C. elegans nervous system and found Hox expression in 97 (32%) of the 302 neurons in adult hermaphrodites. Our results are generally consistent with previous high-throughput expression mapping and single-cell transcriptomic studies. Detailed analysis of the fate markers for these neurons revealed that Hox genes regulate the differentiation of 29 (25%) of the 118 classes of C. elegans neurons. Hox genes not only regulate the specification of terminal neuronal fates through multiple mechanisms but also control subtype diversification along the A-P axis. The widespread involvement of Hox genes in neuronal differentiation indicates their roles in establishing complex nervous systems.Hox genes encode evolutionarily conserved transcription factors that specify regional identities along the anterior-posterior (A-P) axis. Although some Hox genes are known to regulate the differentiation of certain neurons, to what extent Hox genes are involved in the terminal specification of the entire nervous system is unclear. Here, we systematically mapped the expression of all six Hox genes in  C. elegans nervous system in its entirety. We found that the six Hox genes are expressed in 32% of all neurons and five Hox genes regulate the differentiation of 25% of all neuron types through multiple mechanisms and act at various stages of cellular development. Thus, our results suggest that a small number of Hox genes could control the development of a significant portion of the nervous system. Given the conserved functions of Hox genes across species, we suspect that the increasing number of Hox genes may have allowed increased complexity in the nervous system in animal evolution.The nervous system contains an extraordinary array of neuron types. How this neuronal diversity arises during development and what genes regulate the differentiation of each neuron type are among the major questions of neurobiology. Hox genes are a set of transcription factors highly conserved in the animal kingdom and are involved in setting up the body plan in the embryos. Hox genes are known to regulate the differentiation of some neurons, but their contribution to the overall development of a nervous system is unclear. In this study, we analyzed the activity of the Hox genes in the differentiation of the 302 neurons of the  Drosophila Hox gene, Antp, specifies the identity of the second thoracic segment (T2) and promotes leg formation; the loss of Antp transforms T2 to a more anterior identity and converts the second leg to an antenna, and ectopic expression of Antp in the head converts the antenna to the second leg  and wgIs27[mab-5::EGFP] , whereas the integrated fosmid reporter wgIs756 and the endogenous GFP knock-in we used had extremely weak expression in the MNs, which made cell identification difficult. Results from scRNA-seq confirmed the expression of ceh-13 in seven classes of MNs, supporting Reilly et al.’s observations. Thus, we conclude that ceh-13 may indeed have low level of expression in the MNs, which are below our detection limit.One major discrepancy is that Reilly lin-39, mab-5, egl-5, php-3, and nob-1 were similar between our results and the other studies . Thus, gene expression mapping with fluorescent reporters still provides valuable information.Expression patterns of  studies , althouget al.’s results for most parts except for a few cases where the MN subtype expression were disputed  and scRet al., study, and the scRNA-seq at L4 stage all detected expression in mature and differentiated neurons, we analyzed Hox expression in embryonic neurons using the scRNA-seq data of C. elegans embryos  (sra-11 codes for a GPCR) into lin-39 null mutation but did not detect any change in AIY marker expression (mab-5(gk670) deletion allele , were lost in ~55% of AQR neurons (wdIs51[F49H12.4p::GFP] (F49H12.4 codes for an uncharacterized protein) was completely lost in lin-39(-) mutants  among the six Q descendants expressed 39::EGFP . Previou neurons  and most mutants , suggestlin-39 but expressed mab-5. Mutations in neither lin-39 nor mab-5 affected the expression of A/PQR fate markers in PQR (mab-5(-) mutants were anteriorly displaced, because mab-5 is needed for the posterior migration of QL neuroblast  (lad-2 codes for a L1 cell adhesion molecule) was only lost in SDQR neurons and not affected in the more posterior SDQL in lin-39(-) mutants  was lost in ~90% of SDQR but remained unaffected in SDQL in lin-39(-) mutants  mutants, we observed the mispositioning of SDQL due to the defect in QL migration, but the SDQ markers were expressed in 100% of the displaced SDQL neurons. Because SDQL neuron expresses both lin-39 and mab-5, we examined the SDQ fate in the lin-39(-) mab-5(-) double mutants and found that both lad-2 and flp-12 expression were lost in SDQL, suggesting that the two Hox genes act redundantly to induce the SDQ fate in SDQL neurons. Overall, these results indicate that the specification of the SDQ fate requires the Hox genes.The interneuron SDQL/R and the mechanosensory neuron A/PVM are also derived from the Q lineages.  mutants . The SDQ mutants . The morlin-39 was expressed in AVM and not in PVM. Mutations in lin-39 led to the posterior displacement of AVM neurons but did not affect the expression of TRN fate markers, such as zdIs5[mec-4p::GFP] (mec-4 codes for a DEG/ENaC channel) and uIs115[mec-17p::TagRPF] (mec-17 codes for a tubulin acetyltransferase), in the mispositioned AVM neurons (gcy-37 (encoding a guanylate cyclase), a subtype-specific marker that was expressed in AVM but no other TRNs, was completely lost in 100% of lin-39(-) mutants (For the postembryonic touch receptor neurons (TRNs),  neurons . However mutants . Thus, Llin-39 is also expressed in PVD and PDE neurons. We found that the expression of PVD fate markers otIs138[ser-2p3::GFP] (ser-2 codes for a serotonin receptor) and wdIs51[F49H12.4p::GFP] were lost in 100% of lin-39(-) mutants  mutants. Mutations in ceh-20, a Exd/Pbx homolog and Hox co-factor, led to similar loss of PVD and PDE markers  mutants still expressed the neurotransmitter identity markers, egIs1[dat-1p::GFP] (dat-1 codes for a dopamine transporter) and otIs518[eat-4::mCherry] (eat-4 codes for a vesicular glutamate transporter), respectively, (unkEx83[C34B2.9p::GFP] (C34B2.9 codes for an uncharacterized protein) (lin-39(-) mutants (lin-39(-) mutants , mgIs42 [tph-1::GFP], and otIs33[kal-1p::GFP]  were lost in ~90% of egl-5(-) mutants (egl-5 in regulating the migration and maturation of HSN neurons  was lost in ~88% of PHB neurons in egl-5(-) mutants  reported that egl-5 was not expressed in PHB neurons using muIs13[egl-5::lacZ], whereas we found that the wgIs54[egl-5::EGFP] fosmid reporter was indeed expressed in PHB neurons , and otIs33[kal-1p::GFP] were all lost in >95% of egl-5(-) mutants . flp-11 expression in DVA was not affected by mutations in egl-5, which is not expressed in DVA. Nevertheless, flp-11 expression in PVC was abolished in egl-5(-) mutants  mutants, but the expression of a general cholinergic marker vsIs48[unc-17p::GFP] (unc-17 codes for a vesicular acetylcholine transporter) and a motor neuron marker ctIs43[dbl-1p::GFP] (dbl-1 codes for a BMP-like protein) were not affected  showed any difference in expression between the wild-type and egl-5(-) animals  and is dispensable for the specification of one neuronal fate (LUA) in the tail region.affected . So, EGL animals . The expnd et al . Thus, ekal-1, glr-5, flp-4, flp-10, and unc-47  Hox genes, php-3 and nob-1, are both expressed in the tail neurons DVA, PHC, and PLN, which did not express egl-5. Mutations in either php-3 or nob-1 affected the expression of PHC fate markers, inIs179[ida-1p::GFP] and vsIs28[dop-1::GFP] (ida-1 codes for a type N protein tyrosine phosphatase and dop-1 codes for a dopamine receptor), and the expression of PLN markers, uIs130[lad-2p::GFP] and otIs358[ser-2p2::GFP]  mutants showed higher percentage of cells without the marker expression (nob-1(-) mutants had higher penetrance of tail formation defects than php-3(-) mutants and nob-1 was expressed at higher level than php-3 , ctIs43[dbl-1p::GFP], and ynls40[flp-11p::GFP]  nob-1(-) double mutants resulted in 100% larval lethality and showed no posterior morphogenesis  and PVR fate marker otIs92[flp-10::GFP] did not show any defects in php-3(-) mutants . Their expression domains partially overlapped, showing lin-39 expression in more anterior MNs and mab-5 in more posterior MNs  and otIs453[itr-1p::GFP] (itr-1 codes for an inositol triphosphate receptor); these DA9-specific expressions were lost in egl-5(-) mutants  egl-5(-) double mutants but not in mab-5(-) egl-5(-) double mutants. itr-1 expression was not restored in either double mutant, suggesting that EGL-5 activated glr-4 through repressing lin-39 and activated itr-1 independently of lin-39 and mab-5. Similarly, EGL-5 activated mig-13 (which codes for a Frizzled receptor) through repressing mab-5 and repressed unc-129 (which codes for a bone morphogenetic protein) independently of mab-5 and lin-39 in DA9 neurons  mutants in the middle-body DA neurons  mutants  exclusively among the nine DA neurons, and this expression depended on mab-5  and activated mig-13 in VA12 through repressing mab-5  did not require mab-5. Expression of flp-18 was detected in VA11-12 but not VA1-VA10 neurons, and its expression depended on mab-5 and egl-5 in VA11 and VA12, respectively  acquired MAB-5-dependent molecular features and differentiated into distinct subtypes. Supporting this hypothesis, the most posterior AS neuron that expressed mab-5 (i.e. AS11) also defined a AS subtype by specifically expressing unc-129 among the eleven AS neurons (mab-5(-) mutants.Besides DA-type MNs, we made similar observations in VA-type MNs. For example, EGL-5 repressed ng mab-5 . In VA7-ely Figs  and 6B.  neurons . As expeser-2 by repressing mab-5 in VD13, whereas VD1-12 expressed ser-2 normally  mutants, although DB2-4 expression of unc-129 had variable loss  mutants except for ~50% of loss in VA4-6 neurons . In addition, we found that mig-13 expression in DA2-3 and VA2-6 were not affected by mutations in lin-39; and VD1-12 expression of ser-2 were mostly maintained in lin-39(-) mutants except for variable loss of expression in VD6-8  mutants  mutants, including DA8, DB7, VA11, VB11, AS11, DD5, and VD10-12 neurons  mutants, suggesting that egl-5 actively repressed the expression of Hox cofactors in VD13  were expressed in 97 (32%) of the 302 neurons in adult hermaphrodites. These 97 neurons fall into 29 (25%) neuron types or classes out of the 118 anatomic classes previously defined  and CGZ1 lin-39(n1760); unkEx1 strains by first injecting the fosmid WRM0630bD02 that contains the wild-type lin-39 locus into the MT7236 lin-39(n1760) egl-5(n945) strain to restore vulval development in TU6438 and then mating them with lin-39(n1760) males to obtain CGZ1. After crossing various transgenes into TU6438 and CGZ1, we picked the animals that lost the extrachromosomal array unkEx1 and obtained homozygous Hox mutants with the transgene.To cross into animals carrying the wgIs18[lin-39::EGFP], wgIs27[mab-5::EGFP], wgIs54[egl-5::EGFP], php-3::GFP(unk25), and stIs10286[nob-1::GFP] with mCherry reporter strains for the neurotransmitter identities, including otIs518[eat-4(fosmid)::SL2::mCherry::H2B], otIs544[cho-1(fosmid)::SL2::mCherry::H2B], and otIs564[unc-47(fosmid)::SL2::H2B::mChopti] for glutamatergic, cholinergic, and GABAergic neurons, respectively [otIs181[dat-1p::mCherry] and vsIs97[tph-1p::DsRed2] for the identification of dopaminergic and serotonergic neurons . The resulted stable lines were analyzed for GFP expression patterns at day 1 adult stage. At least two independent lines were obtained for each construct, and at least 40 animals were examined for each line. The percentage of animals showing GFP expression were shown as the mean ± SD of different lines.Promoters upstream of the start codon of the cell fate marker genes were cloned from the N2 genomic DNA and inserted into pPD95.75 vector through Gibson Assembly . To delete specific Chi-square test were used to determine whether there is a significant difference between the wild-type and the mutants . Raw data can be found in lin-39(-) or mab-5(-) mutants, respectively.Fluorescent imaging was done on a Leica DMi8 inverted microscope equipped with a Leica DFC7000 GT or a Leica K5 monochrome camera. To analyze gene expression patterns, we imaged at least 30 day-one adult animals for each strain and calculated the percentages of animals showing the expression of the reporter. Three biological replicates were carried out for each imaging experiment, and S1 Tableet al., 2021  in early embryos and the lack of any expression in larva. (B) The expression of endogenous ceh-13::GFP knock-in, which was generated by CRISPR/Cas9-mediated gene editing, in the embryos at the bean stage and the comma stage. The expression faded away in the 3-fold stage embryos and was not observed in larva. (C) Super weak and variable expression of ceh-13::GFP in the ventral nerve cord motor neurons (arrows) in adults. Scale bars = 20 μm.(A) The expression of the fosmid-based reporter (TIF)Click here for additional data file.S2 Figlin-39 reporter in GABAergic MNs, including VD3-12 and DD2-6. The nuclei of GABAergic MNs were labeled by neurotransmitter identity marker unc-47. (B) The expression of lin-39 in cholinergic MNs, including DA2-5, DB2-7, VA3-8, VB4-9, AS2-8, VC1-3, and VC6. lin-39 was also expressed in VC4 and VC5, which are not cholinergic. Scale bars = 20 μm.(A) The expression of (TIF)Click here for additional data file.S3 Figmab-5 reporter in GABAergic MNs, including VD2-12 and DD2-6. (B) The expression of mab-5 in cholinergic MNs, including DA4-8, DB5-7, VA6-11, VB8-11, AS5-11, VC3, and VC6. mab-5 was also expressed in VC4 and VC5, which are not cholinergic. Scale bars = 20 μm.(A) The expression of (TIF)Click here for additional data file.S4 FigstIs10286[nob-1::GFP] is a translational reporter with unc-54 3’UTR. php-3::GFP(unk25) is a GFP knock-in at the endogenous locus of php-3.Black boxes represent exons and black lines for introns. Brown boxes represent the untranslated regions (UTRs). (TIF)Click here for additional data file.S5 Figlin-39(n1760) mutants, but the expression of TRN fate marker uIs115[mec-17p::TagRFP] was not affected in lin-39 mutants. (B) The displaced AVM also expressed the TRN fate marker zdIs5[mec-4p::GFP] in lin-39 mutants. Scale Bars = 100 μm.(A) AVM was posteriorly displaced in (TIF)Click here for additional data file.S6 FigotIs138[ser-2p3::GFP], the PVD fate marker wdIs51[F49H12.4p::GFP], and dopaminergic identity marker egIs1[dat-1p::GFP] in lin-39(n1760), mab-5(gk670), and ceh-20(u843) mutants. (B) The expression of the PDEsh markers unkEx83[C34B2.9p::GFP] in lin-39 and ceh-20 mutants.(A) The penetrance for the expression of the PVD and PDE fate marker (TIF)Click here for additional data file.S7 Fighttp://cisbp.ccbr.utoronto.ca/.(A) The sequences of functional Hox sites identified in this study , which l(TIF)Click here for additional data file.S8 FigChIP-seq data from the modENCODE project were retrieved from Wormbase (WS283) and visualized using the Jbrowse at the promoter regions of various neuronal fate markers. The signals from the “LIN-39 Combined (GFP ChIP) recalled peaks” (A) and “EGL-5 Combined (GFP ChIP) recalled peaks” (B) tracks were shown. In (A), arrows indicate the peaks that cover the region where the functional LIN-39 sites were found.(TIF)Click here for additional data file.S9 Figdbl-1 and flp-11, were not affected in php-3(ok919) and nob-1(ct230) mutants. The numbers in the parentheses indicate the number of cells examined; 100% indicate all of them showed normal expression. For nob-1(ct230) mutants, animals with severe malformation of the tail were excluded. (B) The expression of DVC fate marker srab-12 and PVR fate marker flp-10 were not affected in php-3 mutants. Scale Bars = 20 μm.(A) The expression of DVA fate markers, (TIF)Click here for additional data file.S10 Figmig-13 in the cholinergic MN subtypes in the middle body region, including DA5-8 and VA7-11 in mab-5(gk670) mutants. Asterisk indicates the position of the vulva. Scale Bars = 20 μm. (B) Variable loss of unc-129 expression in a few anterior DA and DB neurons, including DB2 and DB4, in lin-39(n1760) mutants. Dashed circles indicate the loss of GFP expression in corresponding neurons. Scale Bars = 100 μm. (C) Variable loss of del-1 expression in a few anterior VA and VB neurons, including VA4-5 and VB5-6, in lin-39 mutants. (D) Variable loss of ser-2 expression in very few VD neurons, including VD8, in lin-39(n1760) and unc-30(e191) mutants. Scale Bars = 100 μm.(A) Derepression of (TIF)Click here for additional data file.


Primary varicella-zoster virus (VZV) infection leads to varicella and the establishment of lifelong latency in sensory ganglion neurons. Reactivation of latent VZV causes herpes zoster, which is frequently associated with chronic pain. Latent viral gene expression is restricted to the VZV latency-associated transcript (VLT) and VLT-ORF63 (VLT63) fusion transcripts. Since VLT and VLT63 encode proteins that are expressed during lytic infection, we investigated whether pVLT and pVLT-ORF63 are essential for VZV replication by performing VZV genome mutagenesis using CRISPR/Cas9 and BAC technologies. We first established that CRISPR/Cas9 can efficiently mutate VZV genomes in lytically VZV-infected cells through targeting non-essential genes ORF8 and ORF11 and subsequently show recovery of viable mutant viruses. By contrast, the VLT region was markedly resistant to CRISPR/Cas9 editing. Whereas most mutants expressed wild-type or N-terminally altered versions of pVLT and pVLT-ORF63, only a minority of the resulting mutant viruses lacked pVLT and pVLT-ORF63 coding potential. Growth curve analysis showed that pVLT/pVLT-ORF63 negative viruses were viable, but impaired in growth in epithelial cells. We confirmed this phenotype independently using BAC-derived pVLT/pVLT-ORF63 negative and repaired viruses. Collectively, these data demonstrate that pVLT and/or pVLT-ORF63 are dispensable for lytic VZV replication but promote efficient VZV infection in epithelial cells. Varicella-zoster virus (VZV) is a widespread human alphaherpesvirus and the causative agent of both varicella (chickenpox) and herpes zoster (HZ or shingles) . During True-Late transcripts during lytic VZV infection. However, unlike the single VLT isoform and two VLT63 isoforms expressed during latency, numerous lytic VLT (lytVLT) and VLT63 (lytVLT63) isoforms are produced by the usage of alternative transcription start-sites, read-through transcription and alternative splicing. Whereas lytVLT and lytVLT63-2 encode VLT protein (pVLT), lytVLT63-1 encodes for a pVLT-ORF63 fusion protein, in which canonical pORF63 is C-terminally linked to a N-terminal part of pVLT (118 of 136 aa) . VLT RNA specifically downregulates the expression of RNA 61, which encodes the promiscuous transcriptional regulatory protein ORF61 (pORF61), in co-transfection experiments, suggesting that VLT may function in the establishment or maintenance of latency [lytVLT transcription and the genomic position of VLT antisense to the essential gene ORF61 [Similar to latency-associated transcripts of other alphaherpesviruses, the VLT locus is located partially antisense to ORF61 [the homologue of herpes simplex virus infected cell polypeptide 0 -based mutagenesis or by clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) editing ,14. BAC-In this study, we investigated the role of pVLT and pVLT-ORF63 during lytic VZV infection in epithelial cells using mutant viruses obtained through complementary CRISPR/Cas9 genome editing and BAC mutagenesis. v/v) mixture of DMEM  and Ham’s F12  supplemented with 10% heat-inactivated FCS, and 5% penicillin-streptomycin with L-glutamine . Human melanoma MeWo cells (ATCC HTB-65) and MeWo-Cre cells expressing Cre-recombinase [2 incubator at 37 °C. Human retinal pigmented epithelial ARPE-19 cells were cultured in a 1:1  according to manufacturer’s instructions. DNA was eluted in 200 μL and used for PCR and Sanger sequencing.g at 4 °C. From the aqueous phase, RNA was isolated using the RNeasy Mini kit  according to manufacturer’s instructions, including on-column DNAse I treatment. RNA concentration was determined using a Nanodrop spectrophotometer, and residual DNA was removed using the Turbo DNA-free kit . For cDNA synthesis, maximum 5 µg RNA was reverse transcribed with Superscript IV reverse transcriptase (RT+) and oligo-dT primers . Negative control samples were obtained by performing the same cDNA synthesis reaction in the absence of reverse transcriptase (RT−).VZV-infected cells were harvested in 1 mL Trizol , mixed with 200 μL chloroform and centrifuged for 15 min at 12,000× Taqman quantitative PCR (qPCR) was performed in duplicate on RT- and RT+ cDNA on a 7500 Taqman PCR system using 4× Taqman Fast Advanced Master mix . Primer-probe sets for VZV genes RNA 29, RNA 61, RNA 63, VLT2_3 and VLT3_4 have been described previously [PCR was performed on extracted DNA or cDNA using AmpliTaq Gold DNA Polymerase  using primer pairs as indicated in The pVLT coding sequence was amplified from cDNA of VZV EMC-1 infected ARPE-19 cells with NheI_VLT_Fw and VLT_Rv_BamHI primers , cloned http://crispor.tefor.net, accessed on 13 July 2017) and cloned as sgRNA downstream a human U6 promoter in the pSicoR-CRISPR-PuroR or pSicoR-CRISPR-BlastR vector [crRNA target sequences  were desR vector . All vecg, 15 min) on the ARPE-19 cells. Two days after transduction, medium was replaced for selection medium containing either 0.94 µg/mL puromycin  or 20 µg/mL blasticidin  and stable cell lines were obtained by continuous selection for 3 weeks. To generate ARPE-19 cells expressing two sgRNAs, cells stably expressing the first sgRNA were lentivirally transduced to express the second sgRNA, followed by a second round of antibiotic selection.ARPE-19 cells were seeded one day prior to lentiviral transduction in a 48-well plate at 70% confluency. The next day, lentiviral stocks were diluted 1:5 in culture medium containing 4 µg/mL polybrene and spin-inoculated (1000× ARPE-19 cells stably expressing one or two sgRNA(s) and SpCas9 were inoculated with cell-free VZV EMC-1 (MOI = 0.01) in a 12-well plate. Cell-free virus was harvested at 2 days post infection (dpi) and a 2-fold serial dilution was used to infect fresh monolayers of ARPE-19 cells in a 96-well plate. In case of sgRNA VLT-9, the obtained cell-free VZV was also serially diluted on ARPE-19 cells stably expressing pVLT. After 5–6 days incubation, plates were screened and wells containing a single plaque were passaged on fresh ARPE-19 cells in a 12-well plate. Obtained virus cultures were subjected to another 2 (double sgRNAs) or 3 (sgRNA VLT-9) rounds of plaque purification on ARPE-19 cells. The resulting viruses were analyzed by Sanger sequencing of the VLT target region. 2 and 2.5 µL Omnicleave  and incubated for 1 h at 37 °C and DNA was subsequently isolated by the QIAamp DNA mini kit  as described. Paired-end Illumina sequencing libraries were prepared from 100 ng DNA using the KAPA HyperPlus library preparation kit  according to the manufacturer’s instructions with KAPA Unique Dual-Indexed Adapters Kit (Roche) for multiplexing. Libraries were pooled with unrelated samples and run on an Illumina MiSeq version 3 flowcell (2 × 300 cycles) . Resulting sequencing data were demultiplexed and trimmed to remove adapter sequences and low-quality 3’ nucleotides using TrimGalore . Trimmed sequence data were aligned against the VZV strain Dumas reference genome using bbmap  and parsed using SAMTools [https://github.com/genome/bam-readcount, v1.0.0), with the flag –d 1000000, was subsequently used to identify all reported SNPs and Indels prior to filtering using a custom script, variant_caller_v1.0.py, available at https://github.com/DepledgeLab/NAGATA.ARPE-19 cells were infected with VZV EMC1-pVLTwt, EMC1-pVLTΔ1aa, EMC1-pVLTshift or EMC1-pVLTstop, and at 50–70% CPE infected cells were harvested. The cell pellet was resuspended in 200 µL PBS + 0.1% sodium deoxycholate, 50 µL 25 mM MgClSAMTools  and BEDTsacB gene flanked by 102,501–102,550 and 104,448–104,500 of pOka was PCR amplified from the pST76A-SR plasmid [E. coli harboring pOka-BAC genome [sacB gene (pOka-BACampsac1st). To generate the pOka-BACVLTM1I genome a DNA fragment of 100,798–104,820 of pOka amplified from pBpOka100798-104820VLTM1I plasmid was transformed into GS1783 harboring pOka-BACampsac1st, and followed by negative selection with 5% sucrose. The repaired BAC genome pOka-BACVLTM1IR was similarly generated based on the pOka-BACVLTM1I. Briefly, the 102,551–104,447 region of pOka-BACVLTM1I was replaced by an ampicillin-resistant gene and sacB gene in GS1783 E. coli, resulting in pOka-BACampsac2nd and the pOka-BACVLTM1Irev genome was then generated by transformation of a DNA fragment of 100,798–104,820 of pOka amplified from pBpOka100798-104820 plasmid into GS1783 pOka-BACampsac2nd, followed by negative selection with 5% sucrose. Finally, pOka-BACVLTM1I and pOka-BACVLTM1IR were purified using the Genopure Plasmid Maxi Kit , subjected to restriction fragment length polymorphism analysis using BamHI or EcoRI and Sanger sequencing of the recombined region.The pBpOka100798-104820 plasmid was generated by PCR amplification of the VLT locus  of pOka-BAC DNA [ plasmid  using prC genome , resultiVZV strain pOka-based VLTM1I and its repaired VLTM1IR recombinant viruses were reconstituted in MeWo cells by transfection of pOka-BACVLTM1I and pOka-BACVLTM1IR genomes using Lipofectamine2000 (Thermo Fisher Scientific) and the BAC cassette within the reconstituted viruses was subsequently removed in MeWo-Cre cells ,24.ARPE-19 cells on glass coverslips were inoculated with freshly harvested cell-free VZV EMC-1 edited by sgRNA8-2 and incubated for 3 days at 37 °C. Infected cells were fixed with 4% paraformaldehyde (PFA), permeabilized for 10 min with 0.1% Triton X-100 in PBS, blocked with 5% goat serum diluted in PBS + 0.05% Tween and incubated with monoclonal mouse IgG1 anti-ORF8 antibody  and monARPE-19 cells were plated in a 24-well plate and the next day cell monolayers were infected with 100 PFU/well of cell-free VZV. At 6 dpi, plates were washed and fixed with 4% PFA in PBS. Subsequently, cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min, blocked with 5% normal goat serum in PBS-T for 30 min, incubated with mouse anti-VZV glycoprotein E antibody  diluted 1:3200 in PBS-T overnight at 4 °C. The next day, the plates were washed with PBS-T, incubated for 1 h at room temperature with 1:200 biotinylated rabbit anti-mouse Ig antibody  in PBS-T, washed and incubated for 1 h at room temperature with 1:300 streptavidin-HRP  in PBS-T, washed once in PBS-T and twice with PBS. Finally, the signal was visualized using 3-amino-9-ethylcarbazole (AEC) as a substrate. Infectious foci were measured using the Immunospot S6 Ultimate UV Image Analyzer and foci size was determined using the Immunospot software .2 cell culture.Monolayers of ARPE-19 cells in a 24-well plate were infected with 1000 PFU/well VZV, harvested at 3 dpi and titrated on ARPE-19 cells in 96-well plates using five-fold serial dilutions. After 3 days, infectious virus titers were calculated using the Spearman–Karber formula and expressed as TCID50 per cmARPE-19 cells were plated in a 96-well plate one day prior to infection. Cells were infected with 150 PFU/well cell-free virus and harvested at 24 h post infection (hpi), 48 hpi and 72 hpi. Subsequently, cells were fixed and permeabilized with BD Cytofix/Cytoperm, stained for VZV glycoprotein E  in BD PermWash for 30 min at 4 °C, stained with secondary APC-conjugated goat anti-mouse Ig antibody  for 30 min at 4 °C and finally resuspended for analysis. Frequency of VZV-infected (APC-positive) cells was determined. Experiments were performed on a BD FACSLyric flow cytometer and the data was analyzed using FlowJo software (BD Biosciences).g for 15 min. Proteins were separated on a 4–12% Nu-PAGE Gel (Thermo Fisher Scientific) and transferred onto PVDF membranes (0.2 µm). The membrane was blocked in 5% (w/v) skim milk in PBS + 0.1% Tween 20 at room temperature for 1 h and subsequently stained overnight at 4 °C with polyclonal rabbit anti-pVLT (1:2000) [Mock- or VZV-infected ARPE-19 cells at 5 dpi were incubated in RIPA lysis buffer  on ice for 15 min, sonicated in a water bath for 10 min, and centrifuged at 20,000× p < 0.05, ** = p < 0.01, *** = p < 0.001. If no statistics are given, the difference was not significant.Figures show individual data points or mean +/− SEM, all statistical analyses were performed with Graphpad Prism 9 software using the statistical test indicated in the figure legends, with * = Streptococcus pyogenes Cas9 (SpCas9) endonuclease and a single guide RNA (sgRNA) targeting the genomic region of interest (n = 6 per sgRNA), obtained after a single round of limited dilution and plaque purification, detected insertions or deletions (indels) in all analyzed isolates edited using sgRNA ORF8-2 or sgRNA ORF11-1   E. lytVLT and neighboring VZV transcripts in VZV VLT mutant virus isolates compared to VZV EMC-1 by RT-qPCR. Relative expression levels of lytVLT/lytVLT63 RNAs were reduced for all mutants compared to parental VZV EMC-1, especially in pVLT/pVLT-ORF63(-) isolates, C14 and D23  targets the 3’end of VLT exon 2, and could alter the pVLT/pVLT-ORF63 protein sequence  targets one of the upstream exons of lytVLT/lytVLT63 [lytVLT/lytVLT63 variants remain available , induced efficient CRISPR/Cas9-based VZV genome editing  viruses with genomic deletions were impaired in replication compared to the corresponding pVLT/pVLT-ORF63(+) viruses , we perfn = 47) were conserved between all four VZV isolates and thus likely represent SNPs already present in the parental EMC-1 virus (Short-read Illumina sequencing of VZV pVLTwt, pVLTΔ1aa, pVLTshift and pVLTstop genomes identified 75 SNPs  compared-1 virus D. Anothe-1 virus D. Most S-1 virus E. The ma-1 virus . Most of-1 virus . Importa-1 virus G, in theIn contrast to the viruses with large deletions in the VLT locus A, expresThe currently available anti-pVLT antibody recognizes the first 20 amino acids of pVLT , therebyVZV latency in sensory neurons is defined by selective expression of VLT and VLT-ORF63 fusion transcripts ,10. VLT Our study reports on locus-specific VZV genome editing using CRISPR/Cas9 technology and subsequent purification of mutant virus isolates. Non-essential VZV genes ORF8 and ORF11 could be efficiently targeted in lytically VZV-infected cells and produced infectious mutant progeny virus. The VLT locus, however, proved to be difficult to target, and no genome editing was observed when using 11 of 12 single sgRNAs targeting the VLT locus. It is unclear what causes this resistance of the VLT region to CRISPR/Cas9 genome editing, as targeting essential herpesvirus genes is possible and typically results in the outgrowth of escape mutants that carry in-frame deletions . Many ceThe CRISPR/Cas9 system provides the opportunity to edit clinical isolates , enablinCell cultures are oversimplified representations of the host environment, and thus requirements for survival in vivo can substantially differ from those in vitro. Indeed, the phenotype of single-ORF deletion VZV mutants can be markedly different between cell culture and skin tissue culture or xenograft models ,40,41,42Currently available antivirals and vaccines that counteract and prevent VZV disease, leave the latent VZV burden intact. CRISPR/Cas9, besides being a versatile tool for research, also holds promising therapeutic potential for the treatment of persistent virus infections, as illustrated by the application to excise the integrated SIV genome in vivo ,47. AddiIn conclusion, this study describes the use of CRISPR/Cas9-based genome editing to generate VZV mutants. Specifically, we applied this method to investigate the role of pVLT and pVLT-ORF63 during lytic VZV infection in vitro. Our data provide valuable insight into the effectiveness of VLT-specific sgRNAs or combinations thereof that can be used to optimize the inactivation of latent VZV genomes in in vitro neuronal latency models and could ultimately be applied in the human host.


Dysregulated immune responses are the cause of IBDs. Studies in mice and humans suggest a central role of interleukin (IL)-23-producing mononuclear phagocytes in disease pathogenesis. Mechanistic insights into the regulation of IL-23 are prerequisite for selective IL-23 targeting therapies as part of personalised medicine.We performed transcriptomic analysis to investigate IL-23 expression in human mononuclear phagocytes and peripheral blood mononuclear cells. We investigated the regulation of IL-23 expression and used single-cell RNA sequencing to derive a transcriptomic signature of hyperinflammatory monocytes. Using gene network correlation analysis, we deconvolved this signature into components associated with homeostasis and inflammation in patient biopsy samples.We characterised monocyte subsets of healthy individuals and patients with IBD that express IL-23. We identified autosensing and paracrine sensing of IL-1α/IL-1β and IL-10 as key cytokines that control IL-23-producing monocytes. Whereas Mendelian genetic defects in IL-10 receptor signalling induced IL-23 secretion after lipopolysaccharide stimulation, whole bacteria exposure induced IL-23 production in controls via acquired IL-10 signalling resistance. We found a transcriptional signature of IL-23-producing inflammatory monocytes that predicted both disease and resistance to antitumour necrosis factor (TNF) therapy and differentiated that from an IL-23-associated lymphocyte differentiation signature that was present in homeostasis and in disease.Our work identifies IL-10 and IL-1 as critical regulators of monocyte IL-23 production. We differentiate homeostatic IL-23 production from hyperinflammation-associated IL-23 production in patients with severe ulcerating active Crohn’s disease and anti-TNF treatment non-responsiveness. Altogether, we identify subgroups of patients with IBD that might benefit from IL-23p19 and/or IL-1α/IL-1β-targeting therapies upstream of IL-23. IBD is a complex chronic inflammatory disease caused by the interaction of host and environmental factors in a genetically susceptible setting. Targeting the proinflammatory arm of the immune system such as by blocking tumour necrosis factor (TNF) signalling has proven therapeutically successful in subgroups of patients with IBD. Studies in both mice and humans indicate interleukin (IL)-23 as a key pathogenic proinflammatory factor in IBD. Early-stage clinical trials indicate that blocking IL-23 can induce disease remission in patients with Crohn’s disease (CD) and ulcerative colitis (UC). As part of personalised medicine, mechanistic insights into the regulation of IL-23 are prerequisite for selective IL-23 targeting therapies.We identify autocrine and paracrine sensing of IL-1α/IL-1β and IL-10 as key cytokines that control IL-23-producing monocytes. We determine a transcriptional signature of IL-23-producing monocytes that predicted both disease and resistance to anti-TNF therapy and differentiate this hyperinflammation-associated IL-23 production in patients with severe ulcerating active CD from homeostatic intestinal IL-23 production. We show that exposure of monocytes to whole bacteria induced IL-23 production via acquired IL-10 signalling resistance akin to IL-23 secretion by lipopolysaccharide-stimulated monocytes in Mendelian IL-10 signalling defects.We identify subgroups of patients with IBD that might benefit from IL-23p19 and/or IL-1α/IL-1β-targeting therapies upstream of IL-23.IL23R encoding the IL-23 receptorRORC, STAT3, IRF5, IL1R1, IL6ST, IL12B, TYK2, IL21, JAK2, IFNG, SMAD3 and CCR6.via the anti-CD40.The pathogenesis of inflammatory bowel disease (IBD), which includes Crohn’s disease (CD) and ulcerative colitis (UC) and IBD unclassified (IBDu), is caused by dysregulated innate and adaptive immune responses that drive chronic relapsing tissue inflammation.+ T cells,To predict genetic susceptibility or disease course, individual loci and genetic risk scoresPersonalised medicine targeting the IL-23 axis requires an understanding of the cellular sources, networks and regulation of IL-23. Here we investigate the regulation of IL-23, describe distinct monocyte subsets that express IL-23 and identify IL-1 signalling as the key cytokine for the differentiation of IL-23-producing monocytes. We identify a hyperinflammatory signature of IL-23-producing monocytes in intestinal tissue transcriptomes of patients with IBD and find an additional signature of IL-23 that is associated with lymphocyte cell differentiation in healthy tissue.Detailed methods are outlined in the 10.1136/gutjnl-2020-321731.supp1Supplementary dataIL23A expression by subjecting peripheral blood mononuclear cells (PBMC) from 41 patients with IBD  or disease activity in treatment-naïve or treated patients with IBD.To investigate the regulation of secreted proteins by IL-10, we analysed cell culture supernatants after 16 hours of stimulation . LPS sti10.1136/gutjnl-2020-321731.supp6Supplementary data+ (IL-12p40+IL-23p19+) and IL-12+ (IL-12p40+IL-23p19–) cell frequencies and mean fold change were significantly greater in CD14+ relative to CD14– leucocytes after IL-10 receptor blockade   (+IL-23p19+) production in a subset of CD14+ monocytes .We next sought to investigate IL-10 and IL-23 production at the single-cell level and analysed monocytes cytokine production by flow cytometry. At 16 hours poststimulation, monocytes produced IL-10 but low IL-12p40 and IL-23p19. The synthesis of all three proteins was significantly increased when cells were stimulated with LPS and anti-IL-10R. IL-23 fc=3.5) . These r fc=6.3) . Togethe10.1136/gutjnl-2020-321731.supp7Supplementary data+ monocytes , +CD52 (CAMPATH1) and FCN1+ expressing CD14high monocytes (cluster 0—unstimulated) typical of classical monocytes, while a minority of unstimulated monocytes displayed a CD52+, FCN1+, CD16+ (FCGR3A) CD14low phenotype (cluster 7—unstimulated) characteristic of intermediate or non-classical monocytes in keeping with the flow cytometry data (CD14++IL1Bcells. While these two clusters of cells had very similar profiles (CD163 and CD300E and was enriched for genes associated with ‘T-cell tolerance induction’ (cluster 1—LPS-stimulated), while the second displayed higher CD163 and CD300E expression and an enrichment for ‘monocyte activation’ genes (cluster 2—LPS-stimulated). The three additional monocyte phenotypes that appeared on combined LPS stimulation and IL-10R blockade were demarcated by (1) high expression of type I IFN-responsive genes  and genes linked to antigen processing and presentation  ; (2) high expression of ITGAX and enrichment for genes associated with ‘superoxide generation’ and ‘positive regulation of neutrophil activation’  and (3) expression of proinflammatory genes including  and enrichment of genes associated with ‘Th17 cell lineage commitment’, ‘positive regulation of acute inflammatory response’ and ‘IFN-γ production’ . These three phenotypes in LPS-stimulated anti-IL-10R-treated monocytes were replicated in a second donor  when stimulated with LPS and IL-10R blockade . Otherwise, the pattern of segregated IL-10 and IL-23 protein expression might result from oscillatory expression of IL10 and IL23A mRNA. To distinguish between these possibilities, we performed a dual-colour ELISpot assay using MACS-purified monocytes. This approach captured cytokine production in the individual cells over the full duration of their culture in vitro. In line with a paracrine model where IL-10-producing monocytes regulate IL-23 production in others, we found that IL-10-producing monocytes were distinct from IL-23-producing monocytes  study biopsies  where ILpression . We thertestines . This antestines . Clusterth genes .10.1136/gutjnl-2020-321731.supp12Supplementary data10.1136/gutjnl-2020-321731.supp13Supplementary dataIL23A expression (r=0.35) (CD14 and PDPN), proinflammatory cytokines  and fibroblast activation protein (IL23A expression was most strongly correlated with two modules of ‘immune cell differentiation’ (r=0.7) and ‘lymphocyte differentiation’ (r=0.71) genes (CD14) and lymphoid  cell markers, and enriched for ‘myeloid DC differentiation’ (CD79A and CD4) and differentiation (BACH2)IL23A-associated immune cell differentiation and lymphocyte differentiation modules were significantly enriched for genes belonging to the KEGG ‘Th17 cell differentiation’ pathway  and (r=0.35) . This mo protein  in keepi1) genes . Of thestiation’ . The lym pathway .10.1136/gutjnl-2020-321731.supp14Supplementary dataIL-23 expression was primarily associated with lymphoid cell differentiation in both health and disease. We therefore sought to understand which of the IL-10-regulated monocyte genes were specific to disease-associated inflammation. We correlated the expression of a curated set of IL-10-regulated monocyte genes . We therefore investigated the functional effects of 11 coregulated cytokines on monocyte IL-23 production . Strikin10.1136/gutjnl-2020-321731.supp18Supplementary data10.1136/gutjnl-2020-321731.supp19Supplementary data31-1 configurations could be excluded a priori. We identified an optimal network describing the core network dynamics by ranking the models based on their fit to the data using the Akaike information criterion  emphasises a surprising and underestimated extent of IL-10 non-responsiveness in IBD. In those patients, inflammation is present despite transcription of IL-10 (patients express a large amount of IL-10) and absence of pathogenic variants within the IL-10 receptor .PTGS2 in this subgroup of patients with IBD represents an additional indicator for deregulated IL-10 responses and intestinal antimicrobial immunity.The strong predictive ability of an IL-10-sensitive inflammatory monocyte signature  that induces diverse transcriptional states and cellular functions.Single-cell RNA transcriptomic approaches identified transcriptional signatures in circulating and tissue human monocytes and macrophages,+IL1Β ‘inflammatory monocyte’IL1B+IL23A+ ‘inflammatory macrophage’IL1B+IL23A+ transcriptional signature that is very similar to those described for mononuclear phagocytes isolated from the inflamed intestinal biopsies of patients with CD and UC. This observation demonstrates the relevance of functional studies of peripheral monocytes for understanding the behaviour of these cells following their recruitment into the inflamed intestinal mucosa. Together with the previous findings, our observations suggest that inflammatory monocytes and CX3CR1+IL-1β+ macrophagesRecently Importantly, several cell types including regulatory T cells, B cells, macrophages and DCs produce IL-10IL23A expression was likely derived from terminal ileal MALT because the lymphocyte differentiation to which it was assigned also showed strong correlations with orthologues of genes associated with murine small intestine lymphoid tissue (SILT), including IL22RA2, ITGAXVCAM1, which is a marker of lymphoid-associated villi in humans (IL23A is most likely DCs, tolerogenic CD103+DCIn individuals without intestinal inflammation, our network analysis suggested that n humans . In such+PTGS+IL6+ signature might define an anti-IL-1R1 responsive group of patients with IBD. Interventional studies are required to identify differential effects between IL-23 and IL-1 blockade since both have complex effects on licensing for effector cytokine production and differentiation of Th1 and Th17 cells.Our mathematical model pinpoints IL-1 as a key positive regulator of IL-23 expression in monocytes. Studies in monogenic forms of IBD suggest that IL-1R1 blockade can resolve intestinal inflammation in patients with IL-10 receptor defects,10.1136/gutjnl-2020-321731.supp20Supplementary data


Autoactivity of an executor immune receptor due to mutations in putative ATP hydrolysis motifs requires the full-length allele of the cognate sensor immune receptor. Dear Editor, Ralstonia solanacearum 1 (RRS1-R) and Resistance to Pseudomonas syringae 4 (RPS4) are a pair of Arabidopsis  NLR proteins that function together to recognize two bacterial effectors, PopP2, an acetyl-transferase from Ralstonia solanacearum and AvrRps4 from Pseudomonas syringae pv. pisi. The RRS1-R allele in Arabidopsis accessions Nd-1 and Ws-2 confers AvrRps4 and PopP2 recognition, whereas the Col-0 allele of RRS1 (RRS1-S) confers only AvrRps4, but not PopP2, recognition , leading to pattern-triggered immunity (PTI), or by intracellular nucleotide-binding (NB), leucine-rich repeat (NLR) immune receptors, leading to effector-triggered immunity (ETI). Pathogens suppress PTI by secreting effectors into host cells. These effectors can be recognized by NLRs, often encoded by Resistance (R) genes; the resulting ETI often culminates in the hypersensitive cell death response HR; . NLR proognition . Althougognition . Overexpy RRS1-R . Previouy RRS1-R , less atin planta  induces HR to a similar extent, but overexpression of RPS4(D315E) induces no HR . Most st1/3/4A with RPS4(S270F) or RPS4(D315E) also fails to trigger HR (RRS1-R is the only recessive NLR resistance gene; an RRS1-R/RRS1-S heterozygote loses RRS1-R function . We testigger HR , again digger HR .In summary, we report that in paired NLRs, full executor NLR autoactivity caused by mutations in proposed ATP hydrolysis motifs requires the cognate sensor NLR, showing sensor NLRs have a positive as well as negative function . Future kiaa076_Supplementary_DataClick here for additional data file.


Intense transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Africa might promote emergence of variants. We describe 10 SARS-CoV-2 lineages in Benin during early 2021 that harbored mutations associated with variants of concern. Benin-derived SARS-CoV-2 strains were more efficiently neutralized by antibodies derived from vaccinees than patients, warranting accelerated vaccination in Africa. Genomic surveillance is key to elucidate coronavirus disease (COVID-19) transmission chains and to monitor emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants associated with partial or complete immune escape  were used for this study. To enable rapid prescreening of mutations known to affect the viral phenotype, we used 4 reverse transcription PCR (RT-PCR)–based single-nucleotide polymorphism (SNP) assays  targeting 9 hallmark mutations in 7 S codons of variants of concern (VOCs): B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta)  showed the 69/70 deletion, 58.9% (223/378) the E484K mutation, 33.9% (128/378) the N501Y mutation, 30.4% (115/378) the P681H mutation, 14.8% (56/378) the L452R mutation, and 0.3% (1/378) the K417N or P681R mutation. The K417T or V1176F mutations associated with the Beta and Gamma VOCs were not detected. Approximately 22.2% (84/378) of samples were typeable to 1 of the lineages covered by the VirSNiP assays. According to SNP-based analyses, 14.8% (56/378) of the overall samples showed the mutation pattern of the Alpha variant, B.1.1.7, and 7.4% (28/378) of the B.1.525 variant. Frequent occurrence of the mutations under study suggests that earlier SARS-CoV-2 lineages not carrying those mutations have been replaced in Benin.We used 378 SARS-CoV-2–positive diagnostic respiratory samples tested at the reference laboratory in Benin during January 30–April 2, 2021, for genomic surveillance. All samples with cycle threshold  (Delta) . A totalhttps://www.gisaid.org; accession nos. EPI_ISL_2932532–84 and EPI_ISL_2958658–72). Lineage assignment using the Pangolin COVID-19 Lineage Assigner version 3.0.2 (https://pangolin.cog-uk.io) confirmed SNP-based lineage prediction in all 9 typeable samples selected for whole-genome sequencing (Appendix). Despite robust lineage prediction based on unambiguous SNP-based results, our data demonstrate the limited use of VirSNiP assays for strain designation; however, these assays can detect relevant mutations of currently circulating variants. The 68 Benin-derived near-complete genomes were designated to 10 unique lineages, suggesting higher genetic diversity in Benin than ≈1 year before . A 100% consensus sequence of all 68 Benin-derived sequences showed 229 nonsynonymous nucleotide substitutions across the whole genome; 57 (24.9%) occurred in the S protein (https://covariants.org/shared-mutations). The Benin-derived SARS-CoV-2 strains shared 10 unique S mutations reported in VOCs, although most of those strains were not defined as any VOC other than Alpha . Putative higher fitness mediated by genomic change was consistent with more mutations in predominant lineages than in lineages found at lower frequencies (Definite lineage designation relies on the full genome sequence. We selected 68 (9 typeable and 59 nontypeable) samples according to unique mutational patterns covering the complete period of the study for a NimaGen/Illumina-based whole-genome sequencing workflow (Appendix). All near-full genomes generated within this study were deposited into GISAID  (Appendix). Sampling was approved by the ethics committee of the Benin Ministry of Health  and of Charité-Universitätsmedizin Berlin . We compared neutralization titers with a SARS-CoV-2 strain (B.1.153) from January 2020 and the Beta strain (B.1.351), known to evade antibody-mediated neutralization (Because S mutations, individually or in combination, have been shown to afford viral escape to antibody-mediated immune responses, the high prevalence of variants with large numbers of these mutations circulating in Benin was cause for concern. To investigate whether and to what extent SARS-CoV-2 variants circulating in Benin and West Africa : an A.27lization , panel Alization , panel Blization , highligOur study is limited by patient-derived samples taken an average of 8 days after infection (Our data highlight the importance of ongoing monitoring of population immunity to emerging SARS-CoV-2 variants in Africa and of using serum specimens from local settings for phenotypic characterizations. Vaccination programs in Africa should be accelerated urgently, emphasizing the importance of global access to vaccines.Additional information about mutations associated with SARS-CoV-2 variants of concern, Benin, early 2021


PGK1 mRNA in primary human osteoblasts and hFOB1.19 osteoblastic cells. Forced overexpression of miR-4523, using a lentiviral construct, robustly decreased PGK1 3′-UTR (untranslated region) luciferase activity and downregulated its expression in human osteoblasts and hFOB1.19 cells. Furthermore, miR-4523 overexpression activated the Nrf2 signaling cascade, causing Keap1–Nrf2 disassociation, Nrf2 protein stabilization, and its nuclear translocation as well as transcription activation of Nrf2-dependent genes  in human osteoblasts. By expressing a UTR-null PGK1 construct, miR-4523 overexpression-induced Nrf2 cascade activation was however largely inhibited. Importantly, DEX-induced reactive oxygen species production, oxidative injury, and cell apoptosis were significantly attenuated by miR-4523 overexpression in human osteoblasts and hFOB1.19 cells. Such actions by miR-4523 were abolished by Nrf2 shRNA or knockout, but mimicked by PGK1 knockout (using CRISPR/Cas9 method). In PGK1 knockout human osteoblasts, miR-4523 overexpression failed to further increase Nrf2 cascade activation and offer osteoblast cytoprotection against DEX. Significantly, miR-4523 is downregulated in human necrotic femoral head tissues of DEX-taking patients. Together, PGK1 silencing by miR-4523 protected human osteoblasts from DEX through activation of the Nrf2 signaling cascade.Nuclear-factor-E2-related factor 2 (Nrf2) cascade activation can ameliorate dexamethasone (DEX)-induced oxidative injury and death in human osteoblasts. Phosphoglycerate kinase 1 (PGK1) depletion is shown to efficiently activate Nrf2 signaling by inducing methylglyoxal modification of Kelch-like ECH-associated protein 1 (Keap1). We here identified a novel PGK1-targeting microRNA: microRNA-4523 (miR-4523). RNA fluorescent in situ hybridization, RNA pull-down, and Argonaute-2 RNA immunoprecipitation results confirmed a direct binding between miR-4523 and  For patients with chronic inflammatory and auto-immune diseases, dexamethasone (DEX) and other glucocorticoids are commonly prescribed . HoweverHO1), NAD(P)H quinone oxidoreductase 1 (NQO1), γ-glutamyl cysteine ligase catalytic subunit (GCLC), and the modified subunit GCLM [Activation of nuclear-factor-E2-related factor 2 (Nrf2) cascade is essential to inhibit oxidative stress and cell injuries , 11. Stunit GCLM –18.Previous studies have discovered that phosphoglycerate kinase 1 (PGK1) is a key negative regulator of Nrf2 . PGK1 siMicroRNAs (miRNAs) are conserved small non-coding RNAs (ncRNAs) with ~21–23 nucleotides long , 7, 24. DEX, polybrene, puromycin, JC-1, and MTT were obtained from Sigma-Aldrich Chemicals . Antibodies for HO1 (#70081), NQO1 (#3187), Nrf2 (#12721), Keap1 (#8047), α-Tubulin (#2125), and Lamin B1 (#13435) as well as cleaved-poly (ADP-ribose) polymerase  and cleaved-caspase-3 (#9664) were provided by Cell Signaling Tech . The anti-GCLC antibody (ab55435) was purchased from Abcam . The anti-PGK1 (sc-130335) antibody was from Santa Cruz Biotech . Cell culturing reagents, including fetal bovine serum (FBS), Dulbecco’s Modified Eagle Medium (DMEM), Roswell Park Memorial Institute, and antibiotics, were purchased from Gibco-BRL . RNA reagents, Annexin V, CellROX, propidium iodide (PI), and cell transfection reagents (Lipofectamine 3000 and others) were all provided by Thermo-Fisher Invitrogen . All mRNA primers, viral constructs, and sequences were synthesized by Shanghai Genechem Co.  unless otherwise mentioned.hFOB1.19 human osteoblastic cells were from Dr. Cui at Nantong University and were cultured in DMEM medium plus 8% FBS . IsolatiGAPDH was tested the reference gene/internal control. Data quantification was through the 2Ct−∆∆ method. Expression of mature miR-4523 was normalized to U6 RNA. Primers were listed below: U6 mRNA forward: 5′-CTCGCTTCGGCAGCACATATACT-3′ and reverse: 5′-ACGCTTCACGAATTTGCGTGTC-3′. miR-4523 forward: 5′-GACCGAGAGGGCCTCGGC-3′ and reverse: 5′- GAACATGTCTGCGTATCTC-3′. Other primers were listed in our previous study [TRIzol reagent  was utilized to extract total RNA. cDNA was synthesized via reverse transcription by a Superscript II reverse transcriptase kit (Invitrogen). A SYBR GREEN PCR Master Mix kit was utilized for qRT-PCR assays under the ABI Prism 7900 Fast Real-Time PCR system. For data quantification, the melting temperature was calculated. The detailed procedures of Western blotting and Co-IP assays were described elsewhere , 27. DatFor overexpression, the full-length miR-4523 precursor (pre-miR-4523) sequence was synthesized by Genechem and was inserted into a GV369 lentiviral vector (Genechem). The construct, along with the lentivirus package Helper plasmids, were co-transfected to HEK-293T cells, generating pre-miR-4523-expressing lentivirus (“lv-pre-miR-4523”). The viruses were then filtered, enriched (MOI = 10), and transduced to osteoblasts (cultured in polybrene-containing complete medium). Afterwards, osteoblasts were returned back to complete medium. Puromycin (2.5 μg/mL) was added to select stably expressing cells. For miR-4523 silencing, the sequence that encodes the pre-miR-4523 antisense (antagomR-4523) was sub-cloned into the GV369 lentiviral vector to generate lv-antgomiR-4523 construct (Genechem). The construct was stably transduced to human osteoblasts similarly. The lentiviral microRNA control (“lv-miRC”) and microRNA antisense control (“lv-antaC”) were utilized as controls.Human osteoblasts or hFOB1.19 cells were initially seeded into six-well plates (at 60% confluence) and transfected with the applied miR mimic  using Lipofectamine 3000 .PGK1 3′-UTR (containing miR-4523′s putative binding sites at position 898–905) was synthesized by Shanghai Genechem. The primary human osteoblasts or hFOB1.19 osteoblastic cells were transfected with the plasmid, along with the Renillaluciferase reporter vector and pRL-SV40  [PGK1 3′-UTR luciferase reporter activity was examined through a Promega kit [Using the previously-described protocols , a firef, China)  by Lipofmega kit .5 cells/well), and transfected with an ARE-inducible firefly-luciferase vector . Cells PGK1 was generated by Genechem . The construct was transduced to primary human osteoblasts. Stable cells were selected by puromycin. PGK1 expression was verified by Western blotting assays.The GV369 lentiviral construct encoding the 3′-UTR-null PGK1 mRNA was then examined through qRT-PCR assays.RNA pull-down assay was performed by a Pierce Magnetic RNA Pull-Down Kit using the described protocols , 32. In PGK1 mRNA probes  were transduced to primary human osteoblasts or hFOB1.19 osteoblastic cells (at 37 °C for 12 h). Cells were then rinsed and observed under a fluorescence microscope .RNA-FISH was carried out through fluorescent in situ hybridization kit . In brief, FITC-labeled miR-4523 probes (in green fluorescence. RiboBio) and Cy3-labeled Primary human osteoblasts or hFOB1.19 cells were cultured in 10-cm diameter dishes and were lysed in RIP lysis buffer . Total cell extracts, 800 μg lysates in 800 μL lysis buffer per treatment, were pre-cleared and then incubated with magnetic beads conjugated with the anti-Argonaute-2 antibody  overnight. Next, the beads were washed and incubated with Proteinase K. The purified RNA was subjected to qRT-PCR analysis and normalized to the “Input” controls.The Nrf2 shRNA lentiviral particles (Santa Cruz Biotech) were added to cultured human osteoblasts (cultured in polybrene-contain complete medium). After selection by puromycin stable osteoblasts were established. Nrf2 silencing was always verified by qRT-PCR and Western blotting assays.The primary human osteoblasts were transduced with a CRISPR/Cas9 PX458-PGK1-KO-GFP construct  and washed with PBS. The representative CellROX fluorescence images were taken by Leica confocal microscope. The CellROX intensity was measured under a spectrofluorometer  at 625 nm (red fluorescence).Primary human osteoblasts or hFOB1.19 osteoblastic cells with applied genetic modifications were seeded into 96-well plates (at 4000 osteoblasts per well). A single-strand DNA (ssDNA) ELISA kit  was utilized to examine ssDNA levels, with ELISA absorbance (OD) examined at 405 nm.MTT assay of cell viability, JC-1 assaying of mitochondrial depolarization, thiobarbituric acid reactant (TBAR) assay of lipid peroxidation, apoptosis-related assays, including Annexin V-PI FACS, nuclear TUNEL staining, Histone DNA ELISA, and caspase-3/-9 activity assays, were described in detail in our previous studies , 9 and aAs described in our previous study , human nT test (Excel 2007) was utilized. P values <0.05 were considered statistically significant.All quantitative data were with normal distribution and presented as means ± standard deviation (SD). Statistical analyses were performed through the one-way analysis of variance together with the multiple comparisons with Bonferroni’s post hoc test . When comparing two treatment groups a two-tailed unpaired http://www.targetscan.org) was first consulted to predict miRNAs that can possibly bind to 3′-UTR of PGK1. Retrieved miRNAs were further verified using other miRNA databases, including miRBase, miRNAmap, and miRTarbase. Several candidate PGK1-targeting miRNAs with the context score percentage over 99% and the context++ score <−0.5 were retrieved [PGK1 was analyzed. As shown in Figure PGK1 mRNA downregulation. miR-4523 targets the 3′-UTR (position 898–905) of PGK1  mainly colocalized in the cytosol of primary human osteoblasts   Fig.  were detPGK1 mRNA in human osteoblasts, two mutant miR-4523 mimics, containing mutations at the binding sites to PGK1 3′-UTR  was transduced to lv-pre-miR-4523-expressing human osteoblasts, and it restored PGK1 expression Fig. . Importaion Fig.  were larion Fig. . Similarion Fig.  and blocion Fig.  and P inion Fig. . These rStudies have shown that DEX could induce ROS production and oxidative injury in osteoblasts , 24, 36.Furthermore, DEX induced significant mitochondrial depolarization  reduction Fig. , which wMoreover, DEX treatment induced significant apoptosis activation in lv-miRC-expressing human osteoblasts, evidenced by increases in TUNEL-positive nuclei ratio Fig.  and AnneHO1 and NQO1) . Alternatively, a CRISPR/Cas9-Nrf2-KO-GFP construct was transfected to human osteoblasts. Through FACS sorting of GFP-positive cells and Nrf2-KO screening, single stable cells (namely“koNrf2” osteoblasts) were established. As shown, lv-pre-miR-4523-induced Nrf2 protein stabilization Fig. , ARE actO1) Fig. , were alO1) Fig.  and apopO1) Fig.  were sigO1) Fig. . Nrf2 shO1) Fig. . These rSignificantly restoring PGK1 expression by the UTR-null PGK1 construct see Fig.  almost aHO1 and NQO1 mRNAs  silenced Keap1 to activate the Nrf2 signaling cascade, suppressing ultraviolet (UV) radiation-induced oxidative stress and cell death . SimilarIn human osteoblasts, however, only a few miRNAs were reported to activate Nrf2 signaling and inhibit DEX-induced oxidative injury. Liu et al.  found thPGK1 was reported to negatively regulate Nrf2 cascade activation . ConversNQO1, GCLC, and HO1) in human osteoblasts and hFOB1.19 cells. Conversely, miR-4523 silencing, via lv-antagomiR-4523, increased PGK1 3′-UTR activity and its expression in human osteoblasts. As a result, ARE activity was decreased in lv-antagomiR-4523-expressing human osteoblasts.The biological functions of miR-4523 are largely unknown. We here discovered that miR-4523 is a novel and specific PGK1-targeting miRNA. RNA-FISH, RNA pull-down, and Ago2 RNA-IP experiments demonstrated that miR-4523 directly associated PGK1 in primary human osteoblasts and hFOB1.19 osteoblastic cells. Ectopic overexpression of miR-4523 decreased PGK1 3′-UTR activity, resulting in dramatic downregulation of PGK1 mRNA and protein. Importantly, PGK1 silencing by ectopic overexpression of miR-4523 activated the Nrf2 signaling cascade, causing Keap1–Nrf2 disassociation, Nrf2 protein stabilization, and its nuclear translocation as well as transcription activation and increased expression of Nrf2-dependent genes  or KO (by CRISPR/Cas9 method) completely abolished miR-4523 overexpression-induced osteoblast cytoprotection against DEX. Significantly, restoring PGK1 expression, by the UTR-null PGK1 construct, abolished Nrf2 cascade activation by miR-4523 overexpression. Furthermore, miR-4523 overexpression-induced osteoblast cytoprotection was largely attenuated with PGK1 re-expression. Conversely, CRISPR/Cas9-induced PGK1-KO resulted in constitutive Nrf2 cascade activation and protected human osteoblasts from DEX-induced oxidative injury. Intriguingly, miR-4523 overexpression failed to offer additional cytoprotective activity against DEX in PGK1-KO human osteoblasts. These results clearly showed that PGK1 silencing and subsequent Nrf2 cascade activation should be the primary mechanism of miR-4523-induced osteoblast cytoprotection against DEX.In the present study, we found that miR-4523 levels in necrotic femoral head tissues were significantly lower than those in normal bone tissues. Further studies will be needed to explore the potential pathological mechanisms of miR-4523 downregulation by DEX. Long non-coding RNAs (LncRNAs) are transcripts exceeding 200 nucleotides –42 that PGK1 silencing by miR-4523 protected human osteoblasts from DEX through activation of the Nrf2 signaling cascade.Figure S1


Drosophila, successful expression of extremely large male fertility factor genes on Y-chromosome spanning some megabases due to their gigantic intron sizes is crucial for spermatogenesis. Expression of such extremely large genes must be challenging, but the molecular mechanism that allows it remains unknown. Here we report that a novel RNA-binding protein Maca, which contains two RNA-recognition motifs, is crucial for this process. maca null mutant male flies exhibited a failure in the spermatid individualization process during spermatogenesis, lacked mature sperm, and were completely sterile, while maca mutant female flies were fully fertile. Proteomics and transcriptome analyses revealed that both protein and mRNA abundance of the gigantic male fertility factor genes kl-2, kl-3, and kl-5 (kl genes) are significantly decreased, where the decreases of kl-2 are particularly dramatic, in maca mutant testes. Splicing of the kl-3 transcripts was also dysregulated in maca mutant testes. All these physiological and molecular phenotypes were rescued by a maca transgene in the maca mutant background. Furthermore, we found that in the control genetic background, Maca is exclusively expressed in spermatocytes in testes and enriched at Y-loop A/C in the nucleus, where the kl-5 primary transcripts are localized. Our data suggest that Maca increases transcription processivity, promotes successful splicing of gigantic introns, and/or protects transcripts from premature degradation, of the kl genes. Our study identified a novel RNA-binding protein Maca that is crucial for successful expression of the gigantic male fertility factor genes, spermatogenesis, and male fertility.During spermatogenesis, the process in which sperm for fertilization are produced from germline cells, gene expression is spatiotemporally highly regulated. In  Drosophila, extremely large male fertility factor genes on Y-chromosome containing mega-base-sized introns are expressed only during spermatogenesis, and their successful expression is crucial for spermatogenesis. However, expressing such large genes must be very challenging for cells and the molecular mechanisms that ensure their successful expression remain unknown. In this study, we identified a novel RNA-binding protein encoded in the gene CG5213 that is required for this process. We created mutant flies lacking this protein using a genome-editing technique, and investigated them using genetic, microscopic, molecular, transcriptomic, and proteomic approaches. We found that this RNA-binding protein is crucial for successful expression of the extremely large male fertility factor genes, spermatogenesis, and male fertility. We named the CG5213 gene maca, after the plant maca grown in the high Andes mountains whose root has been traditionally believed and used to increase sperm quality and promote male fertility. By identifying the novel RNA-binding protein Maca, our studies give significant insight into how the special gene expression program operates during spermatogenesis.Sperm is produced from germline cells via the process called spermatogenesis, during which a special gene expression program dedicated to this process operates. In fruit fly  Spermatogenesis is the highly conserved and tightly regulated process where diploid germline stem cells develop into mature, haploid sperm capable of fertilizing oocytes. Spatiotemporally highly regulated gene expression is crucial during spermatogenesis, and transcriptional and post-transcriptional regulation by RNA-binding proteins play key roles. Human male sterility results from abnormal spermatogenesis and is mostly due to chromosomal alterations, Y chromosome microdeletions, and related gene mutations. Mutations in the germline-specific DAZ (Deleted in AZoospermia) family of RNA-binding proteins, which contain a highly conserved RNA recognition motif (RRM) and are crucial regulators of gene expression during spermatogenesis, impair spermatogenesis and cause human male sterility –12.Drosophila has been an excellent model system for spermatogenesis studies  sodium deoxycholate, 1 mM ethylenediaminetetraacetic acid (EDTA), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)) on ice, homogenized using a pestle, and kept on ice for 30 min. The homogenates were clarified by centrifugation at 21000g at 4°C for 10 min. The protein concentration was determined using the BCA protein assay kit (Pierce) and the homogenates were diluted with RIPA buffer to final 0.5 mg/mL protein concentration. The protein homogenate quality was examined using SDS-PAGE followed by Coomassie staining. The homogenates were aliquoted into 100 uL per tube, flash-frozen in liquid nitrogen, and stored in -80°C until use.Proteins in the homogenates were reduced with 2.5 μL of 15 mg/mL Dithiothreitol (DTT) in 100 mM triethyl ammonium bicarbonate (TEAB) for 1 hr at 57°C with mixing. After the pH was adjusted to 8.0 with 500 mM TEAB, proteins were alkylated using 3 μL of 36 mg/mL Iodoacetomide for 10–15 min at room temperature in the dark. Proteins were precipitated by adding 8 x volume of TCA/acetone on ice and incubating at -20°C overnight. Protein pellets were washed once with iced acetone, dried, re-constituted in 95 μL 100 mM TEAB in 5% acetonitrile, sonicated for 20 min, and proteolyzed with 3 μg Trypsin (Pierce) at 37°C overnight. Peptides in each sample (50 μg) were labeled with Isobaric mass tags TMT16pro reagents  by adding a unique TMT 16 plex pro reagent in 20 μL acetonitrile to each sample. The TMT labeled peptide from all samples were combined into total 800 μg combined TMT-labeled peptides, aliquoted into 3 aliquots (266 μg each), and dried.Fractionation by basic reverse phase (bRP) chromatography: 1 aliquot 266 μg of combined TMT labeled dry peptides was re-constituted in 50 μl 50 mM TEAB buffer, cleaned on 500 μL Pierce Detergent Removal Spin Columns (Pierce) and eluted in 50 μL TEAB Buffer. 1950 μL 10 mM TEAB in water were added to the eluted peptides. Peptides were fractionated by basic reverse phase (bRP) chromatography using 85 min gradient from 100% solvent A (10mM TEAB in water) to 100% solvent B (10 mM TEAB in 90% acetonitrile) at a 250 μl/min flow rate on a XBridge C18 Column, 5 μm, 2.1 x 100 mm column (Waters) with a XBridge C18 Guard Column, 5 μm, 2.1 x 10 mm (Waters), using an Agilent HPLC system 1200 series binary capillary pumps. Collected 84 fractions were re-combined into 24 fractions for LC-MS/MS analysis.LC-MS/MS analysis: Peptides in 24 fractions with calculated average amount per fraction 11 μg were re-constituted in 125 μL 2% acetonitrile/0.1% formic acid and 6 μL of calculated average amount / fraction 533 ng was analyzed by nano-LC-MS/MS. Peptides were analyzed on a nano-LC-Orbitrap-Lumos-Fusion-ETD (Thermo Fisher Scientific) in FTFT, interfaced with an EasyLC1200 series using reverse-phase chromatography (2%–90% acetonitrile/0.1% formic acid gradient over 100 min at 300 nl/min) on a 75 μm x 150 mm ProntoSIL-120-5-C18 H column 3 μm, 120 Å (BISCHOFF). Eluted peptiMSs were sprayed into an Orbitrap-Lumos-Fusion mass spectrometer through a 1 μm emitter tip (New Objective) at 2.4 kV. Survey scans (Full ms) were acquired on an Orbi-trap within 375–1600 Da m/z using a Data dependent Top 15 method with dynamic exclusion of 15 s. Precursor ions were individually isolated with 0.7 Da, fragmented (MS/MS) using an HCD activation collision energy 38. Precursor and fragment ions were analyzed at a resolution of 120,000 and 60,000, respectively, and the client setting were used for the other parameters in TMT experimentMSMS data analysis: Tandem MS2 spectra  were processed by Proteome Discoverer (v2.4 ThermoFisher Scientific) using Files RC option . MS/MS spectra were searched with Mascot v.2.6.2  against RefSeq2017_83_Drosophila Melanogaster database, a small database containing enzymes, BSA. And a database containing kl3 proteins with deletions. Trypsin as an enzyme, missed cleavage 2, precursor mass tolerance 5ppm, fragment mass tolerance 0.01Da; Carbamidomethyl on C, TMT 16pro on N-terminus and on K as fixed, Oxidation on M, Deamidation on NQ as variable modifications. Peptide identifications from the Mascot searches were processed within the Proteome Discoverer2.4 and Percolator to identify peptides with a confidence threshold of 1% False Discovery Rate, based on an auto-concatenated decoy database search, and calculate the protein and peptide ratios. Only Peptide Rank 1 were considered. Peptides used for quantifications: unique; Only non-modified peptides were used for normalization. All peptides used for protein roll-up. Precursor sn>1.5, Reporter SN >3, Isolation Interference <30.Database search against RefSeq2017_83_Drosophila Melanogaster identified 6559 proteins at high, medium, and low confidence with at least 1 peptide identified at 1% FDR, Rank1 (Percolator FDR).Lysates of hand-dissected testes and tissues were prepared by homogenizing in RIPA buffer , 0.5% [w/v] sodium deoxycholate, 1 mM ethylenediaminetetraacetic acid (EDTA), 5 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)) ,57,65,66RNA from testes and whole adult flies was prepared using miRVana (Thermo Fisher Scientific) and TRIzol (Thermo Fisher Scientific), respectively. RNAs were treated with Turbo DNase (Thermo Fisher Scientific) and were reverse-transcribed into cDNA using a random hexamer primer and AMV Reverse Transcriptase (NEB). qPCR was performed using SsoAdvanced Universal SYBR Green Supermix on CFX96 (Biorad). PCR was performed using GoTaq Green Master Mix (Promega) or Phusion DNA polymerase (Thermo Fisher Scientific). The primers used are listed in 2PO4, 8.1 mM Na2HPO4, pH 7.4) on a square glass cover slip. A glass microscope slide was placed gently placed over the cover slip to squash the testes. Slides containing squashed testes were snap-frozen in liquid nitrogen and then immediately transferred to a prechilled slide rack filled with ice-cold 95% ethanol  and incubated at -20°C for 10 min. Slides were transferred to a slide rack filled with PBS containing 0.1% Triton X-100 (PBS-T) and 4% formaldehyde  and incubated at room temperature for 7 min. Slides were washed in a slide rack filled with PBS. Slides were transferred to PBS-T and incubated for 30 min at room temperature to permeabilize cell membranes. Slides were washed in PBS for 5 min at room temperature. Positions of squashed testes on slides were marked with a hydrophobic barrier pen. After 30–40 ul of primary antibody diluted in PBS-T  ) was applied to testes, the slides were incubated in a moist, dark chamber for 2 hr at room temperature. Slides were washed with PBS for 5 min at room temperature three times. After 30–40 ul of fluorophore-conjugated secondary antibody diluted in PBS , 1/100; Alexa Fluor 488 Donkey anti-mouse Igg , 1/100) was applied to testes, the slides were incubated in a moist, dark chamber for 2 hr at room temperature. After the slides were washed with PBS for 5 min at room temperature twice, 30–40 ul of VECTASHIELD with DAPI (Vector Labs) was applied to testes. Testes Images were collected with the Zeiss LSM700 confocal microscope at the Johns Hopkins University School of Medicine Microscope Facility.Testes hand-dissected from 2–5 day old flies were placed in a drop of ~5 ul of PBS , 2 mM Vanadyl Ribonucleoside complex , 0.5% BSA ) overnight at 37°C. After washed with 200 ul wash buffer three times for 20 minutes each at 37°C, testes were mounted in 30–40 ul of VECTASHIELD with DAPI (Vector Labs). During these procedures, sample tubes and slides containing testes with FISH probes were covered with aluminium foil to minimize photo-bleaching. Testes Images were collected with the Zeiss LSM700 confocal microscope at the Johns Hopkins University School of Medicine Microscope Facility. The FISH probes used are listed in Testes from 2–5 day old flies were hand-dissected in PBS and fixed in 4% formaldehyde  in PBS for 30 minutes at room temperature. Testes were washed briefly in PBS and permeabilized in 1 mL 70% ethanol overnight on a rocker in a 4°C cold room. After rinsed with 300 ul wash buffer , 10% formamide), testes were hybridized with 100 uL of 50 nM (for satellite repeat. Integrated DNA Technologies) or 100 nM  probes in hybridization buffer , 1 mg/mL N-terminally EGFP-fused Maca and mCherry coding sequences were inserted in pAc5.1B/V5-HisB plasmid. S2 cells were co-transfected with these two plasmids using Effectene transfection reagent kit . Three days after transfection, cells were transferred on a glass slide in a 6 well plate and were grown overnight. After the cell medium was removed, cells were fixed with 1 ml 4% formaldehyde in PBS at room temperature for 30 min. After washed with 1 ml PBS, cells were mounted on glass slides with 40 ul of VECTASHIELD with DAPI (Vector Labs). Images were acquired using the Zeiss LSM700 confocal microscope at the Johns Hopkins University School of Medicine Microscope Facility.Poly-A+ mRNA purification was performed as previously reported. small RS1 FigOregonR wild-type virgin females are shown. Mean +/- SD . P-value <0.05 (Student’s t-test) are indicated by *. Trans-heterozygous mutant flies macanull/Df and macaR1/Df have the macanull or macaR1 allele and the Df(3R)Exel6174 chromosomal deficiency allele uncovering the maca gene.  Female fertility assay. (B) Numbers of eggs laid by test virgin females crossed with OregonR wild-type males and (C) hatching rates from the eggs. Mean +/- SD . P-value <0.05 (Student’s t-test) are indicated by *.(A) Male fertility assay. The numbers of the progeny flies obtained from the crosses between test males and (TIF)Click here for additional data file.S2 Figdj-GFP background. DJ-GFP (green), DAPI (blue). DJ-GFP is expressed in sperm. The apical tip of testis is marked by *. Seminal vesicles are indicated by white dashed line. Scale bars are 100 μm.Confocal imaging of whole testes and seminal vesicles from indicated genotypes in the (TIF)Click here for additional data file.S3 FigConfocal imaging of S2 cells transiently expressing EGFP-Maca. mCherry signal marks the cytoplasm while DAPI signal marks the nucleus. EGFP-Maca resides in the nucleus. Scale bar is 5 μm.(TIF)Click here for additional data file.S4 Fig2(fold-change) and -log10 of protein abundance in macanull/null testes compared with macanull/+ testes determined by mass-spec. Light blue: Proteins with adjusted P-value (= FDR) <0.05 and fold-change < 1 in macanull/null testes compared with macanull/+. Dark blue: Proteins with adjusted P-value <0.05 and fold-change > 1 in macanull/null testes compared with maca+/+, macanull/+, and macanull/null with maca-EGFP rescue transgene, consistently. Full list shown in macanull/null testes compared with macanull/+. Red: Proteins with adjusted P-value <0.05 and fold-change > 1 in macanull/null testes compared with maca+/+, macanull/+, and macanull/null with maca-EGFP rescue transgene, consistently. Full list shown in macanull/null testes compared with macanull/+. (B) Volcano plot showing log2(fold-change) and -log10 of each mRNA in macanull/null testes compared with macanull/+ testes determined by poly-A+ RNA-seq. Light blue: Proteins with adjusted P-value (= FDR) <0.01 and fold-change < 1 in macanull/null testes compared with macanull/+. Dark blue: Proteins with adjusted P-value <0.01 and fold-change > 1 in macanull/null testes compared with maca+/+, macanull/+, macanull/+ with maca-EGFP rescue transgene, and macanull/null with maca-EGFP rescue transgene, consistently. Full list shown in macanull/null testes compared with macanull/+. Red: Proteins with adjusted P-value <0.01 and fold-change > 1 in macanull/null testes compared with maca+/+, macanull/+, macanull/+ with maca-EGFP rescue transgene, and macanull/null with maca-EGFP rescue transgene, consistently. Full list shown in macanull/null testes compared with macanull/+. kl-2, kl-3, kl-5, JYalpha, CG5399, and CG31213, which exhibit significant dysregulation in both protein and mRNA levels in macanull/null testes compared with all the other genotypes consistently, are labeled.(A) Volcano plot showing log(TIF)Click here for additional data file.S5 FigScattered plots of normalized number of reads (reads per million non-rRNA-mapping genome-mapping reads) of (A) miRNAs and endo-siRNA  and (B) sense and antisense piRNAs in testis small RNA-seq. Each dot represents a unique miRNA, endo-siRNA, or piRNA cluster (transposon-sense or transposon-antisense mapping). Mean +/- SD .(TIF)Click here for additional data file.S6 Figkl-2 gene region. Gene structures are shown with exons (cyan), introns (black line), intronic satellite DNA repeats (dashed line in parentheses). Regions targeted by RNA FISH probes used in Figs Poly-A+ RNA-seq normalized read counts  in the (TIF)Click here for additional data file.S7 Figkl-3 gene region. Splicing ratios among (1) exon 12—exon 13, (2) exon 13—exon 14, and (3) exon 12—exon 14 (= exon 13-skipped) in determined by LeafCutter are shown. Gene structures are shown with exons (cyan), introns (black line), intronic satellite DNA repeats (dashed line in parentheses). Regions targeted by RNA FISH probes used in Figs Poly-A+ RNA-seq normalized read counts  in the (TIF)Click here for additional data file.S8 Figkl-5 gene region. Gene structures are shown with exons (cyan), introns (black line), intronic satellite DNA repeats (dashed line in parentheses). Regions targeted by RNA FISH probes used in Poly-A+ RNA-seq normalized read counts  in the (TIF)Click here for additional data file.S9 Figks-1 (ory) mRNA determined by poly-A+ RNA-seq. Mean +/- SD . Adjusted P-values <0.01 are indicated by *. (C) poly-A+ RNA-seq normalized read counts  in the ks-1 (ory) gene region. Gene structures are shown with exons (cyan), introns (black line), intronic satellite DNA repeats (dashed line in parentheses).(A) Relative abundance of Ks-1 (ORY) protein in testes determined by mass spec with TMT labeling. Mean +/- SD . Adjusted P-values <0.05 are indicated by *. (B) Normalized counts (reflecting relative abundance) of (TIF)Click here for additional data file.S10 Figkl-2, (B) kl-3, and (C) kl-5 transcripts in late-stage spermatocytes. Single spermatocyte nuclei (white dashed line) at stage 4 of their transcript expression. Scale bars are 5 μm. Cytoplasmic mRNA granules (kl-granules) are indicated by cyan arrows. (A) kl-2 introns 1–2 (green), exon 8 (red), and DAPI (white). (B) kl-3 exon 1 (blue), introns 6, green), exon 14 (red), and DAPI (white). (C) kl-5 exons 1–6 (blue), introns 6, green), exons 16–17 (red), and DAPI (white).RNA FISH to visualize (A) (TIF)Click here for additional data file.S11 FigRNAikl-2, RNAikl-3, and RNAikl-5. Scale bars are 50 μm. (B) Confocal images of the early-stage individualization complexes. Phalloidin , DAPI (blue). Scale bars are 10 μm. (C) Confocal images of the late-stage individualization complexes. Phalloidin . Scale bars are 10 μm.(A) Confocal images of dissected seminal vesicles stained with DAPI. Motile sperm is absent in (TIF)Click here for additional data file.S12 Figkl-3 mRNA exon 13 skipping and alternative splicing of (B) sxl, (C) tra, and (D) msl2 mRNAs. Whole fly total RNAs were used for RT-PCR.Agarose gel electrophoresis of RT-PCR products to test (A) (TIF)Click here for additional data file.S13 Figkl-3 transcripts expression in  macanull/+ and  macanull/null testes. Exon 1 (blue), Y-loop B 6, green), exon 14 (red), and DAPI (white).  Apical regions of testes including the spermatocyte growth region are shown. The apical tip of testis is marked by *. Scale bars are 50 μm.  Single spermatocyte nuclei (white dashed line) at each stage of kl-3 expression. Cytoplasmic mRNA granules are indicated by cyan arrows. Scale bars are 5 μm.RNA FISH to visualize (TIF)Click here for additional data file.S14 Figkl-5 transcripts expression in  macanull/+ and  macanull/null testes. Exons 1–6 (blue), Y-loop A/C 6, green), exons 16–17 (red), and DAPI (white).  Apical regions of testes including the spermatocyte growth region are shown. The apical tip of testis is marked by *. Scale bars are 50 μm.  Single spermatocyte nuclei (white dashed line) at each stage of kl-5 expression. Cytoplasmic mRNA granules are indicated by cyan arrows. Scale bars are 5 μm.RNA FISH to visualize (TIF)Click here for additional data file.S15 Figmaca-EGFP testis. Maca-EGFP (green), Y-loop A/C (Cy5-(AAGAC)6, blue), Y-loop B 6, red), and DAPI (white). (A) Apical regions of testes including spermatocyte growth region are shown. The apical tip of testis is marked by *. Scale bar is 20 μm. (B) Single spermatocyte nuclei (white dashed line) at late stage. Scale bar is 5 μm.  RNA FISH to visualize Y-loop A/C and kl-2 and kl-3 transcripts in control testis. Single spermatocyte nuclei (white dashed line) at late stage. Scale bar is 5 μm. (C) Y-loop A/C (Cy5-(AAGAC)6, green), kl-2 introns (blue), kl-2 exon 8 (red), and DAPI (white). (D) Y-loop A/C (Cy5-(AAGAC)6, green), kl-3 exon 1 (blue), kl-3 exons 16–17 (red), and DAPI (white). (E-L) RNA FISH to visualize kl-2, kl-3, kl-5, and ks-1 transcripts in maca-EGFP testis. Single spermatocyte nuclei (white dashed line) at late stage. Scale bar is 5 μm. (E) Maca-EGFP (green), kl-2 introns (blue), kl-2 exon 8 (red), and DAPI (white). (F) Maca-EGFP (green), kl-3 exon 1 (blue), kl-3 exon 14 (red), and DAPI (white). (G) Maca-EGFP (green), kl-3 exon 13 (blue), kl-3 exon 14 (red), and DAPI (white). (H) Maca-EGFP (green), kl-5 exons 1–6 (blue), kl-5 exons 16–17 (red), and DAPI (white). (I) Maca-EGFP (green), kl-5 exons 1–6 (blue), ks-1 exons all (red), and DAPI (white). (J) Maca-EGFP (green), Y-loop B 6, blue), kl-3 exon 14 (red), and DAPI (white). (K) Maca-EGFP (green), kl-5 exons 1–6 (blue), Y-loop A/C (Cy3-(AAGAC)6, red), and DAPI (white). (L) Maca-EGFP (green), ks-1 exons all (blue), Y-loop A/C (Cy3-(AAGAC)6, red), and DAPI (white). RNA FISH to visualize Y-loops in (TIF)Click here for additional data file.S16 Figmacanull/+ and  macanull/null testes. Y-loop A/C (Cy3-(AAGAC)6, red), Y-loop B 6, green), and DAPI (blue).  Apical regions of testes including spermatocyte growth region. The apical tip of testis is marked by *. Scale bar is 50 μm.  Single spermatocyte nuclei (white dashed line) at late stage. Scale bar is 5 μm.  RNA FISH to visualize Y-loop A/C and kl-5 and ks-1 transcripts in (E) macanull/+ and (F) macanull/null testes. Y-loop A/C (Alexa488-(AAGAC)6, green), kl-5 exons 1–6 (blue), ks-1 exons all (red), and DAPI (white). Single spermatocyte nuclei (white dashed line) at late stage. Cytoplasmic kl-5 and ks-1 mRNA granules are indicated by cyan and magenta arrows, respectively. Scale bar is 5 μm.(A-D) RNA FISH to visualize Y-loops in  (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


H84T-Banana Lectin (BanLec) CAR-NK cells bind high mannose glycosites that decorate the SARS-CoV-2 envelope, thereby decreasing cellular infection in a model of SARS-CoV-2. H84T-BanLec CAR-NK cells are innate effector cells, activated by virus. This novel cellular agent is a promising therapeutic, capable of clearing circulating SARS-CoV-2 virus and infected cells. Banana Lectin (BanLec) binds high mannose glycans on viral envelopes, exerting an anti-viral effect. A point mutation (H84T) divorces BanLec mitogenicity from antiviral activity. SARS-CoV-2 contains high mannose glycosites in proximity to the receptor binding domain of the envelope Spike (S) protein. We designed a chimeric antigen receptor (CAR) that incorporates H84T-BanLec as the extracellular moiety. Our H84T-BanLec CAR was devised to specifically direct NK cell binding of SARS-CoV-2 envelope glycosites to promote viral clearance. The H84T-BanLec CAR was stably expressed at high density on primary human NK cells during two weeks of ex vivo expansion. H84T-BanLec CAR-NK cells reduced S-protein pseudotyped lentiviral infection of 293T cells expressing ACE2, the receptor for SARS-CoV-2. NK cells were activated to secrete inflammatory cytokines when in culture with virally infected cells. H84T-BanLec CAR-NK cells are a promising cell therapy for further testing against wild-type SARS-CoV-2 virus in models of SARS-CoV-2 infection. They may represent a viable off-the-shelf immunotherapy for patients suffering from COVID-19. The prevalence and pathogenicity of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused an international pandemic that has placed healthcare systems under unprecedented stress. Lack of prior exposure and a high fatality rate of coronavirus disease 2019 (COVID-19) , the infvia an extracellular single-chain variable fragment specific to a protein epitope  (via a single amino acid change (H84T) (The SARS-CoV-2 virus mediates cell entry 2 (ACE2) , 25. The2 (ACE2) –28. In t2 (ACE2) . These g2 (ACE2) , 30. The2 (ACE2) , but wil2 (ACE2) . BanLec e (H84T) . H84T-Bae (H84T) –34. Howee (H84T) . ConsequWe sought to improve H84T-BanLec antiviral activity by combining its exquisite viral targeting and the innate activity of primary NK cells. We designed a H84T-BanLec CAR containing the 4-1BB (CD137) and TCRζ intracellular domains and expressed this artificial chimeric receptor at the NK cell membrane. H84T-BanLec chimeric antigen receptor natural killer (CAR-NK) cells diminished the potency of lentivirus pseudotyped with a SARS-CoV-2 spike protein (S-protein) envelope. Binding of target stimulated NK cell activation, likely driven initially by innate antiviral cell responses, and then boosted by CAR molecular signaling. Glycosylation-directed CAR-NK cells such as H84T-BanLec CAR-NK are activated against SARS-CoV-2 and have the potential to blunt viral infection, potentially through clearance of virally-infected cells or prevention of viral entry.We synthesized the H84T-banana lectin (H84T-BanLec)  sequenceIn order to model SARS-CoV-2 infection, we engineered 293T cells to constitutively express the ACE2 transmembrane protein , 39. We We used SARS-CoV-2 envelope pseudotyping of a replication deficient lentiviral vector  in orderWe next investigated whether H84T-BanLec CAR-NK cells could specifically target S-protein pseudoviral transduced hACE2.293T. NK cells (CAR-NKs or unmodified) were plated with hACE2.293T and freely circulating pseudoviral particles  NK cells are decreased during severe COVID-19  into a pCDH lentiviral backbone . Vesicular stomatitis virus G glycoprotein (VSV-G) Pseudotyped HIV-derived lentiviral particles were then produced using the pPACKH1 HIV Lentivector Packaging Kit  according to the manufacturer’s instructions and used for 293T.hACE2 cell generation. Cells with high ACE2 expression, as validated with flow cytometry, were then isolated via fluorescence-activated cell sorting (FACS). hACE2.293T cells used for cytotoxicity analysis were additionally modified with retroviral vectors to express an enhanced green fluorescent protein (GFP) firefly luciferase fusion gene (GFP.ffLuc)  and grown in Dulbecco’s Modified Eagle Medium , supplemented with 10% Fetal Bovine Serum . High-expressing human ACE2 (hACE2) 293T cells were created by first subcloning hACE2  . GFP-posThe sequence of H84T-BanLec  was syntPeripheral blood mononuclear cells were isolated from healthy donor leucopaks . T cells were then depleted with CD3-microbeads . CD3+ cell depletion was verified with flow cytometry using Phycoerythrin (PE)-conjugated anti CD3  and Brilliant Violet (BV)421- conjugated anti-CD56  antibodies  at a 1:1-ΔCt method . Parental 293T cells served as a control for nonspecific cell transduction. On Day 0, S-pseudoviral titrations  were added and the plate was centrifuged at 800g for 30’ at 32°C. Cells were then incubated at 37°C in 5%CO2. At 48h post-transduction, the viral-containing supernatant was aspirated and 150ug/ml D-Luciferin containing fresh media added. BL was measured and reactive light units (RLU) determined after subtraction of virus-only background. For co-culture assays containing NK cells and pseudovirus transduced target cells, pseudoviral particles were titrated first on hACE2.293T cells and added to achieve 100-200 maximum RLU. NK cells were added to the hACE2.293T and pseudovirus immediately after centrifugation. BL change was calculated as 100*(baseline BLI – co-culture BLI)/(baseline BLI - background). Baseline BL was measured from wells containing only hACE2.293T and pseudovirus. The assay was performed in experimental triplicate per donor.SARS-CoV-2 S-protein pseudotyped replication incompetent lentiviral particles were produced by first transfecting 293T with GeneJuice transfection reagent (Millipore-Sigma) and SARS-Related Coronavirus 2, Wuhan-Hu-1 Spike-Pseudotyped Lentiviral Kit . CAR expression on the surface of transduced NK cells was evaluated 4 and 14 days post-transduction using primary staining with H84T.BanLec Ab  and secoHistidine (His)-tagged recombinant S-proteins: trimera (SPN-C52H9) and D614G trimera (SPN-C52H3) were purchased from ACROBiosystems . A His-tagged receptor binding domain (RBD) was purchased from R&D Systems . 293T, hACE2.293T, NK cells, and H84T-BanLec CAR-NK cells were coated with 50ng of recombinant protein, then stained first with His antibody (R&D Systems), followed by PE-anti-mouse F(ab)2 (R&D Systems) and analyzed using flow cytometry.NK cells were lysed in RIPA lysis buffer with protease (cOmplete) and phosphatase (PhosSTOP) inhibitor cocktails  on ice. Protein quantification was performed using Pierce BCA protein assay kit  and iMark plate reader (Bio-Rad). Electrophoresis was conducted using Novex WedgeWell 10% Bis-Tris Mini Gels (Thermo Fisher) and protein transferred to polyvinylidene difluoride (PVDF) membrane. Western blot analysis was performed with the following antibodies: mouse anti-human CD247 , mouse anti-human phosphorylated CD247 , and rabbit anti-human H84T.BanLec Ab . MembranNK cells were cultured with SARS-CoV-2-pseudovirus transduced hACE2.293T at a 1:1 ratio. Following 48 h of culture, supernatant was harvested and analyzed for interferon (IFN)γ or tumor necrosis factor (TNF)α using enzyme-linked immunosorbent assay (ELISA) kits  according to the manufacturer’s instruction. Conditions without pseudoviral particles were included as controls.NK cells were co-cultured for 48h with hACE2.293T.ffLuc at the indicated effector:target (E/T) ratios. D-luciferin was added to plate and BL measured per well. Mean percentage of specific lysis of triplicate samples was calculated as 100*/(spontaneous death-background). Spontaneous death was measured with control wells containing only target cells.All analyses were performed using GraphPad Prism Software (v9). For comparisons of 2 groups, unpaired two-tailed t tests with Welch correction were used.The original contributions presented in the study are included in the article/CB and IC conceived of the work. CB, DM, and IC developed the methodology. CB, IC, SZ, RR, JS, AM, and JR acquired, analyzed, and interpreted data. CB, IC, and WR wrote and all authors revised the manuscript. All authors contributed to the article and approved the submitted version.CB, IC, and DM have pending patent applications describing the use of H84T-BanLec and H84T-BanLec effector cell targeting of SARS-CoV-2.The remaining 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.


Pulmonary function progressively declines with aging. Forced expiratory volume 1-second (FEV1) and forced vital capacity (FVC) are predictors of morbidity of cardiovascular diseases and all-cause mortality. Reduced pulmonary function has shown association with elevated chronic low-grade systemic inflammation and low muscle strength, and glycosylated hemoglobin (HbA1c). We evaluated whether FEV1 and FVC share common genetic factors with interleukin-6 (IL-6), high-sensitivity C-reactive protein (hsCRP), body mass index (BMI), handgrip strength, plasma glucose, and HbA1c employing correlated meta-analysis (CMA) in up to 3888 individuals (age range: 26-106). CMA tests whether combining genome-wide association (GWA) P-values from two or more traits enhances the ability to detect variants concomitantly influencing such traits. We considered a variant pleiotropic if the univariate GWA P≤1.0x10-02 and CMA P≤5.0x10-08. We identified pleiotropic loci for FEV1 (or FVC), IL-6 and hsCRP within CYCS, CALN1, TRIM9, AXIN2, MICAL3, and CMIP; FEV1 (or FVC) and BMI within CYP2U1, LINC00871-RPL10L-MDGA2, and LOC105372472; FEV1 (or FVC) and grip strength within XXYLT1 and MAL2; FEV1 and FPG within MAF; and FVC and HbA1c within HDHD3. Some loci were reported as GWAS suggestive (P<10-6) for pulmonary function. Additionally, the identified loci harbor genes with roles in pro-inflammatory cytokine production, immune and inflammatory pathway, T-helper signaling pathway, transforming growth factor-beta, and T-cell receptor-alpha enhancer. Our findings suggest that inflammation is a feature of reduced pulmonary function with IL-6, hsCRP, BMI, low muscle strength, glucose, and HbA1c. Pleiotropic genetic associations across these traits may explain part of the correlated genetic architecture between pulmonary function and aging-related traits.