Xanthomonas citri subsp. citri (Xcc) is an economically important disease in most citrus production regions worldwide. Xcc secretes a transcriptional activator like effector (TALE) PthA4 to bind to the effector binding elements (EBEs) in the promoter region of canker susceptibility gene LOB1 to activate its expression, which in turn causes canker symptoms. Editing the EBE region with Cas9/gRNA has been used to generate canker resistant citrus plants. However, most of the EBE-edited lines generated contain indels of 1–2 bp, which has higher possibility to be overcome by PthA4 adaptation. The adaptation capacity of TALEs inversely correlates with the number of mismatches with the EBE. LbCas12a/crRNA is known to generate longer deletion than Cas9. In this study, we used a temperature-tolerant and more efficient LbCas12a variant (ttLbCas12a), harboring the single substitution D156R, to modify the EBE region of LOB1. We first constructed GFP-p1380N-ttLbCas12a:LOBP, which was shown to be functional via Xcc-facilitated agroinfiltration in Pummelo (Citrus maxima) leaves. Subsequently, we stably expressed ttLbCas12a:LOBP in Pummelo. Eight transgenic lines were generated, with seven lines showing 100% mutations of the EBE, among which one line is homozygous. The EBE-edited lines had the ttLbCas12a-mediated deletions of up to 10 bp. Importantly, the seven lines were canker resistant and no off-targets were detected. In summary, ttLbCas12a can be used to efficiently generate biallelic/homozygous citrus mutant lines with short deletions, thus providing a useful tool for the functional study and breeding of citrus.Citrus canker caused by  Streptococcus pyogenes to edit citrus PHYTOENE DESATURASE (PDS) via transient expression in Citrus sinensis. Transgenic expression of SpCas9/gRNA [Staphylococcus aureus [Lachnospiraceae bacterium [Citrus is one of the top fruit crops worldwide. However, citrus production faces many biotic and abiotic challenges including citrus huanglongbing and bacterial canker, droughts, flooding, and freezes ,2,3,4,5.as9/gRNA ,13,14,15s aureus , and LbCacterium  have beeacterium  and Duttacterium  demonstrXanthomonas citri subsp. citri (Xcc). Xcc causes typical hypertrophy and hyperplasia symptoms by secreting a transcriptional activator like effector (TALE) PthA4, a key pathogenicity factor of Xcc, into the nucleus of plant cells. PthA4 binds to the effector binding elements (EBEs) in the promoter region of the canker susceptibility gene LOB1 to activate its expression, thus causing canker symptoms [LOB1 abolishes the canker symptoms caused by Xcc [Citrus maxima) mutant line containing an adenine deletion within EBEPthA4-LOBP, and one biallelic Pummelo mutant line comprising a thymine insertion within one EBEPthA4-LOBP allele and a two-adenine deletion within another allele [C. paradisi) mutant line containing a thymine insertion in one allele and an adenine insertion in another allele [CRISPR-mediated genome editing has been used to generate canker resistant citrus varieties. Citrus canker is an economically important citrus bacterial disease that is present in most citrus producing countries. Citrus canker is caused by symptoms ,19,20. Gd by Xcc . In addid by Xcc ,13,14,21r allele . Our datr allele . Thus, ir allele  and usinr allele ,23.Prevotella and Francisella 1, is a class II/type V CRISPR nuclease. Distinct from Cas9, Cas12a requires T-rich protospacer-adjacent motif (PAM) sequences TTTV  and a 23 nt crRNA [Lachnospiraceae bacterium ND2006, shows the highest efficacy [Cas12a (formerly Cpf1), derived from nt crRNA . Important crRNA . In addint crRNA . Notablynt crRNA ,27. Thount crRNA , CRISPR/efficacy .Arabidopsis [LbCas12a has been used to edit genomes of monocot plants, including rice and maize ,35,36,37bidopsis , consistbidopsis .PthA4-LOBP. ttLbCas12a function was first tested via Xcc-facilitated agroinfiltration in Pummelo leaf. Subsequently, we conducted stable expression of ttLbCas12a in Pummelo and EBEPthA4-LOBP was successfully modified with multiple mutations up to 10 nucleotides deletion. Notably, the ttLbCas12a-mediated mutation rates were 100% in seven transgenic Pummelo lines, including one homozygous line, and the seven edited lines were resistant against citrus canker.In this study, we used ttLbCas12a to modify citrus EBESbfI (5′-AGGTCCTGCAGGTGGCAGACATACTGTCCCACAAATGAA-3′), while CmYLCV-3-BamHI (5′-AGGTGGATCCAGCTTAGCTCTTACCTGTTTTCGTCGT-3′) was amplified from Addgene plasmid pDIRECT_10E (Addgene plasmid #91209) and cloned into SbfI-BamHI-digested GFP-p1380N-Cas9 to produce GFP-p1380N-CmYLCV-Cas9. GFP-p1380N-Cas9 was constructed in our previous work [BamHI and EcoRI digestion . BamHI-LbCpf1-EcoRI fragment was inserted into BamHI-EcoRI-cut GFP-p1380N-CmYLCV-Cas9 to form GFP-p1380N-ttLbCas12a.The CmYLCV promoter was amplified using primer CmYLCV-5-ous work . TemperaXhoI and SacI cut, AtU6-crRNA:LOBP was inserted into XhoI-SacI-digested pUC-NosT-crRNA-cspds from pUC-NosT-crRNA:LOBP. pUC-NosT-crRNA-cspds was constructed previously [EcoRI-NosT-crRNA:LOBP-NosT-PmeI fragment was cloned into EcoRI-PmeI-cut GFP-p1380N-ttLbCas12a to generate GFP-p1380N-ttLbCas12a:LOBP . After eviously . Finally12a:LOBP .A. tumefaciens strain EHA105. Recombinant Agrobacterium cells were cultured for Xcc-facilitated agroinfiltration or epicotyl citrus transformation.The binary vector GFP-p1380N-ttLbCas12a:LOBP was electroporated into Citrus maxima) was grown in a greenhouse at around 28 °C and was pruned for uniform shooting before Xcc-facilitated agroinfiltration. It should be kept in mind that ttLbCas12a performed better at 28 °C [Pummelo  Osbeck x Poncirus trifoliata (L.) Raf.) for further analysis.All explants were inspected for GFP fluorescence six weeks after incubation. GFP-positive sprouted shoots were selected and micro-grafted on ‘Carrizo’ citrange rootstock plants  and 35T-3 (5′-TTCGGGGGATCTGGATTTTAGTAC-3′).Genomic DNA was extracted from the Pummelo leaves treated by agroinfiltration or each transgenic Pummelo line. To analyze ttLbCas12a-mediate LOBP mutations, PCR was carried out using primers LOBP3 (5′-AGGTAAGCTTATTCATATTAACGTTATCAATGATT-3′) and LOBP2 (5′-ACCTGGATCCTTTTGAGAGAAGAAAACTGTTGGGT-3′). The PCR products were sequenced either through cloning and colony sequencing or direct sequencing using primer LOB4 (5′-CGTCATTCAATTAAAATTAATGAC-3′). Ten random colonies for each transgenic Pummelo line were selected for sequencing. Chromas Lite program was used to analyze the sequencing results.A Zeiss Stemi SV11 dissecting microscope equipped with an Omax camera was used to detect GFP fluorescence of the Pummelo leaves treated by Xcc-facilitated agroinfiltration and GFP-p1380N-ttLbCas12a:LOBP-transformed Pummelo, under illumination of the Stereo Microscope Fluorescence Adapter (NIGHTSEA). Subsequently, the Pummelo leaves were photographed with the Omax Toupview software.8 CFU/mL) using needleless syringes. Canker symptoms were observed and photographed at five DPI.Wild type and transgenic Pummelo plants were grown in a greenhouse at the Citrus Research and Education Center, University of Florida. Before Xcc inoculation, all plants were trimmed to generate new shoots. Leaves of similar age were inoculated with either Xcc or XccΔpthA4:dLOB1.5 , but not in #Pumtt7, whose mutation rate was 0  from the target site . The delsertions , the othsertions , which i8 CFU/mL. No canker symptoms were observed on seven transgenic plants (#Pumtt 1–6 and 8). Typical canker symptoms were observed on wild type and #Pumtt7 Pummelo plants at five days post inoculation (DPI)  containing 100% indels were resistant against Xcc infection, which results from EBEPthA4-LOBP disruption  . The ressruption .PthA4-LOBP editing, wild type and transgenic Pummelo plants were treated with XccΔpthA4:dLOB1.5. dLOB1.5 is a designed TALE developed to recognize the sequence 5′ TAAAGCAGCTCCTCCTCATCCCTT 3′  conferred resistance to citrus canker.To further verify that the canker resistance of transgenic Pummelo plants was attributed to EBECCCTT 3′ , a sequeEBEPthA4 . Sanger http://www.rgenome.net/cas-offinder/ (accessed on 12 May 2021)) was used to search the potential off-targets of GFP-p1380N-SpCas9p:PumLOBP crRNA. When up to 3 bp mismatches with the targeting crRNA were used for searching, no potential off-targets were identified  ,14. MostRemarkably, the mutation frequencies of seven ttLbCas12a-transformed Pummelo were 100%, and only one plant was not edited. In a previous study, the highest mutation rate was 55% in LbCas12a-transfomred citrus . Thus, tNo off-targets were identified for ttLbCas12a-mediated genome editing of citrus. Intriguingly, similar scenario was also observed for LbCas12a-mediated genome editing of citrus . This coIn summary,  ttLbCas12a was successfully adapted to generate homozygous/biallelic Pummelo mutant lines; thus, it can be used as a valuable tool for functional study of citrus genes and breeding. Intriguingly, ribonucleoproteins (RNPs) consisting of LbCas12a and crRNA were employed for transgene-free genome editing ,52. Deli


Caenorhabditis elegans olfactory neuron amphid wing “C” cell (AWC), while two opposite cGMP signaling pathways are responsible for odor-sensing in olfactory neuron amphid wing “B” cell (AWB): (1) a depolarizing Gα (GPA-3)- phosphodiesterase (PDE) – cGMP pathway which can be activated by low concentrations of isoamyl alcohol (IAA), and (2) a hyperpolarizing Gα (ODR-3)- GC- cGMP pathway sensing high concentrations of IAA. Besides, IAA induces Gα (ODR-3)-TRPV(OSM-9)-dependent currents in amphid wing “A” cell (AWA) and amphid neuron “H” cell with single ciliated sensory ending (ASH) neurons with different thresholds. Our results demonstrate that an elaborate combination of multiple signaling machineries encode the intensity of olfactory input, shedding light on understanding the molecular strategies on sensory transduction.Various odorants trigger complex animal behaviors across species in both quality- and quantity-dependent manners. However, how the intensity of olfactory input is encoded remains largely unknown. Here we report that isoamyl alcohol (IAA) induces bi-directional currents through a Gα- guanylate cyclase (GC)- cGMP signaling pathway in  Animals sense numerous volatile compounds from environmental cues to locate food sources, avoid predators and pathogens, and communicate with each other . For exaCaenorhabditis elegans offers a powerful model to mechanistically study how the nervous system responds to various odorants at the molecular, cellular and circuit levels. Equipped with a compact nervous system with only 302 neurons and 56 glial cells, C. elegans can sense a vast number of odors and execute a wide range of olfactory-related behaviors such as chemotaxis to food sources, pathogen avoidance, social feeding, olfactory associativelearning and imprinting (C. elegans- amphid wing “A” cell (AWA), amphid wing “C” cell (AWC), amphid wing “B” cell (AWB), amphid neuron “H” cell with single ciliated sensory ending (ASH), and amphid neuron “L” cell with dual ciliated sensory endings (ADL) (The nematode printing . Five pags (ADL) . AWC andgs (ADL) .2+-activated Cl– channels  are employed as olfactory receptors (ORs) . Variouschannels . In Dros binding . Previou elegans . Notably elegans .C. elegans exhibits distinct behaviors to certain odorants depending on their concentrations  triggered similar responses in AWCOFF but not AWCON , and that of IAA removal-induced inward currents was 1.25 ± 0.35 s (n = 12)  , which a elegans , supportunc-13(e51), unc-13(e1091), unc-31(e928), and unc-18(e81) mutants, in which the release of neurotransmitter and/or neuropeptide is blocked (A previous study has reported that AWC is suppressed by other neurons under high concentrations of IAA . We then blocked . Remarka blocked . These runc-13(e51) mutants (unc-13(e51) or unc-31(e928) mutants were equal to that in wild-type worms (We next checked IAA-induced currents in AWB neurons. High concentration of IAA (1%) induced outward currents in AWB and inward currents upon removal, which were similar to those in AWC . Strikin mutants . Additiope worms . Taken todr-3(n2150) or osm-9(ky10) mutants  mutants, suggesting that OSM-9 is required for the IAA-induced olfactory responses in AWA neurons  mutant worms, the cGMP-induced currents were abolished in both AWC and AWB neurons (tax-2(p691) or tax-4(ks11) mutant background, IAA failed to induce any detectable current in either AWC or AWB neurons (tax-2(p691) mutants as compared to wild-type worms and the input resistances were higher (A cyclic nucleotide-gated ion channel (CNG) encoded by two subunits of tax-2 (α-subunit) and tax-4 (β-subunit) is required for olfaction, thermosensation, phototransduction, gustation and O elegans . By dial elegans . In tax- neurons . Further neurons . We alsoe higher , indicatcng-1 (jh111);cng-3 (jh113) double mutants were similar to that of wild-type animals, suggesting that CNG-1 and CNG-3 are not required for olfactory transduction in AWC (Data not shown).Additional CNG channel homologs CNG-1 and CNG-3 are expressed by several amphid neurons including AWC . HoweverC. elegans genome encodes 21 Gα, 2 Gβ, and 2 Gγ subunits. The Gi/o-like Gα protein ODR-3, GPA-2, GPA-3, GPA-5, and GPA-13 have been implicated in mediating olfactory-related behaviors in C. elegans  mutants, and the residual currents were eliminated in odr-3(n1605);gpa-3(pk35) double mutants. In gpa-3(pk35) mutants, the outward currents were slightly reduced, but the inward currents were unaffected ;gpa-3(pk35) double mutants, suggesting that ODR-3 and GPA-3 are also required for the IAA-mediated olfactory responses in AWB (gpa-3(pk35) mutants but kept normal in odr-3(n2150) mutants (gpa-3(pk35) mutants, and the deficit was fully rescued by expressing of ODR-3 specifically in AWB neurons  mutants instead of the outward currents in the wild-type worms (odr-3(n2150) mutants were similar to those induced by 0.01% IAA in wild-type worms  or daf-11(ks67) mutants, suggesting that cGMP synthesis by ODR-1 and DAF-11 is essential for the olfactory transduction in AWC  mutants ;pde-2(nj58);pde-3(nj59);pde-5(nj49) quadruple mutants were not significantly different from those in wild-type worms ;pde-6(ok3410) double mutants, suggesting that cAMP is not required for IAA-sensing in AWC neurons (Data not shown).Decrease of cGMP can be achieved by more degradation, less synthesis, or both. There are six PDEs in ave cGMP . PDEs ha elegans . Howeverpe worms . Howeverpe worms . Loss of animals . These olar cGMP . In addipde-1(nj57);pde-2(nj58); pde-3(nj59); pde-5(nj49) quadruple mutants, the amplitude of 1% IAA addition-induced outward currents was increased and that of 1% IAA removal-induced inward currents was reduced  mutant animals, but not in either pde-1(nj57) or pde-2(nj58) or pde-3(nj59) mutants, suggesting that PDE-5 plays a role in 0.01% IAA-induced responses in AWB neurons (pde-5(nj49) mutants ;pde-2(nj58);pde-3(nj59);pde-5(nj49) quadruple mutants , but did not change the amplitude of ThRCs .Requests for information and for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Lijun Kang (Escherichia coli using standard methods previously described (tax-2(p691), tax-4(ks11), cng-1(jh111);cng-3(jh113), unc-13(e51), unc-13(e1091), unc-31(e928), unc-18(e81), odr-3(n2150), gpa-3(pk35), odr-3(n1605);gpa-3(pk35), odr-1(n1936), daf-11(ks67), osm-9(ky10), pde-1(nj57), pde-2(nj58), pde-3(nj59), pde-5(nj49), pde-1(nj57);pde-2(nj58);pde-3(nj59);pde-5(nj49) and pde-4(nj60);pde-6(ok3410) were provided by the Caenorhabditis Genetic Center (CGC).All nematode strains were cultivated at 20°C on nematode growth medium (NGM) plates seeded with the OP50 strain of escribed . Well-fePodr-1:RFP (AWC and AWB), Podr-1:DsRed2b (AWC and AWB), Pstr-2:DsRED (AWCon), Pstr-1:DsRED (AWB), Psra-6:DsRED (ASH and ASI), Pstr-3:YFP (ASI), Podr-10:mCherry (AWA) and Psrh-220:mCherry (ADL) transgenic strains were used to identify AWC, AWB, ASH, AWA and ADL neurons, respectively. Pstr-1:GCaMP5.0 was constructed for calcium imaging in AWB neurons.Promoters were PCR-amplified from N2 genomic DNA and then recombined with specific donor vector fragments using the In-Fusion PCR Cloning Kit (TaKaRa Inc.). Chemotaxis assays were performed essentially as described . Briefly2, 1 MgCl2, 20 D-glucose and 10 HEPES . The pipette solution constitutes (mM) of 145 potassium gluconate, 2.5 KCl, 5 MgCl2, 10 HEPES, 0.25 CaCl2, 10 glucose, 5 EGTA, 5 Na2ATP and 0.5 NaGTP .Patch-clamp recording was carried out on an Olympus microscope (BX51WI) with an Axon 700A patch-clamp amplifier and Digidata 1320A interface (Axon Instruments). Signals were filtered at 2 kHz using amplifier circuitry, sampled at 10 kHz, and analyzed using Clampex 10.4 (Axon Instruments). Recording pipettes were pulled from borosilicate glass capillaries  to a resistance of 15–20 MΩ on a P-97 micropipette puller (Sutter Instruments). Worms were glued on the surface of a sylgard-coated cover glass. A small piece of cuticle in the head of the worm was cut open and glued to the coverslip to expose the neurons of interest for recording as previously described . OdorantAnimals were immersed in bath solution  and subsequently glued on a glass coverslip with a medical grade cyanoacrylate-based glue  . All odot-test or two-way ANOVA was used to compare data sets. If the data were not normally distributed, the Wilcoxon test was used. P < 0.05 was considered to be statistically significant. Sample sizes were determined by the reproducibility of the experiments and are similar to the sample sizes generally used in the field.Data analysis was performed using GraphPad Prism 6. All data were presented as mean ± SEM. Unpaired two-tailed Student’s The original contributions presented in the study are included in the article/LK and SD designed the experiments. HC conducted the calcium imaging experiments. YL conducted the electrophysiological experiments. YX, JS, ZT, and SL conducted the other experiments. HC and YL contributed equally to this study. YL, HC, and LK analyzed and interpreted the results. YL and LK wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Docetaxel resistance developed in half of castration-resistant prostate cancer (CRPC) patients hinders its long-term clinical application. The current study was designed to investigate the effects of Chinese medicine Zhoushi Qi Ling decoction on the docetaxel resistance of prostate cancer as well as elucidate the underlying molecular mechanism. In our study, Qi Ling significantly decreased viability and colony formation as well as increased apoptosis of docetaxel-resistant (DR) CRPC cells. Qi Ling-treated DR cells exhibited decreased glucose consumption, lactate release and pyruvate production. Moreover, lncRNA SNHG10 was upregulated in DR tissues of CRPC patients and was negatively correlated with the progression-free survival. Bioinformatics analysis indicated miR-1271-5p as the associated miRNA possibly binding with SNHG10. miR-1271-5p up-regulation dramatically decreased the luciferase activity of SNHG10 in DR cells. SNHG10 knockdown sharply increased the expression of miR1271-5p in DR cells. Targetscan predicted TRIM66 as one of the downstream targets of miR-1271-5p. miR-1271-5p up-regulation drastically reduced luciferase activity as well as TRIM66 expression in DR cells. Also, the knockdown of SNHG10 remarkably suppressed the expression of TRIM66 in DR cells. Additionally, Qi Ling treatment reduced SNHG10 and TRIM66, while increased miR1271-5p, in DR cells. In summary, Qi Ling inhibited docetaxel resistance and glycolysis of CRPC possibly via SNHG10/miR-1271-5p/TRIM66 pathway. Despite dramatic progress in therapeutics having been made in recent years, prostate cancer remains one of the leading causes of cancer-related death in American men . As prosThe dysfunction in energy metabolism usually occurs in malignant tumors . In physLong non-coding RNAs (lncRNAs) have long been recognized as transcription by-products of RNA polymerase II with no biological functions , 12. In “Qi Ling” was composed of kushan (150 g), rubescens (300 g), raw astragalus (150 g), turmeric (90 g), psoraleae (150 g), cooked rehmannia glutinosa (150 g), motherwort (150 g), and processed licorice (90 g). It was boiled in hot water for 3–4 hours, then refilled to 1 L.2. Docetaxel-resistant (DR) cell lines (PC3-DR and DU145-DR) were successfully established via exposing monolayer-cultured cells to incremental concentrations of docetaxel  in scheduled procedures according to the methods described previously [PC3 and DU145, as two human prostate cell lines, were purchased from American Type Culture Collection . The cells were maintained in RPMI-1640 medium  containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco) at 37°C with 5% COeviously , 19. TheHuman study was approved by LONGHUA Hospital Shanghai University of Traditional Chinese Medicine. All patients signed consent before surgery. Briefly, 56 docetaxel-free (DR-free) and 44 DR tissues were collected from prostate cancer patients during prostatectomy.Cell survival was determined by colony formation and Cell-counting Kit-8 (CCK-8) assays. CCK-8 assay was conducted using the corresponding detection Kit  according to the manufacturer’s instruction. In brief, equal amounts of DR cells were seeded 24 h before 48-h docetaxel treatment at concentrations from 5 to 400 nM. CCK-8 reagent was added, incubated for 3 h to measure the absorbance at 450 nm. For colony formation assay, 1000 cells per well (in 6-well plate) were seeded 24 h before treated with 10 nM docetaxel. After 10 days of culture, the cells were washed using sterile phosphate-buffered saline and fixed using 4% paraformaldehyde followed by staining with crystal violet. The colonies formed in each group were counted manually.SLC2A1, ATTGGCTCCGGTATCGTCAAC (Forward), GCTCAGATAGGACATCCAGGGTA (Reverse); PFKP, CGCCTACCTCAACGTGGTG (Forward), ACCTCCAGAACGAAGGTCCTC (Reverse); PKM, ATGTCGAAGCCCCATAGTGAA (Forward), TGGGTGGTGAATCAATGTCCA (Reverse); LDHA, ATGGCAACTCTAAAGGATCAGC (Forward), CCAACCCCAACAACTGTAATCT (Reverse); SNHG10, CCAGCTTAGATTCATTGATTCC (Forward), TTAAGTGCACCAGATGCTG (Reverse); TRIM66, GCCCTCTGTGCTACTTACTC (Forward), GCTGGTTGTGGGTTACTCTC (Reverse); miR-1271-5p, CAGCACTTGGCACCTAGCA (Forward), TATGGTTGTTCTCCTCTCTGTCTC (Reverse); U6, GCTTCGGCAGCACATATACTAAAAT (Forward), CGCTTCACGAATTTGCGTGTCAT (Reverse); β-actin, CGTCATACTCCTGCTTGCTG (Forward), GTACGCCAACACAGTGCTG (Reverse). The relative expression of target genes was analyzed using the 2−ΔΔCt method with U6 as the internal control for miR-1271-5p and β-actin for the other genes.Total RNAs were extracted using TriZol reagent . Transcription of cDNA was conducted using PrimeScript RT reagent . qRT-PCR was performed using SYBR Green PCR Kit (TaKaRa) on ABI 7300 system . Primers were used as follows (5′ to 3′): Cells of different groups were collected, then lysed using radioimmunoprecipitation buffer containing protease inhibitor. Equal amounts of proteins were loaded and separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel (10%) followed by being transferred to polyvinylidene fluoride membranes . After blocking with bovine serum albumin (5%) at room temperature for 2 h, membranes were then incubated with corresponding primary antibodies probing TRIM66  and β-actin  at 4°C overnight. After incubation with corresponding horse radish peroxidase-conjugated secondary antibodies  at room temperature for 2 h, the blots were visualized using an ECL kit .Cell apoptosis was evaluated via detecting DNA fragmentation and caspase-3 activity. Caspase 3 activity was determined using a commercial caspase 3/7 assay kit . Luminescence was detected using a microplate reader . DNA fragmentation assay was conducted via assessing the levels of cytoplasmic histone-associated DNA fragment in each group using an ELISA kit for cell death detection  according to the recommended instructions.Cell numbers from different groups were determined before detection. Cells were collected to analyze the pyruvate level using a commercial detection kit (Sigma). Culture medium was harvested and the lactate level as well as glucose after different treatments were measured. The lactate production or glucose consumption was normalized using cell number determined before detection.In order to knock down SNHG10 expression, cells were transiently transfected with two SNHG10-pecific siRNAs, si-SNHG10-1 (GCAACCGCTTTGTTAGTTAAT) and si-SNHG10-2 (GCGCGCGATTATTTCTCTAGA), or negative control, , using Lipofectamine 3000 (Invitrogen). SNHG10 was overexpressed via transfecting pcNDA3.1/SNHG10 vectors conducted through inserting cDNA of SNHG10 into pcDNA3.1 vectors as previous described . CommercLysates collected from cells with SNHG10-knockdown were incubated with biotinylated RNA probes. The miR-1271-5p sequence were biotinylated and a nonsense RNA sequence with biotin label served as the negative control. The enrichment of targeted RNA was determined by RT-qPCR.The luciferase reporter plasmid was constructed via inserting different wild-type (WT) or mutated (MUT) sequences. The target sequences were as follows: SNHG10-WT (Forward) CCTTCTCGAGAGCCTCATCCTACTGCCTT, (Reverse) CCTTGCGGCCGCATTTTGGAAAGAGTTTAA; SNHG10-MUT (Forward) CTGAGCAGCCGGCGCGCGATCGCC, (Reverse) GGCGATCGCGCGCCGGCTGCTCAG; TRIM66-WT, (Forward) CCTTCTCGAGGAGCCAAAAGGAGACTGGGC, (Reverse) CCTTGCGGCCGCTTCTTGGTAAAGAAAAGTGCTGTT; TRIM66-Mut (Forward) CAGTGACTGCCCAGTTCCAACAT, (Reverse) ATGTTGGAACTGGGCAGTCACTG. The above plasmids were co-transfected with negative control or miR-1271-5p mimics, respectively. The luciferase activity was measured using the luciferase reporter assay .t-test, One-way, Two-way ANOVA with appropriate post hoc test. It was considered statistically significant when p value was less than 0.05.Data were presented as mean ± SD. Experiments were repeated in triplicate unless specified. Statistical analysis was carried out using SPSS 16.0 and differences between indicated groups were compared using Student’s As shown in Considering Warburg effect is essential phenomena during the development of tumorigenesis, we hypothesized that Qi Ling might affect the glycolysis of prostate cancer cells. As expected, Qi Ling treatment remarkably reduced glucose consumption, lactate release and pyruvate production of CRPC cells compared to control –2C. Meanin vitro. Results of in vitro assays showed that both DR cell lines presented obvious enhanced mRNA expression of SNHG10 compared with the corresponding counterpart cell lines  was firstly applied and miR-1271-5p was predicted to be associated miRNAs possibly binding with SNHG10. Another online software (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid) was employed to predict sites possibly binding with SNHG10. Site mutagenesis was also performed , Qi Ling treatment + vector (QL-treatment + vector), and QL-treatment + SNHG10-OE groups, respectively. Qi Ling treatment significantly reduced the expression of SNHG10 and TRIM66 meanwhile increased miR-1271-5p in DR cells  and 5B. In this study, we found that Qi Ling impaired the docetaxel resistance and regulated the Warburg effect of CRPC cells. LncRNA SNHG10 was upregulated in DR tissues and was negatively correlated with survival of patients. SNHG10 up-regulated TRIM66 via sponging miR-1271-5p, which might be the molecular basis of the docetaxel resistance of CRPC cells. Finally, we found that Qi Ling might inhibited docetaxel resistance and glycolysis of CRPC via SNHG10/miR-1271-5p/TRIM66 axis.in vitro.Chemotherapeutic resistance is one of the major concerns when clinicians make therapeutic strategies for advanced cancer patients and can eventually result in cancer progression as well as poor prognosis . In receScutellaria baicalensis, is reported to reverse hypoxia-induced 5-fluorouracil resistance, via inhibiting glycolysis of gastric cancer cells [Warburg effect, characterized by enhanced aerobic glycolysis, is the important biochemical feature of cancer cells, meaning the glucose dependence of cancer cells . Warburger cells . In the in vivo, and we will perform animal experiment to make our findings more solid in the future.SNHG10 functions as an oncogenic lncRNA overexpressed in liver cancer and positively regulates carcinogenesis and metastasis . SNHG10 In summary, Qi Ling inhibited docetaxel resistance and glycolysis of CRPC via SNHG10/miR-1271-5p/TRIM66 pathway. This study first revealed the inhibition of Qi Ling on docetaxel resistance and glycolysis of CRPC cells. In addition, this study revealed anti-tumor activity of Qi Ling against prostate cancer, providing experimental evidences for chemotherapy and chemo-sensitivity study of other Chinese medicines.


Scientific Reports 10.1038/s41598-021-84777-7, published online 03 March 2021Correction to: The original version of this Article contained errors in Figure 3, where the bar showing the mineralogical combination of “high clay-low Fe (0-10 cm)” was incorrectly given.The original Figure The original Article has been corrected.


The DNA-PK maintains cell survival when DNA damage occurs. In addition, aberrant activation of the DNA-PK induces centrosome amplification, suggesting additional roles for this kinase. Here, we showed that the DNA-PK-p53 cascade induced primary cilia formation (ciliogenesis), thus maintaining the DNA damage response under genotoxic stress. Treatment with genotoxic drugs  led to ciliogenesis in human retina (RPE1), trophoblast (HTR8), lung (A459), and mouse Leydig progenitor (TM3) cell lines. Upon genotoxic stress, several DNA damage signaling were activated, but only the DNA-PK-p53 cascade contributed to ciliogenesis, as pharmacological inhibition or genetic depletion of this pathway decreased genotoxic stress-induced ciliogenesis. Interestingly, in addition to localizing to the nucleus, activated DNA-PK localized to the base of the primary cilium (mother centriole) and daughter centriole. Genotoxic stress also induced autophagy. Inhibition of autophagy initiation or lysosomal degradation or depletion of ATG7 decreased genotoxic stress-induced ciliogenesis. Besides, inhibition of ciliogenesis by depletion of IFT88 or CEP164 attenuated the genotoxic stress-induced DNA damage response. Thus, our study uncovered the interplay among genotoxic stress, the primary cilium, and the DNA damage response. Cells are continuously exposed to several stresses from endogenous and exogenous sources. To maintain physiological homeostasis, a diverse range of signaling pathways are activated in cells. In vertebrates, members of the phosphatidylinositol 3-kinase-related kinase (PI3KK) superfamily, including ataxia telangiectasia, mutated (ATM), ataxia telangiectasia, mutated and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK), initiate signaling in response to DNA damage .DNA-PK is a heterotrimeric complex composed of a catalytic subunit, DNA-PKcs, and two regulatory subunits, Ku70 and Ku80 . Once DNThe centrosome is composed of two centrioles, the mother and daughter centrioles, and surrounding pericentriolar material  and is tAutophagy is a lysosomal degradation process whereby cells degrade and reutilize old organelles and proteins to maintain metabolic homeostasis . It alsoThe primary cilium plays important roles in development and differentiation. Here, we showed that the DNA-PK-p53 cascade induced primary cilia formation, thus maintaining the DNA damage response under genotoxic stress. Treatment with genotoxic drugs led to ciliogenesis in several cell lines. Upon genotoxic stress, the DNA-PK-p53 cascade contributed to ciliogenesis. Interestingly, genotoxic stress also induced autophagy for ciliogenesis. Thus, our study uncovered the interplay among genotoxic stress, the primary cilium, and the DNA damage response.2. These cells were regularly examined for mycoplasma contamination by immunoblotting, immunofluorescence, and DAPI staining according to the guidelines.Human immortalized retina pigmented epithelial (RPE1) and mouse Leydig progenitor (TM3) cell lines were grown in Dulbecco’s modified Eagle medium (DMEM)-F12, human immortalized trophoblast (HTR8) cells were grown in Roswell Park Memorial Institute (RPMI)-1640 medium, and human adenocarcinomic human alveolar basal epithelial (A459) cells were grown in DMEM. All culture media were supplemented with 10% fetal bovine serum, and all cell lines were cultured at 37 °C in a humidified atmosphere of 5% CODorsomorphin , SBI-0206965 , Caffeine , Ku55933 , 3-methyladenine , cytochalasin D , NCS , HU , 7-hydroxy staurosporine , CPT , pifithrin-α , and chloroquine  were purchased from Sigma, St. Louis, MO. Bafilomycin-A1  was purchased from Enzo, NY, USA. Chk2 inhibitor II  was purchased from Merck Millipore, Darmstadt, Germany. Akt inhibitor IV  was purchased from Cell Signaling, Beverly, MA, USA. Cells were treated with all drugs for 24 h except HU, which added for 72 h to induce prolonged replication stress.The following antibodies were obtained commercially: anti-histone H2AX , anti-ATM (GTX70103), anti-Ku70 (GTX101820), anti-Ku80 (GTX109985), and anti-actin  ; anti-TFE3 (HPA023881), anti-acetylated-tubulin (T7451), anti-γ-tubulin (T5326), anti-α-tubulin (T9026) and anti-TTBK2 (HPA018113) ; anti-pericentrin (ab4448), anti-ATM  and anti-CP110 (ab99338) ; polyclonal anti-IFT88 (13967-1-AP) and anti-ARL13B (17711-1-AP) ; anti-DNA-PKcs (sc-9051), anti-DNA-PKcs , and anti-p53  ; anti-ULK1 (D8H5) rabbit mAb (#8054), anti-ULK1 , anti-AMPK (#2532), anti-AMPK  rabbit mAb (#2535), anti-ATR (#2790), anti-ATR , anti-LC3A/B (D3U4C) XP (#12741), anti-PCM1 , anti-Chk2 (#2662), anti-Chk2 , anti-Akt (#9272), anti-Akt , anti-p44/42 MAPK , anti-p44/42 MAPK , anti-p53 , anti-Chk1 (2360), anti-Chk1 , and anti-HSP70 (#4872) ; anti-CEP290 (A301-659A) (Bethyl Laboratories); anti-OFD1 (NBP1-89355) and anti-CEP164 (NBP1-81445) ; and anti-ATG7  .Cells were grown on glass cover slips at 37 °C before fixation with ice-cold methanol at −20 °C for 6 min. After blocking with 5% BSA for 1 h, the cells were incubated with primary antibodies for 24 h at 4 °C. After extensive washing with PBS, the cells were incubated with fluorescein isothiocyanate- and Cy3-conjugated secondary antibodies  for 1 h in the dark. The nuclei were stained simultaneously with 4′,6-diamidino-2-phenylindole . After extensive washing, the cover slips were mounted on glass slides in 50% glycerol. Fluorescent cells were examined with an Axio Imager M2 fluorescence microscope . Primary cilia were imaged with an Axio Imager M2 fluorescence microscope  and captured using ZEN pro software . Primary cilia images were created and the length of cilia were measured from z-stacks using add-on features of the ZEN pro software.DNA-PKcs, Ku70, ATM, Chk2, IFT88, and CEP164 were depleted in human RPE1 cells using annealed siRNAs with the following target sequences:siDNA-PKcs: 5′-gggcgcuaaucguacugaa [dt] [dt]-3′ ;siKu70: 5′-gaugcccuuuacugaaaaa [dt] [dt]-3′ ;siATM: 5′-aacauacuacucaaagacauu [dt] [dt]-3′ ;siChk2: 5′-aagaaccugaggaccaagaac [dt] [dt]-3′ ;siIFT88: 5′-cgacuaagugccagacucauu [dt] [dt]-3′ ;siCEP164: 5′-caggugacauuuacuauuuca [dt] [dt]-3′ .Scrambled siRNA (5′-gaucauacgugcgaucaga [dt] [dt]-3′) was purchased from Sigma .For siRNA transfections, 10 μl Lipofectamine 2000  was mixed with 500 μl Opti-MEM  for 5 min, and 2 μl siRNA (100 μM) in 500 μl Opti-MEM was added to this mixture which was then incubated at room temperature for 20 min before being layered onto cells in 1 ml DMEM/F12 (100 nM working concentration). Cells were harvested for further experiments 72 h after transfection.To generate recombinant lentivirus, plasmids expressing shRNA or envelope and packaging proteins were cotransfected into 293FT cells , and virus was harvested according to the protocols provided by the Taiwan National RNAi Core Facility. The following short hairpin RNA (shRNA) sequences were introduced into the pLKO.1 vector:pLKO.1-shluc (5′-ccuaagguuaagucgcccucg-3′) and pLKO.1-shATG7 (5′-gccugcugaggagcucuccau-3′).Lentiviruses were collected from media of 293FT cells cotransfected with pLKO.1-derived plasmids and the packaging vectors pCMVdelR8.91 and pMD.G according to the protocols provided by the Taiwan National RNAi Core Facility.The p53 knockout RPE1 cells were kindly gifts from Dr. Won-Jing Wang. The stable p53 knockout RPE1 cells were established and published . Brieflyt-tests and ANOVA for multigroup comparisons, for which a P value of < 0.05 was statistically significant.All experiments were performed in at least three independent biological replicates and all results are expressed as the mean ± S.D. (the standard error) of three independent experiments, more than 100 cells were counted in each individual group. Differences between two groups were compared using unpaired two-tailed To investigate whether genotoxic stress induces formation of the primary cilium, the topoisomerase II inhibitor ETO, a known inducer of DNA double-strand breaks, was used. ETO treatment induced DNA damage, as shown by increased γ-H2AX levels Fig. . Then, pNext, we examined whether ETO induces ciliogenesis in other cell lines. Human immortalized trophoblasts (HTR8) and mouse Leydig progenitor (TM3) cells grew primary cilia during serum starvation Fig. . Upon ETTo examine whether ETO-induced ciliogenesis is a general effect of genotoxic stress, rather than a specific response to ETO, other genotoxic stress inducers were evaluated. Neocarzinostatin (NCS), Hydroxyurea (HU), 7-hydroxy staurosporine (UCN-01), and cisplatin (CPT) induced ciliogenesis in RPE1 cells (Supplementary Fig. Next, we examined whether the DNA damage response contributes to ciliogenesis. We first examined activation of the PI3KK family. Upon ETO treatment, ATM and DNA-PKcs, but not ATR, were activated in a dose-dependent manner in RPE1 and A549 cells Fig. . To deteWe then examined whether DNA-PK activation contributes to ETO-induced ciliogenesis. Treatment of cells with the DNA-PK-specific inhibitor vanillin inhibited ETO-induced ciliogenesis Fig. . To examglued (marker protein of mother centriole, Supplementary Fig. Next, the subcellular localization of activated DNA-PK, as shown by DNA-PKcs phosphorylation, was examined. Under normal conditions, phosphorylated DNA-PKcs was hardly detected in the nucleus, and a mild signal was asymmetrically detected in one centriole at interphase (Supplementary Fig. The downstream effectors of PI3KK were further examined. We first evaluated checkpoint kinases. Chk2, but not Chk1, was activated by ETO treatment in RPE1 Fig.  and A549We then examined p53, as it is a key downstream regulator of DNA-PK and plays an important role in genotoxic stress responses. Upon ETO treatment, total and phosphorylated p53 levels increased in a dose-dependent manner Fig. . InhibitCellular stresses activate autophagy to maintain cell survival or induce apoptosis . AutophaNext, we examined whether ETO-induced autophagy contributes to ciliogenesis. Autophagic degradation was blocked by treating cells with Baf.A1, and the frequency of ciliated cells was counted. ETO induced ciliogenesis, and the frequency of ciliated cells was decreased in Baf.A1-treated RPE1 cells Fig. . This fiWe then examined whether ETO-induced autophagy was regulated by DNA-PK-p53 cascade. ETO treatment increased LC3 II to I ratio in wild-type cells, and this was reduced in DNA-PKcs- or p53-deficient cells Fig. . To furtSome centrosomal and ciliary proteins contribute to nuclear events. We thus ascertained whether ETO-induced primary cilia play a role in the DNA damage response. IFT88 and CEP164 are required for ciliogenesis; thus, the effects of primary cilia on the DNA damage response were examined in CEP164- and IFT88-deficient cells. CEP164 expression was efficiently depleted by siRNA transfection Fig. , which rWe next tested whether removal of genotoxic stress reduced ciliogenesis. RPE1 cells were treated with ETO for 24 h for ciliogenesis followed by washing with PBS and cultured in ETO-free medium for 6 and 24 h Fig. . Six houHere, we showed that genotoxic stress facilitated primary cilium formation via the DNA-PK-p53 cascade and autophagy. Interestingly, DNA-PK-p53 cascade did not activate autophagy and vice versa. Moreover, stress-induced ciliogenesis was crucial for maintaining the DNA damage response Fig. . Thus, oSerum deprivation and genotoxic stress induced primary cilia formation in RPE1 cells. Depletion of DNA-PKcs inhibited ciliogenesis induced by genotoxic stress but not by serum deprivation Fig. . HoweverHere we show that genotoxic stresses induce ciliogenesis; whereas disruption of primary cilia formation reduces DNA damage signaling. It remains unclear how cytoplasmic cilia affect nuclear signals. The role of primary cilia in the maintenance of DNA damage response is supported by previous finds that centrosomes contain several components participate in DNA damage responses . Besidesp53 plays roles in regulating autophagy . In the In summary, we show that the DNA-PK-p53 cascade and autophagy induce primary cilia formation to maintain the DNA damage response under genotoxic stress. Thus, our study deciphered the molecular mechanism by which genotoxic stress induces primary cilium formation and uncovered the novel function of the primary cilium in maintaining the DNA damage response.Supplementary dataSupplementary figure 1Supplementary figure 2Supplementary figure 3Supplementary figure 4Supplementary figure 5Supplementary figure 6Supplementary figure 7Supplementary figure 8Supplementary figure 9Supplementary figure 10Supplementary figure 11


FLT3 mutations are the most frequently identified genetic alterations in acute myeloid leukemia (AML) and are associated with poor prognosis. Multiple FLT3 inhibitors are in various stages of clinical evaluation. However, resistance to FLT3 inhibitors resulting from acquired point mutations in tyrosine kinase domain (TKD) have limited the sustained efficacy of treatments, and a “gatekeeper” mutation (F691L) is resistant to most available FLT3 inhibitors. Thus, new FLT3 inhibitors against both FLT3 internal tandem duplication (FLT3-ITD) and FLT3-TKD mutations (including F691L) are urgently sought. Herein, we identified KX2-391 as a dual FLT3 and tubulin inhibitor and investigated its efficacy and mechanisms in overcoming drug-resistant FLT3-ITD-TKD mutations in AML. KX2-391 exhibited potent growth inhibitory and apoptosis promoting effects on diverse AML cell lines harboring FLT3-ITD mutations and AC220-resistant mutations at the D835 and F691 residues in TKD and inhibited FLT3 phosphorylation and its downstream signaling targets. Orally administered KX2-391 significantly prolonged the survival of a murine leukemia model induced by FLT3-ITD-F691L. KX2-391 also significantly inhibited the growth of 4 primary AML cells expressing FLT3-ITD and 2 primary AML cells expressing FLT3-ITD-D835Y. Our preclinical data highlight KX2-391 as a promising FLT3 inhibitor for the treatment of AML patients harboring FLT3 mutations, especially refractory/relapsed patients with F691L and other FLT3-TKD mutations.The online version contains supplementary material available at 10.1186/s13045-021-01098-y. To the editorFLT3 mutations occur in more than 30% of patients with acute myeloid leukemia (AML) and are associated with short relapse-free and overall survival, including internal tandem duplication (ITD) and point mutations within the tyrosine kinase domain (TKD) , 2. To dWe used molecular docking simulations to computationally screen 1487 small molecule ligands from the L3400 Clinical Compound Library and identified KX2-391 as a candidate FLT3 inhibitor Additional file 2: Figure S1. Computational modeling of KX2-391 binding with wild-type FLT3. (a) An overview of the docking results of KX2-391 with FLT3 (Protein Data Bank: 5X02); two orthogonal views are shown. (b) Close-up of the KX2-391-FLT3 model, highlighting the hydrogen bond formed by residues LEU-616 and GLU-661, the predicted KX2-391 binding site are not included Phe691. Figure S2. KX2-391 disrupted microtubules in MOLM13 cells. MOLM13 cells were treated with dimethyl sulfoxide , 100 nM Paclitaxel, 100 nM Vincristine, 100 nM Colchicine, and 100 or 200 nM KX2-391 for 4 h, and confocal microscopy was used to observe the signal corresponding to α-tubulin (green); DNA was counterstained with DAPI (blue). Figure S3. Vincristine (VCR) does not inhibit FLT3 signaling in FLT3-ITD or FLT3-ITD-TKD cells. Ba/F3 cells expressing FLT3-ITD, FLT3-ITD-D835Y, or FLT3-ITD-F691L, and human cell lines MOLM13 and MV4-11were incubated for 12 h with the indicated concentrations of VCR  and subsequently examined by western blotting using the indicated antibodies. GAPDH was used as a loading control. Figure S4. Potent inhibition of KX2-391 against patient leukemic blast cells harboring FLT3-ITD. Patient-derived AML leukemic blast cells expressing FLT3-ITD (patients 4-6) were incubated for 48 h with the indicated concentrations of KX2-391 and AC220, and the cell viability of blast cells was then determined with CellTiter Glo assays. For each FLT3 inhibitor, the percentage over DMSO control was presented as a mean value, with error bars representing ±SD.


DRB5*01:01:01~DRB1*15:01:01:01 was significantly associated with predisposition  as reported previously. A second risk allele, DPB1*104:01 , independent from the haplotype bearing DRB1*15:01 was newly identified. The allele DRB1*01:01:01 showed significant protection . Two DQB1 alleles, DQB1*03:01  and DQB1*03:03 , defined at two-field level also showed protective effects. The HLA class I block, A*02:01:01:01~C*03:04:01:01~B*40:01:02  and the alleles B*27:05  and B*38:01  showed moderately protective effects independently from each other and from the class II associated factors. By comparing statistical significance of 11 HLA loci and 19 haplotype segments with both untruncated and two-field allele names, we precisely mapped MS candidate alleles/haplotypes while eliminating false signals resulting from ‘hitchhiking’ alleles. We assessed genetic burden for the HLA allele/haplotype identified in this study. This family-based study including the highest-resolution of HLA alleles proved to be powerful and efficient for precise identification of HLA genotypes associated with both, susceptibility and protection to development of MS.Multiple sclerosis (MS) susceptibility shows strong genetic associations with HLA alleles and haplotypes. We genotyped 11 HLA genes in 477 non-Hispanic European MS patients and their 954 unaffected parents using a validated next-generation sequencing (NGS) methodology. HLA haplotypes were assigned unequivocally by tracing HLA allele transmissions. We explored HLA haplotype/allele associations with MS using the genotypic transmission disequilibrium test (gTDT) and multiallelic TDT (mTDT). We also conducted a case-control (CC) study with all patients and 2029 healthy unrelated ethnically matched controls. We performed separate analyses of 54 extended multi-case families by reviewing transmission of haplotype blocks. The haplotype fragment including  Multiple sclerosis (MS) is a chronic debilitating neurological disorder associated with central nervous system inflammation, demyelination, and axonal degeneration . MS is cIn addition to bidirectional influences on risk, HLA allelic and haplotypic heterogeneity has been reported , togetheThe recent advent of cost-effective next generation sequencing (NGS) methods and customized algorithms allow to sequence nearly complete HLA genes including UTRs and introns, to uncover the nucleotide variation on study groups including novel variants, and to assign the least ambiguous HLA genotypes and untruncated haplotypes .th International HLA and Immunogenetics Workshop (IHIW), we generated high-resolution data for 11 HLA genes in 477 MS trio families. We took advantage of recently developed algorithms to track the segregation of NGS HLA genotypes and performed a classical transmission disequilibrium test (TDT) analysis  found in multiple generations that included samples with European ancestry. This study was approved by the University of California, San Francisco Institutional Review Board. The analyses of HLA genotype data with the double-blinded sample IDs were conducted at the Stanford Blood Center and Stanford University under the Stanford University Institutional Review Board (IRB) eProtocol titled, “17th International HLA and Immunogenetics Workshop” (#: 38899).HLA-A, -C, -B, -DRB3 ,]-DRB4, -DRB5, -DRB1, -DQA1, -DQB1, -DPA1, and -DPB1) using MIA FORA NGS FLEX HLA Typing Kits (Immucor Inc.)  database (HLA-DRB1*15:01:01:01. For genotypic ambiguities (e.g. HLA-DPB1*04:01:01:01+HLA-DPB1*04:02:01:02|HLA-DPB1*105:01+HLA-DPB1*126:01), it is possible to identify a single phased allele combination in most of the instances by reviewing all the genotypes in the family and assessing segregation (https://www.niaid.nih.gov/research/immport).We initially processed DNA from the above subjects for typing all alleles at the 11 classical HLA loci (or Inc.) , and seqor Inc.)  and impodatabase . Some indatabase . The alldatabase . We incldatabase , 25, butregation . When thregation . HLA genth IHIW database  format  were dowdatabase  and useddatabase . HaplObsdatabase . SuppleThe HLA genotypes represent a combination of HLA alleles or HLA haplotypes for target HLA loci.HLA-A*02:01:01:01) and two-field (e.g. HLA-A*02:01) allele names for 11 HLA genes (see section Genotyping), and 19 HLA haplotype blocks . We chose these blocks to eliminate false signals resulting from ‘hitchhiking’ alleles.First, we performed TDT with HLA genotypes from trio families using “Genotypic TDT “ function in trio version 3.18.0 R package –29. We uIn addition, we performed a second TDT analysis with HLA genotypes from the trio families using the Transmission/disequilibrium test of a multiallelic marker by Bradley-Terry model (mtdt2) function in gap version 1.2.2 R package –32. We uHLA-DRB1*15:01, we removed all the families that contained at least one family member with the risk haplotype bearing HLA-DRB1*15:01 in the data set. Finally, we performed conditional logistic regression TDT analyses using colGxE function in trio R package in the presence or absence of the haplotype bearing HLA-DRB1*15:01 to determine the effect and its interaction with the other HLA alleles/haplotypes  . We testSixty [(11 loci + 19 haplotypes) x 2 (untruncated and two-field)] different tests were performed for gTDT, mTDT and CC. Each software package for gTDT, mTDT and CC generates different format of output files. To circumvent manual manipulation of the output files, and capture essential information from the output files, we developed a script to summarize the results in a single file using Practical Extraction and Report Language (Perl) programming. We compared gTDT, mTDT and CC results for all observed alleles and haplotypes 12. We uvia the Guo and Thompson test (Python for Population Genomics (PyPop) version 0.7.0  was usedson test , assessison test , using avs. Neutral Zero, Protective vs. Neutral Zero and Neutral Risk Protective vs. Neutral Zero to measure the risk and protective effects  Risk (positive score); 2) Neutral Zero (no risk and protective factor present); 3) Neutral Risk Protective ; 4) Protective Risk ; 5) Protective .  effects .Second, we calculated HLA genetic burden (HLAGB) for each study subject as the sum of the burden of each MS associated HLA allele as described , 40. EacHLA-DPA1*01:03:01:02 (HLA06604) and HLA-DPB1*104:01:01:01 (HLA02046) from IPD-IMGT/HLA Database release version 3.35.0 (HLA-DPA1*01:03:01:03~HLA-DPB1*03:01:01 haplotype (Accession Number: AL662824)  , because1:01 HLA046 from IHLA-DPB1 .HLA-DPA1 and HLA-DPB1 using GTEx Portal database  (We investigated expression quantitative trait loci (eQTL) for g/home/) .HLA-DRB5*01:01:01~HLA-DRB1*15:01:01:01 haplotype block was significantly associated with susceptibility to MS in this dataset   Table 2.HLA-DRB5*01:01:01~HLA-DRB1*15:01:01:01 haplotype, and 252 families had one MS-affected child with the HLA-DRB5*01:01:01~HLA-DRB1*15:01:01:01 haplotype. The HLA-DRB5*01:01:01~HLA-DRB1*15:01:01:01 extended haplotype block in this dataset includes several common class I HLA-C~HLA-B embedded haplotypes: e.g. HLA-C*07:02:01:03~HLA-B*07:02:01, HLA-C*07:01:01:01~HLA-B*08:01:01:01, HLA-C*12:03:01:01~HLA-B*18:01:01:02 and the minor HLA-DQA1~HLA-DQB1 haplotype: HLA-DQA1*01:02:01:01~HLA-DQB1*06:03:01. We observed reduced significance of these extended haplotypes across the telomeric and centromeric segments of the locus . Upon application of conditional logistic regression TDT analyses, and consistent with previous reports .Of 477 trio families, 301 families carried at least one HLA-DPB1*104:01 allele  . We did not observe an interaction between the allele HLA-DRB1*15:01 and the allele HLA-DPB1*104:01. Out of 41 parents carrying the HLA-DPB1*104:01 allele, 7 parents were HLA-DRB1*15:01 carriers, but all of them were present on the other copy of chromosome 6, further confirming that HLA-DPB1*104:01 risk haplotypes are independently segregated to their affected offspring. Interestingly, the closely related allele HLA-DPB1*03:01:01 did not show any significant association  children who also carry the HLA-DRB1*01:01:01 allele. Of 370 parents who carry at least one haplotype bearing HLA-DRB1*15:01, we found 42 (11.3%) parents who also carry the HLA-DRB1*01:01:01 allele. The odds ratio confirms the statistically significant protective effect of HLA-DRB1*01:01:01 allele in the presence of haplotype bearing HLA-DRB1*15:01 . Conditional logistic regression TDT analysis suggested a statistically significant interaction between HLA-DRB1*15:01 and HLA-DRB1*01:01 . When eliminating families with haplotypes bearing HLA-DRB1*15:01 , suggesting that HLA-DRB1*01:01:01 may have protective effects also in the context of non HLA-DRB1*15:01 haplotypes. We did not find statistically significant protective effects mediated by the HLA-DRB1*01:01:01 allele in the context of the HLA-DPB1*104:01 risk effect described above.The allele B1*15:01 , 45. Of HLA-DRB3*02:02:01:02~HLA-DRB1*11:01:01:01~HLA-DQA1*05:05:01:01~HLA-DQB1*03:01:01:03 haplotype fragment showed moderate protective effects for gTDT, mTDT and CC . Individual alleles and smaller haplotype segments showed similar protective effects  . The difference between HLA-C*03:04:01:01 and HLA-C*03:04:01:02 is located in 3’-UTR (rs1049650).The block including e for MS . We deteHLA-B*38:01 showed statistically significant protective effects  patients who had HLA-B*27:05:02 allele. Of 370 parents who had at least one haplotype bearing HLA-DRB1*15:01, we found 28 (7.6%) parents who had the allele HLA-B*27:05:02. These observations indicate the protective effects of HLA-B*27:05:02 in the presence of haplotypes bearing HLA-DRB1*15:01 . When stratification analyses were performed by eliminating trio families with haplotypes bearing HLA-DRB1*15:01, no statistically significant under-transmission for HLA-B*27:05:02 was observed .Another class I protective effect included the allele HLA-DRB5*01:01:01~HLA-DRB1*15:01:01:01; all the siblings not affected with MS carry a haplotype that includes HLA-B*27:02:01 while the affected subject inherited the alternative haplotype including HLA-B*08:01:01:01. This transmission pattern is consistent with HLA-B*27:02:01 disease-mediated protection in this particular family. The second-generation individual H0278DC4 diagnosed with MS inherited both, the protective HLA-B*27:02:01 allele and the high-risk HLA-DRB5*01:01:01~HLA-DRB1*15:01:01:01 haplotype block, further exemplifying the limited penetrance of protective and risk alleles and complex interaction underlying the association signals.A detailed transmission analysis was conducted for haplotype blocks in the extended family cases. Family 2965 exemplifies how protective and susceptibility alleles may interact . The result confirms that subjects who carry the positive scores of the risk HLA alleles/haplotypes are more susceptible to MS than the subjects who do not carry risk and/or protective HLA alleles/haplotypes. Conversely, we observed statistically significant more protective effects in controls compared to no risk/protective factors present (p = 0.00060). In addition, aligned with the trio family results described above, the presence of risk factors predisposes to disease even in the presence of equal number of protective factors (p = 0.00042).HLAGB scores , 40 wereHLA-DRB5*01:01:01~HLA-DRB1*15:01:01:01 block was observed in 52.8% of the affected children in the trio dataset, confirming the role of this block conferring susceptibility to MS at the four-field resolution  for HLA-DPB1*03:01 and ATG (Met) for HLA-DPB1*104:01], resulting in an amino acid difference located in the transmembrane domain GXXXG motif (GFVLG -> GFMLG) sequence . We compared the HLA-DPA1*01:03:01:02 and HLA-DPB1*104:01 reference sequences from IPD-IMGT/HLA database with publicly available reference HLA-DPA1*01:03:01:03~HLA-DPB1*03:01:01 genomic sequences  for HLA-DPB1. The later three SNPs are located within the HLA-DPA1 promoter region or HLA-DPB1 intron 1  , respectively. In contrast, HLA-DQB1*03:02 and HLA-DQB1*03:03 differ only by the replacement of one amino acid at residue 57 located at the PDB. The alleles associated with protection present the same charged amino acid (Aspartic acid) at this residue while the non-protective allele carries the hydrophobic Alanine. The amino acid substitutions at residue 57 of DQB1 have been postulated to play important roles in susceptibility and resistance in insulin-dependent diabetes mellitus (IDDM)  .HLA-B*44:02 is frequently associated with the protective allele HLA-DQB1*03:01, thus protection associated with these alleles was difficult to resolve. The neutral transmission of haplotypes bearing both HLA-B*44:02 and the highly susceptibility allele HLA-DRB1*15:01:01:01 is suggestive that the observed susceptibility conferred by HLA-DRB1*15:01:01:01 over-transmitted in haplotypes bearing multiple alleles of HLA-B is specifically neutralized by the presence of HLA-B*44:02. We conclude that the HLA-B*44:02 protein effect in protection is likely to be an independent factor. Conditional logistic regression TDT analysis suggested a statistically significant interaction between the haplotype carrying HLA-DRB1*15:01 and HLA-B*44:02 .The allele HLA-B*27:05:02 and HLA-B*27:02:01 suggests that antigen presenting features of this allele and closely related alleles may confer protection. The HLA-B*27:05 and HLA-B*27:02 are both serologically recognized as B27, and carry the Bw4 ligand for a killer immunoglobulin-like receptor KIR3DL1. The KIR3DL1 in combination with Bw4 epitope was shown to be protective against MS in African Americans (p = 0.00028)  .HLA-A*02:01:01:01~HLA-B*40:01:02~HLA-C*03:04:01:01, we hypothesize that these three HLA alleles are not the protective factors per se, and other elements exert protection within the 1.45 Mb genomic region that is specific to this class I haplotype block.In the case of protection conferred by the class I haplotype HLA-DRB5*01:01:01~ HLA-DRB1*15:01:01:01 was significantly associated with predisposition as expected. A second independent risk allele, HLA-DPB1*104:01 was newly identified. HLA-DRB1*01:01:01, HLA-DQB1*03:01 and HLA-DQB1*03:03, showed protective effects. The HLA class I block, HLA-A*02:01:01:01~ HLA-C*03:04:01:01~ HLA-B*40:01:02 and the alleles HLA-B*44:02, HLA-B*27:05 and HLA-B*38:01 showed moderately protective effects independently from each other and from the class II associated factors. Altogether, the present study demonstrates the effectiveness of high resolution extended coverage typing for dissecting HLA alleles/haplotypes associated with disease susceptibility using family-based segregation analyses. This is the first MS-HLA family study using NGS. In this study both, susceptible and protective candidate HLA alleles/haplotypes were mapped with more precision by eliminating at the same time false signals resulting from ‘hitchhiking’ alleles . The UCSF DNA biorepository is supported by RG-1611-26299 from the National Multiple Sclerosis Society. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the NIAID, NINDS, NIH or United States Government.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Correlation between antibody-mediated rejection (ABMR) and circulating HLA donor-specific antibodies (HLA-DSA) is strong but imperfect in kidney transplant (KT) recipients, raising the possibility of undetected HLA-DSA or non-HLA antibodies contributing to ABMR. Detailed evaluation of the degree of HLA matching together with the identification of non-HLA antibodies in KT may help to decipher the antibody involved.1R)-Ab, endothelin-1 type-A-receptor (ETAR)-Ab and crossmatches with primary aortic endothelial cells (EC-XM)]. We also analyzed HLA epitope mismatches (HLA-EM) between donors and recipients to explore their role in ABMR histology (ABMRh) with and without HLA-DSA.We retrospectively assessed patients with transplant biopsies scored following Banff’15 classification. Pre- and post-transplant serum samples were checked for HLA and non-HLA antibodies [MICA-Ab, angiotensin-II type-1-receptor (ATh (n = 52) or IFTA (n = 47) were studied. ABMRh patients were HLA-DSApos  or HLA-DSAneg . Pre-transplant HLA-DSA and AT1R-Ab were more frequent in ABMRh compared with IFTA and normal histology cases (p = 0.006 and 0.003), without differences in other non-HLA antibodies. Only three ABMRhDSAneg cases showed non-HLA antibodies. ABMRhDSAneg and ABMRhDSApos cases showed similar biopsy changes and graft-survival. Both total class II and DRB1 HLA-EM were associated with ABMRhDSApos but not with ABMRhDSAneg. Multivariate analysis showed that pre-transplant HLA-DSA  and AT1R-Ab  were independent predictors of ABMRhDSApos.One-hundred eighteen patients with normal histology (n = 19), ABMR1R-Ab is frequently found in ABMRhDSApos patients. However, AT1R-Ab, MICA-Ab, ETAR-Ab or EC-XM+ are rarely found among ABMRhDSAneg patients. Pre-transplant AT1R-Ab may act synergistically with preformed or de novo HLA-DSA to produce ABMRhDSApos but not ABMRhDSAneg. HLA epitope mismatch associates with ABMRhDSApos compared with ABMRhDSAneg, suggesting factors other than HLA are responsible for the damage.In conclusion, pre-transplant AT Not a HLA-DSA , generatreported , 18.1R-Ab), endothelin-1 type A receptor (ETAR-Ab), perlecan, agrin or vimentin, among others, have been reviewed recently . Additionally, we evaluated pre-KT HLA-EM load. We focused on KT patients with biopsies with Banff category 2 diagnosis and compared them with two other Banff diagnosis: category 1 or no abnormalities , as a usual control group, and category 5 or interstitial fibrosis and tubular atrophy (IFTA), damage with not clear pathogenicity to evaluate the potential role of non-HLA antibodies in this case . Ninety-two biopsies fulfilling Banff 2015 categories 3, 4 and 6 were excluded and 142 biopsies achieving categories 1, 2 or 5 were selected. From these 142 biopsies, we selected only one biopsy per patient according to these criteria: the first biopsy obtained after 3 months post-transplantation, unless a biopsy with category 2 diagnosis was available. Five biopsies were excluded due to unsuitable serum samples. Finally, 118 biopsies corresponding to 118 patients were included in the study , and folBiopsies were performed for indication or follow-up (including HLA-DSA detection without graft dysfunction). Processing was undertaken as previously described . All bio1R-Ab and ETAR-Ab were measured using enzyme-linked immunosorbent-based assays  was performed as previously described  using Lud assays  (One Lam5 ECs were incubated 30 min with 100 ul patient serum on ice. ECs were washed three times and incubated with 50 mc of 1:400 diluted FITC-AffiniPure F(ab’)2 Fragment Goat Anti-Human IgG Fc fragment (Jackson ImmunoResearch Laboratories) for 30 min on ice. After three washes, cell fluorescence was analyzed on a FACSCalibur flow cytometer using CellQuest software (BD Biosciences). Gates for forward and side scatter measurements were set on EC, and a minimum of 10,000 events was acquired. Positive EC-XM threshold was set at two standard deviations (50 Median Channel Shift) above the mean of negative control serum tests. EC-XM were only performed in 83 pre and 103 post-KT cases due to insufficient sample.Primary human aortic endothelial cells (ECs) were isolated from aortic rings of explanted donor hearts . EC werehttp://www.epitopes.net) was used to define potential HLA-EM between donors and recipients (www.haplostats.org) selecting the most likely high-resolution typing for HLA-A, B, C, DR and DQ according to three-five highest haplotype frequencies in the population of each one .HLAMatchmaker software according to the July 2020 update  and p-values <0.05 were considered statistically significant.Data are presented as mean ( ± standard deviation), median, interquartile range, or number (percentage) based on data distribution. Comparisons between clinical variables were carried out using Student’s T test for parametric continuous variables and U Mann–Whitney or Kruskal–Wallis test for non-parametric data. Chi-squared or Fisher’s exact tests were used to test categorical variables. Survival analyses were performed using the Kaplan–Meier method using the log-rank test. Logistic regression analysis was used to estimate the odds ratio (OR) for ABMRh, n = 52) and category 5 or IFTA (n = 47). Thirty patients (25.4%) lost their grafts and 13 died with a functioning graft (11%) during the study period. Death-censored graft survival 68 months after the biopsy [IQR 48–80] was worse in ABMRh cases than in those with IFTA or normal biopsies  and higher proteinuria. Finally, IFTA patients were more frequently receiving calcineurin inhibitors and less on mTOR inhibitors , category 2 or ABMR histology (ABMRhibitors Table 1.h and 43 IFTA). We found pre-transplant HLA-DSA in 18 ABMRh (46.2%), 9 IFTA (20.9%) and in two normal histology cases (10.5%) (p = 0.006)  and less isolated class I (27.8%).Pre-transplant serum samples were available for 101 patients  vs. 2/19 normal histology (10.5%) and 5/43 IFTA (11.6%), p = 0.003) . Detection of pre-transplant AT1R-Ab correlated with both persistent preformed HLA-DSA  and de novo HLA-DSA detection , but not with preformed HLA-DSA which cleared after transplant  or no HLA-DSA  .Pre-transplant ATh. Pre-transplant ETAR-Ab and MICA-Ab were present similarly in normal histology, ABMRh and IFTA cases . Of 83 KT recipients tested with EC-XM, only 3/29 ABMRh (10.3%) and 3/39 IF/TA cases (7.7%) had a pre-transplant positive EC-XM , 17 with either HLA-DSA or AT1R-Ab (43.6%) and the remaining 13 did not present any of these antibodies (33.3%).Detection of pre-transplant HLA-DSA and/or ATh patients . Among them, 7.7% were class I, 53.8% class II and 11.5% combined class I&II. HLA-DSA were also detected in 17% IFTA and 15.8% normal histology cases  vs. 15/69 HLA-DSAneg cases (21.7%), p = 0.28].Post-transplant ATh. Post-transplant ETAR-Ab were found in 3/19 normal histology (15.8%), 7/52 ABMRh (13.5%) and 9/47 IFTA cases . MICA-Ab were detectable in 1/19 normal histology (5.3%), 8/52 ABMRh (15.4%) and 6/47 IFTA cases . Two normal histology (11.1%), four ABMRh (9.3%) and two IFTA cases (4.8%) had a positive EC-XM  had no peri-biopsy HLA-DSA. ABMRhDSApos cases were more frequently HLA sensitized, less well DR-matched with their donors and received more frequently a graft from a deceased donor than those ABMRhDSAneg. No differences were found in graft function or immunosuppression at biopsy  we could not identify any of the non-HLA antibodies studied.From 52 patients with ABMRt biopsy Table 2.ver). We observed similar class I and class II HLA-EMver in all three groups of study (data not shown). We compared the load of HLA-EMver between ABMRhDSApos and ABMRhDSAneg patients, finding similar class I but significantly higher class II and DRB HLA-EMver in ABMRhDSApos cases  development prediction. Neither class I HLA-EMver nor HLA-AM were useful tools for class I dnDSA prediction. Class II HLA-EMver were significantly associated with class II dnDSA , but not class II HLA-AM (p = 0.26). The extent of DRB HLA-EMver associated with DRB dnDSA , and the rate of DQB HLA-EMver showed a weak association with DQB dnDSA . Neither DRB nor DQB HLA-AM predicted DRB or DQB dnDSA .The median number of class I and class II HLA-EM in our cohort were 16 (0–36) and 18 (0–46) respectively. Among them, 10 class I and 7 class II HLA-EM were antibody-verified  were independent ABMRhDSApos predictors. DRB HLA-EMver also showed a weak association with ABMRhDSApos ; RD16/0009/0013 (ISCIII FEDER REDinREN) and 201822-10 (Fundació la Marató de TV3). MC received grants of the Sociedad Española de Nefrología and Parc de Salut Mar for a research stay at UCLA Immunogenetics Center . ER is supported by National Institute of Allergy and Infectious Diseases Grants RO1 AI135201. One Lambda provided reagents but had no role in the design of the study or the analyses and writing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Correction to: Immun Ageing 17, 18 (2020)https://doi.org/10.1186/s12979-020-00189-7Following publication of the original article , the autturbidimetric assay kits (The Binding Site).”“Assays for complement factors C3 and C4, C-reactive protein (CRP), and classical pathway activity (CH50) were performed at the clinical laboratory at the University of Alabama at Birmingham, using the respective Optilite The original article  has been


Biosci Rep (2020) 40(7), https://doi.org/10.1042/BSR20201627) would like to correct The authors of the original article “LncRNA HOTAIR contributes Taxol-resistance of hepatocellular carcinoma cells via activating AKT phosphorylation by down-regulating miR-34a” (


Video and gene expression analyses coupled with the RNAi technique reveal microtubule-dependent, neuronal-like nucleokinetic migration in a noradrenergic type of neuroblastoma, providing a glimpse into the mechanism by which noradrenergic neuroblastoma cells may spread. The migrational propensity of neuroblastoma is affected by cell identity, but the mechanisms behind the divergence remain unknown. Using RNAi and time-lapse imaging, we show that ADRN-type NB cells exhibit RAC1- and kalirin-dependent nucleokinetic (NUC) migration that relies on several integral components of neuronal migration. Inhibition of NUC migration by RAC1 and kalirin-GEF1 inhibitors occurs without hampering cell proliferation and ADRN identity. Using three clinically relevant expression dichotomies, we reveal that most of up-regulated mRNAs in RAC1- and kalirin–GEF1–suppressed ADRN-type NB cells are associated with low-risk characteristics. The computational analysis shows that, in a context of overall gene set poverty, the upregulomes in RAC1- and kalirin–GEF1–suppressed ADRN-type cells are a batch of AU-rich element–containing mRNAs, which suggests a link between NUC migration and mRNA stability. Gene set enrichment analysis–based search for vulnerabilities reveals prospective weak points in RAC1- and kalirin–GEF1–suppressed ADRN-type NB cells, including activities of H3K27- and DNA methyltransferases. Altogether, these data support the introduction of NUC inhibitors into cancer treatment research. MYCN amplification and a particular pattern of metastasis (stage 4S), are prone to spontaneous regression and differentiation  type, expressing SE-associated TFs FOSL2 and RUNX2 (MYCN amplification (MNA) correlates with ADRN identity . The silencing or inhibition of RAC1 or ADRN-specific RAC1 guanine nucleotide exchange factor (GEF) KALRN abrogates NUC and, hence, migration. Further analysis with RNAi and chemical compounds reveals kalirin function in coordinating NUC via microtubular (MT) cytoskeleton. These results identify NUC as an important drug target for the development of the migration-specific drug in ADRN-type cells.Cell migration is the process that occurs during normal embryogenesis, wound healing, immune responses, and metastasis. Accumulated evidence suggests parallelism between metastatic dissemination of tumor cells and migratory processes during embryogenesis. A plethora of genes governing migration during embryogenesis are also involved in the metastatic process . Active ntiation . In contpe tumor . NB has nd RUNX2 . MYCN amidentity . Both ADin cells . Both ofin cells . Differein cells . Yet, main cells . Previoupatients . Based oDCX mRNA correlated positively with mRNAs for ADRN SE–associated TFs, GATA3, and PHOX2B, but not MES FOSL2, suggesting ADRN-specific expression  and nascent neurons of the central nervous system (CNS), DCX specifically regulates nuclear translocations or NUC c in pseudo 3-D assay.Video 3Download videoIMR-32_NM treated with colcemid and cytochalasin B.Video 4DCX siRNA, or SOX11 siRNA at 48 and 72 h after transfection (movie start).Download videoIMR-32 treated with control siRNA, Video 5RAC1 siRNA (bottom left), kalirin-GEF1 inhibitor#1 (bottom middle), or kalirin-GEF1 inhibitor#2 (bottom right).Download videoIMR-32_NM treated with vehicle (top left), ROCK inhibitor (top middle), RAC1 inhibitor (top right), DCX- or LIS1-KD  of DCX- and LIS1-KD IMR-32 RNA-seq-resolved expression profiles showed that there was no induction of the MES program (DCX- and LIS1 RNAi. DCX-KD and LIS1-KD transcriptomic profiles showed remarkable overlap (DCX-KD and LIS1-KD transcriptomes (Table S1). Gene signatures related to non-sense–mediated decay (NMD) and mRNA transport, mitochondrial function, oxidative phosphorylation (oxphos) signature, as well as genes related to cell contraction and Ras signalling regulation were specifically depleted in DCX-KD and LIS1-KD cells, respectively . This analysis showed that up-regulated, but not down-regulated genes in the DCX-KD cells had an affinity toward transcriptomic profiles of stage 4S, stages 1|2 and MYCN-nonamplified tumors -KDUP ∩ stage 4S DOWN; DCX(LIS1)-KDDOWN ∩ stage 4S UP ). GSEA showed that mis–down-regulation engaged TP53 targets and mis–up-regulation engaged CHEK2 neighbourhood in LIS1-KD cells , 19p13 shutdown in relapse DTCs and DCX-KD cells could imply similar regulatory mechanisms. We retrieved the RNA-seq data from NB DTCs and reanalysed with the following settings: no logFC cutoff; P-values ≤ 0.05; DTCTOTAL versus Tumor (TU)TOTAL; DTCMNA versus TUMNA; DTCRELAPSE versus TURELAPSE to identify whether similarities between transcriptomic profiles of DCX-KD IMR-32 and NB DTC go beyond the 19p13 genes. DCX-KDUP gene set showed splitting when mapped onto NB DTC transcriptome profiles, whereas DCX-KDDOWN showed a match with NB_DTCDOWN  using gse49710 signature (Table S1), showed similar splitting (The plotting of nuclear positions (NCn-CCn or |NC-CC|) showed that nuclei were less present in the periphery of the cells after  LIS1-KD . Stainin LIS1-KD  and γ-tur Dcx-KD , that isKD cells . Accordiectively . Yet, ceootprint . Altoget overlap . Particuectively  and S2B.blastoma  . Interrogation of DCX co-expression signature for the general TFs involved in neuronal migration . SOX11 is mutated in Coffin-Siris syndrome . Also, SOX11-KD IMR32_NM had different morphology, as compared with the DCX-KD cells  . We thought that identification of NUC RAC1-GEF(s) could help to refine RAC1 suppression, and hence, inhibition of NUC migration in the context of NB. As previous studies showed, three GEFs, T-lymphoma and metastasis gene 1 TIAM1, triple functional domain protein TRIO and TRIO homologue kalirin, are inhibited by NSC23766 ; therefore, we checked their expression by WB, which revealed a reduction of kalirin and TIAM1 levels in SOX11-KD IMR-32 cells  -induced neuronal differentiation model, SK-N-BE(2)c, and observed induction of kalirin-9 and kalirin-12 mRNAs and proteins as well as suppression of kalirin-8 after RA treatment. We also noticed TRIO transcripts and proteins’ strong induction, whereas TIAM1 mRNA levels remained unchanged  . Given tn IMR-32 . On the K-N-BE2c , whereasK-N-BE2c  did not  ADRN NB . Given t ADRN NB , we checTaken together, these results suggest that kalirin is involved in RAC1 activation in ADRN-type NB, particularly in its most aggressive, MNA sub-type.MYCN-nonamplified NB69, which was confirmed by cell tracking in IMR-32_NM and NB-S-124  and TP53 targets, gene sets pertaining to mitochondrial function and cortical cytoarchitecture were depleted in kalirin–GEF1–inhibited cells  included up-regulation of ZNF702P, ETV1 , and SIAH1 , as well as down-regulation of KIF6, SCUBE1, EHMT1, and MPDZ  , whereas KIF6 was highly expressed in stage 4 and MNA NB , we checked the lineage affinity of ZNF702P mRNA, which revealed ADRN type–specific ZNF702P expression (ZNF702P mRNA (Table S4).Analysis of RNA-seq–resolved profiles of kalirin-GEF1–inhibited IMR-32 showed that the up-regulated genes had an affinity with transcriptomes of stage 4S, ages 1|2  and S10Ad IMR-32 ; and TP5MR-32_NM , althougadherent . The up-KD cells , left. Ted cells . As ZNF7pression . Pseudogpression ; therefopression , which rCombined analyses of differentially expressed genes from RAC1- and kalirin–GEF1–inhibited IMR-32.Table S4 Video 8TP53 siRNA.Download videoIMR-32_NM treated with MIR181A2 and MIR3609, were down-regulated in kalirin–GEF1–inhibited cells (Table S3), which could explain the up-regulation of miR-181 and miR-3609 targets. These two miRNA genes, together with MIR573 and MIR873, were present in profile overlaps after at least two of the NUC inhibitory treatments used in this study , suggesting essential functions of these miRNAs in NUC regulation. Yet, ARE-positive genes potentially targeted by miR-573 were up-regulated in kalirin–GEF1–inhibited cells . Also, the expression of predicted miR-573 targets showed affinity to stage 4S and stages 1|2.   was reased cells  and S11Dve genes . The exp targets . miR-873KD cells . PotentiKD cells  were locKD cells  and S11G overlap , left, w overlap , right.DCX-KD cells c and observed only minor interaction with the MT drugs, vincristine and paclitaxel . This suKD cells , implyinOur data provide evidence that gene up-regulation driven by kalirin–GEF1 inhibition operates via miRNA-dependent posttranscriptional mechanisms. Most importantly, our analyses suggest that suppressors of epigenetically driven gene down-regulation might interact with kalirin–GEF1 inhibitors synergistically, which should be checked in future experiments.Intact NUC is necessary for neuronal positioning, but its relevance to cancer, particularly to cancer cell migration, has not been elucidated. In this study, we present evidence that NUC is active in ADRN-type NB. Previous studies revealed the RHOA-ROCK pathway’s functionality in NB migration . Our stuLis1 KO and Dcx KO mice. Also, only a few cases have been reported when individuals with lissencephaly had heterotopias and/or hypoganglionosis in the PNS . This complete uncoupling from proliferation together with the observation that mis-expression induced by kalirin-GEF1 inhibition mainly concerns down-regulated genes allow us to consider prospective synthetically lethal . Mouse anti-Golgi apparatus (NB37-100) was from Merck Millipore. Rabbit anti-RAC1/2/3 (2465), rabbit anti centrin-2 (2091), rabbit anti-DCX (4604), and rabbit anti-βIII-tubulin (D71G9) antibodies were purchased from Cell Signalling. Mouse anti-RAC1 antibody (#ARC03) was from Cytoskeleton. Protein G-Agarose was purchased from Santa Cruz Biotechnologies . β-actin antibody and sheep anti-goat HRP-conjugated antibody were from Santa Cruz Biotechnologies. HRP-conjugated antibodies against mouse or rabbit and the Cy3-conjugated anti-goat antibody were obtained from Jackson Immunoresearch Laboratories. The Alexa 488–conjugated goat anti-rabbit antibody (ab150077) and Cy5.5-conjugated goat anti-mouse (ab6947) were obtained from Abcam. RA and Calcein AM were purchased from Sigma-Aldrich. RAC1 inhibitor NSC23766 and cytochalasin B were purchased from Sigma-Aldrich. TRIO/kalirin-GEF1 inhibitors, ITX3 methylene]-thiazolobenzimidazol-3(2H)-one), and NPPD (1-(3-nitrophenyl)-1H-pyrrole-2,5-dione), were purchased from Sigma-Aldrich and Matrix Scientific, respectively. RHOA inhibitor, rhosin, was from Sigma-Aldrich. All the reagents were first dissolved in the medium before application to the cells. ROCK inhibitor, Y27632, was obtained from StemCells Technologies. Colcemid was from Thermo Fisher Scientific.Rabbit anti-kalirin-SP antibody (MBS821543), goat-kalirin-STYV antibody (ab52012), and rabbit kalirin–GEF2 antibody (TA590559) were purchased from MyBioSource, Abcam, and Acris. Rabbit anti-TRIO antibody (A304-269A) was from Bethyl Laboratories, rabbit anti-TIAM1 antibody (ST1070) was from Calbiochem, rabbit anti-SOX11 was from Millipore, mouse (GTU88), and rabbit anti-γ-tubulin (DQ-19) antibodies were from Sigma-Aldrich. The rabbit polyclonal antibody, SOX11-PAb, was custom made (Absea Biotechnology) against the immunogenic peptide p-SOX11RSVSTSSS Preprint2 using gelatin-coated cellware. Spheroids were generated by coating 96 well flat bottom plates with 1% agarose. 5,000 cells were seeded per each well and allowed to form spheroids for 72 h. For cell viability Alamar Blue (AbD Serotec) was used according manufacturer’s instructions. Fluorescence was detected using the FluorStar Optima microplate fluorescence reader (BMG Labtech).Neuroblastoma cell lines were maintained in RPMI1640 supplemented with 10% fetal bovine serum. NB-S-124 cell line was established from infiltrated bone marrow aspirate from 1-y-old patient  pENTR223.1 entry clone was retrieved from DKFZ clone repository (DKFZ). Kalirin-7 cDNA was subcloned using Gateway technology (Thermo Fisher Scientific) into the pTREX31 vector (Thermo Fisher Scientific). Stable SOX11 expression was achieved with transducing SOX11 cDNA sequences were into pLenti6.3/TO/V5-Dest (Thermo Fisher Scientific) and pLVX-Tet3G (Clontech). For siRNA transfection, cells were seeded in six-well tissue culture plates 24 h before transfection. 100 nM of siRNA non-targeting control  or siRNA SOX11 (Dharmacon) were transiently transfected using DharmaFect 2 (Thermo Fisher Scientific) according to the manufacturer’s guidelines. SOX11 expression was induced by doxycycline addition (0.1 μg/ml); and induction was assessed by WB for SOX11 (Preprint). mCherry-gamma-tubulin-17, CyPet-RAC1, and DCX-RFP (KALRN (sc-18592), LIS1 (sc-35814), DCX (sc-35214), HNRNPK (sc-38282), and DISC1 (sc-60539) were purchased from Santa Cruz Biotechnologies. DCX siRNA used in the DCX-RFP experiments was from Thermo Fisher Scientific. RAC1 siRNAs were prepared by chemical synthesis (Sigma-Aldrich) using sequences provided by ROCK1 siRNA  and ROCK2 siRNA  were purchased from Sigma-Aldrich, SOX11 siRNA was from Thermo Fisher Scientific. Transfection with a Silencer Select Negative Control siRNA #1 (Thermo Fisher Scientific) was used as controls.Human or SOX11 Preprint DCX-RFP  construcLIS1 (QT00013447), DCX (QT00008540) and SOX11 (QT00221466). ROCK1 primers were retrieved from PrimerDB . Amplification of cDNA by real-time PCR was quantified using SYBR Green (Thermo Fisher Scientific) as previously described  with thePrimerDB . ROCK2 pPCR fragments were purified using the QIAquick PCR Purification Kit (QIAGEN). Sequencing of RT-PCR products was performed with primers used in the PCR reaction . FinchTVNeuroblastoma cells were seeded into 96-well plates (Platypus) coated with fibronectin/collagenIV and grown to confluence. A single wound was then created in the cell monolayer and migration of the cells from the edge of the wound was analysed. The area between the wound edges was measured using ImageJ software . The area of the wound in the control cells was set as 100%, and the relative change was calculated as a percentage of the initial area.Time-lapse images of living cells were captured using Ti-e Eclipse (Nikon) equipped with an incubation chamber. Neuroblastoma cell lines were cultured in RPMI-1640 medium (Thermo Fisher Scientific) as described above on plastic-bottomed chamber (Ibidi) pre-coated with Collagen IV/Fibronectin/Laminin (Sigma-Aldrich). Cells were analysed with a 10×, 20× and 40× objective lens. Optimal time-lapse intervals  were chosen to capture nucleokinetic or mesenchymal migration. Imaging data were stored as files in NIS-Elements software (Nikon). Object segmentation was performed using ilastik . The tim2, and 1% Nonidet P-40 supplemented with protease inhibitors cocktail) and then lysed. Insoluble material was removed by centrifugation for 10 min at 9,500g. Lysates were incubated with 1 μg of PAK-PBD or Rhotekin-RBD (Cytoskeleton) for 16 h at 4°C. Beads were washed, and the proteins were eluted with Laemmli sample buffer and analysed by immunoblotting with anti-RAC1 antibody according to the manufacturer’s instructions.Rho GTPase activation was analysed by a modification of a protocol described previously . Cells wCells growing on slides were fixed with 4% paraformaldehyde in PBS or ice-cold methanol for tubulin/Golgi Apparatus-compatible staining, washed with PBS, and permeabilized with 0.1% Triton X-100. After blocking with 5% fetal bovine serum and 1% BSA, the slides were incubated with primary antibodies against kalirin-PH and kalirin-STYV, TRIO, TIAM1, βIII-tubulin, γ-tubulin, and Golgi apparatus, washed with PBS and incubated with secondary antibodies: Cy3-conjugated donkey anti-goat, Alexa Fluor 488–conjugated sheep anti-rabbit or Cy5.5–conjugated goat anti-mouse. Triple immunostainings with anti–kalirin–STYV antibody were processed with Cy3-conjugated donkey anti-goat antibody first. Alexa Fluor 555–phalloidin staining was performed according to the manufacturer’s instructions (Thermo Fisher Scientific). The slides were counterstained with DAPI (1 μg/ml) for 5 min. Phalloidin-stained cells were photographed within 30 min after the staining procedure.Western blotting was performed as described previously . Briefly6 cells . Direct cell lysis for each sample was achieved by incubation for 30 min in 950 μl RIPA I on ice . Tissue disruption, formaldehyde fixation, and sonication of tumor material were performed according to a previously published protocol  procedure, and library preparation were performed as described previously , startinprotocol . ApproxiSingle-end reads were aligned to the hg19 genome using Bowtie2 (version 2.1.0). Only uniquely aligned reads were kept. Binary Alignment Map files of aligned reads were further processed using the deepTools3 . Input fhttp://r2.amc.nl). We used publicly available gene expression omnibus (GEO) datasets: Neuroblastoma custom/AG44 , Neuroblastoma RPM/SEQC , Neuroblastoma Versteeg , Neuroblastoma Hiyama , and Normal Peripheral Glial Cells . Differentially expressed genes between clinically-relevant dichotomies in primary neuroblastoma (gse49710) were obtained by GEO2R tool (http://www.ncbi.nlm.nih.gov/geo/geo2r). Gene correlation graphs, “correlated with a single gene” data, “Parametric Gene set Enrichment” data were extracted from the R2 database. Venn diagrams were generated with the GeneVenn an BioVenn web applications c treated with vehicle and RA were generated as two one-color replicates using the whole-genome oligonucleotide microarray platform from Agilent (Agilent Technologies) as previously described . Raw mic2 transformation. Statistical significance was investigated by the rank-based Kruskal––Wallis test (https://www.jstor.org/stable/2280779?seq=1#page_scan_tab_contents) under no assumption of the underlying distribution. No filtering of data was applied based on fold-change or P-value cutoffs because of the low sample sizes available in the experiment.RNA was depleted from ribosomal RNAs using the Ribo-Zero rRNA Removal Kit (Illumina) according to the manufacturer’s protocol. RNA libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer’s protocol with the following changes: RNA was fragmented for 20 min at 94°C followed by first strand cDNA synthesis for 10 min at 25°C, 50 min at 42°C, and 15 min at 70°C. Size selection of adapter-ligated DNA was performed with a bead:DNA ratio of 0.4  removing index primer and short fragments. Quality, quantity, and sizing (∼320 bp) of the RNA library were analysed using a DNA High Sensitivity DNA chip run on a 2100 Bioanalyzer (Agilent Technologies). Libraries were sequenced (50 bases single-end) on the Illumina sequencing platform. The normalized gene expression values were grouped into controls and intervention samples. Fold-change was calculated both by means and trimeans (n = 3) and followed by a logKALRN exon expression analysis. The reads were aligned to hg19 reference using STAR 2.5.2 tool  from previous NB study  was appl5.2 tool . Further5.2 tool .EGAS00001005023.All sequencing data from this publication were deposited to the European Genome-phenome Archive , the ide