However, whether post-developmental enhancement of collagen expression could also increase the lifespan is unknown. Recently, we described a method to induce the expression of a target gene using catalytically dead Cas9 (dCas9)-engineeredC. elegansvia ingestion of bacteria expressing a pair of promoter-specific single guide RNAs (sgRNA). Here, we clonedcol-120promoter-specific sgRNA oligo pair into L4440-Biobrick-sgRNA and fed these bacteria to dCas9::VP64 transgenicC. elegans. We observed a similar percentage of lifespan extension by post-developmentally dCas9-induced expression ofcol-120, as previously reported through transgenic overexpression ofcol-120. Consistent with this result is that induction of another previously shown longevity-promoting collagen,col-10,also increased lifespan. Furthermore, we found an enhanced resilience to heat stress and increased expression ofhsp-16.2upon dCas9-activatedcol-120expression. Together, these results provide an orthogonal method to validate longevity by enhancingcol-120expression and point towards a potential role of collagen enhancement in thermotolerance.Transgenic overexpression of collagen C. elegans,there are 181 collagens, categorized as cuticular and basement membrane collagens . Upon longevity-promoting interventions, such as reduced insulin/IGF-1 receptor signaling (rIIS), some key collagens, includingcol-120andcol-10, showed prolonged expression during aging . Both collagens,col-120andcol-10,are expressed during larval development, with the highest expression at L4 but show a rapid decline in expression in young adults . This progressive and age-associated decline ofcol-120mRNA and protein levels is delayed by rIIS . Moreover, transgenic overexpression of collagencol-120orcol-10is sufficient to increase the lifespan ofC. elegans . However, whether post-developmental enhancement ofcol-120orcol-10is sufficient to increase lifespan is unknown.Collagens form an extensive part of the extracellular matrix in all metazoansC. elegans, such as using a heat-shock inducible promoter or a Tetracycline-controlled transcription system (Tet/Q) . Yet, repeated heat shocks or tetracycline-analog doxycycline used for induction of the Tet/Q hybrid system can increase the lifespan ofC. elegans. To overcome this with an orthogonal method, we employed a recently developed technique of feeding bacteria containing promoter-specific sgRNA for inducing the transcriptional expression of the endogenous target genes in a transgenic dCas9::VP64C. elegans  . The pair of sgRNAs were cloned in L4440_BioBrick-sgRNA plasmid to induce expression inE. coli. Similar to RNAi, sgRNA production is induced by ITPG . The simple cloning of specific sgRNAs and inducing gene transcription through feeding bacteria expressing the sgRNAs makes this technique easy to adapt and feasible, similar to RNAi methods.Several methods exist to force gene expression in.2022) . In bri.2022) . The taet al.(2022), we cloned thecol-120(3+4 sgRNA pair) orcol-10 promoter-specific sgRNA oligo sequences in two consecutive sgRNA cassettes driven by T7 promoter with BioBrick flanking region in the L4440_BioBrick-sgRNA vector. The construct was transformed intoHT115E. colibacteria, and through the bacterial delivery of the sgRNA to the modified dCas9::VP64C. elegansstrain, the expression ofcol-120orcol-10was induced . Therefore, we asked whether higher collagen expression levels could be protective against heat stress. To address this, we fedcol-120sgRNA bacteria to dCas9 transgenic animals that drive GFP expression under the heat-shock promoterhsp-16.2(Phsp-16.2::GFP), placed them for two hours at 33 °C, and let them recover for six hours at 20 °C before quantifying GFP fluorescence. We found increased levels ofhsp-16.2by dCas9-activated expression ofcol-120 plates at 20 °C. MIR249,risIs33[K03A1.5p::3xFLAG::SV40-NLS::dCas9::SV40-NLS::VP64::HA +unc-119(+)] and MIR276,risIs33[K03A1.5p::3xFLAG::SV40-NLS::dCas9::SV40-NLS::VP64::HA +unc-119(+)];gpIs1[hsp-16.2p::GFP] .E. colistrains used in this study areOP50(streptomycin resistance) for general maintenance ofC. elegans,HT115(DE3) (ampicillin and tetracycline resistance) for inducing the target gene sgRNA, and DH5α (ampicillin resistance) for cloning the target gene sgRNA plasmid construct.OP50seeded plates were prepared by spottingOP50culture grown overnight at 37 °C from a single colony in LB broth with streptomycin antibiotic (100 µg/mL). sgRNA constructs containingHT115bacteria primary culture were prepared by inoculating a single colony in 5 mL LB broth with ampicillin (50 µg/mL) and tetracycline (12.5 µg/mL) antibiotic and incubated at 37 °C overnight. The next day, 25 mL of LB broth with ampicillin (50 µg/mL) was added to the primary culture and let it grow at 37 °C for 3 h. Cultures were then concentrated for 15 min at 3500 rpm at room temperature. The pellet was resuspended in 5 mL LB broth with ampicillin (50 µg/mL) and Isopropyl-β-D-thiogalactopyranoside (IPTG) (3 mM), and the homogenous mix was spotted on the NGM plates. Plates were dried for one day before use.col-10andcol-120sgRNAsCloning ofcol-10andcol-120promoter-specific sgRNA target sequences were designed according to the stringent rules described in Fischeret al.(2022). Briefly, six best 20 nucleotide sequences flanked with NGG (sgRNA) were chosen from 50 to 400 bp region upstream of the TSS (Transcription start site) of thecol-10orcol-120gene, with >50 off-target scores and numbered based on their relative distance from the respective TSS, with sgRNA 1 being closest and 6 being furthest away from the TSS. Scr (scrambled) sgRNA sequences were randomly chosen 20 nucleotides with ≥50 % GC content that did not match toC. elegansgenome upon BLAST.col-10 orcol-120(3+4) promoter-specific sgRNA oligo pairs were successfully cloned into the L4440_BioBrick-sgRNA vector. Annealed oligos with appropriate overhangs of BbsI and BsaI restriction sites were ligated to the vector with BbsI and BsaI sites. Ampicillin antibiotic resistance was the selection pressure for positive ligated colonies.E. coliDH5α cells were used as the cloning host, andHT115(DE3) bacteria were used for selecting and propagating the positive clone.Primers for theLifespan assayOP50-seeded NGM plates  until the L4 stage. 30-40 L4 animals were then transferred to scr or sgRNAcol-10orcol-120HT115bacteria seeded NGM plates in replicates of five. 120 µL FUdR  was top coated onto these plates. Survival of animals was assessed by gently prodding their posterior end with platinum wire to score live or dead. Unhealthy animals with vulval rupture and those crawling on the sides or under the agar were censored from the population throughout lifespan scoring. Lifespan Scoring was started at day 7 of adulthood, L4 being taken as t=0 timepoint. Survival curves were plotted using GraphPad Prism 9.2.0. Statistical analysis was performed using OASIS 2.0 software online. Lifespan summary for individual experiments is provided in Extended Data.After hypochlorite treatment, eggs obtained from gravid MIR249 adults were maintained onFluorescence Microscopycol-120HT115bacteria seeded NGM plate until young adult. Young adults were heat-shocked at 33 °C for 2 h. After recovery from shock for 6 h at 20 °C, approx. 30 animals were mounted onto a 2 % agarose pad and anesthetized with 20 mM levamisole for imaginget al.2021). Statistical analysis of the quantified data was done using GraphPad Prism software (GraphPad Prism 9.2.0). The experiment was performed in three biological batches, with n>80 total animals per condition.Gravid MIR276 adults were treated with sodium hypochlorite solution to obtain eggs. The eggs were grown on scr or sgRNAHeat resistance survival assayOP50-seeded NGM plates until the L4 stage. L4 animals were fed with scr and sgRNAcol-120HT115bacteria until adulthood day 3 at 20 °C. Day 3 animals were shifted to 35 °C to initiate heat stress. Survival of the animals was scored every 2-3 h by gently prodding their tails with a platinum wire. Survival curves were plotted using GraphPad Prism 9.2.0. Statistical analysis was performed using OASIS 2.0 software online. Survival summary for individual experiments is provided in Extended Data.MIR249 (or MIR276 forcol-120gene expression by qRT-PCRcol-120sgRNA and scr until young adult (YA) or grown onOP50till L4 and then fed withcol-120sgRNA and scr until day 3 of adulthood. Animals were then collected at YA and Day 3 for RNA isolation. RNA isolation was done using Trizol (Ambion #15596018) method and purified by RNA clean and concentrator kit (Zymo Research #R1017) with in-column DNase (Ambion #AM2222) digestion. 1 µg of RNA was used for first strand cDNA synthesis (SuperScript III by Invitrogen #11752-250) according to the manufacturer’s instructions. Gene expression levels were determined by quantitative real-time PCR (qRT-PCR) using the PowerUp SYBR Green MasterMix (Applied biosystems by Thermo Fischer Scientific #A25742) and QuantStudio PCR system (Applied Biosystems by Thermo Fischer Scientific) according to the manufacturer’s specifications. Relative gene expression, normalized to the geometric mean of two housekeeping genescdc-42andpmp-3, was determined between animals treated withcol-120sgRNA and scr. Statistical analysis was performed using GraphPad Prism 9.2.0. All the primers used are listed in Extended Data.Gravid MIR249 adults were treated with hypochlorite solution to obtain eggs. Eggs were either fed withTRizol reagent (Ambion by Life Technologies) #15596018RNA clean and concentrator kit (Zymo Research #R1017)DNase I (RNase-Free) (Ambion #AM2222)SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen #11752-250)PowerUp SYBR Green MasterMix (Applied biosystems by Thermo Fischer Scientific #A25742)10.22002/d1ffs-a0b42Description: Extended Data File. Resource Type: Dataset. DOI:


To counteract diverse pathogens, vertebrates evolved adaptive immunity to generate diverse antibody repertoires through a B lymphocyte-specific somatic gene rearrangement process termed V(D)J recombination. Tight regulation of the V(D)J recombination process is vital to generating antibody diversity and preventing off-target activities that can predispose the oncogenic translocations. Recent studies have demonstrated V(D)J rearrangement is driven by cohesin-mediated chromatin loop extrusion, a process that establishes genomic loop domains by extruding chromatin, predominantly, between convergently oriented CTCF looping factor-binding elements (CBEs). By deleting and inverting CBEs within a critical antibody heavy chain gene locus developmental control region and a loop extrusion chromatin-anchor at the downstream end of this locus, we reveal how these elements developmentally contribute to generation of diverse antibody repertoires. H, D, and JH gene segments located in separate clusters across the Igh locus. RAG endonuclease initiates V(D)J recombination from a JH-based recombination center (RC). Cohesin-mediated extrusion of upstream chromatin past RC-bound RAG presents Ds for joining to JHs to form a DJH-RC. Igh has a provocative number and organization of CTCF-binding elements (CBEs) that can impede loop extrusion. Thus, Igh has two divergently oriented CBEs (CBE1 and CBE2) in the IGCR1 element between the VH and D/JH domains, over 100 CBEs across the VH domain convergent to CBE1, and 10 clustered 3′Igh-CBEs convergent to CBE2 and VH CBEs. IGCR1 CBEs segregate D/JH and VH domains by impeding loop extrusion-mediated RAG-scanning. Downregulation of WAPL, a cohesin unloader, in progenitor-B cells neutralizes CBEs, allowing DJH-RC-bound RAG to scan the VH domain and perform VH-to-DJH rearrangements. To elucidate potential roles of IGCR1-based CBEs and 3′Igh-CBEs in regulating RAG-scanning and elucidate the mechanism of the ordered transition from D-to-JH to VH-to-DJH recombination, we tested effects of inverting and/or deleting IGCR1 or 3′Igh-CBEs in mice and/or progenitor-B cell lines. These studies revealed that normal IGCR1 CBE orientation augments RAG-scanning impediment activity and suggest that 3′Igh-CBEs reinforce ability of the RC to function as a dynamic loop extrusion impediment to promote optimal RAG scanning activity. Finally, our findings indicate that ordered V(D)J recombination can be explained by a gradual WAPL downregulation mechanism in progenitor-B cells as opposed to a strict developmental switch.Immunoglobulin heavy chain variable region exons are assembled in progenitor-B cells, from V H, D, and JH gene segments  (Igh) spans 2.7 megabases (Mbs) on chromosome 12 with over 100 VHs interspersed within a several Mb distal portion  (Igh V(D)J recombination from a recombination center (RC) formed within highly transcribed chromatin that spans DQ52, the four JHs, and the intronic enhancer (iEμ) J exon -B cells from germline Vsegments . V(D)J rsegments . RAG ints (RSSs) . RSSs cos (RSSs) . After Rs (RSSs) . The mouIgh contains numerous CBEs with remarkable relative orientation. The IGCR1 element, which lies just upstream of the most distal D segment (DFL16.1) has two divergently oriented CBEs, namely upstream CBE1 and downstream CBE2 , in an orientation-specific manner , 7. In t1218eam CBE2 . The lonCR1 CBE1 . Ten conIgh CBEs , 28 lie ain CBEs , 26. Thiain CBEs , 29–31.Igh locus for substrates  can be viably arrested in the G1-cell cycle phase in which V(D)J recombination occurs -based studies revealed the V-B cells –42. VH ll access . Recent -B cells . In this-B cells –47. WAPLH domain . Abelsonn occurs . IntroduD domain , 22, 24. joining , 22, 24.ated CBE . In thiscus CBEs , 22. Neustal VHs , 22. ThuIgh CBEs during V(D)J recombination, many questions remain. While an early study indicated that IGCR1 CBEs act synergistically to segregate the VH domain from the D/JH domain (Igh CBEs confine Igh class switch recombination (CSR) activity to the Igh J-Seq (+CD43highIgM− pro-B cells from BM of wild-type (WT) 129SV controls and previously generated IGCR1/CBE1&2−/− mice  (H4 bait primer was used for HTGTS-V(D)J-Seq analyses in this study to eliminate potential confounding effects of rearrangements within extrachromosomal deletion products . Such analyses are useful for examining effects of potential regulatory element mutations. For example, finding a decrease in total reads between two samples in the absence of differences in the junction profile, reflects decreased RAG or RC activity without changes in long range scanning patterns (To gain insight into factors that determine primary bone marrow (BM) pro-B cell V(D)J-Seq . For the−/− mice . We perfable S1) . A JH4 bproducts . For thepatterns .H locus, which results in highly reproducible utilization of the different VHs (H5-2 (“VH81X”) and three immediately upstream VHs with RSS-associated CBEs are much more highly utilized than any VHs further upstream JH rearranged alleles (Inset), likely reflecting steady-state distributions within pro-B cells entering the compartment and successively generating DJH and VH-to-DJH rearrangements before leaving the compartment. Inactivation of IGCR1 by mutational inactivation of CBE1 and CBE2 , consistent with selection against productive VH5-2 rearrangements . Strikingly, however, in CBE1&2−/− pro-B cell populations as compared to WT pro-B cell populations, the absolute level of VHDJH rearrangements is greatly increased with the vast majority utilizing VH5-2, while the absolute level of DJH rearrangements and upstream VH rearrangements is, correspondingly, decreased .WAPL downregulation in mouse pro-B cells , 44 allorent VHs B and C. upstream B and C.  alleles B, Inset,and CBE2 , 25, 32,pertoire D and E. ngements . This fiH RC and VH5-2, which allows VH5-2 to dominate VH-to-DJH rearrangements due to its robust CBE-mediated interaction with the DJH RC J-seq studies also revealed another notable finding. Despite the dramatically reduced levels of upstream VH rearrangements in CBE1&2−/− pro-B cells, the upstream VHs beyond the several most proximal VHs, relative to each other, have rearrangement junction patterns (i.e. relative levels compared to each other), that were nearly identical to those of WT pro-B cells (H and I). Thus, VH5-2, and to a lesser extent immediately upstream VHs, dominate initial rearrangements in the absence of IGCR1 CBE activity, and, in doing so, terminate most RAG upstream scanning. However, RAG scanning that does proceed beyond VH5-2 continues through the remainder of the VH locus, with similar VH usage patterns as those of WT cells. Overall, these findings indicate that, while most VH5-2 rearrangements in CBE1&2−/− pro-B cells occur before WAPL downregulation, a small fraction of CBE1&2−/− pro-B that do not form VH5-2 rearrangements on one or both Igh alleles undergo upstream VH-to-DJH recombination events at normal frequencies when WAPL-downregulation reaches appropriate levels.The above findings support the notion that, in the absence of IGCR1 CBE activity in normal pro-B cells, RAG scanning continues between the DJe DJH RC . MoreoveH domain, we applied the highly sensitive 3C-HTGTS chromatin interaction assay to explore interactions of the RC-based iEμ enhancer element with upstream and downstream Igh locus chromatin domains in WT −/−rag2 and IGCR1/CBE1&2−/−−/−rag2 cultured pro-B cells derived from the corresponding mouse lines. RAG-deficient cells must be used for such assays to eliminate confounding effects of V(D)J recombination events on such interactions  . RC interactions with transcribed PAIR element-associated sequences are considered a hallmark of loop extrusion-mediated VH locus contraction in pro-B cells  , 22. AmoFig. S3) , 54, 55 -B cells . In this-B cells , which ascanning . Althoug-B cells B, they s-B cells A and B, −/−−/−rag2 cultured pro-B cells exhibit greatly increased RC interaction with proximal VH5-2 and the 3 proximal VHs immediately upstream, which, based on prior studies . Strikingly, nearly all major further upstream interaction peaks were also present in chromatin from CBE1&2−/−−/−rag2 pro-B cells, mostly at similar relative levels to those in WT −/−rag2 pro-B cells J recombination control, we replaced CBE1 or CBE2 with their inverted sequences to generate a CBE1inv or CBE2inv allele in mouse 129SV ES cells  (Methods). We employed HTGTS-V(D)J-Seq to assay JH4-based utilization of the various VHs in pro-B populations harboring IGCR1 CBE deletion or inversion mutations . In contrast, CBE2−/− pro-B cells had very modestly increased VH5-2 rearrangements . These findings indicate that CBE1 and CBE2 cooperatively provide the full impact of IGCR1 scanning impediment activities and unequivocally demonstrate that CBE1 plays a much more dominant role. CBE1inv/inv BM pro-B cells also have significantly increased levels of proximal VH5-2 utilization relative to WT; but to a much lower extent than CBE1−/− pro-B cells, indicating that ability of CBE1 to impede RAG scanning is dampened, but not abrogated, when inverted . In contrast, CBE2inv/inv pro-B cells were very similar to WT pro-B cells with respect to utilization of VH5-2 and upstream VHs . Consistent with our findings for IGCR1/CBE1&2−/− pro-B cells .We have previously generated CBE1−/− mice . To asseA and B) , 49  and VH5-2  locales. In CBE1−/− pro-B cell populations, we observed robust, albeit somewhat diminished, interactions between the iEμ/RC bait and the proximal VH-CBEs compared to interactions in CBE1&2−/− pro-B cells . In CBE2−/− pro-B cells, we observed a modest increase in the interactions between the iEμ/RC bait and the proximal VH-CBEs . These findings indicate that IGCR1 CBEs play a cooperative role in impeding loop extrusion-mediated proximal VH-CBEs and RC interactions and, again, that CBE1 has a more dominant role. In contrast, when CBE1 or CBE2 was inverted, interactions between proximal VH-CBEs and iEμ/RC showed only very modest changes compared to those when IGCR1 CBEs are in normal orientation .We performed 3C-HTGTS on RAG-deficient IGCR1/WT, IGCR1/CBE1&2v-Abl pro-B cell lines provide a very useful system for studies of RAG chromatin scanning process  WAPL-degron v-Abl cells were arrested in G1 and then left untreated or treated with Dox plus IAA to degrade WAPL (v-Abl cells activated V(D)J recombination leading to robust D-to-JH recombination, very low-level VH5-2 to DJH recombination, and extremely low-level upstream VH-to-DJH recombination , as v-Abl lines have high WAPL levels and RAG scanning is impeded at IGCR1 . This reduction was associated with dramatically decreased distal DFL16.1-JH and, to a lesser degree, most other DJH absolute rearrangement levels  as expected . In both untreated and WAPL-depleted 3′CBE− lines, we observed a further 25% decrease in DJH rearrangement levels from their baseline levels. We observed a similar 25% decrease in VHDJH rearrangements in untreated 3′CBE− lines and a nearly 50% decrease in WAPL-depleted 3′CBE lines .WAPL-depletion also led to RAG-scanning and utilization of Vexpected . The abs−v-Abl cells baiting from iEμ/RC . These studies revealed, as previously described . Many of the sequences that contribute to these peaks were highly transcribed including the well-characterized PAIR elements . In contrast, peaks in the proximal 7183/Q52 region that are dominant in untreated v-Abl cells are mainly associated with proximal VH RSS-CBEs and were significantly diminished by WAPL depletion . Notably, in 3′CBE− cells, these same major interaction peaks, including those associated with proximal VHRSS-CBEs in untreated and those associated with the upstream VH regions in treated and untreated cells, remain robust and largely correspond to those in the same locations as in the WT line . While the intensity of some peaks in the untreated or WAPL-depleted 3′CBE−v-Abl cells were somewhat diminished compared those of the untreated or WAPL-depleted WT v-Abl cells, when viewed at high resolution they are clearly still associated with same transcriptional or CBE impediments . Upon the deletion of 3′Igh CBEs, multiple CBEs downstream of the 3′Igh CBEs appear to gain robust interactions with the iEμ/RC (Hs and 3′Igh CBEs and transcription-based (RC and γ1-γ2b enhancer) loop extrusion impediments J-Seq findings to reveal a likely mechanism by which the ordered transition from D-to-JH versus VH-to-DJH rearrangement is regulated . Our findings indicate that robust D-to-JH rearrangements occur in WT pro-B cells before WAPL is sufficiently down-regulated to allow scanning to pass IGCR1 or proximal VH-associated CBEs. In CBE1&2−/− pro-B cells, robust iEμ/RC interactions with proximal VHRSS-associated CBEs promote their robust rearrangements and suppress scanning to upstream VHs. Our finding that low-level rearrangements of upstream VHs in CBE1&2−/− pro-B cells have normal RAG-scanning patterns is consistent with these rearrangements occurring after WAPL-downregulation in the cells that have not formed proximal VHDJH rearrangements on both alleles. In CBE1&2−/−−/−rag2 pro-B cells, lack of V(D)J recombination upon WAPL downregulation allows the iEμ/RC to scan into the upstream VH domain where it reaches normal impediments in a substantial fraction of the cells. In this context, the relatively robust contribution of VH5-2 and immediately upstream VHs to the WT pro-B repertoire indicates that these VHs are dominantly utilized in normal pro-B cells until WAPL levels are sufficiently down-regulated Finally, in support of this model, proximal VHs are poorly utilized in v-Abl cells in which RAG is introduced after complete WAPL-depletion (HTGTS-V(D)J-Seq analyses provided a deep analysis of V-B cells . In addiegulated . Our oveepletion  Fig. 4)H repertoH or DJH recombination centers is strongly impeded by CTCF bound IGCR1 CBEs until they are neutralized by WAPL downregulation J recombination  . We propose that DQ52 recombination, after WAPL depletion, may be less affected, because it accesses RAG by diffusion (versus scanning) from its RC location J junctions in WAPL-depleted 3′CBE− versus WAPL depleted WT v-Abl cells, their VH utilization patterns across the VH locus were nearly identical. These findings support the proposal that the 3′Igh CBEs help maintain RC activity by reinforcing its impediment activity for the RAG scanning process  J recombination patterns .A previously described pLNTK targeting vector  containiv-Abl-kinase-transformed pro-B cell line (v-Abl pro-B cells) was derived by retroviral infection of BM pro-B cells derived from rag2−/− mice as described , and deriving v-Abl lines as described  used to generate HTGTS-V(D)J-seq libraries is listed in SI Appendix, Table S5. HTGTS-V(D)J-seq libraries were prepared as described  .Pro-B cells used in HTGTS-V(D)J-seq experiments were purified from WT or IGCR1-mutated mice as described . 2ug proescribed . HTGTS-V2183883) . All libv-Abl cells was performed as described (H4 coding end primer (C57BL/6 background) used to generate HTGTS-V(D)J-seq libraries. All libraries were normalized to total reads or junctions, and the VHDJH and DJH junctions are described in SI Appendix, Table S3. D usage from the VHDJH joins was analyzed by the VDJ_annotation pipeline.RAG recombination and treatment of WAPL-degron escribed , and thev-Abl pro-B cells for 3C-HTGTS were prepared as described  or paired-end 300-bp sequencing on a Mi-Seq (Illumina) machine. Data were processed as described previously . In addiAppendix 01 (PDF)Click here for additional data file.


C. elegans. Here, we characterize a short BioPart  for PVQ-specific expression. Pnlp-17::mScarletshowed bright, persistent, and specific expression in hermaphrodite and male PVQ neurons from multicopy arrays and single-copy insertions starting from the comma stage. We generated standardized Pnlp-17cloning vectors withgfpandmScarletcompatible with single-copy or array expression for PVQ-specific transgene expression or identification. To facilitate gene synthesis, we have incorporated Pnlp-17as a standard BioPart in our online transgene design tool (www.wormbuilder.org/transgenebuilder).Synthetic biology relies on standardized biological parts (BioParts), and we aim to identify cell-specific promoters for every class of neuron in While BioParts have been extensively used in single-celled organisms, they are less commonly used in multicellular organisms .C. elegansis an exception, with standardized vector kits and detailed documentation distributed informally to the scientific community by the Fire laboratory (https://www.addgene.org/kits/firelab/) playing an essential role in facilitating transgene design and reproducibility between different laboratories.Synthetic biology is an interdisciplinary field that aims to apply engineering principles to the design and construction of biological systems. One key aspect of synthetic biology is the use of standardized and interchangeable biological parts, known as BioParts, to build biological circuitsC. elegansgenome is compact with relatively short intergenic regionsC. elegansPVQ neurons, a class of interneurons involved in regulating locomotion and feeding behaviorsA collection of BioParts composed of neuron-specific promoters would be expected to be useful for many different experiments, e.g., as a starting point for genetic screens , imaging neuronal activity , or controlling neuronal activity using optogeneticsnlp-17promoter, which is specific to PVQ neuronsnlp-17promoter fused to a red fluorophore (mScarlet)and a standard 3' UTR (tbb-2) (Pnlp-17::mScarlet). TransgenicC. eleganswith Pnlp-17::mScarletin extra-chromosomal arrays). We included a piRNA interference (piRNAi) fragment targetinghim-5in the injection mix). To generate standardized reagents, we cloned thenlp-17promoter into two vectors, containing eithergfpormScarlet, that are compatible with single-copy transgene insertions using MosTImScarlet reporter insertion and observed readily visible expression on a fluorescence dissection microscope at high magnification (intensity = 6 out of 6) . We characterized the onset of fluorescence in transgenic embryos and observed expression beginning in the comma stage. The main characteristics of thenlp-17promoter are summarized in.In this study, we focused on a shortened version of themScarlet(pSAH49) andgfp(pSAH50) should be useful for identifying PVQ neurons or for cell-specific transgene expression, and we have deposited the plasmids with Addgene . The constructs were engineered with restriction sites for N-terminal (ApaI) and C-terminal (SmaI) transgene fusions or exchange of the fluorophore (BsaI). The vectors contain a 5' decoy sequence to minimize misexpression , are compatible with MosTI single-copy insertion, and can be used for extra-chromosomal arrays. The short promoter is particularly useful for gene synthesis; to facilitate transgene design, we have incorporated thenlp-17promoter sequence into our online transgene design app (www.wormbuilder.org/transgenebuilder) .The two standard vectors,These design principles and reagents constitute the first of a series of planned BioParts for neuronal transgene expression. Ultimately, we aim to generate compatible and easily synthesized BioParts that allow cell-specific expression in every neuronal class, with the goal of easy cell identification and genetic control of every cell with optogenetics toolsMolecular biologynlp-17promoter tested by Lorenzo et al. (2020) from 339 basepairs to 300 basepairs and removed any BsaI, Esp3I, ApaI, SmaI, and EcoRV restriction sites. We also modified the last four basepairs of the promoter to the consensus start site (aaaaATG).We generated non-clonal synthetic transgenes by gene synthesis  and clonal plasmids by Golden-Gate cloningQuantifying promoter strengthttTi5605location on Chr. II. We quantified the fluorescence on a dissection stereo microscope , objectives 1x and 10x, using an mTomato filter set, and an LED light source , or on an upright compound microscope (LEICA DM2500 LED), 40x oil immersion objective, with a Rhodamine filter set (Leica 11504205), and a mercury metal halide bulb (Leica EL6000). We quantified fluorescence intensity visually in ten L4 animals at different magnifications. We scored both males and hermaphrodites with no obvious differences in expression. The quantification was performed by eye and scored on a scale from 1 to 6, with 1 being the dimmest and 6 being the brightest. The following criteria were used for scoring.Dissection microscope. Score = 6: fluorescence visible with 1x objective and zoom = 1 (lowest). Score = 5: fluorescence visible with 1x objective and zoom = 8 (highest). Score = 4: fluorescence visible with 10x objective and zoom = 1. Score = 3: fluorescence visible with 10x objective and zoom = 8.Compound microscope. Score = 2: fluorescence visible with 20x air objective. Score = 1: fluorescence visible with 40x oil immersion objective. Score = 0: fluorescence not visible at 40x oil immersion objective.We quantified the fluorescence intensity of mScarlet reporter constructs in transgenic animals with stable extra-chromosomal arrays  or single-copy MosTI insertions at theOnset of promoter expressionWe screened at least 15 embryos from transgenic animals at various stages  on NGM plates for the first visible expression on a dissection microscope. We observed no obvious difference in the onset of expression between transgenic animals with extra-chromosomal arrays and single-copy transgenes.MicroscopyTransgenic animals were mounted on 2% agarose pads and anesthetized with an M9 solution containing 50 mM of sodium-azide. We imaged animals with a Leica THUNDER Imaging System, with a 20x oil immersion objective, capturing four image stacks, two for hermaphrodites and two for males. After imaging, we generated maximum intensity projection on Leica LAS X software. We carefully screened for misexpression in additional neurons other than PVQ but did not observe any.Transgenic animalsunc-119animals (CFJ42). The injection mix contained 10 ng/µl of a non-clonal PVQ specific fragmentnlp-17p::mScarlet::tbb-23'UTR(Twist Bioscience), 10 ng/µl pCFJ108 (unc-119ce-rescue) linearized with ApaLI, 10 ng/µl pCFJ782 (hygromycin resistance) linearized with EcoRVhim-5) linearized with ApaLI, and 60 ng/µl GeneRuler 1 kb plus DNA ladder (ThermoFisher SM1331) for a final concentration of 100 ng/µl. After injection, three independent stable transgenic lines were maintained and scored for mScarlet expression.Extra-chromosomal arrays. Transgenic animals carrying extra-chromosomal arrays were generated by injecting intonlp-17p::mScarlet::tbb-23'UTR) were inserted by MosTI into Chr II  following standard protocolsnlp-17p::mScarlet) MosTI targeting vector, 10 ng/µl pSEM238 (histamine negative selection)mlc-p1::gfp, co-injection marker) , 15 ng/µl pCFJ782 (hygromycin resistance)unc-119rescue. A single clonal rescued worm with no visible fluorescence from the co-injection marker was picked to generate a stable line.MosTI insertions. Single-copy transgenes with pSAH49 . The version described in this manuscript has been archived at https://github.com/christianfj/transgenebuilder_v2023_06_13 and available as Extended Data.In silicomolecular biology design was performed with "A plasmid Editor" (ApE)(Biorender.com). The figure was generated with Adobe Illustrator (version 27.4).Plasmidsnlp-17p::mScarlet::tbb-2 (MosTI vector) (Addgene #200325)pSAH49 -nlp-17p::gfp::tbb-2(MosTI vector) (Addgene #200326)pSAH50 -him-5piRNAi fragment (Addgene #159818)pMNK54 -unc-119cbr-(+)rescue marker (Addgene #200367)pCFJ108 -snt-1prpl-3::ce-HisCl1::(Addgene #161515)pSEM238 -rpr-1p::sgRNA targeting thettTi5605location Chr. II (Addgene #159822)pSEM318 -tbb-2mlc-1p::gfp::cbr-(Addgene #159897) pSEM231 -rps-27rps-0p::hygromycinR::(Addgene #190933)pCFJ782 -gpd-2::tagRFP-t::smu-1hsp-16.41p::cas9::(Addgene #191382)pMDJ231 -StrainsN2- (wildtype)CFJ42-kstSi42[unc-119(-)] II;unc-119(ed3)] III (available from CGC)Promoter sequenceTransgene sequences10.22002/qs7eh-g0669Description: Online application for designing transgenes. . Resource Type: Software. DOI:


Nature Communications 10.1038/s41467-020-16618-6, published online 04 June 2020Correction to: The original version of this Article contained errors in Supplementary Fig. Updated Supplementary Information


Arabidopsis thaliana, stomata are composed of two guard cells that control the aperture of a central pore to facilitate gas exchange between the plant and its environment, which is particularly important during photosynthesis. Although leaves are the primary photosynthetic organs of flowering plants, floral organs are also photosynthetically active. In the Brassicaceae, evidence suggests that silique photosynthesis is important for optimal seed oil content. A group of transcription factors containing MADS DNA binding domains is necessary and sufficient to confer floral organ identity. Elegant models, such as the ABCE model of flower development and the floral quartet model, have been instrumental in describing the molecular mechanisms by which these floral organ identity proteins govern flower development. However, we lack a complete understanding of how the floral organ identity genes interact with the underlying leaf development program. Here, we show that the MADS domain transcription factor AGAMOUS (AG) represses stomatal development on the gynoecial valves, so that maturation of stomatal complexes coincides with fertilization. We present evidence that this regulation by AG is mediated by direct transcriptional repression of a master regulator of the stomatal lineage, MUTE, and show data that suggests this interaction is conserved among several members of the Brassicaceae. This work extends our understanding of the mechanisms underlying floral organ formation and provides a framework to decipher the mechanisms that control floral organ photosynthesis.In  Arabidopsis thaliana before and after fertilization. Stomata control gas exchange between the plant and the environment, which facilitates photosynthesis and transpiration. We show that stomatal formation coincides with fertilization of the flower but does not depend upon it. Instead, the activity of a master regulator of stomatal development is directly controlled by a flower-specific factor. We present evidence that this regulation is conserved in several members of the mustard plant family (Brassicaceae) and suggest that the timing of stomatal formation on the female reproductive organs may be modified between species, which may have adaptive benefits. Given that floral organs are derived from leaves, this work also broadens and deepens our understanding of how the underlying leaf developmental program is rewired during flower development.Photosynthesis supports the growth and development of plants and is mainly associated with leaves. However, other organs, including floral organs, are photosynthetic and contribute to the energy requirements of the plant, which has attracted the interest of crop breeders. Despite its importance, very little is known about the establishment and molecular regulation of floral organ photosynthesis. Here, we describe the developmental progression and regulation of stomatal formation on the female reproductive organs of  Arabidopsis thaliana, flowers are composed of four types of floral organs: sepals, petals, stamens, and carpels. The sepals contain high levels of chlorophyll and bear stomata, making them leaf-like in appearance. In contrast, mature petals and stamens lack substantial concentrations of chlorophyll . SP. SPAG, S t-test) . Betweenst) Figs  and 2I.  t-test) . Mean FAstage 12 . Althougion data , the substage 13 . TogetheAG activity would result in the differential expression of master regulators of stomatal development, we harvested stage ~10–13 gynoecia from L-er wild-type plants harboring a fully functional copy of AG and those that were homozygous for the weak ag-10 mutation . Th. ThSPCH, tissues . This comutation . At 2 d  t-test) . MUTE annduction . Taken tFAMApro:2xYFP reporter into the ag-10 background and imaged gynoecia at late stage 12 and stage 13 using confocal laser scanning microscopy ..AG activMUTE first intron are required for AG and SEP3 binding. We synthesized probes with the same sequence as the region of the MUTE first intron that contains the two CArG motifs (AtMUTE_i1) and incubated them with the AG and SEP3 proteins  or simultaneously (mCArG_1+2), which were intended to disrupt protein binding  eliminated all shifted bands  and were ~4-fold more abundant on ag-10 gynoecial vales compared to L-er, as determined by cell-size analysis   and late-stage stomatal cells were 14 to 16 times more abundant on ag-10 gynoecial valves than L-er controls     Tables. t-tests)  Tables. t-tests)  Tables.ag-10 mutant gynoecia, we also perturbed AG activity through dynamic induction of the AG-amiRNA   (AG through static mutation (ag-10) or through independent dynamic perturbation using the AG-amiRNA could promote the precocious formation of stomatal cells on gynoecial valves.To confirm the stomatal phenotypes we observed of the G-amiRNA . Late-stt-tests)  Tables. t-tests) . These dFRUITFULL (FUL), does not bear stomata on the silique  dATP by a Klenow fill-in reaction of 5`-overhangs. Probe sequences are shown in in vitro via the TNT SP6 Quick Coupled Transcription/Translation System (Promega) following manufacturer’s instructions. Plasmids for in vitro transcription/translation of SEP3 and SEP3ΔC have been generated previously [in vitro transcription/translation of AG, SHP1, and SHP2, coding sequences of AG (X53579.1), SHP1 (NM_115740.3), and SHP2 (NM_129844.5) were synthesized via the GeneArt gene synthesis service (Thermo Fisher Scientific) and cloned into pTNT (Promega) using EcoRI and SalI restriction sites. The composition of the protein-DNA binding reaction buffer was essentially as described previously with final concentrations of 1.6 mM EDTA, 10.3 mM HEPES, 1 mM DTT, 1.3 mM spermidine hydrochloride, 33.3 ng μL-1 Poly dI/dC, 2.5% CHAPS, 4.3% glycerol, and 6 μg μL-1 BSA [in vitro transcribed/translated protein were co-incubated with 0.1 ng of radioactively labelled DNA probe in a total volume 12 μL. Binding reactions were incubated overnight at 4°C and loaded on a polyacrylamide  0.5× TBE gel. The gel was run at room temperature with 0.5× TBE buffer for 3 h at 7.5 V cm–1, and afterwards vacuum-dried and exposed onto a phosphorimaging screen to quantify signal intensities. For size comparison a radioactively labelled DNA ladder  was applied. Original gels are shown in Electrophoretic mobility shift assays were performed essentially as described previously . DNA proeviously . For genμL-1 BSA . For eacS1 Fig(A) A scanning electron micrograph of a gynoecium at stage 13 (anthesis) with the valves (green), replum (red), style (blue) and stigma (yellow) false colored. (B) Simplified model of stomatal development on leaves. The bHLH transcription factors, SPEECHLESS (SPCH), MUTE, FAMA, SCREAM (SCRM) and SCRM2 coordinate the progression of stomatal development. SPCH is subject to post-translational regulation by a mitogen activated protein kinase (MAPK) cascade, which is activated by receptors, such as TOO MANY MOUTHS (TMM), ERECTA (ER) and ER-LIKEs (ERLs). The secreted peptides, EPIDERMAL PATTERNING FACTOR1 (EPF1) and EPF2 bind to these receptors to activate them at different stages of stomatal development. EPF-LIKE9/STOMAGEN (STOM) competes with EPF1/EPF2 binding to suppress activation of the receptors. The dotted line from the ERL1-TMM complex to the SCRM/2-MUTE complex indicates an unknown mechanism of repression. (C-F) Scanning electron micrographs of (C) a stage 12 gynoecium, (D) a stage 10 abaxial sepal, (E) a stage 11 abaxial anther, and (F) a stage >17 silique. Asterisks indicate presence of stomatal lineage cells. Red box in (F) highlights the size of style tissue in comparison to valve tissue, both of which bear stomata. Scale in (C) 100 μm, (D-E) 20 μm, and (F) 200 μm.(TIF)Click here for additional data file.S2 Fig(A-B) The progression of the stomatal lineage on wild-type gynoecia and siliques as determined by (A) morphological assessment and (B) by cell size analysis. (C-D) Scanning electron micrographs of (C) a gynoecium from an emasculated flower 5 days after anthesis, (D) a magnification of the valve in (C). Scale in (C) 200 μm, (D) 20 μm.(TIF)Click here for additional data file.S3 FigAGpro:AG-GFP ag-1 plants. Arrowheads indicate accumulation of AG-GFP in the replum. Scale is 100 μm.(A-C) Maximum intensity projections of stitched confocal laser scanning z-stack micrographs of (A) early stage 12, (B) late stage 12, and (C) stage 13 gynoecia from (TIF)Click here for additional data file.S4 FigAG-amiRNAi (OPpro:AG-amiRNA/35Spro:GR-LhG4) FAMApro:2xYFP at (A-B) stage 12 and (C-D) stage 13 gynoecia after  dexamethasone or  mock treatments at the times indicated. Scale is 100 μm.(A-D) Maximum intensity projections of stitched confocal laser scanning z-stack micrographs of (TIF)Click here for additional data file.S5 FigMUTEpro:MUTE-GFP in (A-C) a wild-type L-er background and (D-F) an ag-10 background using confocal microscopy. Arrowheads indicate the presence of fluorescent foci. (G-O) Images of MUTEpro:MUTE-GFP (G-L) stage 11 gynoecia in (G-I) a wild-type L-er background, (J-L) an ag-10 background, and (M-O) stage 12 gynoecia in a wild-type L-er background. Each panel is a sample from a different plant. Scale is 100 μm.(A-F) Images of early stage 12 gynoecial valves of (TIF)Click here for additional data file.S6 FigMUTE first intron identified as bound by SEP3 (SEP3_rep1-4), an AG-SEP3 complex (SEP3AG_rep1-3), or an AG-SEP3Δtet complex (SEP3del_AG_rep1-2). CArG_1 and CArG_2 are highlighted in purple boxes.Sequences mapping to the (TIF)Click here for additional data file.S7 Figag-10 gynoecia and (B) an enlarged segment of the gynoecial valve depicting the presence of stomatal linage cells with symmetric cell divisions (late-stage GMCs) and mature stomatal cells with a central pore. (C-E) Scanning electron micrographs of gynoecia at anthesis of (C) AlcRpro:AG-amiRNA/35Spro:AlcR 5 d after 6 h EtOH vapor treatment, (D) untreated AlcRpro:AG-amiRNA/35Spro:AlcR and (E) L-er 5 d after 6 h EtOH vapor treatment. Scale bars for images of whole gynoecia are 100 μm. Scale bars for magnifications are 20 μm. Purple, blue, and red highlights indicate early, mid, and late-stage stomatal lineage morphology, respectively. (F) Index of early, mid, and late stomatal lineages based on morphology from scanning electron micrographs from stage 13 gynoecial valves of indicated genotypes. Each dot represents an individual sample. NT, no treatment.(A) A stage 13 (TIF)Click here for additional data file.S8 FigSHP1 and SHP2 mRNAs as determined by RT-qPCR in L-er and ag-10 stage 10–13 gynoecia. (B-D) Levels of (B) SPCH, (C) MUTE, and (D) FAMA mRNAs as determined by RT-qPCR in L-er, ag-10, ful-1, and ful-1 ag-10 stage 12–13 gynoecia. Each dot represents the technical mean of an individual independent biological replicate.(A) Levels of (TIF)Click here for additional data file.S9 Fig-er, (B) shp1-1 shp2-1, (C) ag-10, and (D) ag-10 shp1-1 shp2-1. Some sepals, petals and stamens have been removed to allow visualization of gynoecium. Scale is 1 mm. (E-F) Levels of (E) SPCH and (F) TMM mRNAs in L-er, shp1 shp2, ag-10, ag-10 shp1 shp2 stage 10–13 gynoecia as determined by RT-qPCR. Each dot represents the technical mean of an independent biological replicate.(A-D) Flowers at anthesis of (A) L(TIF)Click here for additional data file.S10 FigAtMUTE_i1, (B) mCArG_1, (C) mCArG_2, (D) mCArG_1+2, (E) CrMUTE_i1, and (F) EsMUTE_i1 probes.(A-F) Protein-DNA gel shift assays using combinations of AG, SEP3, SEP3ΔC, SHP1, and SHP2 protein and two replicates of (A) (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.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S10 Table(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Fanconi Anemia (FA) is an inherited bone marrow (BM) failure disorder commonly diagnosed during school age. However, in murine models, disrupted function of FA genes leads to a much earlier decline in fetal liver hematopoietic stem cell (FL HSC) number that is associated with increased replication stress (RS). Recent reports have shown mitochondrial metabolism and clearance are essential for long-term BM HSC function. Intriguingly, impaired mitophagy has been reported in FA cells. We hypothesized that RS in FL HSC impacts mitochondrial metabolism to investigate fetal FA pathophysiology. Results show that experimentally induced RS in adult murine BM HSCs evoked a significant increase in mitochondrial metabolism and mitophagy. Reflecting the physiological RS during development in FA, increase mitochondria metabolism and mitophagy were observed in FANCD2-deficient FL HSCs, whereas BM HSCs from adult FANCD2-deficient mice exhibited a significant decrease in mitophagy. These data suggest that RS activates mitochondrial metabolism and mitophagy in HSC. FA is an inherited BM failure with hematologic disease onset around school age . Yet, muRecently, we and others have highlighted mitochondrial metabolism as a critical component for maintenance in adult quiescent HSCs , 7. MitoTo understand how mitochondrial metabolism affects FA pathophysiology, we hypothesized that RS during FL HSC expansion modulates mitochondrial metabolism. Using pharmacologically induced RS, we observed temporary alterations in MMP and increased mitophagy. We then analyzed FL and BM HSCs from Fancd2 knockout (Fancd2 KO) mice. Strikingly, MMP and mitophagy were elevated in FANCD2-deficient FL HSCs. In contrast, mitophagy was downregulated in adult BM HSC. Taken together these observations indicate that the pathophysiological RS in FANCD2-deficient fetal HSCs boosts mitochondrial metabolism and mitophagy.C57/BL6 background Fancd2 KO mice  were kinHSC immunophenotyping analysis was conducted using antibodies for EPCR, CD150, CD48 (HM48-1), c-Kit (2B8), Sca-1  and Lineage (Gr-1 (RB6-8C5), Ter119 (Ter-119), B220 (RA3-6B2), CD4 (RM4-5), CD8 (53-6.72), Mac-1 (M/70) excluded when analyzing FL HSCs). MMP was measured with tetramethylrhodamine (TMRE) 200nM and used 488nm lasor/585-642nm detector. mtROS was measured with MitoSoxRed  and used 488nm lasor/585-642nm detector. Lysosome acidification was detected with Lysotracker Red  and used 638nm lasor/660-720nm detector. Mitophagy activity was detected with Mitophagy detection kit  and used 488nm lasor/780-860nm detector.To perform intracellular staining, cells were fixed and penetrated using IntraPrep (A07803 Beckman) and stained with anti-p4EBP  1:200.All data were measured by CytoFlex FACS analyzer (Beckman Coulter). Analysis is performed with FlowJo software.Newly synthesized protein was measured. E13.5 FL cells or Mom’s BM were cultured with 10mM O-propargyl-puromycin (OPP) in IMDM 10%FBS for 40min, harvested and staind antibodies of HSC . After washed, cells are fixed and penetrated using IntraPrep (A07803 Beckman). By using Click-iT Plus OPP Alexa Fluor 488 (AF488) Protein Synthesis Assay Kit , stained OPP with AF488 and subsequent to FACS to measure the intensity (used 488nm lasor/525-540nm detector).HSC (CD150+ CD48- Lin- Sca-1+ c-Kit+) or HSPCs (Lin- Sca-1+ c-Kit+) were sorted by FACS AriaII or III (BD) and fixed with 4%PFA. Cells were permeabilized with 0.5% Triton for 15min and stained with anti-Tomm20 (ab78547 or ab289670) 1:200 or anti-ssDNA  1:50. Secondary stains were performed with anti-rabbit-AF488 1:1000 or anti-rat-AF488 1:1000. DAPI 1:1000 or 7-AAD 1:200 were used for nuclear staining. Data correction was performed with Mark-II (Amnis) and analysis was performed with IDEAs software (Amnis).Mice were intraperitoneally injected 5-FU with one shot (200mg/kg body weight).HSCs were sorted by FACS Aria II or III (BD) and subjected to ex vivo culture. 2000 HSCs per 1 well of 96-well dish are cultured with SF-03 medium (Sekisui) supplemented with 100ng/ml SCF , 100ng/ml TPO  and 0.1% BSA. Aphidicolin was diluted with DMSO for 50mg/ml and used as 1:5000-10000.HSCs were sorted by FACS Aria II or III (BD) and 500 cells are seeded onto 3ml of mouse methylcellulose media  with APH (50-100ng/ml) or imTOR of Rapamycin  or iTgf-β of SD-208 . The media was equally divided into three 3.5cm dishes and incubated at 37°C. Colony numbers were counted after 14 days.Statistical analyses were performed with t-test and one-way ANOVA (if the parameter is over 2) by using prism software (GraphPad).+ CD48- LK Hematpoietic Stem and Progenitor Cells (HSPCs)  . After 6 (HSPCs) S1A–C, wWe next analyzed the mitochondrial morphology in HSPCs. Alterations in mitochondrial morphology and distribution in HSCs have been previously reported in myelodysplastic syndrome (MDS) and leukemia; mitochondria in HSCs from MDS models diffuse and spread out while mitochondria in leukemic stem cells (LSC) exhibit polarity and are concentrated , 17. Imain vivo RS drastically alters MMP, mtROS, mitophagy and mitochondrial distribution in adult HSPCs.Adequate levels of mitophagy and lysosomal activity help maintain HSPCs potential –20. WhenWe next investigated changes in mitochondrial activity in cultured HSCs subjected to aphidicolin (APH) which induces RS by inhibiting DNA polymerase . After 7Next, we focused on whether Fancd2 deficiency affected mitochondrial metabolism in quiescent adult BM HSCs, which do not exhibit spontaneous RS . While tFetal HSCs show increased mitochondrial metabolisms compared to adult BM HSCs . Here, wmTOR signaling is closely associated with mitochondrial metabolism and lysosome biogenesis in HSCs . We thusin vivo link of RS with mitochondrial metabolism and mitophagy. In 5-FU-treated mice, prominent mitochondrial changes were observed at day 6 after administration, the phase when HSCs are subjected to proliferation (In this study, we extended our observations on the unique fetal HSC phenotype in FA. We showed that fetal RS, in the context of FA results in a concurrent increase in mitochondrial metabolism and mitophagy. In contrast, adult quiescent BM HSCs show lower levels of mitochondrial metabolism and decreased mitophagy in Fancd2 KO. These observations provide an feration . Both FLferation . mtROS lWe previously described the role of RS in decreasing FA FL HSCs . MetformPrevious reports showed defective mitophagy as a non-canonical phenotype of FA cells and illustrate the role of several FA proteins in mitophagy , 11. OurThe direct correlation between RS, mitochondrial metabolism and mitophagy is difficult to explain. Our data indicate that mTOR signaling and mTOR-related lysosomal biogenesis may sense RS and increase overall metabolic activity including c-Myc  and riboThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by TWMU animal experiment committee.MM-K and NO performed experiments. MM-K, KF, SA, PK, AN-I wrote manuscript, MG, AI, KS and AN-I funded research. All authors contributed to the article and approved the submitted version.


C. elegans, the interneuron RIM plays important yet complex roles in locomotion behavior. Here, we show that RIM both promotes and suppresses distinct features of locomotion behavior to fine tune motor output. This dual function is achieved via the excitation and inhibition of the same motor circuit by electrical and chemical neurotransmission, respectively. Additionally, this bi-directional regulation contributes to motor adaptation in animals placed in novel environments. Our findings reveal that individual neurons within a neural circuit may act in opposing ways to regulate circuit dynamics to fine tune behavioral output.How neural circuits drive behavior is a central question in neuroscience. Proper execution of motor behavior requires precise coordination of many neurons. Within a motor circuit, individual neurons tend to play discrete roles by promoting or suppressing motor output. How exactly neurons function in specific roles to fine tune motor output is not well understood. In  Animals execute a wide range of behavior, which rely on the vast number of neurons in the brain. Control of motor output is an essential feature of the nervous system in nearly all animals, and the successful execution of even the simplest motor behavior requires precise coordination of many individual neurons . For exaC. elegans has emerged as a highly valuable model to investigate the mechanisms by which neural circuits control behavior. C. elegans possess a simple nervous system composed of 302 neurons, approximately 7,000 chemical synapses, and 900 electrical junctions .TQ1164: glc-3(ok321).TQ2225: glc-4(ok212).TQ2384: glc-2(gk179).TQ2580: avr-14(ad1302).TQ2581: avr-15(ad1051).TQ3032: lite-1(xu7); xuEx1040[Pnmr-1::GCaMP3.0 + Pnmr-1::DsRed2b].TQ6292: lite-1(xu7); xuEx2167[Pcex-1::GCaMP6f + Pgcy-13::sl2:mcherry].TQ6346: glc-1(pk54).TQ6744: lite-1(xu7); xuEx2257[Punc-4::tetx::sl2::yfp].TQ6745: lite-1(xu7); xuEx2257[Punc-4::tetx::sl2::yfp]; xuEx1932.TQ6875: xuEx1932; xuIs219[Podr-2b(3a)::yfp + Punc-122d::gfp].TQ7103: xuEx2595[Pgcy-13::tdc-1 sense+antisense+Pgcy-13::DsRed].TQ7119: xuEx1899[Punc-4::DsRed]; xuEx856[pBS-77::5’UTR + avr-14::sl2::yfp].TQ7283: xuEx2693[Pgcy-13::Chrimson::sl2::yfp].TQ7313: xuEx2717[Pgcy-13::avr-14(genomic+cDNA)::sl2::YFP]; avr-14(ad1302).TQ7262: xuEx2675.TQ7264: xuEx2677.TQ7267: xuEx2680.TQ7269: xuEx2682.TQ7274: xuEx2687.TQ7280: xuEx2593[Pgcy-13::eat-4 sense+antisense+Pgcy-13::DsRed].TQ7281: avr-14(ad1302)I; xuEx2593[Pgcy-13::eat-4 sense+antisense+Pgcy-13::DsRed].TQ7324; xuEx2693[Pgcy-13::Chrimson::sl2::YFP]; inx-1(tm3524).TQ7326: xuEx2693.TQ7325: xuEx2693[Pgcy13::chrimson::mcherry]; unc-9(e101).TQ7327: xuEx2730; xuEx2693[Pgcy-13::chrimsom::mcherry].TQ7365: xuEx2765; avr-14(ad1302).TQ7332: xuEx2793; xuEx1040[Pnmr-1::GCaMP3.0 + Pnmr-1::DsRed2b].TQ7399: xuEx2795; xuEx2766; avr-14(ad1302).TQ7340: xuEx2751[Pnpr-4::chrimson::mcherry]; xuEx2793.TQ7441: unc-9(e101); inx-1(tm3524); xuEx2693[Pgcy-13::Chrimson::sl2::yfp].TQ7348: xuEx2751[Pnpr-4::chrimson::mcherry].TQ7433: xuEx1040[Pnmr-1::GCaMP3.0 + Pnmr-1::DsRed2b]; xuEx2766; lite-1(xu7).TQ7568: unc-7(e5); inx-1(tm3524); xuEx2693[Pgcy-13::chrimson::sl2::mcherry].TQ7553: unc-7(e5); inx-1(tm3524); xuEx2751[Pnpr-4::chrimson::sl2::mcherry].TQ7582: xuEx2860[Pnmr-1::unc-7(cDNA)::sl2::mcherry] + xuEx2823[Pgcy-13::chrimson::sl2::mcherry]; inx-1(tm3524); unc-7(e5).TQ7710: xuEx2887[Pnmr-1::sl2::mcherry2 + Pinx-1 L::sl2::YFP].TQ7711: xuEx2888[Pnmr-1::sl2::mcherry2]+ xuEx856[pBS-77::5’UTR + avr-14::sl2::yfp].TQ8002: xuEx2316[pgcy-13::tdc-1(s + as) + pgcy-13::dsRed2b + pnlp-12::dsRed2b].TQ8003: xuEx2323[pgcy-13::eat-4RNAi + pgcy-13::sl2::CFP + pnlp-12::dsRed2b].TQ8004: xuEx2320[pgcy-13::unc-31(s + as) + pgcy-13::sl2-CFP + pnlp-12::dsRed2b].Promoters used in this research include: Pnpr-4: AVA, AIN; Popt-3: AVE; Pgcy-13: RIM; Pnmr-1: AVA, AVE, AVD, RIM, PVC; Punc-4: a-type motor neurons ; Plgc-55: AVB, SMD, RMD; Pacr-5: DB/VB motor neurons.Laser ablation: laser ablation was performed on Olympus BX51 upright microscope equipped with a Micropoint system  was delivered from a home-made LED light source to activate Chrimson to trigger behavior. Animal behavior were recorded and analyzed using the Wormlab system (MBF Bioscience). Each trial included five animals and at least five trials were performed for each group. Reversals were scored as positive responses if the animal stopped forward movement and initiated a reversal lasting at least half of one head swing upon light stimulation.escribed . BrieflySpontaneous reversal frequency was analyzed using an automated single worm tracking system as described previously . Day 1 aCalcium imaging: to eliminate intrinsic response to blue light that excites GCaMP  worms, suggesting that neuropeptide signaling may not play a major role in mediating the suppression effect of RIM on reversal probability  worms  transgene in the avr-14 rescue background, and found that this abolished the rescue effect of the avr-14 transgene  worms . In addi neurons . Furthernt worms . These rransgene , indicatOur results show that RIM neurons can both promote reversal initiation as well as suppresses reversal probability through interactions with the interneurons AVA/AVE. We next wondered how RIM regulates the activities of AVA/AVE neurons. To address this, we examined how RIM ablation affects the calcium activities in AVA/AVE neurons in freely-moving worms. In mock-ablated controls, the calcium spikes in AVA/AVE neurons were coupled with reversals, with reversals initiating upon calcium increase in AVA/AVE, and terminating once calcium traces peaked and began to drop . In RIM-Given our observations that RIM can bi-directionally promote and suppress reversal behavior, we next wondered whether this dual function of RIM contributes to reversal-related complex behavior under more natural conditions. One possible application of this function could be to facilitate motor adaptation after food removal. In the presence of food .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vaccination remains crucial for protection against severe SARS-CoV-2 infection, especially in aged population.®) or 30 µg of BNT162b2 (Comirnaty®). The primary endpoint was the rate of 2-fold antibody titre increase 14 days post-vaccination measured by quantitative enzyme-linked immunosorbent assay (Anti-RBD-ELISA). Secondary endpoints included changes in neutralising capacity  against wild-type and 25 variants at 14 days and up to 12 months.We evaluated, with a randomised controlled, adaptive, multicentre phase II study, the safety and immunogenicity of a 3rd vaccination dose (1st booster) in individuals ≥75 years . Participants were randomized to either a full-dose (100µg) of mRNA-1273  were included in the analyses for immunogenicity after day 14. The primary endpoint of a 2-fold anti-RBD IgG titre increase 14 days post-vaccination was reached in all subjects. A 3rd full-dose mRNA-1273 vaccination provided higher anti-RBD IgG titres (GMT D14 7090 [95% CI 5688 – 8837] BNT162b2 vs. 10711 [95% CI 8003 – 14336] mRNA-1273). A pattern showing higher neutralising capacity of full-dose mRNA-1273 against Wuhan wild-type so as for 23/25 tested variants was observed.Third doses of either BNT162b2 or mRNA-1273 provide substantial antibody increase 14 days post-vaccination, with full-dose mRNA providing higher antibody levels and an overall similar safety profile for ≥75 subjects. Additional booster doses should be prioritised, particularly in aged people and others at high-risk. In-depth data on waning of immune response is needed to assess duration of protection, and evaluation of vaccine-dosage for individuals at risk may be reconsidered. With the data on full-dose (100µg) mRNA-1273evaluation of vaccine-dosage for individuals at risk may be reconsidered, despite the small sample size.Jannik Stemler, -, Ministry of Education and Research (BMBF): Grant/Research Support Oliver A. Cornely, MD PhD, DZIF: Advisor/Consultant|DZIF: Board Member|DZIF: Grant/Research Support|DZIF: Honoraria|DZIF: Stocks/Bonds


Cmpd10357, at a dose of 5 mg/kg, significantly prolonged survival in our B-ALL xenograft mouse model, with a median survival time of 49.0 days compared with 45.5 days in the control group (p < 0.05). The cytotoxicity of Cmpd10357 demonstrated caspase-independent, nonapoptotic cancer cell demise associated with the nuclear translocation of apoptosis-inducing factor (AIF). The cytotoxicity of Cmpd10357 in B-ALL cells was inhibited by Necrostatin-1 but not by Necrosulfonamide. These studies suggest that an AIF-mediated, caspase-independent necrosis mechanism of Cmpd10357 in B-ALL could be used in combination with traditional apoptotic chemotherapeutic agentsB-cell acute lymphoblastic leukemia (B-ALL) is the most common type of cancer found in children. Although the overall survival rates are now >80%, 15%–20% of pediatric patients relapse, with survival rates subsequently dropping to 5%–10%. Cmpd10357, 3-amino-5-arylamino-6-chloro-N- (diaminomethylene) pyrazine-2-carboximide, is a highly potent, cell-permeant compound recently shown to have cytotoxic effects on solid tumors, including human breast cancer and high-grade gliomas, independent of their proliferative status. Cmpd10357 demonstrated concentration-dependent cytotoxicity in two human B-ALL cell lines, JM1 and Reh, at half-maximal inhibitory concentrations (IC B-cell type acute lymphoblastic leukemia (B-ALL) is the most common type of cancer found in children. Although survival rates for B-ALL are >80% largely owing to the development of combination chemotherapy regimens, relapse occurs in 15%–20% of pediatric patients, and survival rates after relapse can drop to 5%–10% . TherefoCmpd10357 is a substituted pyrazine-2-carboximide compound that has demonstrated its cytotoxicity in glioma . In thisCmpd10357, 3-amino-5-arylamino-6-chloro-N-(diaminomethylene) pyrazine-2-carboximide, was provided by Fredric Gorin’s and Pamela Lein’s research unit  [6]. CmpμM, respectively, on the basis of a similar study [The receptor-interacting serine/threonine-protein kinase 1 (RIPK1) inhibitor, Necrostatin-1 (Thermo Fisher Scientific), or the MLKL inhibitor, Necrosulphonamide , was added to culture media at 30 and 2 ar study ,8 with Chttps://www.cellosaurus.org/CVCL_3532) and Reh (https://www.cellosaurus.org/CVCL_1650), were purchased from ATCC. Cells were maintained, grown, and counted as described in the Two human precursor B-ALL cell lines, JM1 -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays were performed as described in the 2O2), and caspase activity was measured using a Caspase-Glo 3/7 assay kit (Promega) according to the manufacturer’s instructions.Cells were treated with Cmpd10357 or hydrogen peroxide  mice using our institutionally approved animal care protocol.The JM1 leukemia mouse model was created by intratibial injection of 2 × 106 JM1 cells per mouse were randomly enrolled into two groups: group 1, vehicle control with 80% polyethylene glycol 400 (PEG)  in phosphate-buffered saline (PBS) (n = 8), and group 2, Cmpd10357 (n = 8). The vehicle control group was dosed by daily intraperitoneal (IP) administration for 2 weeks. A 250-mM Cmpd10357 stock solution was dissolved in 80% PEG and administered intraperitoneally daily at 5 mg/kg for 2 weeks. Mice were monitored daily and euthanized when they showed signs of leukemia in accordance with Institutional Animal Care and Use Committee (IACUC) policy on humane end points. Leukemia cells were harvested from the leukemia-infiltrated bone marrow for experiments. JM1 leukemia cells were confirmed by flow cytometry using an anti-human leukocyte antigen (HLA)-ABC antibody (Biolegends and BD Biosciences) for phenotyping. Statistical significance for survival time was determined by the log-rank test. Kaplan-Meier survival curves were plotted for the two groups. Analyses were performed using Prism 8.3 software (GraphPad).Cmpd10357 was compared with a vehicle control for overall survival in JM1 leukemia model mice. Sixteen mice injected with 2 × 106 cells were denatured in the radioimmunoprecipitation assay (RIPA) lysis buffer (VWR Life Science). For nuclear fractions, cell pellets from 3–5 × 106 cells were denatured using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific), and nuclear fractions were collected according to the manufacturer’s instructions. Immunoblotting and analysis were performed as described in For total cell protein extractions, cell pellets from 1–2 × 1050) of 3.2 and 3.3 μM, respectively . Cell viability was measured using the MTS assay. Cmpd10357 demonstrated concentration-dependent cytotoxicity in both JM1 and Reh with half-maximal inhibitory concentrations (ICectively .p < 0.05)  . Upon eu < 0.05)  and E. N2O2 control  for 6, 8 and 24 hours. No significant increase in expressions, except P-eIF2a in JM1 at 24 hours, is observed in Cmpd10357 treated cells. Actin was used for normalization of protein amount. All data are presented as the mean ± SD. * p<0.05.Supplemental Figure 1


CD46 predispose to atypical hemolytic uremic syndrome (aHUS) with low penetrance. Factors driving immune-dysregulatory disease in individual mutation carriers have remained ill-understood. In addition to its role as a negative regulator of the complement system, CD46 modifies T cell-intrinsic metabolic adaptation and cytokine production. Comparative immunologic analysis of diseased vs. healthy CD46 mutation carriers has not been performed in detail yet. In this study, we comprehensively analyzed clinical, molecular, immune-phenotypic, cytokine secretion, immune-metabolic, and genetic profiles in healthy vs. diseased individuals carrying a rare, heterozygous CD46 mutation identified within a large single family. Five out of six studied individuals carried a CD46 gene splice-site mutation causing an in-frame deletion of 21 base pairs. One child suffered from aHUS and his paternal uncle manifested with adult-onset systemic lupus erythematosus (SLE). Three mutation carriers had no clinical evidence of CD46-related disease to date. CD4+ T cell-intrinsic CD46 expression was uniformly 50%-reduced but was comparable in diseased vs. healthy mutation carriers. Reconstitution experiments defined the 21-base pair-deleted CD46 variant as intracellularly—but not surface-expressed and haploinsufficient. Both healthy and diseased mutation carriers displayed reduced CD46-dependent T cell mitochondrial adaptation. Diseased mutation carriers had lower peripheral regulatory T cell (Treg) frequencies and carried potentially epistatic, private rare variants in other inborn errors of immunity (IEI)-associated proinflammatory genes, not found in healthy mutation carriers. In conclusion, low Treg and rare non-CD46 immune-gene variants may contribute to clinically manifest CD46 haploinsufficiency-associated immune-dysregulation.Mutations in The online version contains supplementary material available at 10.1007/s10875-023-01547-y. CD46 gene is located within a cluster of complement regulatory genes at chromosome 1q3.2 , anti-CD46-FITC  or via a two-step stain of anti-CD46 GB24 mAb  and rat anti-mouse IgG1-FITC . Mouse IgG1 isotype-PE  and mouse IgG2a-PE  were used for isotype control staining. For ex vivo activation marker analysis, cells were laid in culture for 36 h and surface-stained with anti-CD3-Brilliant Violet 510 (UCHT1), anti-CD4-APC (SK3), anti-CD8-Brilliant Violet 421 (SK1), anti-4-1BB-PE-Cy7 (4B4-1) (from Biolegend), Fixable Viability Dye eFluor 780 and anti-OX40-PE (ACT-235) (from eBioscience).Freshly purified or stimulated CD4Intracellular FoxP3 staining was performed on PBMCs with eBioscience FoxP3/transcription factor staining kit (eBioscience/Thermo Fisher) with anti-human FoxP3-PE (PCH-101) and rat IgG2a isotype control-PE (eBR2a) (from eBioscience), with prior surface co-staining of anti-CD127-APC (A019D5), anti-CD25-FITC (BC96), anti-CD8-Brilliant Violet 421 (SK1), anti-CD4-PE-Cy7 (SK3) and anti-CD3-Brilliant Violet 510 (UCHT1) (from Biolegend).+ T cells were resuspended in 100 μl pre-warmed FACS buffer and 100 μl warm staining solution was added (FACS buffer containing mitotracker red or green and mitotracker deep red stain (100nM each)  utilized as the WT CD46 query.A brouser-adapted Alphafold2 (ColabFold v1.5.2)  -based s+ T cells stimulated for 36 h were assessed using the LEGENDplex human Th1 panel (Biolegend) according to the manufacturer’s instructions and analyzed on the Accuri C6 cytometer (BD). Data analysis was conducted using the FlowJo 10.0.8 software (FlowJo).Accumulated cytokine levels in the supernatant of purified CD4vs. WT CD46 GFP reporter/CMV-promotor pRP[Exp]-EGFP/Puro-CAG>hCD46[NM_172359.2] vectors  was performed on a CD46-knocked out human myeloid HAP-1 cell line . Cells were maintained in Iscove's Modified Dulbecco's Medium (IMDM) (Gibco/Thermo Fisher) supplemented with 10% fetal bovine serum (FBS)  and 1% penicillin/streptomycin (Gibco/Thermo Fisher). To obtain the del21bp CD46 vector, the WT plasmid was subjected to KOD-plus-mediated site-directed mutagenesis  and isolated for single colonies with the following primers:Fw5'-CAGTAGCAATTTGGAGCGAGGTTTTGTGTACACCAC-3'Rev5'-GTGGTGTACACAAAACCTCGCTCCAAATTGCTACTG-3'A transient CD46 overexpression of 21 base pair-deleted (del21bp) CD46 region to confirm correct mutagenesis. 1.0 × 105 of CD46-knocked out HAP-1 cells were transfected with WT and/or del21bp CD46 vectors using Lipofectamine 3000 (Thermo Fisher) in 24-well flat-bottomed plates  for 24 hours, and subsequently stained with anti-human CD46-PE (clone E4.3) for surface and intracellular expression analysis. Intracellular staining was performed with Cytofix/Cytoperm kit (BD). Samples were analyzed on FACS Lyric cytometer (BD) and data were analyzed with FlowJo 10.0.8 software (BD/FlowJo).The designed mutant plasmid was purified with Endofree Maxi plasmid purification kit  and Sanger-sequenced for the full + T cells were resuspended in serum-free unbuffered RPMI-1640 medium (Sigma Aldrich) and were plated onto Cell-Tak-coated  XF96 Seahorse plates  in triplicates at a density of 2.5x105 viable cells per well. The Seahorse cartridge injection ports were loaded with oligomycin (1 μM), Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone  or rotenone , respectively, and metabolic profiles were measured on a Seahorse XF96 Extracellular Flux analyzer (Seahorse Bioscience).Stimulated CD4basal OCR  = OCR before mitochondrial perturbationbasal respiration = basal/total OCR – OCR after rotenone injectionNon-mitochondrial respiration = OCR after rotenone injectionATP coupled respiration = basal/total OCR – OCR after oligomycine injectionLeak respiration = OCR after oligomycine injection – OCR after rotenone injectionmaximal respiratory capacity = OCR peak after FCCP injection – OCR after rotenone injectionbasal ECAR = initial ECAR before mitochondrial perturbationmaximal ECAR = ECAR peak after mitochondrial perturbationMetabolic parameters were calculated as follows:https://gnomad.broadinstitute.org) was <0.01. All listed variants had a sequencing coverage >25 with the exception of the TNFRSF4 and STXBP2 variants, where the coverage was <20 but the heterozygous variants were confirmed by integrated genome viewer (IGV).Whole exome sequencing has been performed as recently described . Rare imThe NIHR Bioresource Rare Disease project has provided whole genome sequencing data for more than 10’000 individuals with rare diseases . We selet tests and one-way analysis of variance (ANOVA) were performed on Prism 8.4.3  with a significance value of p < 0.05.Two-tailed unpaired CD46 variant c.475+1G>A (rs753486842)  analysis identified five individuals from a large family that carried a rare heterozygous 1G>A rs75486842  is 0.000004046 with a CADD score of 23.7—predicting functional relevance. This mutation has been previously reported in a pediatric aHUS patient from Italy [CD46 wild-type mRNA compared to examined healthy controls and the family member that did not carry the mutation , flanking glutamic acid (Glu) residue 158  CD4+ T cells  subpopulations  frequencies within normal reference ranges, both diseased mutation carriers had low Treg  vs. CD127hiCD25low CD4+ T cell populations in mutation carrier 5 (SLE) segregated with intracellular FoxP3 positivity, non-discrepant with two unrelated healthy donors  following T cell receptor ligation plus CD28 co-stimulation compared to controls  . However, 36 h following CD4+ T cell activation in vitro, the mean fluorescence intensity (MFI) of mitotracker deep red  was significantly lower upon CD46 co-stimulation in mutation carriers, likely reflecting decreased surface CD46 expression . In contrast, basal and ATP-coupled respiration . In conclusion, the identified CD46 loss-of-expression mutation was directly linked with altered T cell-intrinsic metabolic adaptation following CD46 co-stimulation.A major activity of CD46 during T cell activation is the induction of increased nutrient influx and T cell metabolic adaptation , includiion Fig. a. No difion Fig. b. Next, ion Fig. c were reers Fig. d+e. MaxiMEFV gene, which has been associated with the autoinflammatory Familial Mediterranean Fever [NOD2 gene, known to be positively associated with Crohn’s disease [TNFAIP3) [CD46 mutation carriers  with a CADD score of 22 predicting functional consequences   and inhiTNFAIP3) . None ofCD46 rare predicted functional relevant IEI-gene variants in CD46 mutation carriers, we filtered the NIHR BioResource Rare Diseases project database for individuals carrying rare CD46 variants and co-analyzed functionally likely relevant (CADD Score >15) rare non-CD46 IEI-gene variants  develop. He has currently minor arthritis of the hands under hydroxychloroquine treatment. There is no current clinical manifestation of the anti-phospholipid syndrome under Aspirin treatment. To our knowledge, this is the first report that demonstrates association of a rare CD46 mutation with both aHUS and SLE in different individuals of the same family. The association of assessed .via the timely co-induction of immune-regulative IL-10 secretion in successfully expanded Th1 cells [CD46 mutations has not been performed. Here, we demonstrate that most CD46 mutation carriers, including healthy carriers, have low or low-normal naïve CD4+ T cell frequencies in peripheral blood. While complete lack of CD46 is accompanied by almost absent Th1 responses [CD46 mutation carriers had reduced Treg frequencies may then skew the overall T cell activity towards being pathologically pro-inflammatory. Although we did not observe significant differences in cytokine production by carrier-derived T cells when activated in bulk, it should be noted that we were unable (due to limited sample volume) to carefully dissect the cytokine production of separate T cell subpopulations . We did not find consistent differences in T cell intrinsic CD46 expression in healthy vs. diseased mutation carriers, although we have not further defined the exact CD46 isoform expression profile. We have recently identified the C3–CD46 axis as a key characteristic of CD4+ and CD8+ T cells and macrophages in tissues [In addition to its complement regulating function, CD46 has been recognized as a co-stimulator of T helper cells favoring Th1 induction , 10. CD4h1 cells . So far,esponses , our dat tissues . It may  tissues .CD46 mutations have common variable immunodeficiency (CVID)-like immunodeficiency [None of the CD46 mutation carriers displayed an overt antibody deficiency in circulation although it has been reported that individuals with homozygous ficiency . We haveCD46 mutations [CD46 rare immune gene alleles impacting on disease penetrance. Indeed, in-depth analysis of our diseased CD46 mutation carriers assessed here showed that they carry private, rare and potentially functionally relevant (high CADD scores) variants in non-CD46 IEI-related genes. The proinflammatory effects of the MEFV and NOD2 variants found in the aHUS patient have been discussed above.Incomplete penetrance is commonly observed in IEI, even those that are highly mortal . Severalutations . This wo+ T-cell sustainment via OX40 [TNFRSF4 variants. Effectively, Treg and follicular T helper cells have been demonstrated to be abnormal in human SLE in an OX40/OX40L-dependent manner [+ T cell-intrinsic OX40 expression is up-regulated by CD46 co-stimulation and that the SLE patient had augmented OX40 expression on CD4+ T cells ex vivo.Polymorphisms in OX40/OX40L associated with enhanced protein expression have been described to be associated with SLE , 57 and via OX40 . Like CDt manner , 60. We CFTR) and DCNT4 [SMAD6) and BMP2 amongst others [CD46 mutation carriers experimentally assessed here and show that individuals carrying rare, disease-causing CD46 missense mutations may co-carry up to ten rare, likely functionally relevant mutations in other IEI genes, including genes driving autoinflammation or complement dysfunction [CD46 mutations have previously been connected with CD46 activity: for example, C4 (encoded by the C4A gene) provides C4b as an (extrinsic or intrinsic) ligand for CD46 and CD46 is a strong STAT5 inducer in T cells [Epistasis has been described as the synergistic interaction of genetic loci potentially modifying disease phenotypes and severity. Epistatic interactions have been described for cystic fibrosis  and genetic factors with clinical manifestations in carriers of a disease-associated ESM 1+: activated T cells. d) Quantification of the indicated serum immunoglobulins. e) Functional assessment of the indicated complement pathways using serum of the individual family members. The blue and green dots represent the aHUS and SLE patient, respectively. Closed symbols represent mutation carriers while the open symbol represents the family member lacking the mutated allele. The lines mark normal reference values. Supplemental Figure 4: Purified CD4+ T cells were left non-activated (NA) or stimulated with immobilized agonistic antibodies against CD3, CD3 + CD28 or CD3 + CD46. Supernatants were harvested 36 hours post stimulation and analyzed by cytometric bead array (CBA). Secretion of the indicated cytokines were normalized to anti-CD3-stimulated cells which was set to 1. All bars indicate means ± SD. The ratio of IFN-γ concentration divided by the IL-10 level in supernatants serves as a marker of proinflammatory CD4+ T cell function and is depicted in a separate graph. All bars indicate means ± SD. Analyzed by unpaired t-tests. Supplemental Figure 5: a) Multiple sequence alignment of human TNFRSF4/OX40 and its orthologues. The Arg10 residue of OX40 in humans and other species are colored in pink. b) Functional region prediction of TNFRSF4/OX40 via SignalP-6.0 . c) Representative TNFRSF4/OX40 expression profiles from one donor and quantification of five individual donors. CD4+ cells from healthy human donors were stimulated for 36h with the indicated antibody combinations. TNFRSF4/OX40 expression was measured on viable cells using anti-human CD134 (OX40) APC-conjugated mAb (ACT35). d) Ex vivo expression of OX40 (X axis) and 4-1BB (Y axis) on PBMC-derived viable CD4+ T cells of CD46 mutation carrier 5 aligned with three healthy controls after 36-hour culture. Supplemental Table 1: Clinical history, as assessed with a standardized case report form, of all studied family members. Supplemental Table 2: a) Clinical manifestations of CD46 mutation carrier 6 during the aHUS flares. b) Complement-related diagnostic-lab analysis during the aHUS flares. Supplemental Table 3: a) Autoantibody profiles of CD46 mutation carrier 5 (SLE patient). b) Complement-related lab analysis in the SLE patient over time. Values in red are above the reference range, values in blue are below the reference range. Supplemental Table 4: The British NIHR BioResource Rare Diseases project database was filtered for individuals carrying rare , likely functionally relevant (CADD Score >15) variants in CD46 which have already been described in patients with CD46 dependent disease. In such individuals, we searched for rare , likely functionally relevant (CADD Score >15) variants in other IEI-related genes Supplemental Figure 1: a) Time of hospitalization, requirement of intensive care and duration of peritoneal dialysis during the five flares of aHUS. b) Laboratory parameters during the five flares of aHUS in family member number 6 (aHUS patient). Supplemental Figure 2: (Top) Clinical and laboratory manifestations of SLE in family member 5. X indicates presence of the criteria (Middle) Serum immunoglobulins in the SLE patient. (Bottom) Quantification of different anti-phospholipid autoantibodies Values in red are above the reference range. Supplemental Figure 3: a+b) The indicated lymphocyte and B cell subpopulations of all tested family members were assessed by flow-cytometry. c) The indicated T cell subpopulations in the SLE patient were repeated 6 years following the initial assessment. CM: central memory; EM: effector memory; TEMRA: terminally differentiated effector memory; RTE: recent thymic emigrants; fTh: follicular T helper; Treg: regulatory T cells; MHCIIommittee . IEI genommittee . Genes lESM 2(JPEG 24 kb)ESM 3(JPEG 36 kb)


Androgen receptor (AR) activation and repression dual-functionality only became known recently and still remains intriguing in prostate cancer (PCa). MYC is a prominent oncogene that functionally entangles with AR signaling in PCa. Further exploration of AR regulatory mechanisms on MYC gene transcription bears clinical and translation significance.Bioinformatics analysis of PCa cell line and clinical RNA-Seq and ChIP-Seq (chromatin immunoprecipitation-sequencing) datasets to anchor interactions of AR and MYC transcriptional networks. ChIP-qPCR and 3C (chromosome conformation capture) analyses to probe MYC distal regulation by AR binding sites (ABSs). CRISPR/Cas9-mediated genome-editing to specify functions of ABS within the 8q24-MYC locus on androgen-mediated MYC transcription. Global FoxA1 and HoxB13 distribution profiling to advance AR transcriptional mechanisms.Here we recognize AR bi-directional transcription mechanisms by exploiting the prominent 8q24-MYC locus conferring androgen hyper-sensitivity. At ~ 25 Kb downstream of the MYC gene, we identified an undefined ABS, P10. By chromatin analyses, we validated androgen-dependent spatial interaction between P10 and MYC-Promoter (MYC-Pro) and temporal epigenetic repression of these MYC-proximal elements. We next designed a CRISPR/Cas9-mediated double genomic knock-out (KO) strategy to show that P10-KO slightly lessened androgen-elicited MYC transrepression in LNCaP-AR cells. In similar genomic editing assays, androgen-mediated MYC repression became slightly deepened upon KO of P11, an ABS in the PVT1 gene locus highly enriched in AR-binding motifs and peaks. We also investigated multiple ABSs in the established PCAT1 super-enhancer that distally interacts with MYC-Pro for transactivation, with each KO pool consistently shown to relieve androgen-elicited MYC repression. In the end, we systemically assessed androgen effects in the 8q24-MYC locus and along PCa genome to generalize H3K27ac and BRD4 re-distribution from pioneer factors (FoxA1 and HoxB13) to AR sites.Together, we reconciled these observations by unifying AR dual-functions that are mechanistically coupled to and equilibrated by co-factor redistribution.The online version contains supplementary material available at 10.1186/s12967-023-04429-4. The advancements in chromatin and epigenetics technologies in recent years have enabled the exposure of AR transrepressive functions that remain elusive for decades –3. In coMYC is a prominent oncogene functionally entangled with AR signaling and its overexpression renders androgen insensitivity and invasive PCa transformation –9. ClustMYC could trigger immediate and reversible cancer-driven signals, making it compelling to specify the regulatory mechanisms of the MYC gene . MYC is Our current report was designated to define the mechanisms of androgen-elicited dual-transcriptional functions in global and local contexts. For this purpose, we focused on the 8q24-MYC locus that contains multiple AR binding sites (ABSs) in annotation with PCa-specific enhancers. Using an innovative CRISPR/Cas9-mediated genome-editing strategy, we deleted several key ABSs from the left and right arms of this TAD, respectively. The outcomes of these KO tests were consistent with the concept that MYC transcription is coupled to AR direct activation and indirect repression functions. In the end, we further took bioinformatics to systemically dissect androgen effects in the 8q24-MYC locus and along the PCa genome, demonstrating that H3K27ac and BRD4 are re-distributed from pioneer factors (FoxA1 and HoxB13) to AR sites. Collectively, our findings recognized AR-mediated transcription is intrinsically equilibrated between its dual-functions that are coupled to co-factor exchanges.DHT , G418 , Puromycin , Hygromycin B , Blasticidin , Lenti-gRNA-Neo (G418) vector  and Lenti-gRNA-Puro vector . Transfections were conducted using Lipofectamine 2000 , following the manufacturer’s direction.AR , BRD4 , H3K27ac , H3K4me1 , H3K4me2 , H3K9me1 , H3K9me2 , H3ac , FoxA1 , Med1 , CDK9 , cyclin T1 , p-RNA Pol II-Ser5 , and IgG .LNCaP cells were grown in RPMI medium containing 10% FBS and VCaP cells were grown in DMEM medium containing 10% FBS. LNCaP AR overexpression (OE) stable line (LNCaP-AR or LA) was generated with lentiviral vector expressing Flag-tagged AR and hygromycin B selection; LNCaP-AR Cas9 OE stable line (LNCaP-AR-Cas9 or LAC) was generated based on lentiviral vector expressing Cas9, under Hygromycin B and Blasticidin selection . For CRIThe procedure was reported . BrieflyThe detailed information was included in Additional file The test was conducted using the Fast Virus 1-step kit . The TaqMan primer–probe sets were all from Life Technologies (Applied Biosystems). Primer/FAM-probe sets: MYC (Hs00153408_m1), PSA/KLK3 (Hs02576345_m1), PVT1 (Hs01069041_m1); and the CCAT1 gene TaqMan primer/FAM-probe set: forward primer: GGCCAGCCCTGCCACT; reverse primer: CAGTTTTCAAGGGATTTTAGGAGAA; and probe: ACCAGGTTGGCTCTGTATGGCTAAGCGT. The inventoried internal control is the GAPDH primer/VIC-probe set (4310884E).ChIP-Seq and RNA-Seq datasets used in this study are retrieved from the GEO database, as listed below: LNCaP AR ChIP-Seq, GSE83860 ; LNCaP HRaw ChIP-Seq data were trimmed and then mapped to either Hg19 or Hg38 reference genome as indicated. MACS2  and ucscGenerally, data in bar graphs represent mean ± SD of three biological replicates and are representative of three independent experiments. Two-tailed unpaired Student’s t-test was performed to calculate the statistical significance of two independent groups. P < 0.05 was considered statistically significant: *P < 0.05, **P < 0.01, ***P < 0.001.Recent advancements on chromatin techniques revealed the bi-directionality of AR-mediated transcription: transactivation versus transrepression –3. ConsiThe prominent 8q24-MYC locus is a gene desert featured in residency of MYC and flanking lncRNAs and multiple prostate risk regions , 20. MecMYC is known to have no proximal enhancers and depends on distal elements for transactivation , 19, andTo further expose the mechanisms of androgen-mediated MYC transrepression, we monitored epigenetics of the MYC-Pro and P10 sites in DHT-treated VCaP with ChIP-qPCR analyses. At both sites, androgen stimulated AR and FoxA1 occupancy, enhanced the transrepressive markers H3K9me1 and H3K9me2, and repressed the transactive markers H3K4me1 and H3K4me2  under G418 selection and a downstream gRNA pair (D1/D2) under Puro selection, respectively . As shown in Fig. We next similarly targeted an ABS P11) in the MYC 3′ flanking lncRNA PVT1 region that is enriched in AR binding motifs (ABMs) Fig. A. Consis1 in the The control versus KO pools in androgen-depleted medium were then subjected to DHT treatment as indicated, followed by TaqMan RT-PCR assay for MYC expression Fig. B–E. As sPrevious report established the PCAT1 SE as a robust developmental and locus-wide enhancer that distally engaged with MYC-Pro , 20. We As aforementioned, co-factor re-distribution intrinsically couples with androgen-elicited transcriptional activation and repression. Specifically, each individual androgen-elicited gene and chromatin site exhibited distinct responsive profiling; for example, the magnitude and duration of androgen responsiveness are different among genes in the left arm, middle section, and right arm of the 8q24-MYC locus [To generalize our findings along the PCa genome, we next took global bioinformatics strategy on VCaP datasets. For this purpose, we focused on FoxA1 and HoxB13, two key pioneering factors in PCa AR reprogramming , 32. By The above findings supported that androgen elicited indirect repressive effects by transferring co-activators from FoxA1 and HoxB13 sites to AR sites. To affirm these findings, we next systemically addressed chromatin occupancy of these factors at AR-binding sites along the VCaP genome. As shown, DHT impacted negatively basal-AR binding affinity but positively liganded-AR binding affinity; similar trends were noted on H3K27ac binding affinity at these AR sites, consistent with re-distribution of H3K27ac from androgen-repressed to androgen-activated AR sites  PCa cell lines. As shown in the androgen-refractory LNCaP-abl cells, FoxA1 KD by siRNA had repressive effects on ABS enhancers (particularly the PCAT1 SE) on left arm of this locus and MYC transcription  PCa cells impacted 8q24-MYC gene locus activities and MYC gene expression. Additional information on materials and methods


Antimicrobial Resistance & Infection Control (2023) 12:6910.1186/s13756-023-01265-5The original article [1] contains an error in Affiliation #2. The correct affiliation can be viewed in this Correction article and is as follows:‘Mahidol-Oxford Tropical Medicine Research Unit, Bangkok, Thailand.’


The miR-200b/429 located at 1p36 is a highly conserved miRNA cluster emerging as a critical regulator of cervical cancer. Using publicly available miRNA expression data from TCGA and GEO followed by independent validation, we aimed to identify the association between miR-200b/429 expression and cervical cancer. miR-200b/429 cluster was significantly overexpressed in cancer samples compared to normal samples. miR-200b/429 expression did not correlate with patient survival; however, its overexpression correlated with histological type. Protein–protein interaction analysis of 90 target genes of miR-200b/429 identified EZH2, FLT1, IGF2, IRS1, JUN, KDR, SOX2, MYB, ZEB1, and TIMP2 as the top ten hub genes. PI3K–AKT and MAPK signaling pathways emerged as major target pathways of miR-200b/429 and their hub genes. Kaplan–Meier survival analysis showed the expression of seven miR-200b/429 target genes  to influence the overall survival of patients. The miR-200a-3p and miR-200b-5p could help predict cervical cancer with metastatic potential. The cancer hallmark enrichment analysis showed hub genes to promote growth, sustained proliferation, resistance to apoptosis, induction of angiogenesis, activation of invasion, and metastasis, enabling replicative immortality, evading immune destruction, and tumor-promoting inflammation. The drug–gene interaction analysis identified 182 potential drugs to interact with 27 target genes of miR-200b/429 with paclitaxel, doxorubicin, dabrafenib, bortezomib, docetaxel, ABT-199, eribulin, vorinostat, etoposide, and mitoxantrone emerging as the top ten best candidate drugs. Taken together, miR-200b/429 and associated hub genes can be helpful for prognostic application and clinical management of cervical cancer.The online version contains supplementary material available at 10.1007/s10528-023-10356-2. Cervical cancer is a malignancy of the cervix, with 570 000 cases and 311 000 deaths globally in 2018  changes responsible for carcinogenesis and provided an opportunity to translate these molecular changes as markers for improved clinical management of cervical cancer . We used GEO datasets to strengthen our data as TCGA has fewer normal samples. The GEO datasets analyzed included GSE86100  , respectively. The associations between members of the miR-200b/429 cluster with clinical parameters were tested by MEXPRESS  using UALCAN   of miR-200b/429 targets were undertaken using Enrichr. Gene ontology analysis consisted of enrichment for biological process (BP), molecular function (MF), and cellular components (CC) . The expression of individual members of the miR-200b/429 cluster was evaluated in TCGA-CESC, GSE86100, and GSE105409, datasets consisting of 24 normal and 346 cervical cancer samples. The miR-200b/429 cluster expression was significantly higher in tumor samples compared to normal samples in TCGA-CESC, GSE86100, and GSE105409 datasets , miR-200b 1.53-fold), and miR-429 (1.60-fold) were significantly higher in our small cohort of cervical cancer samples when compared with the normal cervix . The overlapping analysis between 5762 differentially expressed genes (1851 up to and 3911 down) with 510 targets of miR-200b/429 identified 90 differentially expressed genes as targets .The miRNA–miRNA co-expression network constructed using the COMETA tool identified miR-200a, miR-200b, miR-429, and miR-1278 as members of the co-expression as the immediate members of the interaction network . Our analysis has shown the association of hub genes with B-cell, T-cell CD4+, and T-cell CD8+ with a p-value < 0.05 (Supplementary Table 7). To determine the gene expression in the same tissue, we used TCGA-CESC datasets to analyze the gene expression. Next, we performed a Pearson correlation analysis of miRNA and mRNA expression from the TCGA datasets using TACCO , Fms-related receptor tyrosine kinase 1 (FLT1), Insulin-like growth factor 2 (IGF2), Insulin receptor substrate 1 (IRS1), Jun proto-oncogene, AP-1 transcription factor subunit (JUN), Kinase insert domain receptor (KDR), SRY-box transcription factor 2 (SOX2), MYB proto-oncogene transcription factor (MYB), Zinc finger E-box-binding homeobox 1 (ZEB1), and TIMP metallopeptidase inhibitor 2 (TIMP2) as the top ten hub genes of the target network consisting of 10 nodes, 25 edges with a PPI enrichment p-value of 3.42e-14 . We also performed the 10 hub genes systemic analysis of immune infiltrate across the TCGA-CESC cohort .The KM survival analysis showed that the miR-200b/429 cluster expression did not significantly affect patient survival. We next analyzed the prognostic significance of 10 hub genes in patient survival. Interestingly, the differential expression of 7 genes  was shown to influence patient survival as analyzed by the KM plotter Fig. . Next, wWe next investigated the potential of ten hub genes in the induction of cancer hallmarks using CHG online tool. Among the ten hub genes, only data were available for 9 genes. The CHG analysis showed that abnormal expression of hub genes  might facilitate participating in pathways leading to the promotion of growth, sustained proliferation, resistance to apoptosis, induction of angiogenesis, activation of invasion, and metastasis, enabling replicative immortality, evading immune destruction, and tumor-promoting inflammation (Supplementary Table 8).The potentially druggable genes of miR-200b/429 targets were predicted using the DGIdb database. Screening using DGIdb predicted 27 genes and 182 potential drugs. Furthermore, the interaction network of 27 genes using STITCH identified 34 nodes and 41 edges with a PPI enrichment p-value of 2.02e-05. Paclitaxel, doxorubicin, dabrafenib, bortezomib, docetaxel, ABT-199, eribulin, vorinostat, etoposide, and mitoxantrone as the top 10 best candidate drugs emerged out of Pandrug analysis (Supplementary tables 9 and 10).Despite the progress and improvement in diagnosis and prognosis, cervical cancer is one of the key public health problems in many developing and under-developed countries , has been shown to promote cervical cancer cell proliferation and metastasis by inhibiting Forkhead box G1 (FOXG1) —Information about miR-200b/429 cluster. a) Genomic organization of miR-200b/429 cluster. b) Conservation analysis for miR-200b/429, c) Venny analysis of common genes targeted by miR-200b/429 cluster, and d) common genes targeted by miR-200b/429 cluster and significant differential expressed genes in TCGA-CESC datasetsSupplementary file2 (TIF 8037 KB)—a)The PPIN of miR-200b/429 cluster. b) Identification of hub genes by protein-protein interaction analysisSupplementary file3 (TIF 9452 KB)—The pathway enrichment analysis for 10 hub genesSupplementary file4 (TIF 8342 KB)—a and b) Association of miR-200b/429 and hub genes with cervical cancer metastasisSupplementary file5 (TIF 3252 KB)—Overall survival model based on 10 hub genesSupplementary file6 (XLSX 11 KB)—Details of the datasets used in the studySupplementary file7 (XLSX 35 KB)—TCGA_CESC- Sociodemographic and clinical informationSupplementary file8 (XLSX 15 KB)—List of genes targeted by miR-200b/429 clusterSupplementary file9 (XLSX 9 KB)—Common genes targeted by miR-200b/429 clusterSupplementary file10 (XLSX 347 KB)—List of differentially expressed genes in TCGA_CESCSupplementary file11 (XLSX 10 KB)—Common Genes targeted by mir-200b/429 and differential expressed in TCGA_CESC datasetsSupplementary file12 (XLSX 17 KB)—Association of hub genes with B-cell, T-cell CD4+, and T-cell CD8+Supplementary file13 (XLSX 11 KB)—Association of hub genes with cancer hallmarksSupplementary file14 (XLSX 21 KB)—List of Druggable genesSupplementary file15 (XLSX 17 KB)—Gene-drug interactionsBelow is the link to the electronic supplementary material.