S1P1 T236A  decreases TNBC cell migration in vitro and disease invasion in zebrafish xenografts. Pharmacologic disruption of S1P1 T236 phosphorylation, using either a pan-AKT inhibitor (MK2206) or an S1P1 functional antagonist , suppresses TNBC cell migration in vitro and tumor invasion in vivo. Finally, we show that human TNBC cells with AKT activation and elevated phospho-S1P1 T236 are sensitive to FTY720-induced cytotoxic effects. These findings indicate that the AKT-enhanced phosphorylation of S1P1 T236 mediates much of the TNBC invasiveness, providing a potential biomarker to select TNBC patients for the clinical application of FTY720.Hyperactive sphingosine 1-phosphate (S1P) signaling is associated with a poor prognosis of triple-negative breast cancer (TNBC). Despite recent evidence that links the S1P receptor 1 (S1P1) to TNBC cell survival, its role in TNBC invasion and the underlying mechanisms remain elusive. Combining analyses of human TNBC cells with zebrafish xenografts, we found that phosphorylation of S1P receptor 1 (S1P1) at threonine 236 (T236) is critical for TNBC dissemination. Compared to luminal breast cancer cells, TNBC cells exhibit a significant increase of phospho-S1P1 T236 but not the total S1P1 levels. Misexpression of phosphorylation-defective  Breast cancer is the second leading cause of mortality in women worldwide . This heHyperactive sphingosine 1-phosphate (S1P) signaling can induce cancer progression in both solid and hematological malignancies . ProduceEDG1), S1P1 was discovered as one of the immediate early-regulatory genes of endothelial cell differentiation [Among the five S1P receptors, S1P1 has gained attention as the main receptor responsible for vascular development and immune cell regulation . Originantiation . As a GPntiation . Interesntiation . S1P1 hantiation ,16. In antiation ,18,19. Fntiation . In the ntiation . Emerginntiation ,23. HencHere we combine the genetic and biochemical analyses of human TNBC cells with real-time monitoring of tumor cell dissemination in zebrafish xenografts to understand the role and mechanisms of action of S1P1 in TNBC invasiveness. Unlike luminal breast cancer, S1P1 expression was not elevated in human TNBC cells. Instead, we found that AKT-mediated phosphorylation of S1P1 at T236 was the factor most closely related to TNBC invasiveness and distant spread. In addition, FTY720 was sufficient in blocking tumor cell dissemination while inducing cell death for TNBC with an elevated expression of phospho-S1P1 T236 (P-S1P1 T236).AB, Casper, and Casper;Tg(fli1:EGFP).Zebrafish husbandry was performed as described in the aquatic facility at the Boston University School of Medicine, in accordance with our IACUC-approved protocol. Zebrafish lines used in this study include S1P1 cDNA was mutated at the threonine (T) 236 site to alanine (A) using polymerase chain reaction (PCR) for site-directed mutagenesis with the QuickChange II Kit (Agilent). Both S1P1 wild-type and T236A mutant sequences were amplified by PCR and cloned into the PDONR221 gateway entry vector . These entry plasmids were subsequently recombined with the PWPI-DEST-GFP bicistronic lentiviral vector, thus creating PWPI-DESTGFP-S1P1-WT and PWPI-DEST-GFP-S1P1-T236A overexpression plasmids. The PLemiR-RFP lentiviral vector was used for fluorescence tracking of cell lines upon viral infection.The human RFP, Luciferase, S1P1 WT, or S1P1 T236A) was used to infect MCF10A-derived cell lines. Cells were subsequently sorted based on GFP or RFP expression. Overexpression of S1P1 was verified using western blotting.All cell lines used in this study were cultivated according to ATCC recommendations. For virus production, the human embryonic kidney cells (HEK293T) were incubated with the gene-of-interest plasmid, psPAX2, and pMD2.G lentiviral vectors using serum-free OPTI-MEM (Thermo Fisher Scientific) according to standard procedure in the presence of polybrene . HEK293T supernatant containing each type of lentivirus  or 6-well plates , respectively, in triplicates. For the proliferation and viability assays, Cell Titer blue kits  were used according to manufacturer instructions in order to quantify live cells. For wound healing assays, scratches were performed using a pipet tip, washed once with PBS, and replaced with a full growth medium. Images were acquired using an EVOS Xl microscope with a 4× objective (Thermo Fisher Scientific), either directly after the scratch or over time to monitor wound recovery.6 cells per mL in a complete growth media. All cells were either expressing RFP or stained with Cell tracker Orange CMTMR (Thermo Fisher Scientific). About 1 nL of these cells was microinjected into the vascularized area near the perivitelline cavity of the embryo using a needle holder and a microinjection station . These zebrafish xenografts were incubated in the dark at 35 °C for three days and monitored by fluorescence imaging to document tumor cell survival and metastatic spreads. Zebrafish embryos were immobilized using low-temperature melting agarose  for acquiring images or movies using an MVX10 stereomicroscope (Olympus of America). The metastatic cells were quantified using Fiji software based on fluorescent intensity outside the injection area (ImageJ2) or, in some experiments, counted manually.The 48-hpf embryos were dechorionated enzymatically using Pronase  and anesthetized with tricaine . Human TNBC cells were trypsinized, washed, counted, and resuspended to obtain a final concentration of 10 × 10For viability assays, 5000 to 10,000 cells per well were seeded in a 96-well plate with a media containing MK2206  or FTY720  for two days, using cells treated with DMSO  alone as controls. Cell viability was measured by Cell Titer blue assay (Promega) and was presented as a percentage of control cells. Zebrafish transplanted with human TNBC cells were incubated in the presence of MK2206 at 0.3 μM or FTY720 at 2 μM in fish water for three days, using embryos treated with DMSO as controls. Fish water containing the compound was refreshed daily.G:BOX Chemi XT4  and quantified with Fiji software.Breast cancer cells were collected and lysed in RIPA buffer supplemented with Halt proteinase and phosphatase inhibitor cocktail (Thermo Fisher Scientific). The primary antibodies included anti-SPHK1 , anti-EDG1/S1P1 , antiphospho-EDG1/S1P1 T236 , anti-AKT and anti-phospho-AKT S473 , anti-active-Caspase 3 , and anti-β-ACTIN . Secondary antibodies included horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies . Chemiluminescent images were obtained using a ER (ESR1) ≥ 1000, PR (PGR) ≥ 20, HER2 (ERBB2) ≥ 3700 as thresholds, and the data less than the three thresholds were considered as TNBC (n = 41), while the rest were classified as non-TNBC (n = 385).The dataset (GSE2034) for human breast cancer patient samples was obtained from Gene Expression Omnibus (GEO) ), and rep) using a two-tailed Student’s t-test for the probes of interest in gene expression analysis, protein-of-interest to ACTIN ratio for western blot analysis, tumor burden, migration, and invasive spread of TNBC cells. The false discovery rate (FDR) was used to test all the probes in gene expression analysis. The p-values or the FDR-adjusted p-value  less than or equal to 0.05 were considered statistically significant.GraphPad Prism software was used to calculate significant values  . We foun samples a. By con samples b. Intere samples c.Given the recent findings that S1P1 is important for TNBC survival ,23, we nHaving identified a link between the phosphorylation of S1P1 T236 and human TNBC cells, we sought to clarify how the phosphorylation of S1P1 T236 might contribute to disease aggressiveness. We utilized the well-characterized MCF10A-derived model of TNBC progression, consisting of four basal-like/TNBC cell lines with increasing degrees of aggressiveness: nontransformed MCF10A (MI), transformed benign MCF10AT (NeoT), malignant and metastatic MCF10CA1h (MIII), and the most aggressive and invasive MCF10CA1a.cl1 (MIV) . WesternIt has been well demonstrated that zebrafish xenografts can mirror the metastatic properties of human cancer cells in vitro and in mice . MoreoveS1P1 wild-type (WT) or phosphorylation-defective mutant (T236A) on MIII and MIV cell behavior in zebrafish xenografts. A western blot analysis demonstrated elevated S1P1 and P-S1P1 T236 levels in S1P1-overexpressing MIII and MIV cells, compared to control Luciferase-overexpressing cells (S1P1 WT throughout the zebrafish at three dpt, compared to the control cells (S1P1 T236A mutant significantly inhibited the distant spread of the human TNBC cells in zebrafish—the opposite effect from that seen with S1P1 WT overexpression (S1P1 T236A, compared to cells overexpressing either S1P1 WT or Luciferase (To investigate whether phosphorylation of S1P1 T236 directly mediates TNBC aggressiveness, we evaluated the effects of overexpressing ng cells a. Despitng cells , we obseol cells b,c. Stripression b,c. Quanciferase c.Luciferase, S1P1 WT, or S1P1 T236A. Compared to the controls, overexpressing neither S1P1 WT nor S1P1 T236A affected MIII cell proliferation (Luciferase, S1P1 WT, or S1P1 T236A (S1P1 WT in MIII cells led to increased levels of P-S1P1 T236 and more rapid closure of the wound gap than seen in controls (S1P1 T236A mutant in MIII cells significantly inhibited the healing of the wound gap (To understand how increased phosphorylation of S1P1 T236 impacts TNBC invasiveness, we measured the proliferation rate of MIII cells overexpressing feration . Given tP1 T236A d. Our dacontrols a,d,e. Byound gap a,d,e. ThAKT binds directly to the third intracellular loop of the S1P1 receptor and phosphorylates its T236 residue in endothelial cells . To deteLuciferase, S1P1 WT, or S1P1 T236A (S1P1 WT or S1P1 T236A, compared to the vehicle-treated controls (S1P1 WT and not S1P1 T236A led to an increased total of AKT and P-AKT S473 levels, indicating that S1P1 activated its downstream AKT signaling. We then performed a scratch-wound assay of these cells and found that MK2206 treatment significantly impeded the wound healing of MIII cells overexpressing Luciferase, or S1P1 WT, compared with the vehicle-treated cells (S1P1 T236A (S1P1 WT compared to the vehicle-treated controls (Similar to its effect on the parental MIII cells, MK2206 treatment also reduced P-AKTS473 and P-S1P1 T236 levels in MIII cells that overexpress P1 T236A a. Interecontrols a. Overexed cells b,c. InteP1 T236A b,c. In zcontrols . We concFTY720 is an FDA-approved drug for the first-line treatment of multiple sclerosis . PhosphoNext, we transplanted MIII and MIV cells into zebrafish embryos, treated them with FTY720, and monitored tumor cell dissemination over time. Although fish were transplanted with similar tumor burdens , FTY720 SPHK1 yet the decreased expression of SPHK2. This result challenges the concept that high SPHK expression results in hyperactive S1P signaling [S1P1 T236A mutant suppressed migration of TNBC cells, as demonstrated by the wound scratch assays, and distant dissemination as monitored in zebrafish xenografts. Together, our results indicated that P-S1P1 T236 contributes to TNBC aggressiveness by enhancing migration and the distant spread of tumor cells. Thus, increased phosphorylation at the T236 site of S1P1 may serve as a biomarker to predict tumor aggressiveness for TNBC patients. Although most patients with TNBC initially respond well to cytotoxic therapy, they rapidly develop resistance to treatment and often present with metastatic disease. When this occurs, there are limited treatment options available for these patients, underscoring the urgent need to understand the mechanisms that underlie TNBC aggressiveness in order to develop targeted therapy. Our reanalysis of a published microarray dataset showed that compared to other subtypes of breast cancer, TNBC patient samples exhibited elevated mRNA expression levels of ignaling . Among tignaling . In the ignaling ,23. Our Although AKT was shown to phosphorylate S1P1 T236 and promote migration in endothelial cells , its regPrevious studies have shown that MK2206 induces apoptosis in a dose-dependent manner in certain breast cancer cell lines . AdditioFTY720, the FDA-approved drug for multiple sclerosis, can effectively suppress S1P1 signaling through the downregulation of total S1P1 levels . Althoug


H2) blockers are used to treat peptic ulcers and gastric reflux. Chlorquinaldol and chloroxine, which contain an 8-hydroxyquinoline (8HQ) core, have recently been identified as blocking HRH2. To gain insight into the mode of action of 8HQ-based blockers, here, we leverage an HRH2-based sensor in yeast to evaluate the role of key residues in the HRH2 active site on histamine and 8HQ-based blocker binding. We find that the HRH2 mutations D98A, F254A, Y182A, and Y250A render the receptor inactive in the presence of histamine, while HRH2:D186A and HRH2:T190A retain residual activity. Based on molecular docking studies, this outcome correlates with the ability of the pharmacologically relevant histamine tautomers to interact with D98 via the charged amine. Docking studies also suggest that, unlike established HRH2 blockers that interact with both ends of the HRH2 binding site, 8HQ-based blockers interact with only one end, either the end framed by D98/Y250 or T190/D186. Experimentally, we find that chlorquinaldol and chloroxine still inactivate HRH2:D186A by shifting their engagement from D98 to Y250 in the case of chlorquinaldol and D186 to Y182 in the case of chloroxine. Importantly, the tyrosine interactions are supported by the intramolecular hydrogen bonding of the 8HQ-based blockers. The insight gained in this work will aid in the development of improved HRH2 therapeutics. More generally, this work demonstrates that Gprotein-coupled receptor (GPCR)-based sensors in yeast can help elucidate the mode of action of novel ligands for GPCRs, a family of receptors that bind 30% of FDA therapeutics.Histamine receptor 2 (HR H1, is involved in allergic responses and has been extensively characterized both structurally [H2, is involved in gastric acid secretion and gastroesophageal reflux diseases (GERD) [H3, is expressed in the central nervous system and participates in neuronal histamine turnover as well as the modulation of the release of other neurotransmitters, such as dopamine and serotonin [H4, is expressed in peripheral immune cells and is involved in immunomodulation [Histamine receptors belong to the aminergic family of Gprotein-coupled receptors (GPCRs), which includes the serotonin, muscarinic, acetylcholine, adrenergic, and dopamine receptors. Histamine receptors are involved in a variety of cellular processes, from allergies and gastric acid secretion to neurotransmission and immunomodulation . There acturally ,3 and excturally . Histamis (GERD) . Histamierotonin . Histamidulation . H2 blockers, such as cimetidine (Tagamet®) and famotidine (Pepcid®), are used to reduce gastric acid secretion and treat peptic ulcers and acid reflux. The chemical structure of some of these blockers, such as ranitidine (Zantac®), contains a tertiary amine that decomposes into the human carcinogen N-nitrosodimethylamine (NDMA) [H2 activity in mammalian cells [H2 therapeutics. Over the counter HRe (NDMA) , which he (NDMA) . Recentlan cells . ImportaH2 blockers. Limited mutagenesis studies of the HRH2 binding site hamper our ability to understand the mode of action of 8HQ-based blockers. Here, we leverage an HRH2-based sensor in yeast to evaluate the role of key residues in the HRH2 active site on histamine and 8HQ-based blocker binding. First, using molecular docking, we identify the HRH2 residues involved in the binding of therapeutically relevant histamine tautomers and the 8HQ-based blockers. Then, we computationally and experimentally perform an alanine scanning of the HRH2 binding site and find that only HRH2:D186A and HRH2:T190A have residual activity in the presence of histamine. Finally, we determine that 8HQ-based blockers can still inactivate HRH2:D186A by swapping their interaction from D98 to Y250 in the case of chlorquinaldol and D186 to Y182 in the case of chloroxine. The internal stabilization of chlorinated 8HQ-based blockers, where the proton in the hydroxy group interacts with the nitrogen lone pair, is pivotal for interactions with tyrosine. Taken together, this work expands our understanding of histamine and 8HQ-blocker binding to HRH2, and provides evidence that GPCR-based sensors in yeast have utility in elucidating the mode of action of novel ligands for GPCRs.Interestingly, 8HQ-based blockers lack the positively charged amine commonly present in HRMaterials. Luciferase expression was assayed using the NanoGlo® Luciferase Assay System (Promega N1120). Histamine dihydrochloride (Sigma H7250), chlorquinaldol (Selleck S4192), and chloroxine (Selleck S1839) were purchased from the specified vendors. The HRH2 point mutants  were codon optimized for Saccharomyces cerevisiae and commercially synthesized. Tables of plasmids , chlorquinaldol (ZINC119403), and chloroxine (ZINC1131) [H2 (Alphafold: P25021) [H2 was conducted using the PyMOL mutagenesis wizard, with P25021 as the template. Hydrogens were introduced to the ligands using CACTUS (https://cactus.nci.nih.gov/translate/ (accessed on 27 February 2023). The HRH2 binding site was defined by D98, D186, and T190 [ldol ZINC9403, and P25021) ,13. Compand T190 . Ligandsand T190  and PyMOH2 mutant constructionHR. HRH2:D98A, HRH2:Y182A, HRH2:D186A, HRH2:T190A, HRH2:Y250A, and HRH2:F254A were cloned into pESC-HIS3-PTEF-PADH [BamHI/SacII via Gibson assembly to generate pESC-HIS3-PTEF-HRH2_D98A (pPM43), pESC-HIS3-PTEF-HRH2_Y182A (pPM50), pESC-HIS3-PTEF-HRH2_D186A (pPM52), pESC-HIS3-PTEF-HRH2_T190A (pPM53), pESC-HIS3-PTEF-HRH2_Y250A (pPM54), and pESC-HIS3-PTEF-HRH2_F254A (pPM49), respectively. Constructs were sequence-verified using primers LT62/LT63. TEF-PADH  between H2 mutant-based yeast sensors.HR PPY140  [FIG1-NanoLuc [FIG1-NanoLuc and pESC-HIS3-PTEF-PADH (PPY1809)., Δsst2)  was co-t-NanoLuc  and eithHistamine sensing. An overnight culture of PPY2370, PPY2393, PPY2390, PPY2391, PPY2394, or PPY2392 was used to inoculate 50 mL of synthetic complete medium with 2% glucose lacking histidine and leucine (SD(HL−)) to an OD600 = 0.06. After 18 h at 15 °C (150 rpm), the cultures were centrifuged , and resuspended in SD(HL−) to an OD600 = 1. In a white, flat-bottomed, 96-well plate, 190 μL pH = 7 SD (HL−), 8 μL of cells, and 2 μL of histamine , or DMSO as a control were added. After chemical incubation , 20 μL of 1:100 mixture of NanoLuc substrate to NanoLuc buffer were added, and the reaction was incubated for 30 min . Luminescence was read in a Biotek Synergy 2 using the default settings.H2 blocker sensing.HR The histamine sensing protocol was followed except as described. In a white, flat-bottomed, 96-well plate, 188 µL pH = 7 SD (HL−), 8 µL of cells, 2 µL of histamine , and 2 µL chlorquinaldol or chloroxine  were added. For the no chemical control, no histamine or blocker was added, only 4 µL of DMSO. The no-receptor control strain was tested under the same conditions as the HRH2 mutant sensor strains.H2 to histamine.Molecular docking of HR The topological view of HRH2 highlighting its active site is seen in H2:D98A with histamine resulted in no cAMP accumulation [H2:D186A and HRH2:T190A with histamine resulted in 50% and 17% of wild-type HRH2 activation, respectively [mulation . Positioectively . H2, we docked histamine to the AlphaFold structure of HRH2. Notably, although the Cryo-EM structure of HRH2 has been recently elucidated (PDB: 7UL3) [To understand the binding mode of histamine to HRB: 7UL3) , it is iAt pH 7.4, histamine is protonated and present in four tautomer states. The trans tautomers are the predominant form, with the gauche tautomers representing 25% of the population . As showH2.Molecular docking of 8HQ-based blockers to HR Generally, HRH2 blockers carry a positively charged amine that interacts with D98 and are long enough to interact with both ends of the HRH2 binding site. Such is the case for famotidine, whose sulfone end interacts with D98 and Y250 and guanidine group interacts with T190 and D186 [H2 binding pocket. Chlorquinaldol interacts with D98 and Y250 via the hydroxyl group of the hydroxyquinoline ring , Y182 (Y5×39), D186 (D5×3), T190 (T5×461), Y250 (Y6×51), and F254 (F6×55)—and mutated them to alanine. As shown in H2. H2 mutants in a similar fashion to histamine TT1 interacting with wild-type HRH2. Specifically, we do not see the histamine tautomers using two electrostatic interactions via the protonated amine to engage D98 and using the protonated amine in the imidazole ring to interact with Y250. We do observe that in HRH2:D186A all histamine tautomers engage D98 via two electrostatic interactions using the charged amine. The only other instance where D98 is engaged via the charged amine is in HRH2:T190A with histamine GT2. In five HRH2 mutants, D98 is engaged via a single electrostatic interaction with the protonated nitrogen in the imidazole ring, specifically histamine TT2 with HRH2:T190A, HRH2:F254A, and HRH2:Y182A and histamine GT2 with HRH2:F254A and HRH2:Y182A. Of note, in no HRH2 mutant are histamine tautomers able to interact with Y250 via the protonated nitrogen in the imidazole ring. Taken together, the engagement of D98 is possible in some of the single-point mutants, specifically HRH2:D186A > HRH2:T190A > HRH2:F254A = HRH2:Y182A. In none of the HRH2 mutants do the histamine tautomers engage Y250 via the protonated amine, as seen in histamine TT1 with wild-type HRH2.None of the histamine tautomers interact with any of the HRH2 active site and its impact on histamine-driven activationExperimental alanine scanning of the HR. We constructed the six HRH2 alanine mutants—HRH2:D98A, HRH2:D186A, HRH2:T190A, HRH2:F254A, HRH2:Y182A, HRH2:Y250A—and expressed them in a previously developed GPCR-based sensor strain that links GPCR activation to cell luminescence  or Y182 (chloroxine). The results described in this work should aid in our understanding of the mode of action of 8HQ-based blockers and pave the way for the development of improved HRH2 blockers. More generally, this work demonstrates that GPCR-based sensors in yeast are valuable in illuminating the mode of action of novel GPCR ligands. GPCRs are targeted by 30% of FDA-approved drugs . GPCR-ba


Candida glabrata bloodstream isolates obtained from a neutropenic patient during 82 days of amphotericin B (AMB) or echinocandin therapy. For WGS, a library was prepared and sequenced using a Nextera DNA Flex Kit (Illumina) and the MiseqDx (Illumina) instrument. All isolates harbored the same Msh2p substitution, V239L, associated with multilocus sequence type 7 and a Pdr1p substitution, L825P, that caused azole resistance. Of six isolates with increased AMB MICs (≥2 mg/L), three harboring the Erg6p A158fs mutation had AMB MICs ≥ 8 mg/L, and three harboring the Erg6p R314K, Erg3p G236D, or Erg3p F226fs mutation had AMB MICs of 2–3 mg/L. Four isolates harboring the Erg6p A158fs or R314K mutation had fluconazole MICs of 4–8 mg/L while the remaining six had fluconazole MICs ≥ 256 mg/L. Two isolates with micafungin MICs > 8 mg/L harbored Fks2p (I661_L662insF) and Fks1p (C499fs) mutations, while six isolates with micafungin MICs of 0.25–2 mg/L harbored an Fks2p K1357E substitution. Using WGS, we detected novel mechanisms of AMB and echinocandin resistance; we explored mechanisms that may explain the complex relationship between AMB and azole resistance.Whole-genome sequencing (WGS) was used to determine the molecular mechanisms of multidrug resistance for 10 serial  Candida bloodstream infections (BSIs) are the most common nosocomial fungal infections and are associated with high rates of mortality . An. An40]. CDR1 (H58Y), PDH1 (E839D and T1530K), PDR1 , QDR2 (T199M), FLR1 (V254I), FKS3 , and FEN1 (M155T). ERG genes (ERG1–ERG10), FKS1/2, and MSH2 and PDR1 GOF mutations detected by WGS. Of the ERG genes, 11 unique nonsynonymous mutations were detected in ERG2, ERG3, ERG4, ERG6, ERG7, ERG8, and ERG10 throughout the serial isolates, of which 6 SNPs  were observed in all 10 isolates. When the AMB MICs were investigated, three isolates  harboring a frameshift mutation (A158fs) in ERG6 showed strong resistance to AMB (MICs of 8–16 mg/L). The other three isolates showing increased MICs against AMB (2–3 mg/L) harbored ERG6 R314K (isolate 5), ERG3 G236D and ERG4 P227fs (isolate 8), and ERG3 F226fs (isolate 10) mutations.Nonsynonymous mutations in various genes associated with antifungal resistance were compared with the antifungal MICs of the 10 serial isolates (isolates 1–10). All isolates showed the same Msh2p substitution, V239L (associated with ST7 in MLST). All isolates also had the same nonsynonymous mutations in PDR1 GOF mutation (L825P) was observed in the 10 serial isolates. Six FR isolates  showed markedly high MICs (≥256 mg/L) for fluconazole and higher MICs for voriconazole, posaconazole, and itraconazole. However, the fluconazole MICs were 4–8 mg/L in four (isolate 3 and 5–7) isolates that harbored ERG6 A158fs or R314K simultaneously. These four isolates also showed lower MICs for other azoles. With regard to echinocandins, all of the isolates exhibited intermediate to high resistance to at least one of anidulafungin, caspofungin, and micafungin. Of the isolates, isolate 2 and 5, which exhibited MICs > 8 mg/L for three echinocandins, harbored an FKS2 I661_L662insF mutation in combination with FKS1 C499fs. The isolate harboring an F659del HS mutation in FKS2 (isolate 9) showed definitively increased anidulafungin, caspofungin, and micafungin MICs . In contrast, the isolate simultaneously harboring FKS2 F659del and S201fs (isolate 4) was susceptible to anidulafungin and micafungin, and intermediate only to caspofungin. The other isolates  showed slightly increased echinocandin MICs  and harbored a K1357E mutation in FKS2.With regard to azole resistance, a C. glabrata isolates with the same Pdr1p L825P mutation during the course of AMB or echinocandin therapy. All 10 isolates were associated with breakthrough fungemia during the administration of echinocandins (isolates 1 and 4), AMB , or both (isolates 9 and 10). The Fks2p K1357E mutation first appeared after 17 days of micafungin exposure in isolate 1 and it was shared by five subsequent isolates . These six isolates were designated as subpopulation #1. The Erg6p A158fs mutation first appeared after 16 days of AMB therapy in isolate 3 (subpopulation #1–2) and was shared by two subsequent isolates . Clonal subpopulation #1 re-appeared after 6 days of caspofungin therapy and 35 days of AMB therapy with the addition of Erg4p P227fs combined with an Erg3p G236D mutation , and after 19 days of caspofungin and 52 days of AMB therapy with an additional Erg3p F226fs mutation . Overall, echinocandin breakthrough fungemia was caused by two isolates (isolate 1 and 10) of subpopulation #1, which harbored the Fks2p K1357E mutation. On the other hand, Fks2p I661_L662insF and Fks1p C499fs appeared in isolate 2 after 23 days of micafungin therapy; these mutations were shared by isolate 5, so isolates 2 and 5 were designated as clonal subpopulation #2. Clonal subpopulation #2 with an additional Erg6p R314K mutation appeared after 22 days of AMB therapy . In addition, two isolates (isolate 4 and 9) had a unique Fks2p mutation .G. mellonella model, among C. glabrata isolates 1 to 5. In vivo assays in the insect G. mellonella revealed that the 96-h survival rates of G. mellonella larvae infected with four isolates  were relatively higher than survival rates of G. mellonella larvae infected with FR or F-SDD isolates. The mean survival rate in larvae infected with FR isolates (N = 18) was significantly higher than the rate in larvae infected with F-SDD isolates (N = 17) at all four time periods examined .C. glabrata BSI isolates during treatment is a possible cause of treatment failure, but few reports have provided a comprehensive understanding of how C. glabrata genomes can accumulate gene mutations that result in phenotypic resistance to antifungals during an extended course of antifungal therapy. In the present study, all 10 isolates harbored the same Msh2p substitution, V239L, which is known to be associated with both MLST type ST7 [C. glabrata isolates with Pdr1p GOF mutations could lower fluconazole MICs. The development of resistance to type ST7  and hypetype ST7 . All isotype ST7 . Our WGStype ST7 ,42. An iCandida isolates is rare [C. glabrata may be partly due to a lack of detection ability using current CLSI or the European Committee on Antimicrobial Susceptibility Testing reference methods. In this study, the AMB MICs of six isolates were ≥2 mg/L by the ETEST®, but those of all 10 isolates were 0.5–1 and 0.5–2 mg/L by the CLSI M27 method and Sensititre Yeast One® system, respectively (data not shown), in agreement with a previous report [ERG2, ERG3, ERG4, and ERG6 in AMB resistance [ERG6 that resulted in AMB resistance due to a composition change in sterol, which is the target of polyene. Here, we showed that three isolates of C. glabrata harboring a disruptive frameshift mutation (A158fs) in ERG6 exhibited markedly increased MICs (8–12 mg/L) for AMB and that harboring a substitution mutation (R314K) in ERG6 moderately increased the AMB MIC to 2 mg/L. Thus, ERG6 may be involved in AMB resistance in C. glabrata. An I207V mutation in ERG2 was also detected but was found in all isolates (isolates 1–10). Mutations in ERG3 or ERG4 have been found in AMB-resistant Candida albicans [Saccharomyces cerevisiae [C. glabrata [ERG3 G236D and ERG4 P227fs (isolate 8) and ERG3 F226fs (isolate 10) mutations, which may require more supporting evidence. Acquired AMB resistance in  is rare ,44,45,46s report . The limsistance ,49. Prevsistance  in ERG6 albicans ,51,52 anrevisiae , but rarglabrata . In the PDR1 sequences of each C. glabrata isolate with the reference PDR1 sequence of C. glabrata (GenBank accession no. FJ550269) [C. glabrata in Korea harbored FR-specific Pdr1p mutations by excluding MLST genotype-specific Pdr1p amino acid substitutions [PDR1, which may mediate azole resistance in C. glabrata [PDR1 GOF mutation (L825P), while six had a fluconazole MIC ≥ 256 mg/L (FR). All four F-SDD isolates harbored an Erg6p (A158fs or R314K) mutation. A previous study showed that the lower ergosterol content associated with a nonsense mutation in ERG6 may have an indirect effect on susceptibility to azoles by preventing the targeting of efflux pumps to the plasma membrane, thereby favoring the accumulation of these drugs within the cell [ERG6 mutations could lead to defects in ergosterol synthesis and changes in the binding of the efflux pump. In our previous study, FR isolates of C. glabrata exhibited higher mean expression levels of CgCDR1, CgCDR2, and CgSNQ2, compared with F-SDD isolates [CgCDR1, CgCDR2, and CgSNQ2 in five isolates with the same Pdr1p L825P mutation, without  or with  an Erg6p mutation, the expression levels of CgCDR1 and CgSNQ2 in the three FR isolates were relatively higher than levels in the two F-SDD isolates, and similar to the mean expression levels of CgCDR1 and CgSNQ2 in 30 FR isolates harboring Pdr1p mutations. Taken together, our findings indicate that C. glabrata isolates with the same Pdr1p GOF mutations do not always show the same FR result—they can be F-SDD in AMB-resistant isolates with Erg6p mutations.In our previous study, by comparing the itutions . In thisglabrata . Howeverthe cell . The preutation L25P, whilFKS genes is the most convenient way of determining echinocandin resistance mechanisms, mutations occurring outside of these HS regions can also lead to echinocandin therapeutic failure, which confirms the importance of sequencing the entire FKS gene [FKS2, and all isolates (except isolate 9) showed missense or frameshift mutations occurring outside of these HS regions. Of the isolates, two with anidulafungin, caspofungin, and micafungin MICs > 8 mg/L harbored not only the mutation Fks2p I661_L662insF but also Fks1p C499fs. Given that mutations in FKS1 or FKS2 [FKS1 and point HS mutation in FKS2 [C. glabrata strains, the unique HS mutations (I661_L662insF and F659del) in FKS2 found in this study may have different impacts on echinocandin MICs according to the combination of other FKS nonsynonymous mutations. There were also two isolates harboring the mutation FKS2 F659del with or without the upstream FKS2 S201fs mutation. Relatively strong resistance to echinocandins was observed in an isolate harboring a single F659del mutation in FKS2 (isolate 9), but the echinocandin MIC was slightly decreased in an isolate harboring both mutations (F659del and S201fs) in FKS2 (isolate 4). The reasons for the lowered echinocandin MIC in isolate 4 harboring the F659del and S201fs mutations are uncertain. One possibility is that the upstream FKS2 S201fs mutation may affect the downstream FKS2 F659del mutation, but more evidence is needed. Six isolates in subpopulation #1 harboring the Fks2p K1357E mutation  showed micafungin MICs of 0.25–2 mg/L. Although sequencing of the HS regions in FKS gene ,56. In t or FKS2 ,57, or tC. glabrata isolates harboring a single non-HS mutation in an FKS gene showed slightly increased MICs for echinocandins [FKS genes can increase echinocandin resistance. The Fks2p K1357E mutation first appeared as breakthrough fungemia after 17 days of micafungin exposure in isolate 1, and five additional isolates were recovered from HD 99 to HD 160 despite further micafungin (5 days) or caspofungin (19 days) therapy. The role of Fks2p K1357E in echinocandin resistance remains uncertain as this SNP has not been described previously. However, a previous report showed that ocandins . Here, wC. glabrata BSI isolates from a particular geographic area have been reported to comprise a small number of major STs, according to MLST analysis. MLST of Korean BSI isolates showed that ST7 (47.8%) was the most common type, followed by ST3 (22.5%); the remaining isolates exhibited 28 types of minor STs [C. glabrata typically had one Pdr1p amino acid substitution, which were rarely shared by two isolates from the same hospital in the same year, in agreement with a previous report that C. glabrata transmission between patients is rare [C. glabrata in most patients [C. glabrata obtained from our patient, the serial isolates showed significant non-serial phenotypic MIC variations to AMB, azole, or echinocandins. Similarly, phylogenetic analysis by WGS showed substantial genetic diversity, regardless of isolation date and phenotypic antifungal susceptibility pattern  may represent pre-existing mutations, and some new mutations occurred after antifungal drug exposure. Subpopulations with pre-existing mutations are likely to persist in the gut or other mucosal sites and appear in the bloodstream with or without new genetic changes during the long course of antifungal therapy.We postulated that subpopulations with different resistance profiles are likely to have persisted in the gut and alternately invaded the bloodstream under selective pressure, highlighting the adaptability of l agents . In the C. glabrata is unclear, and few studies have been reported thus far [G. mellonella has been used as a host model to study C. glabrata virulence and antifungal efficacy [PDR1 mutations may be more virulent than FR isolates harboring PDR1 mutations. Moreover, our results suggest that our MDR C. glabrata isolates with PDR1 gene mutations have reduced virulence in the G. mellonella model.The fitness cost related to antifungal resistance acquisition by thus far ,62,63. Gefficacy . In the C. glabrata isolates. Some of these newly detected mutations were out of the target region in the gene  or out of the target gene  from our previous study based on conventional sequencing [A notable limitation of this study is that, although many of the detected mutations were located in genes involved in resistance, we did not directly assess their roles in resistance. The reintroduction of mutant alleles into susceptible strains via gene editing would be a useful approach for determining their roles in resistance. We used WGS to detect specific genetic alterations associated with antifungal resistance in serial clonal quencing . In thisC. glabrata BSI isolates obtained from a patient with breakthrough fungemia during extended AMB or echinocandin therapy. Pdr1p GOF and Fksp mutations in C. glabrata may not always have the same effects; they may cause different levels of antifungal resistance, depending on the combination of nonsynonymous mutations present. Fluconazole MICs are lower in C. glabrata isolates with the same Pdr1p GOF mutation than in AMB-resistant isolates with Egr6p mutations. Fks2p HS mutations combined with Fks1p null-function mutations contribute to high-level echinocandin resistance. In addition, Fks2p mutations outside HS regions contribute to low-level echinocandin resistance. Persistent subpopulations of C. glabrata undergoing continuous clonal genetic evolution during long-term antifungal therapy could be responsible for the non-serial multiple antifungal resistance phenotypes of C. glabrata BSI isolates. In conclusion, WGS will improve the detection and monitoring of molecular mechanisms of antifungal resistance.In conclusion, this study provides important perspectives on the utility of WGS for detecting molecular mechanisms of multidrug resistance based on 10 serial


Kinesins support many diverse cellular processes, including facilitating cell division through mechanical regulation of the mitotic spindle. However, how kinesin activity is controlled to facilitate this process is not well understood. Interestingly, post-translational modifications have been identified within the enzymatic region of all 45 mammalian kinesins, but the significance of these modifications has gone largely unexplored. Given the critical role of the enzymatic region in facilitating nucleotide and microtubule binding, it may serve as a primary site for kinesin regulation. Consistent with this idea, a phosphomimetic mutation at S357 in the neck-linker of KIF18A alters the localization of KIF18A within the spindle from kinetochore microtubules to peripheral microtubules. Changes in localization of KIF18A-S357D are accompanied by defects in mitotic spindle positioning and the ability to promote mitotic progression. This altered localization pattern is mimicked by a shortened neck-linker mutant, suggesting that KIF18A-S357D may cause the motor to adopt a shortened neck-linker like state that prevents KIF18A from accumulating at the plus-ends of kinetochore microtubules. These findings demonstrate that post-translational modifications in the enzymatic region of kinesins could be important for biasing their localization to particular microtubule subpopulations. Kinesins were first identified as proteins that transport organelles via microtubules in squid giant axons . Since tSeveral mechanisms of regulation have been identified that work to control the function of individual kinesins. For example, autoregulation, whereby conformational shifts in the kinesin structure result in interactions between the kinesin motor domain and tail, prevent enzymatic activation . This auMany proteins are known to be regulated through PTMs, which can reversibly control their function in a temporal and spatial dependent manner . Large-sModifications have been also reported within the neck-linkers of several kinesins including KIF11, KIF23, KIF20A, KIF26A, KIF3A, KIF2A, and KIF18A . This stKIF18A is a mammalian mitotic kinesin that accumulates on the plus-ends of kinetochore microtubules, where it suppresses microtubule dynamics to promote chromosome alignment and mitotic progression . AdditioSerine 357 (S357) within the neck-linker of KIF18A was identified as a phosphorylation site via mass spectrometry . We showSerine 357 of KIF18A is conserved across kinesin-8 homologs and was previously identified as a site of phosphorylation  Imami e. To studTreating cells with low doses of the microtubule stabilizing drug taxol results in enhanced accumulation of wild-type KIF18A to the plus-ends of kinetochore microtubules . This adTo further examine the altered localization of KIF18A S357D, we analyzed KIF18A localization in both monopolar mitotic cells and interphase cells. Wild-type and S357A KIF18A were primarily localized near the centrosomes of monopolar spindles while KIF18A S357D accumulated at the peripheral edge of monopolar spindles . In inteProper cell division requires the mitotic spindle to maintain a centralized position within the dividing cell. The stabilization of the mitotic spindle depends on contact of peripheral microtubules with the cortex , corticaKIF18A activity is required for chromosome alignment and maintaining spindle length . To examIn addition to causing an increase in unaligned chromosomes, depletion of KIF18A leads to an increase in the time needed to complete mitosis and mitotic arrest in some cell types . To deteKIF18A knockdown in HeLa Kyoto cells results in a population of cells that arrest in mitosis 37.8%, . This frKinetochore microtubules are relatively stable compared to non-kinetochore microtubules within the spindle . The incIf the population of peripheral microtubules that the KIF18A S357D mutant localizes to were stabilized by the motor, we would expect to see an increase in the density of peripheral microtubules in KIF18A S357D expressing cells. To test this, we fixed and stained RPE1 cells for tubulin and measured the tubulin fluorescence at the spindle periphery and within the main body of the spindle. The resulting data indicate that tubulin immunofluorescence was similar within the spindles and spindle peripheries of cells expressing wild type KIF18A or KIF18A S357D –d. HowevAnother possible explanation for the altered localization of KIF18A S357D is that increased motor velocity could allow it to reach the ends of faster polymerizing, non-kinetochore microtubules. To test this hypothesis, we performed single molecule total internal reflection fluorescence microscopy (TIRF) experiments to quantify the velocity of KIF18A wild-type and S357D motors on both taxol stabilized and dynamic microtubules. Results indicate that on both stabilized and dynamic microtubules KIF18A S357D moves at a similar velocity to wild type KIF18A –d. SurprKIF18A has an extended neck-linker (by 3 amino acids) compared to some other kinesins, including kinesin-1. This 17-residue structure is critical for promoting accumulation of KIF18A to the plus-ends of kinetochore microtubules by promoting navigation around microtubule associated proteins, which can serve as obstacles on the microtubule lattice . To deteThis body of work highlights how a single modification within the neck-linker of a kinesin can alter its sub-cellular localization and function. We have demonstrated that KIF18A S357D results in altered accumulation of the motor to peripheral microtubules of the spindle. The altered accumulation is also observed in monopolar mitotic cells and interphase cells, where an increase in KIF18A at the peripheral edge of a cell was observed. Surprisingly, despite this relocalization, the KIF18A S357D mutation only modestly reduces chromosome alignment and spindle length control. This was somewhat surprising since accumulation of KIF18A at the plus-ends of kinetochore microtubules is essential for its role in promoting chromosome alignment and maintaining spindle length . KIF18A In contrast, KIF18A S357D was unable to promote mitotic progression. KIF18A contributes to mitotic progression by promoting kinetochore-microtubule attachment and tension across kinetochores, which are necessary to satisfy the spindle assembly checkpoint . The facThe most striking effect of expressing the KIF18A S357D mutant was in the observed mitotic spindle stabilization defects, which resulted in rotation of the mitotic spindle. Peripheral spindle microtubules play a critical role in contacting the cortex and maintaining mitotic spindle position . The mosInterestingly, the altered localization pattern of KIF18A S357D closely mimics that of a shortened neck-linker mutant, which may indicate that KIF18A S357D causes a kink in the neck-linker of KIF18A and effectively shortens this structure. In support of this idea, Alpha Fold predicts possible charge interactions between S357 and H10 . Charge The extended neck-linker of KIF18A allows for obstacle navigation and subsequent plus-end accumulation . When thIn addition to causing changes in obstacle navigation, it is possible that the S357D mutation may alter KIF18A’s binding preference for the microtubule lattice. Subsets of microtubules within cells can differ in several ways. For example, the microtubule lattice can be either extended or compressed, which impacts the microtubule associated proteins that preferentially bind . Since nOverall, this work highlights how a single PTM within the neck-linker of KIF18A causes altered accumulation to a different microtubule subpopulation in both mitotic and interphase cells. Similar to work focusing on modifications within the motor domain of kinesins  this alt2 in MEM-α medium  containing 10% Fetal Bovine Serum (FBS) . HeLa Kyoto and RPE1 acceptor cells for recombination were maintained in Blasticidin . Once inducible clones were established by recombination, HeLa Kyoto inducible cell lines were maintained in 1 μg/mL puromycin  and RPE1 inducible lines were maintained in 5 μg/mL puromycin .HeLa Kyoto and RPE1 acceptor cells were a kind gift from Ryoma Ohi, University of Michigan. Cells were cultured at 37°C with 5% COFor siRNA transfections conducted in a 24-well plate 5pmol siRNA was incubated in Opti-MEM Reduced Serum Media  with Lipofectamine RNAiMax Transfection Reagent  for 5 minutes at room temperature before adding dropwise to wells. KIF18A siRNA used was a 1:1 mixture of the following two Silencer Select Validated siRNAs (5’ to 3’ sequence): GCUGGAUUUCAUAAAGUGGtt  CGUUAACUGCAGACGUAAAtt . For control knockdown a 1:1 mixture of Silencer Select Negative Control #2  and Silencer Select Negative Control #1 (4390843) was used. For experiments with HURP knockdown the custom siRNA with 5’ to 3’ sequence used was AGUUACACCUGGACUCCUUTT .For fixed cell immunofluorescence experiments cells were seeded on acid-treated 12mm glass coverslips in a 24-well dish and fixed in −20°C methanol  for 3 minutes, −20°C methanol with 1% paraformaldehyde  for 10 minutes, or 1X BRB80, 4mM EGTA  0.5% Triton-X  (MTSB) with 0.5% glutaraldehyde  for 10 minutes. Glutaraldehyde fixed cells were subsequently quenched with 0.1% sodium borohydride  in 1X Tris-Buffered Saline  for 15 minutes two times. After fixation coverslips were washed 2–3 times in 1X TBS for minutes each. Coverslips were then blocked with 20% goat serum in antibody dilution buffer , 0.1% Triton X-100, and 0.1% sodium azide) for 1 hour at room temperature. Prior to incubation with primary antibodies, coverslips were washed two times in 1X TBS for 5 minutes. Primary antibodies were diluted 1:1 in glycerol and stored at −20°C. Prior to incubation on coverslips primary antibodies were further diluted in Abdil at indicated concentrations and incubated for the indicated duration. Prior to secondary antibody incubation coverslips were washed two additional times for 5 minutes in 1X TBS. The following secondary antibodies conjugated to AlexaFluor 488, 594, and 647 were diluted 1:1 in glycerol and stored at −20°C prior to diluting 1:500 in abdil for incubation on coverslips for 1 hour at room temperature . After secondary antibody incubation coverslips were washed an additional two times in 1X TBS for 5 minutes at room temperature prior to mounting in Prolong Gold anti-fade mounting medium with DAPI .2-independent media (Gibco #18045-088) supplemented with 10% fetal bovine serum (Gibco #16000-044). All images were processed and analyzed using ImageJ/Fiji (All fixed and live cell images or movies were acquired on a Ti-E inverted microscope (Nikon Instruments) or on a Ti-2E inverted microscope (Nikon Instruments) both driven by NIS Elements (Nikon Instruments). Images were captured using either a Clara cooled charge-coupled device (CCD) camera (Andor) or Prime BSI scientific complementary metal-oxide-semiconductor (sCMOS) camera (Teledyne Photometrics) with a Spectra-X light engine (Lumencore). Imaging was conducted with the following Nikon objectives: Plan Apo 40X 0.95 numerical aperture (NA), Plan Apo λ 60X 1.42 NA, and APO 100X 1.49 NA. Both microscopes contain environmental chambers held at 37°C. For live cell-imaging, cells were imaged in COgeJ/Fiji .TIRF microscopy experiments with taxol stabilized microtubules were conducted at 37°C using a Ti-E inverted microscope (Nikon Instruments) equipped with an EMCCD camera (Andor), 488 nm and 567 nm lasers, and a 100X Apo TIRF  objective lens (Nikon). TIRF microscopy experiments with dynamic microtubules were conducted at 37°C using an inverted Eclipse Ti-E microscope (Nikon) with dual iXon Ultra Electron Multiplying charge-coupled device cameras and a 100XApo TIRF objective lens  driven by NIS Elements, version 4.51.0.2.Both HeLa Kyoto and RPE1 inducible cell lines were generated using methods described previously . In brieKIF18A wild-type , S357A  were ampGenomic DNA was extracted from resulting HeLa Kyoto and RPE1 inducible cell lines (QIAmp DNA Blood Mini Kit Qiagen #51106) and sequenced through the S357 mutation site (Eurofins) to verify correct incorporation of the desired construct. Inducible constructs were expressed by the addition of 2 μg/mL doxycycline (Thermo Fisher Scientific #BP26531).For quantification of KIF18A expression by immunofluorescence, cells were seeded on 12mm coverslips and endogenous KIF18A was depleted 24 hours later as described above. Simultaneously, expression of GFP-KIF18A constructs was induced with the addition of 2 μg/mL doxycycline. 24 hours post-knockdown and induction coverslips were fixed in −20°C methanol for 3 minutes and stained for ms-gamma tubulin 1:500 , guinea-pig CENP-C 1:25 , and rabbit KIF18A . All primary antibodies were incubated for 1 hour at room temperature with shaking. Random mitotic cells were imaged from each condition. Expression levels were quantified in ImageJ by drawing an ROI centered around but larger than the mitotic spindle and measuring KIF18A intensity. Background levels for each cell were measured by moving the ROI to an area with no cells and measuring the KIF18a intensity. The mean background signal for each image was subsequently subtracted from the mean spindle KIF18A signal. All values were then normalized to the mean background subtracted control KIF18A intensity. Mean and standard deviations are reported for three individual biological replicates. The total number of cells analyzed for each condition was RPE1 control = 61 cells, RPE1 KIF18A knockdown = 62 cells, RPE1 GFP = 64 cells, RPE1 WT = 61 cells, RPE1 S357A = 60 cells, and RPE1 S357D = 64 cells, HeLa Kyoto control = 57 cells, HeLa Kyoto KIF18A knockdown = 62 cells, HeLa Kyoto GFP = 65 cells, HeLa Kyoto WT = 61 cells, HeLa Kyoto S357A = 61 cells, and HeLa Kyoto S357D = 64 cells.For images displaying KIF18A localization in mitosis, cells were seeded on 12mm coverslips. Endogenous KIF18A was depleted 24 hours later as described above, and exogenous GFP-KIF18A was induced simultaneously with the addition of 2 μg/mL doxycycline. 24 hours after induction of GFP-KIF18A and endogenous KIF18A knockdown cells were fixed for 3 minutes in −20°C methanol and subsequently stained for rabbit GFP 1:500  for one hour at room temperature with shaking.For visualization of KIF18A localization in monopolar spindles cells were seeded on 12mm coverslips. Endogenous KIF18A was depleted 24 hours later as described above, and exogenous GFP-KIF18A was induced simultaneously with the addition of 2 μg/mL doxycycline. 24 hours after induction and knock down cells were treated with 100μM monastrol for 2–3 hours to cause spindle collapse prior to fixing in −20°C methanol for 5 minutes. Cells were then subsequently stained for rabbit-gamma-tubulin 1:250 , mouse-EB1 1:50 , and chicken-GFP 1:500  all for one hour at room temperature with shaking.2 Independent Media  with 50nM siR-tubulin , 1:20,000 spy595 DNA , 10uM verapamil , and 2 μg/mL doxycycline and left to incubate at 37 °C for at least 2 hours prior to imaging.For images displaying interphase localization cells were seeded in a glass bottom 35mm dish. Endogenous KIF18A was depleted 24 hours later as described above, and exogenous GFP-KIF18A was induced simultaneously with the addition of 2 μg/mL doxycycline. 24 hours after induction media was exchanged to COFor chromosome alignment and spindle length analysis cells were seeded on 12mm coverslips. Endogenous KIF18A was depleted 24 hours later as described above, and exogenous GFP-KIF18A was induced simultaneously with the addition of 2 μg/mL doxycycline. 24 hours after induction of GFP-KIF18A and endogenous KIF18A knockdown cells were fixed for 3 minutes in −20°C methanol and subsequently stained for ms-gamma-tubulin 1:500 , guinea-pig-CENP-C 1:250  and rabbit-GFP 1:500 . All primary antibodies were incubated on coverslips for one hour at room temperature with shaking. Approximately twenty random metaphase mitotic cells in which both poles of the mitotic spindle were in focus in the same z plane for each condition were imaged. As described previously, , a line 2 Independent Media  supplemented with 2 μg/mL doxycycline for induced conditions. Multiple fields of view for each condition were imaged overnight at 40X magnification, imaging every 3 minutes. To quantify mitotic timing the timepoint at nuclear envelope breakdown and anaphase onset was determined and the difference was calculated to determine time from nuclear envelope breakdown to anaphase onset. Cell that entered mitosis at least one hour prior to the end of mitotic timing movies but never entered anaphase were quantified as cells that never divide. For mitotic timing, median values are reported from three biological replicates for each condition. A Kruskal Wallis Test with Dunn’s Test for Multiple Comparisons was conducted for statistical analysis. For each HeLa Kyoto condition the following cell numbers were analyzed control = 231 cells, KIF18A knockdown = 136 cells, GFP = 154 cells, WT = 162 cells, S357A = 182 cells, and S357D = 177 cells. For each RPE1 condition the following cell numbers were analyzed control = 114 cells, KIF18A knockdown = 95 cells, GFP = 134 cells, WT = 94 cells, S357A = 99 cells, and S357D = 86 cells. For mitotic arrest the percent of cells that never divide is reported from three experimental replicates.To determine mitotic timing, cells were seeded in glass-bottom 24-well dishes. 24 hours later endogenous KIF18A was knocked down as described above and exogenous KIF18A was induced with 2 μg/mL doxycycline. 24 hours after induction and knock down media was exchanged to COFor analysis of the angle between spindle poles cells were seeded on 12mm coverslips. Endogenous KIF18A was depleted 24 hours later as described above, and exogenous GFP-KIF18A was induced simultaneously with the addition of 2 μg/mL doxycycline. 24 hours after induction of GFP-KIF18A and endogenous KIF18A knockdown cells were fixed for 3 minutes in −20°C methanol and subsequently stained for rabbit-KIF18A 1:100 , guinea-pig-CENP-C , and mouse-gamma-tubulin . All primary antibodies were incubated on coverslips for one hour at room temperature with shaking. Approximately 20 random metaphase mitotic cells per condition were imaged at 60X magnification with 41 z stacks recorded at 0.25 μm/step. Using the point tool in ImageJ the two poles of each cell were selected at the z plane where they were in focus and the x, y, and slice information was recorded. A custom macro in ImageJ was created to determine the angle between the two poles for each cell by using the dot product. The mean and standard deviations of the pole angle are reported for three biological replicates for each condition. A one-way ANOVA with Tukey’s Test for Multiple Comparisons was conducted for statistical analysis. For each RPE1 condition the following cell numbers were analyzed control = 81 cells, KIF18A knockdown = 80 cells, GFP = 86 cells, WT = 85 cells, S357A = 82 cells, and S357D = 86 cells.2 Independent Media  supplemented with 2 μg/mL doxycycline for induced conditions. Multiple fields of view per condition were imaged every minute for 30 minutes with 21 z stacks taken at 0.25 μm/step. To quantify spindle rotation one spindle pole of a cell was tracked using MTrackJ in ImageJ and the subsequent track was measured. From track measurements the displacement in microns per minute was calculated. The mean and standard deviations from three biological replicates are reported for each condition. A one-way ANOVA with Tukey’s Test for Multiple Comparisons was conducted for statistical analysis. For each RPE1 condition the following cell numbers were analyzed WT = 12 cells, S357A = 16 cells, and S357D = 24 cells.For quantification of spindle rotation cells were seeded in glass-bottom 24-well dishes. 24 hours later endogenous KIF18A was knocked down as described above and exogenous KIF18A was induced with 2 μg/mL doxycycline. 24 hours after induction and knock down media was exchanged to COFor quantification of EB1 to determine changes in microtubule polymerization cells were seeded on 12mm coverslips. Endogenous KIF18A was depleted 24 hours later as described above, and exogenous GFP-KIF18A was induced simultaneously with the addition of 2 μg/mL doxycycline. 24 hours after induction and knock down cells were treated with 100μM monastrol for 2–3 hours to cause spindle collapse prior to fixing in −20°C methanol for 5 minutes. Cells were then subsequently stained for rabbit-gamma-tubulin 1:250 , mouse-EB1 1:50 , and chicken-GFP 1:500  all for one hour at room temperature with shaking. Approximately 20 random mitotic cells were imaged per condition at 60X magnification with 13 z slices taken at 0.2 μm/step. The ratio of peripheral to spindle EB1 or GFP was determined by drawing an ROI that encompassed the DNA and measuring the GFP and EB1 intensity in that ROI. A 2.5-micron band was then created around the DNA ROI and the EB1 and GFP intensity within that band was measured. The ratio between the two ROIs was then calculated. The mean and standard deviation from three biological replicates is reported. A one-way ANOVA with Tukey’s Test for Multiple Comparisons was conducted for statistical analysis. For each condition the following cell numbers were analyzed WT = 46 cells, S357A = 49 cells, and S357D = 56 cells.For analysis of microtubule density cells were seeded on 12mm coverslips. Endogenous KIF18A was depleted 24 hours later as described above, and exogenous GFP-KIF18A was induced simultaneously with the addition of 2 μg/mL doxycycline. 24 hours after induction and knock down cells were fixed in 0.5% glutaraldehyde in MTSB for 10 minutes and then quenched two times for 15 minutes each with 0.1% sodium borohydride  in 1X TBS. Cells were then subsequently stained for human-anti-centromere (ACA) 1:200 , ms-DM1-alpha 1:150 , and rabbit-GFP 1:400  all overnight at 4°C with shaking. Approximately 20 random metaphase mitotic cells were imaged per condition at 60X magnification with 41 z slices taken at 0.2 μm/step. The mean spindle tubulin intensity was determined by drawing an ROI that encompassed the kinetochore microtubules of the mitotic spindle and measuring the tubulin intensity in that ROI. A 4.5-micron band was then created around the spindle ROI and the tubulin intensity within that band was measured. The background tubulin intensity was measured with an ROI in a region containing no cells and the subsequent measurement was subtracted from both the mean spindle and mean peripheral tubulin intensity for a given cell. The median from a minimum of three biological replicates is reported. A Kruskal Wallis Test with Dunn’s Test for Multiple Comparisons was conducted for statistical analysis. For each condition the following cell numbers were analyzed control = 85 cells, KIF18A knockdown = 84 cells, GFP = 84 cells, WT = 80 cells, S357A = 63 cells, and S357D = 83 cells.2, 0.1 mM ATP, 1X Roche Protease/PPase inhibitor, and 10% glycerol in 1X BRB80) for 15 minutes on ice prior to shearing DNA by passing through a narrow-gauge (No 27) hypodermic needle. The sheared solution was then left on ice for an additional 15 minutes before spinning at 16,000 RCF in a microcentrifuge at 4°C for 20 minutes. The cleared lysate (supernatant) was snap frozen in liquid nitrogen and stored at −80°C prior to use.For analysis of single molecule velocity GFP-KIF18A was isolated as cell lysates by seeding in a 100mm dish and inducing expression of exogenous GFP-KIF18A with the addition of 2 μg/mL doxycycline. Cells were arrested in mitosis with the addition of 100 ng/mL nocodazole overnight prior to obtaining cell pellet. The cell pellet was resuspended in lysis buffer  stabilized with 20 μM taxol  were prepared with a 100:1 ratio of unlabeled GTP tubulin to rhodamine labeled tubulin. The mixture of unlabeled and labeled tubulin was allowed to polymerize at 37°C for 20 minutes prior to stabilizing with 20 μM paclitaxel in DMSO . Experiments were conducted in silanized flow cells. Silanized slides  were prepared by first washing in 100% methanol for 2 hours shaking at room temperatures. Slides were subsequently dried with nitrogen and cleaned with a UV-ozone plasma (Harrick plasma cleaner) for 3 minutes. Slides were then incubated in 99.2% toluene , 0.173% 2-methoxy(polyethyleneoxy)propyltrimethoxysilane  and 0.62% n-butylamine (Acros Organics) with flowing nitrogen gas for 90 minutes. Coverslips were subsequently washed twice in toluene and dried with nitrogen gas before curing for 30 minutes with flowing nitrogen gas. Flow cells chambers were assembled using silanized coverslips adhered to a 25 × 75mm glass slide  with two pieces of double-sided tape which resulted in a ~20μl chamber. Microtubules were linked to PEG-silanized coverslips by incubating chambers with 33 μg/mL monoclonal anti-beta-tubulin III  antibody  for 5 minutes prior to washing with 10 mg/mL BSA for 2 minutes. Flow chambers were then incubated with 1 μM assembled microtubules for 10 minutes. Experiments were conducted in 1X BRB80 containing 10mM DTT, 100mM KCl, and 1X Oxygen Scavenger Mix  in 20μM paclitaxel. Images were collected at 10 frames/sec. Motor velocity was calculated by creating kymographs from individual microtubules in ImageJ, creating a rectangle that bound the sloped line, and measuring the width (run length in pixels) and height (number of time frames) of the resulting rectangle. The subsequent velocity was calculated by multiplying the width (in pixels) by the pixel length and dividing this by the time of the run . For each condition the number of motor runs analyzed was WT = 52, S357D = 106.For analysis on dynamic microtubules rhodamine labeled microtubules (bovine) were prepared as described previously . ExperimTo analyze GFP-KIF18A localization in the presence and absence of HURP cells were seeded on 12mm coverslips. Endogenous KIF18A and HURP was depleted 24 hours later as described above, and exogenous GFP-KIF18A was induced simultaneously with the addition of 2 μg/mL doxycycline. 24 hours after induction cells were treated with 25 μM MG132 for 1–2 hours prior to fixing in 1% paraformaldehyde in −20°C methanol for 10 minutes. Coverslips were subsequently stained for chicken-GFP 1:500 , guinea-pig-CENP-C 1:250 , and rabbit-HURP 1:500 , all for one hour at room temperature with shaking. Approximately 20 random metaphase mitotic cells were imaged for each condition per biological replicate at 60X magnification with 31 z stacks taken at 0.25 μm/step. To quantify the ratio of peripheral GFP-KIF18A to spindle GFP-KIF18A z projected images were rotated so the two spindle poles were horizontal. A 20-pixel wide line twice the length of the distance of the kinetochore mass was centered over the kinetochores and the distribution of GFP-KIF18A was measured using the Plot Profile command in ImageJ. The resulting intensity data was normalized so the line went from zero to one. The average intensity corresponding with the top and bottom quarters of the line (0–0.25 and 0.74–1) was determined and recorded as the peripheral GFP-KIF18A intensity. The average intensity of GFP-KIF18A corresponding to the middle half of the line (0.26–0.75) was determined and recorded as the average spindle GFP-KIF18A intensity. The ratio between peripheral and spindle GFP-KIF18A was then calculated by dividing the peripheral average by the spindle average. A one-way ANOVA with Tukey’s Test for Multiple Comparisons was conducted for statistical analysis. For each condition the following cell numbers were analyzed: WT with HURP = 59 cells, WT HURP KD = 64 cells, sNL with HURP = 46 cells, sNL HURP KD = 55 cells, S357D with HURP = 66 cells, S357D HURP KD = 50 cells.Supplement 1Supplementary Video 1: Stabilization of the microtubules of the mitotic spindle with taxol results in additional accumulation of wild-type KIF18A at kinetochore microtubule plus-ends. Live-cell imaging of RPE1 wild-type GFP-KIF18A inducible cell line. Cells were imaged approximately 24 hours after siRNA treatment to knockdown endogenous KIF18A and induction of GFP-KIF18A with doxycycline. Immediately prior to imaging, cells were spiked with 10 μM paclitaxel.Supplement 2Supplementary Video 2: Stabilization of the microtubules of the mitotic spindle with taxol results in additional accumulation of S357A KIF18A at kinetochore microtubule plus-ends. Live-cell imaging of RPE1 S357A GFP-KIF18A inducible cell line. Cells were imaged approximately 24 hours after siRNA treatment to knockdown endogenous KIF18A and induction of GFP-KIF18A with doxycycline. Immediately prior to imaging, cells were spiked with 10 μM paclitaxel.Supplement 3Supplementary Video 3: Stabilization of the microtubules of the mitotic spindle with taxol does not result in additional accumulation of S357D KIF18A at kinetochore microtubule plus-ends. Live-cell imaging of RPE1 S357D GFP-KIF18A inducible cell line. Cells were imaged approximately 24 hours after siRNA treatment to knockdown endogenous KIF18A and induction of GFP-KIF18A with doxycycline. Immediately prior to imaging, cells were spiked with 10 μM paclitaxel.Supplement 4Supplementary Video 4: Wild-type KIF18A maintains spindle centering during mitosis. Live-cell imaging of GFP-KIF18A wild-type in mitosis. Cells were imaged approximately 24 hours after siRNA treatment and induction of GFP-KIF18A with doxycycline and 3 hours after addition of siR-tubulin to label microtubules.Supplement 5Supplementary Video 5: S357D KIF18A displays defects in spindle centering in mitosis. Live-cell imaging of GFP-KIF18A S357D in mitosis. Cells were imaged approximately 24 hours after siRNA treatment and induction of GFP-KIF18A with doxycycline and 3 hours after addition of siR-tubulin to label microtubules.Supplement 6Supplementary Video 6: In the absence of siR-tubulin wild-type KIF18A still maintains spindle positioning. Live-cell imaging of GFP-KIF18A wild-type in mitosis. Cells were imaged approximately 24 hours after siRNA treatment and induction of GFP-KIF18A with doxycycline.Supplement 7Supplementary Video 7: In the absence of siR-tubulin S357D KIF18A is still unable to maintain spindle positioning. Live-cell imaging of GFP-KIF18A S357D in mitosis. Cells were imaged approximately 24 hours after siRNA treatment and induction of GFP-KIF18A with doxycycline.Supplement 8


Saccharomyces cerevisiae. We predict an SAM-binding pocket near the FeS cluster domain that is conserved across eukaryotes in Dph1 but not Dph2. Site-directed DPH1 mutagenesis and functional characterization through assay diagnostics for the loss of diphthamide reveal that the SAM pocket is essential for synthesis of the décor on EF2 in vivo. Further evidence from structural modeling suggests particularly critical residues close to the methionine moiety of SAM. Presumably, they facilitate a geometry specific for SAM cleavage and ACP radical formation that distinguishes Dph1•Dph2 from classical radical SAM enzymes, which generate canonical 5′-deoxyadenosyl (dAdo) radicals.In eukaryotes, the Dph1•Dph2 dimer is a non-canonical radical SAM enzyme. Using iron-sulfur (FeS) clusters, it cleaves the cosubstrate S-adenosyl-methionine (SAM) to form a 3-amino-3-carboxy-propyl (ACP) radical for the synthesis of diphthamide. The latter decorates a histidine residue on elongation factor 2 (EF2) conserved from archaea to yeast and humans and is important for accurate mRNA translation and protein synthesis. Guided by evidence from archaeal orthologues, we searched for a putative SAM-binding pocket in Dph1•Dph2 from  Both anhamide]) . As the revisiae . The quaw/v) polyacrylamide) and blotted onto PVDF membranes . These were probed overnight at 4 °C with the anti-EF2(pan) and anti-EF2(no diphthamide) antibodies [+ as ADP-ribosyl donors for the DT reaction essentially as previously described with human and yeast EF2 resources [The total yeast cell extracts were generated as previously described , and thetibodies  and devetibodies ,37. Prottibodies  using thesources ,27,40.Pyrococcus horikoshii (Ph) [Candidatus methanoperedens nitroreducens (Cmn) [Methanobrevibacter smithii (Ms) [CmnDph2•Dph2 dimer in complex with SAM demonstrated [MsDph2  ,11, Candns (Cmn)  and Methhii (Ms) . In addinstrated  a subset CmnDph2 A), whichCmnDph2 was aligned to both Dph1 and Dph2 from Saccharomyces cerevisiae (Sc), Arabidopsis thaliana (At), Drosophila melanogaster (Dm), Mus musculus (Mm) and Homo sapiens (Hs)  or instead a branched chain amino acid  at the position of interest   . Except ens (Hs) . CmnDph2Ile-328) B. Of notIle-328) . Thus, o Asp-335 ) to be sCmnDph2•Dph2 dimer, we next aligned the conserved Dph1 residues  to the  at expression levels which were comparable to a wild-type (DPH1) reference  strain (DPH1 deletion strain (dph1Δ) used as a negative control and in association with the G238A, H261A, R370A and D374A substitution mutants , which was found to be DT-sensitive earlier, also appeared unchanged from the wild-type DPH1 cells with regard to their sordarin responses  allowing for cytotoxic expression of the catalytic DT subunit under  control ,21. Upon) strain B. In con mutants B. Intrighenotype B similarhenotype B, an anttifungal B. In add control B. The twesponses B.DPH1 substitutions tested apparently caused defects that are characteristic for a diphthamide loss-of-function (dph1Δ) mutant. This confirms that the majority of the candidate Dph1 residues targeted for mutagenesis are functionally relevant and play important roles in vivo for the Dph1•Dph2 enzyme to decorate EF2 with diphthamide in yeast.As shown previously, resistances toward DT and sordarin are bona fide phenotype diagnostics for failure to initiate or complete diphthamide synthesis on EF2 in yeast cells ,21,43,44+ as an ADP-ribosyl donor, EF2 extracts as ADPR acceptors and an HRP-streptavidin conjugate to detect biotin in the ADPR reaction product [V349A and Q321A extracts were ADP ribosylated by DT, with significantly weak signals seen in the Q321A material (V349A cells and reduced (but not entirely abolished) diphtamide-modified EF2 amounts in the Q321A mutant [G238A, H261A, R370A and D374) and dph1Δ cells lacked detectable ADPR acceptor activity, indicating a loss of diphthamide on EF2 and evasion of the DT attack in these backgrounds  of EF2 by DT in vitro, a reaction known to be strictly dependent on the diphthamide modification. The protocol used DT, biotinylated NAD product ,27,40. Ematerial . These aA mutant ,27,40. Ikgrounds .Q321A mutant produced weak yet detectable ADPR signals on EF2 in the presence of DT (Q321A or V349A) alone with the double carrying both mutations (Q321A and V349A), we found that the two together were phenotypically additive and enhanced the sordarin and DT resistance traits (The fact that the ce of DT  suggestsce of DT B. In an ce of DT  and undee traits A. Since e traits A.DPH1 and dph1Δ) and the mutants  to Western blots using antibodies  and anti-EF2(pan) , the single Q321A and double Q321A V349A mutants produced immune-responsive signals diagnostic for unmodified EF2 and a defect in the diphthamide pathway (Q321A mutant (39%) was weaker than the robust signal of the dph1Δ null mutant (Q321A V349A displayed a pronouced EF2 modification defect (96%) indistinguishable from the dph1Δ control . To acEF2(pan) B) which  pathway B. As wit pathway , the EF2l mutant B. In con control B.G238A, R370A, H261A and D374A mutants produced robust anti-EF2(no diphthamide) Western signals (dph1Δ (or dph2Δ) diphthamide mutants qualifyng the Dph1 residues Gly-238, His-261, Arg-370 and Asp-374  is catalytically vital . OverallS. cerevisiae  and eukaryotic (Dph1•Dph2) enzymes being able to form ACP radicals, dimer asymmetry obviously is not a prerequisite for the underlying SAM cleavage ,14,41. Irom mice  clearly rom mice ,51.


Expression of APOBEC3A and APOBEC3B, but not catalytic mutant controls or APOBEC3H, triggers increased frequencies of TK mutation and similar TC-biased cytosine mutation profiles in the selectable TK reporter gene. Whole genome sequences from independent clones enabled an analysis of thousands of single base substitution mutations and extraction of local sequence preferences with APOBEC3A preferring YTCW motifs 70% of the time and APOBEC3B 50% of the time . Signature comparisons with breast tumor whole genome sequences indicate that most malignancies manifest intermediate percentages of APOBEC3 signature mutations in YTCW motifs, mostly between 50 and 70%, suggesting that both enzymes contribute in a combinatorial manner to the overall mutation landscape. Although the vast majority of APOBEC3A- and APOBEC3B-induced single base substitution mutations occur outside of predicted chromosomal DNA hairpin structures, whole genome sequence analyses and supporting biochemical studies also indicate that both enzymes are capable of deaminating the single-stranded loop regions of DNA hairpins at elevated rates. These studies combine to help resolve a long-standing etiologic debate on the source of APOBEC3 signature mutations in cancer and indicate that future diagnostic and therapeutic efforts should focus on both APOBEC3A and APOBEC3B.A prominent source of mutation in cancer is single-stranded DNA cytosine deamination by cellular APOBEC3 enzymes, which results in signature C-to-T and C-to-G mutations in TCA and TCT motifs. Although multiple enzymes have been implicated, reports conflict and it is unclear which protein(s) are responsible. Here we report the development of a selectable system to quantify genome mutation and demonstrate its utility by comparing the mutagenic activities of three leading candidates—APOBEC3A, APOBEC3B, and APOBEC3H. The human cell line, HAP1, is engineered to express the  A large source of mutation in cancer is attributable to single-stranded DNA cytosine to uracil (C-to-U) deamination by APOBEC3 family enzymes. Chromosomal DNA U’s directly template the insertion of adenine (A), which results in C/G-to-T/A mutations. Chromosomal U’s also trigger error-prone DNA repair processes, which less directly result in C/G-to-T/A mutations, C/G-to-G/C mutations, and more complex events such as insertion/deletion mutations. We report a human cell line for cancer mutation studies, where mutations can be identified selectively or by genomic DNA sequencing. Only APOBEC3A and APOBEC3B proved capable of inflicting the aforementioned types of single base substitution mutations. Interestingly, 70% and 50% of the single base substitution mutations attributable to APOBEC3A and APOBEC3B, respectively, occurred in TCA and TCT trinucleotide motifs preceded by a pyrimidine (T or C). This is significant because most APOBEC3-positive breast tumors exhibit an intermediate percentage of signature mutations in these broader tetranucleotide motifs, which supports the idea that both APOBEC3A and APOBEC3B contribute in a combinatorial manner to the overall mutation landscape in these tumors. CW motifs   relative to catalytic mutant control clones . Similarly, TK sequences derived from A3B-expressing granddaughter clones also harbored a greater number of APOBEC3 signature mutations  relative to catalytic mutant control clones  . Additionally, TK cytosine nucleobases 22 (Q8X) and 635 (R212K) emerged as candidate mutational hotspots in both the A3A- and A3B-expressing clones . No significant differences were found in the number of other SBS or INDEL mutations between A3A- or A3B-expressing clones and controls.We next asked what types of genetic alterations led to inactivation of the TK . In both instances, A was preferred over T at the +1 nucleobase position relative to the mutated C, and this bias was not significantly different between the two enzymes . Similarly, no obvious biases were evident at the +2 or -2 nucleobase positions with all four nucleotides observed at similar frequencies for both enzymes. Moreover, even when pyrimidines and purines were grouped for comparison, A3A did not show an overt preference for C/T (Y) or A/G (R) at the -2 or +2 nucleobase positions . Likewise, A3B also failed to show an overt preference for C/T (Y) or A/G (R) at the -2 or +2 nucleobase positions . These similarities underscore the fact that small mutation numbers are primarily useful for delineating major signature differences such as the shifts described above from a heterogeneous pattern in catalytic mutant- or vector control-expressing cells towards a predominantly 5’-TC focused SBS mutation pattern in A3A- and A3B-expressing cells.We next examined the broader sequence context of the 22 A3A- and 19 A3B-induced APOBEC3 signature mutations that occurred at 5’-TC dinucleotides in A3 construct and compare mRNA levels relative to established breast cell lines and primary breast tumors. All expression values were determined relative to those of the conserved housekeeping gene TBP to be able to compare RNAseq data from different sources . First, as expected from utilizing the same expression vector, mRNA levels of exogenously expressed A3A and A3B are similar . However, these A3A and A3A-E72A mRNA levels in granddaughter clones are over 5-fold higher than the average endogenous A3A expression levels of APOBEC3 signature-enriched breast cancer cell lines BT474 and MDA-MD-453, breast cancer cell lines of the CCLE, or breast tumors of TCGA . In comparison, the A3B and A3B-E255A mRNA levels in granddaughter clones are similar to averages reported for breast cancer cell lines of the CCLE and breast tumors of the TCGA, and approximately 2-fold lower than those of BT474 and MDA-MD-453 . In other words, A3A is overexpressed in this system and A3B approximates levels observed in breast tumors and cell lines. A3H (haplotype I) mRNA levels showed greater variance but only two clones were analyzed by RNAseq and WGS due to negative results above with IF microscopy experiments and TK mutation analysis.Independent granddaughter clones were selected using the workflow described above and RNA sequencing was done to confirm expression of each exogenously expressed A3 genes, as well as AICDA (AID), APOBEC1, APOBEC2, and APOBEC4, were also quantified and compared with those of A3A, A3B, and A3H . Endogenous A3C was expressed at similarly high levels in all granddaughter clones, providing a relatively stable internal control. Endogenous A3F and A3G were expressed at lower but still detectable levels, and endogenous AICDA, APOBEC1, APOBEC2, APOBEC4, and A3D were expressed at very low or undetectable levels. As expected, levels of ectopically expressed A3A, A3B, and A3H mRNA exceeded those in the HAP1-TK-M9 parent clone as well as those in vector expressing granddaughter controls. In addition, protein expression of A3A, A3B, and A3H was confirmed in granddaughter clones by immunoblotting and activity by ssDNA deamination assays . Finally, the integration sites of the TK reporter and MLV-based A3 expression constructs were determined using WGS reads. Data from representative clones demonstrated a single TK-Neo integration site in chromosome 3 between nucleotides 143996100–143996500, which is ~3.5 kbp downstream of the nearest gene (DIPK2A), as well as unique MLV-A3 insertion sites as expected from the independent reactions and low multiplicities of infection that were used initially to establish the clones .The mRNA expression levels of the other four S mother clone prior to transduction with each A3 or control expression construct. Thus, all new SBS mutations had to be present in a significant proportion of reads from the granddaughter clones and absent from the reads from the original mother clone and, as such, must have occurred in the presence of an active A3 enzyme or a catalytic mutant control.To investigate mutational differences genome-wide, Illumina short-read WGS was done for randomly selected granddaughter clones. Mutations unique to each granddaughter were identified by calling SBS variations versus the genomic DNA sequence of the HAP1-TK-M9 mother clone. This approach eliminated any somatic variation that accumulated in the GanS9A Fig). The total number of SBSs in A3B-expressing clones was lower, ranging from 1920 to 2652 . In comparison, the total number of SBSs in catalytic mutant and eGFP control granddaughter clones ranged from 1230 to 2204 . SBS mutations are further broken down into those occurring within NCN and TCW motifs in dot plots in S9B–S9E Fig. Most importantly, analyses of the trinucleotide contexts of all unique SBS mutations revealed strong C-to-T and C-to-G mutation biases in 5’-TCA and 5’-TCT motifs in A3A-expressing clones and weaker, but still significant, mutation biases in the same motifs in A3B-expressing clones . In other words, only A3A- and A3B-expressing granddaughter clones exhibited significant accumulations of APOBEC3 signature single base substitution mutations.In A3A-expressing granddaughter clones, the total number of unique SBSs ranged from 2057 to 5256 . A complementary bioinformatics analysis, non-negative matrix factorization  and “signature B” occurring in all clones regardless of A3 presence or functionality . Signature B is comprised in part of a C-to-A mutation bias, which may be explained by the CA dinucleotide biased mutagenic activity of ganciclovir . Proteins were denatured by boiling samples for 20 min and resolved by SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a PVDF-FL membrane (Millipore Sigma) and blocked in 5% milk in 1X PBS. Primary and secondary antibodies were incubated in blocking buffer, with the addition of 0.2% SDS for fluorescent antibodies. The primary antibodies used were anti-A3A , anti-A3A/B , anti-AWhole cell extract (WCE) assays: ssDNA deamination activities were measured using WCE prepared using 100 μL HED lysis buffer per 1 million cells . Samples were sonicated in a water bath sonicator to ensure complete lysis. A3-containing lysates were incubated at 37°C for 1 hr (A3A and A3B) or 4 hrs (A3H) with purified human UNG2 and a ssDNA substrate containing either a single TCA or a single TCT trinucleotide motif  following established protocols , 102. AfRecombinant enzyme assays: A3A- and A3B-mycHis were prepared from transfected 293T cells as reported , 103–105Propidium iodide (PI) staining was used to assess the ploidy of HAP1 clones relative to THP1  as a confirmed diploid control . Cells wA3 genes relative to the housekeeping gene TBP W mutations are shown in red, other SBSs in black, and INDELs in blue. Composite mutation schematics are shown below for each condition. (B-C) Pentanucleotide logos depicting the -2 and +2 sequence preferences flanking all T(C>T/G)W mutations that accumulated in TK during expression of the indicated constructs.((TIF)Click here for additional data file.S6 FigA) A3A, A3B, and A3H mRNA expression levels relative to those of the housekeeping gene TBP for the indicated HAP1-TK-M9 conditions . RNA-seq data from A3 signature-high breast cancer cell lines (BT-474 and MDA-MB-453), CCLE breast-derived cell lines (n = 52), and TGCA primary breast cancers (n = 1093) for comparison (mean +/- SD). (B) A heatmap depicting mean expression levels of all 7 human APOBEC3 family members, in addition to AICDA, APOBEC1, APOBEC2, and APOBEC4, relative to those of the housekeeping gene TBP . Endogenous A3C provides a consistent internal control.((TIF)Click here for additional data file.S7 FigA) Immunoblots of A3A, A3B, and A3H in the indicated clones. Tubulin (TUB) is a loading control. (B) Deaminase activity of WCE on ssDNA from the same clones .((TIF)Click here for additional data file.S8 FigA table indicating chromosomal locations of representative MLV-A3 insertion sites in HAP-1-TK-M9 clones. In each clone, a single MLV-A3 insertion is positioned in the window between the indicated nucleotides.(TIF)Click here for additional data file.S9 FigA-C) Dot plots showing total numbers of SBS mutations and cytosine mutations in NCN and TCW motifs, respectively, in WGS from individual granddaughter clones . (D-E) Dot plots showing total numbers of cytosine mutations in NCN and TCW motifs, respectively, in WGS from individual A3A and A3B expressing granddaughter clones in comparison to all non-catalytic controls combined .((TIF)Click here for additional data file.S10 FigFig 3.Trinucleotide profiles of all SBS mutations in WGSs from the indicated granddaughter clones . Aggregate profiles for A3A, A3B, and corresponding catalytic mutant controls are shown in (TIF)Click here for additional data file.S11 FigA) Mutation signature profiles extracted from granddaughter clone WGS using an NMF-based approach. The SBSs of each clone resolved into 2 signatures—an APOBEC3-like mutation signature shown in red and a background signature in blue. APOBEC3 mutation enrichment scores are shown above each bar, with significantly enriched values shown in red . The dashed line represents the average level of APOBEC3 signature mutations observed in the two eGFP control clones . (B) Trinucleotide mutation profiles of signatures A and B derived using NMF with the former exhibiting an APOBEC3 SBS signature (C-to-T and C-to-G in TCA and TCT motifs).((TIF)Click here for additional data file.S12 FigBar plots showing the percentage of each of the indicated indel types occurring in WGS from HAP1-TK-M9 granddaughter clones. Total numbers of single T deletions at homopolymers of 6 or more are too numerous to plot on the same axis and are therefore listed here . The cosine similarity of the indel landscape across all conditions is over 0.96 indicating no significant differences.(TIF)Click here for additional data file.S13 FigMethods for additional information). Columns 1 and 2: The A3 or control condition and clone number as listed in A list of the total number of SBS mutations in each HAP1-TK-M9 clone and numbers and frequencies mutations predicted to occur in non-hairpin regions or hairpin loop regions of the genome (see (TIF)Click here for additional data file.S14 FigNUP93-noHP (no hairpin), which has half of the stem replaced by adenines (5’-6-carboxyfluorescein-GCAAGCTGTTCAAAAAAATGA) and is included here as an additional control.Native PAGE (left) and denaturing PAGE (right) analysis of the indicated oligonucleotide substrates. The hairpin substrates migrate faster under native conditions, and their mobility is similar to the linear derivatives under denaturing conditions. The only oligonucleotide not used in biochemical experiments in (TIF)Click here for additional data file.S15 FigA) Trinucleotide profiles of total, clustered, non-clustered single base substitution substitution mutations in representative A3A (top), A3B (middle), and eGFP (bottom) clones. The intermutation distance (IMD) is indicated, together with the expected number number of kataegis events and the actual range of kataegis events observed for clones of each condition determined using SigProfilerClusters . (B) IMDs of A3A (top), A3B (middle), and eGFP (bottom) clones. Green lines are based on actual intermutation distances, and red lines are simulated distributions of intermutation distances. 95% confidence intervals are shown in pink for the simulated IMD distributions.((TIF)Click here for additional data file.


Limosilactobacillus reuteri to poultry has been shown to improve poultry performance and health. Some strains of L. reuteri taxa can produce reuterin, a broad-spectrum antimicrobial compound from glycerol conversion, with high inhibitory activity against enterobacteria. However, little is known about the metabolism of glycerol in the complex chicken cecal microbiota nor the effect of glycerol, either alone or combined with L. reuteri on the microbiota. In this study, we investigated the effect of L. reuteri PTA5_F13, a high-reuterin-producing chicken strain and glycerol, alone or combined, on broiler chicken cecal microbiota composition and activity using the continuous PolyFermS model recently developed to mimic chicken cecal fermentation.Administering probiotic strains of L. reuteri PTA5_F13  were tested in parallel second-stage reactors continuously inoculated with the same microbiota. We analyzed the complex chicken gut microbiota structure and dynamics upon treatment using 16S rRNA metabarcoding and qPCR. Microbiota metabolites, short-chain and branched-chain fatty acids, and glycerol and reuterin products were analyzed by HPLC in effluent samples from stabilized reactors.Three independent PolyFermS chicken cecal microbiota models were inoculated with immobilized cecal microbiota from different animals and operated continuously. The effects of two additional levels of glycerol (50 and 100 mM) with or without daily supplementation of chicken-derived L. reuteri PTA5_F13 resulted in a reproducible increase in butyrate production in the three modelled microbiota (increases of 18 to 25%). Glycerol alone resulted also in a reduction of Enterobacteriaceae in two of the three microbiota, but no effect was detected for L. reuteri alone. When both treatments were combined, all microbiota quantitatively inhibited Enterobacteriaceae, including in the last model that had very high initial concentrations of Enterobacteriaceae. Furthermore, a significant 1,3-PDO accumulation was measured in the effluent of the combined treatment, confirming the conversion of glycerol via the reuterin pathway. Glycerol supplementation, independent of L. reuteri addition, did not affect the microbial community diversity.Supplementation with 100 mM glycerol alone and combined with Enterobacteriaceae that was strengthened when reuterin-producing L. reuteri was spiked daily. Our in vitro study suggests that co-application of L. reuteri PTA5_F13 and glycerol could be a useful approach to promote chicken gut health by enhancing metabolism and protection against Enterobacteriaceae.Glycerol induced a stable and reproducible butyrogenic activity for all tested microbiota and induced an inhibitory effect against The online version contains supplementary material available at 10.1186/s12866-023-03091-6. Campylobacter jejuni and Salmonella enterica that can be transmitted to humans by handling and consuming improperly cooked meat ) . Each treatment was performed for 6 or 8 days until stability, which was defined by less than 10% variations in the daily metabolite concentrations, was reached. Reactor effluents were sampled daily and centrifuged (10 min at 14′000 × g at 4 °C). Bacterial pellet and supernatant were stored at -80 °C and -20 °C for DNA extraction and metabolite analysis, respectively.Model F1 was used to test the effects of two concentrations of glycerol  on microbiota population and metabolite production, whereas models F2 and F3 were used to test the effect of glycerol (100 mM [100G]) and SCFAs , branched-chain fatty acids , intermediate metabolites , 3-HPA and 1,3 PDO were measured during the last three days of the pre-treatment and treatment periods by high-performance liquid chromatography with refractive index detector (HPLC-IR), as presented previously , and expTotal genomic DNA was extracted from the cell pellet of 2 mL of effluent using the FastDNA® SPIN Kit for Soil  and a final elution volume of 100 µL, according to the manufacturer’s instructions. DNA concentrations and quality were determined by absorbance measured at 260 nm using a Nanodrop® ND-1000 Spectrophotometer . Samples were stored at -20 °C before qPCR and 16S rRNA metabarcoding analysis.Quantitative PCR (qPCR) analysis was performed using a Roche LightCycler 480 II . Specific primers Table S targetin16S rRNA amplicon sequencing of bacterial communities in effluents were conducted using an Illumina Miseq platform . Effluent samples were amplified and sequenced using universal primers targeting the V3 (for F1 and F2) or V4 (for F3) region of the 16S rRNA  version 2020.8 was used for analysis of the sequence data . The seq P values less than 0.05 were considered significant. qPCR (log10 transformed) and HPLC-IR data were expressed as mean ± standard deviation of the last 3 days of each fermentation period, corresponding to stable metabolite production as indicated above. Statistical analysis was performed by pairwise comparison between the last 3 days of treatment between CR and TRs during the same period (F1) or between the pre-treatment and treatment periods of the same reactor (F2 and F3) by t-test. Comparison within a reactor was selected for F2 and F3 to account for differences in metabolite composition, qPCR data and 16S data observed between reactors during the pre-treatment periods [GraphPad Prism version 8.0  was used to visualize the data. All statistical analyses were performed with IBM SPSS 28.0.0 , and periods . The DES periods .Additional file 1: Table S1. Primers used for the detection of bacterial groups by qPCR and for the PCR amplification step of 16S rRNA amplicon Illumina sequencing. Figure S1. Daily metabolite concentrations in the effluent of control and treatment reactors during F1 measured by HPLC-IR: control reactor) (A), 50 mM glycerol (50G) supplementation (B), and 100 mM glycerol (100G) supplementation (C). Lactate was below detection limit. Figure S2. Daily metabolite concentrations in the effluent of control and treatment reactors during F2 measured by HPLC-IR: control reactor) (A), L. reuteri (Lbr) supplementation (B), 100 mM glycerol (100G) supplementation (C), and L. reuteri and 100 mM glycerol (Lbr-100G) supplementation (D). Lactate was below detection limit. Figure S3. Daily metabolite concentrations in the effluent of control and treatment reactors during F3 measured by HPLC-IR: control reactor) (A), L. reuteri (Lbr) supplementation (B), 100 mM glycerol (100G) supplementation (C), and L. reuteri and 100 mM glycerol (Lbr-100G) supplementation (D). Lactate was below detection limit. Figure S4. Daily quantification of key bacterial populations in the effluent of control and treatment reactors during F1 measured by qPCR: control reactor) (A), 50 mM glycerol (50G) supplementation (B), and 100 mM glycerol (100G) supplementation (C). Figure S5. Daily quantification of key bacterial populations in the effluent of control and treatment reactors during F2 measured by qPCR: control reactor) (A), L. reuteri (Lbr) supplementation (B) 100 mM glycerol (100G) supplementation (C), and L. reuteri and 100 mM glycerol (Lbr-100G) supplementation (D). Figure S6. Daily quantification of key bacterial populations in the effluent of control and treatment reactors during F3 measured by qPCR: control reactor) (A), L. reuteri (Lbr) supplementation (B) 100 mM glycerol (100G) supplementation (C), and L. reuteri and 100 mM glycerol (Lbr-100G) supplementation (D). Figure S7. Genus taxa with differential relative abundance in reactor effluent during treatment compared to pre-treatment  in modelled cecal microbiota F2. Taxa are ordered according to log2 fold-change. Each ASV affected for a given genus is indicated with a dot. Dots are coloured according to phylum with the color code in the graph legend. Only taxa with more than 2 log2 fold-change and which are significantly differentially abundant  are shown. CR, Control reactor; 100G, 100 mM glycerol ; Lbr, L. reuteri PTA5_F13; Lbr-100G, L. reuteri PTA5_F13 and 100 mM glycerol. Figure S8. Genus taxa with differential relative abundance in reactor effluent during pre-treatment compared with treatment  in modelled cecal microbiota F3. Taxa are ordered according to log2 fold-change. Each ASV affected for a given genus is indicated with a dot. Dots are coloured according to their phylum with the color code in the graph legend. Only taxa with at least 2 log2 fold-change and which are significantly differentially abundant  are shown. CR, Control reactor; 100G, 100 mM glycerol ; Lbr, L. reuteri PTA5_F13; Lbr-100G, L. reuteri PTA5_F13 and 100 mM glycerol.