F-(CH2-CHSi(OMe)3)n-RF (RF-(VM)n-RF)] undergoes the sol-gel reaction in the presence of N-(3-triethoxysilylpropyl)gluconamide [Glu-Si(OEt)3] under alkaline conditions to afford the corresponding fluorinated oligomeric silica nanocomposites containing gluconamide units [RF-(VM-SiO3/2)n-RF/Glu-SiO3/2]. These obtained nanocomposites were applied to the surface modification of glass to provide the unique wettability characteristics such as highly oleophobic/superhydrophobic and highly oleophobic/superhydrophilic on the modified surfaces under a variety of conditions. Such a highly oleophobic/superhydrophobic characteristic was also observed on the modified PET  fabric swatch, which was prepared under similar conditions, and this modified PET fabric swatch was applied to the separation membrane for the separation of the mixture of fluorocarbon oil and hydrocarbon oil. The RF-(VM-SiO3/2)n-RF/Glu-SiO3/2 nanocomposites, which were prepared under lower feed amounts of basic catalyst (ammonia), were found to cause gelation in water. Interestingly, it was demonstrated that these gelling nanocomposites are also applied to the surface modification of the PET fabric swatch to give a highly oleophobic/superhydrophobic characteristic on the surface. On the other hand, the modified glass surfaces treated with the corresponding nanocomposite possessing no gelling ability were found to supply the usual hydrophobic characteristic with a highly oleophobic property. More interestingly, the wettability change on the modified PET fabric swatch from highly oleophobic to superoleophilic was observed, and remained superhydrophobic after immersing the modified PET fabric swatch into water.Fluoroalkyl end-capped vinyltrimethoxysilane oligomer [R F-C(=O)OO(O=)C-RF: RF = fluoroalkyl group] is a useful tool for the synthesis of two-fluoroalkyl end-capped oligomers [RF-(M)n-RF: M = radical polymerizable monomers] [d-gluconamide unit-containing methacrylate monomer can be copolymerized with AIBN  in the presence of methyl acrylate as comonomer to afford the corresponding glycopolymers [F-(CH2-CHSi(OMe)3)n-RF] can undergo the sol-gel reaction under alkaline conditions to afford the corresponding fluorinated oligomeric silica nanoparticles [RF-(CH2-CHSiO3/2)n-RF] [F-(CH2–CHSi(OMe)3)n-RF] can undergo the sol-gel reaction in the presence of N-(3-triethoxysilylpropyl)gluconamide [Glu-Si(OEt)3] under alkaline conditions to afford the corresponding fluorinated oligomeric silica nanocomposites containing gluconamide units [RF-(VM-SiO3/2)n-RF/Glu-SiO3/2]. The modified surfaces treated with these obtained nanocomposites can provide the unique wettability states such as the highly oleophobic/superhydrophobic, highly oleophobic/superhydrophilic, and superoleophilic/superhydrophobic characteristics. These results will be described in this article.It is well known that fluoroalkanoyl peroxide [Ronomers] ,2,3. Theonomers] ,5. In thonomers] ,7,8,9. Ionomers] ,17,18,19onomers] . Similarpolymers . These gpolymers . Therefo/2)n-RF] . These f/2)n-RF] . From thN-(3-triethoxysilylpropyl)gluconamide [Glu-Si(OEt)3] was purchased from AZmax. Co. . Dodecane and 1H-tridecafluorohexane were received from Tokyo Chemical Industrial Co., Ltd. . 25 wt % ammonia was provided by Wako Pure Chemical Industries . Fluoroalkyl end-capped vinyltrimethoxysilane oligomer was prepared according to our previously reported method amino]-9,10-anthracenedione (Quinizarin Green SS) and PET  fabric  were received from Chugaikasei Co., Ltd.  and Unitika Ltd. , respectively. d method . Glass pDynamic light scattering (DLS) measurements were measured by using Otsuka Electronics DLS-7000 HL . Contact angles were recorded using a Kyowa Interface Science Drop Master 300 . Field emission scanning electron micrographs (FE-SEM) and energy dispersive X-ray (EDX) spectra were obtained by using JEOL JSM-7000F . Dynamic force microscope (DFM) was recorded by using SII Nano Technology Inc. E-sweep .F-(CH2CHSi(OMe)3)n-RF; RF = CF(CF3)OC3F7; Mn = 730 (RF-(VM)n-RF)] was added 50 wt % Glu-Si(OEt)3 ethanol solution (1400 mg). The mixture was stirred with a magnetic stirring bar at room temperature for 30 min. 25% aqueous ammonia solution (1.0 mL) was added to this mixture, and then stirred for 5 h at room temperature. Methanol was added to the obtained crude products after the solvent was evaporated off. The methanol suspension was stirred with magnetic stirring bar at room temperature for 1 day. The fluorinated oligomeric silica/Glu-SiO2 nanocomposites were isolated after centrifugal separation for 30 min. The nanocomposite product was washed well with methanol several times, and then was dried under vacuum at 50 °C for 1 day to afford the expected nanocomposites as white powders (774 mg)  containing fluoroalkylated vinyltrimethoxysilane oligomer  proceeded smoothly in the presence of N-(3-triethoxysilylpropyl)gluconamide [Glu-Si(OEt)3] under alkaline conditions to afford the corresponding fluorinated oligomeric silica/Glu-SiO3/2 nanocomposites [RF-(VM-SiO3/2)n-RF/Glu-SiO3/2]. These results are shown in Sol-gel reaction of fluoroalkyl end-capped vinyltrimethoxysilane oligomer  can give a usual oleophobic  with a superhydrophobic property .As shown in 3 from 0.44 to 1.75 mmol, were found to decrease from 133–137 to 0 degrees (superhydrophilic surface) over 15 or 25 min except for Run 8, indicating that the hydrophobic fluoroalkyl segments are replaced by the hydrophilic gluconamide segments in the nanocomposites when the environment is changed from air to water. The relatively higher feed amounts (0.44–0.88 mmol) of Glu-Si(OEt)3 in the nanocomposite preparation illustrated in 3, can supply a highly oleophobic/superhydrophobic characteristic  on the modified surface, due to the lower content of hydrophilic gluconamide segments in the nanocomposites.The water contact angle values on the modified glass surfaces treated with the fluorinated nanocomposites, which were prepared under the greater feed amounts of Glu-Si(OEt)F-(VM-SiO3/2)n-RF/Glu-SiO3/2 nanocomposites possessing the highly oleophobic/superhydrophobic characteristic (Run 2 in F-(VM-SiO3/2)n-RF], for comparison. The results are shown in The fabrication of a superoleophobic surface is, in general, difficult due to the lower surface tension of oils than that of water. Thus, a highly oleophobic (superoleophobic) surface can be realized by lowering the surface energy and enhancing the surface roughness ,31,32,33Run 2 in  and usuaRun 2 in  includinF-(VM-SiO3/2)n-RF/Glu-SiO3/2 nanocomposites possessing a highly oleophobic/superhydrophobic property. Such roughness of surface was also observed in the nanocomposites possessing a similar wettability depicted in F-(VM-SiO3/2)n-RF/Glu-SiO3/2 nanocomposites possessing a usual oleophobic/hydrophobic property and the pristine RF-(VM-SiO3/2)n-RF oligomeric nanoparticles can provide relatively smooth roughness, because these Ra values are 9 and 7 nm, respectively, indicating that such relatively smooth surfaces should supply the usual oleophobic/hydrophobic or oleophobic/superhydrophobic characteristic to the modified surfaces.As shown in F-(VM-SiO3/2)n-RF/Glu-SiO3/2 nanocomposites (Run 2 in H-tridecafluorohexane) and the hydrocarbon oil (dodecane: blue-colored with Quinizarin Green SS), because the modified PET fabric swatch can provide a highly oleophobic/superhydrophobic characteristic on the modified surface, and the results are illustrated in We tried to achieve the surface modification of not only the glass but also the polyethylene terephthalate (PET) fabric swatch by using the RRun 2 in  under siRun 2 in B, comparRun 2 in A. Here, As shown in 3 greater than 0.21 mmol can lead the obtained nanocomposites to a good dispersibility toward water. This finding suggests that the fluorinated nanocomposites can cause gelation toward water through the synergistic interaction between the aggregation of the end-capped fluoroalkyl groups and the intermolecular hydrogen bonding related to the hydroxyl segments in the gluconamide units in the nanocomposites by controlling the sol-gel conditions . In fact, we have succeeded in preparing the RF-(VM-SiO3/2)n-RF/Glu-SiO3/2 nanocomposites, which can cause gelation toward water, by changing the feed amounts of both 25 % aqueous ammonia and methanol from 1.0 to 0.6 and 5.0 to 15 mL, respectively (see As illustrated in vely see .The gelling fluorinated nanocomposites see  were appF-(VM-SiO3/2)n-RF oligomeric nanoparticles is not enough, and the corresponding fluorinated oligomeric nanoparticles were easily released from the modified surface after rubbing the surface with a finger (see F-(VM-SiO3/2)n-RF/Glu-SiO3/2 nanocomposite gels. In fact, FE-SEM images of the modified PET fabric and the pristine PET fabric swatches show that the nanocomposite gels can be uniformly coated on the PET fibers (see F-(VM-SiO3/2)n-RF/Glu-SiO3/2 nanocomposites possessing no gelling ability as in As shown in nger see A. Such sbers see A,B, quitFurthermore, we have measured the dodecane and water contact angle values on the modified PET fabric swatches treated with the nanocomposite gels n-RF/Glu-SiO3/2 nanocomposites  spectra of the modified PET fabric swatch treated with the REDX measurements show that the atomic contents of carbon, oxygen, nitrogen, fluorine, and silicon of the modified PET fabric swatch before and after immersing into water are as follows see .We observe similar atomic values even after immersing into water. In addition, it was verified that fluorine and silicon atoms related to the fluorinated nanocomposites are uniformly dispersed on the modified PET fabric swatch before and after immersing into water by EDX mapping images illustrated in F-(VM-SiO3/2)n-RF/Glu-SiO3/2 nanocomposite gels can be strongly coated into the PET fabric fiber networks due to the gelling ability of the nanocomposites.In this way, it was demonstrated that our present RF-(VM-SiO3/2)n-RF/Glu-SiO3/2] were prepared by the sol-gel reactions of the corresponding oligomers in the presence of gluconamide unit-containing silane coupling agent [Glu-Si(OEt)3] under alkaline conditions. Wettability control between the highly oleophobic/superhydrophobic and highly oleophobic/superhydrophilic states was observed on the modified glass surfaces treated with the RF-(VM-SiO3/2)n-RF/Glu-SiO3/2 nanocomposites. Such wettability can be easily controlled by changing the feed amount ratios of Glu-Si(OEt)3 and RF-(VM)n-RF oligomer for the preparation of the nanocomposites. Lower feed amounts of Glu-Si(OEt)3 can provide a highly oleophobic/superhydrophobic surface; in contrast, a higher feed amount of Glu-Si(OEt)3 enables the modified surface to reveal a highly oleophobic/superhydrophilic characteristic. Such a highly oleophobic/superhydrophobic characteristic was also observed on the modified PET fabric swatch, and this modified PET fabric was applied to the separation membrane to separate the mixture of fluorocarbon oil and hydrocarbon oil.Fluoroalkyl end-capped viniyltrimethoxysilane oligomeric silica nanocomposites containing gluconamide units [RF-(VM-SiO3/2)n-RF/Glu-SiO3/2 nanocomposites were also shown to cause gelation toward water. Especially, the gelling nanocomposites were applied to the surface modification of the PET fabric swatch to exhibit a highly oleophobic/superhydrophobic characteristic on the modified surface. However, interestingly, we can observe the wettability change on the modified PET fabric surface from a highly oleophobic state to a superoleophilic state, while keeping the superhydrophobic characteristic by immersing this modified fabric into water.R


Caenorhabditis elegans and mammals, neural serotonin signaling regulates a range of food-related behaviors. However, the mechanisms that integrate metabolic information with serotonergic circuits are poorly characterized. Here, we identify metabolic, molecular, and cellular components of a circuit that links peripheral metabolic state to serotonin-regulated behaviors in C. elegans. We find that blocking the entry of fatty acyl coenzyme As (CoAs) into peroxisomal β-oxidation in the intestine blunts the effects of neural serotonin signaling on feeding and egg-laying behaviors. Comparative genomics and metabolomics revealed that interfering with intestinal peroxisomal β-oxidation results in a modest global transcriptional change but significant changes to the metabolome, including a large number of changes in ascaroside and phospholipid species, some of which affect feeding behavior. We also identify body cavity neurons and an ether-a-go-go (EAG)–related potassium channel that functions in these neurons as key cellular components of the circuitry linking peripheral metabolic signals to regulation of neural serotonin signaling. These data raise the possibility that the effects of serotonin on satiety may have their origins in feedback, homeostatic metabolic responses from the periphery.The ability to coordinate behavioral responses with metabolic status is fundamental to the maintenance of energy homeostasis. In numerous species including  As a neuromodulator, serotonin regulates numerous behavioral and emotional states. This study shows in the nematode C. elegans, metabolic cues that originate in the organism's periphery modulate neurally regulated serotonergic processes, including feeding behavior and egg laying, revealing a homeostatic signal that informs the nervous system of the organism's nutritional state. In both invertebrate and vertebrate species, behaviors such as feeding, movement, reproduction, and learning are influenced by nutritional and metabolic signals –3. In maC. elegans display a range of behavioral and physiological responses to changes in nutrient availability  in body cavity neurons and found that this manipulation strongly stimulates pharyngeal pumping in both wild-type and acox-1 mutants. Importantly, this feeding enhancement could not be further elevated by addition of serotonin, suggesting a common feeding regulatory circuit (egl-2(gf) did not modulate baseline feeding rates, our data suggest that these neurons likely fine-tune feeding responses rather than governing basal feeding behavior.To our knowledge, body cavity neurons have not previously been implicated in the regulation of feeding behavior. To further study their role, we examined the effect of prolonged activation of body cavity neurons on pharyngeal pumping. Activation of protein kinase C (PKC-1) has been shown to promote synaptic transmission and neuropeptide release from expressing neurons . We exam circuit . As geneacox-1, while activation of these neurons restores serotonin responsiveness to acox-1 mutants. To more directly assess the effects of ACOX-1 activity on body cavity neuron function, we measured intracellular Ca2+ transients from the URX body cavity neurons using the genetically encoded calcium reporter GCaMP5K , SSR1070 flp-8p::mCherry; flp-8p::GCaMP5K. When generating double mutants, genotypes were confirmed by PCR or sequencing. Unless stated otherwise, animals were cultured on NGM agar plates with OP50 E. coli at 20°C. For all experiments, worms were plated as synchronized L1 populations after hypochlorite treatment of gravid adults.nditions . The Briacox-1 tissue-specific rescue lines, full F08A8.1 genomic coding sequence was amplified from wild-type gDNA and was subsequently subcloned into the P221 Gateway entry vector by BP recombination. Transgenic animals were generated by injecting purified plasmids into the gonads of wild-type or mutant animals. Unless otherwise specified, transgenic rescue constructs and an unc-122p::gfp co-injection marker were injected at a concentration of 50 ng/μL. At least two independent and stably expressing lines were maintained and analyzed. To reduce the expression of egl-2 specifically in URX neurons, we used a cell-specific RNAi approach using the flp-8 promoter as described before - adducts in the negative ionization mode (ESI+), or as [M+H]+ ions or [M+Na]+ adducts in the positive ionization mode (ESI+), using a spray voltage of 3 kV. Compound identities were confirmed based on their high-resolution masses (accuracy <1 ppm), MS/MS fragmentation spectra, and/or comparison with authentic standards. Data analysis was carried out using the Bioconductor package XCMS age XCMS ,111. The0). F0 was measured in worms exposed to 10% oxygen during the first 9–13 seconds for each recording and calculated as an average over that period. The number of animals used for each condition is denoted in the figures.Animals were exposed to different oxygen concentrations (10% or 21%) using a microfluidic chamber constructed with the oxygen-permeable poly(dimethylsiloxane) (PDMS) as described . A Valveacox-1 mutants that express Ex were conditioned with 10% butanone for 1 hour and mounted on a 10% agarose pad containing 2 μL of 0.1 mm polystyrene beads followed by immobilization with coverslips. GCaMP imaging for control and conditioned animals was performed as described before [b)/ Fb, and summed to obtain a total integrated fluorescence measurement for each sample.Day 1 wild-type and d before . Brieflyp-values unless otherwise specified. Where only 2 conditions were compared, a two-tailed Student t test was performed. ANOVAs with appropriate posttest corrections were used when comparing multiple conditions.Graphpad Prism 8.0 software package was used to calculate all S1 Fig(A) Knocking down acox-1(F08A8.1) by RNAi suppresses the feeding elevating effects of exogenous serotonin (5 mM 5HT). Animals were treated with RNAi from the L1 stage, and feeding was assayed at day 1 adult stage. Feeding data are normalized to vehicle-treated wild-type animals and are presented as a percentage of wild-type animals. Error bars indicate ±SEM from normalized mean, n = 20 animals per strain. ***p < 0.001 ANOVA (Tukey). (B) Pharyngeal pumping rates were counted over a longer interval (60 seconds) from video recordings. Error bars indicate ±SEM from normalized mean, n = 10 animals per condition. ***p < 0.001 ANOVA (Tukey). See (C) Loss of acox-1 does not influence the transcriptional expression of tryptophan hydroxylase (tph-1) as measured by qPCR. Error bars indicate ±SEM from mean n = 3 independent assays. (D) Relative abundance of 5HT in wild-type and acox-1 mutants, as determined by LC-HRMS. Error bars indicate ±SEM from mean, n = 4 independent experiments. (E) Loss of acox-1 does not grossly alter amphid neuron morphology. DiI staining of amphid chemosensory neurons in wild-type and acox-1 mutants. Images acquired at day 1 adult stage. acox-1, acyl-coenzyme A oxidase 1; HRMS, high-resolution mass spectrometry; LC, liquid chromatography; qPCR, quantitative PCR; RNAi, RNA interference; 5HT, 5-hydroxytryptamine.(TIF)Click here for additional data file.S2 Fig(A) Treating aak-2;acox-1 mutants with 1 μM triacsin C for 60 minutes elevates pharyngeal pumping rates. Day 1 adult animals were treated with to vehicle (0.0002% DMSO) or 1 μM triacsin C for 60 minutes before assessing pharyngeal pumping rates. Error bars indicate ±SEM from normalized mean, n = 15 animals per condition. At the concentration used, triacsin C had no effect on feeding rate. (B) RNAi-mediated inactivation of distinct ACSs suppress feeding defects in acox-1(ok2257) animals. Animals were treated with respective RNAi clones from L1 stage and feeding was assayed at day 1 adult stage. All feeding data are normalized to vehicle and vector RNAi-treated wild-type animals and are presented as a percentage of wild-type rates. Error bars indicate ±SEM from normalized mean, n = 15 animals per strain. *p < 0.05, ***p < 0.001 ANOVA (Tukey). See (TIF)Click here for additional data file.S3 Fig(A)daf-22(m130) animals are still responsive to the feeding elevating effects of 5 mM serotonin, n = 10 animals per condition. (B) Feeding responses of wild-type animals to ascr#18. Animals were exposed to 1 μM and 5 μM ascr#18 from the L1 stage, and pharyngeal pumping rates were determined at day 1 adult stage. n = 15 animals per condition. (C) Feeding responses of acox-1(ok2257) animals to vector and daf-22 RNAi, n = 15 animals per condition. (D) Feeding responses of wild-type and acox-1 mutants to 5 μM ascr#18, n = 15 animals per condition. (E) Feeding responses of wild-type animals to thiamine. Animals were exposed to 0.5 μg/μL thiamine and 1.0 μg/μL thiamine from L1 stage, and pharyngeal pumping rates were determined at day 1 adult stage. n = 15 animals per condition. (F) RNAi-mediated inactivation of FAAH-1 elevates feeding responses of wild-type and acox-1 mutants. Animals were grown on faah-1 RNAi from L1 stage and feeding was assayed at day 1 adult stage, n = 10 animals per condition. All feeding data are normalized to vehicle-treated wild-type animals and are presented as a percentage of wild-type animals. Error bars indicate ±SEM from normalized mean, ***p < 0.001 two-way ANOVA (Tukey). See (TIF)Click here for additional data file.S4 Fig(A)acox-1(ok2257) mutants lay eggs at a later developmental stage than wild-type animals, suggesting that in utero retention time is increased. Histograms indicate the distribution of embryos at each developmental stage. (B) There was no significant difference in the total number of progeny between wild-type, acox-1, egl-2, and acox-1;egl-2 mutants. (C)acox-1 mutants are less responsive to the egg-laying inducing effects of serotonin. Egg-laying response of wild-type and acox-1 mutants in control buffer (M9) or 0.5 mg/mL fluoxetine. Data represent the number of eggs released per animal after a 20-minute exposure to vehicle or drug. Error bars represent ±SEM from mean. n = 15 animals per condition, ***p < 0.001 ANOVA (Sidak). (C-D) Inactivation of egl-2 via RNAi rescues acox-1 egg-laying defects. Representative DIC images of day 1 adults of each genotype. (C) and quantification (D) of eggs retained in utero. Error bars indicate ±SEM from mean, n = 15 animals per genotype. ***p < 0.001 unpaired Student t test. See acox-1, acyl-coenzyme A oxidase 1; DIC, differential interference contrast; egl-2, EGg Laying defective 2; RNAi, RNA interference.(TIF)Click here for additional data file.S5 Fig(A) RNAi-mediated knockdown of egl-2 rescues egg-laying defects associated with aberrant channel activity egl-2(n698) mutants. Representative DIC images acquired from day 1 adults. DIC, differential interference contrast; egl-2, EGg Laying defective 2; RNAi, RNA interference.(TIF)Click here for additional data file.S6 Fig(A-D) Activity of URX neurons in each indicated genotype using Ca2+ imaging by GCaMP5K under the control of the URX-specific flp-8 promoter. Oxygen concentrations in the microfluidic chamber were 10% and 21%, as indicated. (A-B) For each genotype, black traces show the average percent change of GCaMP5K fluorescence (FΔ/F0) and gray shading indicates SEM. The number of animals used for each condition is shown in the figure. (C-D) Individual URX responses are shown for each genotype; each row represents one animal. (E) Maximal (FΔ/F0) values are shown for individual animals in wild-type and acox-1 animals. Bars indicate the average value within each genotype. ***p < 0.001 by Student t test. (F) Individual baseline fluorescence (F0) values at 10% oxygen are shown for individual animals in wild-type and acox-1 mutants. Bars indicate the median value within each genotype; n.s., not significant by Student t test. (G) We imaged mCherry fluorescence in wild-type and acox-1 mutant animals expressing both GCaMP5K and mCherry under the control of the flp-8 promoter. Images were taken in animals exposed to 10% oxygen. (H-I) Activity of RIM neurons in wild-type and acox-1 mutants using Ca2+ imaging by GCaMP3 under the control of the RIM specific cex-1 promoter. (H) The total intensity of RIM GCaMP3 fluorescence in wild-type and acox-1 mutants over a 250-second imaging window. (I) Maximal (FΔ/F0) values for wild-type and acox-1 animals, n = 8 animals. See acox-1, acyl-coenzyme A oxidase 1(TIF)Click here for additional data file.S7 Fig(A-H) Activity of URX neurons in each indicated genotype using Ca2+ imaging by GCaMP5K under the control of the URX specific flp-8 promoter. Oxygen concentrations in the microfluidic chamber were 10% and 21%, as indicated. (A-D) For each genotype, black traces show the average percent change of GCaMP5K fluorescence (FΔ/F0) and gray shading indicates SEM. The number of animals used for each condition is shown in the figure. (E-H) Individual URX responses are shown for each genotype; each row represents one animal. (I) Maximal (FΔ/F0) values are shown for individual animals in wild-type, acox-1, egl-2, and acox-1;egl-2 animals. Bars indicate the average value within each genotype. ***p < 0.001 by Student t test. (J) Individual baseline fluorescence (F0) values at 10% oxygen are shown for individual animals in wild-type, acox-1, egl-2, and acox-1;egl-2 animal mutants. Bars indicate the median value within each genotype; n.s., not significant by Student t test. (K) We imaged mCherry fluorescence in wild-type and acox-1 mutant animals expressing both GCaMP5K and mCherry under the control of the flp-8 promoter. Images were taken in animals exposed to 10% oxygen. See acox-1, acyl-coenzyme A oxidase 1; egl-2, EGg Laying defective 2.(TIF)Click here for additional data file.S8 Fig(A-D) Activity of URX neurons in each indicated genotype using Ca2+ imaging by GCaMP5K under the control of the URX-specific flp-8 promoter. Oxygen concentrations in the microfluidic chamber were 10% and 21%, as indicated. (A-B) For each genotype, black traces show the average percent change of GCaMP5K fluorescence (FΔ/F0), and gray shading indicates SEM. The number of animals used for each condition is shown in the figure. (C-D) Individual URX responses are shown for each genotype; each row represents one animal. (E) Maximal (FΔ/F0) values are shown for individual animals in acox-1 and acox-1; vha-6p::(+)  animals. Bars indicate the average value within each genotype. ***p < 0.001 by Student t test. (F) Individual baseline fluorescence (F0) values at 10% oxygen are shown for individual animals in acox-1 and acox-1; vha-6p::(+) mutants. Bars indicate the median value within each genotype; n.s., not significant by Student t test. (G) mCherry fluorescence in acox-1 and acox-1; vha-6p::(+) mutant animals expressing both GCaMP5K and mCherry under the control of the flp-8 promoter. Images were taken in animals exposed to 10% oxygen. See acox-1, acyl-coenzyme A oxidase 1.(TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Tableacox-1, acyl-coenzyme A oxidase 1; ESI, electrospray ionization; HPLC, high-pressure liquid chromatography; MS, mass spectrometry.(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Using HLA-A2+NY-ESO-1+ myeloma cells and peptides presented by HLA-A2 molecules as a model, both sets of redirected T cells recognized and killed HLA-A2+NY-ESO-1+ myeloma cells in an A2/NY-ESO-1157-specific manner in vitro. Moreover, CAR- and BiTE-activated T cells showed similar functional avidity, as assessed by cytokine production and killing activity, both displaying antitumor reactivity against HLA-A2+NY-ESO-1+ myeloma cells in vivo. Interestingly, cross-reactivity for homologous peptides presented by HLA-A*02:01 and NY-ESO-1157 peptide presented by HLA-A2 alleles was not identical between CAR- and BiTE-redirected T cells, probably due to structural differences of modified antibodies. These results have demonstrated that both antitumor CAR- and BiTE-activated T cells have comparable potential to recognize tumors, while paying attention to unknown off-target reactivity that would differ for each antibody-based modality even if the same scFv was employed.The development of chimeric antigen receptor (CAR) and bispecific T-cell engager (BiTE) has led to the successful application of cancer immunotherapy. The potential reactivity mediated by CAR- and BiTE-redirected T cells needs to be assessed to facilitate the application of these treatment options to a broader range of patients. Here, we have generated CAR and BiTE possessing the same single chain fragment variable (scFv) specific for the HLA-A2/NY-ESO-1 Checkpoint blockade therapy has been shown to induce clinical responses in patients with hematological malignancies such as Hodgkin’s lymphoma, as well as solid tumors6. However, non-specific activation of patients’ T cells induces not only cytotoxic activity against tumors but also unwanted toxicity against normal tissues, resulting in immune-related adverse events9. Therefore, immunotherapy utilizing T cells that can accurately segregate tumor cells from normal cells is needed for safe and effective T-cell therapy.Clinical trials have demonstrated that T-cell therapy can suppress tumor growth even in patients with refractory malignancies10. Peptides derived from these antigens are produced in a proteasome-dependent manner, and are naturally processed and presented by MHC molecules. Antitumor T cells recognize MHC/peptide complexes selectively presented on tumor cells via their T-cell receptor (TCR), and thus exhibit target-specific reactivity. On the basis of this mechanism of T-cell antigen recognition, adoptive transfer therapy has been developed using T cells redirected with TCRs specific for human leukocyte antigen (HLA)/peptide complexes expressed by tumor cells, and clinical trials have shown promise in patients with advanced tumors18. However, TCR-transduced T cells are laborious to generate, and exhibit cross-reactivity induced by the introduced TCR itself and/or mispairing between the introduced TCR and endogenous TCR21. To overcome these issues associated with TCR-T cell therapy, recent technical advances have facilitated the generation of antibody-based antitumor receptors that can activate T cells with tumor specificity. For antibody modification, single chain fragment variables (scFvs) generated on the basis of the primary structure of variable regions derived from monoclonal antibodies specific for tumor antigens expressed as surface proteins or HLA/peptide complexes are utilized. Each chimeric antigen receptor (CAR) encodes an scFv followed by a transmembrane domain derived from a co-stimulatory molecule, such as CD28, 4-1BB, and an intracellular domain of the CD3ζ chain23. One of the bispecific antibodies (BsAb), bispecific T-cell engager (BiTE), possesses an antitumor scFv linked with an anti-human CD3ε scFv24. CAR- and BiTE-redirected T cells recognize and kill tumor cells, and clinical trials using CAR- and BiTE-redirected T cells have shown great success in the treatment of refractory CD19+ malignancies30. In contrast, there are not many CAR and BiTE specific for HLA/peptide complexes expressed by refractory tumor cells that can target intracellular antigens32. To clarify the benefits and risks of antitumor CAR and BiTE, direct comparison of reactivities mediated by CAR- and BiTE-redirected T cells possessing the same scFv recognizing an HLA/peptide complex as a model would reveal in more detail their target-specific reactivity and cross-reactivity by changing specific peptides and HLA alleles. Such knowledge would help facilitate the application of these treatment options to a broader range of patients with refractory malignancies.A series of tumor antigens including tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs) have been identified33. T-cell therapy utilizing CAR and/or BiTE reactive with myeloma cells expressing surface antigens such as BCMA, CD19, CD138, and integrin β7 has advanced, and a series of clinical trials have been ongoing37. Unlike the above surface antigens, NY-ESO-1 is a cancer-testis antigen expressed intracellularly by tumors but not by normal tissues, except for testis lacking the expression of endogenous HLA38. Myeloma cells express NY-ESO-1 abundantly, and importantly, an NY-ESO-1157-165 peptide (SLLMWITQC) presented by an HLA-A*02:01 molecule (A2/NY-ESO-1157) has been demonstrated40. In a clinical trial, adoptive therapy using T cells modified with TCR specific for A2/NY-ESO-1157 in combination with autologous stem cell transplantation successfully induced clinical responses in patients with advanced myeloma15. T cells redirected with A2/NY-ESO-1157-specific CAR have been established and assessed for their anti-myeloma reactivity42, although their precise target-specificity and cross-reactivity are still unknown. In addition, A2/NY-ESO-1157-specific BiTE has yet to be generated or compared for T-cell reactivity with target cells in comparison with CAR-T cells.Multiple myeloma still remains incurable despite the development of new therapeutic agents157-specific scFv. Employing A2/NY-ESO-1157-specific CAR and BiTE and myeloma cells as a model, we investigated the potential target-specific reactivity and cross-reactivity of CAR- and BiTE-redirected T cells for NY-ESO-1157 peptide and its homologous peptides presented by the HLA-A2 molecule as well as HLA-A2+NY-ESO-1+ myeloma cells in vitro and in vivo, and compared them directly.In the present study, to clarify the characteristics of antitumor CAR and BiTE more precisely for expansion of these treatment modalities, we generated both CAR and BiTE carrying the same A2/NY-ESO-1157 CAR. As shown in Fig. nd generation CAR encoding an A2/NY-ESO-1157-specific scFv derived from clone 3M4E5 followed by the CD28 transmembrane domain and cytoplasmic domain of the CD3ζ chain was synthesized. Both A2/NY-ESO-1157 CAR-transduced CD8+ T cells and CD4+ T cells were successfully and reproducibly stained with A2/NY-ESO-1157 tetramer  were cultured in RPMI1640 supplemented with 10% FCS52. Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers using Ficoll-Paque  and stored until use. All the experiments using human samples were approved by the Certified Review Board, Ehime University. All methods were performed in accordance with the relevant guidelines and regulations. Written informed consent was given by all donors.K562 cells, which lack expression of endogenous HLA and NY-ESO-1, were purchased from the American Type Culture Collection (ATCC) and cultured with RPMI1640 supplemented with 10% fetal calf serum (FCS). T2 cells endogenously expressing HLA-A*02:01 as one of the HLA alleles were cultured with RPMI1640 supplemented with 10% FCS. The U266 cell line was a generous gift from Dr. Ryosuke Uchibori and Dr. Keiya Ozawa, Jichi Medical University. Other myeloma cell lines were kindly provided by Dr. Takemi Ohtsuki, Kawasaki Medical University. Myeloma cell lines were cultured in RPMI1640 supplemented with 10% FCS. Plat-A cells  were maintained in DMEM supplemented with 10% FCS, 1 μg/mL puromycin and 10 μg/mL blasticidin, as described previously157-165-specific monoclonal antibody (mAb) (clone: 3M4E5) were linked with a linker sequence to generate the scFv53. A second generation A2/NY-ESO-1157CAR gene encoding the scFv followed by a transmembrane region of CD28 and an intracellular domain of the CD3ζ chain was synthesized by GeneArt (Thermo Fisher Scientific). To detect CAR-transduced T cells, a CAR gene was fused with a truncated nerve growth factor receptor (ΔNGFR) gene via a furin cleavage site, an SGSG spacer sequence, and a codon-optimized P2A sequence56. A BiTE gene was also generated by GeneArt. A human CD3ε-specific scFv derived from a CD3ε-specific mAb (clone: OKT3) was fused with an A2/NY-ESO-1157-specific scFv or an anti-human IgG scFv for control via an SGSG linker sequence. A hexahistidine (6xHis) tag was inserted at the C-terminus of the construct with an SGSG linker sequence for BiTE purification and detection. All genes were individually integrated into a pMX retroviral vector for transduction57.Variable regions derived from immunoglobulin light and heavy chains of an HLA-A*02:01-restricted NY-ESO-1HLA-A*02:01 or HLA-A*02:06 gene to establish K562/A*02:01 (K562/A2) or K562/A*02:06 cells. In some experiments, K562/A2 cells were transduced with the NY-ESO-1 gene to establish K562/A2/NY-ESO-1 cells. U266 cells were transduced with the luciferase gene (SLR: stable luciferase red) to establish U266/SLR cells for in vivo bioluminescence imaging assays. Human peripheral blood T cells were stimulated with 100 IU/mL human IL-2 and 50 ng/mL anti-human CD3ε mAb (clone OKT3) for 2 days before transduction. Then, T cells were cultured in RPMI1640 supplemented with 10% AB type human serum (Sigma Aldrich) and retrovirally transduced with an A2/NY-ESO-1157CAR gene or a control gene  to establish gene-modified T cells as described previously56. To perform the ELISA experiments, NGFR-positive T cells labeled with FITC-conjugated anti-human NGFR mAb (clone ME20.4) were collected by FACSAria II (Becton Dickinson). When needed, further selection of CD8- or CD4-positive T cells was performed using microbeads (Miltenyi Biotec). To confirm the killing activity of CAR-T cells in vitro and in vivo, gene-modified T cells were isolated using PE-conjugated anti-human NGFR mAb (clone ME20.4) in combination with anti-PE microbeads (Miltenyi Biotec). 293 T cells were retrovirally transduced with an A2/NY-ESO-1157BiTE gene or a control BiTE gene to produce supernatants containing BiTE. The BiTEs were purified using a His GraviTrap column and a Vivaspin column  for the following experiments.The experiments were reviewed and approved by the institutional review board at Ehime University. K562 cells were transduced with the 157 monomer and HLA-A*02:01/HIV Gag77 monomer (MBL) were multimerized using PE-conjugated streptavidin (Thermo Fisher Scientific), and utilized for detection of CAR-transduced T cells as described elsewhere56. Myeloma cell lines and K562 transfectants were stained with PE-conjugated anti-HLA-A2 mAb (clone BB7.2). They were also stained with anti-human NY-ESO-1 mAb (clone E978) and PE-conjugated AffiniPure F(ab’)2 goat anti-mouse IgG (Fcγ specific) (Jackson ImmunoResearch). To confirm target-specific binding of BiTE, target cells were stained with purified BiTE in combination with PE-conjugated anti-His mAb (clone GG11-8F3.5.1) for detection. Human CD3+ T cells were collected from freshly isolated PBMCs with anti-CD3 microbeads (Miltenyi Biotec) for in vitro experiments using BiTEs unless otherwise specified. To investigate A2/NY-ESO-1157-specific cytokine production by CAR- and BiTE-redirected T cells, 3.0 × 105 responder cells were incubated with 5.0 × 104 target cells. Brefeldin A (BFA) was added after 2 hours, and the cells were incubated for an additional 4 hours for CAR-T cells and 16 hours for BiTE-redirected T cells to produce cytokines, unless otherwise specified. Then, PE-conjugated anti-human TNF-α mAb (clone Mab11), APC-conjugated anti-human IL-2 mAb (clone MQ1-17H12), and PC7-conjugated anti-human IFN-γ mAb (clone B27) together with other surface markers as described above were utilized for detection. All samples were analyzed using a Gallios flow cytometer (Beckman Coulter) and FlowJo Version 7.6.5 software (Becton Dickinson). ΔNGFR-positive cells were gated to assess CAR-T cells.T cells were stained with PC5-conjugated anti-human CD8 mAb (clone B9.11), FITC-conjugated anti-human CD4 mAb (clone OKT4), and V450-conjugated anti-human NGFR mAb (clone C40-1457). Biotinylated HLA-A*02:01/NY-ESO-15 CAR- and BiTE-redirected T cells against 2.0 × 104 target cells was measured by ELISA. Briefly, anti-human IFN-γ capture mAb (clone 1D1K) was coated onto a Nunc MaxiSorp flat-bottom 96-well plate (Thermo Fisher Scientific) overnight at 4 °C. Collected supernatants were added to the plate and incubated for 2 hours at room temperature. The plate was then incubated with biotin-conjugated anti-human IFN-γ detection mAb (clone 7-B6-1) for 1 hour at room temperature. Then, streptavidin-conjugated alkaline phosphatase was added to each well. One-step pNPP solution (Thermo Fisher Scientific) was utilized for detection. The absorbance at 405 nm was measured by FlexStation3 (Molecular Devices).IFN-γ production by 1.0 × 1058. NY-ESO-1 mRNA was measured by quantitative real-time PCR (qRT-PCR) using QuantiTect SYBR Green PCR kit (Qiagen). Human hypoxanthine phosphoribosyltransferase 1 (hHPRT1) mRNA was utilized as an internal control. NY-ESO-1 mRNA expression was normalized to the levels of hHPRT1 mRNA, and the relative expression values were calculated using the 2−ΔΔCt method. The following primer sets were used for the experiments: NY-ESO-1 forward, TTCTGAAGGAGTTCACTGTGTCC, and reverse, AGGGAGGCTGAGCCAAAAAC; hHPRT1 forward, GGCAGTATAATCCAAAGATGGTCAA, and reverse, GTCAAGGGCATATCCTACAACAAAC.Total RNA and cDNA were prepared as described previously60. Cells were extracted with lysis buffer . After centrifugation at 12,000 rpm for 20 minutes at 4 °C, the supernatant was collected as the lysate. Purified BiTEs were used directly for immunoblotting. Following SDS-PAGE, protein was transferred to a polyvinylidene difluoride membrane and incubated with a blocking buffer (Nakarai Tesque). The membranes were incubated with the indicated primary antibodies  overnight at 4 °C, washed and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Fc specific) secondary antibody (Sigma Aldrich) for 1 h at room temperature. After washing the membrane, the signal was detected using enhanced chemiluminescence .Immunoblotting assays were performed as described previously56. Briefly, 5.0 × 103 target cells were labeled with chromium-51 (51Cr) for 1.5 hours at 37 °C. CAR- and BiTE-redirected T cells were incubated with target cells at various E/T ratios for 5 hours (CAR) and 16 hours (BiTE), respectively, unless otherwise specified. The supernatants were collected and their cpm counts were measured. The percentage of specific lysis was calculated as follows: / × 100 (%).Standard cytotoxicity assays were performed as described previouslyIn-Vivo Science Inc. and subjected to 1.5 Gy of irradiation. Then, two million U266/SLR cells were intravenously injected into NOG mice. After engraftment, five million redirected T cells, control T cells, or T cells with BiTEs were intravenously injected into each mouse. Tumor sizes were measured by AEQUORIA-2D/8600 bioluminescence imaging assays (Hamamatsu Photonics).All the murine experiments in this study were approved by the Ehime University Animal Care Committee. All methods were performed in accordance with the relevant guidelines and regulations. Five-week-old NOD/Shi-scid,IL-2RγΚΟ Jic (NOG) mice were purchased from http://prosite.expasy.org/scanprosite/) was utilized to search for human-derived peptide sequences homologous to NY-ESO-1157 peptide (SLLMWITQC) based on the results of alanine scanning assays. GeneCards (https://www.genecards.org/) was used to search for the expression of proteins containing peptides homologous to NY-ESO-1157. The binding affinity of peptides for HLA-A*02:01 was predicted by netMHC4.0 (http://www.cbs.dtu.dk/services/NetMHC/).The ScanProsite tool  or paired t test (two-sided) was employed to examine whether two groups were significantly different for a given variable. Comparisons between more than two groups were performed by two-way ANOVA with Sidak’s multiple comparisons test. P values of <0.05 were considered statistically significant.Supplementary Information


Caenorhabditis elegans (C. elegans) heterochronic pathway, which regulates developmental timing, is thought to be an ancestral form of the circadian clock in other organisms. An essential member of this clock is the Period protein whose homolog, lin-42, in C. elegans is an important heterochronic gene. LIN-42 functions as a transcriptional repressor of multiple genes including the conserved lin-4 and let-7 microRNAs. Like other Period proteins, levels of LIN-42 oscillate throughout development. In other organisms this cycling is controlled in part by phosphorylation. KIN-20 is the C. elegans homolog of the Drosophila Period protein kinase Doubletime. Worms containing a large deletion in kin-20 have a significantly smaller brood size and develop slower than wild type C. elegans. Here we analyze the effect of kin-20 on lin-42 phenotypes and microRNA expression. We find that kin-20 RNAi enhances loss-of-function lin-42 mutant phenotypes and that kin-20 mutant worms express lower levels of LIN-42. We also show that kin-20 is important for post-transcriptional regulation of mature let-7 and lin-4 microRNA expression. In addition, the increased level of let-7 found in lin-42(n1089) mutant worms is not maintained after kin-20 RNAi treatment. Instead, let-7 is further repressed when levels of kin-20 and lin-42 are both decreased. Altogether these results suggest that though kin-20 regulates lin-42 and let-7 microRNA, it mainly affects let-7 microRNA expression independently of lin-42. These findings further our understanding of the mechanisms by which these conserved circadian rhythmic genes interact to ultimately regulate rhythmic processes, developmental timing and microRNA biogenesis in C. elegans.The  Many organisms exhibit circadian rhythmic behaviors that cycle in accordance with the Earth’s rotation and thus exhibit periodicities of ∼24 hr. These circadian rhythms can be entrained by environmental stimuli and persist in the absence of such cues, are unchanged by variations in temperature, and can be reset by external stimuli. Other fundamental aspects of life are governed by ultradian or infradian rhythms with periods that are either less or greater than the canonical 24 hr circadian rhythm, respectively. Central to these rhythms is the circadian clock, a group of highly conserved genes that function in oscillating feedback loops that are controlled by both transcriptional and post-transcriptional mechanisms.C. elegans, locomotion, resistance to osmotic stress, melatonin biosynthesis, and multiple metabolic variables can be entrained by light cues with a daily periodicity . Specific numbers of seam cells are present during each larval stage, and after the L4-to-adult molt, seam cells exit the cell cycle, fuse and generate a ridged cuticle structure called alae . Thus, heterochronic mutants often display altered seam cell numbers and/or precocious or retarded alae formation .Like the core circadian clock, the heterochronic pathway consists of many genes that successively regulate one another through feedback mechanisms to ultimately promote dulthood . Proper lin-42 is the C. elegans homolog of the Period protein  in mammals  . miRNAs  elegans .let-7 and lin-4 ultimately function as ∼22 nucleotide (nt) RNAs, however they are initially transcribed from the genome by RNA polymerase II into long, primary miRNAs (pri-miRNAs) that are subsequently capped and polyadenylated , to analyze the effect of kin-20 on organismal development, lin-42 phenotypes and expression, and let-7 and lin-4 miRNA biogenesis. We found that kin-20 mutants have significantly fewer progeny and a slower growth than WT N2 worms, though they do not exhibit classical developmental timing defects in alae production. Because kin-20(ok505);lin-42(n1089) worms were lethal, we analyzed the impact of kin-20 RNAi on lin-42(n1089) mutant worms. We found that under-expression of kin-20 enhanced lin-42(n1089) mutant phenotypes including precocious alae formation. Consistent with these phenotypic results, we found that LIN-42A levels were decreased in kin-20(ok505) mutant worms. Like LIN-42, KIN-20 is important for both let-7 and lin-4 miRNA expression. Though it is possible the decrease in let-7 levels in kin-20 mutant worms is dependent on LIN-42A, our results more strongly suggest that KIN-20 impacts expression of the let-7 miRNA independently of LIN-42, since KIN-20 had no effect on a third constitutively-expressed, non-heterochronic miRNA target miR-58.1, and KIN-20 did not impact primary let-7 transcription. In addition, growth of lin-42(n1089) mutant worms on kin-20 RNAi caused a further reduction in let-7 levels. Altogether these results suggest that although KIN-20 regulates both LIN-42 and some miRNAs in C. elegans, KIN-20 mainly does so in a manner unpredicted by the inhibitory function of Doubletime (the KIN-20 homolog) on Period (the LIN-42 homolog) in Drosophila.Here we utilized a large deletion of C. elegans strains were used: wild type (WT) N2 Bristol, kin-20(ok505) (VC398), lin-42(ok2385) (RB1843), and lin-42(n1089) (MT2257). The integrated transgene wls79 contains ajm-1::gfp/MH27:: GFP and scm-1::GFP to allow visualization of seam cells. We crossed animals containing wls79 to VC398 to generate wls79;kin-20(ok505) worms. The integrated strain PQ462 contains 1568 bp of let-7 promoter sequence driving nuclear-localized GFP expression (plet-7B::GFP) .The following E. coliOP50-seeded plates at 25° and collected at the appropriate time point. Larval stages correspond to the timing of development for WT N2 worms based on previously published time course analyses of worm development and molting at 25° (lin-42 mutants develop somewhat asynchronously (Worms were maintained at 15° or 20° and synchronized by standard hypochlorite treatment. Starvation-arrested L1 worms were plated on N2 or kin-20(ok505) worms grown at 15° or 25°. Parental worms were passaged to new plates over the course of the experiment to enable detection of all progeny. Death and dumpy phenotypes were analyzed in at least 500 adult animals grown on RNAi for two-generations as described above. The presence or absence of adult alae on the cuticle was measured in at least 20 synchronized animals after growth at 25° until the L4 or young adult stage. Alae was classified as complete if it extended continuously over all seam cells in three perfectly parallel ridges. Animals that had alae that was not complete and/or did not contain three perfectly parallel ridges were classified as having abnormal alae. Seam cell nuclei were counted in at least 19 synchronized animals grown at 25° until the L4 or young adult stage. Fluorescent microscopy analysis was performed on at least 25 synchronized animals after growth at 25° until the L3 or L4 stage. Fluorescent micrographs were captured under equivalent exposure times.Brood counts were analyzed of synchronized, singled WT t-tests or chi-square tests.Statistical differences of sample phenotypes were analyzed as appropriate by Student’s t-tests or two-way ANOVAs.Total RNA was extracted from synchronized, staged worm populations using TRIzol reagent (Life Technologies), and cDNA synthesis was completed with TaqMan microRNA assays (Thermo Fisher Scientific) or as previously described with random oligos or oligo dT  or a mouse monoclonal antibody against tubulin (Sigma-Aldrich)  of https://doi.org/10.25387/g3.6225893.Reagents will be made available upon request. The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article. Supplemental material available at Figshare: KIN-20 is a homolog of the Period protein kinase Doubletime in Drosophila and casein kinase I ε/δ in mammals  that each have different 5′ and 3′ UTRs of varying lengths and splice patterns. However, the internal sequence (exons 2-4) of all isoforms is shared .-20 gene . Accordis shared . In its s shared . In addis shared . By usinot cycle . By anal L2 molt . This exkin-20 mRNA levels throughout development and between isoforms, as well as the protein expression patterns of KIN-20 are currently unclear. Despite the uncertainty surrounding these finer points, our experiments reveal that overall kin-20 is extremely important, as the fertility of C. elegans significantly drops ∼2 fold in kin-20(ok505) mutant worms relative to WT N2 worms when grown at either 15° or 25° (kin-20(ok505) mutant worms do not show any defects in the timing of alae formation , but do exhibit precocious seam cell exit from the cell cycle during the late L4 stage . In addition, the growth of C. elegans is dramatically slowed in kin-20(ok505) worms relative to WT N2 as measured by animal size and gonad development at 25°  allele and the kin-20(ok505) allele. However, progeny of self fertilized C. elegans that were homozygous for the lin-42(n1089) mutation and heterozygous for the kin-20(ok505) mutation  mutation and heterozygous for the lin-42(n1089) mutation) yielded no progeny that were homozygous for both mutations (Table S2). Growth of lin-42(n1089) worms in the presence of kin-20 RNAi caused a slight, but non-significant increase in the proportion of worms that died by the young adult stage as compared to vector control RNAi  mutant worms display this dumpy phenotype when subjected to kin-20 RNAi as compared to vector control RNAi  worms exhibited abnormal alae formation, as defined by alae that was either incomplete  or was not in three perfectly parallel ridges. At the young adult stage, significantly more animals exhibit complete alae in lin-42(n1089) worms grown on kin-20 RNAi compared to vector control RNAi (lin-42(n1089) worms have complete alae, significantly more lin-42(n1089) worms exhibit precocious alae formation  when grown on kin-20 RNAi compared to vector control RNAi  mutant worms after the L4 molt when grown at 25° , the L3 molt (L3/L4) and the fourth larval stage (L4) at 25° according to size and gonad development (kin-20(ok505) and WT N2 worms  mutant worms relative to WT N2 worms  relative to WT N2 worms  relative to WT N2 worms at L3 and L4 stages  relative to WT N2 worms (P = 0.076), though there is no effect on lin-14 mRNA at a later stage  relative to WT N2 worms at the L1 stage (lin-4 in inhibiting lin-28 mRNA levels (lin-4 levels are significantly reduced in kin-20(ok505) worms  worms , it is sL3 stage . This nolet-7 could be due to a decrease in transcription of the let-7 gene, decreased processing of primary or precursor let-7 RNAs, or decreased stability of mature let-7. To distinguish among these possibilities, we analyzed the levels of these RNAs involved in let-7 biogenesis in the same samples for which mature let-7 levels had also been analyzed. Transcription initiation at two distinct sites yields two primary let-7 transcripts that are both subsequently spliced to yield a third primary let-7 transcript that is ultimately processed into precursor let-7  mutant worms relative to WT (let-7 varied slightly at each stage tested in kin-20(ok505) mutant worms relative to WT, but overall suggested that pre-let-7 levels were either minimally altered or that any impacts on pre-let-7 were negated by its rapid processing into mature let-7 , let-7 promoter driven expression of GFP was stronger at the L4 stage compared to the L3 stage  mutant worms relative to WT N2 worms at both the L3 and L4 stages  worms grown in the presence of vector control or kin-20 RNAi (kin-20 did not cause the growth delay seen in kin-20(ok505) mutant worms . As expected, let-7 levels of worms grown on vector control RNAi were ∼2.5 fold increased in lin-42(n1089) mutant worms relative to WT N2 worms (let-7 were only slightly decreased in WT N2 worms treated with kin-20 RNAi (let-7 levels in a kin-20 mutant (kin-20 levels from kin-20 RNAi treatment (kin-20 levels by RNAi significantly reduced let-7 levels by 10 fold in lin-42(n1089) mutant worms  mutant worms treated with kin-20 RNAi compared to WT N2 worms treated with kin-20 RNAi . Consistent with previously reported results (kin-20(ok505) mutant worms precociously exit the cell cycle at late L4, represented by a decrease in the number of hypodermal seam cell nuclei at this stage . Additionally, kin-20(ok505) mutant worms exhibit wild type timing of alae formation   mutant worms (kin-20(ok505) mutants exhibit altered expression of multiple heterochronic genes including lin-42, let-7 and lin-4  mutant worms (kin-20(ok505), we might expect kin-20(ok505) worms to phenocopy lin-42(ok2385) worms, which delete LIN-42A and contain a large deletion in the C-terminal region of LIN-42B (lin-42(ok2385), kin-20(ok505) worms have a significantly reduced brood size, growth delays, and precocious alae (lin-42(n1089) mutant worms, which contain a large deletion at the N terminus of lin-42, when grown in the absence of kin-20 might be expected to phenocopy lin-42(ox461) worms, which lack all lin-42 isoforms (lin-42(n1089);kin-20(ok505) due to their lethality (Table S2). However, we did analyze the phenotypes of lin-42(n1089) worms subjected to kin-20 RNAi. Despite kin-20 RNAi only decreasing kin-20 levels ∼2-3 fold (lin-42 mutant phenotypes were enhanced after kin-20 RNAi treatment (lin-42(ox461) worms exhibited enhanced larval arrest and growth delays (lin-42(n1089) worms subjected to kin-20 RNAi did not show any significant growth delays . Though the proportion of worms exhibiting some type of precocious alae  did not change in lin-42(ox461) worms, more lin-42(ox461) worms exhibited complete alae  worms subjected to kin-20 RNAi  mutant worms, it is most likely that if KIN-20 regulates the other LIN-42 isoforms it does so in a similar manner to its effects on LIN-42A. Based on its homology with Doubletime, it is also most likely that KIN-20 mediates these effects via phosphorylation.There are three l timing   bursting phenotype  bursting phenotype by causing an ∼2.5 fold increase in let-7 levels  mutant worms showed ∼2 fold decreased levels of let-7 relative to WT (let-7 target lin-41 (let-7 levels would be expected to increase let-7(n2853) lethality, not reduce it as we found  mutant worms  mutant worms  mutant worms (let-7 promoter was not affected since GFP mRNA levels were unchanged in kin-20(ok505) mutant worms  specifically eliminates LIN-42B and C isoforms, reducing kin-20 levels by RNAi in lin-42(n1089) worms will reduce LIN-42A levels and thus should enhance lin-42 knock-out phenotypes. Accordingly, we find that lin-42(n1089) worms subjected to kin-20 RNAi do indeed exhibit enhanced lin-42 mutant phenotypes  mutant worms treated with kin-20 RNAi. If KIN-20 acted solely via LIN-42, we would expect let-7 levels to be the same in lin-42(n1089) worms treated with vector control or kin-20 RNAi. Instead we find that let-7 levels are significantly reduced in lin-42(n1089) worms treated with kin-20 RNAi compared to either lin-42(n1089) worms treated with vector control RNAi or WT N2 worms treated with kin-20 RNAi (lin-42 and kin-20 levels acts to enhance the reduction in let-7 levels. In summary, though these results do not exclude the possibility that the decrease in let-7 levels in kin-20 mutant worms is dependent on LIN-42A, these results strongly support another mechanism, that is independent of LIN-42A, by which kin-20 regulates let-7 levels.Our results suggest that f kin-20 . Becauseenotypes . Since L (kin-20 , we wouln LIN-42 . Second,miR-58.1 , which itionally . To furt-20 RNAi . Thus thKIN-20 as a new, important regulator of LIN-42 and the conserved lin-4 and let-7 miRNAs. These results also highlight several important differences between KIN-20 and its homologs Doubletime and CKIε/δ, and increase our understanding of how rhythmic and developmental processes are regulated in C. elegans.Altogether these results identify


Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with multiple human malignancies. EBV drives B-cell proliferation, which contributes to the pathogenesis of multiple lymphomas. Yet, knowledge of how EBV subverts host biosynthetic pathways to transform resting lymphocytes into activated lymphoblasts remains incomplete. Using a temporal proteomic dataset of EBV primary human B-cell infection, we identified that cholesterol and fatty acid biosynthetic pathways were amongst the most highly EBV induced. Epstein-Barr nuclear antigen 2 (EBNA2), sterol response element binding protein (SREBP) and MYC each had important roles in cholesterol and fatty acid pathway induction. Unexpectedly, HMG-CoA reductase inhibitor chemical epistasis experiments revealed that mevalonate pathway production of geranylgeranyl pyrophosphate (GGPP), rather than cholesterol, was necessary for EBV-driven B-cell outgrowth, perhaps because EBV upregulated the low-density lipoprotein receptor in newly infected cells for cholesterol uptake. Chemical and CRISPR genetic analyses highlighted downstream GGPP roles in EBV-infected cell small G protein Rab activation. Rab13 was highly EBV-induced in an EBNA3-dependent manner and served as a chaperone critical for latent membrane protein (LMP) 1 and 2A trafficking and target gene activation in newly infected and in lymphoblastoid B-cells. Collectively, these studies identify highlight multiple potential therapeutic targets for prevention of EBV-transformed B-cell growth and survival. EBV, the first human tumor virus identified, persistently infects >95% of adults worldwide. Upon infection of small, resting B-lymphocytes, EBV establishes a state of viral latency, where viral oncoproteins and non-coding RNAs activate host pathways to promote rapid B-cell proliferation. EBV’s growth-transforming properties are closely linked to the pathogenesis of multiple immunoblastic lymphomas, particularly in immunosuppressed hosts. While EBV oncogenes important for B-cell transformation have been identified, knowledge remains incomplete of how these EBV factors remodel cellular metabolism, a hallmark of human cancers. Using a recently established proteomic map of EBV-mediated B-cell growth transformation, we found that EBV induces biosynthetic pathways that convert acetyl-coenzyme A (acetyl-CoA) into isoprenoids, steroids, terpenoids, cholesterol, and long-chain fatty acids. Viral nuclear antigens cooperated with EBV-activated host transcription factors to upregulate rate-limiting enzymes of these biosynthetic pathways. The isoprenoid geranylgeranyl pyrophosphate was identified as a key product of the EBV-induced mevalonate pathway. Our studies highlighted GGPP roles in Rab protein activation, and Rab13 was identified as a highly EBV-upregulated GTPase critical for LMP1 and LMP2A trafficking and signaling. These studies identify multiple EBV-induced metabolic enzymes important for B-cell transformation, including potential therapeutic targets. The gamma-herpes virus Epstein-Barr virus (EBV) causes infectious mononucleosis (IM) and is associated with multiple B-cell and epithelial malignancies . EBV is In primary infection, EBV translocates across the tonsillar epithelial barrier to reach the B-cell compartment, which is the reservoir for lifelong infection. Whereas epithelial cell infection typically results in production of infectious virions by the viral lytic cycle, EBV establishes latency in newly infected B-cells. Early in the course of IM, considerable numbers of latently-infected B-cells can be detected in peripheral blood , althougEBV uses a series of latency programs to expand the infected cell reservoir and to reach the memory B-cell compartment, the site of long-term latency , 12. In EBV B-cell growth transformation takes place over at least three phases . Over thOver days 3 to 7 post-infection, viral oncogenes, together with their host targets, drive rapid BL-like cell proliferation, with mitosis occurring as frequently as every 8–12 hours . Over thRemodeling and rapid cell growth place huge demands on lipid and fatty acid biosynthesis. We therefore used a recently constructed proteomic map to gain insights into EBV-driven metabolic pathway remodeling, where multiplex tandem mass tag mass spectrometry was performed on whole cell and plasma membrane fractions of primary human B-cells at rest and at nine timepoints following B95-8 EBV strain infection . To incrHere, we identified that EBV highly induces the mevalonate pathway, which uses glucose-derived acetyl-CoA and NADPH to produce sterols, isoprenoids, cholesterol and fatty acid synthesis in newly infected B-cells. We then investigated how EBV and host transcription factors activate these key anabolic pathways and identified unexpected downstream roles in EBV-driven B-cell proliferation and survival.EBV infection causes dramatic B-cell remodeling that begins early after infection and converts resting B-cells into activated lymphoblasts. Underscoring the need for anabolic pathway upregulation, EBV infection significantly increased cell size over the first week post-infection. Flow cytometry (FACS) analysis identified that newly EBV-infected primary B-cells reach a maximum size at 4 days post-infection (DPI) Figs  and S1, EBV infection induced the eight enzymes that convert mevalonate into cholesterol . EBV alsFurther underscoring key host lipid metabolism roles in early EBV-driven B-cell growth transformation, enrichment analysis also highlighted fatty acid metabolism as highly EBV-induced . EBV uprSREBF2 promoter  are basic-helix-loop-helix leucine zipper transcription factors that bind to DNA sterol response elements to induce expression of mevalonate and fatty acid pathway components –44. Sincpromoter  48, 49], 49SREBFBL cells   retains SREBPs at the endoplasmic reticulum (ER) by binding to the ER retention factor insulin-induced gene 1 (INSIG1) , 51. UpoACLY and SCD in newly-infected primary human B-cells, to a similar extent as reported in other cell types, where the 5 μM dose was also used [ACLY encodes ATP citrate lyase, the main enzyme responsible for cytosolic acetyl-CoA synthesis, whereas SCD encodes the fatty acid biosynthesis enzyme stearoyl-CoA desaturase. 5 μM fatostatin treatment, a dose that was very close to the empirically established half maximal effective concentration (EC50), significantly impaired EBV-mediated primary B-cell outgrowth , in which EBNA2 is fused to a mutant estrogen receptor ligand-binding domain under the control of 4-hydroxytamoxifen (4HT). In the absence of 4HT, EBNA2-HT re-localizes to the cytoplasm and is destabilized [To next investigate EBNA2 roles in sustained cholesterol and lipid biogenesis in transformed cells, we used LCLs with conditional abilized . ConditiHMGCR promoter  complexes transfer GGPP to small GTP-binding protein targets . We askeTo test whether either GGT activity is critical for EBV-driven B-cell outgrowth, we performed chemical epistasis experiments with highly selective chemical antagonists. The GGT-II inhibitor BMS-214662 is 1000-fold selective for GGT-II over GGT-I, whereas the GGT-I inhibitor GGTI-2133 is reported to be 140-fold selective over GGT-II , 68, 69.At these biochemically determined EC50 concentrations, the GGT-II inhibitor but not the GGT-I inhibitor significantly impaired EBV-mediated primary B-cell outgrowth . SimilarRAB13 were most strongly selected against in our 21 day LCL growth and survival screen [RAB13 induction by EBV, since Rab13 abundance was correlated with that of EBNA3C in newly infected B-cells  important for EBV-mediated B-cell outgrowth would be upregulated upon primary B-cell infection. Of the 37 Rab proteins detected in our recent temporal proteomic analysis of EBV-mediated B-cell remodeling, 24 were upregulated at ≥ 1 time point . Of thesl screen . We hypo B-cells  and LCL  protein  Fig 5D)RAB13 wertivation . FollowiRAB13 targeting sgRNAs were strongly selected against in our 21-day CRISPR Achilles heel screen [Immunofluorescence microscopy confirmed that Rab13 was upregulated by four days post EBV infection of primary B-cells . At thisl screen   abrogated Cas9-mediated DNA editing, or control GFP cDNA were stably expressed in Cas9+ GM12878 LCLs. Effects of control versus sgRNA targeting endogenous RAB13 were next tested in a 7-day outgrowth assay. Whereas RAB13 sgRNA depleted endogenous Rab13 and strongly suppressed outgrowth of LCLs with control GFP cDNA expression, RAB13 sgRNAs did not impair outgrowth of LCLs with rescue RAB13 cDNA expression, confirming on-target CRISPR effects (We next tested whether Rab13 exerted a similar role in fully transformed LCLs. A Rab13-targeting single guide RNA (sgRNA) was used to functionally knockout Rab13 expression in Cas9+ GM12878 LCLs. Rab13 depletion markedly reduced LCL live cell numbers in a growth assay . To vali effects .RAB13 targeting by independent sgRNAs on LCL LMP1 and 2A subcellular localization. Interestingly, Rab13 depletion by either sgRNA substantially altered LMP1 and LMP2A distribution: whereas LMP1 and LMP2A formed co-localized foci in LCLs with control sgRNA expression, as is typically observed in LCLs, they instead exhibited a diffuse pattern in LCLs with independent RAB13 sgRNAs , strongly impaired LCL growth and survival , presumaDiverse DNA viruses subvert host lipid biosynthesis pathways to support viral replication, including Kaposi’s sarcoma-associated herpesvirus (KSHV) , 80, humThe studies presented here highlight EBV-induced mevalonate, GGPP, fatty acid biosynthetic and downstream Rab13 pathways as being critical for newly infected B-cell outgrowth . EBNA2 aHMGCR and ACACA promoters. Instead, EBNA2, MYC and SREBPs highly co-occupy these key promoters. LMP2A may nonetheless have critical roles in maintaining cholesterogenic and lipogenic programs at later timepoints by stimulating PI3K/AKT/mTOR flux [LMP1 has been reported to activate lipogenesis via mTOR and SREBP1 in nasopharyngeal carcinoma (NPC) cells, which typically express the latency II program . HoweverTOR flux .We initially suspected that EBV upregulates mevalonate synthesis to supply cholesterol for lipid raft and membrane synthesis in support of B-cell lymphoblast transformation. However, using a chemical epistasis approach , we unexWhile to our knowledge GGPP has not previously been studied in EBV-infected cells, key mevalonate pathway and GGPP roles have been identified in other B-cell contexts. Importantly, CD40-mediated B cell activation upregulates the mevalonate pathway in primary B-cells, where GGPP supports antigen-presenting cell function and T-cell activation by mediating CD80, CD86 and HLA-DR upregulation . HoweverOur results highlight the interplay between Rab13, LMP1 and LMP2A in B-cell transformation and in transformed cell growth and survival. Rab13 has pleiotropic roles, including the regulation of glucose transporter 4 trafficking , macrophGiven that LMP1 and LMP2A can signal from intracellular membranes , 115, itRAB13 mRNA expression [The crucial EBNA3C role in support of LCL Rab13 expression raises the interesting question of how LMP1 and LMP2A traffic and signal in cells with latency II expression where EBNA3C is not expressed, such as in germinal center B-cells, Hodgkin Reed Sternberg (RS) or nasopharyngeal carcinoma tumor cells , 77, 120pression . Since gpression .in vitro can likewise be achieved in humans in vivo [These studies identify multiple EBV-induced metabolic pathway enzymes that may serve as attractive therapeutic targets to prevent or halt EBV-driven lymphoproliferative disease, which could perhaps be targeted by available small-molecule inhibitors as prophylaxis during periods of profound immunosuppression. These could potentially include key druggable enzymes in the mevalonate, geranylgeranylation or fatty acid biosynthetic pathways. Of particular interest are simvastatin and atorvastatin, since the concentrations of these widely used statins at which we observed inhibition of EBV-mediated B-cell outgrowth  in vivo . Simvast in vivo  further  in vivo .MYC allele. In the absence of tetracyclines, high level exogenous MYC is induced. P493-6 cells were maintained continuously in the absence of 4HT or doxycycline to induce a BL-like state of high MYC expression. To grow in the lymphoblastoid cell state (intermediate MYC), P493-6 cells were grown in the presence of both 1 μM 4HT and doxycycline. To induce a low MYC state and consequently G1 growth arrest, cells were grown in the absence of 4HT and presence of 1 μM doxycycline. After 48 hours of growth in any of these conditions, cells were collected for lysate preparation. For selection, 200 μg/mL hygromycin  or 3 μg/ml puromycin (Invitrogen) was used. B95-8 cells and P3HR-1 cells with conditional ZTA-HT alleles responsive to 4HT were kind gifts from Eric Johannsen and Elliott Kieff. All B-cells were cultured in RPMI-1640 (Invitrogen) supplemented with 10% standard FBS and penicillin-streptomycin in a humidified incubator at 37ᵒC and at 5% CO2. All cells were routinely confirmed to be mycoplasma-negative by Lonza MycoAlert assay (Lonza).293T were purchased from American Type Culture Collection (ATCC) and cultured in DMEM with 10% fetal calf serum . GM12878 lymphoblastoid cells were obtained from Coriell. GM12878 Cas9+ cell lines were previously described . The 2-2Discarded, de-identified leukocyte fractions left over from platelet donations collected from volunteer donors following institutional guidelines were obtained from the Brigham and Women’s Hospital Blood Bank. Our studies on primary human blood cells were approved by the Brigham & Women’s Hospital Institutional Review Board. All samples were anonymized.2.Platelet-depleted venous blood obtained from the Dana-Farber Cancer Institute blood bank were used for primary human B cell isolation, following our Institutional Review Board-approved protocol for discarded and de-identified samples. RosetteSep and EasySep negative isolation kits (STEMCELL Technologies) were used sequentially to isolate CD19+ B-cells with the following modifications made to the manufacturer’s protocols. For RosetteSep, 40 μL of antibody cocktail was added per mL of blood and then layered onto Lymphoprep density medium for centrifugation. For EasySep, 10 μL of antibody cocktail was added per mL of B cells, followed by 15 μL of magnetic bead suspension per mL of B cells. After negative selection, the cells obtained were routinely ≥95% positive for CD19, a nearly pan-B cell surface marker (though not strongly expressed on plasma cells). For most experiments, cells were cultured in RPMI-1640 (Invitrogen) supplemented with 10% standard FBS and penicillin-streptomycin. Cells were cultured in a humidified incubator at 37ᵒC and at 5% COBALF5 on total DNA extracted, and cross-compared with levels from B95-8 virus preparations. The plasmid pHAGE-BALF5 was used for generation of standard curves of EBV genome copy number. Calculated genome copy numbers were used to normalize B95-8 and P3HR-1 input DNA amounts. UV irradiation of B95-8 virus supernatants was performed at a cumulative intensity of 3J per square centimeter on ice, to prevent heat-induced virus degradation. EBV was added to purified B-cells at an MOI of 0.1 . We also ensured equal infection by performing quantitative PCR on intracellular DNA extracted from cells and by anti-EBNA1 (OT1x) immunofluorescence microscopy at 24 hours post-infection following at least five washes. Cells were cultured in a humidified chamber at 37˚C in RPMI/10% FCS. For most experiments, virus-containing media was replaced with virus-free media for subsequent experimentation two days post-infection.EBV B95-8 virus was produced from B95-8 cells with conditional ZTA expression and purified by filtration through a 0.45 μm filter , 126. EBAntibodies against the following proteins were used in this study: ACC1 , HMGCR , SCAP , DDX1 , DDX46 , phospho-PI 3-kinase p85α (Tyr 508) , phospho-Syk (Tyr525/526) , RPA1A/B , PGGT1B , MYC , EBNA2 , EBNA3C , RABGGTA , RABGGTB , Rab13 , phospho-mTOR (Ser2448) , TRAF1 , GAPDH , α-tubulin , horse anti-mouse IgG HRP-linked antibody , goat anti-rabbit IgG HRP-linked antibody , goat anti-rat IgG HRP-linked antibody , Alexa Fluor 488 goat anti-mouse , Alexa Fluor 594 goat anti-rabbit , Alexa Fluor 647 goat anti-rat , LMP1 , LMP2A .The following chemicals were obtained from Sigma-Aldrich: fatostatin hydrobromide (Cat#F8932), simvastatin (Cat#S6196), atorvastatin calcium salt trihydrate (Cat#PZ0001), geranylgeranyl pyrophosphate (GGPP) (Cat#G6025), squalene (Cat#S3626), Mevalonate acid lithium salt (Cat #50838), GGTI-2133 (Cat#G5294), BMS-214662 hydrochloride (Cat#BM0008), doxycycline hyclate (Cat#D9891), (Z)-4-hydroxytamoxifen (Cat#H7904). Hoechst 33258 (Cat#H21491) and propidium iodide (Cat#P3566) were obtained from ThermoFisher Scientific.Cells were lysed in a modified TNE buffer  supplemented with protease inhibitor cocktail , 2 mM sodium pyrophosphate, 10 mM β-glycerophosphate and 1 mM PMSF on ice for 15 minutes. The crude cell lysate was then sonicated on ice for 5 minutes using a probe sonicator at the maximum setting and centrifuged at 17,000 x g for 15 minutes. The supernatant was mixed with 5X Laemmli loading buffer and boiled for 10 minutes. Samples were loaded onto and electrophoretically resolved on 12% or 4–20% gradient gels and transferred onto nitrocellulose membranes for 45 minutes at 100 V at 4˚C. Membranes were probed with primary antibodies, typically at a 1:1000 dilution overnight at 4˚C and with HRP-conjugated secondary antibodies in 5% skim milk/PBST for 1 hour. Blots were imaged on Carestream or Li-Cor workstations.Cells were washed once with cold PBS supplemented with 0.5% bovine serum albumin (BSA). Cells were then incubated with a 1:100 dilution of fluorophore-conjugated primary antibody  in PBS with 0.5% BSA for 1 hour. Cells were pelleted and resuspended in 400 μL of PBS, strained into flow cytometry-compatible tubes and processed immediately. Sample processing was performed either immediately or within 24 hours after staining. Flow cytometric data was acquired with a BD FACSCalibur instrument and analysis was performed with FlowJo. For propidium iodide cell cycle analysis, samples were collected over indicated time points and fixed in 70% ethanol overnight. For cell cycle analysis, fixed cells were treated with Staining Buffer  for 30 minutes at room temperature and analyzed by FACS. The FACS data were further analyzed with FlowJo V10.Cells were seeded on glass slide and fixed with 4% PFA solution for 10 minutes. Fixed cells were permeabilized with 0.5% Triton X-100/PBS solution and blocked with 20% newborn goat serum (NGS). Subsequently, cells were incubated with a cocktail of primary antibodies against LMPs and Rab13 for an hour and then a cocktail of secondary antibodies for an additional half hour. Finally, cells were incubated with a solution of Hoechst 33258 (10 μg/mL) for 10 minutes to stain nuclear DNA and dehydrated sequentially from 70% to 90% to 100% ethanol. ProLong anti-fade was applied to the slide and sealed with a No. 1.5 coverslip. Image acquisition was performed at the Brigham and Women’s Hospital core facility with the Zeiss LSM 800 instrument. Image analysis was performed with the Zeiss ZEN Lite (Blue) software. For measurements of cell diameter, differential interference contrast (DIC) microscopy was performed and cells were picked out by eye from the micrographs. Circular ROIs were drawn around the cells and diameters were automatically computed by the ZEN Lite (Blue) software.Newly infected primary human B-cells were seeded at 500,000 per well in a volume of 1 mL and grown for two days before measurements of cell densities were made. For GM12878 lymphoblastoid cells, 100,000 cells were seeded per well in a volume of 1mL and grown for two days prior to measurements of cell densities. For cell density measurements, cells were pelleted and resuspended in the same volume of media and counted with the TC20 automatic cell counter (Bio-Rad). After measurements had been taken, cells were passaged accordingly to give 100,000 cells per mL and grown for two more days. The same procedure was repeated to obtain measurements at subsequent timepoints. At each time point, after passaging, cells were treated with the appropriate inhibitor and/or rescue metabolite(s) at the indicated concentrations. Growth was then calculated using the initial seeding density for normalization.RAB13 (RAB13R) vector, we made use of the Genscript GenParts synthesis service to synthesize a cDNA fragment containing wild-type RAB13 with cytosine 141 mutated to thymidine (c141t) and guanosine 162 mutated to thymidine (g162t), as well as flanking attB1 and attB2 sites. Engineering the c141t and g162t mutations at the PAM sites confers resistance to Cas9-mediated cutting by sgRNA #1 and sgRNA #2, respectively. We also introduced a stop codon at the end of the coding sequence. We performed BP cloning of the gene fragment into a donor vector, pDONR223. The resultant entry vector, pDONR223-RAB13R, was used for LR cloning with pLX_TRC313, a destination vector which carries a hygromycin resistance marker. Because pLX_TRC313 has a C-terminal V5 epitope tag sequence which could interfere with Rab13 function, the engineered cDNA fragment used for BP cloning contained a stop codon at the end of the coding sequence but upstream of the 3’ attB sequence. The expression vector for RAB13 C200M point mutation expression performed as follows. A Gateway entry clone encoding sgRNA-resistant RAB13 with C200 residue mutated to methionine was generated using GenScript’s GenPart synthesis service. Specifically, the codon encoding cysteine at position 200  was mutated to encode methionine (ATG) at the same position. The C200M mutation abolishes the capacity for Rab13 geranylgeranylation at this site as the free thiol group required to react with geranylgeranyl pyrophosphate is absent on methionine. Although a number of earlier studies [C200M stable expression construct with a puromycinc resistance marker using vector MSCV-N-FLAG-HA-IRES-PURO (Addgene plasmid # 41033).Single guide RNA (sgRNA) constructs were generated as previously described  using sg studies , 129 madReverse transcription-quantitative PCR analysis of mRNA abundance was performed on a BioRad CFX Connect Real-time PCR detection system, using Power SYBR Green RNA-to-CT 1-Step Kit (Applied Biosystems) for 40 cycles. Expression values relative to 18S rRNA or GAPDH expression were calculated using CFX Manager Software. Quantitative PCR of viral genome copies utilized host cell GAPDH gene copy number as control. Primer sequences can be found in Full details of how multiplexed tandem mass tag-based proteomic data was obtained were recently reported . Brieflyhttp://www.proteomexchange.org/) via the PRIDE partner repository and can be retrieved with the dataset identifier PXD013034.Pathway Analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID)  version https://github.com/broadinstitute/picard) and uploaded onto a custom session of the WashU Genome Browser as previously described [ChIP-seq raw data was obtained from published LCL datasets for EBNA2   FACS forward scatter cell size measurements of live CD19+ primary human B-cells at the indicated days post-infection (DPI) by B95-8 EBV at a MOI of 0.1. Shown is a representative experiment of n = 3 replicates. Of note, cells were not gated for CD23 as a marker of EBV infection. (B) Propidium iodide (PI) cell cycle and forward scatter cell size FACS analysis are shown for the indicated DPI timepoints from the experiment shown in panel S1A. Data were representative of n = 3 experiments. (C) Propidium iodide (PI) cell cycle analysis for the indicated DPI timepoints from the experiment shown in panels S1A-B. Data were representative of n = 3 experiments. (D) Mean +SEM values of the numbers of cell identified as G1 or as in either G2 or S phases (G2+S) of the cell cycle by the PI analysis at the indicated DPI timepoints from n = 3 replicates, including the experiment presented in panels S1A-C. *, p<0.05; **, p<0.01. (two-tailed t-test).(TIF)Click here for additional data file.S2 Figde novo lipid synthesis pathway conversion of glucose-derived acetyl-CoA into end products. NADPH-dependent acetyl-CoA reduction produces palmitate, which can be directed to one of three routes: (1) oxidation via the fatty acid β-oxidation pathway to produce reducing power in the form of NADH and ultimately, ATP via oxidative phosphorylation; (2) used for post-translational palmitoylation of target protein cysteine residues; (3) condensed with other molecules to produce triglycerides for energy storage and/or phospholipids for membrane biogenesis. Enzymes are indicated in blue. (D) Temporal traces of the DEAD box DNA helicases DDX1 and DDX46 relative protein abundances at the indicated DPI of primary human B-cell EBV infection. Data show the mean + SEM of n = 3 biological replicates.(A) Temporal traces of whole cell LDL receptor (LDLR) relative protein abundances at the indicated DPI of primary human B-cell EBV infection. Data show the mean + SEM of n = 3 biological replicates. (B) Temporal traces of plasma membrane (PM) LDLR relative protein abundances at the indicated DPI of primary human B-cell EBV infection. (C) Schematic diagram showing (TIF)Click here for additional data file.S3 FigSREBF2 locus. Y-axis ranges are indicated for each track. (B) Mean + SEM of input versus day 21 SREBF2-targeting sgRNA abundances from genome-scale CRISPR/Cas9 screen performed in quadruplicate in Cas9+ GM12878 LCLs (71). Each sgRNA targets an independent SREBF2 exon regions. The y-axis value refers to the log2-transformed number of reads for each sgRNA normalized to the total number of reads. (C) Mean + SEM of input versus day 21 SREBF1-targeting sgRNA abundances from genome-scale CRISPR/Cas9 screen performed in quadruplicate in Cas9+ GM12878 LCLs (71). Each sgRNA targets an independent SREBF1 exon regions. The y-axis value refers to the log2-transformed number of reads for each sgRNA normalized to the total number of reads. (D) Dose-response curve analysis of fatostatin on newly-infected primary human B-cell growth and survival. Newly infected primary human B-cells were treated with the indicated doses of fatostatin or DMSO vehicle control for 4–7 DPI. The fatostatin effective concentration 50 (EC50) on newly-infected B-cell outgrowth was determined by GraphPad curve fitting analysis, as shown. (E) ChIP-seq tracks for the indicated transcription factors or H3K27Ac at the LCL ACACA locus, which encodes the ACC1 enzyme. The y-axis value refers to the log2-transformed number of reads for each sgRNA normalized to the total number of reads. (F) RT-PCR analysis of mRNAs encoding the fatty acid synthesis pathway enzymes ACLY or SCD, the cholesterol pathway enzymes HMGCR or FDFT1, LDLR, or the GGT-I subunits FNTA and PGGT1B from in primary human B-cells that were either mock-infected or infected with equal amounts of the non-transforming P3HR-1, UV-irradiated B95-8 or B95-8 EBV strains for four days. Mean values + SEM from n = 3 replicates are shown. *, p<0.05; **p, <0.01 (two-tailed t-test).(A) ChIP-seq tracks for the indicated transcription factors or H3K27Ac at the LCL (TIF)Click here for additional data file.S4 FigHMGCR targeting sgRNAs as indicated. (B) RT-PCR analysis of mRNAs encoding the cholesterol pathway enzymes FDFT1, SQLE, or LDLR from newly infected primary human B-cells treated for DPI 2–7 with the indicated doses of simvastatin or DMSO vehicle control. Mean values + SEM from n = 3 replicates are shown. *, p<0.05; **p, <0.01. (two tailed t-test). (C) Dose-response curve analysis of simvastatin on newly-infected primary human B-cell growth and survival. Shown are relative live cell numbers of EBV-infected primary human B-cells treated with the indicated doses of simvastatin or with DMSO vehicle control from day 4–7 post-infection. Mean + SEM values for n = 3 replicates are shown. The simvastatin effective concentration 50 (EC50) on newly-infected B-cell outgrowth was determined by GraphPad non-linear regression analysis, as shown. (D) Fold change in live cell number of primary human B-cells infected by EBV for the indicated DPI, cultured in the presence of DMSO or atorvastatin (2 μM) from 2 DPI to 7 DPI. Data show the mean + SEM from n = 3 replicates. *, p<0.05; **, p<0.01 (two-tailed t-test). (E) Mean + SEM of CFSE abundance values of primary human B-cells newly infected with B95-8 EBV or GM12878 LCL treated with DMSO, simvastatin (2 μM) and/or GGPP (2 μM), as indicated from days post-infection (DPI) 2–7, or from days in culture (DIC) 2–7 for GM12878 LCLs. Data were collected from n = 3 replicates, a representative one of which is shown in (A) Immunoblot analysis of whole cell lysates from Cas9+ GM12878 LCL expressing control or (TIF)Click here for additional data file.S5 Fig50) on newly-infected B-cell outgrowth not determined. Mean + SEM values for three replicates are shown. (D) Dose-response curve analysis of GGT-II inhibitor BMS-214662 on newly-infected primary human B-cell growth and survival. Shown are relative live cell numbers of EBV-infected primary human B-cells treated with the indicated doses of BMS-214662 or with DMSO vehicle control from day 4–7 post-infection. Mean + SEM values for three replicates are shown. EC50 on newly-infected B-cell outgrowth was determined by GraphPad non-linear regression analysis. (E-F) Mean + SEM of input versus day 21 abundances of FNTA-targeting (left) or PGGT1B-targeting sgRNAs from a genome-scale CRISPR/Cas9 screen performed in quadruplicate in Cas9+ GM12878 LCLs (71). FNTA and PGGT1B encode GGT-I subunits. Each sgRNA targets independent exon regions. The y-axis value refers to the log2-transformed number of reads for each sgRNA normalized to the total number of reads. (G-H) Mean + SEM of input versus day 21 RABGGTA-targeting (G) or RABGGTB-targeting (H) sgRNA abundances from genome-scale CRISPR/Cas9 screen performed in quadruplicate in Cas9+ GM12878 LCLs (71). RABGGTA and RABGGTB encode GGT-II subunits. Each sgRNA targets an independent exon.(A) Immunoblot analysis of GGT-I target Rap1a/b and GAPDH load control levels in newly infected primary human B-cells treated with the indicated concentrations of GGT-I inhibitor GGTI-2133 (GGT-Ii) or DMSO vehicle control DPI 4–7. Loss of prenylation causes higher Rap1a/b electrophoretic mobility, indicated by the *. (B) Immunoblot analysis of GGT-II target Rab13, LMP1 target TRAF1, phospho-mTor S2448 and GAPDH load control abundances in newly infected primary human B-cells treated with the indicated doses of GGT-IIi inhibitor BMS-214662 or DMSO control for DPI 4–7. Loss of prenylation causes higher Rab13 electrophoretic mobility, indicated by the *. (C) Dose-response curve analysis of GGT-I inhibitor GGTI-2133 on newly-infected primary human B-cell growth and survival. Shown are relative live cell numbers of EBV-infected primary human B-cells treated with the indicated doses of GGTI-2133 or with DMSO vehicle control from day 4–7 post-infection. An effective concentration 50 (EC(TIF)Click here for additional data file.S6 FigRABGGTA (A) or RABGGTB (B) targeting sgRNAs. (C) Propidium Iodide cell cycle analysis of conditional EBNA3C-HT 2-2-3 LCLs grown in the presence of 4HT (column 1) or in the absence of 4HT for 14 days (columns 2–4) and then treated with 4HT (1 μM) for 24 hours to re-induce EBNA3C expression, where indicated. Also where indicated, EBNA3C induction was performed in media lacking 10% fetal bovine serum (serum) in order to control for EBNA3C effects on cell cycle. See also (A-B) Immunoblot analysis of whole cell lysates from Cas9+ Gm12878 LCLs expressing non-targeting control or independent (TIF)Click here for additional data file.S7 FigRAB13-targeting sgRNA abundances from genome-scale CRISPR/Cas9 screen performed in quadruplicate in Cas9+ GM12878 LCLs (71). Each sgRNA targets an independent exon(A) Immunofluorescence micrographs of LMP2A, Rab13 and nuclear Hoechst 33528 uninfected versus infected primary human B-cells at 4 DPI. Merged images are shown with white boxes indicating inset images that have been further magnified. Scale bar is indicated (50 μm for single-channel and merged images). Three columns are presented for DPI 4 in order to increase the number of cells displayed at this timepoint. See also (TIF)Click here for additional data file.S8 FigImmunofluorescence micrographs of from newly infected primary B-cells cultured from days 2–7 post infection in the presence of DMSO, simvastatin (2 μM) or GGPP (2 μM) as indicated. Shown are images from n = 3 experiments. Scale bar is indicated (50 μm for single-channel and merged images). See also (TIF)Click here for additional data file.S9 FigImmunofluorescence micrographs of from newly infected primary B-cells cultured from days 4–5 post infection in the presence of DMSO or the indicated concentration of GGT-II inhibitor BMS-214662. Shown are images from n = 3 experiments. Scale bar is indicated (50 μm for single-channel and merged images). See also (TIF)Click here for additional data file.S10 FigRAB13-targeting sgRNAs. LMP1 target TRAF1 mRNA abundance was quantitated with GAPDH used for normalization. Data show the mean + SEM from n = 3 replicates. *, p<0.05 (one-sample t-test). (B) Immunoblot analysis of LMP1 and LMP2A from WCL of Cas9+ GM12878 LCL that express either control or independent RAB13-targeting sgRNAs. Representative blots from n = 2 experiments are shown.(A) Quantitative RT-PCR analysis of TRAF1 mRNA abundance in Cas9+ GM12878 LCL that express either control or an independent (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


The preliminary results from antibacterial assay which were tested against Gram-positive bacteria such as Bacillus subtilis, Staphylococcus epidermidis, Staphylococcus aureus and Gram-negative bacteria such as Escherichia coli, Salmonella typhimurium are also described.This paper provided comprehensive data on spectroscopic and antibacterial activities of thioureido compounds which are relevant with research article entitled “Synthesis, Spectroscopic Studies and Antibacterial Activity of New Lauroyl Thiourea Amino Acid Derivatives” [1]. Based on the reported study, four new thioureido derivatives, namely 3-(3-dodecanoyl-thioureido)propionic acid (R1), 2-(3-dodecanoyl-thioureido)-3-methyl butyric acid (R2), (3-dodecanoyl-thioureido)acetic acid (R3) and 2-(3-dodecanoyl-thioureido)-3-phenyl propionic acid (R4) were characterized by elemental analysis, Fourier Transform Infrared (FTIR),  This re, 2- and ammonium thiocyanate  were dissolved in acetone (25 mL) and stirred for an hour to give white precipitate. Then, β-alanine  was added dropwise and the mixture was heated at reflux until reaction was completed being filtered into ice. This was monitored by thin layer chromatography. The resulting solid was collected by filtration, washed with acetone and dried under vacuum to give 3-(3-dodecanoyl-thioureido)-propionic acid (R1). The synthesis of 2-(3-dodecanoyl-thioureido)-3-methyl-butyric acid (R2), (3-dodecanoyl-thioureido)-acetic acid (R3) and 2-(3-dodecanoyl-thioureido)-3-phenyl-propionic acid (R4) were prepared in similar manner as described for R1, employing Bacillus subtilis ATCC 11774, Staphylococcus epidermidis ATCC 13518 and Staphylococcus aureus ATCC 25923) and Gram-negative  strains using common well diffusion method. Mueller-Hinton media were seeded with bacterial inoculum using cotton swab. Wells of 6.0 mm diameter were bored into the media using sterile cork borer and 90 μL of the diluted compounds at a dose range of 10–0.01 mg/mL were added in each well. Streptomycin  was used as the positive control while methanol served as negative control. All plates were incubated overnight at 37 °C. The antibacterial activity was evaluated by measuring the zone of inhibition (mm) and minimum inhibitory concentrations (MIC). Ligand that have high specific surface area structure gave higher antibacterial activity [The antibacterial activity of compounds R1-R4 were screened against test strains of Gram-positive (