C. elegans a mutation in the mitochondria-localized protein SPD-3 leads to elevated levels of Polo-like kinase 1, causing abnormal chromosome positioning during mitosis.In  Caenorhabditis elegans embryos. SPD-3 mutant embryos contain abnormally positioned mitotic chromosomes, show a delay in anaphase onset and asymmetrically disassemble the nuclear lamina. We found that more PLK-1 accumulated on centrosomes, nuclear envelope, nucleoplasm, and chromatin before NEBD, suggesting that PLK-1 overexpression is responsible for some of the observed mitotic phenotypes. In agreement with this, the chromosome positioning defects of the spd-3(oj35) mutant could be rescued by reducing PLK-1 levels. Our data suggests that the mitochondrial SPD-3 protein affects chromosome positioning and nuclear envelope integrity by up-regulating the endogenous levels of PLK-1 during early embryogenesis in C. elegans. This finding suggests a novel link between mitochondria and nuclear envelope dynamics and chromosome positioning by increasing the amount of a key mitotic regulator, PLK-1, providing a novel link between mitochondria and mitosis.In metazoans, Polo-like kinase (PLK1) controls several mitotic events including nuclear envelope breakdown, centrosome maturation, spindle assembly and progression through mitosis. Here we show that a mutation in the mitochondria-localized protein SPD-3 affects mitotic events by inducing elevated levels of PLK-1 in early  PLK-1 is a crucial mitotic kinase that functions in many aspects of cell division, such as centrosome maturation  mutant, which was previously shown to impact spindle alignment (spd-3(oj35) mutant was isolated in an ethyl methanesulfonate mutagenesis screen in search of cell division mutants and carries a single cytosine-to-thymidine transition, resulting in a leucine-to-phenylalanine change at amino acid 130 (spd-3(oj35) strain is a temperature-sensitive, maternal-effect mutant, which is defective in meiosis (spd-3(oj35) mutant, three additional mutant strains are available (spd-3(ok1817) and spd-3(tm2969), are null spd-3 alleles (spd-3(me85) carries an early stop mutation and has severe meiotic defects (spd-3 mutation in the spd-3(oj35) strain.To further determine the functions of the mitochondrial protein SPD-3 during mitosis, we characterized the lignment . The spdacid 130 . The spd meiosis  and mito meiosis . In addivailable . Two del alleles  and are  defects , Video 1 defects . Thus, wVideo 1spd-3(me85) embryos during mitosis. The WT, (left) and spd-3(me85) (right) embryos coexpressing GFP::β-tubulin, mCherry::γ-tubulin, and mCherry::histone H2B. Fluorescence confocal images were acquired every 15 s by collecting 17 z-planes at 1.0-μm intervals. Sale bar represents 10 μm.Download videoLive-cell imaging of WT and spd-3(oj35), we filmed embryos coexpressing mCherry::γ-tubulin and mCherry::histone H2B (spd-3(oj35) embryos, resulting in a mitotic spindle, that is, misaligned to the A/P axis. Consistent with the previous report (spd-3(oj35) embryos (spd-3(oj35) embryos are shorter in comparison to control spindles (spd-3(oj35) 12.7 ± 1.4 μm) and centrosomes are enlarged in the spd-3(oj35) (spd-3(oj35) mutant embryos. Although the spd-3(oj35) mutant embryos display a similar duration of the time from pronuclear meeting to NEBD (spd-3(oj35) (spd-3(oj35) embryos had not formed metaphase plates at this time point. These results suggest that spd-3(oj35) does not only affect spindle positioning but also spindle length and the timing of mitotic events.To characterize the effects on mitotic spindle alignment in tone H2B . Followitone H2B . This ros report , we obse embryos . Moreove-3(oj35) . Quantif to NEBD , the dur-3(oj35) . We alsoVideo 2spd-3(oj35) embryos during mitosis. The WT (left) and spd-3(oj35) (right) embryos coexpressing GFP::β-tubulin, mCherry::γ-tubulin, and mCherry::histone H2B. Fluorescence confocal images were acquired every 15 s by collecting 17 z-planes at 1.0-μm intervals. Sale bar represents 10 μm.Download videoLive-cell imaging of WT and spd-3(oj35) embryos we noticed that the mutant embryos display an additional unusual phenotype during chromosome positioning and alignment. Instead of forming a normal metaphase plate in the center of the embryo, the individual sets of chromosomes  are initially positioned on the periphery of the respective nuclei, giving rise to a transient diamond–shaped spindle configuration (spd-3(oj35) were still in the SDC state at this point. Once a metaphase plate forms in the spd-3(oj35) embryos, it is significantly wider (spd-3(oj35): 7.3 ± 1.4 μm) in comparison to control metaphase plates. This observation suggests an additional role for SPD-3 during chromosome congression.During our analysis of mitosis in the guration , Video 2guration  as the tguration . In addiC. elegans leads to pronuclear migration defects, and the spindle forms at the posterior, orients incorrectly, and contains unusually short microtubules. It was shown that zyg-9 (RNAi) also leads to a strong decrease in microtubule nucleation rate and a nearly threefold decrease in microtubule growth rate (spd-3(oj35) embryos that could affect spindle and maybe chromosome positioning, we generated a spd-3(oj35) strain that expressed EBP-2, a marker of growing microtubule plus-ends (spd-3(oj35) (157 ± 26) in comparison to control (125 ± 21) (spd-3(oj35) (0.51 ± 0.1 μm/s) in comparison to control (0.61 ± 0.01 μm/s) (spd-3(oj35)) (spd-3(oj35) that grow with a decreased velocity, the approximate total length of microtubules, calculated by growth rate and duration of track, is only marginally different. In addition, the change in microtubule growth rate in the spd-3(oj35) is rather small, that is, in comparison to the ∼threefold reduction in ZYG-9–depleted embryos, suggesting that this might not be the main driver of spindle misalignment.Previous publications have shown that changes in microtubule nucleation and growth rate could affect spindle positioning. As an example, RNAi of ZYG-9 in wth rate , which mlus-ends . We quanlus-ends , Video 325 ± 21) , suggest01 μm/s) . The dur3(oj35)) . OverallVideo 3spd-3(oj35) expressing EBP-2::GFP embryos during mitosis. The WT (left) and spd-3(oj35) (right) embryos expressed EBP-2::GFP. Fluorescence confocal images were acquired at 400 m sec intervals for 1 min. Sale bar represents 10 μm.Download videoLive-cell imaging of WT and spd-3(oj35) showed an increase in the duration from NEBD to anaphase onset, suggesting a potential delay in anaphase onset. As anaphase onset is regulated by the SAC (spd-3(oj35) embryos (spd-3(oj35) with and without Mad2 and Mad3 (spd-3(oj35) after depletion of Mad2 and Mad3. However, we noticed that in spd-3(oj35) embryos, the treatment of mdf-2/Mad2(RNAi) and san-1/Mad3(RNAi) often resulted in chromosome segregation that occurred before or in absence of the formation of a metaphase plate (six out of eight embryos), suggesting defects in chromosome congression or alignment, and an increase in spindle alignment defects (eight out of eight embryos) (spd-3(oj35).Our analysis of the  the SAC , we test embryos , Video 4and Mad3 . This anembryos) . OverallVideo 4mdf-2- and Mad3san-1-depleted embryos during mitosis. The WT (top) and spd-3(oj35) (bottom) embryos coexpressing GFP::β-tubulin, mCherry::γ-tubulin, and mCherry::histone H2B without (left), and with Mad-2mdf-2(RNAi) (middle) or Mad3san-1(RNAi) treatment. Fluorescence confocal images were acquired every 15 s by collecting 17 z-planes at 1.0-μm intervals. The images were processed by maximum projection. Sale bar represents 10 μm.Download videoLive-cell imaging of Mad2spd-3(oj35) could lead to reduced function of cytoskeletal motors, possibly caused by variations in ATP levels, we measured the metabolic rates in spd-3(oj35) embryos by fluorescence lifetime imaging microscopy (FLIM) of NADH autofluorescence (spd-3(oj35) embryos are increased in comparison to WT   and immurescence . FLIM NArescence . NADH aurescence . These mon to WT . This agon to WT .C. elegans (clk-1(qm30) for defects in spindle and chromosome positioning but could not reproduce any of the observed phenotypes of the spd-3(oj35) mutant in respect to spindle and chromosome positioning. We further analyzed the effects of isp-1(qm150), ucr-1 (RNAi), mev-1 (RNAi), or cco-1 (RNAi), which alter the ETC function, resulting in decreased ATP levels (To test if elevated (or reduced) ATP could lead to similar mitotic phenotypes, we examined the effect of depleting proteins that are part of the electron transfer chain (ETC) in  elegans   worms may not directly lead to the observed chromosome-positioning defects. In agreement with this, also treatment of control embryos with mitochondrial inhibitors (spd-3(oj35) phenotype, suggesting that the observed phenotype is not based on misregulation of ATP levels alone.In summary, although the disruption of some mitochondrial genes did affect spindle alignment, it did not mimic the SDC phenotype, suggesting that metabolic perturbations in hibitors  did not spd-3(oj35) mutant, we generated a mutant strain that co-expressed GFP-labeled SP12, an ER lumen marker, and mCherry-labeled histone. Analysis of this strain revealed that the morphology of the ER is severely affected in the spd-3(oj35) mutant (spd-3(oj35) mutant, ER clusters begin to form earlier, at 98.3 ± 15.5 s before NEBD (spd-3(oj35) embryo (spd-3(oj35) embryos (pd-3(oj35), suggesting a potential role for SPD-3 in these processes.As we observed an effect on the timing from NEBD to anaphase, we decided to further investigate the arrangement of ER and nucleus. The ER surrounds and encloses the pronucleus, forming a characteristic bi-membraned nuclear envelope that protects the chromatin. To determine the arrangement of the ER in the ) mutant . The ER ) mutant . In gene) mutant , with mo) mutant . In cont) mutant  and beco) mutant . In the ore NEBD  and are ) embryo . In addi embryos . These rVideo 5spd-3(oj35) expressing GFP::SP12 embryos during mitosis. The WT (left) and spd-3(oj35) (right) embryos coexpressing GFP::SP12 and mCherry::histone H2B. Fluorescence confocal images were acquired every 15 s by collecting 17 z-planes at 1.0-μm intervals. Sale bar represents 10 μm.Download videoLive-cell imaging of WT and spd-3(oj35) worms with rab-5 (RNAi) and yop-1 (RNAi) with the aim to reduce the number of ER clusters in the cytoplasm. Although we did observe fewer and smaller ER clusters in the spd-3(oj35) mutant after rab-5 and yop-1 depletion (yop-1 (RNAi); however, after rab-5 (RNAi), the spindle misalignment was slightly reduced from 22.8 ± 8.1 in spd-3(oj35) to 13.4 ± 6.1 in spd-3(oj35) treated with rab-5 (RNAi) (yop-1(RNAi) and rab-5 (RNAi) lead to a decrease in the duration between NEDB and anaphase onset  could shorten the overall duration of this time period in spd-3(oj35). It is however also possible that the extended duration in spd-3(oj35) is a consequence of disrupting the ER structure. In summary, this data suggest that the SDC phenotype observed in the spd-3(oj35) is unrelated to defects in the ER structure, whereas the duration from NEBD to anaphase onset and the spindle mis-orientation could be in part induced by the ER cluster formation.To determine the potential effects of ER morphology on spindle and chromosome positioning, we analyzed a number of ER mutants that show drastic changes in ER morphology, in particular, the ratio of clusters to tubes. The RAB-5 and YOP-1/RET-1 proteins are important for regulating the ER morphology during mitosis and controlling the kinetics of nuclear envelope disassembly . The endepletion , the SDCepletion . We also5 (RNAi) . Interesse onset . In C. eassembly . As we qER) is changed in the spd-3(oj35) mutant. During C. elegans UPRER, two homologs of the ER-resident heat-shock protein BiP, HSP-3, and HSP-4 are essential for the formation of sheet-like ER structures (hsp-4 gene in C. elegans (ER in spd-3(oj35), we imaged worms expressing the zcIs4 transgene, a transcriptional fusion of the hsp-4 promoter to GFP that can be used to quantify HSP-4 expression levels (zcIs4 reporter is strongly up-regulated in response to treatments inducing ER stress and can thus function as a readout for UPRER activity (spd-3(oj35) mutant worms by quantification of fluorescence levels, indicating an approximately 11-fold decrease in hsp-4::gfp expression  mutant. To further determine whether the mitotic defects could be rescued by increasing HSP-4, we treated spd-3(oj35) mutant with hsp-3 (RNAi) to increase the endogenous hsp-4 transcription (spd-3(oj35) mutant are not prevented by hsp-3 (RNAi) (hsp-3 (RNAi) does not affect ER dynamics in the early embryo. In contrast, when HSP-4 function is reduced by RNAi, the ER accumulates in multiple foci, and the ER morphology is distinctly disrupted (hsp-4 (RNAi), we saw a slight increase in spindle misalignment, from 4.5 ± 1.2 in untreated to 12.3 ± 4.1 in hsp-4 (RNAi)–treated embryos (spd-3(oj35) resulted in severely affected embryos that could not be analyzed, suggesting that hsp-4 (RNAi) reduced the viability of spd-3(oj35). Taken together, our results suggest that perturbation of the mitochondrial protein SPD-3 affects the morphology of the ER and that this might be induced by the UPRER regulatory system. However, the structural changes of the ER are most likely not responsible for the observed chromosome positioning defects but could contribute to the spindle alignment defects in the spd-3(oj35) mutant embryos.Next, as ER dynamics and morphology changes can also be induced by ER stress, we tested whether the ER stress–unfolded protein response (UPRructures . Depleti elegans . To examn levels . The zcIactivity . We founpression . This recription . However3 (RNAi) . Becauseisrupted . We did isrupted . In cont embryos . Depletispd-3(oj35) mutants. As the ER and the nucleus are intimately connected, we generated strains that carry either NPP-22::mNeonGreen (NPP-22::mNG) (spd-3(oj35), consistent with the observed changes in ER morphology surrounding the spindle (spd-3(oj35) mutant background showed that nuclear lamin is disassembled completely around 180 s after NEBD in both WT and spd-3(oj35) embryos (spd-3(oj35) embryos. This asymmetry in nuclear disassembly is not observed in control embryos (spd-3(oj35) mutant.Our analysis of the ER morphology revealed a change in the shape of the pronucleus in 22::mNG)  (Video 622::mNG) , which e spindle  and 3A.  spindle . In addi spindle . Analysi embryos . However embryos , suggestVideo 6spd-3(oj35) expressing NPP-22::GFP embryos during mitosis. The WT (left) and spd-3(oj35) (right) embryos coexpressing NPP-22::GFP and mCherry::histone H2B. Fluorescence confocal images were acquired every 15 s by collecting 17 z-planes at 1.0-μm intervals. Sale bar represents 10 μm.Download videoLive-cell imaging of WT and Video 7spd-3(oj35) expressing LMN-1::GFP embryos during mitosis. The WT (left) and spd-3(oj35) (right) embryos coexpressing LMN-1::GFP and mCherry::histone H2B. Fluorescence confocal images were acquired every 15 s by collecting 17 z-planes at 1.0-μm intervals. Sale bar represents 10 μm.Download videoLive-cell imaging of WT and spd-3(oj35) embryos as it is initiated 101.7 ± 18.0 s and completed 182.0 ± 29.0 s after anaphase onset. Although NE reformation around the segregated chromosomes is a little bit delayed in the spd-3(oj35) embryos, the duration of NE reformation does not differ between control and spd-3(oj35) embryos  embryos  mutant to monitor the localization and expression of PLK-1. To our surprise, this strain revealed excessive amounts of PLK-1 in the spd-3(oj35) mutant localizing to centrosomes, the nuclear envelope, nucleoplasm, and chromosomes (spd-3(oj35) mutant compared with N2 animals by immunoblot (plk-1 was not changed in spd-3(oj35) as assessed by RT-qPCR (spd-3(oj35) mutants have generally increased gene expression, we tested the levels of Aurora B (AIR2) (spd-3(oj35) (P = 0.0664), all measured protein levels of AIR-2 in the spd-3(oj35) were below the levels detected for control embryos, suggesting some potential effect on AIR-2 levels  mutant. Interestingly, the decrease in DNC-2 levels could be involved in the observed spindle positioning defect, as has been shown for dynein depletion (Nuclear disassembly is triggered by phosphorylation of lamin at multiple residues in the head and tail domain by PLK-1 in  elegans . Therefoomosomes . We furtmunoblot . However RT-qPCR . To testB (AIR2) , another-3(oj35) . We foun-3(oj35) . Althoug2 levels . Interes2 levels , in contepletion .Video 8spd-3(oj35) expressing PLK-1::GFP embryos during mitosis. The WT (left) and spd-3(oj35) (right) embryos expressing PLK-1::GFP. Fluorescence confocal images were acquired every 10 s by collecting 31 z-planes at 0.5-μm intervals. The images were processed by maximum projection. Sale bar represents 10 μm.Download videoLive-cell imaging of WT and C. elegans, PLK-1 is recruited to the nuclear pore complexes (NPC) by nucleoporins NPP-1, NPP-4, and NPP-11 before NEBD where the importins α/β IMA-2 and IMB-1 promote the nuclear import of PLK-1, where it phosphorylates nuclear lamin (ima-2 (RNAi) is essential for spindle assembly and nuclear envelope formation in the C. elegans embryo (spd-3(oj35) embryos to about 3 min before anaphase onset. In addition, we detected an overall increased fluorescence of PLK-1::GFP in the paternal pronucleus relative to the maternal pronucleus  embryos (In ar lamin . In addis embryo . To detes embryo . This peonucleus . This reonucleus . In addi mitosis . Surpris embryos . This re embryos . These dspd-3(oj35) worms to ima-2 (RNAi) to prevent nuclear import of PLK-1. In control embryos ima-2 (RNAi) prevents the nuclear import of PLK-1 and thus its localization to kinetochores and chromatin (spd-3(oj35) mutant embryos, PLK-1::GFP still localizes to nucleoplasm and chromatin after ima-2 (RNAi) in embryos (ima-2 (RNAi) supporting the hypothesis that this phenotype is induced by elevated amounts of PLK-1 in the nucleus  worms to short treatments of plk-1 (RNAi) to reduce the elevated levels of PLK-1 to approximately WT levels . We found that the ER morphology and distribution of ER clusters are not different between control and plk-1 (RNAi) in spd-3(oj35) embryos, suggesting that the changes in ER morphology are not induced by elevated PLK-1 levels  embryos  phenotype, changes in the ER structure and a dysregulation of mitotic events in  embryos .spd-3(oj35) embryos. PLK-1 is a master regulator in mitosis with multiple important roles to ensure the proper progression through mitosis such as chromosome alignment (C. elegans PLK-1 was shown to be involved in establishment of polarity (spd-3(oj35) embryos show a number of defects that could be linked to PLK-1 overexpression.Our results revealed an increase in PLK-1 levels in the lignment , anaphaslignment , and cytlignment . In C. epolarity , centrospolarity , nuclearpolarity , and dispolarity . In agreThe involvement of PLK1 in the regulation of the SAC is well established. PLK1, together with MPS1, phosphorylates several proteins involved in the establishment and maintenance of the SAC, such as Bub1, KNL1, and Mad1 and CDC20 . This phspd-3(oj35) embryos exhibit an extended duration of the time between NEBD and anaphase onset. However, this prolonged duration does not appear to be dependent on the presence of SAC proteins. All spd-3(oj35) embryos showed the SDC phenotype and with this a delay in metaphase plate formation with and without SAC proteins (Mad3 and Mad2). Interestingly, our data showed that chromosome and spindle positioning defects were elevated in embryos depleted of SAC proteins (Mad3 and Mad2) and often anaphase was initiated before the formation of a metaphase plate. Conversely, in untreated spd-3(oj35) embryos, with an intact SAC, anaphase onset only happened after a metaphase plate was established. These data suggest that the SAC contributes to reduce the chromosome and spindle alignment defects in the spd-3(oj35) embryos. Interestingly, it was previously shown that in C. elegans, SAC components like SAN-1 and MDF-2 are vital for preventing errors in cell division under extreme stress conditions, that is, anoxia (spd-3(oj35) embryos and further research will be required to address this question.In light of our own experimental findings, we have noted that , anoxia . Under oin spd-3(oj35) embryos could lead to the SDC phenotype affecting kinetochore-microtubule attachments and with this metaphase plate formation. In agreement with this, our data show that reducing PLK-1 levels indeed prevented the SDC phenotype, whereas we were not able to rescue the delay in anaphase onset. This could be due to difficulties reducing PLK-1 levels in the spd-3(oj35) embryos to WT levels. In addition, because PLK-1 is a key regulator of mitosis, already small deviations from the regular levels have major effects on mitosis. It is however also possible that the anaphase delay is independent of PLK-1 levels and rather a consequence of SAC activation.PLK1 was also suggested to play a role in stabilizing the kinetochore–microtubule attachment during prometaphase and it is possible that the elevated levels of PLK-1 spd-3(oj35) after rab-5(RNAi) and yop-1(RNAi). It was previously shown that RAB-5 and YOP-1, together with RET-1 are involved in ER structure and promoting nuclear envelope disassembly. Depletion of either RAB-5 or YOP-1/RET-1 leads to a delay in NEBD (rab-5 and yop-1(RNAi) could shorten the overall duration between NEBD and anaphase onset in spd-3(oj35) to WT levels. It is however also possible that the extended timing between NEBD and anaphase onset in spd-3(oj35) is a consequence of disrupting the ER structure, as it has been suggested that the structure of the ER could potentially affect nuclear disassembly (Along this line we detected a decrease in the duration in the time between NEBD to anaphase onset of  in NEBD . As we qassembly .spd-3(oj35), and the reported reduction in mobility of SUN-1 aggregates and pairing-center binding proteins on the nuclear envelope in spd-3(me85) mutant  mutant  could bespd-3(oj35) (spd-3(oj35).SUN1 is a connector between the cytoskeleton and the chromosomes during pairing and alignment of homologous chromosomes during meiosis. PLK-1 phosphorylates SUN1, which regulates its function and localization . In part-3(oj35) . A decreucr-1 (RNAi), mev-1 (RNAi), and cco-1 (RNAi), which alter the ETC function, resulting in decreased ATP levels, suggesting that mitochondrial ETC pathways could also affect mitotic spindle alignment, possibly even by affecting motor proteins or other energy-dependent processes (Table S1).In addition, we also found spindle alignment defect phenotypes in embryos after RNAi of C. elegans. At this point, we can only speculate, but possible mechanisms include, that is, post-translational modifications  were maintained at 16°C, and L4 hermaphrodites were shifted to RT (∼23°C) overnight and heat-shocked at 25°C for 4 h before analysis. C. elegans strains and RNAi feeding clones are listed in the Supplemental Material. L4 or young adult hermaphrodites were fed with RNAi bacteria and incubated for 6–24 h at RT (23°C) before dissection to obtain embryos for filming. The following alleles and strains were used: LGIII: clk-1(qm30); LGIV: isp-1(qm150), spd-3(oj35). The C. elegans strains used in the study, carrying integrated fluorescent proteins, are outlined in Table S2. They were backcrossed four times with WT animals before being used for genetic analyses.The Bristol strain N2 was used as the standard WT strain. Culturing, handling, and genetic manipulation of ocedures . All C. C. elegans strains carrying integrated GFP or mCherry used in this study are listed in the table aboveThe   were administered by feeding. Feeding vector (L4440) was obtained from the Addgene. The ucr-1, mev-1, and cco-1 RNAi-feeding clones were a gift from Orna Cohen-Fix laboratory , followed by transformation into Escherichia coli HT115(DE3) bacteria. The E. coli HT115(DE3) bacteria containing the feeding vectors were cultured and used to seed RNAi plates (1 mM IPTG and 50 μg/ml ampicillin). L4 hermaphrodites were allowed to feed for 24 or 48 h at RT (∼23°C) and then shifted to 25°C for 4 h before analysis. For plk-1 (RNAi), L4 larvae were cultured at RT overnight and then transferred to plk-1 (RNAi) feeding plates allowed to feed for 6 h at 25°C before analysis.The boratory ; the rabC. elegans RNAi-feeding clones in this study.Primers for Table S3. 2HPO4, 22 mM KH2PO4, 86 mM NaCl, and 1 mM MgSO4). One-cell embryos were mounted on slides with 2% agarose pad, overlaid with a 22 × 22-mm coverslip, and imaged at RT on a wide-field fluorescence microscope  equipped with a CFI Apo TIRF 60 × 1.45 NA oil immersion lens and a CCD camera (iXon 897 Ultra EMCCDs) or on a 3i VIVO spinning-disc confocal microscope  equipped with Zeiss Plan-Apochromat 63x/1.40 oil microscope objective, six laser lines and a Hamamatsu ORCA-Flash4.0 scientific CMOS camera (Hamamatsu) for detection. The microscope was controlled by Nikon NIS-Elements software (Nikon). For z-stacks, images in a 27 μm z-series (1 μm per section) were captured every 15 s . For figure construction, final image panels were scaled for presentation in Prism v9.5.0 (GraphPad).C. elegans embryos was determined using ImageJ. The spindle orientation angle refers to the orientation of the mitotic spindle relative to the anterior/posterior (A/P) axis of the embryo.The angle of spindle orientation (θ) at the onset of anaphase in the one-cell stage of spd-3(oj35) mutant.The width of the metaphase plate was determined by measuring the distance from top end to bottom end at the metaphase plate (perpendicular to the spindle axis) just prior to anaphase onset. For the quantification of distance between chromosomes, we measured the distance between the center of chromosome mass in the male and female pronucleus at time point after NEBD. Once the longest distance was analyzed, this stage was called “SDCs” positioning in The extend of ER cluster formation was assessed visually. Nuclear lamin fluorescence intensity (GFP::LMN-1) was quantified using ImageJ software by drawing a box around the nuclei and subtracting the background fluorescence in an equal-sized box drawn in the cytoplasm . The totTo quantify the PLK-1::GFP fluorescence in the nucleoplasm, a fixed-size box was drawn in nucleoplasm (red box) and in cytoplasm (black box) as background at each time point. The normalized PLK-1 level was calculated as ([integrated intensity in red box—integrated intensity in black box]/integrated intensity in black box)  and S7B.A gamma of 1.5 was applied for images of centrosomal PLK-1::GFP  to visuahsp-4::ghp reporter was measured using ImageJ software by drawing a box around the individual worm for quantification and subtracting the background fluorescence in an equal-sized box drawn over the background fluorescence  microscope, motorized stage for automated scanning, Chameleon Vision-II (Coherent Inc.) ultrafast Ti:sapphire laser with dispersion compensation to maintain pulses at the specimen plane  for multiphoton excitation and a standard set of dry and immersion objectives was used. Three HPM—100-40 hybrid GaAsP detectors (Becker and Hickl) are connected to the nondescanned (NDD) port of the microscope using two T-adapters (Zeiss) with proper dichroics and band pass filters to collect as much fluorescence as possible in the spectral ranges tryptophan channel: 740 nm Ex, 340–380 nm Em, NAD(P)H channel: 740 nm Ex, 460–500 nm Em, and FAD channel: 890 nm Ex, 520–560 nm Em. The three channels also contain a 690 nm short pass filter (Zeiss) in the beam path to avoid excitation background. Three SPC-150 cards (Becker and Hickl) synchronized with the pulsed laser and the Zeiss LSM-780 scan head signals collect the time-resolved fluorescence in TCSPC mode using SPCM (Version 9.74) acquisition software. A motorized stage is used during imaging using Zeiss 40× NA1.3 oil apochromatic objective lens.FLIM data fitting is based on the B&H handbook. We used a two components incomplete model to fit tryptophan, NAD(P)H and FAD/FMN FLIM images. The offset and scattering are set to 0. Shift is optimized to make sure the Chi2 as close as to 1.The FLIM processing followed the previously published article  with an http://rsbweb.nih.gov/ij/) and normalized the signal to signal of α-tubulin.200 adult worms from each strain were transferred to 30 μl of sample buffer , subjected to the freeze-thaw treatment three times in liquid nitrogen and water bath and then boiled in a 95°C-heating block for 5 min. The samples were loaded on 10% SDS–PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane and subjected to Western blot analysis using anti-PLK-1 , P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****). Data distribution was assumed to be normal, but this was not formally tested.Statistical analysis was conducted using Prism v9.5.0 (GraphPad).


COVID-HEROES is a multicentre study of Australian healthcare workers (HCW) assessing 1) incidence of and risk factors for SARS-CoV-2 infection and 2) serological response to SARS-CoV-2 vaccination and infection.We conducted a multi-centre, prospective observational cohort study of Australian HCWs from January 2020 – January 2023, evaluating incidence of, and risk factors for, SARS-CoV-2 infection. HCWs underwent monthly surveys to assess SARS-CoV-2 occupational exposure, vaccination status and infection and serology testing to detect antibody response to vaccination (measured by IgG immunofluorescent antibody titre) or infection (development of SARS-CoV-2 nucleoprotein (NP) IgG antibodies measured by ELISA). Factors associated with infection were identified using logistic regression models.575 participants completed 4882 surveys and 4325 serological tests over 36 months. Median enrolment duration was 8.4 (IQR 2.2-17.4) months. 160/575 (28%) of participants reported SARS-CoV-2 infection diagnosed by polymerase chain reaction (PCR) and/or rapid antigen test (RAT). 101/160 (64%) participants with known infection developed NP antibodies. A further 41 participants, without reported infection developed NP antibodies, indicating unrecognised infection .153/160 (96%) known infections occurred after December 2021, corresponding to the Omicron wave, despite 25-50% of participants reporting occupational exposure to SARS-CoV-2 patients throughout the study period. 571/575 participants developed IgG response following two doses of SARS-CoV-2 vaccine, 6 of whom seroreverted. A logistic regression model of risk factors for HCWs contracting SARS-CoV-2 infection (Table 2) did not identify significant association between occupational exposure and infection .In this 3-year prospective cohort, we report detailed occupational SARS-CoV-2 exposures to assess nosocomial acquisition risk, including symptomatic and unrecognised infection. We demonstrate low rates of infection until community prevalence increased, despite significant workplace exposure, indicating effective use of protective precautions in the Australian healthcare setting.All Authors: No reported disclosures


The severity of disease following infection with SARS-CoV-2 is determined by viral replication kinetics and host immunity, with early T cell responses and/or suppression of viraemia driving a favourable outcome. Recent studies uncovered a role for cholesterol metabolism in the SARS-CoV-2 life cycle and in T cell function. Here we show that blockade of the enzyme Acyl-CoA:cholesterol acyltransferase (ACAT) with Avasimibe inhibits SARS-CoV-2 pseudoparticle infection and disrupts the association of ACE2 and GM1 lipid rafts on the cell membrane, perturbing viral attachment. Imaging SARS-CoV-2 RNAs at the single cell level using a viral replicon model identifies the capacity of Avasimibe to limit the establishment of replication complexes required for RNA replication. Genetic studies to transiently silence or overexpress ACAT isoforms confirmed a role for ACAT in SARS-CoV-2 infection. Furthermore, Avasimibe boosts the expansion of functional SARS-CoV-2-specific T cells from the blood of patients sampled during the acute phase of infection. Thus, re-purposing of ACAT inhibitors provides a compelling therapeutic strategy for the treatment of COVID-19 to achieve both antiviral and immunomodulatory effects.Trial registration: NCT04318314. Cholesterol metabolism is a key element in both viral replication and immune cell signalling. We show that Avasimibe, an inhibitor of the cholesterol esterification enzyme Acyl-CoA:cholesterol acyltransferase (ACAT), potently inhibits SARS-CoV-2 entry and viral RNA replication. Importantly, Avasimibe boosts the expansion of functional SARS-CoV-2-specific T cells isolated from the blood of patients sampled during the acute phase of infection. Our findings show that Avasimibe has combined antiviral and T cell boosting properties in acute SARS-CoV-2 infection. This study reinforces an emerging picture of the importance of cholesterol metabolism in viral replication and T cell immunity, providing new insights on intrinsic anti-viral pathways that may be applicable to other viral pathogens. SARS-CoV-2 is a global health issue associated with over 600 million infections and 6 million deaths by October 2022 . PreventMetabolic syndrome and hyperlipidaemia have been associated with a poorer outcome of SARS-CoV-2 infection and cholesterol-lowering HMG-CoA-reductase inhibitors (statins) may improve COVID-19 survival, highlighting the potential of targeting cholesterol metabolism as a treatment strategy –15. Chol2) clusters, which are commonly associated with disordered regions of the cellular membrane, in a cholesterol-dependent manner [2 clusters, inhibiting SARS-CoV-2 entry [Cholesterol homeostasis is integral to replication of a wide range of viruses, impacting multiple steps in the viral life cycle including entry, replication, assembly and egress . Recent t manner . Anaesth-2 entry . In addi-2 entry .In addition to viral entry, recent genetic screens have implicated cholesterol homeostasis as an essential process in coronavirus RNA replication –34. Likehi) and ACAT inhibition restored these properties, suggesting it may provide beneficial effects on the activated PD-1hi antiviral T cells in acute SARS-CoV-2 infection. Thus, we hypothesized that modulation of cholesterol metabolism by ACAT inhibitors such as Avasimibe (AVS) would inhibit SARS-CoV-2 replication and boost virus-specific T cells to control infection.Acyl-CoA:cholesterol acyltransferase  esterifies free cholesterol; previous work demonstrated that pharmacological inhibition of ACAT reduced hepatitis B and C virus replication ,42, whilS1A Fig). Pre-treating cells with AVS reduced SARS-CoV-2pp infection of both VeroE6 and VeroE6-TMPRSS2 cells . To evaluate whether AVS influenced the lentiviral promoter we transfected lentiviral DNA into VeroE6-TMPRSS2 cells and observed comparable luciferase activity in AVS and DMSO treated cells , demonstrating that AVS has a minimal effect on lentiviral genome expression.SARS-CoV-2 infection is initiated by the viral Spike protein binding to ACE2 at the cell surface. Subsequent cleavage by the transmembrane protease serine 2 (TMPRSS2) triggers fusion of viral and host membranes ,45. In a50 of 1.77μM compared to 0.23μM for Vero-TMPRSS2 cells and no detectable effect on cell viability . The emergence of SARS-CoV-2 variants of concern (VOC) with altered Spike proteins such as Omicron that can evade vaccine protection prompted us to evaluate their sensitivity to ACAT inhibition. AVS inhibited pseudoparticles expressing B.1 (D614G), Delta and Omicron (BA.1) Spike proteins infecting VeroE6-TMPRSS2 and Calu-3 cells . All SARS-CoV-2pp were neutralized with a saturating dose of ACE2-Fc, demonstrating ACE2-dependent infection. To evaluate whether this antiviral activity was dependent on endocytic trafficking we infected cells with pseudoparticles bearing Vesicular Stomatitis Virus G glycoprotein (VSV-G) that are internalized via clathrin-dependent endocytosis and fuse with endosomal membranes [Fig 1C), suggesting a role for ACAT in regulating endocytic trafficking pathways.We extended our observations to Calu-3 cells, a lung epithelial cell line that expresses both ACE2 and TMPRSS2 , and obsembranes . AVS redS1C Fig). Membrane cholesterol can cluster in lipid rafts, cholesterol- and glycosphingolipid-rich microdomains that can be identified by fluorescent-labelled cholera toxin B (CTB) subunit binding to GM1 and visualized by direct stochastical optical reconstruction microscopy (dSTORM). AVS increased the diameter of GM1-enriched domains in VeroE6 cells , consistent with the increase in plasma membrane cholesterol. Next, we assessed whether the increase in GM1-enriched domains altered ACE2 localisation. Quantification of ACE2 and GM1 co-localisation by dSTORM imaging of VeroE6 cells revealed a significant increase with AVS treatment . Collectively, these data highlight a role for AVS to inhibit SARS-CoV-2 uptake by regulating the association of ACE2 with GM1 lipid rafts.AVS has been reported to alter plasma membrane cholesterol levels, showing a reduction in hepatoma cells  and an iFig 2A). AVS treated cells showed a reduction in nucleocapsid staining  compared to control DMSO treated cells. Treating the viral inocula with a neutralizing anti-Spike mAb or ACE2-Fc prevented viral attachment, demonstrating Spike-ACE2 dependent particle attachment . Furthermore, pre-treating the virus with heparin reduced virus attachment by competing for cell surface heparan-sulphate-proteoglycans, previously described as factors involved in SARS-CoV-2 attachment [To further explore the mechanism underlying AVS inhibition of SARS-CoV-2pp infection we quantified viral binding to VeroE6-TMPRSS2 cells using a previously reported protocol . AVS tretachment . TogetheFig 3A). Notably, AVS showed comparable anti-viral activity to the neutralizing anti-Spike mAb FI-3A [Fig 3B). As a control for viral genome replication, cells were treated with the guanosine nucleoside analogue Remdesivir (RDV) . We confirmed that treating infected VeroE6-TMPRSS2 with AVS showed a dose-dependent inhibition of intracellular viral RNA . Finally, we assessed the effect of AVS on SARS-CoV-2 infection of human primary bronchial epithelial cells (PBEC) grown at air-liquid-interface to provide a more physiological model of infection. Treatment with AVS pre- or post-infection reduced viral RNA and infectious virus shed from the apical surface of the cultures . Taken together, ACAT inhibition has an antiviral effect against SARS-CoV-2 infection and RNA replication.To determine whether our observations with lentiviral pp translate to authentic viral infection, we pre-treated Calu-3 cells with AVS prior to infection with SARS-CoV-2 (Victoria 01/20 strain) and observed a significant reduction in intracellular viral RNA and secreted infectious virus . We previously developed a single-molecule-fluorescent-in-situ-hybridisation (smFISH) technique to visualise SARS-CoV-2 RNAs within individual cells [Fig 4B). Quantifying replicon RNA molecules in AVS, RDV or DMSO treated cells showed a significant reduction with both drug treatments . SARS-CoV-2 exploits host membranes to generate structured complexes where the viral genomes replicate during infection. Using the spatial resolution of smFISH, we quantified replication complexes, defined as spatially extended foci containing multiple gRNA molecules in the perinuclear region (Inset), as previously reported [Fig 4D).To delineate the effect of AVS on SARS-CoV-2 entry and replication, we utilised a viral replicon system where the Spike and Membrane protein coding sequences are deleted to prevent the assembly of nascent viral particles. The SARS-CoV-2 replicon encodes both lication . Transielication . Replicoal cells , allowinreported . We foun, collectively demonstrating a role for ACAT in regulating the establishment of SARS-CoV-2 replication complexes.To confirm our observations in the context of authentic viral infection, we infected VeroE6-TMPRSS2 cells with a high MOI of SARS-CoV-2 for 1h and, after extensive washing to remove the unbound viral inoculum, treated the cells with AVS. The infected cells were fixed and imaged at 6h post-infection, consistent with the establishment of viral replication complexes within the eclipse phase of the viral life cycle, allowing us to study primary RNA replication events . During SOAT1 and SOAT2 respectively. We confirmed gene expression of both isoforms in Vero-TMPRSS2, Calu-3 and PBEC cultures, finding higher levels of SOAT1 expression in all three settings . Analysis of previously published RNAseq data from the lung tissue of SARS-CoV-2 infected Golden Syrian Hamsters [Soat1 and Soat2 expression . To understand their respective roles in SARS-CoV-2 infection we transfected siRNAs either individually or in combination to knockdown (KD) SOAT1 and SOAT2 expression in VeroE6-TMPRSS2 or Calu-3 cells prior to infection. KD was confirmed by qPCR measurement of SOAT1 and SOAT2 mRNA respectively . SOAT1 KD had a limited effect on SARS-CoV-2 infection, whereas we observed a significant reduction in virus replication and infectious particles in SOAT2 KD, either in isolation or in combination with SOAT1 . We confirmed this phenotype by overexpressing FLAG-tagged wild-type ACAT1 and ACAT2 along with catalytically inactive mutants in VeroE6-TMPRSS2 cells [S2D Fig) and noted a comparable frequency of cells expressing exogenous wild-type or inactive ACAT1 (H460A) and ACAT2 (H360A) with similar patterns of cytoplasmic expression [Fig 5C). Infection of cells overexpressing wild-type ACAT2, but not ACAT1, resulted in a significant increase in viral RNA and infectious particles . Importantly this augmentation of viral replication was not observed in cells expressing the catalytically inactive ACAT2-H360A mutant, demonstrating that enzymic activity is necessary to promote viral replication. To consolidate these results we evaluated the antiviral activity of an ACAT1 inhibitor Nevanimibe [50 27.4μM) compared to AVS (IC50 4.60μM), consistent with a dominant role for ACAT2 in SARS-CoV-2 replication vanimibe ,58, whicSOAT1 expression in CD4+ and CD8+ T cells during acute SARS-CoV-2 infection compared to healthy controls . As expected SOAT2 was not detected in T cells, as we previously reported [+ and CD8+ T cells by multiparameter flow cytometry. We identified SARS-CoV-2 spike and membrane-specific cytokine production that was significantly above the background seen in PBMC from the same donors without peptide stimulation . AVS increased the frequency of CD4+ T cells producing the antiviral cytokines IFNγ, TNF (or both) and MIP1β in response to either spike or membrane peptides, boosting responses in some patients and inducing de novo responses in others . The response to AVS was heterogeneous, showing a 50-fold increase in the magnitude of IFNγ-producing T cells in one patient and decreased cytokine production in a minority of patients, as previously reported for other in vitro and in vivo immunotherapeutic approaches [+ T cells in individual donors but was less consistent than for CD4+ T cells, resulting in non-significant changes for CD8+ T cell responses across the cohort . CD4+ T cells provide help to activate and differentiate B cells, for example via the interaction of CD40 and CD40L (CD154). AVS increased the SARS-CoV-2-specific expression of CD154 (CD40L) on CD4+ T cells, reflecting an enhanced capacity to co-stimulate CD40 to activate B cells . Consistent with the expansion of functional responses, AVS increased the proliferation of virus-specific CD4+ T cells .Given the potential of AVS to exert immunomodulatory as well as antiviral activity, we examined the effect of AVS on SARS-CoV-2-specific T cell function. Analysing a publicly available single cell (sc) RNAseq data set  showed creported . PBMC isproaches ,62. A siS4H Fig). COVID-19 severity is associated with male sex [S4I Fig), showing the potential of this therapeutic approach for a variety of patients, including those at risk of severe infection.Immunomodulatory therapies for viral infections carry the risk of increasing bystander immune responses and cytotoxic tissue damage; however, we did not detect any significant increase of CD107a mobilization to the cell membrane of perforin-producing T cells, markers of degranulation and cytotoxicity, respectively . This is in line with our previous findings showing that ACAT inhibition preferentially rescues PD-1hi T cells directed against chronic viral infections or tumours and not the highly functional memory responses to cytomegalovirus [S5E Fig). Thus, our data support AVS selectively expanding acutely activated SARS-CoV-2-specific T cells, without affecting memory or non-activated T cells.To ascertain whether AVS only boosts virus-specific effector and early memory T cells during or shortly after infection but not memory T cells, we analysed a second cohort of unvaccinated donors 6 months after SARS-CoV-2 infection  lipid regions [Our findings that ACAT inhibition altered the membrane localisation of ACE2, promoting its association with GM1 clusters and increasing the size of lipid rafts, highlights how manipulation of the intracellular esterified cholesterol content can impact the composition of the cellular membrane. An increasing body of literature shows ACE2 association with ordered (GM1) and disordered . The Royal Free Biobank tissue access for patient benefit (TapB) was approved by the Wales Research Ethics Committee . The Primary Bronchial Epithelial Cell study was reviewed by the Oxford Research Ethics Committee B (18/SC/0361). All study participants gave written informed consent prior to inclusion in the study and all storage of samples obtained complied with the Human Tissue Act 2004.The COVIDsortium cohort was approved by the ethical committee of UK National Research Ethics Service (20/SC/0149) and registered at Lentiviral pseudoparticles were generated by transfecting 293T cells with p8.91 (Gag-pol), pCSFW (luciferase reporter) and a codon optimised expression construct pcDNA3.1-SARS-CoV-2-Spike, as previously described . Delta aNaïve VeroE6 cells were infected with SARS-CoV-2 at an MOI of 0.003 and incubated for 48-72h until visible cytopathic effect was observed. At this point, cultures were harvested, clarified by centrifugation to remove residual cell debris, and stored at -80°C. Viral titre was determined by plaque assay. Briefly, VeroE6 cells were inoculated with serial dilutions of SARS-CoV-2 viral stocks for 2h followed by addition of a semi-solid overlay consisting of 1.5% carboxymethyl cellulose (Sigma-Aldrich). Cells were incubated for 72h, visible plaques enumerated by fixing cells using amido black stain and plaque-forming units (PFU) per mL calculated. For infection of Calu-3 cells with SARS-CoV-2, cells were plated 24h before infection with the stated MOI. Cells were inoculated for 2h after which the residual inoculum was removed with three PBS washes. Unless otherwise stated, infected cells were maintained for 24h before harvesting for downstream applications. Drug treatments were applied as described in individual figure legends, for pre-infection assays drugs were applied to cells before infection and maintained throughout the duration of the experiment.SOAT1 and SOAT2 RNA was quantified by SYBR green (PCR Biosystems) using the following primers, SOAT1_F: 5’-GCT CGT GTT CTG GTC CTA TGT G-3’, SOAT1_R: 5’-TAG AAC ATC CTG TCA CCA AAG CG-3’, SOAT2_F: 5’-CAA TGG ACC CGA CAC ATG GA-3’, SOAT2_R: 5’-GGG GCA GAG GTT TGT CTT GT-3’. These were expressed relative to the housekeeper gene Beta-2-Microglobin using B2M_F: 5’-CTA CAC TGA ATT CAC CCC CAC TG-3’ and B2M_R: 5’-ACC TCC ATG ATG CTG CTT ACA TG-3’.Total cellular RNA was extracted using the RNeasy kit (Qiagen) or Trizol (Thermofisher) according to manufacturer’s instructions. For quantification of viral or cellular RNA, equal amounts of RNA, as determined by nanodrop, were used in a one-step RT-qPCR using the Takyon-One Step RT probe mastermix (Eurogentec) and run on a Roche Light Cycler 96. For quantification of viral copy numbers, qPCR runs contained serial dilutions of viral RNA standards. Total SARS-CoV-2 RNA was quantified using: 2019-nCoV_N1-F: 5’-GAC CCC AAA ATC AGC GAA AT-3’, 2019-nCoV_N1-R: 5’-TCT GGT TAC TGC CAG TTG AA TCT G-3’, 2019-nCoV_N1-Probe: 5’-FAM-ACC CCG CAT TAC GTT TGG TGG ACC-BHQ1-3’. 4 per well). Cells were incubated with fresh EMEM +10%FBS for 1h followed by 1h of incubation in 100μL EMEM +10%FBS with 5μM AVS or equivalent concentrations of DMSO. After washing with 200μL PBS, cholesterol assay reactions were promptly begun by adding 100μL of working solution containing 50μM Amplex red, 1U/mL horseradish peroxidase, 2U/mL cholesterol oxidase and 2U/mL cholesterol esterase  in PBS. Relative cholesterol concentration and the background (no cells) was determined in triplicates for each sample by measuring fluorescence activity with a fluorescence microplate reader  for 2h at 37°C with excitation wavelength of 530nm and an emission wavelength of 585nm. Subsequently, cholesterol level was normalized to the control activity after subtracting the background.To measure the relative changes in plasma membrane cholesterol after treatment with AVS, we developed an Amplex Red-based cholesterol detection assay. Briefly, VeroE6 cells were seeded into 96-well flat culture plates with transparent bottoms to reach confluency  / 0.05% Triton in PBS) for 90min at room temperature. For labelling, cells were incubated with Alexa Fluor 647-CTB (Sigma-Aldrich) for 60min in 5% BSA / 0.05% Triton / PBS at room temperature followed by 5 washes with 1% BSA / 0.05% Triton / PBS for 15min each. Cells were then washed with PBS for 5min. Cell labelling and washing steps were performed while shaking. Labelled cells were then post-fixed with fixing solution, as above, for 10min without shaking followed by three 5min washes with PBS and two 3min washes with deionized distilled water. Images were recorded with a Bruker Vutara 352 with a 60X Olympus Silicone objective. Frames with an exposure time of 20ms were collected for each acquisition. Excitation of the Alexa Fluor 647 dye was achieved using 640nm lasers and Cy3B was achieved using 561nm lasers. Laser power was set to provide isolated blinking of individual fluorophores. Cells were imaged in a photo-switching buffer comprising of 1% β-mercaptoethanol (Sigma-Aldrich), oxygen scavengers  in 50mM Tris (Affymetrix) +10mM NaCl (Sigma-Aldrich) +10% glucose (Sigma) at pH 8.0. Axial sample drift was corrected during acquisition through the Vutara 352’s vFocus system. Images were constructed using the default modules in the Zen software. Each detected event was fitted to a 2D Gaussian distribution to determine the centre of each point spread function plus the localization precision. Pair correlation and cluster analysis was performed using the Statistical Analysis package in the Vutara SRX software. Pair correlation analysis is a statistical method used to determine the strength of correlation between two objects by counting the number of points of probe 2 within a certain donut-radius of each point of probe 1. This allows for localization to be determined without overlapping pixels as done in traditional diffraction-limited microscopy. Cluster size estimation and cluster density were calculated through cluster analysis by measuring the length and density of the clusters comprising of more than 10 particles with a maximum particle distance of 0.1μm.Renilla luciferase and mNeonGreen respectively [2. At an appropriate time post-transfection the cells were fixed with 4% (v/v) paraformaldehyde in PBS for 15min. Coverslips were then washed, and coverslips stored in PBS containing 0.1% (v/v) diethylpyrocarbonate until processing for smFISH.A SARS-CoV-2 replicon (pSC2-Rep-Del-p-RL-7NG) corresponding to the Delta Variant of Concern (VOC) was used. The coding regions for the Spike, membrane and ORF7a proteins were replaced with those of puromycin N-acetyl transferase, ectively . Replicoectively ; were cohttps://github.com/jefflee1103/Lee_Wing-SARS2/tree/main/smFISH/analaysis-codes/Bigfish-pipeline). Tiff files were converted to a numpy array, and individual cells segmented from the image using the Cellpose library. Background signal in the smFISH channel was subtracted with the skimage.white_tophat algorithm . Threshold setting for smFISH spot detection was set specifically for each set of images collected in each session. Replication foci were defined as gRNA smFISH signals with spatially extended foci that exceed the point-spread function of the microscope and intensity of the reference single molecules.smFISH was carried out as previously reported . BrieflyVero-TMPRSS2 cells were pre-incubated with 10μM AVS, 1μg/mL Anti-spike mAb FI-3A, 100U/mL Heparin or 1μg/mL ACE2-Fc for 2h at 37°C. Cells were then infected with SARS-CoV-2 (MOI = 10) at 4°C for 2h, unbound virus was removed by extensive washing in PBS, and samples fixed in 4% paraformaldehyde. For immunofluorescent staining of the viral nucleocapsid cells were permeabilised with PBS 0.1% Triton X-100 for 30min at room temperature. Samples were blocked in PBS 3% bovine serum albumin (BSA) for 1h room temperature. Cells were incubated with 2μg/mL of the human anti-N primary antibody (EY-2A clone)  overnighCells were washed with PBS and lysed using RIPA buffer  supplemented with protease inhibitor cocktail tablets (Roche). Clarified samples were mixed with Laemmli sample buffer, separated by SDS-PAGE and proteins transferred to polyvinylidene difluoride membrane. Membranes were blocked in 5% milk in PBS/0.1% Tween-20 and incubated with anti-TMPRSS2 (Proteintech Europe Ltd), anti-β-Actin (Sigma), anti-SOAT1  or anti-SOAT2 (abcam) and appropriate HRP-conjugated secondary antibodies (DAKO). Chemiluminescence substrate  was used to visualize proteins using a G:Box Imaging system (Syngene).SOAT1, SOAT2 or a scramble control (Ambion) complexed with DharmaFECT4 transfection reagent (Dharmacon) according to manufacturer’s instructions. Transfected cells were seeded and left to recover for 4h after which media was replenished. Cells were infected with a SARS-CoV-2 mNeonGreen reporter virus at an MOI of 0.01 24h post siRNA delivery. Viral replication was assessed through quantification of mNeonGreen fluorescence using a ClarioStar plate reader (BMG-LABTECH). Data was normalised to DAPI staining to account for cell density.Calu-3 or VeroE6-TMPRSS2 were reverse transfected with 50nM of siRNAs directed against VeroE6-TMPRSS2 cells were transfected with 1μg of pBABE-puro constructs containing either wild-type or catalytically inactive mutant ACAT1 (H460A) or ACAT2 (H360A) respectively using Fugene transfection reagent (Promega). Cells were infected with the SARS-CoV-2-mNeonGreen reporter virus and replication quantified as above.Peripheral blood samples were taken from unvaccinated study participants during or after SARS-CoV-2 infection during the first pandemic wave of infections in the UK (March-July 2020). The Acute Cohort was recruited from hospitalized patients at the Royal Free Hospital, London, and SARS-CoV-2 infection was confirmed by PCR . The Memory Cohort (COVIDsortium) was recruited from healthcare workers in London and SARS-CoV-2 infection was confirmed by PCR and/or serology. Samples were taken 5–6 months post infection . More information about COVIDsortium can be found in .For samples taken during acute SARS-CoV-2 infection, PBMC were isolated from EDTA blood using Histopaque-1077 (Sigma-Aldrich) density-gradient centrifugation in Leukosep tubes (Greiner Bio One) according to the manufacturer’s instructions. For the COVIDsortium cohort, PBMC were isolated from heparinized blood samples using Pancoll (Pan Biotech) or Histopaque-1077 Hybri-Max (Sigma-Aldrich) density-gradient centrifugation in SepMate tubes (StemCell) according to the manufacturer’s instructions. Isolated PBMC were cryopreserved in fetal bovine serum  containing 10% dimethyl sulfoxide  and stored in liquid nitrogen prior to cell culture.To examine SARS-CoV-2-specific T cell responses in the blood, PBMC were stimulated with 1μg/mL SARS-CoV-2 peptide pools (Membrane (Mem): 15mer peptides overlapping by 10aa, 43 peptides total; Spike S1: 18-20mer peptides, 18 peptides total. The full peptide sequences can be found in ) in cRPMFor flow cytometry, cells were stained with saturating concentrations of surface antibodies and a fixable viability dye diluted in 1:1 PBS (Invitrogen): Brilliant Violet Buffer (BD Biosciences). Following surface staining, cells were fixed and permeabilized with cytofix/cytoperm (BD Biosciences) followed by an intracellular staining with antibodies in saturating concentrations diluted in a 0.1% saponin-based buffer (Sigma-Aldrich). Full details on fluorescent monoclonal antibodies can be found in rd to 5th order bronchi and cultured in Airway Epithelial Cell medium (PromoCell) in submerged culture.Biopsies were obtained using flexible fibreoptic bronchoscopy from healthy control volunteers under light sedation with fentanyl and midazolam. Airway epithelial cells were taken using 2mm diameter cytology brushes from 3https://www.covid19cellatlas.org/index.patient.html . All data processing steps are described in Wilk et. al, [Processed count matrices with de-identified metadata were downloaded from  et. al,  includinStatistical analyses were performed with Prism 7.0 (GraphPad) as indicated in figure legends  with significant differences marked on all figures. In experiments with a sample size >100 normality was assessed using a D’Agostino-Pearson omnibus normality test. All tests were performed as two-tailed tests, and for all tests, significance levels were defined as not significant (ns) P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.S1 Fig(a) Immunoblot of TMPRSS2 in parental Vero and Vero-TMPRSS2 cells. (b) Vero-TMPRSS2 cells were transfected with the luciferase reporter construct used to generate SARS-CoV-2 Spike pseudo-particles and treated with 10μM of AVS. Luciferase activity was quantified 24h post infection and expressed as relative light units per μg of total cellular protein. (c) Free and total cholesterol levels in VeroE6 cells treated with 5μM Avasimibe (AVS) or DMSO for 60min. Data normalised to mean of DMSO control (n = 4).(PDF)Click here for additional data file.S2 FigSOAT1 and SOAT2 in Vero-TMPRSS2, Calu-3 and PBEC. Data are the delta Ct between SOAT1 or SOAT2 and Beta-2-Microglobin. (c) FPKM read counts of Soat1 and Soat2 from RNAseq data of SARS-CoV-2 infected Golden-Syrian hamster lung samples (n = 5). (d) Western blot of ACAT1 and ACAT2 expression in Vero and Vero-TMPRSS2. (e) VeroE6-TMPRSS2 were infected with SARS-CoV-2_mNeonGreen at an MOI of 0.01 and treated with increasing doses of AVS, Nevanimibe and RDV. Viral replication was assessed by measuring fluorescence and expressed relative to the DMSO control.(a) VeroE6-TMPRSS2 cells were infected with SARS-CoV-2 (MOI 0.01) for 2h, the inoculum removed, and cells treated with AVS or 1μM of RDV. Cells were harvested 24h post infection and intracellular viral RNA quantified by qPCR. Data are representative of n = 4 biological replicates. Statistical significance was determined by ANOVA . (b) qPCR quantification of (PDF)Click here for additional data file.S3 FigExpression of ACAT1 and ACAT2 by cell subset in PBMC taken 2–16 days following symptom onset from donors with severe acute SARS-CoV-2 infection (n = 7) or from healthy control is shown (n = 6). Analysis of publicly available scRNA-seq data from Wilk et al . The ave(PDF)Click here for additional data file.S4 Fig+, CD4+/CD8- or CD4-/CD8+). (b-i) Human PBMC from donors with acute SARS-CoV-2 infection were stimulated with SARS-CoV-2 peptide pools  for 8 days and analysed via flow cytometry. (b) IFNγ production by CD8+ and CD4+ T cells without peptide and after stimulation with Spike and Mem peptide pools. (c-i) PBMC were stimulated with Spike and Mem peptide pools and treated with AVS or DMSO for 8 days and analysed via flow cytometry. The cytokine production in wells without peptide stimulation was subtracted to determine SARS-CoV-2-specific cytokine production in summary data. (c) Fold change of IFNγ production by CD4+ T cells with AVS compared to DMSO (n = 13). (d) SARS-CoV-2-specific IFNγ and TNF production by CD4+ T cells. (e-g) SARS-CoV-2 specific IFNγ (e), TNF (f), MIP1β (g) production by CD8+ T cells (n = 19). (h) SARS-CoV-2-specific upregulation of CD107a and production of perforin (perf) by CD4+ and CD8+ T cells (n = 11). (i) Fold change of SARS-CoV-2-specific (Spike+Mem) IFNγ production by CD4+ T cells from donors during acute infection assessed by sex (left) and correlated with donor age (right). Bars represent the mean of the data set. Doughnut charts indicate fraction of donors with response to AVS (red). Response defined as de novo or increased cytokine production. P values determined by Kruskal-Wallis test (b), Wilcoxon matched-pairs signed rank test (c-h) and Mann-Whitney . Correlation assessed by Spearman correlation .(a) Flow cytometry gating strategy for PBMC Click here for additional data file.S5 Figin vitro peptide stimulation. Bars represent the mean of the data set. Doughnut charts indicate fraction of donors with response to AVS (red). Response defined as de novo or increased cytokine production/CD154 expression. P values determined by Wilcoxon matched-pairs signed rank test.(a-d) Human PBMC from donors 6 months post infection were stimulated with SARS-CoV-2 peptide pools  and treated with Avasimibe (AVS) or DMSO for 8 day. SARS-CoV-2-specific cytokine production by T cells was detected via flow cytometry. The cytokine production and CD154 expression in wells without peptide stimulation was subtracted to determine SARS-CoV-2-specific cytokine production/CD154 expression in summary data. (a-d) SARS-CoV-2-specific IFNγ (a), TNF (b), MIP1β (c) production and CD154 expression (d) from donors 6 months post SARS-CoV-2 infection . (e) IFNγ production by T cells from donors with acute SARS-CoV-2 infection without (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 Acknowledgements(PDF)Click here for additional data file.


Htr1d, Drd2, and Chrna3 lose rhythmicity, but P2rx7 gains rhythmicity; the up-regulation of Htr1d and Drd2 and down-regulation of Chrna3 inhibit adenylate cyclase (AC), and up-regulation of Htr1d, Drd2, and P2rx7 activates protein kinase C (PKC). Together, these results suggest that epilepsy can disrupt the circadian dynamics of the epileptic genes, shed light on possible TLE pathogenesis, and provide potential targets for TLE diagnosis and chronotherapy.Temporal lobe epilepsy (TLE) is a common and severe epilepsy displaying rhythmicity in humans and animals. However, how the circadian clock contributes to TLE remains elusive. A recent circadian analysis of the ventral hippocampal transcriptome of pilocarpine-induced TLE mice revealed as many as 1650 rhythmically expressed transcripts. Here, a comparison of the mouse ventral hippocampal transcriptome with the human epilepsy-related gene set identified 315 possible mouse epilepsy-related genes. Rhythmicity analysis classified them into arrhythmicity, loss-of-rhythmicity, gain-of-rhythmicity, and rhythmicity-maintaining groups. KEGG and GO analyses of these mouse epilepsy genes suggest their involvement in circadian entrainment. In TLE mice,  Temporal lobe epilepsy (TLE) is a common severe neurologic disorder characteristic of recurrent seizures . The potA recent circadian analysis of the ventral hippocampal transcriptome of the pilocarpine-induced TLE mice revealed abnormal expression patterns of genes involved in aerobic glycolysis and oxidative phosphorylation in TLE mice , sheddinHtr1d, Drd2, and Chrna3 involved in the neuroactive ligand-receptor interaction pathway lose rhythmicity, but P2rx7 gains rhythmicity, and their up-regulation or down-regulation results in the down-regulation of adenylate cyclase (AC) and up-regulation of protein kinase C (PKC) in TLE mice, providing potential targets for TLE diagnosis and chronotherapy.Here, we interrogated the ventral hippocampal transcriptome of mice with 661 human epilepsy-related genes we recently compiled  and idenGrin2a (Glutamate ionotropic receptor NMDA type subunit 2A), Gria2 (Glutamate ionotropic receptor AMPA type subunit 2), Kcnj3 (Potassium inwardly rectifying channel subfamily J member 3), and Grin1 (Glutamate ionotropic receptor NMDA type subunit 1), in TLE and control (CTL) mice  mice F. In addTL) mice , implicaBdnf (Brain-derived neurotrophic factor), Gli3 (GLI-Kruppel family member GLI3), Htr2a (5-Hydroxytryptamine receptor 2A), and Gabrb1 (GABA receptor A subunit β1), are up-regulated in TLE mice (Gli3 [Gli3 implicates the down-regulation of PKA. Htr2a activates Ca2+ transporters in postsynaptic cells and interacts with Gq to activate Protein kinase C (PKC), indirectly reducing adenylate cyclase (AC) production [Gabrb1, facilitated by the cAMP pathway, is up-regulated in TLE mice [The expression of 251 arrhythmic epilepsy-related genes in TLE and CTL mice is shown in the heatmap . KEGG anTLE mice . As protce  and Drd2 (Dopamine receptor D2), lose rhythmicity but are up-regulated in TLE mice  also loses rhythmicity but is down-regulated in TLE mice , Pcdh19 (Protocadherin 19), and Aldh2 (Aldehyde dehydrogenase 2 family member) lose rhythmicity with altered expression patterns in TLE mice  and Grm1 (glutamate metabotropic receptor 1), gain rhythmicity. P2RX7 transports the calcium into the cytoplasm to up-regulate PKC [Atp6v0c (ATPase H+ transporting V0 subunit c) and Lepr (leptin receptor)  , gain rheceptor) E.Abcb6 (ATP-binding cassette subfamily B member 6), Rnf13 (ring finger protein 13), and Samd12  are statistically rhythmically expressed in both TLE and CTL mice; however, Samd12 and Abcb6 advance approximately 2–4 h, but Rnf13 delays approximately 3 h in TLE mice (Chd2 (Chromodomain helicase DNA binding protein 2) and Apeh (Acylaminoacyl-peptide hydrolase) are not markedly altered, both genes display lengthened periods with elevated amplitudes in TLE mice , a human epilepsy driver gene, is expressed primarily in the human brain [Pcdh19 loses rhythmicity and is down-regulated in TLE mice (+ transporting V0 subunit c (ATPV0C), encoding c-subunit of the vacuolar ATPase, is another human epilepsy driver gene, as it has been reported to cause intellectual impairment and epilepsy [Atpv0c gains rhythmicity but is up-regulated in TLE mice  of 315 possible mouse epilepsy-related genes are arrhythmic , while tAdcy1 (Adenylate cyclase 1) and up-regulation of Prkch (Protein kinase C eta type) in TLE mice, implicating the reduced AC levels and enhanced PKC levels in TLE mice ) (GEO Accession GSE54652). Briefly, the ventral parts of the hippocampus of wild-type control (CTL) and pilocarpine-induced temporal lobe epilepsy (TLE) mice were collected at six time points each with four replicates in a 4 h interval for a single 24 h period, and total RNAs extracted from these samples were subject to transcriptome analysis using Affymetrix GeneChip Mouse Gene 2.1 ST arrays (no. 902120) [https://www.rdocumentation.org/packages/homologene (accessed on 24 May 2021)). The KEGG enrichment analysis was conducted with the KOBAS database ) [www.biocloud.net (accessed on 8 August 2022)). The rhythmicity of genes was analyzed with MetaCycle-meta2d (https://www.rdocumentation.org/packages/MetaCycle/versions/1.2.0/topics/meta2d (accessed on 27 May 2021)) based on the R program [p-value < 0.05. Gene set enrichment analysis (GSEA) is performed by clusterProfiler in R program [p < 0.05 and cut-off false discovery rate < 0.5. Two-Way ANOVA was conducted to determine the significance of the differentially expressed levels of the genes between CTL and TLE mice with a MATLAB program ‘anova2’, and a t-test was conducted to compare the differential expression of genes at ZT7 between CTL and TLE mice with GraphPad ). The phases, amplitudes, and periods of rhythmicity-maintaining genes were calculated by BioDare2 (https://biodare2.ed.ac.uk/ (accessed on 13 March 2021)) [https://biit.cs.ut.ee/gprofiler/convert (accessed on 26 May 2021)) [https://www.ensembl.org/index.html (accessed on 12 October 2022)). The putative E-box, D-box, and RORE motifs in these promoter sequences were determined with FIMO  under the MEME Suite (https://meme-suite.org/meme/tools/fimo (accessed on 12 October 2022)) [http://jaspar.genereg.net/ (accessed on 12 October 2022)) [The ventral hippocampal transcriptome data of wild-type control (aat5979_Table_S1) and pilocarpine-induced temporal lobe epilepsy (aat5979_Table_S3) mice used in this study were generated by microarray analysis as previously reported [t 2022)) , and GO  program . Genes w program . Gene seh 2021)) . ‘Liner y 2021)) . The 5′ r 2022))  scanned r 2022)) . Only mo


Therapeutic interventions that optimize angiogenic activities may reduce rates of end-stage kidney disease, critical limb ischemia, and lower extremity amputations in individuals with diabetic kidney disease (DKD). Infusion of autologous mesenchymal stromal cells (MSC) is a promising novel therapy to rejuvenate vascular integrity. However, DKD-related factors, including hyperglycemia and uremia, might alter MSC angiogenic repair capacity in an autologous treatment approach.n = 29; Controls n = 9) to identify differentially expressed  messenger RNA (mRNA) and microRNA (miRNA) involved in angiogenesis (GeneCards). Paracrine-mediated angiogenic repair capacity of MSC conditioned medium (MSCcm) was assessed in vitro using human umbilical vein endothelial cells incubated in high glucose and indoxyl sulfate for a hyperglycemic, uremic state.To explore the angiogenic activity of MSC in DKD, the transcriptome of adipose tissue-derived MSC obtained from DKD subjects was compared to age-matched controls without diabetes or kidney impairment. Next-generation RNA sequencing (RNA-seq) was performed on MSC . DKD-MSCcm incubation with injured endothelial cells improved tube formation capacity, enhanced migration, reduced adhesion molecules E-selectin, vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 mRNA expression in endothelial cells. Moreover, angiogenic repair effects did not differ between treatment groups (DKD-MSCcm vs. Control-MSCcm).RNA-seq analyses revealed 133 DE mRNAs (77 upregulated and 56 down-regulated) and 208 DE miRNAs (119 up- and 89 down-regulated) in DKD-MSC versus Control-MSC. Interestingly, miRNA let-7a-5p, which regulates angiogenesis and participates in DKD pathogenesis, interacted with 5 angiogenesis-associated mRNAs (transgelin/MSC from individuals with DKD show angiogenic transcriptome alterations compared to age-matched controls. However, angiogenic repair potential may be preserved, supporting autologous MSC interventions to treat conditions requiring enhanced angiogenic activities such as DKD, diabetic foot ulcers, and critical limb ischemia.The online version contains supplementary material available at 10.1186/s13287-023-03269-9. Tissue hypoxemia from loss of microvascular capillary density is a major contributor to morbidity and mortality in individuals with diabetic kidney disease (DKD), one of the most common causes of kidney failure worldwide . SustainRegenerative cell-based therapies, such as mesenchymal stem/stromal cells (MSC), have considerable potential to boost endogenous repair of injured tissues. MSC release soluble factors, such as extracellular vesicles (EVs), growth factors, and cytokines, which enhance tissue regeneration and stimulate pro-angiogenic signaling –12. In pDespite the promise of MSC in DKD, numerous challenges remain. Chronic hyperglycemia and uremia may damage endogenous repair systems like MSC, disturbing the coordinated network of pro- and anti-angiogenic factors activated by these cells. Autologous  MSC source may be preferable, given reduced risk of allosensitization, particularly for patients that might ultimately require kidney transplantation . HoweverOur previous phase 1 clinical trial showed that intra-arterial infusion of autologous adipose-derived MSC (AD-MSC) improved kidney blood flow and reduced kidney tissue hypoxia as assessed through blood oxygen level dependent magnetic resonance imaging (BOLD-MRI) and improved glomerular filtration rate in atherosclerotic renovascular disease subjects . These f2 and/or abnormal albuminuria with preserved kidney function (eGFR ≥ 60) in the setting of pharmacologically treated Type 1 or Type 2 diabetes mellitus. Controls were age-matched individuals, without diabetes or CKD, undergoing laparoscopic nephrectomy for kidney donation. Adipose tissue was collected from DKD (n = 29) participants in an outpatient surgical suite, while adipose tissue from controls (n = 9) was sampled during their surgical procedure. To minimize bias, those with dialysis dependency, kidney transplant, immunosuppressive therapy, hemoglobin A1c (HbA1c) > 11% (97 mmol/mol), or malignancy were excluded. The Mayo Clinic Institutional Review Board approved all experimental study procedures and all participants provided written informed consent prior to participating.The expression of angiogenesis-related mRNAs and miRNAs and angiogenic potential was characterized in AD-MSC isolated from diabetic kidney disease (DKD) subjects. Eligible individuals age ≥ 18 years were recruited from Mayo Clinic in Rochester, Minnesota, in the Nephrology clinic between November 2015–2019 (IRB: 15-000933), as previously reported . Informe2) in advanced minimum essential medium supplemented with 5% platelet lysate [6 cells were incubated with the antibodies for 30 min at 4 °C according to the manufacturer’s instructions. Following incubation, cell suspensions were washed, pelleted, and resuspended in staining buffer . Thereafter, samples were run on the FlowSight® and analyzed using IDEAS software version 6.2. Initial gates were selected for single cells (aspect ratio intensity versus brightfield), followed by gates of positively stained cells. Thresholds for positivity were established from scatterplots generated by single-stained compensation beads .Abdominal subcutaneous adipose tissue (0.5–2.0 g) was collected from control and DKD subjects to isolate MSC as previously described , 21, 25.t lysate   [MSC (passages 3–5) were cultured to 70% confluence in T-75 flasks, then washed extensively with phosphate-buffered saline, and replenished with serum-free advanced minimum essential medium for 36 h. After serum starvation, MSCcm was centrifuged at 2000×106 MSC) . Thrombo2. MSC were sequenced on an Illumina HiSeq 2000. Comprehensive analysis including alignment, gene and exon quantification, and QC of raw RNA sequencing reads was performed using the Mayo Analysis Pipeline for RNA Seq (MAP-RSeq) version 3.1.3 [p adjusted value < 0.05 and |log2FC|> 1 were considered significantly differentially expressed (DE) genes. GeneCards® database (http://www.genecards.org/) was utilized to screen genes associated with angiogenesis. DE angiogenesis mRNAs-of-interest were visualized on volcano plots and heatmaps created using R package ggplot2.To explore MSC angiogenic regulation in DKD, high-throughput RNA sequencing was performed as previously reported . Brieflyon 3.1.3 , an in-hp value < 0.05).Secondary analyses of high-throughput miRNA analyses on MSC were performed utilizing CAP-miRSeq version 1.1 workflow . CAP-miRwww.ingenuity.com).The miRNA/mRNA interactions were performed by crossing DE MSC mRNAs and miRNAs to predict targets analyzed using microRNA-Target analysis module in Ingenuity Pathway Analysis software  and ran using an Applied Biosystems ViiA7 Real-Time PCR system as previously mentioned [BMP2: Hs00154192, proenkephalin/PENK: Hs00175049, vascular cell adhesion molecule 1/VCAM1: Hs01003372, insulin-like growth factor-binding protein 2/IGFBP2: Hs01040719_m1, thrombospondin 1/THBS1: Hs00962908, integrin subunit beta 8/ITGB8: Hs00174456, glyceraldehyde 3-phosphate dehydrogenase/GAPDH: Hs02786624, miR-let-7a-5p: hsa-let-7a-5p, miR-30c-5p: hsa-miR-30c and U6: U6 small nuclear RNA/snRNA, 715680. mRNAs and miRNAs were normalized to GAPDH and U6, respectively.To validate the expression levels of representative upregulated and down-regulated mRNAs and miRNAs, qPCR was performed on MSC. RNA was isolated from MSC (0.5–1.0 × 10entioned . Fold ch5 cells), respectively, at the time of HG + IS injury. After 6 h, all HUVEC underwent functional assessments in vitro  or were lysed and prepared for qPCR analyses.Angiogenic potential of MSC was assessed in vitro. Briefly, commercially available human umbilical vein endothelial cells  were grown in endothelial cell growth medium . HUVEC w5/well) were seeded onto a 6-well plate and treated as described above. To assess proliferation of HUVEC in groups 1–4, the cells were fixed in 4% paraformaldehyde for 15 min, then stained for Ki67  according to the manufacturers protocol. Treated HUVEC were also assayed for migration capacity via the QCM 24-well Colorimetric Cell Migration  following manufacturer’s protocol. Samples were observed and imaged using an EVOS M5000 imaging system and analyzed via Image J. HUVEC (1.4 × 104/well) were also seeded onto a 96-well plate coated with GelTrex™ matrix  to assess tube formation. Plates were placed into a 37 °C incubator with 5% CO2. After 4 h, HUVEC were stained with calcein-AM  to assess capillary-like structures and observed under an EVOS M5000 imaging system . A total number of tubular structures were manually counted. Gene expression was evaluated in HUVEC using the following probes from Thermo Fisher Scientific . Expression was normalized to TATA-binding protein (TBP: HS00427620).HUVEC  and post hoc pairwise testing were employed to detect differences in co-culture experiments. Statistical significance was accepted at n = 9 controls and n = 29 DKD subjects). Clinical characteristics of each cohort are summarized in Table 2; p < 0.0001), and by design, eGFR was lower in DKD participants  compared to non-diabetic, non-CKD control subjects.In total, 38 participants were included . Of these mRNAs, a total of 133 DE genes were related to angiogenesis. The distribution of DE upregulated and down-regulated genes is displayed in a volcano plot  and VCAM1, and anti-angiogenic genes, including THBS1 and ITGB8. Among the top 15 DE down-regulated genes several were pro-angiogenic genes, including interleukin (IL)13 receptor subunit alpha-2, C-X-C motif chemokine ligand (CXCL)3, IL33, PENK, matrix metalloproteinase (MMP)10, CXCL8, BMP2, and MMP3 as well as an anti-angiogenic gene, MMP12. Expression patterns of several mRNAs were subsequently confirmed by qPCR , involved in phosphorylating cAMP-response element-binding protein (CREB) that promotes angiogenesis by regulating vascular endothelial growth factor (VEGF) receptor-1(VEGFR1). Notably, let-7a-5p targeted five mRNAs  were DE in DKD-MSC. Expression patterns of miRNAs were subsequently confirmed by qPCR   compared to VCAM1−-MSC. In in vivo studies, exogenous delivery of VCAM1+-MSC increased perfusion in the preclinical ischemic hindlimb model versus VCAM1−-MSC. Likewise, VCAM1 mRNA was upregulated in our DKD-MSC, which could potentially influence pro-angiogenic repair activity observed in vitro.Angiogenic activity is dependent on the intricate balance between pro-angiogenic factors  and factors that inhibit angiogenesis  . DiabeteTHBS1 gene, is a secreted anti-angiogenic protein that modulates cell migration and adhesion by regulating vascular nitric oxide signaling [Contrarily, anti-angiogenic genes were upregulated (e.g. THBS1) and pro-angiogenic genes were down-regulated (e.g. BMP2) in DKD-MSC in our study, which could disturb angiogenic activity. Thrombospondin 1 (TSP1), encoded by the ignaling , 38. In ignaling , MSC harignaling , invasioignaling  and induignaling . OverallTAGLN, THBS1, LOXL4, COL4A1, and COL8A1. Let-7a-5p has pro-angiogenic properties with increasing sprout formation of hypoxic HUVEC [TAGLN, a regulator of angiogenesis [LOXL4, a contributor to the vascular permeability in diabetes [COL4A1 and COL8A1) that influence angiogenesis plus THSB1 an inhibitor of angiogenesis [Given that miRNA regulate gene activity, we further assessed the relationship between miRNA and angiogenesis-related mRNA in DKD-MSC. In our study, 208 differentially expressed miRNAs were identified among which 14 miRNAs targeted differentially expressed angiogenesis-related mRNA. Interestingly, let-7a-5p was upregulated in RNA-seq analysis  expression, key molecules associated in early vascular injury [. [, and angiopoietin-2 were evident in both control- and diabetic MSC infused into ischemic tissue 2 weeks-post administration, indicating that diabetic MSC still possess paracrine angiogenic repair capacities albeit limited. Hence, a therapeutic boost of autologous MSC infusion could still help enhance angiogenic repair capacity in patients with DKD.We further examined MSC angiogenic repair capacity in vitro. After an in vitro injury mimicking a DKD microenvironment, endothelial cells lost tube formation capacity and increased adhesion molecule  and down-regulated (C) differentially expressed (DE) messenger RNAs (mRNAs) in diabetic kidney disease (DKD)-mesenchymal stromal cells (MSC), as well as microRNAs (miRNAs) (D). ELISA of TSP1 in MSC conditioned medium (MSCcm) (B). BMP2, bone morphogenetic protein 2; PENK, proenkephalin; VCAM1, vascular cell adhesion molecule 1; IGFBP2, insulin-like growth factor-binding protein 2; THBS1/TSP1, thrombospondin 1; ITGB8, integrin subunit beta 8.Additional file 2: Fig. S2. Representative images of capillary-like tubes formed by non-injured human umbilical vein endothelial cells (HUVEC) or in the presence of high glucose (HG) plus indoxyl sulfate (IS) injury and either Control-MSC conditioned medium (cm) or DKD-MSCcm. Images were acquired at 40X resolution and were not enhanced. (A). 4′,6-diamidino-2-phenylindole (DAPI) nuclear DNA (A). Nuclear staining (blue), proliferation marker Ki67 protein staining (red). Quantification of proliferation ability (A) and migratory function (B). Thrombospondin (THSB1) gene expression in HUVEC groups (C).


Fig. 1PET68Ga]Ga-RM2 PET/CT imaging on compassionate use basis. The PET scan showed a small subcarinal, distant lymph node metastasis and a highly suspicious lesion in the left proximal femur, both not detected on conventional imaging . Given the upstaging from UICC stage II to stage IV [A 43-year-old patient with newly diagnosed cancer of the right breast  underwent preoperative a [68Ga]Ga-RM2 PET/CT is a promising technique for staging of primary BC with positive ER status [68Ga]Ga-RM2 PET/CT might improve risk stratification and, ultimately, prognosis of patients with primary ER-positive BC.[R status . In this


Dok-3 tumor suppressor gene led to malignant progression in the intestinal benign tumor model ApcMin/+ mice. However, Dok-3 gene expression was undetectable in epithelial tumor cells and the transplantation of bone marrow cells lacking the Dok-3 gene–induced malignant conversion of epithelial tumor cells in ApcMin/+ mice, indicating a previously unrecognized tumor cell–extrinsic mechanism. Moreover, the Dok-3 loss–induced tumor invasion in ApcMin/+ mice required CD4+ and CD8+ T lymphocytes, but not B lymphocytes. Finally, whole-genome sequencing showed an indistinguishable pattern and level of somatic mutations in tumors irrespective of the Dok-3 gene mutation in ApcMin/+ mice. Together, these data indicate that Dok-3 deficiency is a tumor-extrinsic driving force of malignant progression in ApcMin/+ mice, providing a novel insight into microenvironments in tumor invasion.The fundamental difference between benign and malignant tumors lies in their invasive ability. It is believed that malignant conversion of benign tumor cells is induced by a tumor cell–intrinsic accumulation of driver gene mutations. Here, we found that disruption of the This study uncovers tumor cell–extrinsic cues that can induce malignant conversion of benign tumors without intensifying mutagenesis in tumors, a novel concept potentially providing a new therapeutic target in malignancy. Benign tumors become malignant when they acquire the ability to breach epithelial basement membrane and invade into neighboring tissues, which is the first essential step of the invasion-metastasis cascade followed by intravasation, extravasation, formation of micrometastases, and colonization of distant organs. Metastasis is estimated to be responsible for around 90% of cancer-associated mortality, yet the underlying mechanisms of the invasion-metastasis cascade remain poorly understood .Apc promotes the invasiveness of intestinal tumors through recruitment of matrix metalloproteinase (MMP)-expressing CCR1+ bone marrow–derived cells to the invasion front  deficiency in myeloid cells induces tumorigenesis in various tissues and malignant progression of colorectal tumors through reactive oxygen species–mediated epithelial and/or tumor mutagenesis, including mutations in the driver genes: and Tp53 , indicatand Tp53 . Moreoveand Tp53 . However/Dok-2/Dok-3 triple knockout mice on a C57BL/6 and 129/Sv mixed background develop histiocytic sarcoma, an aggressive malignancy of macrophages  and Csf1op mice  were obtained from Jackson Laboratory. Rag1 knockout (stock no. 002216), Cd4/8 knockout (stock no. 002664), Tcrd knockout (stock no. 002120), and Igh6 knockout (stock no. 002399) mice were obtained from Dr. Hiroshi Kiyono  through the Jackson Laboratory. Dok-3 knockout mice on a C57BL/6 and 129/Sv mixed background were provided by Dr. Brian Seed . ApcMin/+ mice (C57BL/6J-ian Seed . Mice frn = 3).For histopathological analysis, tissues were fixed with 4% paraformaldehyde, paraffin embedded, and serially sectioned at 4 μm thickness. The paraffin sections were stained with hematoxylin and eosin (H&E). H&E-stained sections were analyzed using an optical microscope CX-33 (Olympus). Histologic assessment was reviewed in consultation with a pathologist. For IHC, paraffin sections prepared at 4 μm thickness were deparaffinized, rehydrated, and subjected to antigen retrieval by heating the sections at 90°C in HistoVT One . After inactivating endogenous peroxidases with 3% hydrogen peroxide and blocking in 2.5% normal goat or horse serum, the sections were incubated with a primary antibody against Ki67 (NeoMarkers), E-Cadherin, or CD3ε . ImmPRESS Reagent Anti-Rabbit IgG and ImmPACT DAB (Vector Laboratories) were used for detection. After counterstaining with hematoxylin, the slides were mounted. For immunofluorescence, tissues were fixed in 4% paraformaldehyde, optical cutting temperature-embedded, snap-frozen in liquid nitrogen, and sectioned at 6 μm thickness. The frozen sections were incubated with the primary antibodies specific for CD45, CD11b, Gr1, B220 (BioLegend), Cytokeratin, or α-smooth muscle actin , followed by Alexa Fluor 488–conjugated or Alexa Fluor 594–conjugated goat secondary antibodies (Molecular Probes/Thermo Fisher Scientific). After counterstaining with 4ʹ,6-diamidino-2-phenylindole (DAPI), the slides were mounted. All bright-field and fluorescence images were captured using a BZ-9000 microscope (Keyence). The mean Ki67-labeling indices were calculated as the number of Ki67-positive cells per total number of tumor cells by counting at ×120 in five independent microscopic fields per tumor (Apc mice). Irradiation was performed using an IBL-437C instrument  at 9.5 Gy.Nucleated bone marrow cells were collected from 8-weeks-old wild-type (WT) and Dok-3 knockout mice and intravenously injected into lethally irradiated ApcMin/+ mice , or organoids  and Gapdh , Il1b , Il6 , Il7a , Cxcl1 , Cxcl2 , Cxcl10 , Ccl11 , Tnfa , Cox-2 . Semiquantitative RT-PCR analysis was performed with GoTaq (Promega) and the following primer sets: Dok1 , Dok2 , Dok3 , Gapdh .Total RNA was extracted from cells, tumors (Apc mice or Apc/Dok3 mice (n = 6 for each genotype) were stained with the following fluorochrome-conjugated antibodies (BioLegend): allophycocyanin (APC)-conjugated anti-CD3ε (145-2C11); APC/Fire 750-conjugated anti-CD11c (N418); Alexa Fluor 488–conjugated anti-Foxp3 (MF-14); Alexa Fluor 700–conjugated anti-F4/80 (BM8); Brilliant Violet (BV421)-conjugated anti-CD4 (RM4-5); Brilliant Violet (BV510)-conjugated anti-SiglecF (E50-2440) and anti-B220 (RA3-6B2); Brilliant Violet (BV605)-conjugated anti-CD11b (M1/70); FITC-conjugated anti-Ly6C (HK1.4) and anti-Gr1 (RB6-8C5); PE-conjugated anti-Ly6G (1A8), anti-TCRγ/δ (GL3), and anti-CD45 (30-F11); phycoerythrin-cyanine7 (PE-Cy7)-conjugated anti-CD8a (53-6.7). Antibody-stained cells were acquired with FACSAria (BD Biosciences) and analyzed with FACSDiva software Version 8.0.2 (BD Bioscience).Mononuclear cells from size-matched pooled intestinal tumors (containing five tumors ≥2 mm in diameter) of 6-month-old n = 3 mice for Apc or Apc/Dok3). Genomic DNA was extracted using lysis buffer  followed by phenol-chloroform extraction and isopropanol precipitation. Extracted genomic DNA was processed at Macrogen using Truseq DNA PCR-Free library preparation (Illumina). For whole-genome sequencing (WGS), tumor DNA samples were sequenced to an average depth of 100 × coverage, and matched non-tumor (tail) DNA samples were sequenced to an average depth of 30 × coverage, with 150 bp paired-end reads on Illumina Novaseq 6000 instruments. The Illumina platform generates raw images and base calling through an integrated primary analysis software called RTA . The BCL/cBCL  binary was converted into FASTQ using Illumina package bcl2fastq2-v2.20.0. The demultiplexing option (–barcode-mismatches) was set to perfect match .Size-matched pooled ileal tumors (containing 9–12 tumors ≥2 mm in diameter) and mouse tails were used for genomic DNA extraction (https://github.com/Genomon-Project/genomon-docs/tree/v2.0). GRCm38/mm10 was used as the mouse reference genome. Candidate mutations in a tumor sample were identified with the following procedure: A candidate with (i) the Fisher exact P ≤ 0.01; (ii) 0.1 < strand ratio < 0.9; (iii) ≥5 variant reads in the tumor sample; (iv) the variant allele frequency (VAF) in the tumor sample ≥0.02; and (v) the VAF in the matched normal sample <0.1 was adopted. ANNOVAR (https://annovar.openbioinformatics.org/en/latest/) was used to annotate the detected variants.Candidate somatic mutations were detected through the Genomon pipeline  or percentage (Fisher exact and χ2 tests). P < 0.05 was considered statistically significant.Data were analyzed by Student Sequencing FASTQ data files from WGS have been deposited in the Japanese Genotype-phenotype Archive, which is hosted by the DDBJ, under accession number DRA015135.Apc mice), a mouse model of intestinal tumorigenesis bearing a heterozygous mutation in the tumor suppressor gene Apc  was significantly greater than that of Apc mice  and exhibited no deep invasion beyond the smooth muscle layer (muscularis propria), 56% of the tumors in Apc/Dok3 mice were invasive and 11% of the tumors penetrated beyond the muscularis propria and reached the serosal surface  remained noninvasive (82%) and exhibited no deep invasion beyond the muscularis propria (Apc mice (Apc mice transplanted with Dok-3–deficient bone marrow cells (Apc: BMT Dok-3 knockout) developed invasive tumors in higher proportion (60%) than Apc: BMT WT, including some reaching the serosal surface -induced colonic benign tumors  was significantly lower than that in Apc/Dok3 mice  or CD8+ T cells (35%) or CD4+ and CD8+ T cells (13%) was significantly lower than that in Apc/Dok3 mice , which is involved in a wide range of malignant progression  by directly regulating tumor-associated macrophages  lacking Dok-3 were absent in the expected numbers at weaning regardless of Apc mutation status (Csf1op/op mice for reasons not understood.It has been shown that Th2-type CD4rophages , which arophages . Althougrophages . Namely,n status  save forApc enhances expression of the inflammatory chemokine CCL9 to promote malignant progression of the Apc mutant tumors by recruiting MMP-expressing CCR1+ bone marrow–derived cells as mentioned earlier (Apc/Dok3 mice (Tumor-intrinsic accumulation of genetic alterations is reported to drive malignant progression by shaping the inflammatory microenvironment. For instance, the loss of Smad4 in tumor epithelial cells mutated in  earlier . Howeverok3 mice  and 5, sTogether, our findings uncover previously unrecognized tumor cell–extrinsic cues that can induce malignant conversion of benign tumors irrespective of exacerbating gene mutations or inflammatory responses in tumors. However, the current study does not sufficiently address mechanisms, including how T cells and potentially other stromal cells promote tumor invasion, nor the clinical relevance of the proposed concept. Thus, further studies are required for better understanding stromal cell–driven malignant progression and developing new therapeutic strategies targeting such malignant progression.Figure S1Loss of Dok-1/-2 enhances intestinal tumor growth in Apc mice.Click here for additional data file.Table S195 genes significantly mutated in human colorectal cancer.Click here for additional data file.Figure S2Loss of Dok-1/-2 has no significant effect on invasiveness of tumors in Apc mice.Click here for additional data file.Table S2Dok-3 KO mice bearing Csf1 homozygous mutations are not observed at the expected Mendelian ratios at weaning.Click here for additional data file.Figure S3Assessment of invasion depth.Click here for additional data file.Figure S4Stromal reaction associated with invasive tumors of Apc/Dok3 mice.Click here for additional data file.Figure S5Characterization of invasive tumors in Apc/Dok3 mice.Click here for additional data file.Figure S6Dok-3 deficiency causes malignant progression of colorectal tumors of Apc mice.Click here for additional data file.Figure S7mRNA expression of Dok-1 and Dok-2, but not Dok-3, is detectable in tumor epithelial cells.Click here for additional data file.