To provide an applicable method to remove m6A modification at specific loci, an m6A editing system called “targeted RNAdemethylation by SunTag system (TRADES)” is engineered. In this system, the targeting element dCas13b is fused to ten copies of GCN4 peptides which can recruit multiple scFv‐fusion RNA demethylase to demethylate the target m6A site. Owing to this design, TRADES is more flexible to the indistinct m6A sites for its wide editing window. By site‐specific demethylation of messenger RNA (mRNA) SON A2699, the lifetime of SON RNA is successfully prolonged in HeLa cells. Meanwhile, TRADES negligibly influences the lifetime of other non‐targeted transcripts. TRADES also can regulate the gene expression of target transcript in an m6A‐dependent manner. Moreover, the interference occuring for HBV and HIV replications demonstrates that the TRADES system holds potential in viral life cycle regulation and clinical applications.N6‐methyladenosine (m 6A) demethylase conjugates system enables the site‐specific demethylation of m6 Ain target RNA. It provides convenience to the functional research for its wide editing window and good on‐target capability. Targeted RNA demethylation by the SunTag System successfully regulates the RNA stability, gene expression, and viral life cycles in cells through an m6A‐dependent manner.A dCas13b‐SunTag and N6‐methyladenosine (m In general, m6A modification tends to occur at the consensus RRACH motif  and enrich in the vicinity of stop codons of mRNAs. Owing to the findings of m6A demethylases FTO and ALKBH5 which endow m6A with dynamic features, m6A is rapidly being studied and uncovered to play a significant role in various biological processes such as stem cell self‐renewal and differentiation, control of heat shock response, and circadian clock controlling, as well as in RNA fate and functions such as mRNA stability, alternative splicing, mRNA nuclear export, translation, primary microRNA processing, and secondary structure switching. Further investigations also indicate that m6A is related to cancer and immune system.As the most abundant internal (non‐cap) modification in mRNA and lncRNA of all higher eukaryotes, N6‐methyladenosine  system has been widely used in nucleic acid engineering which showed high potential to address this requirement. The DNA‐targeting Cas protein, the family of Cas9, can be programmed to aid the modification and detection of DNA in their endogenous environment. Moreover, an inactivated version of Cas9 (dCas9) nuclease which retains the on‐target ability with sgRNA, but do not break double‐stranded DNA, can be fused with other functional enzymes to facilitate the site‐directed modification of the genome. The RNA‐targeting Cas proteins, for example, the Cas13 family proteins, have provided a simple and effective way to study and regulate endogenous RNA transcripts. The nuclease LwaCas13a can be programmed to degrade target RNA transcripts efficiently. An in vitro nucleic acid detection platform called “SHERLOCK” also took advantage of the great cleavage activity of Cas13a. Meanwhile, catalytically inactive PspCas13b (dCas13b) fused to ADAR2 deaminase domain (ADAR2DD) was used to edit reporter and endogenous transcripts.However, most of current research which relies on globally affecting mironment.24 Mor6A enzymes was developed for programmable m6A editing, which opens a new era to elucidate the precise role of RNA modification. Rcas9 was also chosen as the RNA‐targeting protein to realize sequence‐specific m6A demethylation. However, these technologies still need to be improved for two reasons. First, additional PAMer DNA with multiple 2′‐OMe modifications should be co‐transfected via two kinds of transfection reagents that increase the cost and complexity. Second, the distinct m6A distribution is still lacking for the imperfect m6A sequencing methods, which results many difficulties for sgRNA design in some low‐resolution sites using the fused Cas9‐m6A enzyme conjugates with fixed linker. Another approach to manipulate RNA methylation status on specific loci is to engineer a fusion between m6A enzymes and the customizable RNA binding protein PUF, which also suffered from the indistinct m6A distribution.Recently, the fusion of CRISPR‐dCas9 and mfication.29 Rca6A editing toolkit, here we developed a method named “targeted RNAdemethylation by SunTag system (TRADES)” by using dCas13b, RNA demethylase and SunTag system. SunTag, containing 10 copies of GCN4 peptide which can efficiently recruit scFv‐fusion effector protein, is a signal amplification system. It has been successfully applied in fluorescence imaging, targeted demethylation of specific DNA loci and base editing at endogenous DNA sites. The base editing method named “BE‐PLUS” revealed that SunTag system could increase editing window and enhance fidelity, which encourages us to combine this system with RNA epigenetic editing technology. We found that the TRADES system shows wider editing window than direct dCas9‐effector protein conjugates. Thus, the TRADES may be more flexible and potential to target the indistinguishable m6A sites. We demonstrated that the TRADES could regulate the stability and expression of target RNA in an m6A‐dependent manner. Moreover, the interference occurs for HBV and HIV replications demonstrates that the TRADES system holds the potential in viral life cycle regulation and clinical applications.For these limitations and the need to expand the RNA m22.1Figure 6 short peptide linker was anchored between two function proteins. The linker is critical to adjust the conformation of the recombinant protein to ensure protein activity. Finally, the sgRNA sequence was cloned into pc0043 vector which has U6 promoter. The sgRNA designates the target site through hybridizing to the bases around it and recruiting the dCas13b‐GCN4 conjugates. In each experiment, a panel of sgRNAs with varied spacers was screened toward each different target RNA. Spacer is the region of the sgRNA that hybridized to the target sequence . Previous work has shown that m6A demethylation caused by FTO is prominent in the cell nucleus. As the proportion of cytoplasmic FTO increases, m6A demethylation in HeLa decreases. So nuclear localization signal (NLS) sequence was cloned into dCas13b‐GCN4 and scFv‐effector protein‐GBI.We engineered the TRADES system consisting of three parts Figure 1A. Firstecreases.34 So All plasmids were transfected into HeLa cells and their expressions were verified. The western‐blot results showed that these recombinant proteins could be expressed in HeLa cells . The nuclear localization of these recombinant proteins was investigated using western‐blot from the separated nuclear and cytoplasmic fractions. Because Lamin B is located at nuclear while alpha‐Tubulin mostly located in cytoplasm. Thus, Lamin B and alpha‐Tubulin were selected as references for the separation of nuclear and cytoplasm. As shown in Figure S3, Supporting Information, these recombinant proteins were located in the nuclear mostly.x‐axis represents distance along the line and the y‐axis is the pixel intensity. As shown in Figure S4B, Supporting Information, the GCN4‐dCas13b has a good co‐localization with scFv‐GGS6‐ALKBH5. Furthermore, we conducted the co‐immunoprecipitation (Co‐IP) experiments to confirm that GCN4‐dCas13b could specifically capture scFv‐GGS6‐effector proteins in transfected cells. Briefly, we transfected HeLa cells with GCN4‐dCas13b and ScFv‐GGS6‐FTO or ScFv‐GGS6‐ALKBH5 as indicated. The co‐transfection ratio of GCN4‐dCas13b and scFv‐effector protein‐GB1 is 1:1. Two days after transfection, we collected cells to carry out the Co‐IP experiments. As shown in Figure S5A,B, Supporting Information, GCN4‐dCas13b did capture the scFv‐fused demethylases, FTO and ALKBH5. Moreover, the flow through (FT) of the Co‐IP experiment was collected and analyzed by western‐blot. As shown in Figure S5C,D, Supporting Information, the FT from the anti‐His (His‐GCN4‐dCas13b) Co‐IP showed negligible free scFv fused demethylase while the FT from the anti‐IgG (negative control) Co‐IP contained lots of scFv fused demethylase, which re‐confirmed that the scFv‐GGS6‐effector proteins were recruited by GCN4‐dCas13b.To investigate whether GCN4‐dCas13b conjugates could recruit scFv‐effector proteins, the co‐localization of GCN4‐dCas13b and scFv‐effector protein was identified by immunofluorescence staining. The results showed that the dCas13b‐GCN4 (red) had a co‐localization with the scFv‐effector proteins (green). DAPI nuclear stain (blue) confirmed the nuclear localization of these recombinant proteins . By using the plot profile tool of Image J software and Co‐localization Finder plugin, we have displayed two‐dimensional graphs of the intensities of pixels along a line within the merge image. The 2.26A sites were chosen to verify the feasibility of TRADES.We then evaluated the demethylation effect of the TRADES method on the endogenous transcripts. 28S ribosome RNA (rRNA) and mRNA SON transcripts with precise m6A level of rRNA, which indicated that rRNA is not the major substrate of ALKBH5. So FTO was chosen to be the demethylation element for 28S rRNA. The linker of the demethylation part and the spacer length of sgRNA were investigated first. After TRADES treatment, the A4220‐containing fragment in 28S rRNA was isolated from total RNA and digested for LC‐MS/MS analysis followed the published protocol. The results showed that scFv‐XTEN‐FTO had a much better demethylation efficiency on 28S rRNA fragment than that of scFv‐GGS6‐FTO. The difference may due to the influence of linker on the protein structure and activity. So scFv‐XTEN‐FTO and sg1‐28S rRNA were picked out for further experiments . And we compared the demethylation effect of GCN4‐dCas13b and dCas13b‐GCN4 version TRADES to 28S rRNA. As shown in Figure S6B, Supporting Information, GCN4‐dCas13b collaborated better with scFv‐GGS6‐FTO than dCas13b‐GCN4. On the other hand, a base‐editing tool called BE‐PLUS which also utilized the Sun‐tag system showed that GCN4‐dCas9 performed more effective than dCas9‐GCN4. Based on our initial result and previous report, we chose GCN4‐dCas13b for further experiments in this study. As shown in Figure 6A level of A4220‐containing fragment decreased by 25% with TRADES system compared to direct FTO over‐expression without sgRNA and mutant FTO control (FTOH231A D233A) with sgRNA. While the m6A level of total RNA in all treatment groups barely changed  the single‐base elongation activity of DNA polymerases and 2) the nick ligation efficiency of ligases. As a result, the less m6A modification would cause higher relative ligation products. Moreover, SELECT could detect the m6A level of transcripts with low abundance because it employs qPCR for amplification and quantitation.For mRNA and lncRNA, which are less abundant compared with rRNA, the m ligases.36 As 6A modification sites which stand closely. The target A2699 and adjacent A2759, A2789 and A2819 in SON gene were all fit for RRACH motif  group has the best demethylation effect to SON A2699 than other groups. So scFv‐GGS6‐FTO and sg4‐SON were selected for further experiments. Then, the m6A level of target A2699 and adjacent A2759, A2789 and A2819 were measured by SELECT after TRADES treatment. As shown in Figure 6A level on A2699 and A2759. The demethylation effect of Mut (sg‐SON) on SON A2759 is close to WT (sg‐SON). We speculated that the unanticipated demethylation effect of FTO‐inactive TRADES to SON is caused by endogenous m6A demethylase. Upon binding to sgRNA, the secondary structure of SON RNA may change to a status which is more convenient for the access of endogenous m6A demethylase. For the SON A2789 and A2819, we detected them in a mixed manner because the sequences of the Up Probe and Down Probe of these two sites are the same in SELECT detection. As shown in Figure 6A ratio of adjacent SON A2789 and SON A2819 barely changed. Altogether, these results showed that TRADES was effective on SON A2699.Messenger RNA SON A2699 was selected as target site of TRADES. SON has multiple mf Figure . First, 2.36A‐seq and RNA‐seq were performed to evaluate the on‐target ability and demethylation effect of TRADES on SON. To investigate whether the m6A peaks we identified contain the m6A consensus sequence of RRACH, we analyzed the top 996 significant peaks and found such motif in 882 peaks . Moreover, m6A peaks were enriched in the vicinity of the stop codon . Figure S7C, Supporting Information, showed a significant correlation of gene expression level between two biological replicates. Collectively, these indicated the high quality of our m6A‐seq and RNA‐seq data. As shown in Figure S8, Supporting Information, pair‐wise comparison of m6A abundance and gene expression level showed few differences which suggests that TRADES has few effects to non‐targeted genes. Moreover, the m6A coverage of WT (sg‐SON) at targeted SON A2699 site showed a strong decrease compared to the control which indicates the great demethylation effect of TRADES . Collectively, TRADES exhibited the capability of site‐specific demethylation and showed low effects toward other untargeted genes.m6‐ALKBH5 (sg4‐MALAT1) had the best demethylation rate compared with others . So sg4‐MALAT1 and scFv‐GGS6‐ALKBH5 were selected for further analyses. There are three known m6A sites  on lncRNA MALAT1 transcript from HeLa  and mutant control (ALKBH5H204A). While the methylation status of nearby m6A sites barely changed after TRADES‐based editing of A2577.We then utilized SELECT to measure the demethylation effect of TRADES to lncRNA MALAT1 A2577. We screened a panel of sgRNAs targeting MALAT1 A2577 and the effector proteins. The results showed that scFv‐GGSa Figure 2A. To de6A enzyme conjugates, we then designed three sgRNAs with 2, 12, 30 nucleotides away from MALAT1 A2577 site, respectively  “reader” protein. Upon binding to YTHDF2, mRNA from the translatable pool transferred to mRNA decay sites, such as processing bodies. SON, which was known to be m6A modified and had been validated for binding by YTHDF2, was selected as the target gene to investigate whether its stability could be influenced after TRADES treatment.mg bodies.11 SONFigure  The lifetime of SON has a larger fold change by TRADES treatment than YTHDF2 knockdown samples. Then, the results of RNA lifetime profiling were verified by RT‐qPCR. The transcription of WT (sg‐SON), WT (sg‐control), and Mut (sg‐SON) were inhibited with actinomycin D at the same time points as indicated. Indeed, demethylation of the target RNA prolonged the half‐life of SON transcript than these two controls (WT (sg‐control) and Mut (sg‐SON)), which further confirmed the effect of TRADES system . To determine the on‐target ability of TRADES, the pairwise comparison of genes halftime under WT (sg‐Control) and WT (sg‐SON) treatment was conducted. The results revealed that TRADES negligibly influenced the lifetime of other non‐targeted transcripts  group compared with WT (sg‐Control) from RNA‐seq data Figure 3A, whichFigure 9 The s Figure . The cums Figure . While s So, we sought to investigate whether targeted demethylation of ASB2 by TRADES system could influence its expression. Briefly, the dual‐luciferase system which contains firefly luciferase (Fluc) reporter bearing ASB2 3′‐UTR that have intact or mutant m6A sites and renilla luciferase reporter as the control were used as targets for demethylation treatment , and A1907 site contains m6A methylation. This m6A site is encoded in the lower stem of the epsilon stem loop present in all HBV transcripts and is conserved across all known HBV genotypes.Encouraged by the results of programmable demethylation in various kinds of RNA, we intended to investigate the capability of TRADES to viral RNA. HBV is a DNA virus that completes its life cycle through an RNA intermediate called pregenomic RNA (pgRNA). The previous report has shown that there is a distinct menotypes.236A abundance of HBV A1907 in treatment and control groups by SELECT. As shown in Figure 6A were reduced by sgRNA targeting HBV than that of control. To investigate the influence of TRADES to HBV replication, cellular circular DNA (cccDNA) was isolated and quantified by micro‐drop digital PCR analysis. As shown in Figure H231A D233A transfection groups. Collectively, these results suggest that m6A methylation of HBV transcripts negatively regulates HBV cccDNA replication, which is consistent with the conclusions of previous report. Thus, we experimentally determined that our method of site‐directed removal of m6A modification from HBV pgRNA could modulate HBV replication level.To determine whether TRADES could affect HBV replication through epigenetic regulation, we applied TRADES to HBV‐expressing cells. Then we calculated the ms report.23 Thu So we wondered whether TRADES could show potential clinical application to inhibit viral replication by defined m6A demethylation. First, we designed a panel of sgRNAs to HIV‐1 HXB2, which has the m6A sites in RRE RNAs. Then, HEK293T  cells were infected with the HIV‐1 HXB2 virus. After 12 h infection, the cells were transfected by plasmids in the TRADES system. Next, viral replication was monitored by western‐blot analysis of p24. As shown in Figure 6A level of HIV HBX2 was substantially reduced by TRADES compared to direct ALKBH5 overexpression without sgRNA(WT(sg‐Control)) and mutant control (Mut (sg‐HIV)). Thus, we speculated that the influence of TRADES to HIV replication was due to its site‐specific demethylation effect to HIV HBX2. Altogether, these results suggest that TRADES exhibited high potentiality in some clinical applications especially viral infections.Intriguingly, previous research demonstrated that ALKBH5 silencing caused a striking increase in viral replication of HIV.21 So 36A plays an important role in various biological processes as well as in RNA fate and functions. However, most of current research investigate the function of m6A modification through affecting m6A level globally and permanently. So we still lack a flexible approach to remove m6A with spatial and temporal specificity. Recently, some effective RNA m6A editing methods were reported by CRISPR‐dCas9 conjugates and other programmable RNA binding proteins, but with sufficient space for improvement.As the most abundant RNA modification, mproteins,31 but6A‐dependent manner. Moreover, targeted demethylation of HBV pgRNA sufficiently promoted the replication of HBV. Site‐specific demethylation of HIV‐1 HXB2 inhibited the replication of HIV. These studies demonstrate that TRADES system holds the potential in viral life cycle regulation and clinical applications.In this study, we developed and validated a programmable RNA site‐specific demethylation system–TRADES that relies on the fusion of dCas13b and ten copies of GCN4 peptides which could recruit multiple scFv‐fusion RNA demethylase to demethylate RNA transcript at specific loci. TRADES features wide editing window and good on‐target ability. By site‐specific demethylation of SON, we successfully prolonged the lifetime of SON RNA in HeLa cells. Meanwhile, TRADES negligibly influenced the lifetime of other non‐targeted transcripts. TRADES also could regulate the gene expression of target transcript in an m We expected that other programmable RNA‐guided RNA target effectors with smaller size and low immunogenicity may overcome these limitations. Moreover, directed evolution of the effector protein perhaps endows TRADES with higher activity.Several optimizations remain with the current TRADES system. Such as the size of dCas13b is too large for viral packaging and protein delivery. And continuous delivering of dCas13b which has the bacterial origin may cause immune response.39 We 6A at specific loci in endogenous environment. Because of the excess GCN4 peptides to eliminate the unrecruited effector protein, TRADES may cause less perturbation to other endogenous RNA transcripts than forced expression of effector protein alone. Compared to the dCas9‐demethylase conjugates, TRADES showed wider demethylation window. This difference could be due to the recruitment of multiple copies of demethylase. Based on this, TRADES is more flexible to the indistinct m6A sites for its wide editing window, which indeed expanded the RNA m6A editing toolkit. Collectively, the efficiency and wide editing window of TRADES suggests that TRADES may be a useful strategy for functional research and clinical application.Generally, TRADES provided a convenient tool to study the functional importance of m4H231A D233A) were amplified from pcDNA3‐FTO and pcDNA3‐mutFTO by PCR  and then subsequently cloned into psT1374‐scFv‐APOBEC‐UGI‐GB1 to produce psT1374‐scFv‐XTEN‐FTO (WT or Mut)‐GB1 or psT1374‐scFv‐GGS6‐FTO (WT or Mut)‐GB1.The wild type (WT) FTO coding region sequence (CDS) and mutant (Mut) FTO‐CDS (FTO6‐GB1 to produce psT1374‐scFv‐GGS6‐ALKBH5 (WT)‐GB1 by ClonExpress II One Step Cloning Kit . ALKBH5H204A mutation was introduced by site‐directed Mutagenesis Kit.The wild type ALKBH5‐CDS was amplified from pPB‐ALKBH5 by PCR and then subsequently cloned into psT1374‐scFv‐GGSFor the co‐localization and Co‐immunoprecipitations assay, the His tag in pst1374‐scFv‐gene‐GBI plasmids was replaced by the Cut‐His sequence (Table S1).6 linker Oligo DNA purchased from GeneCreate Co., Ltd. was annealed and then inserted into pFlag CMV2 dPspCas13b‐delta984‐1090 to produce pFlag CMV2 dPspCas13b‐delta984‐1090‐GGS6. The GCN4 DNA sequence was amplified from psT1374‐dCas9‐GCN4 by PCR and then cloned into pFlag CMV2 dPspCas13b‐delta984‐1090‐GGS6 to produce pFlag CMV2 dPspCas13b‐delta984‐1090‐GGS6‐GCN4. The location GCN4 and dPspCas13b‐delta984‐1090 in pFlag CMV2 dPspCas13b‐delta984‐1090‐GGS6‐GCN4 was also exchanged to produce pFlag CMV2 GCN4‐GGS6‐dPspCas13b‐delta984‐1090. For the co‐localization assay, the Flag tag in pFlag CMV2 dPspCas13b‐delta984‐1090‐GGS6‐GCN4 and pFlag CMV2 GCN4‐GGS6‐dPspCas13b‐delta984‐1090 was replaced with His tag. For cloning the guide RNAs, the top and bottom oligonucleotides were annealed and inserted into pc0043 vector.The dPspCas13b‐delta 984‐1090 DNA sequence was amplified from pC0050‐CMV‐dPspCas13b‐longlinker‐ADAR2DD (WT) by PCR and then cloned into pFlag‐CMV2 vector to produce pFlag CMV2 dPspCas13b‐delta984‐1090. The GGSThe primers used for subcloning are listed in Table S1, Supporting Information. .The dPspCas13b plasmid (pC0050‐CMV‐dPspCas13b‐longlinker‐ADAR2DD(WT)) and sgRNA‐expressing plasmid (pC0043‐PspCas13b crRNA backbone) was a gift from Prof. Feng Zhang . psT1374‐dCas9‐GCN4 (Addgene plasmid no.113026) and psT1374‐scFv‐APOBEC‐UGI‐GB1 (Addgene plasmid no.113029) were gifts from Prof. Xingxu Huang. pcDNA3‐FTO and pcDNA3‐mutFTO were gifts from Prof. Chuan He. pPB‐ALKBH5 was a gift from Prof. Guifang Jia. pFlag‐CMV2 plasmid was a gift from Prof. Xiaolian Zhang. ASB2‐3′‐UTR (wild‐type and mutant) and pRL‐TK were gifts from Prof. Jianjun Chen. pNL4‐3.E‐R‐(CopGFP) and HIV‐env plasmids were gifts from Prof. Binglian Sun. All plasmid sequences were verified by Sanger sequencing.6‐dCas13b, scFv‐effector protein‐GB1 and sgRNA‐expressing plasmid was 1:1:2. HepG2.215 cell line was kindly provided by Prof. Ying Zhu (Wuhan University College of Life Sciences) and maintained in DMEM supplemented with 10% FBS. Rattail collagen  should be used overnight in cell culture container before cell passage. Each plasmids  and pc0043‐HBV sgRNA) were transfected into HepG2.215 cells (the transfection ratio was 1:1:2) using NEOFECTTM DNA transfection reagent  according to the manufacturer's protocol. HEK293T cell line was kindly provided by Professor Binglian Sun and maintained in DMEM supplemented with 10% FBS and puromycin (2 µg mL−1). Each plasmids  and pc0043‐HIV sgRNA) were transfected into HEK293T  cell line (the transfection ratio was 1:1:2) using NEOFECTTM DNA transfection reagent  according to the manufacturer's protocol.HEK293T (ATCC) and HeLa (ATCC) cells were maintained using Dulbecco's modified Eagle's medium (DMEM)(high glucose) supplemented with 10% Fetal Bovine Serum (FBS), and 1% penicillin/streptomycin . Plasmids transfection were achieved using lipofectamine 3000 (Invitrogen) following the manufacturer's protocol. The co‐transfection ratio of GCN4‐GGS−1). Two days after transfection, the virus‐containing supernatant was collected and filtered. The tilter of HIV‐1 HXB2 virus was measured by immunofluorescence. The HEK293T  cell line was then infected with HIV‐1 HXB2 virus and 12 h later, the cells were centrifuged to remove the virus, washed with PBS and resuspended in DMEM supplemented with 10% FBS and puromycin (2 µg mL−1) for further analysis.HIV‐1 HXB2 virus was produced by transfection of HEK293T cells with the pNL4‐3.E‐R‐(CopGFP) and HIV‐env plasmids (the transfection ratio was 3:1) using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. After 5 h incubation, the medium was replaced with DMEM supplemented with 10% FBS and puromycin  at room temperature for 10 min. The cell lysate was collected and then boiled at 95 °C for 10 min. The appropriate amount of protein was loaded onto SDS PAGE gels. The separated proteins were transferred onto a PVDF membrane (Millipore) in an ice‐bath for 2 h. Then the PVDF membrane was blocked in 5% (w/v) BSA  in TBST  at room temperature for 1 h. The blot of protein was stained as indicate for at least 12 h at 4 °C. The blot was washed four times with TBST at room temperature for 5 min each, then stained with 1:5000 HRP‐conjugated Affinipure Goat Anti‐Rabbit IgG(H+L)  or HRP‐conjugated Affinipure Goat Anti‐Mouse IgG(H+L)  in 5% BSA (w/v) in TBST for 1 h at room temperature. The blots were washed four times with TBST at room temperature for 5 min each time and imaged on Molecular Imager ChemiDocTM XRS+ Imaging System (Bio‐Rad) after incubation with Rhea ECL .For western‐blotting, HeLa cells were plated in 12‐well plates and transfected with 1 µg plasmid. After 48 h transfection, cells were washed with PBS and lysed in 50 µL 1 × SDS loading buffer  following the manufacturer's protocol. mRNA was extracted using Oligo d(T)25 Magnetic Beads  followed by genomic DNA removal using TURBO DNase  treatment. Then RNA was purified by RNA Clean & Concentrator Kits . mRNA concentration was measured by NanoDrop. Briefly, to isolate a region of 28S rRNA that contains A4220, biotin labelled oligodeoxynucleotide complementary to C4170–C4210 nucleotide region of 28S rRNA (28S probe: 5′ Biotin‐CTCGCCTTAGGACACCTGCGTTACCGTTTGACAGGTGTAC) was purchased from Takara or obtained by DNA solid‐phase synthesis. To purify synthesized DNA oligo, Crude DNA products were dissolved in DEPC water.The samples were separated on Agilent HPLC system (Thermo Fisher Scientific). Target DNA were evaporated to dryness. The molecular weight was confirmed byLC−ESI mass spectrometry (Sangon Biotech). To obtain RNA: DNA hybrid, 4 µg 28S probe was incubated with about 33 µg of total RNA in 0.3 volumes of hybridization buffer , final 72 µL. The hybridization mixture was boiled at 90 °C for 7 min in water‐bath, slowly cooled down to room temperature overnight. Single‐stranded RNA and DNA were digested with mung bean nuclease  and RNaseA  over 1 h at 37 °C in 1 × mung bean nuclease buffer . Then wash 30 µL streptavidin C1 beads three times with 500 µL IP buffer , and combine C1 beads in 100 µL IP buffer with 150 µL RNA‐DNA hybridization, rotate at room temperature for 1 h, and wash 3 times with 500 µL IP buffer, twice with 500 µL H2O, then add 30 µL H2O, heat at 70 °C, 5 min. It was quickly put on ice for 2 min, and the supernatant was removed to a new tube from magnetic stand; this was the 40 nt RNA fragment.The isolation of a defined 28S rRNA was followed by the published protocols.35 Bri−ΔCt. All assays were performed with at least three biological replicates and three technical replicates. The qPCR primers used in this study are listed in Table S2, Supporting Information. RNA was reverse transcribed to cDNA using the EasyScript One‐Step gDNA Removal and cDNA Synthesis SuperMix . All qPCR reactions were performed as 20 µL reactions using 2 × Hieff qPCR SYBR Green Master Mix  and amplified on a CFX‐96 Real‐Time System . Expression levels were obtained by subtraction the housekeeping gene (GAPDH or HPRT1) Ct value from target Ct value and normalizing to the non‐targeting sgRNA (sg‐Control). Relative abundance was determined using 2Cytoplasm and nucleus were separated using Nuclear and Cytoplasmic Extraction Kit  following the manufacturer's protocol. The separation efficiency was validated by western‐blot. The following antibodies wereused for western blot analysis: anti‐Flag , anti‐LaminB1 , anti‐GAPDH , anti‐Tubulin .−1 DAPI  for 15 min. Cells were then washed five times and imaged.Cells were fixed with 4% paraformaldehyde  in PBS at room temperature for 10 min. Cells were then washed with PBS three times and permeabilized with 0.1% TritonX‐100 at room temperature for 10 min. Cells were washed again three times with PBS and blocked for 3 h with 3% (w/v) BSA in TBST at room temperature. Cells were then incubated with primary antibodies  in 3% (w/v) BSA for 1 h at room temperature. After washing five times with PBS, cells were incubated with secondary antibodies  in 5% (w/v) BSA for 1 h. After washing five times with PBS, cells were incubated with 1 µg mLThe cells were imaged immediately by confocal laser scanning microscope . For DAPI imaging, the excitation was 405 nm, and the emission filter was 447 nm (w60); for Alexa Fluor 488 imaging, the excitation was 488 nm, and the emission filter was 525 nm (w50). for Alexa Fluor 561 imaging, the excitation was 561 nm, and the emission filter was 600 nm (w52).m NaCl and 2 mm of ZnCl2 at 37 °C for 2 h, which was followed by addition of NH4HCO3  and Shrimp alkaline phosphatase  and additional incubation at 37 °C for 2 h. Samples were centrifuged at 12 000 × g at room temperature for 20 min; 3 µL of solution was loaded into Shimadzu LC‐MS/MS system . Nucleosides were quantified by using retention time and nucleoside to base ion mass transitions of 282.1 to 150.1 (m6A), and 268 to 136 (A).50 ng of mRNA, 28S rRNA fragment or total RNA was digested by nuclease P1  in 25 µL of buffer containing 10 m First, 1 µg total RNA were mixed with 40 nm Up Primer, 40 nm Down Primer and 5 µm dTTP  in 17 µL 1 × CutSmart buffer . The RNA and primers were annealed by incubating mixture at a temperature gradient: 90 °C for 1 min, 80 °C for 1 min, 70 °C for 1 min, 60 °C for 1 min, 50 °C for 1 min, and then 40 °C for 6 min. Subsequently, a 3 µL of mixture containing 0.01 U Bst 2.0 DNA polymerase , 0.5 U SplintR ligase  and 10 nmol ATP  were added in the former mixture to the final volume 20 µL. The final reaction mixture was incubated at 40 °C for 20 min, denatured at 80 °C for 20 min and kept at 4 °C. Afterward, quantitative real‐time PCR (qPCR) reaction was amplified on a CFX‐96 Real‐Time System  . The 20 µL qPCR reaction was composed of 2 × Hieff qPCR SYBR Green Master Mix (Yeasen), 200 nm qPCRF primer, 200 nm qPCRR primer, 2 µL of the final reaction mixture and ddH2O. qPCR was run at the following condition: 95 °C, 5 min;  × 40 cycles; 95 °C, 15 s; 60 °C, 1 min; 95 °C, 15 s The procedure was adapted from the previous report.36 Fir6A‐immunoprecipitation. Briefly, nearly 5 µg mRNA was incubated with m6A antibody  and diluted into 500 µL IP buffer . The mixture was rotated at 4 °C for 2 h, following Dynabeads Protein A  was added into the solution and rotated for another 2 h at 4 °C. After four times washing by IP buffer, the m6A IP portion was eluted twice by 100 µL m6A‐elute buffer  with incubating and shaking at 4 °C for 1 h. Finally, m6A IP mRNA was recovered by RNA Clean & Concentrator Kits and the RNA concentration was measured with Qubit RNA HS Assay Kit . Sequencing libraries of input and IP samples were prepared using NEBNext Ultra II RNA Library Prep Kit. mRNA m6A profiling was generated from two biological replicates.Total RNA was isolated by TRIzol reagent (Invitrogen). PolyA‐RNA was isolated by two rounds of ployA selection with Oligo d(T)25 Magnetic Beads  from total RNA and followed by additional TURBO DNase treatment. mRNA was purified by RNA Clean & Concentrator Kits and then fragmented to about 100 nt using RNA Fragmentation Reagents . The fragmented mRNA was purified by using RNA Clean & Concentrator Kits. While 100 ng mRNA was saved as input sample, the rest mRNA was used for m−1 at 6, 3, and 0 h before cell scraping collection. Total RNA was isolated by Trizol and analyzed by RT–PCR and RNA‐seq. For RNA‐seq, two rounds of mRNA selection were conducted and followed by TURBO DNase treatment. Then the mRNA was fragmented to about 100 nt. Sequencing libraries were prepared using NEBNext Ultra II RNA Library Prep Kit. mRNA lifetime profiling was generated from two biological replicates.HeLa cells were seeded into 6‐well plates to get 50% confluency after 12 h. Cells were transfected by identical plasmids for overall 48 h. And actinomycin D was added to 5 µg mL Briefly, 100 ng wild‐type or mutant ASB2‐3′‐UTR, 50 ng GCN4‐dCas13b, 50 ng ScFv‐GGS6‐FTO (WT or Mut), 100 ng sgRNA and 20 ng pRL‐TK were co‐transfected into HEK‐293T cells in 24‐well plate. 48 h post transfection, the relative luciferase activities were accessed by Dual‐Luciferase Reporter Assay System . Each group was repeated in three biological replicates and two technical replicates.The dual‐luciferase reporter assay was performed as reported previously with some modification.38 Bri6 mL−1. Three plasmids  and pc0043‐HBV sgRNA) were co‐transfected into HepG2.215 cells at the ratio of 1:1:2, and the samples were collected 24 h later. The cellular DNA from each sample were treated with DNeasy Blood &Tissue Kit (QIAGEN GERMANY), then genomic DNA was removed by Plasmid‐Safe ATP‐Dependent DNase  enzyme according to protocol. While small circular DNA could not be digested. Thus, cccDNA copy number was quantified using the QX200 Droplet Digital PCR system  according to previous report. Briefly, 20 µL ddPCR mixture consisted of 10 µL of 2 × ddPCR supermix for probes (Bio‐Rad), 950 nmol L−1 of cccDNA selective primers, 250 nmol L−1 of HBV cccDNA Taqman probe (5′‐FAMTCACCTCTGCCTAATCATCTCTAMRA‐3′), and 7 µL of DNA template. The mixture was loaded into the DG8 cartridge, 70 µL of droplet generation oil was added, and droplets (approximately 1 nL per droplet) were then formed in the droplet generator (Bio‐Rad). Generally, each sample could generate up to 20 000 stable water‐in‐oil droplets. Next, the droplets were transferred to a 96‐well PCR plate (Eppendorf) and amplified on a C1000 thermal cycler (Bio‐Rad) with a thermal profile beginning at 95 °C for 5 min, followed by 45 cycles of 94 °C for 30 s and 60 °C for 60 s, 1 cycle of 98 °C for 10 min, and ending at 4 °C. After amplification, the plate was loaded on the droplet reader (Bio‐Rad), and data were analyzed by QuantaSoft analysis software version 1.7.4 (Bio‐Rad) on the basis of Poisson distribution. For cell counting and normalization, β‐actin copy number in each DNA sample without PSAD treatment was also simultaneously determined with a pair of primers (5′‐ACTGTGCCCATCTACGAGG‐3′ and 5′‐CAGGCAGCTCGTAGCTCTT‐3′) and a probe (5′‐FAMCGGGAAATCGTGCGTGACTAMRA‐3′) in neighboring wells.HepG2.215 cells were seeded with 6‐well plate and the cell density was 1 × 10s report.41 Brim Tris‐HCl, pH 8.0, 150 mm NaCl, 2 mm EDTA, 1% NP‐40, 1 mm PMSF, protease inhibitors) for at least 5 min. Collect the lysates into EP tubes and incubate the lysates on ice for 30 min, pipetting every 5 min. After incubation, centrifuge the lysates at 15 000 × g for 10 min at 4 °C. Collect the supernatant and discard the pellet. Incubate 4 µg indicated antibody with 200 µL supernatant for 1 h at 4 °C. Immunoprecipitations were performed with anti‐His mouse antibody  or anti‐IgG mouse antibody . Then the supernatant‐antibody mix was bound to magnetic Dynabeads Protein A (Life Technologies) overnight at 4 °C, the immunoprecipitations were washed five times with Lysis buffer. The following antibodies were used for western‐blot analysis: anti‐Flag , anti‐His .Cells were washed twice with cold PBS and then lysed with 500 mL cold Lysis Buffer (50 m (version 2.5) with parameters “‐m 18 –max‐n 3.” Reads that cannot be aligned to human rRNA sequences by using bowtie2 with default parameters were mapped to human genome (GRCh38 and GENCODE.v29) by using STAR with parameters “–alignEndsType EndToEnd.” Uniquely mapped and proper‐paired alignments were filtered out by using bamtools with parameter “‐tag NH:1 ‐isProperPair true” and PCR amplification bias was eliminated by using picard  with parameters “MarkDuplicates REMOVE_DUPLICATES = true.” For m6A‐seq data, the fragment coverage of each base of all transcripts was calculated by custom script. The peak calling algorithm was modified from Ma et al. To calculate the enrichment score using the average fragment coverage of the window instead of the read count. The peak sequences in the length range of 50–200 nt were analyzed by using findMotifs.pl script of Homer (version 4.10) with parameters “‐rna ‐norevopp ‐mask ‐len 5,6,7” to find out the m6A motifs. For mRNA lifetime profiling data, we calculated the FPKM (Fragments Per Kilobase per Million) or all transcripts. The FPKM of 3 and 6 h were normalized to the FPKM of 0 h to calculate relative abundance and the relative abundance of 0 h were arbitrarily adjusted to 1. In order to meet the law of degradation, the relative abundance of 3 and 6 h were corrected as follows: 1) screeing out transcripts with length greater than 500‐nt, fragment count greater than 100 and FPKM greater 0.1 as representative transcripts; 2) sorting the relative abundance of 3 and 6 h of representative transcripts from small to large, respectively; 3) taking the 95th percentile value as normalization factor, respectively; 4) normalizing the relative abundance of 3 and 6 h to the corresponding factor. After these normalizations, it was ensured that the relative abundance of most genes at 3 and 6 h was lower than that at 0 h. we calculated the halftime of transcripts by linear fitting with the following formula (two replicates and three time points), where T represents the halftime, A0 represents the relative abundance of 0 h and always equal to 1, and tA represents the corrected relative abundance of 3 or 6 h:All samples were sequenced by Illumina HiSeq XTen with paired end 150‐bp read length. The adapters were trimmed by using cutadapt42 . Correlation analyses were performed using the Pearson correlation test. p < 0.05 was considered statistically significant. Symbol “*” represent p < 0.05, “**” represent p < 0.000, “***” represent p < 0.000001 and “NS” represent p > 0.05.All data are expressed as the mean ± standard error of the mean (SEM). For LC‐MS/MS, Dual‐Luciferase assay, qPCR quantification studies and western‐blot, The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Severe Acute Respiratory Syndrome-coronavirus 2 (SARS-CoV-2) is a new virus with a global pandemic. Yet, no vaccine or efficient treatments are found against the disease. The viral RNA dependent RNA Polymerase (RdRP) is a suitable target for developing antiviral agents. SARS-CoV-2 RdRP was employed to test its binding activity with some drugs.Using some docking methods, RdRP was targeted by Milbemycins (MMs), Ivermectin (IMT), Baloxavir Marboxil (BM), and Tadalafil (TF), a phosphodiesterase type 5 inhibitor.MM-A3 5-oxime (MMA35O), MM-A3 (MMA3), MM-A4 5-oxime (MMA45O), IMT, BM, and TF showed the highest binding affinity to RdRp.The drugs used in the present computational investigation are effective against the SARS-CoV-2 RdRP with high affinity values especially, milbemycins, ivermectin, and Baloxavir marboxil, which could further be studied in laboratory and clinical trials for saving millions of lives around the world. In a compassionate-clinical use of RDS in hospitalized COVID-19 patients breathing ambient air or on oxygen support, clinical improvement was seen in 36 of 53 patients (68%) 4 which is still a humble recovery especially in the case of more severe and critical patients. Like RDS, FPV  is a nucleoside analogue used as an antiviral drug that halts the replication of the viral RNA due to its incorporation in the newly-made RNA strand of wide-range viruses 5. FPV was recorded to induce inhibition to seasonal and avian influenza viruses even those strains that are resistant to neuraminidase inhibitors as approved using cell culture and experimental animals 6.A few drugs have been used for treating Coronavirus Disease 2019 (COVID-19) patients such as Remdesivir (RDS) and Favipiravir (FPV) which are considered as nucleotide and nucleoside, respectively, inhibitors that interfere with the assembly of the viral RNA. RDS is used for the treatment of Ebola 7, docking methods in the present work were employed to enhance production of drugs that bind with high affinities to the binding sites of the enzyme. This might block the activity of the enzyme, halt the viral RNA building, and eventually stop the viral replication.Due to the availability of the RdRP 3D structure https://www.rcsb.org/), after removing cofactors from the structure using PyMOL 8. MMs, members of the macrocyclic lactone family, were selected according to a report that IMT, a member of the same family, was effective in minimizing the replication of the COVID-19 in cell culture 9. For BM, the selection was according to its inhibition effects on the polymerase-based initiation of mRNA of the influenza virus 10. In the case of TF selection, it was previously reported to prevent infection of a cell line by the Human Immunodeficiency Virus (HIV) 11. PyRx-Python Prescription (0.8) and Autodock vina plugin 12 were used for the prediction of the affinity values. In details, Standard Database Format (SDF) files of the ligands were downloaded from https://pubchem.ncbi.nlm.nih.gov/ and were converted into PDB file type via the use of BABEL plugin in the PyRX software. IMT was downloaded from the http://www.chemspider.com/ due to unavailability of the 3-dimensional structure in the https://pubchem.ncbi.nlm.nih.gov/. Then, both protein and ligand files were prepared for the docking process using PyRx software for removing waters, in which the PyRx software gets rid of water molecules automatically before generating pdbqt files. Grid boxes were optimized for free searching by the ligands on the whole enzyme molecule (Active and non-active sites). The dissociation constant (Kd) is predicted via auto dock built in the PyRx software that converts the affinity energy values  into Kd according to the following equation:kcal/mol)DeltaG: Affinity energy (R: Gas constantK=25°C)T: Room temperature  between the RdRP and the ligands were visualized using Discovery Studio Visualizer v20.1.0.19295 (BIOVIA). The images were generated using Discovery Studio Visualizer v20.1.0. 19295 (BIOVIA), Microsoft PowerPoint, and GIMP v2.10.18.The outcomes of the present investigation shown in The results showed that the palm subdomain in motif B of the RdRP is engaged by the MMA35O . The bon7. This activity is probably due to different directions taken by the major components; RNA template, NTPs, and product hybrid, involved in the building of the new viral RNA strands. The finding of the present study revealed that the palm subdomain in the motif B of the RdRP is bound by the MMA35O. The palm subdomain (Residues T582-P620 and T680-Q815) is an important part of the core region of the RdRP that participates in the stability of the enzyme structure along with fingers subdomain (Residues L366-A581 and K621-G679) and a thumb subdomain (Residues H816-E920) 7. Hypothetically, the presence of the MMA35O in the bound region may increase the chances of halting the enzyme activity especially when this region is considered adjacent to the passage of the RNA building materials. MMA3 showed its binding to the F motif of the RdRP enzyme. The F motif is a critical part through which the RNA template is expected to enter to the active sites in the motifs A and C to engage in the process of the RNA replication. This attachment region is located in the active groove that is used as an RNA template entry passage. Typically, this means that the occurrence of the ligand in this pocket might enhance the chance of disrupting the replication of the viral RNA strand.The active sites of the RdRP, also known as nsp12, are located in the motifs A to G 7.Another molecule that expressed binding to RdRP is MMA45O. The drug attached to the binding sites in the motifs B and F. The bound ligand was seen sitting in the groove, RNA template passage entry to the motif A and C, clamped by the F motif. Such occasion might interfere with the activity of the RdRP, thereby stopping its activity and eventually finishing the cycle of viral replication. Motif A (Residues 611-TPHLMGW-DYPKCDRAM-626) has a conserved divalent-cation-binding residue (D618) which is similar to that in other viral RdRP such as in HCV (D220) and poliovirus (D233). IMT revealed binding to motif C and F. Motif C consists of residues of 753-FSMMILSDDAVVCFN-767 including the catalytic residues of 759-SDD-761 13. Since the target of this drug is the viral polymerase, this might be a great drug of choice for treating COVID-19 patients. The TF showed strong binding activity to the polymerase of the virus. Tadalafil is an inhibitor to the phosphodiesterase type 5 component utilized to manage erectile dysfunction 14. This could be applied as a great drug of choice for treating COVID-19 cases especially when this drug is used to treat pulmonary arterial hypertension 15.In the present study, Baloxavir marboxil displayed successful binding to the active sites in the B and F motifs of the RdRP. Baloxavir marboxil suppresses the activity of the viral replication of the influenza virus by inhibiting the polymerase PA subunit in vitro, in vivo, and in clinical trials to find whether they are effective against the SARS-CoV-2 (COVID-19) virus and thereby, quickly helping to manage the pandemic around the world.The current work provided a group of drugs that could be further studied


During the summer, benthic temperatures show high-frequency fluctuations (2 to 8 °C) associated with internal waves that advected cool, nutrient-rich water across the reef. The interaction between local water flow and reef morphology resulted in a highly heterogenous physical environment even within isobaths that likely influenced the growth regime of H. tuna. Variability in H. tuna isotopic values even among closely located individuals suggest biological responses to the observed environmental heterogeneity. Although isotopic composition of reef carbonate material can be used to reconstruct past temperatures (T(°C) = 14.2–3.6 ; r2 = 0.92), comparing the temperatures measured across the reef with that predicted by an isotopic thermometer suggests complex interactions between the environment and Halimeda carbonate formation at temporal and spatial scales not normally considered in mixed sediment samples. The divergence in estimated range between measured and predicted temperatures demonstrates the existence of species- and location-specific isotopic relationships with physical and environmental factors that should be considered in contemporary as well as ancient reef settings.On Conch Reef, Florida Keys, USA we examined the effects of reef hydrography and topography on the patterns of stable isotope values (δ Halimeda  are important benthic autotrophs in subtropical and tropical waters worldwide3 where they can be found from the shallow subtidal to depths exceeding 100 m6. In addition to being important primary producers in many ecosystems, they are an import aragonitic sediment source via reproduction, physical fragmentation, and herbivory10. In some reef settings, calcareous algae can contribute more than 60% of the total carbonate deposition11 and in the Florida Keys, species of Halimeda can contribute more reef sediment than either coral or coralline algae12. Because of the conspicuous sedimentary particles made by Halimeda sp., they have produced an important paleontological record since the early Miocene15 and have proven useful for environmental reconstructions and biostratigraphy21.Calcareous macroalgae of genus Halimeda, can provide information regarding the integrated chemical and physical environmental conditions of the surrounding seawater during precipitation following the foundational work of Urey and colleagues25. The analysis of variations in δ18O, particularly when integrated with additional data from elemental ratios such as strontium:calcium and barium:calcium, have been used to imply natal origin in pelagic larvae26, infer variability in marine salinity27 as well as estimate oceanographic paleo temperature time series28. The degree to which the carbonate isotopic values are in equilibrium with the surrounding seawater depends upon the physical thermodynamic kinetics during calcification—diffusion, hydration and hydroxylation of CO2—and the degree of metabolically driven fractionation associated with general physiology, respiration and photosynthesis . Ultimately, the preserved isotopic signature depends upon both these exogenic and endogenic kinetic factors, the distribution of isotopes in the Halimeda fragments, the contribution of Halimeda to the total sediment record, and any syndepositional and later diagenetic effects30.The stable isotopic composition of sedimentary fragments composed from calcifying organisms, like Halimeda consists of a holdfast comprised of unorganized coenocytic filaments, and a series of branching segments with lengthwise oriented filaments and a laterally displaced cortex with a semi-isolated intercellular space31. The crystallization of aragonite is mediated by photosynthetic uptake of CO2 (see review in33) and occurs in three stages in the intercellular space. There is first the growth of fine-grained aragonite into the filament walls, followed by the growth of aragonite needles perpendicular to the filaments and into the intercellular space. The final step involves the crystallization of any remaining space with irregularly arranged aragonite needles36. Most evidence suggests that oxygen isotopes in Halimeda aragonite are precipitated in thermal isotopic equilibrium with the surrounding seawater38. Because the resulting 18O isotopic value is determined by physical environmental conditions (i.e. temperature) during calcification, it is believed that Halimeda, under favourable circumstances, can be used to reconstruct seawater temperature in shallow-water environments analogously to the widespread use of benthic foraminifera from deep sea sedimentary environments37.An individual 2 into biogenic pathways that influence δ13C values of Halimeda aragonite39. Fractionation of carbon reservoirs associated with photosynthesis and respiration as well as during calcification lead to variable disequilibrium in δ13C values depending upon light level, growth stage and environmental conditions which can vary substantially in both time and space in a reef ecosystem41. Recent research has also examined the varying and differential effects of temperature, pH and light intensity on photosynthetic rates and calcification in Halimeda spp. in light of ocean acidification and warming49, although their coincident biogenic effects on the isotopic carbon signature has only received limited study to date51.In contrast to stable oxygen, metabolic processes affect isotopic fractionation and the incorporation of COHalimeda tuna isotopic composition sampled over the irregular surface of a coral reef in the Florida Keys. The site chosen was Conch Reef, within the Florida Keys Marine Sanctuary, which has been the location of several decades of detailed study associated with NOAA and the National Undersea Research Center  thermistors along the reef from June through September in 2003 and 2004, as well as on a less dense spatial scale almost continuously from 1992 to 201055. These studies have shown that during approximately half the year, May through September, the water column and near bottom hydrography of Conch Reef is strongly influenced by the episodic incursion of cool, sub-thermocline water from offshore of the reef slope, driven by strong semidiurnal internal tides and higher frequency internal wave activity . The offshore water masses interact with the reef bathymetry producing a temporally and spatially heterogeneous temperature and nutrient environment both across and within reef isobaths. Most importantly, the water flux creates areas with persistent warm and cool temperature anomalies relative to mean thermal conditions, that are closely associated with the heterogeneous reef topography. The pooling of cool, dense water constrained within pockets and depressions of low relief can remain for several hours following individual incursion events, and the events typically occur up to twice per day. This implies that there could be spatially distinct geochemical signals of the thermal anomalies detected in variability of isotopic values in calcifying benthic algae. This further implies that it may be important to recognize the sources and magnitude of inherent spatial variability when attempting to interpret patters of isotopic values through time, for example in the case of reconstructing past temperature patterns from historical to ancient reef sediments.In the present study we describe temporal and spatial variability in both seawater temperature and in ter Fig. . Details57, and high frequency upwelling appears to be a significant overall nutrient source for the outer reef slopes62. Sampling of the water column seaward of Conch reef, indicated that cooler offshore water had a nearly linear increase in total inorganic nitrogen with decreasing temperature54 while at specific locations on the reef, integrated cooling degree-hours below 26 °C could be used as an index of net nutrient exposure and was strongly correlated with tissue δ15N in benthic macroalgae51. During the summer, the thermocline seaward of Conch reef was often relatively shallow (50 m depth), and upward incursions of cool (22–26 °C) water onto reef slopes occured frequently. The relatively high concentrations of subsurface nitrate (5–20 μmol L−1) and soluble reactive phosphate (0.1–2.0 μmol L−1) below the thermocline58 indicated the potential for the offshore nutrient pool to be an important source of nutrients reaching the reef slope, especially at depths of > 10 m as incoming internal wave bores mixed subthermocline water with warm surface waters.The cool water intrusions are also associated with increased levels of nutrients and greater plankton densities, up to an order of magnitude higher than background levels, along with temperature fluctuations as great as 8 °C within minutes with durations of multiple hours60.During the winter months, the water temperatures at Conch Reef can be on average up to 8 °C colder than the summer maximum temperatures and occasional atmospheric cold fronts can cause surface temperatures to occasionally drop below 20 °C for short periods . Cooling during the fall and winter (October through January) was associated with the breakdown of water column stratification and a reduction in subsurface thermal variability due to internal waves. The thermal structure and current flow patterns impacting Conch Reef are thus compounded by the localized seasonal variability in stratification that modulates high-frequency variation associated with internal wave productionHalimeda tuna, collected at multiple locations across Conch Reef, for comparison to measured patterns in benthic temperature that resulted from interactions of local hydrography and bathymetry. Because we compared the spatial distribution of the isotopic signatures and benthic temperature parameters as 3-dimensional maps of the landscape, rather than as a time series from a single spatial location, we used a geospatial analysis technique in order to quantify the similarity in the landscape maps. This provided more details on potential sources of heterogeneity in contemporary carbonate biogeochemistry that differentially affect isotopic values of δ18O and δ13C from sessile autotrophs, like Halimeda, that improve our understanding of the importance of integrating environmental variation and spatial heterogeneity into analysis of paleocarbonates and modelled paleoclimate indicators. We also used the δ18O values to compare models of the predicted seawater temperature during H. tuna carbonate formation to the observed spatial and temporal variation of benthic water temperature across the study site. The enhanced resolution of variability in both isotopic values and environmental conditions allows for a more accurate calibration for δ18O-based paleothermometers using Halimeda for new, or existing, paleorecords in the Florida Keys or other locations with highly variable physical environments.Here we describe spatial patterns in the stable isotopic signature of 57. Temperature variability is greatest at the base of the reef slope and the deeper depths are associated with the lowest mean temperatures because not all cold incursions advect all the way up the reef slope . There also can be slight water column variation in salinity, between 35 and 36.5 PSU in near-surface waters less than 100 m deep. Salinity time series measured on the reef slope at 21 m, can sometimes show both increases and decreases in salinity on order 0.1 to 1 PSU on the same time scale as the large fluctuations in temperature associated with internal waves54 (and Leichter unpublished data). However, the small density differences due to salinity variations are not as important as the large density changes due to temperature variation that then control the density-driven and topographically-constrained flow of water across the benthos57.The benthic water temperature on Conch Reef varies about 8 °C with season and depth Fig. . The yeaope Fig. A–C. The As shown by the temperature anomalies in Fig. 18O values across depths from 3 to 80 m as determined by ANOVA  = 1.54, p = 0.24). The mean and S.D. of δ18O was 0.93 ± 0.05, 0.97 ± 0.06, 0.93 ± 0.05, 0.96 ± 0.15, 0.86 ± 0.17, for the 3, 40, 65 and 80 m samples respectively. This result is similar to the reported seawater δ18O of + 1.0 estimated for this region from the Global Seawater Database63. There was a significant difference in the water collected from approximately 100 m depth with a mean and S.D. of 0.63 ± 0.16 using a Tukey HSD post hoc test.There were no significant differences in the seawater δH. tuna isotopic composition are complex  + c(δ18Ocarb − δ18Osw)2 where T is the predicted temperature, δ18O carb is the δ18O signature of the carbonate, δ18Osw is the δ18O signature of the seawater in which the carbonate precipitated, and a, b, and c are constants particular to the specific carbonate-forming organisms sampled and the environment in which they were living. The constants used were from temperature calibrations for late Holocene Halimeda samples from the Caribbean Virgin Islands34 using the model of Grossman and Ku64. Miocene Halimeda reef samples21 as well as aragonite collected from the coral Porities sp. collected from the Galapagos69. The predicted results in Fig. Using the measured δels Fig.  and then18O predicted temperature . For the δ13C spatial distribution  and predicted metrics do not show as close a fit with the raw bathymetry  measure of map similarity between the predicted temperature and the measured temperature maps, indicated the closest fit , NFK = 0.61, between δure Fig.  and the ure Fig. A, with dion Fig. B, the maH. tuna, that can be sensitive to relatively small changes in the physical environment. On coral reefs, temperature variability is intrinsically important because of the physiological stresses on the sessile benthic community occurring at both low and high temperatures69 and also because it can often be inversely correlated with the concentration of dissolved nutrients (like on Conch Reef), which is critical for the growth of autotrophs. Similar physical forcing has been identified in other coral reef habitats73 and in a wide variety of other shallow water marine environments77. In the case of Conch Reef, mapping seawater temperature and thermal anomalies onto the reef topography reveals distinct locations on the reef with persistent cooling and warming while spatial mapping of H. tuna isotopic signatures also reveals both large and small scale heterogeneity in biogeochemical patterns.The high degree of physical and biological variation within isobaths suggests that simple interpretations of apparent depth effects on biological parameters may be problematic when considering ancient or contemporary systems where extensive sampling within depths is often absent due to logistic or experimental design limitations. This variation, on spatial scales from cm to many meters and likely resulting from processes that vary on temporal scales from minutes to seasons can be of significant ecological importance to benthic sessile organisms, including benthic algae like 52. For example, temperature anomalies evident in Fig. Halimeda is supported by recent experimental evidence by other researchers. Smith et al.78 and Vroom et al.3 measured higher growth rates of H. tuna on the reef slope at Conch Reef with increasing depth. In response to a series of nutrient addition experiments across depths at Conch Reef78, Smith et al. found that H. tuna at 21 m showed no change in growth rate, but showed an increased growth response at 7 m depth suggesting that the shallower individuals live in nutrient-poor conditions. The heterogenous spatial patterning in H. tuna δ13C , found evidence of an approximately 4 °C cooler time period roughly 4,000 years before the present in the Northeast Caribbean. Holmes applied the Grossman and Ku64 paleothermometer model originally formulated for δ18O in benthic foraminifera aragonite whose precipitation is considered to be in close equilibrium to seawater80. The Halimeda fragments from the Virgin Islands had a δ18O range from − 2.5 to − 2.0 ‰ (mean 2.15 ‰), yielding predicted temperatures from 26.7 to 28.9 °C which, when assuming the seawater δ18O to be close to 0 ‰, was within the range of measured values for the area. Holmes34 also reported Halimeda δ18O values of − 3.3 to − 3.0 ‰ for Marquesas Keys in Florida, corresponding to predicted temperatures of 29.8–31.0 °C, also within the range of measured temperature at that location.In a paleoecological study, HolmesH. tuna δ18O ranged from − 5.06 to − 2.07 with mean − 3.36 ‰. Following Holmes34 and using the equation of Grossman and Ku64 and a seawater δ18O of + 1.0 estimated for this region from the Global Seawater Database63 (similar to the water samples collected during this study), yields predicted temperatures ranging from 30.0 to 41.4 °C with mean of 34.9 °C  at a reef location which undergoes similar yearly temperature fluctuations of 6 to 8 °C and similar cold water upwelling as found along reefs in the Florida Keys.On Conch Reef, by contrast, 50) are imbedded in the time-averaged 18O calcification signature sampled from H. tuna which has a lifespan anywhere from 1 to > 12 months at Conch Reef3 and maximal growth during summer months coincident with the period of maximum temperature variation. Using the spatially sampled temperature field and δ18O signatures sampled here  calculated using a least squares regression suggests a temperature calibration T (in °C) = 21.0−2.6  − 0.24 2 with r2 = 0.26. It should be noted that this calibration uses the temperature data collected only during the summer months. A better representation of the general temperature field may be calculated from the long time series  for the binned mean temperature data from 1997–2006 (r2 = 0.92) which may be useful for other temperature reconstructions. The overestimates produced by the Holmes34 and Brachert et al.21Halimeda calibrations and the similarity to the Porities aragonite calibration from the upwelling Galapagos reefs is also shown.The large seasonal and daily temperature fluctuations present at Conch Reef, and the heterogeneous physical conditions experienced by the benthos, even along isobaths, Fig.  and50 ar18O values in the H. tuna aragonite may be lower (less enriched in 18O) than would be expected based on estimates of the average seawater δ18O for the Florida Straits and known temperatures at the site. If we assume a lower value of seawater δ18O, − 1.5 ‰, rather than + 1.0 approximated from63 we attain a more reasonable range of predicted temperatures, 22.8–33.8 °C with mean of 27.5 °C. Under the modelling assumptions, the relatively low (less enriched) δ18O values we find for H. tuna aragonite imply that seawater δ18O is approximately 2.5‰ lower than expected based on observed values for the offshore waters of the Florida Keys. Fresh water input is a possible source for low seawater δ18O values27. Thus, the relatively positive δ18O signatures for H. tuna at Conch Reef may be indicative of freshwater input into this system, perhaps via frequent heavy rainfall and potentially from inshore or submarine groundwater sources with more negative levels of δ18O89. However, groundwater input has not been identified in prior tracer experiments85, benthic temperature and salinity data54, or radium isotopic measurements86 and the effects of well-mixed rainfall at the reef front would be negligible. Salinity can vary along with temperature during the arrival of internal waves, however salinity remains in the range of full ocean salinity (e.g. 35 to 36.5 PSU at the site), and does not indicate significant impact of low salinity water at the depths of the reef surface57 (and J. Leichter unpublished data). Although there were indications of lower δ18O seawater values in offshore water collected at or below 100 m, (0.6 vs 1.0), this small difference does not have a large influence in predicated temperatures, and usually, the water that advects onto the reef front is often well mixed and from much shallower depths57.An additional way to interpret disparity in the predicted temperature data could be that the measured δHalimeda samples collected here, in a paleoenvironmental context, it is also important to consider potential syndepositional and diagenetic effects on Halimeda fragments in the sediment. These effects can be significant and under some conditions can occur even within 500 days of burial30. The coupled dissolution and re-precipitation of carbonate can lead to a shift in isotopic signature towards equilibrium with the sediment pore waters and away from that of the seawater from which it originally precipitated. The possible isotopic shift towards pore water equilibrium and the complexities in biologically-mediated fractionation during fragment formation suggests that co-occurring inorganically precipitated marine cements within paleocarbonate samples, may provide fidelity for oceanographic geochemical conditions than do the skeletal fragments bound within87 .Additionally, although it is of minimal importance in the contemporary 38, summarized most cogently, “Vital effects must not be seen merely as a vexation in attempting to reconstruct the environment, however. The task is to extract some of the wealth of ecologic information  which is hidden in the disequilibrium patterns of the isotopic signals.” In the case of Halimeda, its rapid growth, robust aragonitic form and distinctive fragmentation products create a carbonate sediment amenable to paleoenvironmental reconstruction, with certain caveats. Evidence suggests that the Halimeda δ18O signature is precipitated in equilibrium with seawater, hence its use as paleothermometer, but, as evidenced here, both δ18O and δ13C values can show high variability over short spatial scales and within a single depth isobath. A linear model fit to the δ18O data, even if sampled along a single depth isobath, may not be easy to interpret, and a mixed or homogenized sample that incorporates the full range of variability underestimates the heterogeneity that may be a real and important component of a dynamic environment. Paleocarbonate samples that show large heterogeneity in both δ18O and δ13C within a single sedimentary horizon may be indicative of reef settings that have a variable thermal, salinity, and nutrient regime associated with internal waves and tides like at Conch reef.Wefer and Berger90. The shallow fore reef at approximately 8–10 m depth and the fore reef both slope gradually (ca. 2–5% slope) to approximately 16 m depth where the slope becomes steeper (ca. 8–15% slope) down to the base of the reef at 30–32 m depth. In some locations, a series of 1–2 m height parallel coral spurs separated by sand channels transect the fore reef slope. At the base of the fore reef, a gradual (1–5%) sand slope extends seaward of the reef for 20–30 km into the deeper channel of the Straits of Florida. At the Conch Reef study site currents are typically alongshore, 0.1–0.5 ms−1 flowing towards the northeast with tidal reversals to the southwest57. Flows are stronger (0.5–2.0 ms−1) offshore of the reef tract and driven primarily to the northeast by the Florida Current55. The surface tides at Conch Reef are mixed semidiurnal with mean amplitude of approximately 0.5–0.75 m57.The data were collected as part of a series of studies at Conch Reef in the Florida Keys, USA , with the National Undersea Research Center, Fig. Halimeda tuna was conducted in conjunction with temperature and current recording instrumentation deployment during a saturation diving project at the NOAA Aquarius habitat. For the high temporal frequency component of the study, the primary apparatus deployed was an extensive temperature-sensor array capable of synchronized, high precision, autonomous sampling for extended periods, known as the Benthic Oceanographic Array (BOA92). The system consisted of 100 temperature sensors arranged in 10 arrays of 10 elements spaced serially along cables at 15 m intervals. The BOA system allows synchronous sampling of all sensors at a 5-s interval with a resolution and accuracy of 0.007° and 0.04 °C, respectively. Sensor nodes were anchored to the reef by attaching them to 25 cm spikes driven into the bottom or tied onto dead coral substrate. The 10 array cables were connected to a submersible junction box at the BOA control and power supply located at the base of the reef slope at approximately 33 m depth, adjacent to an acoustic Doppler current profiler (ADCP) (RDI Workhorse 600 kHz) with pressure sensor. The ADCP sampled at 1.33 Hz and stored 1-min averages in 1 m vertical bins. Detailed ADCP data are presented in Leichter et al.57. An additional BOA strand was fixed to a vertical mooring near the BOA junction box in order to provide water column temperature measurements in approximately 2.5 m intervals between 3 and 30 m depth.Systematic benthic sampling of the macroalgae In addition to the high-frequency sampling described above, benthic temperature was measured continuously from 1992 to 2010 at 10, 20, 30 m depth with SBE39 (Seabird Electronics) temperature recorders with 0.001 °C resolution. Data were sampled at 2 to 10 min intervals and averaged in 10 min bins for the long-term data set.The entire BOA sensor array and the reef topography was mapped in situ using compass board, calibrated measuring tape, and digital depth gauge see Fig. . This prH. tuna, consisting of entire plants and their segments attached to the holdfasts (excluding loose or dead segments), were hand collected by divers at each node position of the BOA array, rinsed in de-ionized water, and then dried and prepared for isotopic analysis. H. tuna is common at this Conch Reef location and samples could usually be collected within 0.5 m of a node. The H. tuna samples were divided into two equal parts, half treated with 0.1% HCl to remove calcium carbonate and the remaining half treated with 0.1% NaOH to remove the organic component. The treated samples were then powdered with mortar and pestle and weighed subsamples were placed in tin capsules for isotopic analysis.Mixed samples of 13C at the Isotrace laboratory of University of Otago by combustion in a Carlo Erba NC2500 elemental analyzer to reduce the samples to CO2 and N2. The isotopic ratio of CO2 and N2 were measured by a Europa Scientific 20–20 ANCA Mass Spectrometer operating in continuous flow mode. Delta values were reported against the international standards Vienna Pee Dee Belemnite (VPDB) and atmospheric nitrogen (AIR) for δ13C and δ15N respectively. Normalization was applied with a 3-point calibration from 2 glutamic acid international reference materials and a laboratory EDTA standard  for carbon  and nitrogen . Results were expressed in standard delta notation where, for example, δ13C = [Rsample/Rstd) − 1]*1000 where Rsample = 13C/12C and Rstd = 13C/12C of Peedee belemnite limestone. Analytical precision was quantified by comparing results with quality control standards EDTA-OAS and IAEA MA-A-1 (Copepod). All measured values for the quality control standards were in the range of accepted values.Each algal subsample (1–5 mg) of organic material was analyzed for δH. tuna were independently analysed for δ18O. Aliquots (2–3 mg) of ground samples were reacted with 103% H3PO4 under vacuum overnight. The carbon dioxide produced by this reaction was purified and analysed on a Thermo Advantage isotope ratio mass spectrometer (IRMS) in continuous-flow mode for carbon and oxygen ratios. The same procedure was followed for NBS  18 and NBS 19 carbonate standards. Results are reported in the standard delta notation against the standard mean ocean water (SMOW). Consensus values for laboratory standards were obtained from internal laboratory calibration records against primary reference materials, VSMOW, GISP and SLAP (Isotrace – University of Otago).Samples of calcium carbonate from 54). Nisken samples (n = 3–15) were collected at approximately 3, 10, 30, 55, 80, 100 m depth. δ18O from the samples was determined by the method of equilibration93 with CO2. Half of a millilitre of a subsample was equilibrated with 12 ml of 0.3% CO2 in helium on a Thermo (Bremen) GasBench preparation unit for 18 h at 25.0 ± 0.1 °C. The equilibrated gas was measured in 10 replicates with a Thermo Advantage isotope ratio mass spectrometer (IRMS) in continuous-flow mode and values more than 1 standard deviation from the average were excluded. The filtered average was corrected to the international VSMOW-SLAP isotope scale using a three-point calibration provided by three laboratory standards analysed before and after every batch of 84 samples. In addition, a control sample was measured at every 12th position to correct for instrumental drift. Consensus values for the laboratory standards have been obtained from 6-year internal laboratory calibration records against primary reference materials, VSMOW, GISP and SLAP, external 6-member interlaboratory comparison exercise and by back-calculation from the ~ 170 member IAEA interlaboratory comparison exercise, WICO2012. The laboratory standards and their consensus values are as follows: ICE (δ18OVSMOW = − 32.097 ± 0.075‰), TAP (δ18OVSMOW = − 11.432 ± 0.038‰), SEA (δ18OVSMOW = − 0.029 ± 0.036‰).Seawater samples were collected from the reef slope around the BOA array using diver operated 5L Nisken bottles and from deeper waters offshore of the study site during CTD casts from a small vessel  measure which produces a statistic that represents the difference in average similarity between different spatial maps99. All computations are contingent on the reasonable a priori assumption that the map data are spatially autocorrelated101. NFK is an extension of the original Kappa algorithm and is based on a misclassification matrix between map cells65 but in addition, uses the weighted inputs of neighbouring cells to compute the Kappa statistic based on fuzzy set theory. By doing so, the measure of cell similarity is continuous across the map based on the value and distance of neighbouring cells (in this case with a Gaussian weighting in a 12 cell neighbourhood). The final NFK statistic is able to quantify the level of map difference and can model a human estimate of map ‘similarity’ more closely than the original Kappa103. The computed NFK statistic varies between 0 and 1 with greater values indicating a higher degree of similarity between different maps .In order to perform similarity comparisons between the spatial maps of benthic temperature and


Glioblastoma (GBM) is the most aggressive brain tumor and most resistant to therapy. The identification of novel predictive biomarkers or targets to counteract chemoresistance, requires a better understanding of the GBM primary response to therapy. The aim of our study was to assess the molecular response of GBM to the standard of care chemotherapy by temozolomide (TMZ). We established a comprehensive map of gene expression changes after treatment and discovered that GBM cells elicit a coordinated gene expression program after chemotherapy that differs between sensitive and resistant cells. We found that a novel class of genes expressed as long non-coding RNAs (lncRNAs) is involved in gene regulatory circuits in GBM and could represent novel markers of GBM patient prognosis. By shedding light on the involvement of the non-coding genome in GBM, our results may provide new mechanistic insight on lncRNAs and their importance in chemoresistance. Resistance to chemotherapy by temozolomide (TMZ) is a major cause of glioblastoma (GBM) recurrence. So far, attempts to characterize factors that contribute to TMZ sensitivity have largely focused on protein-coding genes, and failed to provide effective therapeutic targets. Long noncoding RNAs (lncRNAs) are essential regulators of epigenetic-driven cell diversification, yet, their contribution to the transcriptional response to drugs is less understood. Here, we performed RNA-seq and small RNA-seq to provide a comprehensive map of transcriptome regulation upon TMZ in patient-derived GBM stem-like cells displaying different drug sensitivity. In a search for regulatory mechanisms, we integrated thousands of molecular associations stored in public databases to generate a background “RNA interactome”. Our systems-level analysis uncovered a coordinated program of TMZ response reflected by regulatory circuits that involve transcription factors, mRNAs, miRNAs, and lncRNAs. We discovered 22 lncRNAs involved in regulatory loops and/or with functional relevance in drug response and prognostic value in gliomas. Thus, the investigation of TMZ-induced gene networks highlights novel RNA-based predictors of chemosensitivity in GBM. The computational modeling used to identify regulatory circuits underlying drug response and prioritizing gene candidates for functional validation is applicable to other datasets.  Since the introduction of temozolomide (TMZ)  in 2005 As studies of the transcriptional changes induced by TMZ mainly assessed long-term changes following the selection of resistant clones, the primary impact of TMZ on the GBM transcriptome remains elusive . MoreoveAdvances in next-generation sequencing (NGS) have revealed the importance of long non-coding RNAs (lncRNAs) as major components of the human transcriptome . LncRNAsIn this project, using sequencing of small and long RNAs, we established the transcriptomic changes induced by TMZ in patient-derived GBM stem-like cells , differi8 nM vs. 2.7–3.7 × 105 nM for the MGMT negative NCH421k and NCH601)  ,26 and o NCH601) . Since T NCH601) a. The ba NCH601) b, wherea NCH601) . DDR act NCH601) b, while  NCH601) b. As exp NCH601) . Taken t NCH601) c from hi NCH601) c.To determine the immediate transcriptional response of GBM to TMZ, we carried out RNA sequencing from cells treated for 24 h with TMZ or vehicle. From the same RNA samples, we performed small RNA sequencing, to allow the direct comparison of small RNA, mRNA and lncRNA expression levels, an essential experimental set-up for accurate data integration. Principal component analysis (PCA) identified individual variations between the respective GBM lines as the main determinant of transcriptome variability . In bothSOCS3, CSMD1, and SULF2  in chemoresistance [We then asked if there was a coordinated TMZ-regulation of coding genes leading to statistical functional pathway enrichment . Althougsistance ,30. ENSG00000224272, ENSG00000233230, ENSG00000233695 (or GAS6-AS1), and ENSG00000246263  revealed 133 lncRNAs as putative prognostic markers in GBM . Four of00246263 a. Using 00246263 b and dis00246263 , and conal brain c. We theal brain  and the al brain . In summA common challenge in noncoding RNA research is to predict the biological significance of newly identified molecules and select key genes for functional validation. As noncoding RNAs exert major functions in transcriptional regulation, we sought to uncover regulatory networks underlying the GBM response to TMZ. To this aim, we developed a pipeline based on available information on lncRNA and mRNA associations with transcription factors (TFs) and miRNAs. To build such a global gene regulatory network of mRNAs, ncRNAs (miRNAs and lncRNAs), and TFs, we first retrieved molecular association data from several databases: ChipBase, StarBase, and miRcode databases a 34,35,,35,36. TNext, we mapped our experimental dataset of TMZ-induced DEGs on the RNA interactome and extracted specific networks. In these networks, mRNAs coding for transcription factors were labeled as TFs. These analyses resulted in cell line-specific subnetworks composed of RNA-RNA and/or TF-RNA interactions. Cell line subnetworks were visualized as circular layouts b using cWe next analyzed the connectivity between the DEGs. We identified complex subnetworks in NCH601 and NCH421k that displayed a large number of DEGs. In contrast, only a small subnetwork was detected in NCH644, which displayed relatively few TMZ-associated DEGs, with some of its components being also present in NCH601 networks. We then measured the connectivity of each node  by calculating the in-degree (number of interactions that target a node) and out-degree of each node (number of out-going edges from a node). Notably, miR-19a-3p and miR-19b-3p displayed high out-degree values in almost all cells, suggesting a prominent regulatory role for these miRNAs in TMZ treatment response. z-value) and identified common promoter motifs as for example YBX1, NRF1, and SP1  involving TFs, miRNAs, mRNAs, or lncRNAs a. Such lWe focused on the composition of motifs involving lncRNAs, as such motifs were only present in the most sensitive GSCs (NCH601). The 22 lncRNAs formed 104 loops by interaction with a limited number of regulators  with mRNAs. We investigated the TMZ associated gene regulatory networks and selected mRNA-miRNA and miRNA-lncRNA pairs for the seven miRNAs involved in TMZ regulatory loops b: let7e-The transcriptional response to chemotherapeutic drugs has so far been mostly analyzed at the level of mRNAs, yet, a wealth of information is present in non-coding RNAs, making up more than 90% of the human genome . Here, wOur study provides a comprehensive overview of the RNA regulatory circuits induced by TMZ and lays the basis for exploring non-coding RNA function in GBM tumorigenesis and chemosensitivity. The analysis of regulatory network identified key TF-miRs molecular interactions, as the crosstalk between mir-19 and MYC . Mir-19,Our work has revealed that a large number of lncRNAs are differentially regulated by TMZ in chemo-sensitive and resistant GSCs, as well as in non-malignant NSCs, and identified key lncRNAs that may be linked to the regulation of DDR, apoptosis, and EGFR signaling in GBM, as part of co-expression networks with mRNAs and other small RNAs. Although several DNA repair mechanisms are involved in the repair of TMZ-induced lesions, TMZ resistance in the clinic has so far been associated mainly with the activity of MGMT and the selection of resistant clones that account for tumor recurrence . To dateThe glioblastoma stem-like cell cultures NCH421k, NCH601 and NCH644 were generated in the lab of Dr Christel Herold-Mende   and charTM BrdU Flow Kit  according to the manufacturer’s instructions. The following conjugated antibodies were used: anti H2AX-Phospho Ser139-Alexa Fluor 647 , anti-ATM-PhosphoSer1981-PE , Alexa Fluor® 647 Mouse IgG1 κ Isotype Control , Mouse IgG1 Negative Control, clone Ci4, PE conjugate .DNA was counterstained with DAPI (1 ug/mL) and anti–BrDU-PerCP-Cy5.5 . Data were acquired on a FACS AriaTM SORP cytometer (BD Biosciences) and analyzed with Diva (BD Biosciences) and FlowJo software version v10.5.3. TMZ or vehicle were added 24 or 6 h prior to analysis. For proliferation assay, BRDU (Bromodeoxyuridine / 5-bromo-2’-deoxyuridine) incorporation was performed for 6 h before the end of the experiment. Prior to fixation, cells were dissociated and incubated with the IR-LIVE/DEAD® Fixable Dead Cell Stain . Cells were fixed, permeabilized and stained with the BD PharmingenTotal RNA was extracted with Trizol and quantified using Nanodrop. Three biological replicates were used per experimental condition. RNA quality was checked using a bioanalyzer . Total RNAs were depleted from Ribosomal RNAs using RiboMinus™ technology. RNA-seq libraries were prepared according to the Illumina standard protocol using TruSeq Stranded RNA Kits. Small RNA-Seq libraries were generated from total RNA using TruSeq Small RNA Library Prep Kit. Single stranded sequencing reads were performed on HiSeq2500 instrument . Base calling was performed with CASAVA 1.8.2 pipeline (Illumina). Fastq data and processed counts of RNA-seq  and small RNA-seq  are available through Gene Expression Omnibus (GSE98128). The associations of TFs with genes, miRNAs, or lncRNAs were obtained from ChipBase . AssociaPrediction of lncRNAs as potential prognostic markers were reported in , analyzeIn conclusion, this study provided a comprehensive map of transcriptome regulation upon TMZ in GSCs displaying different drug sensitivity. The transcriptional response to TMZ is a coordinated program of coding and non-coding RNAs, orchestrated by regulatory loops, some of them being oppositely modulated in sensitive and resistant GBM cells. Importantly, we uncovered a subset of largely unknown lncRNAs potentially involved in essential pathways of tumorigenesis and drug resistance. Several TMZ regulated lncRNAs display prognostic value in GBM patients. This data resource, the systems approaches, and novel RNA targets identified in this study open the way for understanding lncRNA function in GBM.


C. elegans, unc-59 and unc-61 . Here, with live imaging of green fluorescent protein (GFP) fluorescence, we characterize UNC-59/Septin expression and localization throughout C. elegans development. Using CRISPR/Cas-9, we endogenously tagged C. elegansunc-59, which is most similar to human septins 1,2,4,5, and 7 , with GFP for visualization and live imaging using high resolution confocal microscopy. We first show the localization of UNC-59 at the cleavage furrow ) during a time lapse of cell divisions in early 2- to 4-cell stages of embryogenesis  and throughout embryogenesis . Septins are also important for gonad morphogenesis and distal tip cell (DTC) migration  where UNC-59 protein is detected throughout gonad development in the rachis ) and DTCs . We highlight UNC-59/Septin localization in the DTC ) at the L2 and L3 stages where it is organized into bundles  and ring structures . The two bilateral sex myoblast cells express UNC-59 during their posterior to anterior migration in the L2 and early L3 stage  and continue to express UNC-59 in these cells as they differentiate into vulval muscles in the late L3 to early L4 stages . Lastly, we show UNC-59/Septin expression and localization in tissue not previously reported: in the pharynx  ; in the seam cells, both in bundles and at the cleavage furrows, beginning in the L1 stage  and continuing throughout development and into the adult; and in sperm surrounding an embryo that has exited the spermatheca .Septins are cytoskeletal proteins involved in cytokinesis, morphogenesis, and cell migration. Misregulation of septin expression occurs in neurodegenerative diseases and cancers (Angelis and Spiliotis 2016). Originally discovered in yeast, there are 13 mammalian septins and two in unc-59(qy88[unc-59::GFP::3xflag::AID+loxP]), pDD282 (GFP-C1^SEC^3xFlag^AID) was modified (Addgene 66823). Primers to generate the homology arms (amplified from gDNA): 5’ F: tacgactcactatagggcgaattgggtaccacaactagtCGTAATGTTCATTATGAGAATCRISPR constructs were generated using a self-excising cassette (SEC) for drug (hygromycin) selection as described previously . Guide plasmids were generated using plasmid pDD122 (Peft-3::Cas9 + ttTi5605 sgRNA); Addgene 47550. SG: AAGAAACGAATGGGCGGTCTCGG. To generate 5’ R: TGGGACAACTCCAGTGAACAATTCTTCTCCTTTACTCATGTTTCGATTAAACAATCCtAatCCtCCCATTCGTTTCTT3’ F: AAATCAAGCGGTGGCCCGGAGGCGGCGGCGTTCGTGAATAATTCCCTCATTTTTTAAACG3’ R: agggaacaaaagctggagctccagcggccgctttgcatgCATGTCTTTGTAATGCTGTGGThe first two codons of GFP were removed to generate a direct fusion after a recommendation from Amy Gladfelter at UNC Chapel Hill.


Dear Editor,., 2020[Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded non-coding RNA molecules, which is 20-25 base pairs in length. siRNAs can regulate the expression of genes, by a phenomenon known as RNA interference (RNAi). Based on the phenomenon, the siRNA based therapeutics have been developed and implemented for anticancer, antiviral, and genetic diseases .., 2020[In December 2019, WHO reported the outbreak of a novel coronavirus, designated as SARS-CoV-2 or severe acute respiratory syndrome-related coronavirus. This virus has currently spread across 212 countries which resulted in 2,416,135 active cases of infection, and approximately 165,939 mortalities, as per WHO  which coded for twenty-seven structural and nonstructural proteins . At theTo date, various delivery systems for siRNA have been identified which are broadly classified into nanoparticles based carriers and viral vectors. In 2003, siRNA based drug was developed by Sirnaomics, Inc.  for the outbreak of SARS-CoV and H5N1 influenza. Besides, in 2017, six siRNA have been developed by Alnylam Pharmaceuticals (USA) and Vir Biotechnology against infectious diseases. Further, Alnylam Pharmaceuticals (USA) has designed and synthesized over 350 siRNA targeting highly conserved regions of the available SARS-CoV-2 genome  dendrimer nanocarriers for the potential aerosol-based delivery system of siRNA onto lung epithelial cells (Conti et al., 2014[For SARS-CoV-2, the ciliated cells of the human lungs are the primary site for viral infection, with reports indicating viral transmission via contact, droplets of saliva or fomites from the infected person. Therefore, techniques designed for optimal delivery of drugs onto the lung epithelial cells can provide better and timely results. In this context, Conti and co-researchers have demonstrated an Despite the pandemic outbreaks of COVID-19 and the high rate of transmission in humans, there is no specific treatment for the COVID-19 at present. Thus, for the treatment of COVID-19 siRNA based therapy can be developed against the novel coronavirus SARS-CoV-2, where siRNAs can hit the highly conserved region of SARS-CoV-2 RNA and also can act as an inhibitor to suppress the genetic disorders of the lungs. This approach could help to achieve a better treatment goal that can reduce the pandemic threat of COVID-19.The authors declare no conflict of interest.