Candida albicans have significantly increased in recent years. C. albicans transition from yeast to germ tubes is one of the essential factors for virulence. In this study we noted that Lee’s medium, commonly used to induce filamentation, contained 500-fold more biotin than needed for growth and 40-fold more biotin than is typically added to growth media. Thus, we investigated the effects of excess biotin on growth rate and filamentation by C. albicans in different media. At 37 °C, excess biotin (4 µM) enhanced germ tube formation (GTF) ca. 10-fold in both Lee’s medium and a defined glucose-proline medium, and ca. 4-fold in 1% serum. Two biotin precursors, desthiobiotin and 7-keto-8-aminopelargonic acid (KAPA), also stimulated GTF. During these studies we also noted an inverse correlation between the number of times the inoculum had been washed and the concentration of serum needed to stimulate GTF. C. albicans cells that had been washed eight times achieved 80% GTF with only 0.1% sheep serum. The mechanism by which 1–4 µM biotin enhances GTF is still unknown except to note that equivalent levels of biotin are needed to create an internal supply of stored biotin and biotinylated histones. Biotin did not restore filamentation for any of the four known filamentation defective mutants tested. C. albicans is auxotrophic for biotin and this biotin auxotrophy was fulfilled by biotin, desthiobiotin, or KAPA. However, biotin auxotrophy is not temperature dependent or influenced by the presence of 5% CO2. Biotin starvation upregulated the biotin biosynthetic genes BIO2, BIO3, and BIO4 by 11-, 1500-, and 150-fold, respectively, and BIO2p is predicted to be mitochondrion-localized. Based on our findings, we suggest that biotin has two roles in the physiology of C. albicans, one as an enzymatic cofactor and another as a morphological regulator. Finally, we found no evidence supporting prior claims that C. albicans only forms hyphae at very low biotin (0.1 nM) growth conditions.Due to the increased number of immunocompromised patients, infections with the pathogen  Candida albicans  is an important fungal pathogen of humans. As such, there has been intense interest in the virulence factors which make C. albicans such a versatile pathogen, and of these virulence factors the one which has garnished the greatest attention is the ability to interconvert between the yeast and hyphal morphologies. In his monumental work on Candida and Candidiasis, Odds [is, Odds  listed 3C. albicans hyphae in vitro include: (i) Lee’s medium which contains eight amino acids [N-acetyl-d-glucosamine [l-proline [N-acetyl-d-glucosamine (mGPP) [Media commonly used to produce no acids ; (ii) seno acids ,2; (iii)cosamine ,4; (iv) -proline ; and (v)e (mGPP) , all at e (mGPP) .2; (v) l-methionine; (vi) N-acetylglucosamine; and (vii) a neutral pH or the absence of an acidic pH.Current research has focused on the signal transduction mechanisms and transcription factors which connect these external triggers with the induction of hyphae-associated genes . SudberyHowever, there are still many unanswered questions regarding the identity of the hyphal triggers, like which component(s) of Lee’s medium and serum actually induce hypha formation. These questions persist despite evidence that for serum both glucose  and bactC. albicans is a biotin auxotroph [C. albicans [C. albicans in a biotin-free glucose-phosphate-proline (GPP) medium through three growth cycles (ca. 20 buddings) were ca. 60%, 40%, and 20%, respectively, of those achieved in a biotin replete medium. The presence of an internal biotin reservoir, possibly biotinylated histones, was indicated by the same decreasing cell yields being observed with and without added neutravidin in the biotin-free medium. Neutravidin is a strong biotin-binding protein.uxotroph  and our albicans  showed tS. cerevisiae needs biotin or the biotin analogs d-desthiobiotin, biocytin, or biotin-d-sulphoxide [The present paper studies the role of added biotin in germ tube formation at 37 °C. The interactions of biotin with yeasts date back to the discovery of biotin by Kӧgl and Tӧnnis . They idlphoxide .S. cerevisiae in that many strains only contain a partial biosynthetic pathway. Presumably the pathway was lost in a distant ancestor and then rebuilt by horizontal gene transfer, gene duplication, and neofunctionalization [Biotin biosynthesis is a variable trait in lization . HoweverS. cerevisiae required an external supply of KAPA, DAPA, desthiobiotin, or biotin while two earlier precursors, pimelic acid and pimeloyl-CoA, would not suffice. C. albicans is also a biotin auxotroph [C. albicans [C. albicans grown on excess (1–4 µM) biotin contained biotinylated histones even though cells grown on 100 nM biotin did not [C. albicans and that the biotin present in both Lee’s Medium and serum is in part responsible for the ability of these media to achieve germ tube formation.7-keto-8-amino-pelargonic acid and 7,8-diamino-pelargonic acid are also called 8-amino-7-oxononanoate and 7,8-diaminononanoate respectively. Thus, Phalip et al.  found thuxotroph  and Fireuxotroph  found thalbicans ,6 and wealbicans . Signifi did not . The preC. albicans strain SC5314 was obtained from the American type culture collection, Rockville, MD and strain A72 was obtained from Prof. Patrick A. Sullivan, then at Otago Univ., Dunedin, New Zealand. The HLC52 (efg1/efg1), JCK19 (cph1/cph1), and HLC54 (efg1 cph1/ efg1 cph1) mutants were from Prof. Gerald Fink and CR216 (cdc35/cdc35) was from Prof. Deborah Hogan ; samples were stored at 4 °C overnight for further use [ah Hogan . Cells wther use .600 of 0.1 (1 × 106 cells per mL). All OD600 values were recorded on a Spectra Max Plus microplate reader . Cells were tested in vitro with 2.5 mM N-acetylglucosamine at 37 °C [Cells were prepared for growth experiments by diluting the overnight culture in 50 mL of YPD, GPP, or GSB to an ODC. albicans cells were inoculated at 1 × 107 cells per ml (OD600 ~ 1.0) in prewarmed 25 mL flasks and supplemented with or without biotin (0–4 µM) or desthiobiotin (0–4 µM) and incubated with shaking (225 rpm) at 37 °C for 4 h. Every 30 min, a sample was analyzed microscopically. A total of 100–300 cells were counted in each experiment and the percentage of cells that had formed germ tubes was determined. Data from three independent experiments are presented. The effect of biotin in Lee’s medium was also observed in separate experiments conducted with and without l-proline [C. albicans cells were grown at 1 × 106 cells per ml in supplemented media  with or without biotin (0 or 100 nM) in 250 mL flasks. The cells for the first population were grown for 30 h at 30 °C or 37 °C (225 rpm). After 30 h, the cells were used as inoculum (1:100) and transferred to a fresh media supplemented with or without biotin (0 or 100 nM) and this population was counted as the second population. After 30 h growth, another 1:100 dilution gave the third population. For each population 5 mL samples were taken every 5 or 6 h, analyzed microscopically, and prepared for dry-weight measurement.The dry-weight measurement was done using 5 mL samples that had been washed twice with distilled water (before drying). After measuring the initial weight of the weighing dish, the washed samples were dried in the drying oven for 6–8 h at 150 °C. Then, the samples were cooled in the desiccator. After constant measurement of the dry-weight of each sample, the data were recorded. Data obtained were from three independent experiments.C. albicans SC5314 in 50 mL of biotin free glucose-salts medium pH (5.6) for 25 h at 30 °C. The cells were harvested, washed three times with KP buffer, and stored at 4 °C prior to use. The GS plates were prepared with Noble agar supplemented with 0, 5, 50, or 4000 nM biotin. Noble Agar was chosen to minimize the contaminating biotin provided by the agar. YPD agar plates were also used as controls. The inoculum was diluted to 6 × 102 cells per ml so that a 100 µL cell suspension gave ca. 60 CFU/plate after they had been spread with 3–4 glass beads. Triplicate plates were incubated 4–5 days under four environmental conditions: (i) 30 °C; (ii) 37 °C; (iii) 30 °C with 5% CO2; and (iv) 37 °C with 5% CO2. Both 5% CO2 cultures were incubated in a dual chamber VWR 18401R incubator . After 5 days all colony diameters were measured and averaged and then three colonies per plate were scraped off with a spatula and weighed.Biotin deficient inocula were prepared by growing C. albicans grown in synthetic complete (SC) medium was reinoculated into SC medium with and without biotin until they reached mid log phase. The cells were harvested and total RNA were extracted using a phenol extraction method as described by Kohrer and Domdey [® III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen™) according to the manufacturer’s recommendation using oligo dT primers. Based on 1:1 ratio production of cDNA, a hundred ng of resulting sample was subjected to quantitative PCR using iQ™ SYBR® Green Supermix in a Biorad iCycler iQ real time PCR detection system. Each cDNA preparation was normalized using CaCDC36 as an internal control. The primers used for genes under study, BPL1, BIO2, BIO3, BIO4, BIO32, and CDC36 in this study are listed in For gene expression analysis, a preculture of d Domdey  and thenBPL1, BIO2, BIO3, BIO4, and BIO32 were performed by using PSORT II [mTPs predictions on PSORT II  server, PSORT II  server, PSORT II  server aPSORT II  server. C. albicans are remarkably diverse in their chemical composition. One feature which stands out is the concentration of biotin present, ranging from 20 µg/L in GPP up to 200 µg/L in RPMI 1640 and 1000 µg/L (4 µM) in Lee’s Medium [S. cerevisiae [The chemically defined media commonly used to achieve germ tube formation (GTF) in revisiae . Thus, tl-pro, a known trigger for GTF, had been omitted (l-pro is not necessary for biotin to exert this activity.In Lee’s medium we observed a dose-response relationship between % GTF and biotin (0 to 4 µM) such that the high biotin grown cells initiated GTF earlier and by 2 h had formed ca. 10-fold more germ tubes a. This r omitted b, and fo omitted c. These 7 cells at 37 °C after 5 h , imidazole, and Mg/MnCl2 [GTF can also be achieved without growth in a differentiation medium composed solely of Mg/MnCl2 ,6. Bioti∆efg1, ∆cph1, and ∆cdc35 (adenylyl cyclase) deletion mutants of C. albicans. However, none of these mutants exhibited GTF in 2.5 mM GlcNAc, either with or without 4 µM biotin. These results are consistent with earlier reports that these mutants did not form germ tubes in Lee’s medium. Rocha et al. [C. albicans mutants defective in adenylyl cyclase were unable to switch from yeasts to hyphae under all in vitro conditions analyzed, including Lee’s medium. Similarly, the hyphal defective mutants cph1/cph1 [efg1/efg1 [efg1/efg1 and cph1/cph1, and the double mutant efg1cphl/efg1cph1 did not form hyphae in Lee’s medium at 37 °C. These experiments collectively show that high biotin acts before adenylyl cyclase and the two transcription factors for hyphal associated genes, Efg1p and Cph1p.The previous series of experiments showed that biotin enhanced GTF in all growth media not already maximally stimulated by something else. However, as yet we do not know biotin’s mode of action. One approach to this question tests a series of mutants defective in hyphal formation to see if high biotin restored their hyphal forming ability. Accordingly, we examined the a et al.  showed tph1/cph1  and efg1fg1/efg1  were unafg1/efg1  showed tBIO2, BIO3, and BIO4 increased 11-, 1500-, and 150-fold, respectively, in the biotin starved cells, while BIO32 remained effectively unchanged  was also increased 5-fold in the biotin starved cells  and colony diameter and dry weight were recorded after 5 days  where the colony diameters with biotin (7 ± 0.3 mm) were twice as large  at 29 °C [Our data that high biotin stimulates GTF seems at variance with the work of Yamaguchi ,43,44 whamaguchi  were unuat 29 °C , even that 29 °C  that an C. albicans SC5314 or A72 at either 30 or 37 °C. C. albicans 6713 itself was no longer available. It is possible that high and low biotin GTF are separate and distinct phenomena because the levels of biotin employed are 10,000–fold different. It is also possible that low biotin GTF is characteristic of strain 6713 and not typical of other C. albicans strains. For instance, C. albicans 6713 could have had reduced levels of heat shock protein 90 (Hsp90) because strains engineered with reduced Hsp90 levels form hyphae in serum at 30 °C as well as 37 °C [C. albicans 6713 would now be reclassified as C. dubliniensis, a closely related species known to produce fewer hyphae under all induction conditions tested [We replicated the experimental conditions described by Yamaguchi  in all ras 37 °C . Finallys tested . These pC. albicans. These signals are important because yeast-mycelial dimorphism was the first virulence trait recognized in pathogenic fungi and as such it is somewhat perplexing that the actual germ tube triggers are still uncertain for Lee’s medium [C. albicans with a view towards establishing high biotin as a factor that enhances germ tube formation (GTF). Biotin may eventually be shown to be a trigger for GTF, but at present, our data only prove that it is an enhancer of GTF. Three well studied conditions for GTF were shown to be biotin dependent: Lee’s medium, 1% serum, and GPP  is provided by the observation of Mock et al. [e medium  must be k et al.  that bioC. albicans SC5314 and WO-1 contain distinct biotin clusters including BIO2, BIO3, BIO4, and BIO5 [C. albicans genes which were significantly upregulated during infection; both BIO2 and BIO4 were upregulated ca. 2.5-fold. Similarly, BIO2 was upregulated ca. 107-fold on shifting from low iron to high iron growth conditions [C. albicans. For both yeasts, only BIO2 is predicted to be mitochondrion-localized. Presumably BIO2 has to remain mitochondrial because, as in A. thaliana [C. albicans does not contain BIO1 (pimeloyl-CoA synthase) or BIO6, the alternate method of synthesizing KAPA found in biotin prototrophic sake yeasts [Despite being a biotin auxotroph, and BIO5 . With reand BIO5  catalogunditions . Figure thaliana , the bioe yeasts . We antiC. albicans is both a biotin auxotroph and a successful opportunistic pathogen. How is this possible? Part of the answer was shown previously [C. albicans contains an internal biotin reservoir, possibly in the form of biotinylated histones. We have now shown that high biotin (4 µM) is a previously unknown enhancer of germ tube formation (GTF) and its presence in both serum and Lee’s medium [C. albicans has a cluster of four genes (BIO2–5) encoding the latter half of the biotin synthetic pathway [BIO2p is localized to the mitochondria. Based on our findings, we suggest that biotin has two roles in the physiology of C. albicans, one being known as an enzymatic cofactor and another as a morphological regulator.eviously  in that s medium  is essen pathway , implyin


However, the involvement of connexin 30 in astroglial polarization, which is known to control cell morphology, remains unexplored. We found that connexin 30, independently of gap-junction-mediated intercellular biochemical coupling, alters the orientation of astrocyte protrusion, centrosome and Golgi apparatus during polarized migration in an in vitro wound-healing assay. Connexin 30 sets the orientation of astroglial motile protrusions via modulation of the laminin/β1 integrin/Cdc42 polarity pathway. Connexin 30 indeed reduces laminin levels, inhibits the redistribution of the β1-integrin extracellular matrix receptors, and inhibits the recruitment and activation of the small Rho GTPase Cdc42 at the leading edge of migrating astrocytes. In vivo, connexin 30, the expression of which is developmentally regulated, also contributes to the establishment of hippocampal astrocyte polarity during postnatal maturation. This study thus reveals that connexin 30 controls astroglial polarity during development.Astrocytes undergo intense morphological maturation during development, changing from individual sparsely branched cells to polarized and tremendously ramified cells. Connexin 30, an astroglial gap-junction channel-forming protein expressed postnatally, regulates  Summary: Connexin 30 sets the orientation of astroglial motile protrusions during polarized migration in vitro and contributes in vivo to the establishment of hippocampal astrocyte polarity during postnatal maturation. Remarkably, the two main astroglial Cxs, Cx43 and Cx30, show different developmental expression patterns: in mice, expression of Cx43 starts around embryonic day 12, whereas that of Cx30 is detectable from postnatal day 16 (P16) . Strikintrusions . Interestrusions . In addia fibers . Such ada fibers , 2006.in vitro scratch assay. This effect is independent of GJ-mediated biochemical coupling and is mediated by modulation of a laminin/β1-integrin/Cdc42 signaling pathway  astroglial processes. This study thus identifies Cx30 as an important regulator of astrocyte polarization during development.In contrast and despite the late postnatal onset of Cx30 expression, little is known about the contribution of Cx30 to astroglial maturation during brain development. Similar to other Cxs, Cx30 interacts with the actin and tubulin cytoskeletons . In addi pathway . Cx30 in pathway . ImportaFig. S1). Following scratching of the cell monolayer, wound-edge control astrocytes polarized and initiated directed migration by extending tubulin-rich protrusions perpendicularly to the wound : 40.7±4%, n=187 cells, n=352 cells; Cx30: 35.4±4%, n=153 cells, n=418 cells; Golgi apparatus: 40.7±4.8%, n=503 cells, Fig. S1). These data demonstrate that Cx30 alters the polarization of migrating astrocytes independently of GJ-mediated intercellular biochemical coupling.To investigate a role for Cx30 in the polarization of migrating astrocytes, we first used the scratch-induced migration assay in cultured astrocytes  transfecn=6; Cx30: 1.06±0.08, n=6 cultures, 2, n=14 cells; Cx30: 0.074±0.011 per µm2, n=15 cells, Mann–Whitney, P<0.01) and size of laminin puncta . Remarkably, during polarized migration, laminin puncta extended up to the leading edge of astroglial protrusions where they accumulated, as shown by immunocytochemistry in GFP-labeled cells (Migrating astrocytes contribute to the deposition of extracellular matrix (ECM) molecules, including laminins, which are essential for integrin-mediated cell polarization . We thused cells C. We foued cells C,D.Fig.n=15 cells; Cx30: 5.2±0.7, n=9 cells, Laminin locally binds receptors, including β1 integrins, which are trafficked to the cell leading edge where they are essential for proper astrocyte polarization . To expln=19 cells; Cx30: 89.3±7.6 a.u., n=15 cells; n=3 cultures, Integrin-mediated signaling promotes astrocyte polarization through the rapid activation and polarized recruitment of the small Rho-GTPase Cdc42 at the leading edge of migrating astrocytes . We thusin vivo the expression of laminins at synaptic sites. To do so, we prepared synaptosomal extracts from hippocampi of wild-type juvenile mice , and further performed laminin proteins immunoblot assays. We observed that control hippocampal synaptosomal extracts contained laminins, especially laminin β2 -rich stem processes to polarize perpendicularly to the pyramidal cell layer  at two developmental stages [postnatal day 10 (P10) and 30 (1 month)]. Moreover, to estimate the preferential orientation of s.r. astrocytes with regards to the pyramidal layer, we measured a polarity index corresponding to the ratio of crossing points between GFAP-positive processes and parallel or perpendicular axis to the pyramidal layer . This modulation of astroglial cell polarity was independent of GJ-mediated intercellular biochemical coupling, as astroglial polarity was intact in astrocytes from 1-month-old Cx30T5M mutant mice with defective Cx30 channels  was sufficient to fully rescue a normal polarity phenotype in s.r. astrocytes from 1-month-old animals, whereas GFP infection alone had no effect  in CA1 s.r. astrocytes from hippocampal slices via patch-clamp whole-cell recording. We found that wild-type CA1 astroglial GJ-mediated networks were preferentially oriented perpendicularly to stratum pyramidale (s.p.)  were group housed on a 12 h light/dark cycle. −/−Cx30 (T5M/T5MCx30 mice (Experiments were carried out according to the guidelines of the European Community Council Directives of January 1st 2013 (2010/63/EU) and of the local animal welfare committee . All efforts were made to minimize the number of animals used and their suffering. Mice , Cx43 mouse monoclonal , GFAP rabbit polyclonal , GFP chicken polyclonal , α-tubulin rabbit polyclonal , β-actin mouse monoclonal , PSD95 mouse monoclonal , synapsin I mouse monoclonal , GM130 mouse monoclonal , γ-tubulin mouse monoclonal , laminin rabbit polyclonal , laminin β2 mouse monoclonal , Cdc42 mouse monoclonal , paxillin mouse monoclonal , vinculin , MAP2 mouse monoclonal , IB4-647 . The HRP-conjugated primary rabbit anti-GAPDH antibody  has been used as loading control. The following HRP-conjugated secondary antibodies were used: goat anti rabbit IgG  and goat anti-mouse IgG . The following fluorescent dye-conjugated secondary antibodies were used in appropriate combinations: goat anti-mouse IgG conjugated to Alexa 488 or 647 , goat anti-rabbit IgG conjugated to Alexa 488 or 555  and goat anti-chicken conjugated to Alexa 488 .Immunohistochemistry and quantifications were performed as previously described . Brieflyz projections were reconstructed using ImageJ software. Alternatively, primary astrocyte cultures were fixed at room temperature with 4% paraformaldehyde for 10 min, washed three times with PBS and incubated 1 h with 10% non-immune goat serum (Zymed) in the presence of 0.1% Triton-X100 before proceeding as described previously.After several washes, brain slices were mounted in fluoromount and examined with an inverted confocal laser-scanning microscope . Stacks of consecutive confocal images taken with a 63× objective at 600-1000 nm intervals were acquired sequentially with two lasers (argon 488 nm and helium/neon 543 nm) and Western blotting and quantification were performed as previously described . ShortlyFig. S2A) were prepared as previously described , as previously shown , whereas processes crossing vertical lines were scored with a lateral orientation towards the pyramidal cell layer. A ratio between axial/lateral processes was finally measured, which reflects the polarity index of the cell. A polarity index of 1 indicates no polarity, whereas an index larger than 1 indicates preferential perpendicular orientation toward the pyramidal layer.escribed . Confocain vivo Golgi apparatus reorientation was directly adapted from the method of scoring Golgi repositioning upon scratching  to the scratch were considered properly polarized. Random orientation was thus scored at 33%.The protocol used for analysis of ratching . Confocaz-stack thresholded images of the sulforhodamine-B-labeled CA1 s.r. astrocyte networks were manually delimited with best-fit ellipses. The length of the major/minor ellipse axis were measured and its ratio (major/minor) provided an estimation of the polarity (or anisotropy) of astroglial networks. A polarity index value larger than one indicated preferential perpendicular orientation with respect to the pyramidal layer.The spatial properties of GJ-mediated inter-astrocytic coupling were analyzed using the ImageJ software. Confocal Analysis of Cdc42 relocalization was adapted from Similarly, astrocyte monolayers were transfected with β1 integrin-GFP (provided by F. Coumailleau) and Cx30. 8 h after wounding, then cells were fixed with 4% PFA and immunolabeled for Cx30. Regions of interest were drawn at the leading edge of migrating astrocytes and the intensity of β1 integrin-GFP was quantified using ImageJ software and compared with control cells.The analysis of laminin puncta number and size, as well as axial distribution was adapted from a previous study . Briefly2) were washed once in ice-cold PBS and rapidly mixed with 1 ml of ice-cold lysis buffer  to yield a protein concentration of 400-600 µg/ml. An aliquot was further taken to perform both BCA protein assay and Cdc42 immunoblot on the total Cdc42 fraction. The mix was then clarified by ultracentrifugation , and the supernatant was incubated with GST-p21-activated kinase (PAK) binding domain (PBD) affinity beads at 4°C for 1 h on a rotator. The beads were next ultracentrifugated , washed and resuspended in wash buffer . After a second step of ultracentrifugation , 10-20 µl of Laemmli 2× was added to resuspend the beads, and the proteins were boiled for 2 min. A western blot for Cdc42 was finally performed on the total and PBD-bound sample fractions with an appropriate dilution of Cdc42 antibodies (1:150). The intensity of activated GTP-bound Cdc42 was normalized to the total Cdc42 levels, and GAPDH  as well as Ponceau staining (GTP) were used as loading controls.The Cdc42 pull-down assay was adapted from a commercial kit (Cytoskeleton) . Brieflyg for 15 min at 4°C and the pelleted nuclear fractions discarded. The supernatants were further centrifuged at 200,000 g for 30 min at 4°C and the new pellets were washed and resuspended in ice-cold homogenization buffer, while the supernatant crude cytosols were withdrawn. Finally, crude membrane pellets were centrifuged one last time at 200,000 g for 30 min at 4°C and resuspended in 100 µl of HEPES lysis buffer . Samples were briefly sonicated and the protein concentration of synaptosomal crude membrane fraction was next determined using a Bradford protein assay, and further processed for immunoblot as described above.For the isolation of synaptosomal crude membrane fractions, freshly dissected hippocampi were first lysed in 500 µl of ice-cold homogenization buffer  using a mechanical Potter-Elvehjem homogenizer. The homogenates were then centrifuged at 1000 in vivo gene transfer, a transgene composed of GFP and Cx30 cDNA separated by a P2A sequence in a single open reading frame was placed under the control of a GFAP-specific promoter in an AAV shuttle plasmid containing the inverted terminal repeats (ITR) of AAV2. The control vector expressed GFP only. Pseudotyped serotype 9 AAV particles were produced by transient co-transfection of HEK-293T cells, as previously described  were anesthetized with a mixture of ketamine  and xylazine  in 0.9% NaCl and placed on a stereotaxic frame under body temperature monitoring. AAVs were diluted in PBS with 1% BSA at a concentration of AAV-GFP 1.2×1013 vg/ml and AAV-GFP-Cx30 0.5 x1013 vg/ml, and 1 µl of virus was stereotaxically injected bilaterally into the hippocampi at a rate of 0.2 μl/min, using a 29-gauge blunt-tip needle linked to a 2 µl Hamilton syringe (Phymep). The stereotaxic coordinates to Bregma were: antero-posterior, −1.7 mm; lateral: ±1.5 mm; and dorso-ventral, −1.7 mm. At the end of the injection, the needle was left in place for 5 min before being slowly removed. The skin was sutured, and mice recovery was checked for the next 24 h. After 2 weeks, the mice were sacrificed and processed for immunohistochemistry.With the dose of AAVs and injection conditions used in this study, no astrogliosis was observed. Astroglial morphology was analyzed at a distance of ∼150–200 μm from the injection site and the images were taken at the same distance.2, 1.3 MgSO4, 1 NaH2PO4, 26.2 NaHCO3 and 11 glucose, saturated with 95% O2 and 5% CO2) for at least 1 h at room temperature before the experiments. Slices were transferred in a submerged recording chamber mounted on an Olympus BX51WI microscope equipped for infrared-differential interference (IR-DIC) microscopy and were perfused with aCSF (2 ml/min). Whole-cell patch-clamp recordings were obtained from s.r. astrocytes using 4-6 MΩ glass pipettes filled with (in mM) 105 K-gluconate, 30 KCl, 10 HEPES, 10 phosphocreatine, 4 ATP-Mg, 0.3 GTP-Tris and 0.3 EGTA . Astrocytes were patched at similar distances from the stratum pyramidale (∼200 µm) and were identified by their small somata, low input resistance and resting membrane potential, passive membrane properties (linear IV relationship), lack of action potential and extensive gap junctional coupling. For intercellular dye coupling experiments, the internal solution contained biocytin (7 mg/ml), which diffused passively in astrocytes patched in current-clamp mode for 30 min. Recordings were acquired using a MultiClamp 700B amplifier (Molecular Devices), digitized at 10 kHz, filtered at 2 kHz, stored and analyzed on a computer using Clampex10.3 and Clampfit10.3 Software (Molecular Devices). Biocytin was obtained from Sigma, and all other chemicals were from Tocris.Acute transverse hippocampal slices (400 µm) were prepared as previously described . Slices in vitro, ex vivo and in vivo, respectively). For cellular analysis performed in astrocyte cultures or in vivo, n refers to the number of cells analyzed from five fields per independent experiment. For biochemical experiments performed in astrocyte cultures, n refers to the number of cultures; when performed in vivo, n refers to the number of mice. For intercellular coupling experiments in astrocyte cultures, n refers to the number of cultures; when performed ex vivo, n refers to the number of hippocampal slices. Statistical significance for within-group comparisons was determined by one-way ANOVA (followed by Dunnett's post-test), whereas unpaired t-tests were used for between-group comparisons, unless otherwise stated. Statistical analysis was performed in GraphPad InStat.All data are expressed as mean±s.e.m. Data were obtained from at least three independent experiments that were performed in astrocyte cultures, hippocampal slices or mice, according to the type of experiment performed (


STAT3) promoter methylation, mRNA expression and the invasiveness or recurrence of PAs and patient clinical characteristics.Pituitary adenoma (PA) is a benign brain tumor that can cause neurological, endocrinological and ophthalmological aberrations. Till now there is a need to identify factors that can influence the tumor invasiveness and recurrence. The aim of this study was to evaluate the associations between the signal transducer and activator of transcription 3  was performed.Study participants comprised of 102 subjects with a diagnosis of PA: 54 functioning and 48 non-functioning, 58 invasive and 30 non-invasive PAs and 14 relapses. The bisulfite treatment of tumor DNA and methylation-specific polymerase chain reaction (MS-PCR) method was used to determine the STAT3 gene promoter was methylated. A gender of male and patient group older than 60 years were significantly associated with reduced STAT3 mRNA expression . However, no more statistical differences were found between STAT3 promoter methylation, mRNA expression and patient clinical characteristics or PA invasiveness or recurrence.In 10.78% (11/102) of PA tissues STAT3 gene promoter methylation and mRNA expression changes in PAs.Further investigations are needed to clarify the influence of  Pituitary adenomas (PA) arise from adenohypophyseal cells and account for 10–15% of all intracranial neoplasms. About 0.2% of all PAs with subarachnoid, brain or systemic metastasis are considered to be malignant [Surgical resection is the first-line of treatment for PAs, except for PRL-producing adenomas. Residual or recurrence tumors require re-operation, medical treatment or radiation . HoweverCyclin D1, C-myc), tumor suppressor genes , cell cycle regulator genes , and epigenetic factors  [DAP-kinase gene was associated with highly invasive and/or metastatic pituitary tumors [GADD45G) leads to gene silencing and is associated with the development of somatotropinomas, prolactinomas, and non-functioning adenomas [In many cancers, including PAs, epigenetic changes have been involved in development of tumors . Pituitaication) , 6. For y tumors . Also, tadenomas .STAT3) gene, a member of the STAT family of proteins that mediates cytokine signaling and nuclear transcription [in mouse tumor models STAT3 is integrally involved in tumorigenesis, including apoptosis, cell cycle progression, tumor angiogenesis, invasion and metastasis [STAT3 gene DNA and mRNA expression changes in invasive or recurrence PAs.One of the potential marker for PA diagnosis and prognosis could be signal transducer and activator of transcription 3  to determine the mRNA expression changes in PAs. These epigenetic alterations were then correlated with clinical features of the patients and the invasiveness, recurrence of PAs.To this purpose, we performed methylation-specific polymerase chain reaction (MS-PCR) to evaluate methylation status of The study was performed in the Neuroscience Institute, Lithuanian University of Health Sciences between 2010 and 2016. After surgical resection tumor tissues were frozen in nitrogen liquid and the following clinical characteristics were gathered: gender, age at the time of the operation, relapse, tumor size, PA activity, invasiveness, hypersecretion of PRL, GH, IGF-1, ACTH or more than one hormone and diagnoses of Cushing syndrome, acromegaly or prolactinoma.Study participants comprised of 102 patients . Clinical findings showed that the endocrinological features were: 54 functioning and 48 non-functioning adenomas. Functioning adenomas were: 4 GH-secreting adenomas, 2 IGF-1-secreting adenomas, 36 PRL-secreting adenomas, 1 ACTH-secreting adenoma and 11 adenomas secreting more than one hormone. Also, magnetic resonance imaging findings and the Hardy classification, modified by Wilson was used to quantify the sphenoid sinus invasion and suprasellar extension [For the DNA methylation studies, 102 frozen PA tissues were used. First, DNA was extracted by 10% “UtraPure™ ”SDS , proteinase K , followed by phenol–chloroform  and 96% ethanol  precipitation. Subsequently, according to the manufacturer’s instructions, genomic DNA was modified with sodium bisulfite using EZ DNA methylation kit™  and eluted in 40 μL of nuclease-free water.The MS-PCR was performed in 15 μl of a mixture containing 10 pmol of each primer , 7.5 μL Maxima® Hot Start PCR Master Mix  with Hot Start Taq DNA polymerase and nuclease-free water. MethPrimer online tool [The MS-PCR products were analyzed on a 2% agarose gel with ethidium bromide  by horizontal electrophoresis.STAT3 gene mRNA expression analysis, total RNA was extracted from the same set of PA samples (102 samples) that has been used for STAT3 gene methylation analysis with Trizol reagent , according to the manufacturer’s instructions and stored at −80 °C. RevertAid H Minus M-MuLV Reverse Transcriptase  and random hexamer primers  were used to produce the first-strand cDNA. Total reaction volume was 20 μl. Negative controls without reverse transcriptase were prepared as above.For the STAT3 and β-actin was performed in a Real-Time PCR System “Applied Biosystems 7500 Fast” with SYBR Green chemistry. The qRT-PCR was carried out in a 12 μl of mixture which consisted of 6 μl Maxima SYBR Green/ROX qPCR Master Mix (2X) , 15 ng of the cDNA, nuclease-free water and STAT3 gene-specific primers that were designed according to the published data [β-actin with primers designed according to the published data [GAPDH TaqMan assay  was used to verify the expression of another endogenous control in PAs. The PCR amplification was performed according to the manufacturer’s protocol. All sample measurements were performed in triplicate. Healthy human brain RNA sample (RHB) “FirstChoice Human Brain Reference RNA”  was used for standard curve design and the parameters were: for STAT3: efficiency 92.68%, R2 = 0.99, slope − 3.51; for β-actin: efficiency 100.08%, R2 = 0.99, slope − 3.32.Quantitative real-time PCR for hed data : 5′-CATAhed data : 5′- AGA-ΔΔCt calculations, where ΔΔCt = Sample x - RHB [Finally, results were presented as 2rols)RHB .STAT3 gene mRNA expression and the methylation status, also the clinical factors: gender, age, relapse, Cushing syndrome, acromegaly, prolactinoma, invasiveness, secreting and non-secreting PAs and hormone groups. The associations among STAT3 gene promoter methylation and clinical characteristics of PA patients were assessed with Chi-square test and p < 0.05 values were considered statistically significant.Statistical analyses were conducted with the SPSS Statistics 19  and GraphPad Prism 7  software packages. The Mann-Whitney test was used to find the associations between STAT3 gene methylation status was performed in 102 PA samples by the MS-PCR method. The representative chart of MS-PCR products that were separated by horizontal electrophoresis and visualized under UV illumination is shown in Fig. STAT3 gene was methylated in 10.78% (11/102) and non-methylated in 89.22% (91/102) of PA samples. These results indicate that STAT3 has low methylation status in PAs.STAT3 gene promoter methylation and patient clinical characteristics: gender, age at the time of operation, relapse, PA function, invasiveness and diagnoses of Cushing syndrome, acromegaly or prolactinoma. To this purpose, Chi-square test was performed. However, statistical analysis showed no significant differences (Table STAT3 gene. Only in 12.07% (7/58) invasive PAs STAT3 gene was methylated and non-methylated in 87.93% (51/58) cases but with no significance .Next, we tried to find out the associations between es Table . In addiSTAT3 gene promoter might be strongly methylated. Thus, the hormone distribution of PAs in methylated and non-methylated STAT3 gene promoter groups was performed. The analysis showed that with methylated STAT3 promoter, the quantities of PRL (4/36), IGF-1 (0/2), ACTH (0/1) and more than one hormone secreting PAs (0/11) hypersecretion were highly reduced as well as the non-secreting PAs (5/48)  and the group of non-methylated STAT3 gene .To investigate the STAT3 mRNA expression and patient clinical characteristics. We found that gene mRNA expression was lower with males (36/89 cases) than females  and with patients older than 60 years .Next, we assessed the associations between STAT3 gene mRNA expression values were categorized into four levels. Values 0.5-fold lower than the gene mRNA expression median were indicated as “low” expression level (N = 37), expression values 0.5-fold higher - “high” level (N = 33), values ranging in between “low” and “high” were referred to as “medium” mRNA expression level (N = 19). In 13 samples there were no STAT3 mRNA expression. Then we analyzed PRL, IGF-1, GH, ACTH, more than one hormone and non-secreting PAs distribution in these expression levels. As was shown in Fig. STAT3 mRNA expression level . Similarly, GH (2/4) and more than one hormone hypersecretion (6/11) - at “high”STAT3 mRNA expression level . The IGF-1 hormone hypersecretion (1/2) mostly occured at “high”STAT3 mRNA expression level and with no gene expression .For further investigation STAT3 mRNA expression is associated with PA invasiveness .In our last series of result analysis, we took the biggest group of hormonally active PAs (PRL-secreting PAs) and non-secreting PAs and asked if ess Fig. . HoweverSTAT3 gene may be a promising target because it is aberrantly activated in a wide variety of human cancers and plays crucial roles in tumor cell proliferation, survival, invasion and tumor-promoting inflammation [Pituitary adenoma is a benign brain tumor that could invade the sphenoid, cavernous sinus, or dura mater and some of them are described as aggressive, with a high proliferation rate and short postoperative time to recurrence , 19. Theammation –26.STAT3 gene promoter methylation and mRNA expression in 102 PA patients and to determine the associations with patient clinical data. We determined 10.78% (11/102) STAT3 promoter methylation frequency. Also, in our study invasive PAs showed low STAT3 methylation status. Only in 12.07% (7/58) invasive PAs, STAT3 promoter was methylated . However, the mechanism of STAT3 expression changes in PA proliferation and invasion has not been reported yet, making it necessary to further research on the pro- and anti-tumorigenic effects of STAT3 gene activation.We conducted this study to examine the prognostic significance of f tumors –26 and tf tumors . Increasf tumors –23. Moref tumors , in colof tumors , as wellf tumors . French f tumors . TherefoP < 0.001) [STAT3 mRNA expression level  made a research of STAT3 expression in somatotroph adenomas. They found that STAT3 expression was significantly enhanced in somatotroph adenomas (67% ± 5%) as compared with non-secreting pituitary tumor expression  . Also, t< 0.001) . Our resvel Fig. , making STAT3 gene promoter methylation and mRNA expression in 102 PA samples and analyzed the relationships between STAT3 gene epigenetic changes and patient clinical characteristics: age at the time of operation, gender, diagnoses of prolactinoma, acromegaly or Cushing syndrome, PA activity, recurrence and invasiveness. Our data have revealed that STAT3 gene mRNA expression is significantly lower with male gender and patients older than 60 years. However, no more significant results were found. To this purpose, our designed STAT3 gene promoter methylation analysis and mRNA expression cannot be considered as a prognostic marker in PAs or further examination including new methylation sites investigation for STAT3 methylation-specific PCR is required to estimate the influence of epigenetic changes in PAs.In conclusion, this is the first study that has demonstrated the


Aethina tumida, are parasites of social bee colonies native to sub-Saharan Africa and have become an invasive species at a global scale. Reliable Polymerase Chain Reaction (PCR) diagnosis of this mandatory pest is required to limit its further spread and impact. Here, we have developed SHB primers, which amplify for 10 native African locations and 10 reported introductions, but not for three closely related species . We also show that adult honey bee workers can be used as matrices for PCR-based detection of SHBs. The sensitivity of this novel method appears to be 100%, which is identical to conventional visual screenings. Furthermore, the specificity of this novel approach was also high (90.91%). Since both sensitivity and specificity are high, we recommend this novel PCR method and the new primers for routine surveillance of hives in high-risk areas.Small hive beetles (SHBs),  Aethina tumida Murray (Coleoptera: Nitidulidae), are parasites of social bee colonies native to sub-Saharan Africa [A. tumida feed on honey, pollen, bee larvae, as well as dead adult bees [Apis mellifera Linnaeus , n Africa . Larval ult bees ,2,3. Fee Apidae, ), which  Apidae, .Recently, SHBs have emerged as an invasive species and were first noticed in November 1996 in South Carolina in the United States . In its A. tumida and tested whether SHB DNA can be detected on adult workers of infested and non-infested honey bee colonies.Many countries have now included measures to slow down the spread of the SHB ,8. This Apis mellifera colonies from 10 native locations in Africa  and from 10 SHB introductions . Aethina concolor, Aethina flavicollis, and Aethina inconspicua specimens were sampled in Australia and South Korea. All Aethina samples were preserved in 70% Ethanol, transported at room temperature room temperature room temperature and stored at −20 °C in the laboratory until further analyses. All Aethina sampling sites covered the natural range of A. m. adansonii, A. m. capensis, A. m. scutellata, A. m. unicolor, A. m. simensis, A. m. ligustica, A. m. iberiensis, and A. m. mellifera.Adult SHBs were sampled from 20 infested A. m. adansonii) per colony were sampled from SHB infested colonies (N = 10) and non-infested colonies (N = 10) by shaking brood frames into envelopes. SHB infestation status of colonies was evaluated by visual screening of bottom boards [A. m. carnica) were also sampled from 10 colonies at two apiaries (Aare and Liebefeld). These latter samples were used as negative control templates. Collected bees were preserved in RNA later, transported at RT and stored at –20 °C in the laboratory until DNA extraction.In Nigeria, 100 adult honey bee workers . Crushed samples were homogenized with a TissueLyser  for 25 s at 20 1/s frequency using a Qiagen Retsch®MM 300 mixer mill  and centrifuged for 2000 rpm. 50 µL of the supernatant was taken from the homogenate and used for DNA extraction using innuPrep DNA Mini Kit  by following the manufacturer’s recommendations. The DNA yield and purity of each sample were checked using a Spectrophotometer Thermo Scientific™ Nanodrop 2000 .N = 100) from each colony were crushed in filter grinding bags  containing 20 mL TN buffer  and homogenized with a homex6 . Aliquots of 50 μL of the homogenate in each case were used for DNA extraction using innuPrep DNA Mini Kit  by following the manufacturer’s recommendations. After DNA extraction, a spectrophotometer Thermo Scientific™ Nanodrop 2000 was used to quantify the DNA of each pooled sample.Prior to DNA extraction collected bee samples were carefully visually screened to ensure there are no SHBs  or beetle parts. Pooled bee samples  each primer (reverse and forward), 0.125 µL (0.63 units) Taq DNA polymerase, 2 µL tenfold-diluted DNA) in a Biometra® Thermal Cycler  as follows: 95 °C for 2 min, 35 cycles of 95 °C for 20 s, 56 °C for 30 s, 72 °C for 30 s, and 72 °C for 2 min. Positive and negative controls were included in each PCR (DNA of a previously identified SHB specimen from Nigeria (based on both morphometrics and DNA sequence data or millipore water). Aliquots of the PCR products were electrophoresed on a 2 % (w/v) agarose gel. To resolve any ambiguities associated with a lack of amplification products, the universal primers LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) were used to amplify the COI region [Taq DNA polymerase inhibitors. The primer sensitivity was determined with a PCR series using the same primer concentration, but decreasing DNA concentrations .Eight SHB species-specific primer sets were developed using the exon 1 region of Cytochrome c oxidase subunit I (COI) genome (GenBank accession number: NW_017855158.1) with the online tool primer3Plus . All priI region . The detTaq DNA polymerase and 2 μL DNA (ranging from 56 to 255 ng/μL). All reactions were carried out in a Biometra® Thermal Cycler using the following cycling parameters: 95 °C for 2 min, followed by 35 cycles of 95 °C for 20 s, 56 °C for 30 s, 72 °C for 30 s, and 72 °C for 2 min. DNA of the identified SHB specimen from Nigeria (=positive control) and millipore water for molecular biology applications instead of DNA template (=negative control) were included in each PCR series. Aliquots of the PCR products (10 μL) were electrophoresed on a 2% (w/v) agarose gel. All the Swiss samples were also tested with lys-1 F (5′-ACCCGATAATTCAACGACGA-3′) and lys-1 R (5′CATTCGACCCTGGTTCATTT-3′) primers [Se) and specificity (Sp) of the visual screening and of the novel PCR method were calculated following [A = number of true positives, B = number of false positives, C = number of true negatives, D = number of false negatives.SHB species-specific primers were tested in PCR runs for all extracted bee DNAs. Conventional PCRs were carried out in a volume of 25 μL containing 15.88 μL millipore water, 5 μL 5× reaction buffer, 1 μL (0.4 μmol/L) of each primer (reverse and forward), 0.125 μL (0.63 units) of  primers  to excluA. concolor, A. flavicollis, and A. inconspicua . PCR with the specific primer pair AT430F/AT622R and series of diluted crude DNA from a single SHB specimen were used to test the sensitivity. The primer pair yielded positive results for 0.1 ng/µL of the diluted SHB DNA  and AT622R (5′-TAGTTCCACTAATACTAAGAGCCCC-3′)) proved to be reliable for SHB detection and identification, because all tested SHB individuals yielded amplification products of the expected size 193 bp  illustrates that the DNA quality was adequate and that the absence of a PCR product for the SHB primers was due to absence of : 193 bp A,B. From: 193 bp A. Simila: 193 bp B. None o: 193 bp C. The PCThe data clearly show that the developed primers reliably detect SHB in all tested populations, even at low DNA concentrations and to separate it from three species of the same genus. Since rapid detection and reliable identification of SHB is crucial to limit further distribution of this species , these dOur results also show that adult honey bee workers can be used as matrices for PCR-based detection of SHB’s DNA. The sensitivity of this novel PCR diagnostic method appears to be 100%, which is identical to conventional visual screenings . Further


Background: Diallyl trisulfide (DATS), a bioactive sulfur compound in garlic, has been highlighted due to its strong anti-carcinogenic activity.Objective: The current study investigated the molecular mechanism of garlic-derived DATS in cancer cells. Additionally, we explored possible molecular markers to monitoring clinical responses to DATS-based chemotherapy.Design: EJ bladder carcinoma cells were treated with different concentration of DATS. Molecular changes including differentially expressed genes in EJ cells were examined using immunoblot, FACS cell cycle analysis, migration and invasion assays, electrophoresis mobility shift assay (EMSA), microarray, and bioinformatics analysis.Results: DATS inhibited EJ cell growth via G2/M-phase cell cycle arrest. ATM-CHK2-Cdc25c-p21WAF1-Cdc2 signaling cascade, MAPKs, and AKT were associated with the DATS-mediated growth inhibition of EJ cells. DATS-induced inhibition of migration and invasion was correlated with down-regulated MMP-9 via reduced activation of AP-1, Sp-1, and NF-κB. Through microarray gene expression analysis, ANGPTL4, PLCXD1, and MMP3 were identified as candidates of molecular targets of DATS. Introduction of each gene to EJ cells revealed that ANGPTL4 was associated with the DATS-induced inhibition of cell growth, migration, and invasion.Conclusions: ANGPTL4 regulates DATS-mediated inhibition of proliferation, migration, and invasion of EJ cells, and thus, has potential as a prognostic marker for bladder cancer patients. Bladder cancer is one of the most common cancers of the human genitourinary system worldwide. According to the American Cancer Society, bladder cancer accounts for approximately 5% of new cancer cases, and in 2016 an estimated 77,000 cases were diagnosed, among which 16,390 patients died due to the disease in the USA [Allium sativum) is renowned as a medicinal food in traditional and alternative medicine. Epidemiological studies support a positive association between intake of Allium vegetables and low incidence of cancers [Garlic , also known as Cdc2, and its activating partner cyclin B1 [Cell-cycle progression is regulated by cell-cycle checkpoints at the G1, S, and GM phases . The G2/yclin B1 . Activatyclin B1 . In addiyclin B1 ,11. Furtyclin B1 ,13. ThusAngiopoietin-like protein 4 (ANGPTL4) is an endogenous inhibitor of lipoprotein lipase that is regulated by fatty acids through PPAR regulatory pathways . AlthougAlthough the inhibitory effects of DATS on cancer cell proliferation have been well demonstrated, the underlying molecular mechanisms remain largely unclear. In this study, we investigated the mechanism of DATS-mediated inhibition of proliferation, migration, and invasion of EJ bladder cancer cells through comprehensive analysis of signaling pathways, cell cycle regulation, and transcription factor-associated MMP-9 regulation. Microarray analysis identified ANGPTL4 as a crucial factor associated with the DATS-mediated anti-tumor effect in EJ cells.DATS (SMB00289) was purchased from Sigma-Aldrich . Antibodies were purchased from Santa Cruz Biotechnology  or Cell Signaling Technology . The nuclear extract kit and electrophoretic mobility shift assay (EMSA) gel shift kit were obtained from Panomics . cDNAs of ANGPTL4, PLCXD1, MMP3, and vectors pOTB7 and pCNS were obtained from the Korean Human Gene Bank. The human bladder carcinoma cell line EJ was purchased from the American Type Culture Collection . Detailed information on cells and materials is available in the Supporting Information.EJ cells were seeded in 6-well plates and treated with DATS  for 24 h. The cells were detached from the plates by treatment with 0.25% trypsin containing 0.2% EDTA . Fifty microliters of detached cells were mixed with 50 μL of 0.4% trypan blue (Sigma-Aldrich) by gentle pipetting, after which 20 μL of the mixture was loaded into each chamber of a hemocytometer, and the cells were counted.2 incubator, the supernatant was removed and 100 uL of DMSO was added. After incubation for 1 hr, cell proliferation was determined by measuring the absorbance at 540 nm on a microplate reader. Cell morphology was analyzed using phase-contrast microscopy.Cellular proliferation was measured by the MTT -2,5-diphenyltetrazolium bromide) assay as described previously with some modification . In briev/v)) was added dropwise and resuspended the cell pellets and stored at 4°C overnight. The cells were then pelleted and resuspended in 1 ml of 1 × PBS containing RNase A (100 μg/mL) and propidium iodide (100 μg/mL), followed by incubation at 37°C for 30 min. Cell-cycle distribution was analyzed by a flow cytometer  supplied with BD Cell FIT software.After treatment with DATS  for 24 hr, the cells were collected and washed twice with 1 × PBS. To determine cell cycle distribution, 5 mL of ice-cold ethanol (70% (5) were plated in 2 mL of medium in each well of a 6-well plate and grown to 90% confluence. The surface area of the cells was scratched with a 2-mm-wide tip. After washing with PBS three times, the plate was incubated with culture media in the presence or absence of DATS for 24 h. The rate of cells migrating into the scratched area was measured and photographed using an inverted microscope at a magnification of 40 ×.Cells (3 × 104) were suspended in serum-free medium and seeded in the upper chamber. Medium supplemented with 10% FBS was added to the lower chamber as a chemo-attractant. After 24 h of incubation, cells in the lower chamber were stained and photographed.Invasion assays were performed using an invasion assay kit  according to the manufacturer’s instructions. Cells , containing 15 uL of Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific). After incubation at room temperature for 5 min, the mixture was carefully added to the cells at 90% confluence in 60 mm culture plates and incubated for 8 hr, followed by changing medium to 1 × DMEM. After incubation for additional 16 h, cells were treated with different concentration of DATS  for the indicated period. Then, the cells were investigated by immunoblotting, FACS, wound-healing migration and Boyden chamber invasion assays, zymography, and EMSA. After incubation for the indicated period, the cells were investigated by immunoblotting, FACS, wound-healing migration and Boyden chamber invasion assays, zymography, and EMSA.After harvesting, cells were washed and resuspended in a buffer to prepare nuclear extracts for EMSA. For more details, please refer to the Supporting Information.Total RNA was isolated from EJ cells treated with or without DATS using TRIzol reagent (Thermo Fisher Scientific). RNA quality was validated by 1% agarose gel electrophoresis.Biotin-labeled cRNA for the hybridization was prepared according to the manufacturer’s recommended procedure. For detailed procedures, please refer to the Supporting Information.p < 0.05.Where appropriate, data are represented as the means ± standard errors (SEs). Data were evaluated by factorial ANOVA and Fisher’s least significant difference test where appropriate. Statistical significance was considered at 50 concentration was observed. To examine whether DATS-mediated growth inhibition is associated with cell-cycle arrest, DATS-treated EJ cells were subjected to flow cytometric analysis  and hemanalysis –h). As ctionally –h). Thes2/M-checkpoint in EJ cells, the activation of ATM, phosphorylation of CHK1/CHK2 kinases, and inactive phosphorylated forms of Cdc2 kinases were initially analyzed by immunoblotting (To examine the effect of DATS on the Gblotting ). DATS tblotting , left pablotting , left pablotting , right pblotting , right pblotting , right pSeveral lines of evidence have demonstrated that DATS leads to MAPK phosphorylation ,21 and Ain vitro wound healing and invasion assays. The wound-closure and invasive rates were reduced dose-dependently by DATS treatment , suggreatment , the gelreatment ). These p < 0.05 were regarded differentially expressed. Hierarchical clustering was employed to analyze systematic variations in mRNA expression in DATS-treated groups as compared to untreated groups. DATS treatment led to differential expression of 2,285 genes (p  <  0.05), of which 1,121 were upregulated and 1,164 were downregulated .Although the cellular mechanism of DATS has been intensively studied, genomic approaches such as microarray analysis are infrequent. We performed a comparative expression analysis of EJ cells treated with or without DATS  to identify genes associated with the mechanism of action of DATS using a microarray harboring 47,323 probe sequences derived from RefSeq genes. mRNAs with fold changes ≥1.5 or ≤-1.5 and egulated ). The geegulated  shows thd PLCXD1 . The 1NFRSF11B . FigurNFRSF11B  shows thd CLDN11  and 4. d CLDN11 . AmongBecause ANGPTL4, PLCXD1, and MMP3 were upregulated by DATS treatment, we aimed to evaluate whether introduction of these genes would affect the proliferation of EJ cells. To this end, EJ cells were transfected with ANGPTL4, PLCXD1, or MMP3 cDNA or an empty vector (EV) as a control, followed by treatment with DATS. The transfection efficiency of the three genes was confirmed by immunoblotting . As shown in To investigate whether ANGPTL4 participates in the DATS-mediated inhibitory action on migration and invasion of EJ cells, we transfected cells with ANGPTL4 cDNA or EV followed by DATS treatment. As demonstrated in 2/M phase as compared to DATS alone , we pTS alone –n). On tTS alone –i). Next ANGPTL4 ) when co ANGPTL4 ). Accord ANGPTL4 ). Next,  ANGPTL4 ), but no ANGPTL4 ). These Finally, we examined the effect of ANGPTL4 on DATS-mediated inhibition of cell migration and invasion by monitoring MMP-9 activity using gelatin zymography. MMP-9 expression in EJ cells transfected with ANGPTL4 cDNA followed by DATS treatment was further decreased at 24 h as compared to DATS treatment alone or DATS treatment of cells transfected with EV ,b). EMSAIn the present study, we investigated how DATS regulates the cellular physiology of EJ bladder cancer cells. In addition, we generated a comprehensive dataset of molecular targets of DATS using microarray technology. Finally, based on the microarray analysis, we identified ANGPTL4 as a key regulatory factor of DATS-mediated inhibition of proliferation, migration, and invasion of EJ cells.2 as well as prometaphase stages [2/M arrest in cancer cells was largely unknown. Therefore, we focused on filling in this knowledge gap using the EJ bladder cancer cell line. First, we observed that DATS treatment significantly inhibited the proliferation of the EJ cells through G2/M cell-cycle arrest. G2/M transition is regulated by Cdc2, whose activity is dependent upon its association with regulator cyclin B1 [2/M, which was dictated by the inhibition of cell growth-mediated activation of the ATM-CHK2-Cdc25c-p21WAF1 pathway, resulting in Cdc2 activation.Previous studies have shown that cancer cells treated with DATS are arrested at Ge stages ,6,7. Inte stages . A recene stages . Howeveryclin B1 . Howeveryclin B1 ,31. On tyclin B1 . In addiThe importance of MAPK and AKT signaling pathways in regulating cell proliferation under stress conditions has been widely investigated ,11. PrevIt is well known that metastasis is a major factor in disease progression, which accounts for the majority of cancer deaths ,13. PrevANGPTL4, PLCXD1, and MMP3) selected for further analysis, we found that ANGPTL4 participates in the inhibition of proliferation, migration, and invasion induced by DATS in EJ cells. Despite the fact that the effects of this protein in tumorigenesis and tumor suppression have been controversial, our data indicated that ANGPTL4 alone exhibits no effect on growth, migration, and invasion of EJ cells. Moreover, we verified that ANGPTL4 is involved in the ATM/CHK2/Cdc25c/p21WAF1/Cdc2 cascade, which led to the G2/M-phase cell-cycle arrest. We also confirmed that ANGPTL4 is a critical cofactor implicated in the MAPK  and AKT signaling pathways in the DATS-mediated growth inhibition of EJ cells. In addition, our data demonstrated that ANGPTL4 further enhanced the inhibitory effect of DATS on MMP-9 expression by further suppressing NF-κB, AP-1, and Sp-1 activities. Thus, this study identified ANGPTL4 as a novel potential regulatory factor in the suppression of proliferation, migration, and invasion of DATS-treated bladder carcinoma EJ cells.Although the inhibitory effect of DATS on the proliferation and metastasis of bladder cancer cells has been demonstrated, the involved molecular players remained largely unidentified. Therefore, we utilized microarray analysis to identify differentially expressed genes upon DATS treatment. After gene expression profiling of DATS-treated and untreated groups, we selected the 20 most highly up- and downregulated genes in the categories of BP and MF –4. TheseTaken together, ANGPTL4 might be one of the upstream regulators in DATS-mediated signaling cascades, and thus a prognostic marker for DATS-treated bladder cancer patients associated with muscle invasiveness. Further studies should examine the roles of other genes in the inhibition of proliferation, migration, and invasion of bladder cancer cells following DATS treatment.Click here for additional data file.


Flaviviral infections including dengue virus are an increasing clinical problem worldwide. Dengue infection triggers host production of the type 1 IFN, IFN alpha, one of the strongest and broadest acting antivirals known. However, dengue virus subverts host IFN signaling at early steps of IFN signal transduction. This subversion allows unbridled viral replication which subsequently triggers ongoing production of IFN which, again, is subverted. Identification of downstream IFN antiviral effectors will provide targets which could be activated to restore broad acting antiviral activity, stopping the signal to produce endogenous IFN at toxic levels. To this end, we performed a targeted functional genomic screen for IFN antiviral effector genes (IEGs), identifying 56 IEGs required for antiviral effects of IFN against fully infectious dengue virus. Dengue IEGs were enriched for genes encoding nuclear receptor interacting proteins, including HELZ2, MAP2K4, SLC27A2, HSP90AA1, and HSP90AB1. We focused on HELZ2 (Helicase With Zinc Finger 2), an IFN stimulated gene and IEG which encodes a promiscuous nuclear factor coactivator that exists in two isoforms. The two unique HELZ2 isoforms are both IFN responsive, contain ISRE elements, and gene products increase in the nucleus upon IFN stimulation. Chromatin immunoprecipitation-sequencing revealed that the HELZ2 complex interacts with triglyceride-regulator LMF1. Mass spectrometry revealed that HELZ2 knockdown cells are depleted of triglyceride subsets. We thus sought to determine whether HELZ2 interacts with a nuclear receptor known to regulate immune response and lipid metabolism, AHR, and identified HELZ2:AHR interactions via co-immunoprecipitation, found that AHR is a dengue IEG, and that an AHR ligand, FICZ, exhibits anti-dengue activity. Primary bone marrow derived macrophages from HELZ2 knockout mice, compared to wild type controls, exhibit enhanced dengue infectivity. Overall, these findings reveal that IFN antiviral response is mediated by HELZ2 transcriptional upregulation, enrichment of HELZ2 protein levels in the nucleus, and activation of a transcriptional program that appears to modulate intracellular lipid state. IEGs identified in this study may serve as both (1) potential targets for host directed antiviral design, downstream of the common flaviviral subversion point, as well as (2) possible biomarkers, whose variation, natural, or iatrogenic, could affect host response to viral infections. For these reasons, investigations have focused on exploiting antiviral properties of the host protein interferon alpha (IFN) in a DENV/human cell line model, toward identification of preclinical leads for broad-acting HDAV development. While the antiviral properties of IFN have been known since the 1950s , Zika virus (ZIKV), and yellow fever virus , or genes required for the antiviral effect of IFN against HCV . We defined positive hits as those genes for which knockdown led to 1.5-fold or more increase of percent infection, compared to negative controls, for 2 or more unique siRNAs. We found that 56 of the 120 HCV IEGs were also DENV IEGs, required for full antiviral effects of IFN against DENV  supernatant from HELZ2 knockdown cells generated more plaques than supernatant from vector controls  vs. vector control (APM) cells  for high resolution lipid analysis  possible eventual targets for host directed antiviral design and/or (2) candidate biomarkers for study in variable DENV clinical outcomes.In this study, we have performed a targeted RNAi screen seeking genes required for IFN to suppress both HCV and DENV, leading to identification of 56 HCV/DENV IEGs. These HCV/DENV IEGs included multiple nuclear receptor interactors , prompting further examination of IEG—nuclear receptor interactions. HELZ2 target gene knockdown was validated at the mRNA and protein levels, and IEG status confirmed outside of screening conditions. Impact of IFN on HELZ2 promoter and protein localization was then examined, revealing that IFN activates both HELZ2 isoforms, and IFN stimulation is associated with increased levels of nuclear HELZ2. Lifecycles assays measuring effect of HELZ2 knockdown on DENV via early-infection RNA ISH, qRT PCR, western blot, immunofluorescence, and supernatant plaque assay reveal that HELZ2 appears to exert anti-DENV activity from early infection onward. In our prior study of HELZ2 effect on HCV, we found that HELZ2 does not affect entry, as measured by a pseudoparticle assay, but does affect post-entry lifecycle stages  identified AHR interactions with DNA from genes in the triglyceride pathway  with lipase maturation factor 1, whose gene product regulates TAG metabolism through lipoprotein lipases Peterfy, , during FICZ production from tryptophan. However, there have been reports of FICZ production in skin affected by vitiligo  supplemented with 15% (volume/volume) fetal bovine serum  and 2 mM L-glutamine (Gibco). Medium was prepared on a weekly basis to maintain maximum L-glutamine activity. C636 mosquito salivary gland cells (ATCC CRL-1660) were cultured in 10% EMEM (ATCC #30-2003) supplemented with 10% (volume/volume) fetal bovine serum  and 2 mM L-glutamine (Gibco) with no antibiotics. Cells were maintained at 28°C with 5% CO2.Huh7.5.1 hepatoma cells  and HeLa MAGI cells  were maintained at 37°C in humidified air containing 5% CODengue New Guinea C 2 was purchased from ATCC (ATCC VR1584). Virus was propagated by inoculating C636 mosquito cells with 1 ml viral stock for 1 h, followed by addition of 9 ml EMEM with 2% FBS with no antibiotics, at 28°C. Viral supernatant was harvested 4–8 days following inoculation, and clarified by centrifugation at 1,500 rpm × 15 min at 4°C. Intracellular virus was harvested by scraping remaining cells into 1 ml remaining medium, and then alternately exposing cell mixture to dry ice and 37°C water bath for periods of 5 min, repeated 3–5 times, followed by centrifugation at 1,500 rpm × 15 min and aliquoted for storage at −80°C.DENV IEG Screen was performed at the Harvard ICCB-L Screening Facility. Four unique siRNA duplexes  targeting each of 120 HCV IEGs  were purchased from Dharmacon/GE Lifesciences and reverse transfections performed, as described above, in HeLa and Huh7.5.1 hepatocytes.For HELZ2 and AHR, knockdown of target gene product was confirmed at the mRNA and protein level using qRT PCR and western blot, respectively. For qRT PCR, HeLa cells were reverse transfected in 24 well dishes, followed by RNA isolation using QIAshreddor (Cat. 79654), then RNeasy RNA isolation kit including genomic DNA eliminator columns (Cat. 74134). RNA was then reverse transcribed to DNA , followed by qRT PCR using human TaqMAN probes against HELZ2 (huHELZ2 Hs 00375699) and AHR (huAHR Hs 00169233), normalized using GAPDH huGAP (Hs 02758991). For western blot, HeLa cells were reverse transfected in 24 well dishes followed by protein isolation using Cell Signaling Protein Isolation buffer with PMSF, then protein quantification using either Bradford or micro BCA method (Thermo Scientific #23235), then western blot. Western blot quantification was performed using image analysis software developed in our laboratory, GELQuant, which is available to the public upon request.Intracellular total cellular and viral RNA was isolated using QIAshredder™ (Qiagen), followed by removal of genomic DNA and RNA isolation using RNEasy plus kits (Qiagen), all according to manufacterer's protocols. RNA was stored at minus 80°C, then reverse transcribed by random priming using the High capacity cDNA reverse transcription kit (Applied Biosciences) and quantified by real-time PCR using the DyNAmo HS SYBR green qPCR kit . qRT-PCR results were interpreted using delta/delta CT (PMID 11328886), using GAPDH as an internal control. Gene specific primers are provided in Datasheet Cells were plated 24–48 h prior to drug treatment. When the cells were confluent, they were given fresh medium containing either 1,600 IU/mL interferon, 10 um fenofibrate (Sigma F6020), or no drug as a negative control. The cells were incubated in their respective medium at 37°C for 24 h.The cells were harvested using trypsin and pelleted at 500 g for 5 min. The pellets were then resuspended in ice-cold PBS and pelleted again at 500 g for 5 min. Subcellular fractionation was then performed using the Thermo Scientific Subcellular Protein Fractionation Kit [product #78840] per the manufacturer's instructions. Protein samples were stored at minus 80°C.Protein concentration was determined by Bradford assay kit (BioRad ##5000116). Twenty microliters of the least concentrated sample was then used for western blot analysis; the more concentrated samples were normalized to this first sample such that the same amount of protein was loaded for each (20 ug). Deionized water was added as needed to bring each sample volume to 20 uL. Samples were mixed with 7 uL NuPage sample reducing agent and blue dye mixture and incubated for 10 min at 80°C to denature the proteins. Samples were then run on a NuPage gel in 1x MES SDS running buffer at 180 V for 1 h, with LifeTechnologies' HiMark™ Pre-Stained High Molecular Weight Protein Standard.A tank transfer onto nitrocellulose membranes was performed using 1x NuPage Transfer buffer with 20% methanol at 4°C and 30 V for 2 h. Membranes were then blocked for 1 h at room temperature in 5% milk in 1x TBST. The membranes were then cut horizontally and primary antibodies were added, in 5% milk in 1x TBST.Membranes were incubated with primary antibodies overnight at 4°C with gentle mixing. They were then washed 5 times for 5 min each wash at room temperature with gentle mixing in 1x TBST. Following the washes, the membranes were blocked for 1 h at room temperature in their respective secondary antibodies (see diagram above) at a concentration of 1:5,000 in 5% milk in 1x TBST. Five more 5 min washes in 1x TBST were performed prior to development.ThermoScientific SuperSignal chemiluminescent substrate was required to visualize HELZ2, SP1, and Na+/K+ ATPase. For actin, tubulin, and histone H3, standard ThermoScientific ECL substrate was used. Membranes were allowed to sit in their respective substrate mixtures for 5 min before development. Proteins were visualized on blue X-Ray films; for each membrane, exposures of 2, 5, 10, 30 s and 2 min were performed. When bands appeared were too dark for quantitative analysis even after 2 s, repeat exposures were performed after allowing the membranes to sit in the dark room for several minutes to allow luminescence to fade.Bands were scanned using an Epson scanner on the color photo setting. Quantitative analysis was performed on the resulting images using an original program. Each image was 3,504 × 2,550 pixels with horizontal and vertical resolution each 300 dpi.Background color density was determined manually for each scan by selecting a rectangular portion of the image that was known not to have been exposed to any signal. Color density was calculated by dividing the total color value of the region by the area of the region.Rectangular regions containing bands were manually selected from each scan for analysis. Each selected region was divided by the program into rectangular subregions of equal width containing the bands themselves. A vertical cross-section from the center of each subregion was taken for manual analysis, and the program plotted the color values for each pixel of the cross section moving from top to bottom. In order to be considered valid for quantitative analysis, a cross-section needed to display a clear maximum color-value at some vertical coordinate; cross-sections that rose to a color-value plateau instead were considered to have saturated the X-Ray film's capacity to accurately record a signal and were quantified from a film developed with a shorter exposure.For all bands considered quantifiable, the program moved from left to right generating vertical cross-sections at every horizontal coordinate. The program then determined the peak color-value for each vertical cross section. These peak values were then averaged to produce the final “signal” value for the band. These signal values were assumed to correspond to the amount of protein generating a given band and were used in analysis.HELZ2 signal values were normalized to their respective β–actin signal values. For each compartment, the HELZ2:actin ratio for the cells that received no drug treatment was considered to be baseline, and the HELZ2:actin ratios for the interferon- and fenofibrate-treated cells were normalized to the non-treated value to produce fold-change values. Normalization was not performed between compartments as β–actin is not necessarily expected to be present in the same concentration in different subcellular compartments. When three replicates of the experiment had been performed, these fold-change values were averaged and standard deviations of the mean were calculated for the interferon and fenofibrate conditions. Fold-change values were not calculated for the cytoskeleton as HELZ2 signal was not significantly above background for these samples.Screen hits were analyzed using Ingenuity Pathway Analysis (IPA). HELZ2 promoter search for IFN stimulated response elements (ISREs) was performed using the Position Specific Scoring Matrix (PSSM) AGTTTCAGTTTC /ThioMac-LPS-Expression(GSE23622)/Homer, BestGuess:ISRE(IRF)/ThioMac-LPS-xpression(GSE23622)/Homer(1.000)10.512507 −1.194927e+02 087179.0,1722.0,789.4,104.0,0.00e+000.653653653653654 0.001001001001001 0.2292292292292290.116116116116116   0.008 0.288 0.676 0.028   0.001 0.003 0.001 0.995   0.001 0.027 0.001 0.971   0.001 0.001 0.025 0.9730.003003003003003 0.975975975975976 0.0190190190190190.002002002002002   0.583 0.007 0.24 0.170.0790790790790791 0.393393393393393 0.4714714714714710.056056056056056   0.016 0.001 0.001 0.982   0.001 0.001 0.004 0.9940.00899100899100899 0.044955044955045 0.0039960039960040.942057942057942   0.001 0.881 0.01 0.108FIMO discovered 3 ISREs in the Helz-Beta promoter at marginal significance.p-value cutoff of 0.0001 was employed, and revealed 3 ISREs in Helz2-beta promoter. No ISREs were discovered for Helz2-alpha at p-value cutoff of 0.0001. The actual corrected p-values  ranged from 0.143 to 0.581 (Datasheet A These putative ISREs are highlighted in yellow in Datasheets q = 0.581).V$ISRE_01|2058 (+)|cAGTTTCGATTCtgg (q = 0.143).V$ISRE_01|5166 (−)|tggAAAGTGAAATTg  of long isoform, −19,832/+23 bp of short isoform] were inserted into KpnI/NheI sites of the pGL4.10 luciferase reporter vector (Promega) employing a recombinant engineering strategy as previously described  and Protein A beads  were then prepared in quantity adequate for 3.5 ul multiplied by (the number of samples multiplied by number of Ab per sample + 0.5). For example, for two Ab, two samples, 2 × 2 = 4 + 0.5 = 4.5 × 3.5 ul = 15.75 ul protein G beads. Protein G and A beads were combined into a single microfuge tube, followed by addition of 1 ml of 0.5% BSA, then placement onto magnet. Bead mixture was then divided into separate microfuge tubes for each antibody (500 ul/tube), followed by addition of 500 ul more BSA to each tube. Ten micrograms of appropriate antibody was then added to each tube and samples placed at 4°C shaker for 5–6 h. Antibodies used were HELZ2 (abcam 129781), AHR (enzo BML-SA550-0100), and isotype control .Each 10 cm dish was then rinsed in 1xPBS, trypsinized, then cells centrifuged at 2,000 rpm for 5 min, followed by aspiration of trypsin and pellet resuspension in cell signaling lysis buffer . Each sample was then incubated on ice for 30 min with vortexing every 10 min. Samples were then spun at 16,000 rcf at 4°C for 10 min, followed by collection of supernatant containing protein for further processing. Protein samples, maintained at all times on ice, were then quantified by the uBCA method.Beads were then retrieved from 4°C rotator, placed on magnet and washed twice with 1 ml 0.5% BSA. Four hundred microliters CHiP incubation buffer was then added to each tube, and 200 ul of each Ab:bead mixture distributed to a new tube for incubation with the appropriate protein sample. One hundred micrograms of the appropriate protein was then added to each Ab:bead mixture, followed by filling to 1,500 ul total volume per tube, with CHiP incubation buffer. Samples were then incubated overnight at 4°C on shaker.The following morning, samples were retrieved from 4°C, then washed thrice for 5 min at 4°C in 1x PBS with 0.5% triton x-100. Beads were then replaced onto magnet, followed by aspiration of wash, then addition of 100 ul Laemmli buffer (Laemmli-BOLT LDS-#B0007 Novex), then heating to 100°C for 8 min then loading of 20 ul of each IP lysate for western blot. During wash steps, original protein samples were retrieved from −80 and 20 ug prepared for gel loading , and heated to 100°C for 8 min. Western blot was then processed as above.1% triton x-100, 2 mM EDTA, 150 mM NaCL, 20 mM tris-HCL, pH 8.1.ITE was obtained from Tocris (# 1803) and diluted to 10 um in DMSO. FICZ was obtained from ENZO (# BML-GR206-0100) and diluted to 0.3517 um in DMSO. Both reagents were stored at −20°C. Huh7.5.1 or HeLa cells were plated at 4,000 cells per well in 96 well black clear bottomed plates (Corning Costar #3603), followed 24 h later by treatment with AHR agonist with or without IFN for 24 h, then addition of virus concentrate (25 ul) on top of medium, then 48 h incubation, then fixation, permeabilization, and staining for imaging/analysis for percent infection.Huh7.5.1 cells were plated in 10 cm dishes overnight then were treated with mock or IFN 1,600 IU/l for 24 h, followed by harvest of supernatant and cells for LC MS according to published methods for Intracellular Metabolite Extraction/Lipid Method  for 10 cm Dish  for 1–2 s. H2O was then siphoned off. Plates were then inverted , and Liquid Nitrogen poured directly onto under-surface of plate using Liquid Nitrogen-resistant basin. Liquid Nitrogen was then allowed to evaporate (5–10 s). Plate was then turned and 5 ml of ice-cold 2-Propanol  poured to evenly cover surface of 10 cm plate, which was left for approximately 15 s. Samples were then aspirated 3 × 1.25 ml into 3, respectively, labeled sample tubes .Plates were 1.25 ml was then aspirated into collective  15 ml tube (pooled lysate), and after mixing , 1.25 ml aspirated each into tubes labeled as “PL (pooled lysate) 1, PL2” etc. . Samples were then spun at 14,000 rpm × 10 min at 4°C, then aliquotted in aliquots of 1 ml into conical Eppendorf tubes, labeled as above, transferred on dry ice, spun in a speedvac (Thermo Scientific) to dry down, and resuspended in 100 ul Lipid IS-IS. Finally, 65 ul was then placed into glass tubes for mass spectrometry.Deproteinized extracts were subjected to reverse phase chromatography using a HPLC C4 column (Grace) and mobile phases . The samples were injected directly onto the C4 column that was eluted at a flow rate of 350 μL/min with initial conditions of 80% mobile phase A and 20% mobile phase B for 2 min, followed by a 1 min linear gradient to 20% mobile phase A, then a 12 min linear gradient to 0% mobile phase A, maintained for 10 min, then a 1 min linear gradient to 80% mobile phase A, maintained for 9 min. Injection volume was 10 ul. The multiplexed liquid chromatography (LC) system comprised of a 1,200 Series pump (Agilent Technologies) and an HTS PAL autosampler (Leap Technologies) equipped with two injection ports and a column selection valve.The LC system was connected to a 4,000 QTrap triple quadropole mass spectrometer (Applied Biosystems/Sciex) run in positive ion mode, in full scan mode (Q1 MS). MS analyses were carried out using electrospray ionization (ESI).Metabolite peaks were integrated using Sciex MultiQuant software. All metabolite peaks were manually reviewed for peak quality in a blinded manner. In addition, pooled cellular extract samples were interspersed within each analytical run at standardized intervals every 10 injections, enabling the monitoring and correction for temporal drift in mass spectrometry performance. The nearest neighbor flanking pair of pooled plasma was used to normalize samples in a metabolite-by-metabolite manner. Internal standard peak areas were monitored for quality control and individual samples with peak areas differing from the group mean by more than two standard deviations were reanalyzed.Huh7.5.1 cells were plated in 10 cm dishes at 80–90% confluence then treated with IFN or mock for 24 h, followed by nuclear extraction for CHiP seq.Preblock and binding of antibody to magnetic beads. Dynal beads were washed in block solution then resuspended in block solution with antibody followed by overnight incubation at 4°C. Beads were then washed in block solution then resuspended in 0.5% BSA/sample. Nuclei Preparation. Cross-linked cells were thawed on ice, followed by isolation of nuclei using Covaris kit according to manufacturer's protocol, then shearing . Post-Shearing. Sheared samples were transferred into cold 1.5 mL Lo-bind microcentrifuge tubes and centrifuged followed by transfer of supernatant containing sheared chromatin to a new cold 1.5 mL Lo-bind tube, removal of ~50 μL for Whole Cell Extract and storage at minus 20°C, and removal of ~900 μL for IP or storage at minus 80°C. ChIP. Antibody/Bead Solution was then added to each IP sample, followed by overnight incubation at 4°C. Crosslink Reversal: Beads were then precipitated with dynamag, then washed with Low salt buffer + protease inhibitor then High Salt buffer + protease inhibitor then 1X LiCl + protease inhibitor then 1X TE Buffer + 50 mM NaCl (100 uL/10 ml) followed by elution at 65°C for 45 min, with vortexing every 5 min, then centrifugation (to spin down beads) then removal of supernatant and transfer to new lo-bind microcentrifuge tube, and reversal of crosslinks at 65°C for 6 h. Digestion of Cellular Protein and RNA was then performed using RNase A followed by Proteinase K. DNA was then extracted with 400 μL Phenol:Chloroform:IsoamylOH. DNA concentrations were measured with Qubit.Sequencing was performed on Illumina HiSeq 2,500 instrument, resulting in ~40 million single-end 50 bp reads per sample. Reads were aligned against the hg19 reference genome using BWA  selected based on manual inspection of resulting peak calls. Regions of significant enrichment were generated by merging adjacent significantly enriched windows separated by 1 Kb or less. In addition, we called broad regions of enrichment using the peak caller SPP , we also compared input-normalized coverages over all annotated TSS ±1 Kb windows. This resulted in the identification of additional regions of enrichment not previously discovered through peak calling alone.t-test  was used for determination of statistical significance.IEG hits were selected based upon 1.5x rescue of DENV infection, compared to non-targeting control, on 2 or more plates, for 2 or more siRNA duplexes. Out-of-screen confirmation siRNA transfection data was compared using means with standard error of the means (SEM). qRT PCR data was analyzed using the δδCT method, and data was compared using means with SEM. Students This study was carried out in accordance with the recommendations of American Association for Laboratory Animal Care. The protocol was approved by the MGH IACUC Committee .DF, HP, DC, MB, JO, TT, SY, SK, SC, XS, and CY designed and performed the experiments. DF, HP, WL, DC, MB, KJ, AA, RS, JO, RG, TT, SY, TS, SK, SC, CY, XS, and RC analyzed and interpreted the data. DF wrote the manuscript then HP, WL, DC, MB, KJ, AA, RS, JO, RG, TT, SY, TS, SK, SC, XS, CY, and RC performed critical review and edits of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, these compounds have not been compared on an unbiased, raw-signal level. Here, we directly compare several in vivo SHAPE acylation reagents using the simple primer extension assay. We conclude that while multiple SHAPE technologies are effective at measuring purified RNAs in vitro, acylimidazole reagents NAI and NAI-N3 give markedly greater signals with lower background than 1M7 for in vivo measurement of the RNA structurome.Recent advances in SHAPE technology have converted the classic primer extension method to next-generation sequencing platforms, allowing transcriptome-level analysis of RNA secondary structure. In particular, icSHAPE and SHAPE-MaP, using NAI-N RNA molecules have the inherent ability to fold into complex structures through base-pairing and three-dimensional interactions, allowing them to interact with all types of molecules, while still containing genetic information . For decDimethylsulfate (DMS) RNA methylation, for example, is used to identify base-paired adenosine and cytosine nucleotides . DMS hasIn contrast, SHAPE (selective 2′-hydroxyl acylation and primer extension) is unique in that it interrogates all four nucleobases, enabling structure measurements at single-nucleotide resolution. Furthermore, there has been a recent expansion of SHAPE to the intracellular context. For the most part, this has been the result of novel chemical and biochemical designs that enable robust RNA modification inside cells. For these reasons, SHAPE is now regarded as a gold standard chemical strategy to measure RNA secondary structure . Chemica3, while SHAPE-MaP uses 1M7 or NMIA  and the 5S ribosomal RNA (rRNA). All four electrophiles generated robust cDNA truncations for these RNAs in vitro . A major difference between the in vivo and in vitro conditions for SHAPE mapping are the cell walls and membranes encasing the transcriptomes of these organisms. We therefore hypothesized that lack of 1M7 signal from in vivo experiments comes from the impermeability of 1M7 to cell barriers, and therefore, permeabilization of these barriers would increase 1M7's acylation signal. To this end, we compared the SHAPE signal of mouse ES cells modified with NAI-N3 and 1M7 in “intact” and “permeabilized” conditions, effectively live and dead cells, respectively. Briefly, to permeabilize cells we incubated them in a dilute, nonionic detergent (0.05% NP-40) for 5 min at 25°C. While NAI-N3 samples produced comparable SHAPE signals between the intact and permeabilized conditions, 1M7 modified samples had increased signal after cell permeabilization  as compared with NAI (−1.3) and NAI-Nlization C. 1M7 molization C. These By implementing a traditional approach to interpreting SHAPE modification, we were able to directly compare the RNA acylation capacity of four SHAPE electrophiles on in vivo and in vitro modified RNA. FAI and NAI derivatives were specifically designed and synthesized to have properties amenable for labeling of RNAs inside living cells, such as longer half-lives and higher solubilities . Comparitrans-acting RNAs and proteins  supplemented with 15% FBS, 1% PenStrep, 1% MEM NEAA, 1% GlutaMax, 0.2% β-mercaptoethanol, and 0.01% LIF. HST08 E. coli cells were grown in LB medium with ampicillin at 37°C.V6.5 mouse embryonic stem cells were grown at 37°C with 5% COE. coli RNA was extracted by bacterial lysis as stated in 2, 100 mM NH4Cl, 4 mM β-mercaptoethanol, and 16% (wt/vol) sucrose supplemented with 15 µL of 50 mg/mL lysozyme.Mouse ES cell RNA was collected from cells by TRIzol and chloroform preparation, followed by RNeasy Mini column (Qiagen). 2, and 100 mM NaCl. Samples were then modified for 5 min at 37°C with either the DMSO control or the SHAPE reagents FAI, NAI, NAI-N3 at 100 mM final or 1M7 at 10 mM final. Modified RNA was then cleaned up and eluted in pure, RNase-free water using RNeasy Mini columns (Qiagen) before primer extension.Purified RNA from both mouse and bacteria cells was folded by a 95°C denaturation, a quick cool to 4°C, followed by 5 min incubation at 37°C in 100 mM HEPES pH 7.5, 6 mM MgCl3, 10 mM 1M7, and 100 mM 1M7. Cells were modified for 5 min at 37°C. Media were removed, and RNA was purified by TRIzol and chloroform preparation followed by RNeasy Mini Column (Qiagen).RNA was modified and collected from mESCs using the same conditions as previously published for mammalian cell modification . Old medE. coli cells were modified with SHAPE reagent using the conditions as written in 3 at 30 mM and 1M7 at 5 mM, all with a final DMSO concentration of 3% (v/v) in LB. Modified RNA was extracted from bacteria by lysis as described earlier, followed by TRIzol chloroform preparation and RNeasy Mini column (Qiagen).Mouse U1 snRNA: 5′-CCCACTACCACAAATTATGCAG-3′Mouse 5S rRNA: 5′-AAAGCCTACAGCACCCGGTAT-3′E. coli 5S rRNA: 5′-TGCCTGGCAGTTCCCTACTC-3′32P using OptiKinase and [γ-32P]–ATP. RNA was reverse transcribed into cDNA with SuperScript III at 55°C for 25 min. After cDNA synthesis, the remaining template RNA was degraded by adding NaOH and heating for 5 min at 95°C. The samples were then run across a 35-cm-long 8% Urea PAGE gel. The gel was dried for 2 h on a vacuum gel drier and then exposed for 24 h.RT primers were synthesized by IDT, with basic desalting purification, and then radiolabeled with The signal-to-background (S/B) ratio reported for each lane is the arithmetic mean of the ratios  of three bands. Signal is defined as mean pixel intensity of a band as measured by ImageJ . Background is defined as mean pixel intensity of a low intensity region next to the signal band. The bands were in the same position for all lanes within a blot and were chosen based on presence in the greatest number of SHAPE modified condition lanes , but not in the unmodified  condition.