solution from each well was transferred to the new 96-well plate and the absorbance was measured at 430 nm and a reference wavelength of 630 nm using a multi-detection microplate reader Multiskan GO. The spheroid viability was calculated using the following formula:Spheroids were formed using the magnetic 3D Bioprinting method as previously stated elsewhere . Cancer A—mean of absorbance of medium of spheroids treated with fractions;0—mean of absorbance of blank ;ANC—mean of absorbance of negative control (spheroids with no treatment).Ap < 0.05 were considered statistically significant.All data are presented as mean values (M) ± standard deviation (SD). The statistical significance of obtained results was established by one-way ANOVA with Tukey’s and Duncan’s post-hoc tests. The Pearson correlation method was used to evaluate correlations between data. IBM SPSS Statistics version 26.0 package, Microsoft Office Excel 2016, and GraphPad Prism 8 software were used for statistical analysis and visualization. Values of wax) and fruits (Fwax) (Crude cuticular wax extracts of lingonberry leaves (Ls (Fwax)  were evawax and Fwax, respectively. These results were in line with the study of Dashbaldan et al. [Vaccinium fruits, except bilberry, were also distinguished by the predominant isomer of ursolic acid [wax, compared to Lwax. The complex of ursolic and oleanolic acid made up 86.5% of the total triterpenoids in the cuticular wax extract of fruits. A slightly lower contribution of these acids  was found earlier in Finnish and Polish entire lingonberry fruits [wax and Fwax and were detected for the first time in cuticular waxes of lingonberry raw materials.Triterpenoid acids were the major group determined in the cuticular wax extract of fruits accounting for 87.0% of the total identified triterpenoids. A significantly lower (5.3-fold) amount of triterpenoid acids was found in the cuticular wax extract of leaves. Ursolic acid prevailed in both samples as the principal compound, constituting 84.1% and 81.6% of identified triterpenoid acids in Ln et al. , whereinlic acid ,39,40. Oy fruits . Other twax and the major group in Lwax, accounting for 11.8% and 53.0% of total identified triterpenoids, respectively. This is consistent with previous results [wax was α-amyrin , followed by fernenol and uvaol, while Fwax was characterized by the abundance of lupeol . As found by other researchers [wax , while Lwax did not contain this compound. This is distinguishable from extracts from entire leaves, in which 1.0–6.9% friedelin contribution to neutral triterpenoids was reported [Neutral triterpenoids  comprised the second prevailing group of triterpenoids in F results , whereinearchers ,41 main earchers  indicatereported ,41.The content of phytosterols in cuticular waxes of lingonberry fruits and leaves was very low, only 0.4–1.2% of the total triterpenoids. Previous data  indicatem/z values as reported for plant sterols earlier [m/z at 129, which is one of the base peaks of phytosterols, having a double bond on the B ring [m/z 218 and 189, which are abundant in pentacyclic triterpene alcohols [m/z at 320, indicating the presence of ester group in C-17 [m/z at 203, which is a typical mass ion fragment of oleanane and ursane skeletons [wax and Fwax, while the derivative of triterpenoid acids III was determined only in Fwax.Results of GC-MS analysis  confirme earlier . Furthere B ring . Exact ie B ring ,42. Thisalcohols . In the alcohols ,41 it isalcohols ,42,44 in in C-17  and m/z keletons . Derivatwax than that in Fwax. Chlorophyll also could be one of the main residuals in Lwax since it cannot be removed during the extraction with chloroform [wax contained a considerable amount of phenolic acids precursors—benzoic and trans-cinnamic acids (71.27 ± 10.79 and 2.81 ± 0.25 mg/g DW), while 86-times lower content of these acids was found in Lwax. Furthermore, trace levels of p-coumaric, sinapic, and ferulic acids were detected in both cuticular wax extracts. Klavins et al. [The authors of ,47 suggeloroform . AdditioFirst fractions from cuticular wax extracts of lingonberry leaves and fruits (1L and 1F), collected at the beginning of semi-preparative HPLC analysis, were pure from phenolic acids residuals, which were eluted with void volume due to higher polarity. They contained the following triterpenoids: three triterpenoid acids  and one neutral triterpenoid (fernenol) . FurtherFractions 2L and 2F were characterized by the complex of oleanolic and ursolic acids. It is extremely difficult to separate these structural isomers from each other due to very similar chemical properties. Therefore, the pharmacological effects of the combination of oleanolic and ursolic acids are usually studied and commonly used in clinical practices ,51. Low Vaccinium plants did not distinguish by the abundance of uvaol [Third fractions of cuticular wax extracts were enriched by neutral triterpenoid uvaol, which made up 87.0% and 80.1% of 3L and 3F, accordingly. The second prevailing compound was erythrodiol, which has a similar chemical structure and molecular weight to uvaol, thus they are usually analyzed together . Ratios of uvaol ,54. The Parkia biglobosa (Jacq.) G. Don [Zanthoxylum monogynum A.St.-Hil. [Grewia flava DC. [Fractions 4L and 4F contained one principal neutral triterpenoid—lupeol. Fraction 4F was pure and no other triterpenoids were detected, while 4L was characterized by the presence of impurities—lanosterol (1.5% to identified triterpenoids) and one unidentified compound (unknown phytosterol I). Results indicate that due to differences in lingonberry matrixes, cuticular waxes of lingonberry fruits seem to be a better source for the isolation of lupeol. Furthermore, the richness of this compound in lingonberry fruits could be noted. The content of lupeol in the cuticular wax of lingonberry fruits was the highest among tested Northern berries . Recentl) G. Don , ZanthoxSt.-Hil. , and roolava DC. .Fifth fractions of cuticular wax extracts of leaves and fruits differed from each other by predominant compounds. Fraction 5L was distinguished by the high content of β-amyrin, which comprised 87.3% of identified compounds, and significantly lower levels of the following sitosterols: β-sitosterol (9.5%), taraxasterol (2.7%), and lanosterol (0.6%). Whereas the main component of 5F was β-sitosterol, accounting for 52.1% of identified compounds; followed by β-amyrin (42.5%), taraxasterol (3.7%), and lanosterol (1.7%). The sum amount of phytosterols was 4.2 times higher in the fifth fraction of fruits than that of leaves. Two more unidentified compounds (unknown phytosterol II and III) were found in samples 5L and 5F. The complex of β-sitosterol and lanosterol from cuticular waxes of lingonberry fruits was fractionated previously and obtained ratio of these phytosterols was found to be 1:1.1 .The last fraction of cuticular waxes of leaves (6L) can be regarded as a pure fraction of α-amyrin as no more compounds were identified. α-Amyrin was earlier highlighted as a phytochemical marker of lingonberry leaves, which highest content was found in old lingonberry leaves (collected from the bottom of the stem) with an increasing tendency during colder months . The las50 values could not be determined for all tested fractions, r = 0.805–0.905, p < 0.05) towards different cancer cells were determined. In general, higher cytotoxicity was determined towards HT-29 and CaKi-1 cell lines than against IGR-39 cells. Cytotoxicity against endothelial cells was also correlated with cytotoxicity against cancer cells  with the lowest correlation to the CaKi-1 cell line. Tissue selectivity is a major issue of conventional chemotherapeutic antitumor drugs [r = 0.661–0.882, p < 0.05).Cuticular wax extracts of lingonberry leaves and fruits and obtained fractions were found to reduce the viability of human cancer cells at concentrations of 12.5 and 25.0 µg/µL . The higor drugs . Agents or drugs . Cytotoxwax) was 3.5–5.1 times more active against different cancer cells than that of leaves (Lwax). The observed difference might be explained by the significantly higher contribution of triterpenoids to the wax extract of fruits as discussed previously. Fwax showed higher selectivity than Lwax with up to 1.6-fold higher cytotoxicity to cancer cells than for normal endothelial cells. The obtained EC50 values towards cancer cells were in a range of 12.5–17.7 µg/mL for Fwax, and >25.0 µg/µL for Lwax. Cuticular wax extracts enriched with triterpenoids were found to be considerably more active against the same cancer cells than crude extracts of entire lingonberry raw materials enriched with phenolic compounds as determined earlier. Previously obtained EC50 values were in a range of 1100–1500 µg/mL for the crude extract of entire lingonberry fruits and 120–170 µg/mL for the leaves [At a concentration of 25 µg/µL, crude cuticular wax extract of fruits . The higher contribution of the predominant compound fernenol in 1L when compared to 1F implies the potency of this compound in targeted therapy of melanoma. Previously, fernenol showed the highest fungicidal activity among several tested triterpenoids [Fraction 1F was among the most active fractions and at a concentration of 25 µg/µL reduced cancer cells viability by 94.5–98.1%. Furthermore, this fraction was distinguished by the highest selectivity—observed cytotoxicity to normal cells was 4.9–14.4 times lower than for cancer cells. Counterpart fraction 1L at a concentration of 25 µg/µL was about 10-fold less active against cancer cell lines with the greatest differences towards HT-29 cell line. Maslinic, corosolic, and betulinic acids have been reported to be promising compounds for the chemoprevention or treatment of colon cancer by inhibiting proliferation and inducing apoptosis in HT-29 and other colon cancer cell lines ,61,62, trpenoids  and signrpenoids .50 values were calculated for 2F (EC50 = 4.4–6.5 µg/mL), which distinguished from 2L by a lower ratio of oleanolic:ursolic acids (lower contribution of ursolic acid). Ursolic and oleanolic acids are among the most commonly investigated triterpenoids with well-documented anticancer properties. However, most studies have focused on the activity displayed with either oleanolic or ursolic acids alone. According to the literature reviewed [There was no significant difference between cytotoxicity at a concentration of 25 µg/µL against cancer cell lines between 1F and fractions 2F and 2L, which were enriched with oleanolic and ursolic acids. Slightly higher activity at a lower tested concentration (12.5 µg/µL) and up to 2-times lower ECreviewed ,67,68,69reviewed . Authorsreviewed ,72 disclreviewed , it is p50 of 3L ranged from 9.9 µg/mL towards CaKi-1 up to 14.8 µg/mL against the IGR39 cell line. Moreover, this uvaol-rich fraction was characterized by relatively good selectivity to HT-29 and CaKi-1 cancer cell lines. Cytotoxicity values towards these cancer cell lines were 2.8–6.3 and 2.0–3.5 times higher than for normal EC at different concentration levels. Results of Bonel-Pérez et al. [50 = 14.1 µg/mL after 72 h of incubation) supported by strong anti-migratory capacity. Similar profound activity towards the HepG2 cell line was observed for erythrodiol (IC50 = 8.3 µg/mL after 72 h of incubation) [Counterpart fractions 3F and 3L showed different activity against cancer cells with significantly stronger activities of the fraction of leaves at both concentration levels. This seems to be dependent on the higher contribution of uvaol and purity of erythrodiol:uvaol complex. The greatest difference in cytotoxicity was observed against the HT-29 cell line at the concentration level of 25 µg/mL—the activity of 3L was up to 20.1-fold higher compared to 3F. The observed ECz et al.  showed tubation) , which wubation) . It was ubation) .wax, Lwax, 1F, 1L, 2F, 2L, and 3L. Fraction 4F showed slightly (up to 1.3 times) higher activity than 4L, which was characterized by impurities of phytosterols. The EC50 values of 4F and 4L were reported to be greater than 25.0 µg/mL (>58.6 µM for 4F as for pure fraction of lupeol). Several researchers suggested that lupeol has stronger cytotoxicity towards other than our tested cancer cell lines, namely human glioblastoma U87MG.ΔEGFR (EC50 = 13.6 µM), colon colorectal cancer HCT116 (IC50 = 19.6 µM) [50 = 27.6 µM), multiple myeloma RPMI 8226 (IC50 = 37.5 µM) [Fractions, which were enriched with lupeol (4F and 4L) were significantly more active against HT-29 and CaKi-1 cell lines compared to IGR-39. However, higher than 15% (14.6–41.6%) cytotoxicity towards cancer cells was only observed at a concentration of 25 µg/mL, and cytotoxicity of 4F and 4L was significantly weaker when compared to F19.6 µM) , T-lymph37.5 µM)  cell lin37.5 µM) .p < 0.05) than found of Fwax, Lwax 1F, and 1L. Moreover, 5L and 5F fractions were approximately twice more active towards the CaKi-1 cell line than the normal EC. It has previously been concluded that β-sitosterol, the principal phytosterol of 5L and 5L, has a strong potential for suppressing neoplastic transformation in experimental renal cancer and can be used in a variety of diseases without significant toxicity [Significantly lower cytotoxicity was found for 5L compared to 5F, which was distinguished by the higher contribution of phytosterols. The strongest cytotoxicity was observed towards renal CaKi-1 cell line—5L and 5F reduced viability of these cells by 49.8–64.8% at a concentration of 25 µg/mL. At the lower tested concentration (12.5 µg/mL) cytotoxicity against this cell line was even higher  of 6L towards HT-29 was compatible with EC50 = 19.5 µg/mL of Lwax, which also contained α-amyrin as prevailing neutral triterpenoid. Gonçalves Pereira et al. [50 = 9.9 µM and 8.5 µM, respectively) than that of β-amyrin, β-sitosterol, and friedelin derivatives. Another study [Mesua ferrea L. stem bark, which was enriched with α-amyrin .A pure fraction of α-amyrin from the cuticular wax of lingonberry leaves (6L) was characterized by the highest activity towards the HT-29 cell line. At a concentration of 25 µg/mL, the cytotoxicity of 6L towards the HT-29 cell line was 1.8-fold higher than that of 6F, which besides α-amyrin, contained residuals of β-sitosterol and friedelin and 2.1-fold greater than that of 5L, which in addition to phytosterols contained β-amyrin as prevailing triterpenoid. These results support the superiority of α-amyrin compared to β-amyrin or phytosterols in terms of cytotoxicity against colon cancer HT-29 cells. Nevertheless, a concentration of 25 µg/mL seems to be not selective against cancer cells when compared to EC and further cytotoxicity studies with more specific normal colon fibroblast are needed. The obtained ECa et al.  reporteder study  providedwax, 1F, and 2F) on HT-29, IGR39, and CaKi-1 cancer spheroid growth at concentrations of 5 and 25 µg/mL are depicted in r = 0.921–0.984, p < 0.05), indicating that our results obtained in the 2D model are also reliable for initial screening.The effects of the most promising samples (Fr = 0.635–0.830), however, correlations were not significant (p > 0.05). The authors in [wax) had no significant effect on the size of spheroids, a significant reduction  was observed in the viability of IGR-39 3D culture at both tested concentrations (5 and 25 µg/mL). In general, Fwax was distinguished by the lower activity when compared to tested fractions (1F and 2F), with respective 1.2-fold and 1.5-fold weaker inhibition of cancer spheroids growth and viability on average. This suggests that particular triterpenoid mixtures from lingonberry waxes are more potent as anti-cancer agents than crude unpurified extract.This study showed that spheroid diameter positively correlated with spheroid viability  and the actual IGR-39 spheroid size (by 58.1%) at a concentration of 25 µg/mL. These findings seem to support our previous statements about the strong cytotoxic activity of 1F and its possible application in the treatment of colon and renal cancer. Butkeviciute et al.  noted th50 values of this fraction as determined previously (4.4–6.5 g/mL). Piet and Paduch [Similar to 1F, profound anticancer activity was observed in fraction 2F, namely a mixture of oleanolic:ursolic acids (1:2.2) obtained from the cuticular wax of fruits. At a concentration of 25 g/mL, this fraction reduced cell viability by 73.7–85.2% and inhibited spheroids growth by 31.4–62.9%. Moreover, the clear disintegration of spheroids affected by 2F could be seen at the end of the experiment. A significant reduction in viability (by 33.9%) of IGR39 spheroids was found at a lower tested concentration (5 g/mL), which was comparable with the ECd Paduch  reportedd Paduch  implied 50 values ranging from 4.4 to >25 µg/mL. Cuticular wax extract of lingonberry fruits (Fwax) and fractions—containing maslinic, corosolic, and betulinic acids plus fernenol (1F) and complex of oleanolic:ursolic acids (2F)—were further evaluated for cytotoxicity in 3D cancer cell cultures, showing the ability of these samples to initiate disintegration of cancer spheroids and reduce their viability and growth. Our findings support the use of lingonberry cuticular waxes as a source for the isolation of particular triterpenoids or their complexes, which could be used in the development of new therapeutic candidates for the management of colon, melanoma, or renal cancer diseases.This study first fractionated triterpenoids from cuticular waxes of lingonberries to identify anticancer agents. Chromatographic analyses (HPLC-PDA and GC-MS) indicated the presence of five triterpenoid acids, eight neutral triterpenoids, three phytosterols, and eight minor unidentified triterpenoids in cuticular waxes of lingonberry leaves or fruits. As a result of semi-preparative HPLC fractionation, 12 fractions enriched with particular triterpenoids or their mixtures were obtained in total. Cytotoxicity studies carried out by MTT assay towards HT-29, IGR39, and CaKi-1 cancer cells, as well as normal endothelial cells, highlighted the most efficient fractions and their predominant compounds, with EC