Setaria italica L.) is an important model crop for studying tolerance to various abiotic stressors. Therefore, examining the response of foxtail millet to salt stress at the molecular level is critical. Herein, we discovered that SiDi19-3 interacts with SiPLATZ12 to control salt tolerance in transgenic Arabidopsis and foxtail millet seedlings. SiDi19-3 overexpression increased the transcript levels of most Na+/H+ antiporter (NHX), salt overly sensitive (SOS), and calcineurin B-like protein (CBL) genes and improved the salt tolerance of foxtail millet and Arabidopsis. Six SiDi19 genes were isolated from foxtail millet. Compared with roots, stems, and leaves, panicles and seeds had higher transcript levels of SiDi19 genes. All of them responded to salt, alkaline, polyethylene glycol, and/or abscisic acid treatments with enhanced expression levels. These findings indicate that SiDi19-3 and other SiDi19 members regulate salt tolerance and other abiotic stress response in foxtail millet.Salt stress is an important limiting factor of crop production. Foxtail millet ( In theSiDi9-3 under salt stress, we overexpressed SiDi19-3 in the hairy roots of ‘Yugu1’ seedlings using K599 agrobacterium-mediated transformation. Notably, the SiDi19-3 transcript levels in transgenic foxtail millet seedlings were higher than those in the control   A. The 35eedlings B–D, demoSiSOSs, SiCBLs, and SiNHXs  in foxtail millet. In comparison, chr3 contained three SiDi19 genes, chr5 had two, and chr1 had only one SiDi19 gene. Based on their chromosomal placement, the SiDi19 genes were named SiDi19-1—SiDi19-6  analysis, we examined the transcriptional levels of in seeds A. Furthecis-elements in the promoters of SiDi19 genes to determine whether any SiDi19 genes other than SiDi19-3 operated in response to abiotic stresses (SiDi19 promoters. An auxin-responsive element (TGA) was predicted in the promoters of SiDi19-1, -3 and -5. Two or three ethylene response elements (ERFs) were predicted in the promoters of SiDi19-2 and -3. More than one drought responsive element (DRE/MBS) was predicted in the promoters of SiDi19-1, -2, -4, and -6. One or two low-temperature responsive elements (LTRs) were predicted in the promoters of SiDi19-2 to -5. One to three salicylic acid-responsive elements (TCA/SARE) were predicted in the promoters of SiDi19-1 and -3, respectively. More than one wound responsive element (WRE) was predicted in the promoters of all SiDi19 genes except SiDi19-2. These results suggest the potential roles of SiDi19 genes in hormone-mediated abiotic stress responses.We initially examined the stresses . Two or SiDi19 genes at 3 and 24 h after appropriate treatments. As depicted in SiDi19 genes. For example, all SiDi19 genes were induced by NaCl, NaHCO3, and ABA treatments at 3 or 24 h, and all SiDi19 genes, except SiDi19-6, were induced by PEG. Furthermore, SiDi19-3 and -4 indicated the highest inducible levels in response to NaCl and ABA treatments. These results indicate the redundant roles of SiDi19 genes in response to multiple ABA-mediated abiotic stresses in foxtail millet.Furthermore, we detected the transcript levels of the Arabidopsis [Di19 genes function in response to abiotic stress is unknown. In this study, we discovered that SiDi19-3 favorably regulates the salt tolerance in foxtail millet and Arabidopsis /SCaBP8 (CBL10) complexes activate the transporter activities of SOS1 and NHXs [SOS, CBL, and NHX genes  cultivar ‘Yugu1’ and Arabidopsis Col-0 were employed. Foxtail millet seeds underwent sodium hypochlorite sterilization, were washed three times, and then were germinated for three days at 25 °C. Seven-day-old ‘Yugu1’ seedlings were exposed to Hoagland solution containing 200 mM NaCl, 50 mM NaHCO3, 100 μM ABA, or 20% (w/v) polyethylene glycol 6000 (PEG6000) for 3 and 24 h, respectively, for inducible expression analyses. The seedlings were collected and frozen in liquid nitrogen as soon as possible. Roots, stems, and leaves from 14-day-old seedlings, panicles from 2-month-old seedlings, and seeds of ‘Yugu1’ were collected and immediately frozen in liquid nitrogen for tissue expression analyses. The previously described techniques were used to grow foxtail millet seedlings with transgenic hairy roots [For investigation of gene expression and function, the foxtail millet  as previously described . Full-le35S::SiDi19-3-NYFP and 35S::SiPLATZ12-CYFP constructs were transformed into Agrobacterium tumefaciens GV3101. The 35S::P19 vector was co-transformed into three-week-old N. benthamiana leaves with 35S::SiDi19-3-NYFP and 35S::SiPLATZ12-CYFP, 35S::SiDi19-3-NYFP and CYFP, NYFP, 35S::SiPLATZ12-CYFP, NYFP, and CYFP. After 28 h of culture, the transformed tobacco leaves were monitored for reconstituted YFP fluorescence using an LSM880 confocal microscopy . The primers used in this experiment are listed in For these assays, the coding regions of SiDi19-3 and SiPLATZ12 were amplified and cloned into pSPYNE-35S (NYFP) and pSPYCE-35S (CYFP) vectors to fuse them with the N- or C-terminal YFP, respectively. The resultant SiDi19-3 and SiPLATZ12 were cloned into pGEX-4T-1 and pCold-TF vectors to generate SiDi19-3-GST and SiPLATZ12-His constructs, respectively, as previously described [E. coli cells. SiDi19-3-GST and SiPLATZ12-His proteins were induced by IPTG and were then used for protein purification using the His-Tagged Protein Purification Kit  and BeyoGold GST-tag Purification Resin . Next, SiDi19-3-GST or GST proteins were incubated with SiPLATZ12-His protein in 300 μL of binding buffer  for 1 h at 4 °C in a continuously rotator. The proteins were detected using immunoblotting with anti-GST or anti-His antibodies after elution from the beads. Signals were detected using a Chemiluminescence Imaging System . The primers used in this experiment are listed in For these assays, the CDSs of escribed . SubsequThe transgenic hairy-roots harboring pSiDi19-3:SiDi19-3-GFP were obtained using the previously described techniques  to analySiDi19-3 was amplified and cloned into PROKII vector. Based on an earlier study [A. tumefaciens K599. Positive transformants were inoculated into 20 mL of liquid solution and cultured with continuous shaking for 12 h. The culture was then centrifuged at 6000× g for 5 min. Subsequently, the cells were resuspended in 1/2 MS liquid medium to a final concentration with OD600 = 1.0. Three-day-old shoot tips were cut and incubated with A. tumefaciens containing 35S::SiDi19-3 or the empty vector at 28 °C for 10–20 min with continuous shaking. The transformed shoot tips were transferred onto 1/2 MS solid medium supplemented with 100 mg/L timentin overnight to induce root formation [Under the control of the 35S promoter, the full-length CDS of er study , the recormation . ApproxiArabidopsis containing 35S::SiDi19-3 was simultaneously generated using the floral-dipping method as previously described [3 transgenic Arabidopsis plants were selected using 1/2 MS medium supplemented with 50 mg/L kanamycin and confirmed by RT-qPCR using the primers listed in Transgenic escribed . T3 tranArabidopsis were used to BLAST the foxtail millet genome in Phytozome V12 (https://phytozome.jgi.doe.gov (accessed on 19 April 2022)) to identify the SiDi19 genes. The isoelectric point and molecular weight were predicted using ExPASy Proteomics Server (http://expasy.org/ (accessed on 19 April 2022)). An evolutionary tree was generated using MEGA6 software and a phylogenetic evolutionary tree was constructed using neighbor-joining analysis and bootstrap method, with 1000 replicates. Di19 genes from Oryza sativa, Arabidopsis, Sorghum bicolor, Zea mays, and Glycine max were previously identified [http://gsds.cbi.pku.edu.cn/ (accessed on 19 April 2022)). MEME (https://meme-suite.org/meme/tools/meme (accessed on 19 April 2022)) was used to analyze the conserved motifs. The cis-elements in the promoters of the SiDi19 genes were predicted using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 19 April 2022)).Di19 family members from entified ,16,29. TSiACTIN7 and 18S rRNA were used as two normalizing expression values within the calculation leading to the relative expression values in foxtail millet [AtGAPDH and AtUBQ10 were used as two normalizing expression values within the calculation leading to the relative expression values in Arabidopsis [Total RNAs was extracted from the indicated tissues and seedlings treated with the indicated stimuli using RNAiso Plus . Two micrograms of total RNAs were used to synthesize cDNAs using the PrimeScriptTM RT reagent kit  according to the manufacturer’s instructions. Further, RT-qPCR was performed using ChamQ Universal SYBR qPCR Master Mix  in a three-step program on a CFX96TM Real-Time PCR Detection System . l millet ,44. AtGAbidopsis ,42. Thre


Protease-activated receptors (PARs) and their activating enzymes have been postulated to play a role in inflammatory bowel disease (IBD) pathogenesis, but the specific roles of PAR2 in disease initiation and progression remain unclear. PAR2 activation has both pro-proliferative and pro-migratory effects and could be involved with the restoration of the epithelial barrier following injury. We previously showed that PAR2 activation increases epithelial wound healing through ERK, PI3K, and JNK pathways. However, the role of Src kinase and RhoGTPases, which PAR2 also activates, is not known.We hypothesized that PAR-2 activation induces wound healing in intestinal epithelial cells through Src and Rac1 activity.Circular wounds were made in T84 colonic epithelial cell monolayers. Wounded monolayers were treated with the PAR2 activating peptide, 2-furoyl-LIGRLO , or the inactive control reverse-sequence peptide, 2-furoyl-OLRGIL , and live-cell imaging was used to record wound healing over a 24-hr period. Proliferation and apoptosis were measured using EdU and TUNEL assays, respectively. The mechanism of action was evaluated using inhibitors of Src (PP2), EGFR (PD153035), MLCK (ML-7), ROCK (Y-27632), Rac1 (NSC 23766), and Cdc42 (ML141), and western blot (WB) was used to confirm the protein levels of p-Src (Y416), p-ERK1/2, p-JNK, and p-PI3K. For immunofluorescence, images of E-cadherin and F-actin were taken to capture the entire wound border and surrounding cells.p<0.05). PAR2 activation had no effect on proliferation at the wound edge and did not affect apoptosis but did enhance lamellipodia/filopodia formation (p<0.001). These findings indicate that PAR2 reprograms the cells toward a migratory rather than a proliferative phenotype. When we investigated the mechanisms of action, the Src tyrosine kinase inhibitor, PP2, blocked PAR2-induced wound healing (p<0.0001). PAR2 activation increased Src phosphorylation  and the immunofluorescence showed a rise in actin cable formation at the wound edge and a reduction in lamellipodia/filopodia formation in the group treated with PP2 when compared to 2fLI (p<0.001). Although PAR2 activation increases the phosphorylation of ERK1/2 and JNK, this did not happen through Src. Furthermore, PAR2 did not increase the phosphorylation of PI3K as confirmed by WB analysis. Inhibition of EGFR (PD153035), MLCK (ML-7), ROCK (Y-27632), and Cdc42 (ML-141) did not alter PAR2-induced wound healing (p>0.05). In contrast, Rac1 inhibition by NSC23766 completely abrogated the PAR2-induced wound healing (p<0.05).PAR2 activation by 2fLI promoted wound healing compared to 2fO or vehicle control at the 24-hr time point (PAR2 activation drives wound healing via Src tyrosine kinase and Rac1 activities. These findings provide a further mechanism whereby PAR2 can participate in the resolution of intestinal wounds in gastrointestinal inflammatory diseases.None Declared


In the current work, spatial and quantitative analysis of tumor burden were performed using simultaneous [18F]FDG-PET/MRI to monitor therapeutic responses to a novel silicified cancer cell immunotherapy in a mouse model of disseminated serous epithelial ovarian cancer. Tumor progression was validated by bioluminescence imaging of luciferase expressing tumor cells, flow cytometric analysis of immune cells in the tumor microenvironment, and histopathology. While PET demonstrated the presence of metabolically active cancer cells through [18F]FDG uptake, MRI confirmed cancer-related accumulation of ascites and tissue anatomy. This approach provides complementary information on disease status without a confounding signal from treatment-induced inflammation. This work provides a possible roadmap to facilitate accurate monitoring of therapeutic responses to cancer immunotherapies.Current imaging approaches used to monitor tumor progression can lack the ability to distinguish true progression from pseudoprogression. Simultaneous metabolic 2-deoxy-2-[ Growing evidence that ovarian cancer induces tumor-specific immune responses supports the development of novel immunotherapies for this disease ,2. We re18F]FDG-PET can detect cancer cells based on elevated glucose consumption and is readily available in clinics. Critically, [18F]FDG-PET and MRI can be performed simultaneously, and have been shown clinically to be equivalent to [18F]FDG-PET/computed tomography for detecting abdominal metastases FDG-PET/MRI as a clinically applicable strategy for differentiating tumor growth versus treatment-associated immune responses, we analyzed treatment responses to our silicified cancer immunotherapy using an established preclinical mouse model of serous epithelial ovarian cancer. Imaging results were compared with immune cell measurements using flow cytometry, gross anatomy, tissue histopathology, and tumor burden using bioluminescence imaging. In luciferase-labeled tumors, bioluminescence provides a specific measure of tumor burden that is not confounded by inflammation, providing a measure of true tumor progression. Integrated [18F]FDG-PET with structural MRI, including structural T2-weighted imaging and T1-mapping, was used to monitor therapeutic responses to vaccination in female mice with existing BR5-akt-Luc2 metastatic ovarian cancer. To evaluate the utility of FDG-PET/MRI was completed in the four mouse groups. Mice were administered [18F]FDG by either retro-orbital (RO) or tail vein (TV) injection, with no statistical difference in imaging results between injection methods , as shown in FDG-PET images (independent and overlayed) for cancer-no Tx or cancer-vaccine mice, acquired during simultaneous imaging, are shown in 18F]FDG-PET measurements of percent injected dose per gram (%ID/g), a standard measure of FDG accumulation in preclinical studies, and standardized uptake values (SUV), a standard measure of FDG accumulation in clinical studies,  images from the four study groups are shown in 1 relaxation provides a measure of ascites accumulation, while T2 relaxation enables anatomical identification and co-registration of PET with MRI. [18F]FDG-PET/MRI provides a measure of tumor accumulation that correlates with tissue anatomy.Representative variable repetition time (VTR) Tstudies, . Regardil groups D, with cMilky spots in the omentum are metastatic niches for ovarian cancer, with adipocytes required for subsequent cancer spread . Examina18F]FDG-PET MIP , whereas [18F]FDG-PET was useful in the identification of metabolically active tumors. The identify of tumor was validated by bioluminescence, supporting detection of true tumor progression. Furthermore, spatial location of tumor metastasis was supported by gross anatomy. [18F]FDG-PET/MRI, gross anatomy, tissue weights, and T cell presence. As stated previously, pseudoprogresssion is an apparent increase in tumor size following immune therapy that is attributed to immune cell infiltration. [18F]FDG-PET/MRI imaging of tumor-challenged vaccinated mice was distinct from that of untreated mice supporting a lack of a confounding signal from treatment-induced inflammation. The most abundant immune cell type present in ascites fluid in untreated tumor- bearing mice was pmnMDSC. Tumor MDSC have higher glycolytic rates than their normal cell counterparts [18F]FDG uptake by immune cell post vaccination. T cells on the other hand, specifically CD8+ T cells, are large consumers of glucose and have been shown to contribute to [18F]FDG uptake in mouse models of inflammation FDG uptake. [18F]FDG uptake in naïve vaccinated mice was similar to untreated controls, indicating that immune cell infiltration did not significantly contribute to [18F]FDG uptake. A limitation of our study was that we initiated therapy 4 days post cancer challenge. Future studies will delay vaccination until later stages of tumor progression, likely leading to higher levels of immune cell infiltration.For contrast, enhanced T therapy ) is due  therapy . A strenTen per cent buffered formalin, tetramethyl orthosilicate (TMOS), hydrochloric acid (HCl), sodium chloride (NaCl), and MPL from salmonella enterica serotype were purchased from Sigma-Aldrich . Phosphate-buffered saline (PBS) was purchased from Thermo Fisher Scientific . Fetal bovine serum (FBS) was purchased from ATCC ; 0.05% EDTA trypsin solution, and penicillin-streptomycin were purchased from Life Technologies Corporation . Dulbecco’s Modified Eagle’s Medium (DMEM) was obtained from Caisson Labs . XenoLight D-Luciferin Potassium Salt was purchased from Perkin Elmer . Endotoxin-free CpG ODN 1826 was purchased from InvivoGen  and linear, MW 25,000 polyethyleneimine (PEI) from Polysciences . Endotoxin free cell culture grade water was purchased from GE Healthcare . b (M5/14.15.2) AF532, CD127 (A7R34) BV650, CD3 (500A2) Pacific Blue, NKp46 (29A1.4) PE/Dazzle 594, CD49a (Ha31/8) BV711, CD49b (HMa2) AF647, CD25 (PC61) PE/Cy5, B220 (RA3-6B2) APC-Cy5.5, and CD200R (Ox110) PerCP-eFlour 700. Antibodies, Fc receptor blockers (anti-CD16/CD32 (clone 2.4G2)), mouse IgG (31205), LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit, and Far-Red Cell Stain Kit were purchased from eBioscience™/Thermo Fisher Scientific , Biolegend , or BD Biosciences .CD3 (17A2) APC-eFluor 780, CD4 (GK1.5) APC or PE-Fire, CD8a (53-6.7) eFluor 450 or PerCP, CD44 (IM7) PerCP-Cyanine5.5, CD62L  FITC, Ly6c (HK1.4) BV421, CD45 (30-F11) BV510, CD11b (M1/70) BV570, CD11c (HL3) BV786, Ly6g (1A8) Spark Blue 550, F4/80 (T45-2342) APC, NK1.1 (PK136) BV750, I-Aakt cell line, generated on a Friend leukemia virus B (FVB) background, was lentivirus transduced to constitutively express firefly luciferase FDG uptake time was 45 min. During the scan, the depth of anesthesia was monitored via animal respiration rate and core body temperature was maintained with a forced-air warming system. PET/MRI was performed using a 7-Tesla Biospec 70/30 MRI  equipped with a 72 cm RF volume coil, a 20 cm gradient coil with 300 mT/m gradient strength, and a 3-ring PET insert . Whole-body PET was acquired in list-mode for 40 min and MRI was performed simultaneously. MRI included a VTR T1-map  followed by T2 . PET images were reconstructed as a single static frame using a maximum likelihood expectation maximization (MLEM) algorithm with 12 iterations. Corrections were applied for scatter, randoms, decay, and partial volume. Reconstructed PET image dimensions were 320 × 320 × 600 and the voxel size was 0.25 mm isotropic.A schematic for the simultaneous PET/MRI protocol is shown in 1 relaxation maps. PET data were registered to MRI data based on the calibrated scanner transformation matrix, which allows automatic alignment of PET and MRI. Vivoquant software version 3.5p2  was used to perform manual ROI analysis on coregistered T2-MRI/[18F]FDG-PET/T1-map-MRI data for quantification of abdominal [18F]FDG uptake and T1 signal. Coregistered images were cropped just inferior to the heart and superior to the bladder in order to exclude bright areas of [18F]FDG signal in those regions. All ROIs were drawn on the coronal view of the T2 image, using the sagittal and axial views for reference. Abdominal ROIs were delineated using a semi-automated thresholding technique based on the T2-MRI signal intensity, starting ventrally, and extending dorsally to just above the kidneys  were obtained for the abdomen. FDG-uptake values were calculated as two different normalized metrics: (1) activity concentration normalized to injected dose (%ID/g) and activity concentration normalized to injected dose and subject body weight (SUV).Paravision 360 software  was used to generate T kidneys A. Abdomit-tests were used for single comparisons. Data collected from individual subjects is displayed, along with mean and the standard deviation included as error bars. For MRI analysis, comparison between multiple groups was carried out with the Welch ANOVA with multiple comparisons performed using Dunnet’s T3. The Welch version of ANOVA does not assume that all the groups were sampled from populations with equal variances. Ordinary ANOVA with Tukey’s for multiple group comparisons were performed for flow cytometry and PET data. GraphPad Prism version 9  was used for all statistical analysis. Unpaired, parametric, two-tailed 2, T1-map, [18F]FDG-PET) and provides anatomic and metabolic information for monitoring therapeutic responses to cancer immunotherapy.Hybrid PET/MRI with simultaneous acquisition offers a method for accurate co-registration of multiple imaging parameters (TPatent applications US20220125825A1 and US20200276286A1 are associated with the silicified cancer cell technology.


To determine neutralizing activity against the severe acute respiratory syndrome coronavirus 2 ancestral strain and 4 variants of concern, we tested serum from 30 persons with breakthrough infection after 2-dose vaccination. Cross-variant neutralizing activity was comparable to that after 3-dose vaccination. Shorter intervals between vaccination and breakthrough infection correlated with lower neutralizing titers. However, booster vaccinations with 1 dose of mRNA vaccine after priming with an initial 2 doses induce high levels of serum neutralizing activity against Omicron (https://www.medrxiv.org/content/10.1101/2022.01.18.22269452v2), and emerging evidence suggests that breakthrough infections with non-Omicron SARS-CoV-2 variants also elicit cross-neutralizing serum activity against Omicron (The B.1.1.529 (Omicron) variant of concern of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) carries a high number of nonsynonymous mutations in the spike glycoprotein, relative to that of the ancestral (wild-type) strain (Wu01). Those mutations result in a strong immune evasion phenotype, as demonstrated by severely reduced serum neutralization after vaccination or previous infection with ancestral variants in most persons (https://www.comirnaty.com) or heterologous vaccination with ChAdOx1  and BNT162b2  in 20 persons with non-Omicron  SARS-CoV-2 infection after 2-dose mRNA vaccination with BNT162b2  against Wu01 was 6.3-fold higher after breakthrough infection . This difference in serum neutralizing activity was particularly pronounced against the Beta  and Omicron  variants, each of which exhibits substantial immune escape. The boosting effect of non-Omicron breakthrough infections was highly variable (50) showed a strong correlation with the interval between second vaccination and diagnosis of breakthrough infection   , panel DIn summary, we found that Omicron and non-Omicron SARS-CoV-2 breakthrough infections elicit cross-variant neutralizing antibodies. Our results suggest that short vaccination-to-infection intervals correlate with lower neutralizing titers, which may be relevant for recommendations concerning additional booster vaccination of persons who experience early breakthrough infections after initial immunization.Additional methods for study of cross-variant neutralizing antibodies after SARS-CoV-2 breakthrough infections.


Enterobacter cloacae sequence type 1 (ST1) isolate AVS0889, which was recovered from a river in Switzerland in 2021. The genome consists of a 4.95-Mbp chromosome and five plasmids, including a large plasmid (90.8 kb) harboring a disrupted mcr-10 gene.Here, we report the complete genome sequence of colistin-resistant  Enterobacteriaceae strains harboring the plasmid-mediated colistin resistance gene mcr-1 in 2015 (mcr (mobilized colistin resistance) variants (mcr-2 to mcr-10) have been observed in various species  and a positive PCR result for mcr-10, was isolated in November 2021 from a water sample collected from the river Lorze in Switzerland . The water sample (100 mL) was filtered through a 0.45-μm membrane filter (Millipore). The filter was incubated in 10 mL enterobacteria enrichment (EE) broth (BD) at 37°C for 24 h. One loopful of the EE broth was spread on cystine-lactose-electrolyte-deficient (CLED) agar (Oxoid) supplemented with 4 mg/L colistin, 10 mg/L vancomycin, and 5 mg/L amphotericin and was incubated at 37°C for 24 h. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)  was used for preliminary genus identification. Different PCR primers were used to detect mcr-1 to mcr-10 genes . DNA was isolated from a subculture obtained from a single colony that had been grown for 24 h at 37°C on sheep blood agar. Genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen). Libraries were prepared using the Nextera DNA Flex library preparation kit (Illumina) and sequenced on the Illumina MiniSeq platform (2 × 150 bp). Illumina reads were trimmed with fastp v0.20.1 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc). For long-read sequencing, libraries were prepared using the SQK-LSK109 kit and sequenced on the MinION platform with a FLO-MIN106 flow cell (Oxford Nanopore Technologies [ONT]). Base calling, demultiplexing, and barcode trimming were performed with guppy v4.2.2 (ONT), and quality was assessed with LongQC v1.2.0  and 320 Mbp of short-read data  using the Unicycler v0.4.8 pipeline (https://github.com/tseemann/mlst). Resistance genes and plasmid replicons were identified using abricate v1.0.1 (https://github.com/tseemann/abricate)  with the ResFinder (esFinder  and PlasesFinder  databaseesFinder . DefaultdnaA) and the circular plasmids pAVS0889-a , pAVS0889-b , pAVS0889-c , pAVS0889-d , and pAVS0889-e , with a GC content of 54.7%. AVS0889 was identified by rMLST and MLST as Enterobacter cloacae sequence type 1 (ST1). Two chromosomally encoded antimicrobial resistance genes were detected, namely, blaCMH (AmpC β-lactamase) and fosA (fosfomycin thiol transferase). In addition, plasmid pAVS0889-b harbored a disrupted mcr-10 gene. This gene contained a 29-bp insertion at position 64 and an IS26 insertion element inserted near the 5′-terminal end. No complete mcr genes were detected, suggesting that the phenotypic colistin resistance of AVS0889 is mediated by one of the various known intrinsic resistance mechanisms , CP092043 (pAVS0889-a), CP092044 (pAVS0889-b), CP092045 (pAVS0889-c), CP092046 (pAVS0889-d), and CP092047 (pAVS0889-e) under BioProject number PRJNA803974. The sequencing data were deposited in the NCBI Sequence Read Archive (SRA) under the accession numbers SRR17902521 (Illumina reads) and SRR17902520 (ONT reads).The complete genome sequence of AVS0889 has been deposited in GenBank under the accession numbers


C. elegans WDFY-3 protein is important for cargo selection during selective autophagy and for regulating axon termination. The C-terminal region of WDFY-3 contains BEACH, WD repeats, and FYVE-like domains, all of which are required for selective autophagy. WDFY-3 also contains a large N-terminal region that is relatively uncharacterized. Currently,wdfy-3(ok912)is the only mutant allele that has been characterized for this gene. This allele features a small deletion that is predicted to disrupt the C-terminal region of the protein. Here, we used CRISPR Cas9 to produce a newwdfy-3(cue30) allelethat is a near complete deletion of the coding region. We report that, unlike the existingwdfy-3(ok912)allele, this newwdfy-3(cue30)null allele causes a weak overextension phenotype in the PLM axon. Like the existingwdfy-3(ok912)allele, the newwdfy-3(cue30)null allele can suppress PLM axon termination defects caused by anfsn-1null allele. Creating and characterizing newwdfy-3 alleles will increase our understanding of this gene and could help elucidate more of the gene’s conserved functions.The WDFY3gene  encodes a scaffold protein that is required for selective autophagy and has been associated with autism . WDFY3 functions as an adaptor that links ubiquitinated cargo to the core autophagy components, thereby recruiting ubiquitinated targets into autophagosomes for eventual destruction in autophagolysosomes. Large scale genome sequencing studies have revealed a significant association betweenWDFY3and autism . Consistent with this observation,WDFY3is required for the normal development of axon tracts in mice . Moreover, genetic analysis inC. eleganshas indicated thatwdfy-3regulates axon targeting by functioning in a genetic pathway withegl-19, an ortholog of theCACNA1Cautism-associated gene . However, the mechanism through which WDFY-3 affects axon targeting remains mostly unknown.The humanwdfy-3(cue30)allele. This newwdfy-3(cue30)allele is a near complete deletion of the coding region ofwdfy-3, except for the last three exons that make up the FYVE-type domain at the C-terminus of the protein . This is unlike the previously studied loss-of-functionwdfy-3(ok912)allele, which contains a small deletion that removes part of the BEACH domain in the C-terminal region . The removal of the BEACH domain in thewdfy-3(ok912) allele also causes a frameshift mutation, likely resulting in the disruption of the WD repeats and FYVE-type domains . However, the large N-terminal region of WDFY-3 may not be affected by this mutation.As a first step in further exploring the role of WDFY-3 in axon targeting, we used CRISPR Cas9 to create the newwdfy-3(cue30)allele affects axon targeting, we examined the PLM axon. The PLM cell body resides in the tail and extends an axon anteriorly along the lateral body wall. In wildtype worms, the PLM axon terminates at the midbody, prior to reaching the ALM cell body . We found that our newwdfy-3deletion allele causes PLM axon termination defects that are significantly greater than those observed in wildtype . Interestingly, thewdfy-3(ok912)allele does not cause PLM axon termination defects. Together, these observations suggest the possibility that thewdfy-3(ok912) allele might not be a null allele and that the uncharacterized N-terminal region of WDFY-3 could have functions important for axon guidance. Future investigations could test this hypothesis through the analysis of additional targeted deletions inwdfy-3.To determine how our newwdfy-3(ok912)allele can suppress axon termination defects caused by anfsn-1null allele . To determine if our newwdfy-3(cue30)allele also has this property, we analyzed PLM axon termination inwdfy-3(cue30);fsn-1(null)double mutants. We found thatwdfy-3(cue30)also suppresses overextension caused byfsn-1 . However, this suppression did not bring the overextension penetrance back to wildtype levels like thewdfy-3(ok912) allele . These observations are consistent with our previous report of a genetic interaction betweenwdfy-3andfsn-1 .Our prior work demonstrated that thewdfy-3(cue-30)mutation. Unique guide RNAs were designed to cut in two places to excise most of the coding region of thewdfy-3 gene without perturbing the function of nearby genes or the non-coding UTR region ofwdfy-3. Guide RNAs used were upstream: GCGATTATTGGATTATCTCG & downstream: AGTTTTGAACACGTGCGACG.A CRISPR editing technique was used to create theC. elegansstrains were cultured and maintained on nematode growth medium (NGM)-agar plates using standard methods at 20°C . Axons were labeled and observed as previously described . Briefly, animals were mounted on a 5% agarose pad and observed with a 40x objective. PLM neurons were visualized with themuIs32transgene which encodesPmec-7::gfp+lin-15(+)and is expressed in all mechanosensory neurons .muIs32[mec-7p::gfp+ lin-15(+)]II;fsn-1(gk429) IIIAGC52:muIs32[mec-7p::gfp+ lin-15(+)]II;wdfy-3(ok912) IIAGC135:muIs32[mec-7p::gfp+lin-15(+)]II;wdfy-3(cue30) IVAGC209:muIs32 [mec-7p::gfp+ lin-15(+)]II;fsn-1(gk429) III; wdfy-3(ok912) IVAGC259:muIs32 [mec-7p::gfp+ lin-15(+)]II;fsn-1(gk429) III; wdfy-3(cue30) IVAGC260:


AZD7442—a combination of 2 human, extended–half-life, SARS-CoV-2–neutralizing monoclonal antibodies (mAbs) (tixagevimab/cilgavimab)—has received US Food and Drug Administration emergency use authorization for COVID-19 prevention in immunocompromised individuals. We evaluated the effect of AZD7442 in prevention and treatment settings in Syrian hamsters challenged with SARS-CoV-2.5 plaque-forming units) in prevention; OR control mAb (5 mg) or AZD7442 (0.5–5 mg) 1 day after IN SARS-CoV-2 challenge in treatment. The impact of AZD7442 on lung viral RNA and pathology and AZD7442 serum levels was assessed on Days 3 and 7 post infection. Body weight was recorded daily through Day 7.Hamsters received intraperitoneal isotype control mAb (2 mg) or AZD7442 (0.002–2 mg) 1 day before intranasal (IN) SARS-CoV-2 challenge  levels were seen with doses ≥0.2 mg AZD7442. Concomitantly, increased serum levels of AZD7442 were observed. By Day 7, infectious virus titer and sgmRNA fell below the level of detection (LOD) at all doses tested. Moreover, AZD7442 at doses ≥0.2 mg protected hamsters from weight loss versus controls. Lung pathology scores  were generally dose dependent, with mean scores of < 2 for AZD7442 versus 10 for controls, indicating less SARS-CoV-2–induced inflammation and alveolar damage in hamsters given AZD7442. Lower AZD7442 doses were associated with mean pathology scores similar to controls. With AZD7442 treatment, infectious virus titers were below the LOD at Day 3 post infection and at Day 7 for sgmRNA, for all doses tested. Mean lung pathology score was <2 for AZD7442 versus 12 for controls. AZD7442 doses ≥0.5 mg protected against weight loss relative to controls.In a SARS-CoV-2 challenge model, AZD7442 administered as prevention or treatment led to significantly lower lung viral loads and improved lung pathology, without weight loss. There was also no evidence that AZD7442 mediated antibody-dependent enhancement of disease or infection.Yueh-Ming Loo, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Patrick M. McTamney, PhD, AstraZeneca: Employee Richard Roque, B.S., AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Yingyun Cai, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Mark T. Esser, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds.


In response to high carbohydrate demand from nodules, legumes display autoregulation of nodulation (AON) to restrict nodules to the minimum number necessary to sustain nitrogen supply under current photosynthetic levels. AON mutants super-nodulate and typically grow smaller than wild-type plants under ambient CO2. Here, we show that AON super-nodulating mutants have substantially higher biomass under eCO2 conditions, which is sustained through increased photosynthetic investment. We examined photosynthetic and physiological traits across super-nodulating rdn1-1 (Root Determined Nodulation) and sunn4 (Super Numeric Nodules) and non-nodulating nfp1 (Nod Factor Perception) Medicago truncatula mutants. Under eCO2 conditions, super-nodulating plants exhibited increased rates of carboxylation (Vcmax) and electron transport (J) relative to wild-type and non-nodulating counterparts. The substantially higher rate of CO2 assimilation in eCO2-grown sunn4 super-nodulating plants was sustained through increased production of key photosynthetic enzymes, including Rieske FeS. We hypothesize that AON mutants are carbon-limited and can perform better at eCO2 through improved photosynthesis. Nodulating legumes, especially those with higher nitrogen fixation capability, are likely to out-perform non-nodulating plants under future CO2 conditions and will be important tools for understanding carbon and nitrogen partitioning under eCO2 conditions and future crop improvements.Legumes are generally considered to be more responsive to elevated CO The rdn1-1 mutant has a 103-kb deletion that includes RDN1 as well as 17 other annotated genes 2-carboxy-D-arabinitol 1,5-bisphosphate to the fully carbamylated enzyme, as described by Ruuska et al. [For biochemical analysis of leaves, leaf discs of 0.353 cma et al. .v/v) glycerol, 8% SDS, 4% bromophenol blue, 0.5% betamercaptoethanol, and incubated at 95° C for 5 min. Samples were loaded on a leaf area basis and separated by polyacrylamide gel electrophoresis (Nu-PAGE 4–12% Bis-(2-hydroxyethyl)-amine-tris(hydroxymethyl)-methane (Bis-Tris) gel, Invitrogen, Life Technologies Corporation, Carlsbad, CA) in running buffer (pH 7.3) containing 50 mM 2-(N-morpholino)ethanesulfonic acid, 50 mM trisaminomethane, 0.1% sodium dodecylsulfate (w/v), and 20 mM ethylenediaminetetraacetic acid. Proteins were transferred to a nitrocellulose membrane and probed with antibodies against Rieske FeS protein  at 1:3000 dilution, and Rubisco small subunit protein  at 1:10,000 dilution. Quantification of Western blots was performed with Image Lab software .In order to isolate leaf proteins, protein extract derived from the active Rubisco assay was diluted into 4x SDS Sample buffer containing 0.25 M tris-HCl pH 6.8, 40%  at 663.2 nm and 646.8 nm against an 80% acetone blank. Chlorophyll A content was determined from sample absorbance according to Lichtenthaler [2) were oven-dried at 80 °C, weighed, and then ground to powder in a ball mill. Percentage of N and C was determined on the ground tissues using an Isoprime isotope-ratio mass spectrometer (Elementar).Additional leaf discs from where photosynthetic measurements were conducted were used for measurements of leaf carbon (C) and nitrogen (N) content. Leaf discs . Where two-way ANOVA results were significant, a Tukey’s multiple comparisons test was used to identify significant pair-wise differences arising from CO2 treatment or plant genotype. p-values < 0.05 were considered significant.All statistical analyses were completed in GraphPad Prism


Formulated Tat/pDNA-Ca2+ nanoparticles mainly use macropinocytosis for intercellular delivery, and their macropinocytic uptake was persisted in mitosis (M-) phase and highly activated in DNA synthesis (S-) phase of cell-cycle. Over-expression or phosphorylation of a mitochondrial chaperone, 75-kDa glucose-regulated protein (GRP75), promoted monopolar spindle kinase 1 (MPS1)-controlled centrosome duplication and cell-cycle progress, but also driven cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca2+ nanoparticles. Further in vivo molecular imaging based on DF (Fluc-eGFP)-TF (RFP-Rluc-HSV-ttk) system showed that Tat/pDNA-Ca2+ nanoparticles exhibited highly suicide gene therapy efficiency in mouse model xenografted with human ovarian cancer. Furthermore, arresting cell-cycle at S-phase markedly enhanced delivery performance of Tat/pDNA-Ca2+ nanoparticles, whereas targeting GRP75 reduced their macropinocytic delivery. More importantly, in vivo targeting GRP75 combined with cell-cycle or macropinocytosis inhibitors exhibited distinct suicide gene therapy efficiency. In summary, our data highlight that mitochondrial chaperone GRP75 moonlights as a biphasic driver underlying cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca2+ nanoparticles in ovarian cancer.Practice of tumor-targeted suicide gene therapy is hampered by unsafe and low efficient delivery of plasmid DNA (pDNA). Using HIV-Tat-derived peptide (Tat) to non-covalently form Tat/pDNA complexes advances the delivery performance. However, this innovative approach is still limited by intracellular delivery efficiency and cell-cycle status. In this study, Tat/pDNA complexes were further condensed into smaller, nontoxic nanoparticles by CaThe online version contains supplementary material available at 10.1186/s12951-022-01530-6. Current treatments of ovarian cancer (OC) mainly choose surgery and chemotherapy, resulting in limited clinical benefits . Tumor-tIntroduction of cell-penetrating peptides (CPPs), typically HIV-Tat-derived peptide (Tat), to noncovalently complex therapeutic gene incorporated in plasmid DNA (pDNA) for intracellular delivery represents an innovative approach for targeting malignancy . Noncova2+ has been widely used in therapeutic gene delivery [2+ precipitates with pDNA to form condensed particles amenable to cellular transfection. Previous studies revealed that Ca2+ has a synergic effect on DNA condensation along with other kinds of CCPs [2+ to DNA-complexes. Theoretically, a strong affinity between Tat and pDNA is required to form condensed and stable particles for yielding efficient uptake. On the other hand, a relatively lower affinity between Tat and pDNA is desired to facilitate pDNA release after endocytosis [2+ addition controlling the delicate formulation of Tat/pDNA nanoparticles should be explored.As a stabilizer of DNA-carrier complexes, divalent Cadelivery . When us of CCPs –13. Thisocytosis . Becauseocytosis , optimalMacropinocytosis is an evolutionarily conserved endocytic route that mediates non-specific fluid-phase uptake of extracellular particles and solutes. It is now well appreciated that active macropinocytosis is a hallmark of certain tumors, and macropinocytosis is markedly upregulated by various oncogenic pathways or nutrient-deficient microenvironments , 15. Com2). This simple formulation (Tat/pDNA-Ca2+ nanoparticles) exhibited advantages, including smaller size, positive charge, safer and highly suicide gene therapy efficiency. The Tat/pDNA-Ca2+ nanoparticles was delivered by cell-cycle-dependent macropinocytosis in OC cells, and their uptake was driven by expression or phosphorylation of moonlighting chaperone GRP75. Targeting GRP75 blocked monopolar spindle kinase 1 (MPS1)-controlled centrosome duplication and cell-cycle progress, but also reduced the active macropinocytosis of Tat/pDNA-Ca2+ nanoparticles at cell-cycle mitosis (M-) and DNA synthesis (S-) phases. In vivo targeting GRP75 combined with cell-cycle or macropinocytosis inhibitors underlies distinct therapy effect in mouse model xenografted with human ovarian cancer.In this study, pDNA was complexed with Tat peptide and condensed into nanoparticles by addition of calcium chloride  efficiently condensed Tat/pGL3 or Tat/pGL3-DiYO-1 complexes into nanoparticles even when the N/P ratio reached to 1 , and possessed small particle size (252–512 nm) at N/P ratio of 10  and phosphates (DNA) can control particle size [2+ nanoparticles is compatible with the results from AFM and TEM checking images, and irregular granules with asymmetric morphology were frequently observed  can reduce aggregation and yield of more monodisperse lipoplexes [2, whereas Tat/pDNA-Ca2+ nanoparticles with optimal CaCl2 concentration (113 mM) were relatively stable in mouse serum and serum-contained culture media  marker Tfn-AF647 and lipid raft marker CTxB, but co-localized with the macropinocytosis marker Dextran  since macropinocytic capacity is determined by the properties of cells [2+ nanoparticles with irregular and asymmetric morphology  are prone to stimulate macropinocytosis boosted in OC cells  Posof cells , 37. Our Posof cearticles , which i2+ nanoparticles with Dextran in unperturbed cells showed that they were still internalized via macropinocytosis in different sub-phases of cell-cycle  -induced S-phase and persisted in PTX(paclitaxel)-induced M-phase  do take place during normal mitosis when checking asynchronous cells under advanced microscopy (e.g. Electron microscopy) –46. Conscal Fig. B also sung Figs. A, B. Litcal Fig. B. Since 2+ nanoparticles. Cyclebase-based predication showed that high-expression of GRP75 frequently appeared at S-phase and G2(gap phase 2)/M boundary. High-expression of GRP75-bound mitotic kinase MPS1 was mainly distributed from S- to M-phase. However, high-expression of GRP75-bound, MPS1-recruited mitochondrial gatekeeper VDAC1 was mainly presented at S-phase. Remarkably, high-expression of macropinocytosis driver Arf6 was scarcely distributed at S-phase, G2/M boundary and early G1-phase (gap phase 1)  cells, but significantly enhanced in GRP75-OE or phosphorylation-activation (T62D/S65D) cells  reporter gene, and Tat/TF -Ca2+ nanoparticles were introduced by tail vein injection for suicide gene therapy . Skov3 labeled with DF reporter gene were created by our lab , 61. LenMouse anti-GRP75 antibody (Ab) (sc-133137) and anti-VDAC1 (sc390996), rabbit anti-B-actin Ab (sc-7210) were obtained from Santa Cruz. Rabbit anti-Arf6 Ab (381658) was obtained from Zenbio. Mouse anti-RFP Ab (31101ES60) was obtained from Yeasen. Mouse anti-β-actin Ab (66009), anti-r-tubulin Ab (66320), and rabbit anti-GRP75 Ab (14887), anti-r-tubulin Ab (15176), anti-MPS1 Ab (10381), and goat anti-mouse Ab-AF488 (A-11001), anti-mouse Ab-AF647 (SA00006-1), anti-mouse Ab-AF594 (SA00006-3), anti-rabbit Ab-AF488 (SA00006-3) were obtained from Proteintech. HIV-Tat-derived peptide (GRKKRRQRRRPPQC) was obtained from GL Biochem Ltd. DiYO-1 (17580) and DiYO-3 (17581) were obtained from AATbio. Luciferase Reporter Gene Assay Kit (11401ES60), Coelenterazine H (40906ES03), D-Luciferin (40901ES02), TUNEL apoptosis Kit (40306ES), cell apoptosis kit (40306ES) and polybrene (40804ES) were obtained from Yeasen. Annexin V-FITC apoptosis detection kit (C1062L) was obtained from Beyotime. GCV, (HY-13637), MKT077 (FJ-776), RO3306 (HY-12529) and EIPA hydrochloride (HY-101840A) were obtained from MCE. SP-9000 SPlink Detection Kit (SP-9000) was obtained from ZSGB-BIO. Lovastatin (SL8280), GCB (G8970). PTX (SP8020), Sucrose (S8271) and HU (H8420) were obtained from Solarbio. 70 kDa Dextran-RD (D1841), BCA protein assay Kit (23225), Hochest33342 (B2261), propidium Iodide (P4170), puromycin (A1113803), lipofectamine 3000 (L3000015) and other chemicals were obtained from Thermo Fisher.Synchronizing cells at different sub-phases of the cell-cycle were performed by the previous report with some modifications , 62 . Resulted mixtures were equilibrated at 4 °C, centrifuged, washed with PBS, and the pellet contained the target nanoparticles.Tat/pDNA-Caescribed , 12. Bri2+ nanoparticles was negatively stained with 2% uranyl acetate dihydrate and checked under the transmission electron microscopy (Philips Tecnai G2 20 S-TWIN). Mean particle size and the zeta potential of the nanoparticles were determined by DLS . Stability of the nanoparticles was tested by electrophoretic retardation and DNase I protection assays. All these characterizations were similarly performed as previously described [Morphology of Tat/pDNA-Caescribed , 7.2+ nanoparticles (10 ug pDNA) diluted in 2 mL incomplete medium with/without cell-cycle inhibitors  at 37 °C for 4–14 h transfection. Before changing to complete medium, one aliquot of cells was thoroughly washed by PBS/0.5 M NaCl, lysed and determined the fluoresce intensity by the fluorescence microplate reader (FLUOstar Omega). The RFU (relative fluoresce units) was normalized for protein content. The other aliquot of cells were changed with complete medium for an additional 44 h growth, and cells were lysed, transferred for the luciferase expression analysis. The light emission was determined with signal integration over 10 s at 25 °C. The activity  was normalized for protein content.Tat/pDNA-Ca2+ nanoparticles was measured by confocal imaging analysis as previously described [Cellular uptake of Tat/pDNA-Caescribed , 7. CellTo compare the uptake in synchronized culture to that in non-synchronized one, care was taken to ensure that a similar number of cells were exposed to nanoparticles. In addition, prior to addition of fluorescent particles, various cell-cycle arrested conditions were achieved via 24 h pre-incubation with serum-free media containing lovastatin, GCB, RO3306, and PTX. These drugs were made in DMSO, diluted in PBS, and the final concentration of DMSO was less than 0.4%.Centrosome re-duplication assay was performed as previously described . BrieflyCentrosome from asynchronously and synchronously growing cells was isolated by sucrose density-gradient centrifugation method as previously described , 66. ProNOD/SCID female mice  were randomized into 12 groups (6 mice per group), and injected subcutaneously with 2 × 105 DF-labeled Skov3 cells into the right armpit. OC-bearing mice were firstly treated with Tat/pDNA-Ca2 + nanoparticles (1 mg/kg) by tail vein injection every week, then followed by intratumoral injections of EIPA (10 mg/kg) or MKT077 (7.5 mg/kg). Combined intratumoral injections of cell-cycle inhibitors, lovastatin (12.5 mg/kg), GCB (35 mg/Kg), Ro3306 (4 mg/kg), PTX (10 mg/kg), were parallel performed once a week in the additional nanoparticle groups. All mice were administered intraperitoneally with GCV (50 mg/kg) from day 11 till the end of experiment  cells and the distribution of the aforementioned nanoparticles in living mice was tracked by bioluminescence imaging Fig. B as prevTumor and other organ tissues from sliced mice were isolated, fixed, and processed for tissue paraffin sections as previously described . To dete4 cells in triplicate per condition. Tumor tissues were fixed for 24 h in 4% paraformaldehyde, and then processed to cryosections. In situ fluorescent TUNEL staining was done by using TUNEL apoptosis detection kit. Images were obtained by fluorescent microscopy (Olympus) and quantified with ImageJ software (NIH).Cell death was assessed by using annexin V-PI apoptosis detection kit followed by flow cytometry analysis. Data was recorded from 1 × 10Cyclebase 3.0 , a multiAll data were obtained from three independent experiments, and are presented as mean ± SD. For two-sample comparisons against control, unpaired Student’s t-test was used. One-way analysis of variance with Dunn’s multiple comparison was used to evaluate the statistical significance among multiple groups. Graphs were created using GraphPad Prism 8.0 software.Additional file 1. Table S1. Mean size and zeta potential of Tat/pDNA-Ca2+ nanoparticles.Additional file 2: Fig. S1. Granularity and electric potential analysis of Tat/pGL3 and Tat/pGL3-Ca2+ particles. Fig. S2. High-concentration, long-term treatment with Tat/pGL3-Ca2+ nanoparticles triggers necrotic apoptosis. Fig. S3. Stability characteristics of Tat/pDNA-Ca2+ nanoparticles in culture media or mice serum. Fig. S4. Unpackaging of Tat/pDNA complexes or Tat/pDNA-Ca2+ nanoparticles by heparin displacement of pDNA. Fig. S5. Tat/pGL3-Ca2+ nanoparticles mainly use macropinocytosis for uptake. Fig. S6. EIPA treatment inhibited the uptake and expression of Tat/pGL3-Ca2+ nanoparticles. Fig. S7. Tat/pDNA-Ca2+ nanoparticles do not interfere with sub-phase distribution of cell-cycle. Fig. S8. Construction of recombinant lentiviral plasmids for GRP75 over-expression (A) and knock-down (B). Fig. S9. Highly expression or phosphorylated activation of GRP75 promotes centrosome duplication in Cos7 cells, and GRP75 mainly localizes in duplicated centrosome. Fig. S10. Highly expression or phosphorylated activation of GRP75 promotes itself and Mps1 co-translocating to centrosome in Cos7 cells. Fig. S11. GRP75 and Mps1 co-localized with r-tubulin only in duplicating centrosome. Fig. S12. Quantification of apoptotic cells in ovarian tumor with different treatments. Fig. S13. H&E staining of hearts, livers, spleens, lungs, kidneys and tumor tissues from mice with different treatments.


Notably, regardless of dramatic increases in humoral immunity after booster vaccination, T cell responses targeting S protein from either wild type or Omicron remained stable before and after booster vaccination in all three serotype vaccinees. No severe cases were observed even in the sero-decay group during the Omicron epidemic in Shanghai. Our results thus illustrate that unlike fluctuating humoral responses, viral-specific T cell responses are extremely stable after booster vaccination. Sustained T cell responses might be dedicated to the rapid restoration of antibody responses after booster vaccination.BBIBP-CorV exerts efficient protection against SARS-CoV-2 infection. However, waning vaccine-induced humoral immune responses after two-dose vaccination have significantly undermined durable immuno-protection. In this study, we have demonstrated that although anti-spike (S) antibody responses in BBIBP-CorV vaccinees exhibited three serotypes after 6 months, including  The Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a huge burden on public health worldwide . It is wHowever, real-world data reveal that vaccine-induced antibody titres as well as neutralizing activity wane over time in most of the available SARS-CoV-2 vaccines, especially in elders and those with clinical risk groups ,7. In mode novo deficiency in S-nAb), seropositive with sustained S-nAb at 6 months (Vac-Pos/SS) and seropositive with decayed S-nAb at 6 months (Vac-Pos/SD). Interestingly, three groups exhibited comparable S protein-specific T cell responses to wild-type (WT) SARS-CoV-2 and B.1.1.529 (Omicron) variant of concern (VOC) demonstrated by antigen-specific interferon (IFN)- γ release and cytokine production in CD4+ and CD8+ T cells. More importantly, booster vaccination with a third dose of BBIBP-CorV remains an efficient way to restore the activity of humoral immunity against SARS-CoV-2 WT and Omicron VOC with stable specific T cell immune responses.In this study, we enrolled 84 BBIBP-CorV-vaccinated subjects from the previous study  to deterde novo deficiency in S-nAb), seropositive with sustained S-nAb at 6 months (Vac-Pos/SS) and seropositive with decayed S-nAb at 6 months (Vac-Pos/SD). All participants had no documented history of SARS-CoV-2 infection. This study was approved by the Medical Ethical Committee at Shanghai Ruijin Hospital (RJHKY2021-12). The study adheres to the principles of the Declaration of Helsinki and written informed consent was obtained from every participant. This study was unblinded and not randomized.The objective of this study was to investigate the vaccine-induced cellular immunity against SARS-CoV-2 and variants in vaccinees without detectable neutralizing antibodies. A prospective cohort study of health care workers (HCWs) in Shanghai Ruijin Hospital was initiated in 2021 with a focus on the immuno-protection of BBIBP-CorV (NCT04795414). From January 2021, HCWs were offered vaccination with 2 doses of BBIBP-CorV at an interval of 21 days, and 84 participants were invited for the follow-up study in which immune profiles upon vaccination were studied. From September 2021, 38 HCWs in the study were invited for a follow-up study after booster vaccination with the homologous vaccine. We also enrolled 12 individuals without vaccination as control (non-Vac). Venous blood samples were collected and separated to obtain serum and peripheral blood mononuclear cells (PBMCs). Serum samples were used to measure neutralizing antibodies. PBMCs were used for interferon-γ releasing assay and flow cytometry. Based on S-nAb levels at 1 and 6 months after two-dose BBIBP-CorV vaccination, the participants were subgrouped into seronegative  and 6 months (6mpp) after the second dose, while peripheral mononuclear cells were obtained only at 6mpp after the second dose. Additional serum and PBMC samples were obtained at 28 days (28dpb) after booster vaccination. Participants of this study had no history of previous SARS-CoV-2 infection, as confirmed by the absence of spike/nucleocapsid-specific antibodies before vaccination at baseline.3EDTA tubes by density gradient centrifugation. Briefly, PBMCs were isolated by Ficoll-paque plus density gradient (GE) centrifugation at 2000 rpm for 20 min at room temperature. PBMCs were washed 2 times in phosphate-buffered saline and subsequently frozen in liquid nitrogen in 90% fetal bovine serum  with 10% DMSO (Gibco) for further immune profile analysis.Serum was collected from whole blood in 5-ml tubes without anticoagulant, centrifugated at 2500 rpm for 15 min, aliquoted, and stored at −20°C for further experiments. PBMCs were isolated from blood collected in K50) was defined as the serum dilution at which the relative light units (RLUs) were reduced by 50% compared with the virus control wells. Serial dilutions of heat-inactivated sera (six dilutions in a 4-fold step-wise manner) were incubated with 250 TCID50 SARS-CoV-2 pseudoviruses expressing the reporter gene luciferase for 1 h together with the virus control and cell control wells before seeding HEK293T-ACE2 cells in 96-well plates . After 48 h of incubation in 5% CO2 at 37°C, the supernatant was removed, and the luminescence was measured using the Luciferase Assay System  according to the manufacturer’s instructions. The IC50 values were calculated by generating a three-parameter non-linear regression curve fit in GraphPad Prism v.7.0. The lowest serum dilution tested was 1:4. When no neutralization was observed, we set the pVNT50 value at 2.A pseudovirus-based neutralization assay was performed as previously described. The lentivirus-based SARS-CoV-2 pseudoviruses express a luciferase reporter gene and bear the S protein from the wild-type strain . The 50% inhibitory concentration (IC5 PBMCs were incubated with 10 μg/mL SARS-CoV-2 Spike (S) protein (Genscript). Phytohemagglutinin (PHA) (Sigma Aldrich) at a final concentration of 2.5 μg/mL served as a positive control, while the culture medium was a negative control. Stimulated cells were incubated for 20 h at 37°C. Biotin-labelled detection antibody incubated with IFN-γ secreting cells at 4°C overnight. Antibody-labelled plates were incubated with streptavidin-labelled horseradish peroxidase (HRP) at 37°C for 1 h after discarding the supernatant. AEC solution was applied to develop coloration. Plates were then placed in the dark for 30 min. The coloration was terminated by thoroughly rinsing the PVDF membrane with demineralized water. ELISpot Reader  was used for counting and quantity control after drying out. The number of antigen-specific IFN-γ producing cells was calculated as spot forming units (SFUs) per 2.5 × 105 cells.Human IFN-γ ELISpot Kit  was used for antigen-specific IFN-γ releasing detection. Following the manufacturer’s instruction, a 96-well PVDF plate (Millipore) was conjugated with an anti-IFN-γ antibody overnight at 4°C. 2.5 × 106 PBMCs were stimulated with SARS-CoV-2 S protein in 200 μl in 96-well U-bottom plates  in a 5% CO2 incubator at 37°C for 20 h. To detect antigen-specific intracellular cytokine levels, GolgiStop (BD Bioscience) was added 14 h after ex vivo stimulation. Cells were additionally stimulated with a combination of phorbol 12-myristase 13-acetate (50 μg/ml) and ionomycin (500 μg/ml) as a positive control. After stimulation, cells were stained for phenotypic lymphocyte markers.PBMCs were thawed in RPMI 1640 medium (Gibco) supplemented with 10% FBS (Gibco), and Penicillin Streptomycin . Subsequently, 1 × 10The immune profiles of the BBIBP-CorV vaccinated cohort were detected by high-dimensional flow cytometry as described previously . BrieflyP values < 0.05 were considered statistically significant.Prism v.7.0  was used for statistical analyses. Data were represented by a median with an interquartile. Statistical differences were assessed by the Mann–Whitney test for the data with variance homogeneity and by the Kruskal–Wallis test for those with non-variance homogeneity. n = 47, 70.1%) whose pVNT50 titres were more than 4, and the Vac-Pos/SD group  whose pVNT50 titres were less than 4, while pVNT50 titres of the Vac-Neg group (n = 15) remained less than 4  from Shanghai Ruijin Hospital have been previously enrolled in a study of vaccine efficacy evaluation after two-dose BBIBP-CorV vaccination . We colln = 69) group was 246.5 geometric mean neutralization titre (GMT) : 202.8–299.6), whereas Vac-Neg (n = 15) exhibited S-nAb deficiency (p = 0.1574) at 28 dpp. At 6mpp both Vac-Pos/SS and Vac-Pos/SD groups exhibited the attenuation of S-nAb pVNT50 titres with different trends. GMT values of the Vac-Pos/SS group decreased from 273.4 to 57.59 (95% CI: 37.72–87.95), whereas those of the Vac-Pos/SD group decreased from 197.5 to 1.713 (95% CI: 1.392–2.107) (de novo S-nAb deficiency (the Vac-Neg group), Vac-Pos group vaccinees experienced dramatic attenuation of S-nAb after 6 months of two-dose vaccination among which 31.9% (22/69) became seronegative, which indicates the impairment of long-term maintenance of S-nAb after two-dose vaccination.It was obvious that at 28dpp after the second dose vaccination the average pVNT50 values of S-nAb in the Vac-Pos (p = 0.2104), as well as between Vac-Pos/SS and Vac-Pos/SD groups  (p = 0.8402) whereas SFUs from the non-Vac group were undetectable with a significant difference when compared to the other three groups  of S-specific interferon-gamma (IFN-γ) releasing cells were comparable between Vac-Pos/SS : 0–489.1) and Vac-Neg  subjects . Whilst no difference in the frequencies of total CD4+ T cells among three groups , Vac-Neg vaccinees showed higher proportions of CD45RO-CD27+ naïve CD4+ T cells (TN) when compared to Vac-Pos/SS (p = 0.0263) or Vac-Pos/SD (p = 0.0211) subjects and lower proportions of CD45RO-CD27- effector CD4+ T cells (Teff) , which were similar to those in the non-Vac group. There was no difference in the frequencies of CD4+ TN cells (p = 0.6755), CD4+ Teff cells (p = 0.4999), and CD45RO+ memory CD4+ T cells (TM) (p = 0.8955) between Vac-Pos/SS and Vac-Pos/SD groups (+ T cells involved in B cell activation and differentiation including CD40L and programmed death-1 (PD-1). CD40L is inducibly expressed on activated CD4+ T cells to provide costimulatory signals for B cell activation, whereas PD-1 on activated T cells exerts as an inhibitory regulator for CD4+ T cell function [+ T cells among three groups were comparable, while all were higher than those in the non-Vac group   or waned (Vac-Pos/SD group) after 6mpp, indicating fewer variations of T cell immunoreactivity after BBIBP-CorV vaccination.We further determined S protein-specific cytokine production in CD4n = 7), Vac-Pos/SS (n = 25), and Vac-Pos/SD (n = 6) vaccinees who finished booster immunization at 9 months after regular two-dose BBIBP-CorV primary vaccination. Sera and PBMCs were collected at 28 days post booster (28dpb)  (p = 0.0210 to those before booster vaccination), which was comparable to pVNT50 values of the Vac-Pos group at 6mpp  (p = 0.9352) . Moreover, both Vac-Pos/SS and Vac-Pos/SD vaccinees showed a dramatic increase in S-nAb titres after booster vaccination as well where GMT values of Vac-Pos/SS vaccinees reached 489.6 from 46.27 at 6 mpp (p = 0.0313) and those of Vac-Pos/SD vaccinees increased from 1.532 to 288.6 at 6mpp (p < 0.0001). When we calculated the seroconversion rates at 28dpb which was defined as a change of titres from seronegative at 6mpp to seropositive at 28dpb with a four-fold increase in pVNT50 titres, 71.43% (5/7) of the participants showed the seroconversion within the Vac-Neg group, 100% (6/6) in the Vac-Pos/SD group and 76% (19/25) in the Vac-Pos/SS group  is highly recommended to restore the efficacy of BBIBP-CorV vaccines . Owing t+), naïve  B cells, CD38++CD19+ B cells as terminally differentiated B cell subset, and double negative , resting naïve , activated naïve , and DN2 (CD38-CD27-IgD-CD11c+ CD19+) cells for EFB pathway (+ B cells (+ CD19+ B cells after booster vaccination (p = 0.0496) (p = 0.0156) in the Vac-Neg group  pathways and under certain circumstances by extrafollicular B cell (EFB) differentiation pathways , which ip = 0.0151) and Vac-Pos/SD (Median SFUs: 467.8) (p = 0.0411)  when compared with those at 6mpp (Median SFUs: 616.5 for WT)  from WT and Omicron, there are nearly 31 nucleotide variations in RBD. This is associated with immune escape from S-nAb induced by WT isolate-derived vaccines  and alsoAlthough we have profiled antibody neutralizing activity and S protein-specific T cell responses at 6 months after two doses of BBIBP-CorV vaccination and 28 days after booster vaccination, our relatively small sample size and the large loss of follow-up between 28 days and 6 months after vaccination may limit further information on vaccine-induced immunity. Moreover, whether similar levels of T cell responses to WT and Omicron strains are due to cross-reactivity or epitope spreading needs to be further defined.Collectively, with a well-designed prospective sample collection, we demonstrate that BIBP-CorV vaccination induces equivalent anti-SARS-CoV-2 specific T cell responses no matter whether neutralizing antibody responses are present or absent. After booster vaccination, antibody neutralizing activity to WT isolate is restored with less efficacy to Omicron, whereas T cell responses to WT and Omicron isolates are still comparable. This was partially consistent with the good prevention of severe infection during the Omicron epidemic. Therefore, unlike fluctuated viral-specific humoral immune responses T cell responses induced by the BIBP-CorV vaccine are more stable at different stages of vaccination.Click here for additional data file.


Caenorhabditis elegans mitochondrial chaperone hsp-6 activates detoxification and RNA interference defense pathways. Mitochondrial mutations increase C. elegans longevity, and mutations that disable RNA interference suppress this longevity increase. Mitochondria also serve as a signaling hub for mammalian immune responses to viruses. The conserved DNA helicase gene arip-4 is required for the increase in antiviral RNAi caused by mitochondrial dysfunction. But an arip-4 mutation extends the life span of the hsp-6 mitochondrial mutant, suggesting that the futile activation of detoxification pathways is deleterious to health when the mitochondrial dysfunction is caused by mutations.Eukaryotes surveil and respond to mitochondrial dysfunction caused by toxins or mutations, triggering antiviral and immune responses as well as mitochondrial repair. A mutation in the  Caenorhabditis elegans mitochondrial status is coupled to defense responses such as drug detoxification, immunity, antiviral RNA interference (RNAi), and regulation of life span. A cytochrome p540 detoxification gene, cyp-14A4, is specifically activated by mitochondrial dysfunction. The nuclear hormone receptor NHR-45 and the transcriptional Mediator component MDT-15/MED15 are required for the transcriptional activation of cyp-14A4 by mitochondrial mutations, gene inactivations, or toxins. A genetic screen for mutations that fail to activate this cytochrome p450 gene upon drug or mutation-induced mitochondrial dysfunction identified a DNA helicase ARIP-4 that functions in concert with the NHR-45 transcriptional regulatory cascade. In response to mitochondrial dysfunction, ARIP-4 and NHR-45 protein interaction is enhanced, and they relocalize from the nuclear periphery to the interior of intestinal nuclei. NHR-45/ARIP-4 also regulates the transcriptional activation of the eol-1 gene that encodes a decapping enzyme required for enhanced RNAi and transgene silencing of mitochondrial mutants. In the absence of arip-4, animals were more susceptible to the mitochondrial inhibitor antimycin. Thus, ARIP-4 serves as a transcriptional coactivator of NHR-45 to promote this defense response. A null mutation in arip-4 extends the life span and health span of both wild type and a mitochondrial mutant, suggesting that the activation of detoxification pathways is deleterious to health when the mitochondrial dysfunction is caused by mutation that cannot be cytochrome p450-detoxified. Thus, arip-4 acts in a pathway that couples mitochondrial surveillance to the activation of downstream immunity, detoxification, and RNAi responses.Surveillance of  Caenorhabditis elegans are mutations that reduce mitochondrial function  associates with viral RNA sensor retinoic acid–inducible gene I (RIG-I) or melanoma differentiation–associated protein 5 (MDA5) to trigger the downstream nuclear factor kappa B, as well as other interferon immune signaling . In addiC. elegans and plants by its antiviral activities that detect and cleave foreign nucleic acids such as viruses  is highly conserved across most eukaryotes. It deploys 22- to 26-nt single-stranded small interfering RNAs (siRNAs) produced by Argonaute proteins and the Dicer dsRNA ribonuclease, as well as RNA-dependent RNA polymerases in a large fraction of eukaryotes but in a small fraction of animal genomes . These s viruses , 13. The viruses , 15. In  viruses ; similar elegans , 18. In ral RNAi .C. elegans, the mitochondrial unfolded protein response (UPRmt) is a mitochondria-to-nuclear communication channel that is activated by various types of mitochondrial dysfunction, including natural toxins such as oligomycin or mutations in mitochondrially localized nuclear-encoded proteins  glycosylating, sulfate and glutathione S-transferase (GST) genes that add hydrophilic groups to generally hydrophobic toxins, and ABC transporters that excrete the modified toxins , a hypomorphic mutation in the mitochondrial chaperone HSP-6/mtHSP70.Surveillance of mitochondrial integrity is essential for maintaining cellular homeostasis and is coupled to a complex signaling network that induces mitochondrial recovery, drug detoxification, and immunity. Although almost all of the known mitochondrial stress–related transcriptional responses required the UPR factors . There ad toxins . Differed toxins . The manfunction , 25. A futations . For exacyp-14A4p::gfp in hsp-6(mg585) revealed an allele mg691, a Trp371 to Stop (TGG to TAG) nonsense mutation in rad-26 (cyp-14A4p::gfp in the hsp-6(mg585) mitochondrial mutant. However, many of the EMS mutations will be located in introns or intergenic regions, and many are not predicted to cause amino acid substitutions (rad-26(mg691), it is necessary to reconstruct a similar mutation in the absence of the other EMS lesions traditionally by multiple backcrossing but recently using the CRISPR to engineer only that single lesion in an unmutagenized background.A genome-wide ethyl methanesulfonate (EMS) mutagenesis screen for mutants that fail to induce n rad-26 A and B. rad-26 in this F2 mutant isolated after a mutagenesis screen was the causative mutation for the failure to induce cyp-14A4p::gfp in hsp-6(mg585), CRISPR-Cas9 editing was used to generate just a single independent null allele of rad-26(mg697) with 28 base pairs inserted that caused a frameshift (rad-26(mg697) mutant also disrupted induction of cyp-14A4p::gfp in the hsp-6(mg585) background (rad-26 requirement for the activation of cyp-14A4 expression was not limited to the hsp-6(mg585) mitochondrial mutant: The complex III natural toxin antimycin isolated from Streptomyces bacteria also activates cyp-14A4p::gfp, and this activation is dependent on rad-26 gene activity , these two proteins are not orthologs but paralogs. They are distant from each other on the phylogenetic tree of these DNA helicases (S. cerevisiae Rad26 is responsible for transcription-coupled nucleotide excision repair (C. elegans (C. elegans rad-26 encodes a conserved Snf2-like DNA-dependent ATPase orthologous to human ARIP4 (androgen receptor–interacting protein) , we rename this C. elegans gene arip-4.The 1,274 amino acid proteins encoded by  strands . There a strands . Althougelicases E. S. cern repair , and its elegans E. The phprotein) E. Becauscyp-14A4 transcriptional regulatory function of C. elegans arip-4, and with its genetic and biochemical interaction with the C. elegans NHR NHR-45 (see below), human ARIP4 is a transcriptional coregulator for NHRs  animals, the intranuclear patterns were different (hsp-6(mg585) mitochondrial mutant, ARIP-4::GFP was localized to the nuclear interior (We first tested whether fication . The gutll types . To obse protein . The pro protein A. Intereifferent A. In theinterior A.vha-6 promoter to drive expression. In the hsp-6(mg585) mitochondrial mutant, NHR-45::mScarlet showed a similar shift of localization away from the nuclear periphery  mitochondrial mutant  and hsp-6(mg585); nhr-45(mg641) mutants (hsp-6(mg585) mutant but are not up-regulated in the hsp-6(mg585); nhr-45(mg641) mutant (cyp-14A4 and ugt-8 and two immunity genes clec-144 and cnc-9. Their expression levels were examined by quantitative reverse transcription PCR (RT-qPCR). All four genes were up-regulated 10–30 fold in the hsp-6(mg585) mitochondrial mutant, and the induction was decreased more than 70% in either the nhr-45(mg641) or the arip-4(mg697) mutant  mutant D, sugges) mutant E. Thus, mt signaling, loss of nhr-45 had no effect on mitochondrial homeostasis, for example, activation of hsp-6 gene expression  mutant was not affected by either nhr-45(mg641) or arip-4(mg697)  E, which C. elegans ARIP-4 is conserved, and the corresponding lysine in ARIP-4 and the K296A substitution was engineered (cyp-14A4p::gfp. Wild-type ARIP-4 was able to rescue the arip-4(mg697) mutant and could mediate the induction of cyp-14A4p::gfp caused by hsp-6(mg585); to our surprise, the ARIP-4(K296A) mutant was also able to rescue the arip-4(mg697) mutant and allow induction of cyp-14A4p::gfp (arip-4 is able to rescue the failure to activate cyp-14A4p::gfp in the hsp-6(mg585); arip-4(mg697) double mutant, suggesting that ARIP-4 regulation of cyp-14A4 is autonomous to the intestine. But, surprisingly, ARIP-4 regulated detoxification and immune responses are independent of its DNA helicase activity.As a DNA helicase, we expected that the ATPase activity of ARIP-4 would be essential for its function in regulating transcription. The ATPase activity of ARIP4 was disrupted with a lysine-to-alanine mutation at the amino acid 310 (K310A), the ATP-binding site . The ATPgineered F. The fuA4p::gfp G and H. C. elegans also promotes an antiviral RNAi response through activation of the DRH-1/EOL-1 pathway (hsp-6(mg585) mutant causes release into the cytosol of mRNAs transcribed from the mitochondrial genome, with none of the hallmarks of RNA splicing or other cytoplasmic RNA modifications. These mitochondrial mRNAs are likely to be recognized by DRH-1, the C. elegans ortholog of mammalian RIG-I or MDA5, as not transcribed, not spliced, not capped, and not modified in the nucleus (eol-1. mRNA-seq comparisons of hsp-6(mg585) mutant and hsp-6(mg585); nhr-45(mg641) double mutant strains demonstrated that the upregulation ofeol-1 expression requires nhr-45 (eol-1 in the hsp-6(mg585) mutant is mediated by nhr-45, and to test whether it is also mediated by arip-4, a transcriptional fusion reporter eol-1p::gfp including the eol-1 promoter, GFP, and the eol-1 3'UTR was employed. The fluorescent signal of eol-1p::gfp was almost undetectable in wild-type animals treated with control RNAi and was significantly increased in the hsp-6(mg585) mitochondrial mutant with control RNAi (nhr-45 RNAi or arip-4 RNAi abolished the activation of eol-1p::gfp in the hsp-6(mg585) mitochondrial mutant (hsp-6(mg585) mitochondrial mutant enhances RNAi so that transgenes are now recognized as foreign and silenced in mitochondrial mutants (see below). The eol-1p::gfp transcriptional fusion reporter is a transgene that is subject to RNAi-based transgene silencing. Therefore, RT-qPCR was performed to accurately evaluate the gene expression of eol-1 from its normal chromosomal location in these mutants. The mRNA level of eol-1 in the hsp-6(mg585) mutant was increased 30 fold compared with that in wild type (. This induction eol-1 mRNA in hsp-6(mg585) is 2-3× decreased (but still 10x induced relative to wild type) in the hsp-6(mg585); nhr-45(mg641) or hsp-6(mg585); arip-4(mg697) double mutant, and decreased another 3× in the hsp-6(mg585); arip-4(mg697); nhr-45(mg641) triple mutant . T. TC. elerol RNAi A and B. l mutant A and B. ild type C. This ie mutant C. Thus, eol-1 is critical for the enhanced RNAi caused by hsp-6(mg585)  mitochondrial mutant shows enhanced RNAi when tested with particular dsRNAs expressed from Escherichia coli. For example, a dsRNA targeting the lir-1 gene causes a far milder phenotype  in wild type than in a variety of enhanced RNAi mutants such as hsp-6(mg585)   (eol-1(mg698) null mutant suppresses the enhanced RNAi of the hsp-6(mg585) mutant: the hsp-6(mg585); eol-1(mg698) double mutant showed 17% lethality treated with lir-1 RNAi (nhr-45(mg641) or arip-4(mg697) mutations moderately suppressed the enhanced RNAi phenotype in hsp-6(mg585) mutant: RNAi of lir-1 caused 36% and 43% lethality in hsp-6(mg585); nhr-45(mg641) and hsp-6(mg585); arip-4(mg697) double mutants, respectively, compared with 66% lethality for the hsp-6(mg585) single mutant (nhr-45 and arip-4 were synergistic: In the hsp-6(mg585); nhr-45(mg641); arip-4(mg697) triple mutant, 21% of animals were lethal after lir-1 RNAi treatment (The upregulation of 6(mg585) . Enhance6(mg585) . We test lethal) D. The leRelated) . The eolr-1 RNAi D 18). n. neol-1 e mutant D. Mutatireatment D.his-44 that encodes a histone 2B gene. Because the DNA sequences of the large histone 2B gene family have a high DNA sequence similarity, RNAi of his-44 in enhanced RNAi mutants may promiscuously target multiple histone 2B genes to cause lethal arrest. RNAi of his-44 causes 9% arrest in wild type but 72% arrest in the hsp-6(mg585) mutant with enhanced RNAi ; eol-1(mg698) double mutant animals, 41% in hsp-6(mg585); nhr-45(mg641) double mutant animals, 46% in hsp-6(mg585); arip-4(mg697) double mutant animals, and 22% in hsp-6(mg585); nhr-45(mg641); arip-4(mg697) triple mutant animals arrested when treated with his-44 RNAi (hsp-6(mg585) mitochondrial mutant requires nhr-45 and arip-4 for the induction of eol-1 to enhance RNAi.Another enhanced RNAi dsRNA is ced RNAi E. Mutati-44 RNAi E. Thus, C. elegans transgenes are silenced in a wide variety of enhanced RNAi mutants, most likely because their assembly during DNA transformation into tandem multicopy extrachromosomal elements marks them as probable foreign genetic elements (rol-6(su1006), a dominant amino acid substitution mutation of a hypodermal collagen, causes 99% of wild-type transgenic animals to have a rolling phenotype (Rol). But if the hsp-6(mg585) mitochondrial mutant is crossed into the strain, only 29% of the hsp-6(mg585) mutant animals harboring the tandem array transgene is Rol (eol-1 null mutation suppresses this hsp-6 transgene silencing: The hsp-6(mg585); eol-1(mg698) double mutant is 80% Rol (hsp-6(mg585); nhr-45(mg641) or hsp-6(mg585); arip-4(mg697) double mutant, the rol-6(su1006) transgene causes 46% or 47% Rol, respectively, and in the hsp-6(mg585); nhr-45(mg641); arip-4(mg697) triple mutant, animals are 70% Rol (hsp-6(mg585) mitochondrial mutant , nhr-45(mg641), or arip-4(mg697) mutation (hsp-6(mg585) mutant require nhr-45 and arip-4.Multicopy elements . A chrome is Rol F. An eol 80% Rol F. In the 70% Rol F. To surl mutant G and H. mutation G and H. C. elegans NHR-45/MDT-15 pathway mediates detoxification of mitochondrial toxins, for example, the Streptomyces mitotoxin antimycin or the pathogenic P. aeruginosa (arip-4(mg697) animals were treated with 6.25 μg/ml antimycin, the sensitivity of the arip-4(mg697) mutant was dramatically increased relative to wild type  mutants were more susceptible to P. aeruginosa  mitochondrial mutant  or aged (day 10 adults) animals. Wild-type animals move at approximately 200 μm/s on day 1, but by day 10, they move 3× more slowly at about 60 μm/s. The movement speed of hsp-6(mg585) mitochondrial mutant animals was 59 μm/s at day 1, 30% that of wild type, as would be expected for a mutation that affects mitochondrial production of ATP. At day 10, hsp-6(mg585) mitochondrial mutant animals continued to show about 1/3 the motility of wild type with about 20 μm/s (arip-4(mg697) did not affect locomotion at day 1 but moved at double the speed (128 μm/s) at day 10 (arip-4 null mutation attenuated the locomotion defects caused by hsp-6(mg585) with speed of movement increased 49% on day 1 and 150% on day 10  mutant to render the animals healthier. For example, cytochrome p450 proteins are heme proteins that require iron, often in limiting supply. The nearly 100× induction of cyp-14A4 caused by mitochondrial dysfunction may usurp iron from mitochondrial biogenesis, for example. Similarly, secreted antibacterial proteins pass through the endoplasmic reticulum where multiple cysteines are oxidized to disulfides, an energetically expensive modification. Animals bearing mitochondrial mutations that do not mount impotent but stressful detoxification and immune responses because of the second arip-4 mutation paradoxically fare better. Consistent with this, moderate mitochondrial disruption caused by hypomorphic mutations of essential mitochondrial genes or gene inactivations of nonessential mitochondrial genes extends life span (hsp-6(mg585) (21 d) was substantially increased compared with that of wild type (17 d) (arip-4(mg697) single mutant, with a median survival of 18 d, marginally increased the life span compared with wild type, the hsp-6(mg585); arip-4(mg697) double mutant lived much longer (24 d) than hsp-6(mg585)  B. While 6(mg585) B, showinarip-4(mg697) was extremely low before day 17  mutant background, emphasizing the deleterious effects of impotent pathogen defense responses triggered by mutations that cannot be detoxified.It was notable that the mortality of e day 17 B, which e day 17 A. The cohsp-6(mg585) mitochondrial mutant, which identified the nhr-45 mutant, also revealed the arip-4(mg691), a nonsense mutation in arip-4 (C. elegans arip-4 encodes a conserved Snf2-like DNA-dependent ATPase that is orthologous to human ARIP4 (androgen receptor–interacting protein), which also interacts with a NHR, the androgen receptor. We showed that tagged ARIP-4 and NHR-45 proteins colocalize in the nucleus to coregulate cyp-14A4 as well as other genes involved in drug detoxification and antibacterial immunity and that ARIP-4 protein physically interacts with the NHR-45 protein in a similar manner that its human ortholog ARIP4 binds to the NHRs, such as the androgen receptor  mitochondrial mutant are moderately suppressed by the nhr-45(mg641) or arip-4(mg697) single mutant but strongly suppressed by the nhr-45(mg641); arip-4(mg697) double mutant (nhr-45(mg641) or the arip-4(mg697) mutant do not completely abolish the upregulation of eol-1 gene expression in hsp-6(mg585) mutant, while the induction of eol-1 gene is almost absent when both nhr-45 and arip-4 were mutated quinazoline-2,4-diamine, which may be a ligand of NHR-86 ::mscarlet, the plasmid was injected into unc-119(ed3) following the miniMOS protocol (arip-4(mg697), we chose dpy-10(cn64) as the co-CRISPR marker . The cDNA was generated by the ProtoScript® II First Strand cDNA Synthesis Kit . qPCR was performed toward cyp-14A4, ugt-8, clec-144, cnc-9, and hsp-6 with act-1 as control by the iQ™ SYBR® Green Supermix .For estimation of the mRNA level of Around 20,000 worms of each genotype were synchronized by bleach preparation to L1 larvae, grown to the L4 stage, collected, and frozen by liquid nitrogen. Worm lysates were generated by the TissueLyser with steel beads  and resuspended in 500 μl lysis buffer . The lysate was centrifuged at 1,000 g for 10 min at 4°C to remove the pellet debris. And the supernatant was divided into two parts: 30 μl to mix with the NuPAGE™ LDS sample buffer  as the input and 450 μl to mix with 20 μl GFP-nAb magnetic agarose , which was pretreated twice by the washing buffer . The mixture was rotated for 2 h at 4°C to allow the binding. The beads were washed three times with the washing buffer and resuspended in 50 μl 1X NuPAGE™ LDS sample buffer. The input and the beads were heated at 70°C for 10 min. Samples were loaded onto the NuPAGE™ 4 to 12% Bis-Tris protein gels  and run with the NuPAGE™ MES SDS running buffer . After semidry transfer, the PVDF membrane  was blocked with 5% nonfat milk and probed with anti-GFP  or anti-FLAG  primary antibody and goat anti-mouse IgG HRP secondary antibody . The membrane was developed with the SuperSignal™ West Femto Maximum Sensitivity Substrate  and visualized by the Amersham Hyperfilm .arip-4(mg697) animals, 6.25 μg/ml antimycin was used. Survival was examined on a daily basis, and the survival curve was generated by GraphPad Prism.For each experiment, 30 L4 larvae were treated with different concentrations of antimycin A , and each experiment was repeated three times. To determine the sensitivity of wild-type animals, a twofold serial dilution was performed to antimycin starting from 25 μg/ml. For comparing the sensitivity of wild-type and P. aeruginosa PA14 was grown at 37°C overnight, seeded on the NGM plates, and incubated at 37°C for another night. One hundred L4 larvae were picked onto P. aeruginosa lawn and grown at 25°C. Survival was examined on a daily basis, and the survival curve was generated by GraphPad Prism.Fifty L4 larvae were picked and grown at 20°C for 10 d. On day 1 and day 10, worms were picked onto bacteria-free NGM plates and photographed directly by the Zeiss AX10 Zoom.V16 microscope. The movement of worms was recorded by continuing picturing every 0.5 s for 30 s in total. The speed of movement was analyzed and calculated by the AxioVision (Zeiss).Animals were synchronized by egg laying and grown until the L4 stage as day 0. Adults were separated from their progenies by manually transferring to new plates. Survival was examined on a daily basis, and the survival curve was generated by GraphPad Prism.


BMC Emergency Medicine (2022) 22:7010.1186/s12873-022-00616-6The original article [1] contained errors affecting correct attribution of the citations to their respective references which has since been corrected.


H1-2 heavy chain (HC) and, substantially, a human Vκ1-33 light chain (LC). Thus, primary humanized BCR repertoire diversity in these mice derives from immensely diverse HC and LC antigen-contact complementarity-region-3 (CDR3) sequences generated by non-templated junctional modifications during V(D)J recombination. Immunizing the human VH1-2/Vκ1-33-rearranging mouse model with SARS-CoV-2 (Wuhan-Hu-1) spike protein immunogens elicited several VH1-2/Vκ1-33-based neutralizing antibodies that bound RBD in a different mode from each other and from those of many prior human patient-derived VH1-2-based neutralizing antibodies. Of these, SP1-77 potently and broadly neutralized all SARS-CoV-2 variants through BA.5. Cryo-EM studies revealed that SP1-77 bound RBD away from the receptor-binding-motif via a CDR3-dominated recognition mode. Lattice-light-sheet-microscopy-based studies showed that SP1-77 did not block ACE2-mediated viral attachment or endocytosis, but rather blocked viral-host membrane fusion. The broad and potent SP1-77 neutralization activity and non-traditonal mechanism of action suggest this antibody might have therapeutic potential. Likewise, the SP1-77 binding epitope may further inform on vacccine strategies. Finally, the general class of humanized mouse models we have described may contribute to identifying therapeutic antibodies against future SARS-CoV-2 variants and other pathogens.SARS-CoV-2 Omicron sub-variants have generated a world-wide health crisis due to resistance to most approved SARS-CoV-2 neutralizing antibodies and evasion of vaccination-induced antibodies. To manage Omicron sub-variants and prepare for potential new variants, additional means of isolating broad and potent humanized SARS-CoV-2-neutralizing antibodies are desirable. Here, we describe a mouse model in which the primary B cell receptor (BCR) repertoire is generated solely through V(D)J recombination of a human V A humanized antibody from a recently-developed mouse model potently neutralizes SARS-CoV-2 variants by inhibiting membrane fusion. SARS-CoV-2 Omicron and its sub-variants, including the recently emergent BA.5 sub-variant, have spread worldwide and remain the cause of a public health crisis (Binding of the SARS-CoV-2 spike protein to its obligate angiotensin-converting enzyme 2 (ACE2) receptor on the target cell surface initiates infection , thereby, directly impeding its binding to the ACE2 receptor (de novo by non-templated V(D)J junctional modifications during V(D)J recombination J recombination center (H1-2 due to its association with the CTCF-binding element (CBE) downstream of the mouse VH5-2 (H1-2 accessibility to the V(D)J recombination apparatus and leads to its dominant usage . We also retained the mouse Ds, as mouse Ds are highly related to a subset of human Ds and, in any case, contribution of D segments to CDR3 are often unassignable due to extensive non-templated junctional diversification  that targets MHC II-complex antigens (+ B cells were fluorescence-activated single-cell sorted from each mouse and their IgH (HC) and IgL (LC) variable region exons were sequenced . These experiments identified 9 separate spike-specific B cell lineages, each of which expressed BCRs with unique sets of VH1-2 and Vκ1-33-associated CDR3s .We immunized the human Vantigens . Immunizantigens . All micantigens . At 3 weed CDR3s . In each50: 38 ng/ml), VHH7-7-53 (IC50: 68 ng/ml), and SP1-77 (IC50: 20 ng/ml) (50: 20 ng/ml) and Iota (K484E) (IC50: 43 ng/ml) VOCs (50: 43 ng/ml), Epsilon (IC50: 62 ng/ml), Iota (K484E) (IC50: 43 ng/ml) and Delta (IC50: 96 ng/ml) VOCs (50: 6.5 ng/ml), BA.2 (IC50: 33 ng/ml), BA.3 (IC50: 7 ng/ml), BA.4/BA.5 (IC50: 16 ng/ml) and BA.2.12.1 (IC50: 8 ng/ml) . Finally, we expressed the other four antibodies from the SP1 clonal lineage, each of which has unique pattern of somatic hypermutations compared to SP1-77. All had similar broad and potent neutralization activities .SARS-CoV-2 pseudovirus neutralization assays revealed that 3 of the RBD-binding monoclonal antibodies potently neutralized the SARS-CoV-2 G614 virus including VHH7-5-82  confirmed interactions of the Fabs of the 3 RBD-binding neutralizing antibodies with SARS-CoV-2 spike protein . We further imaged Spike-Fab complexes using negative-stain electron microscopy (NSEM) to elucidate binding characteristics  projected into a ring-like structure formed by the HC CDR3  from a neighboring protomer . The int HC CDR3 . The int CDR3  and S309 neutralizing antibodies. The RBD epitope targeted by SP1-77 overlapped very modestly with that of the LY-CoV1404, which is currently approved for therapeutic use . The RBDractions . Notablyractions .When the SP1-77 footprint was projected onto modeled RBDs from different SARS-CoV-2 variants , it was To address the SP1-77 neutralization mechanism, we applied a lattice light sheet microscopy (LLSM) approach to monitor target cell infection by a chimeric vesicular stomatitis virus in which the coat glycoprotein was replaced by the SARS-CoV-2 (Wuhan strain) S protein (VSV-SARS-CoV-2) (50 ~ 15 ng/ml) . Pre-incubation of VSV-SARS-CoV-2 Atto 565 with 500 ng/ml of SP1-77 did not appreciably impact its binding to the Vero TMPRSS2 cell surface J recombination of these single human VH and Vκ sequences in association with diverse CDR3 sequences generated by V(D)J junctional diversification mechanisms. Immunization of this mouse line with SARS-CoV-2 (Wuhan-Hu-1) spike protein immunogens elicited several humanized VH1-2/Vκ1-33-based SARS-CoV-2-neutralizing antibodies that bound RBD in a different mode from each other and from those of many prior human patient-derived VH1-2-based neutralizing antibodies. Of these three antibodies, two (VHH7-5-82 and VHH7-7-53), like many other previously described SARS-CoV-2 neutralizing antibodies, bound an epitope overlapping with the RBM. In this regard, our LLSM-based mechanism of action study directly demonstrated that VHH7-5-82 neutralized SARS-CoV-2 by blocking its binding to the host cell. The third antibody, SP1-77, bound RBD on the opposite side from the RBM. SP1-77 showed much broader neutralizing activity than the other two, and potently neutralized all major SARS-CoV-2 variants through the recently emergent BA.5 variant. Cryo-EM studies revealed that SP1-77 binding epitope on the SARS-CoV-2 RBD was different from those of previously characterized neutralizing antibodies. However, future mutagenesis studies are needed to more precisely define residues that are critical for SP1-77 binding to the RBD. The LLSM assay further revealed that SP1-77 did not inhibit SARS-CoV-2 binding to the host cell or its endocytosis. Instead, SP1-77 inhibited a downstream step in the SAR-CoV-2 infection process that involved fusion of the virus membrane with that of the host cell.We have engineered the human Vin vivo, it would have potential to be a therapeutic against current and newly-arising VOCs. Also, SP1-77 might be useful in a cocktail with other neutralizing antibodies, such as LY-CoV1404, that potently neutralizes all tested VOCs through an ACE2-blocking mechanism. In addition, the non-traditional neutralization mechanism of SP1-77 may inspire the design of new vaccine strategies to more broadly target BA.5 and other VOCs.The SP1-77 neutralization mechanism we described provided further insight into how a non-ACE2 blocking antibody can potently neutralize SARS-CoV-2. Besides the S309 neutralizing antibody mentiond above, several other Omicron sub-variant neutralizing antibodies that have binding epitopes outside of the RBM have been reported J rearrangements. We then immunized the mice with SARS-CoV-2 spike protein constructs and analyzed their antibody responses. We found that the mice indeed could make humanized antibodies that potently neutralized SARS-CoV-2. We focused on characterizing one of these antibodies that broadly and potently neutralized all major SARS-CoV-2 variants at least through the recently emergent BA.5 variant. We performed Cryo-EM studies that revealed that this antibody binds to an overall epitope on the SARS-CoV-2 Spike protein receptor binding domain that was different than any found to date. We further used a lattice-light-sheet-microsopy-based assay to reveal that this antibody inhibits a downstream step in the SARS-CoV-2 infection process that involves fusion of the virus membrane with that of the host cell.The main objective of this study was to develop a humanized mouse model that through appropriate immunizations could be used to generate antibodes that broadly and robustly neutralize SARS-CoV-2 VOCs. The specific hypothesis tested was that such mice could be generated by programming them to produce sufficiently diverse antibody precursor repertoires through diversification mechanisms associated with the rearrangment of just a single human antibody heavy and light chain variable region sequence out of the 100 s present in the human genome. We generated such mice through standard gene targeted mutational H1-2IGCR1∆ ES cells (129/Sv and C57BL/6 F1 hybrid background)  inserted 50 bp downstream of human Vκ1-33 segment. The replacement was mediated by homologous recombination using a PGKneolox2DTA.2 (Addgene #13449) construct and two guide RNAs that targeted the mouse Vκ3-2 segment. The Cer/sis regulatory element in this ES cell line was deleted via the use of two guide RNAs. A human TdT gene was expressed in this mouse model. The human TdT cDNA was inserted into the first intron of mouse Rosa26 gene by homologous recombination using a CTV (Addgene #15912) construct. The sequences of guide RNAs used for targeting were listed in table S9.All the genetic modifications were introduced into previously generated VH1-2mVH∆/IGCR1∆/Vκ1-33CS∆ -rearranging mouse was generated by blastocyst injection of the ES cells described above and several rounds of breeding to get germline transmission and homozygous mice. All mouse experiments were performed under protocol 20-08-4242R approved by the Institutional Animal Care and Use Committee of Boston Children’s Hospital. More details on this protocol can be found in the supplementary materials.The VH1, JH2, JH3 and JH4 were mixed to capture the Igh heavy chain repertoire in one library. Likewise, four 4 bait primers that target mouse Jκ1, Jκ2, Jκ4 and Jκ5 were mixed to capture the Igκ light chain repertoire in one library. The sequences of mouse JH and Jκ primers were as same as the previously reported (Splenic B cells were purified from 5-8 week old mice by MACS Microbeads according to the manufacturer’s protocol. 10 ug of genomic DNA from purified splenic B cells was used for generating HTGTS-repertoire-seq (HTGTS-rep-seq) libraries as previously described (HexaPro Spike immunogen was produced and purified as previously described (+ B cells were selected for the phenotype B220+ (PE-Cy7: BD 552772), IgD− (BV711: BioLegend 103255), IgG1+ (BV605: BD 563285), IgG2a+ (BV605: BD 564024), Spike+ (Alex647) or RBD+ (Alex647). SARS-CoV-2 spike and RBD were conjugated with Alex647 fluorescence (Thermo Fisher A30009). Single cell RT-PCR was performed as described previously (+ B cell was sorted into 96-well plate. Single cell cDNA was generated by reverse transcription (Thermo Fisher 18080093) using a primer mixture that specifically target Cu, Cγ1, Cγ2a and Cκ. VH1-2 and Vκ1-33 V(D)J exons were amplified by two rounds of nested PCR and identified by sanger sequencing. Primer sequences used are listed in table S9.Mouse spleen samples from immunized mice were processed for single B cell sorting as previously described (The antibody expression constructs containing the heavy-chain and the light-chain variable region exons (table S3), with human constant region sequences  at the C terminus were made by Genscript. Monoclonal antibodies were generated using the Expi293 expression system (Thermo Fisher Scientific) and purified by high-performance liquid chromatography (HPLC) coupled with HiTrap Protein A HP columns (Cytiva). Fabs were made as previously described (ELISA was preformed as previously described (50) compared to virus control wells. More details on this protocol can be found in the supplementary materials.The pseudovirus neutralization assay in 293 T/ACE2 cells was performed at Duke  were performed in the Duke Regional Biocontaiment Laboratory BSL3  as previously described with virus-specific modifications  proteins were performed using a Biacore S200 instrument (Cytiva) and as described previously  protein Wuhan-Hu-1 strain (VSV-SARS-CoV-2) was previously described . Each virus trajectory was visually examined for localization to the cell surface, internalization into the cell, and spreading of the Atto565 dye fluorescence. The virus is 80-100 nm, which made it a diffraction limited object. The spot limited by the wavelength of the dye being emitted was 300 nm. The endosome was 600 nm and larger. The fusion of the viral envelope to the endosomal membrane was observed by a spreading of the fluorescent signal from a point spread limited object to 500-1000 nm over the endosomal membrane.The single virus tracking and image analysis was performed as previously described .