Caenorhabditis elegans, alterations in intestinal physiology caused by pathogen colonization and sensation of microbial metabolites may lead to activation of pathogen aversive behaviors ranging from aversive reflexes to learned avoidance. However, the neural circuitry between chemosensory neurons that sense pathogenic bacterial cues and the motor neurons responsible for avoidance-associated locomotion remains unknown.Altering animal behavior to reduce pathogen exposure is a key line of defense against pathogen attack. In C. elegans, we found that backward locomotion was a component of learned pathogen avoidance, as animals pre-exposed to Pseudomonas aeruginosa or Enterococcus faecalis showed reflexive aversion to drops of the bacteria driven by chemosensory neurons, including the olfactory AWB neurons. This response also involved intestinal distention and, for E. faecalis, required expression of TRPM channels in the intestine and excretory system. Additionally, we uncovered a circuit composed of olfactory neurons, interneurons, and motor neurons that controls the backward locomotion crucial for learned reflexive aversion to pathogenic bacteria, learned avoidance, and the repulsive odor 2-nonanone.Using C. elegans connectome and computational modeling in uncovering new neuronal regulators of behavior.Using whole-brain simulation and functional assays, we uncovered a novel sensorimotor circuit governing learned reflexive aversion. The discovery of a complete sensorimotor circuit for reflexive aversion demonstrates the utility of using the The online version contains supplementary material available at 10.1186/s12915-022-01424-x. Caenorhabditis elegans exhibits behavioral immunity in the form of pathogen avoidance strategies that improve its survival , JN1715 peIs1715 [str-1p::mCasp-1 + unc-122p::GFP], JN578 peIs578 [npr-9p::casp1 + npr-9p::Venus + unc-122p::mCherry], NY2078 ynIs78 [flp-8p::GFP], NY2087 ynIs87 [flp-21p::GFP], PR680 che-1(p680), PY7502 oyIs85 [ceh-36p::TU#813 + ceh-36p::TU#814 + srtx-1p::GFP + unc-122p:DsRed], PY7505 oyIs84 [gpa-4p::TU#813 + gcy-27p::TU#814 + gcy-27p::GFP + unc-122p::DsRed], and ZM6804 hpIs270 [rig-3p::FRT::stop::FRT::ChR2(H134R)::wCherry + nmr-1p::FLP + lin-15(+)], ZM7297 hpIs331 [lgc55Bp::tomm20::miniSOG::SL2::RFP + lin-15(+)]. The following transgenic strains were generated for this study using standard microinjection protocols [flp-8p::GFP]; flp-8p::ced-3 (p15)::nz + flp-32::cz::ced-3 (p17) + unc-122p::rfp and AY179 ynIs87 [flp-21p::GFP]; flp-21p::ced-3 (p15)::nz + ncs-1p::cz::ced-3 (p17) + unc-122p::rfp. AWB::ChR2 (raxIs15 [str-1p::ChR2::sl2gfp] was a gift from Meng C. Wang’s laboratory. C. elegans hermaphrodites were maintained on E. coli OP50 at 20 °C.The following rotocols : AY178 yflp-8p::ced-3 (p15)::nz), pJL13 (pPD95.75 flp-32p::cz::ced-3 (p17)), pJL14 (pPD95.75 flp-21p::ced-3 (p15)::nz), and pJL15 (pPD95.75 ncs-1p:: cz::ced-3 (p17)) plasmids. The pJL12 plasmid was constructed by cloning a 2019 bp upstream-promoter region of the flp-8 gene into a ced-3 (p15)::nz backbone vector, via SphI-BamHI restriction sites. The pJL13 plasmid was constructed by cloning a 2085 bp upstream-promoter region of the flp-32 gene into a cz::ced-3 (p17) backbone vector, via SphI-BamHI restriction sites. The pJL14 plasmid was constructed by cloning a 4109 bp upstream-promoter region of the flp-21 gene into a ced-3 (p15)::nz backbone vector, via SphI-BamHI restriction sites. The pJL15 plasmid was constructed by cloning a 3116 bp upstream-promoter region of the ncs-1 gene into a cz::ced-3 (p17) backbone vector, via SphI-BamHI restriction sites. recCaspase plasmids were a gift from Martin Chalfie, Addgene plasmids # 16080 and # 16081, Addgene, MA ). The plates were then incubated overnight at 37 °C. The plates were cooled to room temperature for at least 30 min before seeding with animals. Synchronized young gravid adult hermaphroditic animals grown on E. coli OP50 were washed with M9 buffer and transferred outside the bacterial lawns, and the number of animals on and off the lawns were counted at 24 h for P. aeruginosa and 4 h for E. faecalis. Three 3.5-cm-diameter plates were used per trial in every experiment. Occupancy index was calculated as Non lawn/Ntotal.Lawn avoidance assays were carried out as previously described . BrieflyP. aeruginosa on SK agar or E. faecalis on BHI agar were made as described above for avoidance assays. Young gravid adult hermaphroditic animals grown on E. coli OP50 were washed with M9 and transferred to the training plates and allowed to roam for 24 h on P. aeruginosa and 4 h on E. faecalis. They were then transferred to the appropriate assay plates. For cross-training with odors . Plates without ATR were used as a control. Animals were allowed to feed overnight for 16 h, and then placed under an epifluorescent microscope equipped with an EGFP filter. Forward crawling animals were stimulated with one second of blue light, and a positive response was recorded if an animal moved backward during the stimulation.Animals expressing the blue-light-activated cation channel channelrhodopsin-2 (ChR2) in specific neurons were grown to L4 larvae and then transferred to plates with P. aeruginosa and 4 h for E. faecalis using the Leica LAS v4.6 software, and the diameter of the intestinal lumen was measured using ImageJ software. For quantification of fluorescent bacteria, fluorescent images were acquired using the Leica LAS v4.6 software, and ImageJ software was used to first draw a region of interest around the bacteria in the intestines of animals and then to measure fluorescence intensity in the region.Fluorescence imaging was carried out as described previously . BrieflyC. elegans nervous system were carried out using a local copy of the Neural Interactome, pulled from https://github.com/shlizee/C-elegans-Neural-Interactome. Simulations were run for at least 12 s and neuronal activity for each simulation was saved into a numpy file. Simulated data was loaded directly with the Python Numpy package into a Jupyter Notebook and converted to a Pandas DataFrame. Python scripts were written to visualize individual neuronal activity over time as rows on a heatmap and graphs were generated using the Matplotlib and Seaborn libraries. The first two neurons of the VD, DD, VA, DA, and AS motor neuron classes were chosen as representatives of each class to aid visualization.Simulations of the x-y correlation plots, individual dots represent measurements for individual animals, and the red line depicts a simple linear regression. All experiments were performed in triplicate on at least three separate days, resulting in the sample sizes listed in the figure legends. One- or two-way ANOVA with subsequent group comparisons were performed as indicated in the figure legends. In the figures, ns denotes not significant, and asterisks (*) denote statistical significance as follows: *≤0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001, as compared with the appropriate controls.The statistical analysis was performed with Prism 9 (GraphPad). All bar graphs depict the mean of the population, with individual dots representing individual trials. Error bars represent the standard deviation of the population. For Additional file 1: Fig. S1. Motor neurons show oscillations upon AWB and command interneuron simulated stimulation. (A) The same data as in Fig. Additional file 2: Fig. S2. AWC, ASI, and ASE neurons do not lead to oscillations in motor neurons important for backward locomotion. Heatmaps (above) and waveforms (below) of VD, DD, VA, DA, and AS motor neuron activity in the Neural Interactome upon 0.9 nA stimulation of the CIs and 5.0 nA stimulation of AWC (A), ASI (B), and ASE (C).Additional file 3: Fig. S3. The response to SDS and dodecanoic acid are ASH mediated and can be modeled in the Neural Interactome. (A) Response index to buffer, 0.6 mM SDS, or 1mM dodecanoic acid for animals with no neurons ablated  or ASH neurons ablated (ASH(-), purple). Two-way ANOVA with subsequent comparison to the WT groups was performed. Error bars depict standard deviation. N = 15 for buffer, 10 for SDS, and 25 for dodecanoic acid . (B) Diagram of the circuit for reflexive aversion to SDS and dodecanoic acid. Arrows represent chemical synapses, while jagged lines represent electrical synapses. (C) Heatmap (above) and waveforms (below) of activity for the motor neurons VD, DD, VA, DA, and AS (rows) upon 5.0 nA stimulation of ASH neurons and 0.9 nA stimulation of the CIs in the Neural Interactome.Additional file 4: Fig. S4. AUA and RMG neurons are required for motor neuron oscillations upon AWB and command interneuron stimulation in simulations of the C. elegans nervous system. (A) The same data as in Fig. Additional file 5: Fig. S5. AIB, AVB, or SMB neuron ablation does not diminish oscillations in backward locomotion-associated motor neurons. Heatmaps (left) and waveforms (right) of activity of motor neurons (rows) upon 5.0 nA stimulation of AWB neurons and 0.9 nA stimulation of the CIs with AIB (A), AVB (B), or SMB (C) neurons ablated in the Neural Interactome. (D) Response index to P. aeruginosa for both naïve (gray) and trained (green) animals with either no neurons ablated  or AIB (JN578) or AVB (ZM7297) neurons ablated. For ZM7297 animals, the miniSOG ablation protocol was followed as in Fig. N = 25  for all groups.ablated N, WT or AAdditional file 6: Fig. S6. Genetic ablation of AUA and RMG neurons. (A) Representative fluorescent micrographs of NY2078 ynIs78 [flp-8p::GFP] (left) and AY178 ynIs78 [flp-8p::GFP]; flp-8p::ced-3 (p15)::nz + flp-32::cz::ced-3 (p17) + unc-122p::rfp (right) animals. (B) Representative fluorescent micrographs of NY2087 ynIs87 [flp-21p::GFP (left) and AY179 ynIs87 [flp-21p::GFP]; flp-21p::ced-3 (p15)::nz + ncs-1p::cz::ced-3 (p17) + unc-122p::rfp (right) animals. White, filled arrows point to intact AUA or RMG neurons, while white, unfilled arrows point to the lack of AUA or RMG neurons. GFP-positive neurons were counted for both intact and ablated animals (pink circles), showing that only the targeted neurons were ablated, with other neurons left intact. AUA intact: 4 GFP neurons; AUA ablated: 2 GFP neurons; RMG intact: 15 GFP neurons; RMG ablated: 13 neurons. Scale bars are 50 μm.Additional file 7: Fig. S7. Correlation of intestinal distention and learned reflexive aversion for P. aeruginosa but not E. faecalis exposure. (A) PA14::GFP relative fluorescence in the intestine (x-axis) and the trained response index to P. aeruginosa (y-axis) were measured in individual animals (dots), and linear regression was performed (red line). (B) Same as A but with intestinal diameter on P. aeruginosa (x-axis). (C) Same as A but with E. faecalis. (D) Same as B but with E. faecalis. R2 and P-values are shown next to linear regression lines in red.Additional file 8: Data. Combined raw data, with each figure on a separate spreadsheet.


Blocking either viral replication with Remdesivir23 or the downstream IFN stimulated cascade with anti-IFNAR2 in vivo in the chronic stages of disease attenuated the overactive immune-inflammatory response, especially inflammatory macrophages. Here, we show SARS-CoV-2 infection and replication in lung-resident human macrophages is a critical driver of disease. In response to infection mediated by CD16 and ACE2 receptors, human macrophages activate inflammasomes, release IL-1 and IL-18 and undergo pyroptosis thereby contributing to the hyperinflammatory state of the lungs. Inflammasome activation and its accompanying inflammatory response is necessary for lung inflammation, as inhibition of the NLRP3 inflammasome pathway reverses chronic lung pathology. Remarkably, this same blockade of inflammasome activation leads to the release of infectious virus by the infected macrophages. Thus, inflammasomes oppose host infection by SARS-CoV-2 by production of inflammatory cytokines and suicide by pyroptosis to prevent a productive viral cycle.Severe COVID-19 is characterized by persistent lung inflammation, inflammatory cytokine production, viral RNA, and sustained interferon (IFN) response all of which are recapitulated and required for pathology in the SARS-CoV-2 infected MISTRG6-hACE2 humanized mouse model of COVID-19 with a human immune system Two hallmarks of severe COVID-19 are a sustained interferon (IFN) response and viral RNA persisting for months28. This chronicity is recapitulated in SARS-CoV-2 infected MISTRG6-hACE2 humanized mice19. Copious interleukin (IL)-1β, IL-18 and lactate dehydrogenase (LDH) correlate with COVID-19 severity in patients, suggesting a role for inflammasome activation and pyroptosis in pathology29. Here, we show that human lung macrophages are infected by SARS-CoV-2. Replicating SARS-CoV-2 in these human macrophages activates inflammasomes and initiates an inflammatory cascade with a unique transcriptome, results in pyroptosis, and contributes to the downstream type-I-IFN response. Blocking either viral replication, the downstream IFN response or inflammasome activation in vivo during the chronic phase of the disease attenuates many aspects of the overactive immune-inflammatory response, especially the inflammatory macrophage response, and disease.Acute SARS-CoV-2 infection resolves in most patients but becomes chronic and sometimes deadly in about 10–20%30. To test whether a viral RNA-dependent type-I IFN response was a driver of chronic disease, we treated SARS-CoV-2 infected MISTRG6-hACE2 mice with Remdesivir23 and/or anti-IFNAR2 antibody , inflammatory  and anti-viral  response, and interferon stimulated genes (ISGs)   program in pneumocytes39 that was reversed in infected MISTRG6-hACE2 mice by anti-IFNAR2/Remdesivir combination therapy, restoring self-renewal and differentiation programs (19 (and humans36)  , d, emph2, IRF7)  showed t2, IRF7) . As macr2, IRF7) . All mac2, IRF7) . These I2, IRF7) . Yet, kee spared , highligprograms . Overallumans36) .40 in lung tissue or in sorted lung epithelial cells or human immune cells from infected MISTRG6-hACE2 mice  but only few total lung epithelial cells were infected with SARS-CoV-2  and subgenomic viral RNA (sgRNA)CE2 mice –d. SurprCE2 mice . AlthougRS-CoV-2 . StrikinRS-CoV-2 ,g. No moRS-CoV-2 . To addroV-2 . To test the role of antibody-mediated viral entry to macrophages, we treated infected mice with monoclonal antibodies (mAb)45 against SARS-CoV-2-Spike protein early (35hpi) when effects of endogenous antibodies are minimal or late (7dpi)  as proof of concept, or late  as a possible therapeutic with anti-CD16 antibody. Anti-viral antibody levels in lung tissue were sufficient to mediate viral uptake and positively correlated with mNG levels at 4dpi  . Indeed,e (7dpi) . Immune  at 4dpi , i. With at 4dpi .in vitro. Indeed, SARS-CoV-2 were taken up by BMDMs and replicated in these cells as measured by mNG signal  was reduced by CD16, ACE2, or RdRp blockade  with SARS-CoV-2 G signal  and highG signal . This waG signal . As in vblockade ,o. SgRNAblockade , furtherat 48hpi –r.in vitro , chemokines of interstitial macrophages , inflammatory cytokines , complement genes  and ISGs  . Interestingly, this strong network of CXCL10 specific gene signature was no longer restricted to AMs later in infection as different macrophage subsets continuously differentiate, evident in high IL7R expression by developing lung macrophages50 (CXCL10+ and AM) at all timepoints  . Further, IFI27) . In linemepoints .Morphological analysis of sorted mNG+ cells revealed the appearance of membrane bubbles, a characteristic of pyroptosis and prompted us to investigate inflammasome activation as part of the inflammatory cascade initiated by infection. Inflammasomes are dynamic multiprotein complexes in which specific NOD-like receptors (NLRs) and adaptor molecules are assembled to activate caspases, the central effector proteins. We sorted mNG+ and mNG− human immune cells, mNG+ epithelial cells, and mouse immune cells  and assaOnce inflammasome complexes are formed, active caspase-1 cleaves and proteolytically activates the proinflammatory IL1-family cytokines, IL-1β and IL-18, typically elevated and characteristic of severe COVID-19 in patients. IL-18 levels in blood and lungs were significantly elevated in SARS-CoV-2 infected mice and correlated well with proportions of infected (mNG+) macrophages . AlthougFinally, we assayed for pyroptosis by detecting LDH and gasdermin D (GSDMD) in serum. GSDMD, a substrate of active caspase-1 and pore-forming executer of pyroptosis, and LDH, released by pyroptosis, were particularly enriched in serum of infected mice at late timepoints 14dpi-, furtherAll aspects of inflammasome activation were also recapitulated in vitro when BMDMs were infected in vitro with SARS-CoV-2. Active caspase-1 in infected BMDMs was dependent on viral replication was inhibited by Remdesivir . High leTo determine the role of viral infection on the inflammatory macrophage response, we first blocked viral entry and replication in vivo and measured inflammatory cytokines and chemokines. Blocking viral entry (CD16 or ACE2 blockade) or inhibiting viral replication (Remdesivir) all reduced IL-18, IL-1RA, and CXCL10 levels, paralleling mNG levels –e. DepleFinally, to assess the causal role of NLRP3 and caspase-1 activation in inflammasome mediated inflammation and disease, we treated mice with caspase-1 and NLRP3 inhibitors . As expein vitro but did not impact macrophage infection, measured as mNG+ macrophage frequency, and reduced the inflammatory response to infection , the exact mechanism of NLRP3 activation is still poorly understood29. Activation of other NLRs may also contribute to the process, as inhibition of caspase-1 was stronger than NLRP3 alone. Finally, there may be other mechanisms that enhance SARS-CoV-2 infection or the downstream inflammatory response in human macrophages that are unexplored here.Viral RNA and particles can be detected by a variety of innate immune sensors. Inflammasome sensor NLRP3 is both upregulated and activated by replicating SARS-CoV-2. NLRP3 inflammasome can directly sense viral replication/RNA or can rely on other viral RNA sensors like MDA5 or RIG-I18. Targeting inflammasome pathways in patients may provide alternative therapeutic options for resolving chronicity in COVID-19. However, the increased virus production seen upon inflammasome blockade could pose a significant risk to the benefit of wholesale inhibition of the pathway. The findings from our study and its implications provide alternative therapeutic avenues to be explored in the clinic and may guide novel therapeutic developments and prompt clinical trials to investigate combinatorial therapies that target viral RNA, inflammasome activation or its products and sustained IFN response.A role for inflammasome driven hyperinflammation in COVID-19 pathophysiology in patients is now recognized All data that support the findings of this study are available within the paper, its supplementary Information files, and source files. All 10x Genomics single cell RNA sequencing and bulk RNA sequencing data that support the findings of this study are deposited in the Gene Expression Omnibus (GEO) repository with accession codes GSE186794 and GSE199272.Rag2−/− IL2rg−/−129xBalb/c background supplemented with genes for human M-CSF, IL-3, SIRPα, thrombopoietin, GM-CSF and IL-6 knocked into their respective mouse loci62. MISTRG6 mice are deposited in Jackson Laboratories and made available to academic, non-profit, and governmental institutions under a Yale-Regeneron material transfer agreement . Instructions on obtaining the material transfer agreement for this mouse strain will be available along with strain information and upon request. CD1 strain of mice acquired from Charles River Laboratories were used for cross-fostering of MISTRG6 pups upon birth to stabilize healthy microbiota. All mice were maintained under specific pathogen free conditions in our animal facilities  under our Animal Studies Committee-approved protocol. Unconstituted MISTRG6 mice were maintained with cycling treatment with enrofloxacin in the drinking water . All animal experimentations were performed in compliance with Yale Institutional Animal Care and Use Committee protocols. For SARS-CoV-2–infected mice, all procedures were performed in a Biosafety Level 3 (BSL-3) facility with approval from the Yale Institutional Animal Care and Use Committee and Yale Environmental Health and Safety.MISTRG6 was generated by the R. Flavell laboratory by combining mice generated by this lab, the laboratory of Markus Manz and Regeneron Pharmaceuticals based on the Fetal liver samples were cut in small fragments, treated for 45 min at 37 °C with collagenase D , and prepared into a cell suspension. Human CD34+ cells were purified by performing density gradient centrifugation , followed by positive immunomagnetic selection with EasySep™ Human CD34 Positive Selection Kit (StemCell). For intrahepatic engraftment, newborn 1–3-day-old pups were injected with 20,000 fetal liver CD34+ cells in 20 μl of PBS into the liver with a 22-gauge needle (Hamilton Company). All use of human materials was approved by the Yale University Human Investigation Committee.63 was purchased from Vector Biolabs (AAV9-CMV-hACE2). Animals were anaesthetized using isoflurane. The rostral neck was shaved and disinfected. A 5-mm incision was made, and the trachea was visualized. Using a 32-G insulin syringe, a 50-μl injection dose of 1011 genomic copies per milliliter of AAV-CMV-hACE2 was injected into the trachea. The incision was closed with 4–0 Vicryl suture and/or 3M Vetbond tissue adhesive. Following administration of analgesic animals were placed in a heated cage until full recovery. Mice were then moved to BSL-3 facilities for acclimation.AAV9 encoding hACE241. All infection experiments were performed in a BSL-3 facility, licensed by the State of Connecticut and Yale University. Mice were anesthetized using 20% vol/vol isoflurane diluted in propylene glycol. Using a pipette, 50 μl of SARS-CoV-2-WA1 or SARS-CoV-2-mNG (1–3×106 PFU) was delivered intranasally.SARS-CoV-2 isolate USA-WA1/2020 was obtained from BEI reagent repository. SARS-CoV-2 mNG was obtained from Dr. P.Y. Shi (UTMB)22 for 3 consecutive days starting at 7dpi  antibody early and late. For early CD16 blockade studies mice were treated with anti-CD16 antibody at 2dpi with a single dose (20μg per mouse) and euthanized at 4dpi. For late CD16 blockade studies mice were treated with anti-CD16 antibody at 7dpi and 11dpi and euthanized at 14dpi. For depletion experiments mice were treated with anti-CD16  antibody with a daily dose of 20μg for 3 days starting 1dpi. Rabbit IgG, monoclonal [EPR25A] Isotype Control (ab172730) and Mouse IgG1 kappa Isotype Control (P3.6.2.8.1) were used.19 presumably due to differences between mouse and human ACE2 that limit viral entry and replication42. Mouse IgG2 Isotype was used as control.Infected MISTRG6 (without AAV-hACE2) mice were treated with monoclonal antibody against human ACE2  for 3 days i.p. with a daily dose of 20μg starting at 1dpi. In these mice only, epithelial cells were not infected or infected poorly with SARS-CoV-2 with undetectable titers using standard plaque assaysin vitro and in vivo in mouse adapted SARS-CoV-2 infection and ancestral stain of SARS-CoV-2/WA164.Infected MISTRG6-hACE2 mice received a mixed cocktail of monoclonal antibodies clone 135 (m135) and clone 144 (m144) at 20mg/kg at 35hpi or 7dpi. Monoclonal recombinant antibodies (mAbs) used in this study were cloned from the convalescent patients (whose plasma was used for in vitro studies infecting BMDMs) and had high neutralizing activity against SARS-CoV-2 67. For caspase-1 inhibitor experiments, infected MISTRG6-hACE2 mice treated with VX-765 (Invivogen) at a dose of 8 mg/kg on days 6, 8, 10,12 post infection and euthanized on day 1467. Control infected mice were treated with PBS.For NLRP3 inhibitor experiments, Infected MISTRG6-hACE2 mice were treated with MCC950 (R&D Systems) at a dose of 8 mg/kg intraperitoneally on days 6, 8, 10,12- post infection and euthanized on day 1468. Plaques were resolved at 48 hours after infection by fixing in 10% formaldehyde for 1 hour followed by staining for 1 hour in 0.5% crystal violet in 20% ethanol. Plates were rinsed in water to visualize plaques. Multiple dilutions of lung homogenates were used to quantify Infectious titers (minimum number of plaques that can be quantified= 10 per ml of lung homogenate or ml of supernatant). Viral titers from supernatants of bone-marrow derived macrophage cultures were determined by plaque assay in Vero ACE2+TMPRSS2+ (sensitive) cells following the same protocols described for lung homogenates. VERO C1008  were obtained from ATCC. Vero ACE2+ TMPRSS2+ cells were obtained from B. Graham (NIAID). None of the cell lines were authenticated or tested for mycoplasma contamination.Mice were euthanized in 100% isoflurane. Approximately half of the right lung lobe was placed in a bead homogenizer tube with 1 ml of DMEM+2% FBS. After homogenization, 300 μl of this mixture was placed in 1mL Trizol (Invitrogen) for RNA extraction and analysis. Remaining volume of lung homogenates was cleared of debris by centrifugation . Infectious titers of SARS-CoV-2 were determined by plaque assay in Vero E6 (standard) or Vero ACE2+TMPRSS2+ (sensitive) cells in DMEM 4% FBS, and 0.6% Avicel RC-58169. The primer-probe sequences were as follows: E_Sarbeco_F primer: ACAGGTACGTTAATAGTTAATAGCGT (400 nM per reaction). E_Sarbeco probe _P1: FAM-ACACTAGCCATCCTTACTGCGCTTCG-BBQ (200nM per reaction); E_Sarbeco_R primer ATATTGCAGCAGTACGCACACA (400 nM per reaction); E leader specific primer sgLead-F: CGATCTCTTGTAGATCTGTTCTC (400 nM per reaction).RNA was extracted with the RNeasy mini kit (Qiagen) per the manufacturer’s protocol. SARS-CoV-2 RNA levels were quantified using the Luna Universal Probe Onestep RT-qPCR kit (New England Biolabs) and US CDC real-time RT-PCR primer/probe sets for 2019-nCoV_N1. For each sample, 1 μg of RNA was used. Subgenomic viral RNA was quantified using primer and probe sets targeting E gene as has been previously describedYale pathology kindly provided assistance with embedding, sectioning of lung tissue. A pulmonary pathologist reviewed the slides blinded and identified immune cell infiltration and other related pathologies. Paraffin embedded lung tissue  sections were deparaffinized in xylene and rehydrated. After antigen retrieval with 10 mM Sodium Citrate pH 6 and permeabilization with 0.1% Triton-X for 10 min the slides were blocked with 5% BSA in PBS with 0.05% Tween 20 for an hour. Then the samples were stained with primary antibodies against SARS-CoV-2-dsRNA; SARS-CoV2-RNA-dependent RNA Polymerase, SARS-CoV-2-Spike, human CD68, human ACE2 their isotype controls diluted in 1%BSA overnight at 2–8 °C. The next day, the samples were washed and incubated with fluorescent secondary antibodies. After washes, samples were treated with TrueBlack lipofuscin autofluorescence quencher for 30 seconds and mounted on DAPI mounting media (Sigma). Images were acquired using Keyence BZ-X800 Fluorescence Microscope or Nikon ECLIPSE Ti Series Confocal Microscope. Pseudo-colors were assigned for visualization.70. Lungs were harvested, minced, and incubated in a digestion cocktail containing 1 mg/ml collagenase D (Sigma) and 30 μg/ml DNase I (Sigma-Aldrich) in RPMI at 37°C for 20 min with gentle shaking. Tissue was then filtered through a 70 or 100-μm filter. Cells were treated with ammonium- chloride-potassium buffer and resuspended in PBS with 1% FBS. Mononuclear cells were incubated at 4°C with human (BD) and mouse  Fc block for 10 min. After washing, primary antibody staining was performed at 4°C for 20 min. After washing with PBS, cells were fixed using 4% paraformaldehyde. For intracellular staining, cells were washed with BD permeabilization buffer and stained in the same buffer for 45 min at room temperature. Samples were analyzed on an LSRII flow cytometer (BD Biosciences). Data were analyzed using FlowJo software.All mice were analyzed at approximately 9–14 weeks of age. Single cell suspensions were prepared from blood, spleen, bronchioalveolar lavage (BAL) and lung. Mice were euthanized with 100% isoflurane. BAL was performed using standard methods with a 22G Catheter (BD). Blood was collected either retro-orbitally or via cardiac puncture following euthanasia. BAL was performed using standard methods with a 22G Catheter (BD)For cell sorting experiments, single cell suspensions from digested lungs were stained with antibodies against human CD45, mouse CD45, mouse EPCAM and sorted using BD FACS Aria II which is contained in a Baker BioProtect IV Biological Safety Cabinet. Cell viability was assessed with DAPI when applicable.For imaging flow cytometry, cells from SARS-CoV-2 infected humanized mice were sorted based on: human immune cells (hCD45+); mouse immune cells (mCD45+) or epithelial mouse cells (EPCAM+). A- mNG+ epithelial cells (SARS-CoV-2-mNG+ mCD45(PE)− EPCAM(APC)+ hCD45(PB)−; B-total mouse immune cells (mCD45(PE)+ EPCAM(APC)− hCD45(PB−); C- mNG+ human immune cells (SARS-CoV-2-mNG+ mCD45(PE)− EPCAM(APC)− hCD45(PB)+); D- mNG− human immune cells (SARS-CoV-2-mNG− mCD45(PE)-EPCAM(APC)− hCD45(PB)+). These sorted cells  were fixed in 4% paraformaldehyde (PFA) for at least 30 minutes. Fixed sorted cells  were permeabilized, stained with unconjugated primary antibodies for ASC , NLRP3 , then stained with secondary antibodies . Cells data were acquired using an ImageStream X MKII (Amnis) with 63X magnification and analyzed using Ideas software (Amnis). ASC, NLRP3 specks were gated and quantified based on fluorophore intensity/max pixels. For FLICA-Caspase1 colocalization, macrophages were pretreated with FLICA prior to sorting.6 per ml concentration for 6 days in 5% CO2 incubator at 37°C. Media supplemented with 10% FBS was replenished with new media every 3–4 days. Prior to infection, cells were monitored for granularity, elongated morphology, and stronger adherence to the plate. Purity was confirmed by flow cytometry. Human macrophages were then cultured with SARS-CoV-2-mNG in presence or absence of COVID patient plasma, healthy plasma, monoclonal antibodies , Remdesivir, anti-CD16 antibody, anti-ACE2 antibody, control isotype antibody, caspase-1 inhibitor (VX-76571) or NLRP3 inhibitor (MCC950).Using aseptic techniques under sterile conditions, bone marrow cells were isolated from femurs of immune-reconstituted MISTRG6 mice. For differentiation into human macrophages, bone marrow cells were incubated in media supplemented with 10% FBS, 1% penicillin/streptomycin and recombinant human M-CSF (50ng/ml), GM-CSF (50ng/ml) and IL-4 (20ng/ml) at 1×10Ex vivo lung macrophage cultures: To enrich for human macrophages and monocytes, lung cells from uninfected MISTRG6 mice were sorted based on CD11B and human CD45 expression. These cells were then incubated with GM-CSF and IL-4 for 48 hours to mature macrophages. Non-adherent cells were aspirated prior to culturing with SARS-CoV-2.in vitro or lung macrophages ex vivo were cultured with a viral inoculum at 10^4 PFU of SARS-CoV-2-mNG (~ MOI=0.1). These macrophage cultures were then incubated at 37 C, 5% CO2 for 24, 48 and 72 hours at which time cells were harvested. Cells were dissociated from culture plate with 10 mM EDTA or Accutase (ThermoFisher) cell dissociation reagent (10–20 minutes). For studies pertaining to the mechanism of viral entry, viral replication and inflammasome activation, infected macrophages were treated with Remdesivir (10uM), anti-CD16  and anti-ACE2 (10 μg/ml), caspase-1 inhibitor  and NLRP3 inhibitor  in culture. Cells were stained when applicable and fixed for 30 min with 4% PFA. Convalescent plasma samples from the top 30 neutralizers in a cohort of 148 individuals were pooled to create a mixture with an NT50 titer of 1597 against HIV-1 pseudotyped with SARS-CoV-2 S protein45. We used this pooled serum at a concentration of 5μl-plasma/ml for in vitro experiments and refer to it as COVID patient plasma. Healthy plasma was collected from healthy volunteers and pooled prior to COVID-19 pandemic and used at a concentration of 5μl-plasma/ml. Monoclonal antibodies (a mix of clones 135 and 144) were used at 4 μg per ml concentration.Bone-marrow derived macrophages (BMDMs) Single cell suspension from in vitro cultures or enzymatically dissociated lungs were washed and stained for viability with Zombie Aqua ((Biolegend- 423101) in PBS (1:400) for 15 min at 4°C. Without washing the cells, cell surface antibody cocktail was added, and cells were incubated for another 15 minutes. Cells were then washed with PBS twice and resuspended in Annexin V binding buffer. Cells were stained with Annexin V PE (1:400) in binding buffer for 15 min at 4°C. Cells were then washed with Annexin V buffer and fixed in 4% PFA.Single cell suspension from in vitro cultures or enzymatically dissociated lungs were resuspended in RPMI 10% FBS with FLICA substrate (BioRad-FLICA 660 caspase-1 kit- ICT9122) and cultured for 1h (for microscopy) or 30 min (for flow cytometry) at 37°C. Cells were then washed twice with PBS and stained with Zombie Aqua and Annexin V as described. Cells were then fixed with 1x Fixative (provided in BioRad-FLICA caspase-1 kit) for at least 1 hour not exceeding 16 hours. Cells were kept at 4°C until further staining and analysis. FLICA 660 caspase-1 kit uses a target sequence (YVAD) sandwiched between a far-red fluorescent 660 dye .LDH levels were measured from freshly collected supernatant of infected cells (BMDMs) or freshly collected serum using CyQUANT LDH Cytotoxicity Assay (ThermoFisher- C20300) following manufacturer’s instructions under BSL3 conditions.For this study we have acquired six control uninfected, and two SARS-CoV-2 infected deidentified lung (4 different cuts) specimens as paraffin embedded tissues from autopsies of individuals admitted to Yale New Haven Hospital. Lungs were fixed in 10% Formalin (see Human IL-18 (Sigma or RND), human CXCL10 (RND), human IL-1RA(Abcam), Human Gasdermin D (MyBioSource) were quantified from supernatants of BMDMs infected (or not) with SARS-CoV-2-mNG or from serum or lung homogenates of SARS-CoV-2-mNG infected (or not) MISTRG6 or MISTRG6-hACE2 mice following manufacturer’s instructions. Human IL-1β was quantified from supernatants of BMDMs infected with SARS-CoV-2-mNG using cytometric bead array for human IL-1B (BD) following manufacturer’s instructions. Human anti-Spike-RBD IgG (Biolegend) was quantified from sera and lung homogenates of infected or uninfected MISTRG6-hACE2 mice.All antibodies used in flow cytometry were obtained from Biolegend, unless otherwise specified. Antibodies against the following antigens were used for characterization or isolation of cells by flow cytometry:CD45, CD45, CD45, CD326, F4/80 .CD45, CD45, CD3, CD14,CD16, CD16, CD19, CD19, CD33, CD20, CD20, CD206, CD206, CD86, CD123, CD11B, CD11C, HLA-DR, HLA-DR, HLA-DR, CD183, CD335-NKp46, CD4, CD8, CD8, CD68.Anti-dsRNA antibody  was purchased from Sigma (Cat# MABE1134) or Antibodies online (Cat# Ab01299–23.0). Polyclonal SARS-CoV-2 RNA-dependent RNA Polymerase antibody was purchased from CellSignaling (Cat# 67988S). Monoclonal SARS-CoV-2 RNA-dependent RNA Polymerase antibody was purchased from Kerafest (Cat# ESG004). Anti-Spike (Spike 1) antibody  was obtained from GeneTex. Anti-Spike (Spike 2) antibody  was obtained from ThermoFisher.Mouse anti-human PE-Cy7 CD16(Clone 3G8) was purchased from Biolegend (Cat# 302016). Rabbit anti-human ASC was purchased from Santa Cruz (Cat# sc-22514-R). Goat anti-human NLRP3 was purchased from Abcam(Cat# ab4207). Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody was purchased from ThermoFisher (Cat# A-31573). Donkey anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody  was purchased from ThermoFisher (Cat# A-10040). Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody was purchased from ThermoFisher(Cat# A-21447).45. Anti-IFNAR2 antibody was purchased from PBL Assay science (Cat# 21385–1).Monoclonal antibody against human CD16 used in blocking experiments were purchased from Abcam (SP175). Monoclonal antibody against human ACE2 was purchased from Abcam. Anti-CD16 antibody used in depletion experiments was purchased from ThermoFisher (3G8). Monoclonal antibodies (clones 135 and 144) were acquired from M. Nussenzweig as has been previously describedDDX58, IL6, IFITM3, IRF7, IFIH1, IFNA6, IFNG and HPRT1 were purchased from Sigma.RNA was extracted with the RNeasy mini kit (Qiagen) per the manufacturer’s protocol High-Capacity cDNA Reverse Transcription Kit was used to make cDNA. Quantitative reverse transcription PCR (qRT-PCR) was performed using an SYBR FAST universal qPCR kit (KAPA Biosystems). Predesigned KiCqStart primers for 72, annotated and counted with HTSeq73, normalized using DESeq274 and graphed using the Broad Institute Morpheus web tool. Heatmaps visualize normalized counts of duplicates as min-max transformed values, calculated by subtracting row mean and diving by SD for each gene. Rows (genes) were clustered by hierarchical clustering (one-minus Pearson) or K-means clustering as indicated in the figure legends. Differential expression analysis was also performed with DESeq2. For IFN-stimulated gene identification, http://www.interferome.org was used with parameters -In Vivo, -Mus musculus or Homo sapiens -fold change up 2 and down 2.RNA isolated from homogenized lung tissue, also used for viral RNA analysis, was prepared for whole tissue transcriptome analysis using low input (14dpi) or conventional (28dpi) bulk RNA sequencing. Libraries were made with the help of the Yale Center for Genomic Analysis. Briefly, libraries were prepared with an Illumina rRNA depletion kit and sequenced on a NovaSeq. Raw sequencing reads were aligned to the human-mouse combined genome with STAR19. Duplicates from each condition/time point were pooled in equal numbers to ensure 10000 cells were encapsulated into droplets using 10X Chromium GEM technology. Libraries were prepared in house using Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10X Genomics). scRNA-seq libraries were sequenced using Nova-Seq. Raw sequencing reads were processed with Cell Ranger 3.1.0 using a human-mouse combined reference to generate a gene-cell count matrix. To distinguish human and mouse cells, we counted the number of human genes (nHuman) and mouse genes (nMouse) with nonzero expression in each cell, and selected cells with nHuman > 20 * nMouse as human cells. The count matrix of human cells and human genes was used in the downstream analysis with Seurat 3.275. Specifically, this matrix was filtered to remove low quality cells, retaining cells with > 200 and < 5,000 detected genes and < 20% mitochondrial transcripts. We then log normalized each entry of the matrix by computing log (CPM/100 + 1), where CPM stands for counts per million. To visualize the cell subpopulations in two dimensions, we applied principal component analysis followed by t-SNE, a nonlinear dimensionality reduction method, to the log-transformed data. Graph-based clustering was then used to generate clusters that were overlaid on the t-SNE coordinates to investigate cell subpopulations. Marker genes for each cluster of cells were identified using the Wilcoxon test (two-tailed) with Seurat. For the adjusted P values the Bonferroni correction was used. In this analysis, uninfected: 438 cells, 4dpi: 336 cells, 14dpi: 793 cells, 28dpi: 1368 cells were included.Sorted human lung immune cells  were stained with TotalSeq  antibodies (Biolegend) prior to processing for droplet based scRNA-seq. 10X Chromium GEM technology. Single cell transcriptomes and associated protocols of 4dpi lungs  were previously described76, a targeted, multiscale approach that quantifies a local DA measure for each cell for comprehensive and accurate comparisons of transcriptomic distributions of cells. DA measure defined by DA-seq. shows how much a cell’s neighborhood is enriched by the cells from either uninfected or infected lungs. DA-seq analysis of our data revealed that T cells, monocytes and macrophages were responsible for most of the chronic infection driven changes. Red coloring signify enrichment at 28dpi lungs and blue coloring mark enrichment in uninfected lungsTo identify differentially abundant (DA) subpopulations not restricted to clusters, we applied DA-seq75 in Seurat to remove batch effects. We then performed principal component analysis and retained top 30 PCs as the input to tSNE, a nonlinear dimensionality reduction method, to embed the data onto 2-dimensional space for visualization. Graph-based clustering with a resolution of 0.8 was then used to generate clusters that were overlaid on the t-SNE coordinates to investigate cell subpopulations. Marker genes for each cluster of cells were identified using the Wilcoxon test (two-tailed) with Seurat . After cell type identification, we separated out macrophage populations from all DPIs, and applied the same procedures as described above to re-preprocess and visualize the data. Clusters were redefined based on a resolution of 0.3.To combine cells from different DPIs , we applied the integration methodin vivo studies, each dot represents a biologically independent mouse.Unpaired or paired t-test  was used to determine statical significance for changes in immune cell frequencies and numbers while comparing infected mice to uninfected control mice or treated mice to untreated mice. To determine whether the viral RNA quantification is statistically significant across treatment groups or timepoints, Mann-Whitney, two-tailed test was used. Unpaired t-test (two-tailed) or ratio paired t-test (two-tailed) was used to determine whether the viral titer quantification of the untreated condition is significantly different from the treated groups. For Pearson’s test, significance was deemed using t-test. The test statistic is based on Pearson’s product-moment correlation coefficient cor and follows a t distribution with length(x)-2 degrees of freedom. For Spearman’s test, p-values are computed using algorithm AS 89 with exact = TRUE. All micrographs presented in the study were representative of at least 3 animals or specimens. Each experiment was repeated independently at least two times. All attempts yielded similar results. In Supplementary Table 1Supplementary Table 2Supplementary Table 4Supplementary Table 5Supplementary DiscussionSupplementary Table 3


Dear Editor,n = 121; 49%), chemotherapy  with (n = 24) or without (n = 79) achieving complete remission (CR) or CR with incomplete count recovery (CRi), and allogeneic stem cell transplant ; 1- and 3-year survival rates were 66% and 32% for ASCT, 37% and 19% for chemotherapy-treated patients achieving CR/CRi but were not transplanted, and 8% and 1% in the absence of both ASCT and CR/CRi, respectively [Blast-phase transformation in myeloproliferative neoplasms (MPN-BP) occurs at rates of 3.9, 2.6, and 9.3%, after median follow-up periods of 8.2, 9.9, and 3.2 years, in polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), respectively –3. A Mayvs 33.3%) and overall survival (9.56 vs 5.95 months) compared to 7 + 3 [Reported chemotherapeutic regimens for MPN-BP included either intensive acute myeloid leukemia (AML)-like induction chemotherapy with 7(cytarabine)+3(daunorubicin/idarubicin), or less intensive treatment with hypomethylating agent (HMA) based combinations with venetoclax (Ven) or ruxolitinib; associated CR/CRi rates were 59% (AML-like induction), 43% (HMA-Ven) and 8% (HMA-ruxolitinib) [The current study was conducted under an Institutional Review Board-approved minimum risk protocol that allowed retrospective extraction and analysis of data from MPN-BP patients receiving CPX-351, as first-line therapy, outside the context of a clinical trial at the Mayo Clinic. Study patients were seen between 2018 and 2021 and follow-up was updated in December 2022. Diagnosis of MPN-BP required the presence of ≥20% blasts in either the peripheral blood or bone marrow . All patJAK2 mutated; other mutations included IDH1/2 (n = 7), ASXL1 (n = 4), TP53 (n = 3), TET2 (n = 3), N/KRAS (n = 2) and SRSF2 (n = 1), EZH2 (n = 1) and U2AF1(Q157) (n = 1). Cytogenetics were abnormal in 8 (67%) of patients, with adverse karyotype in 42%. Details on patient characteristics at time of treatment initiation, response rates and subsequent treatments are provided in Table A total of12 consecutive patients with MPN-BP  received CPX-351 in the upfront setting. Antecedent MPN included PV/post-PV MF (50%), ET/post-ET MF (25%) and PMF (25%). All 12 study patients were IDH2 and BCOR mutated, achieved CR following two cycles of CPX-351 induction therapy. He was bridged to ASCT following one cycle of CPX-351 consolidation and remains relapse-free thirty-two months after leukemic transformation. Patient 2 was a 72-year-old male with antecedent post-PV MF, FLT3-ITD, NPM1 and IDH2 mutated with normal karyotype, received one cycle of CPX-351 plus midostaurin and achieved CR. Subsequently, he received two cycles of high dose cytarabine plus midostaurin consolidation followed by ASCT and remained disease-free but succumbed to organizing pneumonia/respiratory failure thirteen months following diagnosis. Patient 3 was a 74-year-old female with antecedent post-ET MF, harboring ASXL1, NRAS, RUNX1, SRSF2. BCOR, PHF6 mutations, with normal karyotype, achieved CR following induction therapy with CPX-351. Subsequent treatments included CPX-351 consolidation and azacitidine maintenance, following which disease relapsed thirteen months after diagnosis; received Ven plus cytarabine (2 cycles) with achievement of CR for 12 months, followed by second relapse treated with HMA-Ven without response.Treatment-emergent toxicity included neutropenic fever in 7 (58%) patients, resulting in death in one patient. Responses following CPX-351 induction therapy included CR in 3 (25%) patients, partial remission (PR) in 1 (8%) patient while none achieved CRi. Patient 1 was a 61-year-old male with antecedent post-PV MF and trisomy 8, n = 4), Ven plus cytarabine (n = 1), FLAG-IDA (n = 1), and enasidenib (n = 1). Three of 5 patients treated with HMA-Ven or cytarabine achieved CR, one of whom was bridged to ASCT, and an additional patient achieved CR and underwent ASCT following salvage therapy with FLAG-IDA. Notably, two patients attained CR with ivosidenib and decitabine plus Ven, respectively, after failure of second line and subsequent therapies, and also proceeded to ASCT (Table n = 7) sepsis (n = 2) and respiratory failure (n = 1). Median overall survival was 11 months  and was longer in six patients that were transplanted .Second-line therapy following failure of CPX-351 was pursued in 7 patients and included HMA-Ven (The current retrospective experience with CPX-351 in patients with MPN-BP was not overtly different than previous observations in similar patients receiving standard 7 + 3 induction or HMA-Ven , 5. The Checklist


DLX6-AS1 expression was elevated in the myocardial tissues of IR injury rats, while DLX6-AS1 deficiency alleviated IR-induced myocardial injury in rats by reducing inflammatory response and cell apoptosis. Moreover, rat embryonic cardiomyocyte cell line H9c2 was subjected to hypoxia reoxygenation (HR). DLX6-AS1 was upregulated in the HR-treated H9c2 cells, and DLX6-AS1 enhanced the expression of F-box and WD40 repeat domain-containing 7 (FBXW7) by sponging miR-204-5p. Inhibition of DLX6-AS1 inhibited inflammatory response and cell apoptosis in H9c2 cells via miR-204-5p/FBXW7 axis. In conclusion, this work demonstrates that DLX6-AS1 accelerates myocardial IR injury through regulating miR-204-5p/FBXW7 axis. Thus, this work provides a novel ceRNA DLX6-AS1/miR-204-5p/FBXW7 axis in myocardial IR injury, and DLX6-AS1 may be a potential target for the treatment of myocardial IR injury.Myocardial ischemia-reperfusion (IR) injury is the restoration of blood flow post ischemia, which threatens the human life. Long noncoding RNA distal-less homeobox 6 antisense 1 (DLX6-AS1) has been found to take part in the IR-induced cerebral injury. Here, we determined the functional role of DLX6-AS1 in IR-induced myocardial injury. We ligated the left anterior descending coronary artery of rats to induce IR injury. IR injury rats exhibited severe tissue damage and increase of infraction size. The levels of lactate dehydrogenase (LDH), creatine kinase (CK), proinflammatory factors including MCP-1, IL-6, and IL-1 Acute myocardial infarction (AMI) is a common and frequently occurring disease in the cardiovascular system . Timely Long noncoding RNA (lncRNA) has been proposed as a key regulator of various biological processes . Recent lncRNA and microRNA (miRNA) are widely involved in competitive endogenous RNA (ceRNA) model. lncRNA relieves the inhibitory effect of miRNA on the downstream target mRNA by sponging miRNA . Many miin vitro.In this work, we constructed a rat model of IR injury and determined the impact of DLX6-AS1 inhibition on IR injury in rats. Next, we further verified the mechanism of action of DLX6-AS1/miR-204-5p/FBXW7 axis in myocardial IR injury Wistar male rats with 14 weeks were purchased from the Shanghai SLAC Laboratory Animal Co., Ltd (China). All protocols were carried out under the authorization of the Ethics Committee of Zhongshan Hospital of Fudan University.IR injury rat model was established by ligation of the left anterior descending (LAD) coronary artery as previously described . Briefly12 vg/mL of AAV9-sh-DLX6-AS1 at multiple sites of myocardium  using a microsyringe. Rats were injected with AAV9-sh-Scramble as control. The LAD coronary artery of rats was ligated after 24 hours post the injection. The AAV9-adenovirus particles containing short hairpin RNA (shRNA) specifically targeting DLX6-AS1 (AAV9-sh-DLX6-AS1) and scrambled RNA (AAV9-sh-Scramble) were obtained from GenePharma .For induce knockdown of DLX6-AS1, rats were injected with 1 × 10μm) of myocardial tissues were subjected to dewaxing and hydration. Then, the sections were stained with hematoxylin and eosin sequentially using Hematoxylin-Eosin (HE) Staining Kit . For detection of apoptotic cells in myocardial tissues, the paraffin sections were stained with TUNEL Apoptosis Assay Kit . The nucleus was stained with DAPI . Finally, the pathological changes and apoptotic cells in myocardial tissues were observed under optical or fluorescence microscope.Paraffin sections  following the protocol of kit. The absorbance was examined on a microplate reader .The levels of lactate dehydrogenase (LDH), creatine kinase (CK), monocyte chemoattractant protein-1 (MCP-1), IL-6, and IL-1μg/mL penicillin/streptomycin at 37°C and 5% CO2. For inducing IR through hypoxia reoxygenation (HR) treatment, H9c2 cells were cultured in serum- and glucose-free DMEM in a hypoxic chamber for 6 h, followed by incubation in DMEM containing 10% FBS in normal oxygen environments.The rat embryonic cardiomyocyte cell line H9c2  was cultured in Dulbecco's Modified Eagle Medium  containing 10% fetal bovine serum  and 100 The pcDNA3.1 carrying DLX6-AS1 (pc-DLX6-AS1) and small interfering RNA specially targeting DLX6-AS1 (si-DLX6-AS1) were used for overexpressing or knocking down DLX6-AS1. The oligonucleotides miR-204-5p mimic and miR-204-5p inhibitor were used to upregulate and downregulate miR-204-5p. The plasmids pc-DNA, NC-mimic, and inh-NC were served as control. All the plasmids were obtained from the GenePharma. H9c2 cells were transfected with the plasmids utilizing Lipofectamine 2000 Transfection Reagent .β-forward: ACA GAT GAA GTG CTC CT; IL-1β-reverse: AAG ATA GGT TCT TCT TCA; GAPDH-forward: GTC AGC CGC ATC TTC TTT TG, GAPDH-reverse: GCG CCC AAT ACG ACC AAA TC; U6-forward: CTC GCT TCG GCA GCA CA, U6-reverse: AAC GCT TCA CGA ATT TGC GT. The relative expression of lncRNA and mRNA was normalized to GAPDH, and miRNA expression was normalized to U6. The results were analyzed using 2-∆∆CT method for quantification.Myocardial tissues and H9c2 cells were treated with TRIzol reagent (Invitrogen) to extract total RNA. PrimeScript RT Master Mix  was utilized to synthesize complementary DNA from total RNA. The relative expression of mRNAs was assessed by using TB Green Premix Ex Taq II (TaKaRa). The primer sequence (5′-3′) was shown as follows: DLX6-AS1-forward: CCA CCC ACT GAG AGA AGA GG, DLX6-AS1-reverse: CCT CCA AGC AAT TGT CCA GT; FBXW7-forward: GTT CCG CTG CCT AAT CTT CCT, FBXW7-reverse: CCC TTC AGG GAT TCT GTG CC; miR-204-5p-forward: TCC GTA TCC TAC TGT T, miR-204-5p-reverse: GCA GGG TCC GAG GTA TTC; MCP-1-forward: TCT GGG CCT GTT GTT CAC AGT, MCP-1-reverse: TGC TGC TGG TGA TTC TCT TGT AGT; IL-6-forward: CTC TCC GCA AGA GAC TTC CAG, IL-6-reverse: TTC TGA CAG TGC ATC ATC GCT; IL-1μg/mL dilution), or GAPDH  4°C for 12 h and then incubated with goat antirabbit HRP-IgG  at 37°C for 1 h. These antibodies were obtained from Abcam . The data was analyzed by Image J software. The expression of protein was normalized to the internal reference gene GAPDH.Total Protein Extraction Kit  and BCA Protein Assay Kit  were utilized to extract proteins from myocardial tissues or H9c2 cells and measure protein concentration. Proteins from each sample were separated by SDS-PAGE gel electrophoresis and then transferred onto the PVDF membranes. The membranes were stained with the primary antibody, Bax , Bcl-2 , FBXW7  or mutant type (Mut) of DLX6-AS1 and FBXW7 were constructed by GenePharma. H9c2 cells were transfected with WT/Mut of luciferase reporter vectors and miR-204-5p mimic or NC-mimic using Lipofectamine 2000 Transfection Reagent. Finally, Luciferase Assay System  was used to assess the relative luciferase activity of H9c2 cells.μg anti-pan-Ago IgG 2A8  and conjugated with Dynabeads Protein G  at overnight rotating at 4°C. IgG1 isotype control  coated with Dynabeads Protein G was used as control. Subsequently, the cell lysates were incubated with 50 μL glycine (pH 2.3) at room temperature for 15 min to elute protein-RNA complexes from the beads. The protein-RNA complexes were lysed with proteinase K. Finally, miRNeasy Mini Kit  was used to purify RNA following the manufacturer's recommendations. The purified RNA was served as template to detect DLX6-AS1 and miR-204-5p expression by performing qRT-PCR. Cell lysates were served as Input group.RIP assay was carried out to determine whether DLX6-AS1 and miR-204-5p interacted with Ago-2 to form a RNA-induced silencing complex (RISC) according to reported work . H9c2 ceFlow cytometry was done to measure apoptotic cells in H9c2 cells utilizing Annexin V-FITC Apoptosis Detection Kit  on a BD FACSCalibur . H9c2 cells were cultured for 24 h. After washing with PBS for 3 times, cells were stained with Annexin V-FITC and PI in the dark. Finally, the apoptotic cells were detected by flow cytometry analysis.t-test (between two groups) and one-way ANOVA (among multiple groups) were utilized to analyze the statistical differences. P < 0.05 indicates significant differences.Each assay was performed for 3 times. The representative data were presented and expressed as mean ± standard deviation. Statistical analysis was carried out utilizing SPSS 22.0 statistical software . Two-tailed Student's β were also enhanced in the serum of IR rats as compared with sham-operated rats  E3 ubiquitin ligase complex. In recent years, a lot of work has found that miR-223-3p targets FBXW7 to regulate myocardial inflammation and apoptosis after myocardial infarction . AdditioThis work demonstrated that lncRNA DLX6-AS1 promoted FBXW7 expression by sponging miR-204-5p, which contributed to accelerating myocardial IR injury. Thus, this work provides a novel ceRNA DLX6-AS1/miR-204-5p/FBXW7 axis in myocardial IR injury, and DLX6-AS1 may be a potential target for myocardial IR injury treatment.


An animal model that can mimic the SARS-CoV-2 infection in humans is critical to understanding the rapidly evolving SARS-CoV-2 virus and for development of prophylactic and therapeutic strategies to combat emerging mutants. Studies show that the spike proteins of SARS-CoV and SARS-CoV-2 bind to human angiotensin-converting enzyme 2  to mediate viral entry. Several hACE2 transgenic (hACE2Tg) mouse models are being widely used, which are clearly invaluable. However, the hACE2Tg mouse model cannot fully explain: (1) low expression of ACE2 observed in human lung and heart, but lung or heart failure occurs frequently in severe COVID-19 patients; (2) low expression of ACE2 on immune cells, but lymphocytopenia occurs frequently in COVID-19 patients; and (3) hACE2Tg mice do not mimic the natural course of SARS-CoV-2 infection in humans. Moreover, one of most outstanding features of coronavirus infection is the diversity of receptor usage, which includes the newly proposed human CD147 (hCD147) as a possible co-receptor for SARS-CoV-2 entry. It is still debatable whether CD147 can serve as a functional receptor for SARS-CoV-2 infection or entry.scid IL2Rgammanull (NSG) background. In this hCD147KI-NSG mouse model, the hCD147 genetic sequence was placed downstream of the endogenous mouse promoter for mouse CD147 (mCD147), which creates an in vivo model that may better recapitulate physiological expression of hCD147 proteins at the molecular level compared to the existing and well-studied K18-hACE2-B6 (JAX) model. In addition, the hCD147KI-NSG mouse model allows further study of SARS-CoV-2 in the immunodeficiency condition which may assist our understanding of this virus in the context of high-risk populations in immunosuppressed states. Our data show (1) the human CD147 protein is expressed in various organs  in hCD147KI-NSG mice by immunohistochemical staining and flow cytometry; (2) hCD147KI-NSG mice are marginally sensitive to SARS-CoV-2 infection compared to WT-NSG littermates characterized by increased viral copies by qRT-PCR and moderate body weight decline compared to baseline; (3) a significant increase in leukocytes in the lungs of hCD147KI-NSG mice, compared to infected WT-NSG mice.Here we successfully generated a hCD147 knock-in mouse model (hCD147KI) in the NOD-hCD147KI-NSG mice are more sensitive to COVID-19 infection compared to WT-NSG mice. The hCD147KI-NSG mouse model can serve as an additional animal model for further interrogation whether CD147 serve as an independent functional receptor or accessory receptor for SARS-CoV-2 entry and immune responses.The online version contains supplementary material available at 10.1186/s13578-022-00822-6. SARS-CoV-2 is the novel coronavirus that causes Coronavirus-Disease of 2019 (COVID-19) and has become a global pandemic and devastated millions. While there are many similarities between SARS-CoV-2 and its sister virus SARS-CoV . ACE2 rePlasmodium falciparum  at room temperature for 1 h before being stained with primary rabbit anti-RBD antibody (diluted 1:400 in blocking buffer) overnight at 4 °C. Then, slides were washed three times with PBS for 5 min before being stained with Alexa Fluor 488 conjugated goat anti-rabbit secondary antibody  at room temperature for 1 h. Slides were washed three times and then mounted using Prolong Gold Antifade with DAPI  and sealed before imaging.The16-bit and 8-bit images were acquired of stained lung tissue from infected and control mice. 20 random fields were imaged per mouse. To obtain final quantification, image processing was done with Python and NumPy library . Matrix Homogenized lungs suspensions from lungs of SARS-CoV-2 infected mice were rinsed and resuspended in PBS prior to staining with the following anti-mouse antibody cocktail for 30 min on ice: CD45-BV510 , CD11b-FITC , CD11C-Pe/Cy7 , Ly6C-PerCP/Cy5.5 , Ly6G-Alexa Fluor 700 . Stained samples were then rinsed with PBS and resuspended in 4% PFA and allowed to fix for 72 h in accordance with biosafety requirements before analyzing using a BD LSRFortessa™ X-20 Cell Analyzer. Flow data was quantified using FlowJo (Tree Star). Bioinformatic analysis was performed using GraphPad Prism 5.0 (GraphPad).het-NSG males precluding the generation of a hCD147KIhomo-NSG mouse. Thus, hCD147KIhet-NSG mice were used for the purpose of evaluating SARS-CoV-2 sensitivity.We developed a mouse model in which hCD147 was expressed in normal cells and tissue under the control of the endogenous Bsg/Cd147 gene promoter mimicking physiological expression  was inserted in frame with mouse Ace2 using CRISPR-Cas9 in exon 1. The mouse Ace2 5’UTR and the first 15 amino acids of mouse Ace2 (MSSSSWLLLSLVAVT) encoding the leader sequence were retained and the remainder of the sequence was replaced with the hACE2 cDNA sequence. Cas9 protein complexed with sgRNA containing the spacer sequence 5ʹ-GAGCAGTAGTAACAGCAACA-3ʹ and plasmid targeting vector was microinjected into NSG zygotes. Founders were screened for integration of human ACE2 cDNA, then screened for proper targeting to the mouse locus by PCR using primers external from and internal to the targeting vector. PCR fragments were purified and sequenced by the Sanger method to confirm the cloning junctions. Founders were bred to WT NSG mice and N1 progeny were confirmed to receive the targeted allele. Thus, the hCD147KI-NSG and hACE2KI-NSG mice were successfully generated using CRISPR/Cas9 technology.A similar strategy was used to generate a physiological expression model for the human angiotensin-converting enzyme 2 (hACE2) downstream of the mouse het-NSG mice, we further verified human CD147 protein expression in these mice. Organs were harvested from adult hCD147KIhet-NSG mice and WT-NSG littermates and stained for human CD147 protein by IHC  in knock-in mice compared to WT-NSG mice where no staining was visualized. Interestingly, we observed that red blood cells in the lung also expressed strong human CD147 staining indicating successful integration of the protein into erythrocyte precursors. The strongest staining was associated with bronchioles in the lungs relative to surrounding parenchyma, in condensing spermatids in the testes, and in mucosal villi in the intestines. Comparable tissue expression levels of human CD147 in hCD147KIhet-NSG mice to that of mouse CD147 in WT-NSG mice was observed in sampled tissues as well 2Prlmn/J, 034,860) line. Expectedly, we observed ruffling of the fur and significant body weight drops  hACE2, the putative receptor for SARS-CoV-2 entry (hACE2KI-NSG), and for (2) CD147, the potential accessory or co-receptor for coronavirus entry (hCD147KI-NSG). Next, we characterized the hCD147KI-NSG mice by flow cytometry and IHC and confirmed physiological distribution of the hCD147 protein across a variety of tissues in hCD147KI-NSG mice. Finally, we compared the ability of live SARS-CoV-2 virus to infect both knock-in models and observed significant amounts of viral RNA in lung tissues by sm-FISH and qRT-PCR.Key to the development of successful and effective vaccines to SARS-CoV-2 infections and treatments for COVID-19 patients is the understanding SARS-CoV-2 infectivity and pathogenesis. The fundamental mechanism underlying SARS-CoV-2 entry remains poorly understood. Previous studies show that the spike proteins of SARS-CoV , 44 and However, while these models have recapitulated some aspects of the COVID-19 disease course, such as lung inflammation , cytokinCoronaviruses are known to have a high diversity of entry receptors, which includes the newly proposed human CD147 (hCD147) as a receptor for SARS-CoV-2. CD147 is a transmembrane glycoprotein with multiple functions in normal lung, immune cells, and diseased tissues . It has Bsg and Ace2, respectively. The human keratin 18 promoter was used to overexpress hACE2 receptor in the epithelial cells of the most commonly used K18-hACE2-B6 model. Specifically, over-expression of human ACE2 was identified in airway epithelia and the epithelia lining the liver, kidney and gastrointestinal tract in K18-hACE2-B6 mice [The hCD147KI-NSG and hACE2KI-NSG mice developed in this study utilize the natural promoter for mouse -B6 mice . However-B6 mice , a major-B6 mice .CRISPR/Cas9 protein complexed with gRNA, along with the hCD147 or hACE2 plasmid constructs, were microinjected directly into NSG mouse zygotes, in order to generate a scarless knock-in of the human cDNA in frame within the first 22 amino acids of the mouse CD147 protein. Therefore, the human CD147 receptor and human ACE2 receptor are both expected to follow the same pattern of expression as the endogenous mouse receptor equivalents under normal conditions in hCD147KI-NSG and hACE2KI-NSG mice, respectively. Since the ACE2 gene is located on the X chromosome in humans and mice, we elected to utilize homozygous male and female hACE2KI-NSG mice  as controls for in vivo infection experiments to eliminate the possibility for varying mouse ACE2 protein expression between homozygous hACE2KI-NSG male and heterozygous hACE2KI-NSG female mice.Our data supports the hypothesis that human CD147 plays an accessory role in SARS-CoV-2 infection. We confirmed strong expression of the human CD147 protein in the bronchiolar airway cells in hCD147KI-NSG mice compared to lung parenchymal cells. We have also confirmed expression in extra pulmonary organs such as the trachea. This protein distribution pattern may help explain why newer variants can replicate faster and more efficiently in the upper airway compared to lower airway regions . ImportaAdditionally, CD147 is an important immune-modulator  making iThe limitations of the current study include small sample sizes and lack of exact molecular mechanisms. To better assess the possibility that the human CD147 receptor contributes to overall SARS-CoV-2 infection, we are currently generating a double knock-in hCD147KI-hACE2KI-NSG mouse model to evaluate synergistic effects between the two receptors. In concordance with our previous results, we hypothesize that this mouse model will be more likely to develop significant morbidity and demonstrate a heightened sensitivity to SARS-CoV-2 infection. Additionally, future experiments will be able to compare the natural course of SARS-CoV-2 infection in immunocompromised hCD147KI-NSG and immunocompetent hCD147KI-B6 mice and will further elucidate the role of the human CD147 receptor during the SARS-CoV-2-induced immune response. While our results do not provide clarity on the mechanism by which this receptor enhances viral entry or exacerbates viral replication and persistence, we confirm here that this model can set the stage for future inquiry and examination.The hCD147KI model offers several strengths to the scientific community as it will better capture other nuances of the COVID-19 disease. (1) This model will allow researchers to study hemodynamic instability and increased thrombosis risk following COVID-19 infection as the hCD147 protein is expressed in circulating erythrocytes. (2) The NSG background will allow scientists to study how adoptive transfer can either dampen or exacerbate COVID-19-induced cytokine storm that is often seen in severe disease. Additionally, as the NSG background has been used to study diabetes , our modCurrently available animal models available for the study of SARS-CoV-2 infection cannot explain many aspects of COVID-19 disease as it presents in humans such as increased thrombosis risk, increased risk of severe disease in patients with Diabetes or other comorbidities, and other clinical sequelae associated with SARS-CoV-2 infection. It has been suggested that due to its ability to serve as an entry receptor for other viruses including other coronaviruses, CD147, also known as Basigin, was recently proposed as a viral entry receptor for SARS-CoV-2. Here, we have successfully developed and validated expression of the human CD147 receptor in immunocompromised NSG mice for the purposes of evaluating whether this protein can also serve as an additional functional or accessory receptor for SARS-CoV-2 viral entry and subsequent immune responses. Our data show that similar to the newly generated hACE2KI-NSG mice, hCD147KI-NSG mice are also more sensitive to SARS-CoV-2 infection compared to WT-NSG mice characterized by a moderate body weight decline post-infection correlated with persistent viral presence in the lungs for up to 7 days post-infection. However, the effect may not be induced by direct receptor binding via CD147. The hCD147KI-NSG mouse model generated in this study will help further elucidate the molecular mechanism(s) of CD147 in SARS-CoV-2 infection.Additional file 1: Figure S1. Similar distribution of transgenic hCD147 protein by immunohistochemistry in various tissues in hCD147KIhet-NSG mice relative to WT-NSG mice. Tissues from wild-type NSG mice (left) were stained for mouse CD147 protein using rabbit anti-mouse primary antibody  and tissues from hCD147KIhet-NSG mice (right) were stained for human CD147 protein using mouse anti-human primary antibody . Images were taken using an Olympus Inverted Light Microscope. Scale bar represents 100 µm. Figure S2. Specificity of anti-human CD147 and anti-mouse CD147 antibodies tested against human and mouse cell lines. Representative contour plots of CD147 expression on BNL 1ME A.7R.1 (top) and HepG2 (bottom) cells using antibodies targeting either mouse CD147 protein, human CD147 protein, or a combination of both antibodies (far right). Relative percentages are listed, and significant shifts highlighted in red. Gating was determined based on donkey anti-goat and mouse isotype IgG antibody background staining. Figure S3. Reduction of body weight 2 days post-infection in SARS-CoV-2 infected hACE2KI-NSG and K18-hACE2-B6 mice, and slight body weight reduction in infected hCD147KI-NSG mice. Average body weight loss as a percent of original body weight in WT-NSG (n=3), hCD147KI-NSG (n=3), hACE2KI-NSG (n=4), and K18-hACE2-B6 (n=3) mice following intranasal infection with the TCID50 dose of SARS-CoV-2 virus (1×105 PFU in 25ul per nostril). Error bars represent standard error measure (SEM). Statistical significance was determined using an unpaired one-tailed Student t test. *indicates significance of p<0.05. Figure S4. Trends in increased viral presence 2 days post-infection in the lungs of SARS-CoV-2 infected hCD147KI-NSG, hACE2KI-NSG, and K18-hACE2-B6 mice. Quantification of total SARS-CoV-2 viral copies in the lungs of infected mice at day 2 post-infection by qRT-PCR represented as total N-gene RNA copies per lung lobe (left) and as N-gene log-2 fold-change relative to WT-NSG mice (right). Mean values (red) are listed above each group. Each data point represents the average of two duplicate qRT-PCR reactions from one mouse lung RNA preparation. Statistical significance was determined using an unpaired one-tailed t test. Figure S5. Increased viral presence 2 days post-infection in the lungs of SARS-CoV-2 infected hCD147KI-NSG, hACE2KI-NSG, and K18-hACE2-B6 mice by sm-FISH imaging. Representative sm-FISH images at 20X (1st column) and 63 ×  overlay) magnifications showing SARS-CoV-2 RNA detection in lung sections from WT-NSG (1st row), hCD147KI-NSG (2nd row), hACE2KI-NSG (3rd row), and K18-hACE2-B6 (4th row) mice, 2 days post-infection. Blue: DAPI; green: SARS-CoV-2 RNA. Scale bar represents 50 µm. Figure S6. Increased leukocytosis 2 days post-infection in the lungs of SARS-CoV-2 infected hCD147KI-NSG, hACE2KI-NSG, and K18-hACE2-B6 mice. Quantification of various immune cell subpopulations in the lungs of SARS-CoV-2 infected mice was carried out by flow cytometry. Leukocytes were enumerated as a percentage of total live cells. Subsequent immune subpopulations are shown as percentages of total CD45+ cells. Error bars represent standard error measure (SEM). Statistical significance was determined using an unpaired one-tailed Student t test. * indicates significance strength where *signifies p < 0.05, **signifies p < 0.01, and ***signifies p < 0.001). Figure S7. H&E and immunohistochemical staining of human CD147 and SARS-CoV-2 Spike protein Receptor Binding Domain (RBD) in infected hCD147KIhet-NSG and hACE2KIhomo-NSG mouse lungs. Representative fields of H&E (top row), human CD147 protein (middle row), and SARS-COV-2 Spike protein RBD (bottom row) in infected WT-NSG (left column), hCD147KIhet-NSG (middle column), and hACE2KIhomo-NSG (right column) mice lung tissues harvested 7 days post infection. Scale bar represents 20 µm. Figure S8. Immunohistochemical staining and quantification of SARS-CoV-2 spike protein in lungs of infected WT-NSG, hCD147KIhet-NSG, and hACE2KIhomo-NSG mice. (A) Representative fields of lung tissues from infected WT-NSG (top row), hCD147KIhet-NSG (middle row), and hACE2KIhomo-NSG (bottom row) mice stained for SARS-CoV-2 Spike protein Receptor Binding Domain (RBD) (1st column), human CD147 (2nd column), DAPI (3rd column), differential interference contrast (DIC) (4th column), merged image (5th column). Images were acquired with an Olympus confocal microscope using a 60 × oil objective. Scale bar represents 20 µm. (B) Representative images for quantification of SARS-CoV-2 Spike protein Receptor Binding Domain (RBD) mean and total fluorescent intensity in infected lung tissues. An example of an 8-bit rescaled image (left) and its corresponding transformation after threshold cutoff was applied (right) are shown of a single field from an hACE2KIhomo-NSG mouse lung 7 days post-infection. Scale bar represents 20 µm. (C) Relative quantification of mean and total fluorescent intensity of SARS-CoV-2 Spike protein Receptor Binding Domain (RBD) in infected lung tissues of hCD147KIhet-NSG, and hACE2KIhomo-NSG mice compared to control WT-NSG mice. Violin plots of mean (left) and total (right) fluorescent intensity values obtained from at least 20 individual fields per mouse lung tissue section from infected hCD147KIhet-NSG and hACE2KIhomo-NSG mice compared to control WT-NSG mice are shown. Statistical significance was determined using an unpaired two-tailed Student t test. The average mean (red) and total (black) fluorescence intensity value are listed beneath each violin plot. Table S1. Genotyping primers, RNA-FISH probes, and qRT-PCR primers and probes. A complete list of primers used to genotype hCD147KI-NSG and hACE2KI-NSG mice (top) is shown, followed by a list of the primers and molecular beacon probe used for SARS-CoV-2 quantification in infected lung tissues by Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) (bottom left) and Single Molecule RNA Fluorescent in-situ Hybridization (sm-FISH) (bottom right). CFR = Cal Fluor Red 610, BHQ-2 = Black Hole Quencher 2, underlined nucleotides in the molecular beacon probe indicate the 5ʹ and 3ʹ arm regions of the probe.


Drosophila. They demonstrated that modulating miR-34 and its downstream target, Eip74EF, showed beneficial effects on an age-related disease using a Drosophila model of Spinocerebellar ataxia type 3 expressing SCA3trQ78. These results imply that miR-34 could be a general genetic modifier and therapeutic candidate for age-related diseases. Thus, the goal of this study was to examine the effect of miR-34 and Eip47EF on another age-related Drosophila disease model.In 2012, Liu et al. reported that miR-34 is an age-related miRNA regulating age-associated events and long-term brain integrity in Drosophila eye model expressing mutant Drosophila VCP (dVCP) that causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), or multisystem proteinopathy (MSP), we demonstrated that abnormal eye phenotypes generated by dVCPR152H were rescued by Eip74EF siRNA expression. Contrary to our expectations, miR-34 overexpression alone in the eyes with GMR-GAL4 resulted in complete lethality due to the leaky expression of GMR-GAL4 in other tissues. Interestingly, when miR-34 was co-expressed with dVCPR152H, a few survivors were produced; however, their eye degeneration was greatly exacerbated. Our data indicate that, while confirming that the downregulation of Eip74EF is beneficial to the dVCPR152HDrosophila eye model, the high expression level of miR-34 is actually toxic to the developing flies and the role of miR-34 in dVCPR152H-mediated pathogenesis is inconclusive in the GMR-GAL4 eye model. Identifying the transcriptional targets of Eip74EF might provide valuable insights into diseases caused by mutations in VCP such as ALS, FTD, and MSP.Using a  Multisystem proteinopathy (MSP) is a dominantly inherited autosomal disease with differing degrees of severity, ranging in different combinations of inclusion body myopathy (IBM), Paget’s disease, frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS) in the same family members , 2. ArouDrosophila melanogaster. When miR-34 was knocked out, life spans were reduced, and aging deficits were present including an increase in misfolded proteins and accelerated neurodegeneration. When miR-34 was overexpressed, the lifespan of adult Drosophila increased. Therefore, miR-34 may potentially protect against neurodegeneration and extend the lifespan by reducing disease-associated protein misfolding. Indeed, they tested miR-34 against Drosophila expressing the polyQ domain of mutant Ataxin-3 (SCA3trQ78) causing spinocerebellar ataxia and found that inclusion formation was slowed, and neurodegeneration was suppressed  sc[*] v[1] sev[21]; P{y[+ t7.7] v[+ t1.8] = VALIUM20-mCherry}attP2: siRNA for mCherry.29353 y[1] v[1]; P{y[+ t7.7] v[+ t1.8] = TRiP.JF02515}attP2: siRNA for Eip74EF (FBgn0000567).63790 w[*]; P{w[+ mC] = UAS-DsRed-mir-34.L}3/TM3, Sb[1] Ser[1]: mir-34.41158 w[1118]; P{y[+ t7.7] w[+ mC] = UAS-LUC-mir-34.T}attP2: mir-34.61386 w[*]; P{y[+ t7.7] w[+ mC] = UAS-mCherry.mir-34.sponge.V2}attP40; P{y[+ t7.7]w[+ mC]=.UAS-mCherry.mir-34.sponge.V2}attP2/TM6B, Tb[1]: mir-34 sponge.R152H/CyO, and GMR-GAL4 were gifts from Dr. J. Paul Taylor.GMR-GAL4, UAS-dVCPDrosophila were crossed and grown on Bloomington Stock Center standard cornmeal food at 25℃. Progeny were collected at two days old.Drosophila eye pictures were taken using a Leica Z16 APO with the DMC2900 camera and a ring light.http://estimationstats.com [Eye phenotype quantification was performed with ilastik  and Flyntats.com . AlthougR152H and miR-34 pathways. We used a previously published Drosophila model expressing mutant Drosophila VCPR152H (dVCPR152H) in eyes with GMR-GAL4 [R152H was expressed in Drosophila eyes, severe eye degeneration was observed as published [To test our hypothesis, we examined genetic interactions between dVCPDrosophila expressing dVCPR152H in their eyes  were crossed with Drosophila lines carrying a construct for RNAi of Eip74EF (Bloomington 29353), miR-34 overexpression , and miR-34 sponge (Bloomington 61386). mCherry siRNA control line (Bloomington 35785) was used as a negative control. Eip74EF siRNA expression significantly rescued abnormal eye phenotypes generated by dVCPR152H compared to the degenerated eyes of control siRNA (mCherry siRNA) expressing Drosophila in both males  and neurons (ELAV-GAL4/Nsyb-GAL4) may be performed. In addition, we only observed siRNA-mediated phenotypic changes. Increased expression of Eip74EF or a validated human ortholog may provide further supporting evidence for our findings. Finally, we used different fly lines from previous literature claiming protective effects of miR-34 on neurodegeneration. Thus, further studies into the interactions between miR-34 and dVCPR152H with different miR-34 lines may be necessary or a different eye-specific GAL4 line (Rh1-GAL4) may need to be used to avoid the lethality.While the data is meaningful in the


Physical function predicts health-related quality of life. The biological mechanisms underlying declines in physical function with age remain unclear. We examined the plasma proteomic profile associated with longitudinal changes of physical functions measured by gait speed and grip strength in community-dwelling adults. We applied aptamer-based platform to assay 1,161 plasma proteins on 2,871 participants  in Cardiovascular Health Study (CHS) in 1992/1993 and 1,550 participants  in Framingham Offspring Study (FOS) in 1991-1995. Gait speed and grip strength were measured annually for 6 years in CHS and at cycles 7 (1998-2001) and 8 (2005-2008) in FOS. The associations of individual protein levels (log-transformed and standardized) with longitudinal changes of gait speed and grip strength in two populations were examined separately by linear mixed effect models. Meta-analyses were implemented using random effect models with a Bonferroni correction for multiple testing. We found that plasma levels of 18 and 12 proteins were associated with changes in gait speed and grip strength, respectively (Bonferroni-corrected p < .05). The proteins most strongly associated with gait speed decline were growth/differentiation factor 15 (GDF-15) , pleiotrophin (PTN) (1.29E-08), and metalloproteinase inhibitor 1 (TIMP-1) (2.02E-08). For grip strength decline, the strongest associations were for GDF-15 (1.39E-07), carbonic anhydrase III (6.60E-07), and TIMP-1 (3.21E-06). Several statistically significant proteins are involved in the alternative complement pathway, extracellular matrix remodeling or immune function. These novel proteomic biomarkers may inform our understanding of the pathophysiology of functional decline.