Using linkage mapping, association analysis and gene editing, we determined that N2 carries an insertion in the collagen gene col-182 that acts as a recessive enhancer of rol-1 rolling. From population and comparative genomics, we infer the insertion is derived in N2 and related laboratory lines, likely arising during the domestication of Caenorhabditis elegans, and breaking a conserved protein. The ancestral version of col-182 also modifies the phenotypes of four other classical cuticle mutant alleles, and the effects of natural genetic variation on worm shape and locomotion. These results underscore the importance of genetic background and the serendipity of Brenner’s choice of strain.Genetic background commonly modifies the effects of mutations. We discovered that worms mutant for the canonical  C. elegans, an experimental organism with properties ideal for probing the molecular basis of development and neurobiology  V, BE13: sqt-1(sc13) II, BE22: rol-1(sc22) II, BE44: dpy-8(sc44) X, BE93: dpy-2(e8) II, BE108: sqt-2(e108) II, CB61: dpy-5(e61) I, CB91: rol-1(e91) II, CB224: dpy-11(e224) V, CB768: bli-2(e768) II, CB769: bli-1(e769) II, CB1166: dpy-4(e1166) IV, CB2070: bli-1(e935) rol-1(e91) II, CB4856: Hawaiian wild type, COP1834: col-182(knu732) X, EG7993: oxTi412 [eft-3p::TdTomato::H2B] X, EG8951: oxTi1015 [eft-3p::GFP::NLS + NeoR] X, N2: laboratory wild type, QG2797: rol-1(e91) II; ajIR6  X, QG2798: rol-1(e91) II; oxTi412 [eft-3p::TdTomato::H2B] oxTi1015 [eft-3p::GFP::NLS + NeoR] X, QG2804: mIs12rol-1(e91) II, QG2952: bli-1(e769) II; col-182(knu732) X, QG2954: dpy-11(e224) V; col-182(knu732) X, QG2953: rol-1(e91) II; col-182(knu732) X, QG2955: dpy-4(e1166) IV; col-182(knu732) X, QG2956: bli-1(e935) rol-1(e91) II; col-182(knu732) X, QG2957: rol-1(sc22) II; col-182(knu732) X, QG2958: dpy-2(e8) II; col-182(knu732) X, QG2960: dpy-5(e61) I; col-182(knu732) X, QG2961: sqt-2(e108) II; col-182(knu732) X, QG2962: bli-2(e768) II; col-182(knu732) X, QG3070: sqt-3(sc8) V; col-182(knu732) X, QG3072: sqt-1(sc13) II; col-182(knu732) X, QG3074: dpy-10(cn64) II; col-182(knu732) X, QG3076: dpy-8(sc44) col-182(knu732) X, SP419: unc-4(e120) rol-1(e91) II, TN64: dpy-10(cn64) II, and WE5241: ajIR6  X.We used the following strains: BE8: col-182(knu732) X was generated by Knudra Biosciences (now InVivo Biosystems), using the CRISPR/Cas9 homology-directed repair protocol of col-182anc. In the N2 background, Cas9 was directed to sites on each side of the target insertion with sgRNA sequences CTGGATATAGTTGTTCCCGG and CAAGGAGAGATCGGACGCGA, and repair was directed by the DNA oligo AGGACGGATGCCGCGTCTGCCCACCAGGACCACCGGGTA- CTACCGGTTACCCAGGTCAGCCTGGCCCCCAGGGTCTCC- CAGGTAACCAAGGAGACCAAGGTGAAATTGGTCGCGAA- GGATCACCAGGAGCCCAGGGAGCTCCAGGAG. This oligo precisely excises the 8-bp insertion sequence from the N2 col-182 gene and simultaneously alters 16 additional sites, all silent codon positions, to prevent recutting by Cas9.COP1834 rol-1(e91) mutation in these strains by Sanger sequencing using primers RolF3: CAAATTCGACAAAGCGACAA and RolR3: GAGCATCGTAAGGCTGGAAA. We verified the presence of the col-182(knu732) mutation by Sanger sequencing using primers X12.636F: TAGGCAAACTTGCTGCACAC and X12.636R: GAGACAGGCTGGAAATGAGC.We verified the presence of the unc-4(e120) rol-1(e91) II in the N2 background. As described in rol-1 on chromosome II and heterozygous on the X chromosome, with one X chromosome from N2 and the second from the strain of interest (SOI). If the SOI carries the dominant suppressor of rol-1, then these animals are expected to show suppressed rolling behavior.We used visible markers to generate animals that are homozygous mIs12[GFP] rol-1 II males to SOI hermaphrodites to generate mIs12rol-1 / + + II; SOI X unc-4rol-1 II hermaphrodites. For each SOI, we tested six GFP-positive nonUnc hermaphrodite progeny of this cross for rolling behavior in the second day of adulthood. These animals are mostly unc-4 + rol-1 / + mIs12rol-1 II; SOI/N2 X. A small fraction of the phenotyped animals could be rol-1 heterozygotes due to rare recombination events between mIs12 and rol-1 in the Specifically, we crossed We used this approach to test 5 recombinant inbred advanced intercross lines . We also tested 8 wild isolates by this approach , along with N2 and LJS1, which are laboratory-adapted strains.rol-1(e91) II; CB4856 X and rol-1(e91) II; oxTi1015oxTi412 X. The latter strain carries integrated single-copy transgenes on the X expressing fluorescent proteins, tdTomato::H2B from X:11.049 Mb and GFP::NLS from X:13.480 Mb [Wormbuilder; WBVar00083599 at X:12,579,121, and recombinant AM_GNR13 placed it to the left of SNP WBVar1602269 at X:12,662,633.To fine-map the suppressor, we crossed WBVar01981458 indel, TGGGTAAACATCGGCTCCAT and TGTTCTGCACGGGAAAAGAT; WBVar00083599 SNP, CGACATCCAAAGTTTTTGAGACT and GAGAAAGTGTTATGGGCATGG; WBVar01602269 SNP, CGTGTGTTTCCGTTGTGAAT and TTCAGTGTTCATCGCAATCTG.Genotyping primers used for mapping were: C. elegans isotypes were downloaded as the 20180527 CeNDR release soft-filtered vcf. We tested for a match between variants in the recombination-mapping interval  and rol-1 suppression phenotype in the panel of N2, CB4856, LSJ1, and the seven other tested wild isolates.Variant data for 330 Caenorhabditis genus were downloaded from the Caenorhabditis Genomes Project. RNAseq data from young adults of C. elegans wild isolates were downloaded from the NCBI SRA: CB4856 PRJNA437313  and two species (C. quiockensis and C. sulstoni) with extremely long predicted ortholog sequences potentially deriving from annotation errors were excluded. The CGP C. remanei col-182 protein sequence contains a large deletion that is not present in the WormBase version, and we opted to use the latter. Collagen triplet stability scores shown in Figure 2B were obtained from  double mutant in parallel at low population densities for several generations and performed synchronous egg lays to generate animals for phenotyping. For most strains we observed these synchronized animals three days later as young adults. For sqt-2(sc108), because the heterozygous phenotype (Rol) is different from the homozygous Squat phenotype (Sqt), we also scored sqt-2/+ animals. For rol-1bli-1 and rol-1bli-1; col-182 animals, we followed 200 of each genotype through the third day of adulthood. Videos of rol-1 worms  were taken on day four of adulthood. Ten worms were picked to fresh seeded plates and imaged for 8 min at 12 frames per minute. Videos of sqt-3 worms  were taken on day two of adulthood. Twenty worms were picked to fresh seeded plates and imaged for five minutes at 12 frames per minute. Video samples are in Files S3-S9.For each tested mutation, we grew the mutant line and rol-1(sc22), and sqt-3(sc108)), each with the ancestral and derived col-182 allele. Conditions and worm tracking have been described previously  or sqt-3(sc8), separately, using plate means. Traits retained for sqt-3 , ordered by absolute loading on the discriminant function, were log transformed curvature (S), width (F), kink, width (S) variance, and acceleration (F). Traits retained for rol-1  were log transformed circularity, velocity variance, width (F), kink (F), and run length (F) variance. R code for this analysis is in File S14.We show univariate and multivariate  panel we genotyped the N2 indel from existing sequence data , the rol-1 and sqt-3 suppression discriminant functions that are linear combinations of five traits , and the single traits with the highest loading in each discriminant function, curvature and track circularity (log transformed). Trait values were best linear unbiased predictions (BLUPs) extracted from linear mixed effects models . Genome-wide significance was declared against a null distribution of >1000 test statistics generated by permuting lines within populations and retaining the minimum observed p-value. We used a relative permissive false discovery rate (FDR) of 0.2. Quantile-quantile plots showed statistics were well calibrated for length/width and the Rol/Sqt discriminant functions at To test for col-182 . The nine significantly linked transcripts were tested for functional enrichment using the WormBase Enrichment Analysis Suite (We extracted data for 199 recombinant inbred advanced intercross lines (RIAILs) from https://github.com/lukemn/cuticle. Strains are available upon request. File S1 details all supplemental files, File S2 contains genotyping data from col-182 and SNPs in the CeMEE  rol-1(e91) II worms (N2 background) with wild isolate strain CB4856, isolated in Hawaii in 1972  allele into an X-chromosome substitution strain, which carries the CB4856 X chromosome but is otherwise N2, and confirmed that the resulting rol-1(e91) II CB4856 X strain is strongly, but incompletely, suppressed for rolling. The worms retain a slight helical twist and can be distinguished from wildtype N2 and CB4856, but the dramatic rolling and circling behaviors of rol-1(e91) are absent (Videos: CB91 rol-1(e91); N2 X vs. QG2797 rol-1(e91); CB4856 X in Files S3 and S4). Suppression of rol-1 is also observed in X chromosome heterozygotes, consistent with a dominant suppressor on the CB4856 X and explaining the pattern of segregation distortion we observed in the unc-4rol-1 crosses.We hypothesized that the CB4856 X chromosome carries a dominant suppressor of rol-1 alleles with known effects on phenotype at present, and we found that the second, sc22, is also suppressed by the CB4856 X chromosome. Although the sc22 molecular lesion is unknown, e91 and sc22 mutants show distinct phenotypic profiles, including temperature sensitivity .We performed complementation testing with a panel of N2-CB4856 recombinant inbred advanced intercross lines  with breNext we used integrated single-copy fluorescent marker transgenes  deletion in the gene col-182 (WBVar01928355). Like rol-1, col-182 is one of the 181 collagen genes in the C. elegans genome , the 8-bp deletion is present in every strain except for the lab strains N2, LSJ1, and ECA252. These three strains are all derived from a single isolate of C. elegans sampled by L. N. Staniland in 1951 . CRISPR-Cas9 conversion removed the 8-bp insertion and altered 16 additional base pairs, each a synonymous third codon position change in the predicted ancestral reading frame. We then confirmed that this ancestral allele col-182anc suppressed rol-1, both the e91 and sc22 alleles (videos: CB91 rol-1(e91)vs. QG2953 rol-1(e91); col-182anc, and BE22 rol-1(sc22)vs. QG2957 rol-1(sc22); col-182anc, Files S3 and S5-S7). We generated quantitative data for the sc22 allele (selected arbitrarily) by tracking young adult hermaphrodites and extracting an array of statistics describing locomotion, size, and shape . Reduction of this data by multidimensional scaling into two orthogonal axes provided quantitative confirmation that the ancestral col-182anc allele suppresses some of these phenotypes in the rol-1 background . Although we observed no gross phenotypic effect of col-182 on dpy-8(sc44), dpy-10(cn64), or sqt-1(sc13), we observed partial suppression of rolling in sqt-3(sc8), which we quantified by worm tracking (sqt-3(sc8)vs. QG3070 sqt-3(sc8col-182) anc, Files S8,S9). Suppression was qualitatively and quantitatively distinct to that of rol-1; near complete for the single measure of worm width, but again highly variable and generally weak for other measures of locomotion and morphology (rphology .sqt-2(sc108) exhibits right-handed rolling as a heterozygote in the N2 background, and did so as well in the col-182anc background. However, sqt-2 heterozygotes showed slowed development in the N2 col-182 background, while the ancestral allele suppressed the developmental delays. Finally, alleles of several additional genes involved in cuticle development – collagens dpy-2(e8), dpy-4(e1166) and dpy-5(e61), and thioredoxin dpy-11(e224) – showed no gross phenotypic modification in the col-182anc background.col-182 in two genetic backgrounds  panel, a collection of recombinant inbred lines derived from the pooled standing genetic diversity of 14 wild isolates and two N2-related strains  complex . However, none are known to be involved in cuticle development and collectively they show no enrichment for any particular tissue. Thus the col-182 mutation in N2 appears to have limited effects on gene expression in young adult hermaphrodites, at least under ordinary laboratory conditions.Several of the alleles that arose during  and genetic sensitivity. Around 4% of the worm genome is dedicated to expressing, processing, and assembling the collagens, cuticulins, glycoproteins and other components of the multilayered extracellular matrix . To thiscol-182 in the worm cuticle and its mode of interaction with other collagens remains obscure. The derived N2 insertion represents an evolved enhancer of rol-1 rolling, and the ancestral col-182 modifies to a variable extent the phenotypes from other classical mutant alleles of bli-1, bli-2, sqt-2 and sqt-3, but not obviously those of dpy-2, -4, -5, -8, -10 and -11, or sqt-1.In the absence of molecular and structural data, the precise role of rol-1, for which suppression by ancestral col-182 was strongest, shows strong stage specificity, being around 30-fold enriched in L4 . But bli-1, bli-2 and rol-1 show generally similar patterns and levels of transcriptional activity over the life-cycle, with very low expression in embryonic and early larval stages. The Blistered phenotype is thought to be due to defects in struts linking basal and cortical layers of the adult cuticle, and of six Blistered mutants, three are enzymes rather than structural components.The expression of cuticular genes during worm development offers no clear insight. Of the tested genes, only sqt-1, -2 and -3 are all highly expressed collagens that interact genetically, with similar stage specificity from L2 onward . sqt-3 is unique among collagens in its essentiality  mutants in the ancestral col-182 background. This may be explained by the extreme specificity of allelic interactions among collagen mutants, and sqt mutants in particular. The sqt-1(sc13) allele tested is a recessive C-terminal C>Y substitution, altering cross-linking (sqt-2(sc108) is an N-terminal R>C substitution of unknown structural effect. Alleles of sqt-1 vary markedly in their type, severity, temperature sensitivity, and degree of dominance of phenotypes, as well as inter- and intragenic interaction effects; from near wild-type for a null allele, to left or right rolling, abnormal hermaphrodite tail or male rays, or variation in body length. Enrichment in CeMEE interaction statistics for worm length/width over a small region spanning sqt-1 (col-182 may exist.-linking , while sdpy-2, -5, -8, -10 (col-182 likely plays a role, apparently redundant under laboratory conditions, in one or both of the strut-anchored adult cuticular layers. The oft-touted genetic simplicity of C. elegans breaks down somewhat when considering the cuticle, and targeted biochemical and structural analysis, together with epistasis analysis encompassing natural genetic variation, will be required to clarify the precise role of col-182 and the majority of other collagens with no known function in the N2 background.Lastly, no grossly visible interactions were seen for collagens involved in annuli formation and shape C. elegans (C. elegans and core components of the worm geneticist’s toolkit.Effects of genetic background are ubiquitous in complex genetic systems wherever they are carefully considered. Studies mixing natural with domesticated genetic variation have amply shown the importance of genetic interaction on the phenotypic outcome of allelic effects in


Near infrared photoimmunotherapy is a newly developed and highly selective cancer treatment that employs a monoclonal antibody conjugated to a photo-absorber dye, IRDye700DX, which is activated by 690 nm light. Cancer cell-targeted near infrared photoimmunotherapy selectively induces rapid necrotic/immunogenic cell death only on target cancer cells and this induces antitumor host immunity including re-priming and proliferation of multi-chronal T-cells that can react with cancer-specific antigens. Interleukin-15 is a type-I cytokine that activates natural killer-, B- and T-cells while having minimal effect on regulatory T-cells that lack the interleukin-15 receptor. Therefore, interleukin-15 administration combined with cancer cell-targeted near infrared photoimmunotherapy could further inhibit tumor growth by increasing antitumor host immunity. In tumor-bearing immunocompetent mice receiving this combination therapy, significant tumor growth inhibition and prolonged survival was demonstrated compared with either single therapy alone, and tumor infiltrating CD8+ T-cells increased in number in combination-treated mice. Interleukin-15 enhances therapeutic effects of cancer-targeted near infrared photoimmunotherapy.Near infrared photoimmunotherapy (NIR-PIT) is a newly developed and highly selective cancer treatment that employs a monoclonal antibody (mAb) conjugated to a photo-absorber dye, IRDye700DX, which is activated by 690 nm light. Cancer cell-targeted NIR-PIT induces rapid necrotic/immunogenic cell death (ICD) that induces antitumor host immunity including re-priming and proliferation of T cells. Interleukin-15 (IL-15) is a cytokine that activates natural killer (NK)-, B- and T-cells while having minimal effect on regulatory T cells (Tregs) that lack the IL-15 receptor. Here, we hypothesized that IL-15 administration with cancer cell-targeted NIR-PIT could further inhibit tumor growth by increasing antitumor host immunity. Three syngeneic mouse tumor models, MC38-luc, LL/2, and MOC1, underwent combined CD44-targeted NIR-PIT and short-term IL-15 administration with appropriate controls. Comparing with the single-agent therapy, the combination therapy of IL-15 after NIR-PIT inhibited tumor growth, prolonged survival, and increased tumor infiltrating CD8+ T cells more efficiently in tumor-bearing mice. IL-15 appears to enhance the therapeutic effect of cancer-targeted NIR-PIT. Both the US Food and Drug Administration (FDA) and the Pharmaceuticals and Medical Devices Agency (PMDA) in Japan have given fast track status to this agent. Near infrared photoimmunotherapy (NIR-PIT), which is a new type of cancer therapy that induces highly selective cell death on targeted cells only in NIR light exposed tumor beds, by employing a monoclonal antibody (mAb) conjugated to a silica-phthalocyanine photoabsorbing dye, (IRDye700DX: IR700) ,2. The aIn this study, we sought to augment the effects of tumor targeted NIR-PIT using interleukin-15 (IL-15). We employed CD44 as a tumor target, which is a well-known marker of cancer stem cells  and is eIL-15 has recently emerged as a candidate immune-activator for the treatment of cancer . IL-15 i2 of NIR light on day 0. IR700 fluorescence signal in tumors decreased due to dispersion of fluorophore from dying cells and partial photo-bleaching in all cases       D. CD44-ter test) D. Tumor er test) E. The coer test) E. These nk test) F. Thus, In order to assess the density of tumor infiltrating T cells, an important indicator of antitumor immune activity, multiplex immunohistochemistry (IHC) was performed in MC38-luc tumors . With ILThe cell density of Tregs significantly increased in stroma 7 days after IL-15 administration and combination therapy. This was thought to be a secondary effect in response to the rapid increase of CD8 T cells.We also tested the distribution of Granzyme B (Gzmb) expressing cytotoxic cells. With the combination of IL-15 and CD44-targeted NIR-PIT, the number of Gzmb-positive CD8 T cells in the tumor tissue significantly increased at day 7 after the therapy . With IL2 of NIR light on day 0. IR700 tumor fluorescence signal decreased due to dispersion of fluorophore from dying cells and partial photo-bleaching     (The NIR-PIT regimen and imaging protocol are depicted in leaching B. Tumor er test) C. The coer test) C, and sher test) C. In thenk test) D. This c2 of NIR light on day 0. IR700 tumor fluorescence signal decreased due to dispersion of fluorophore from dying cells and partial photo-bleaching.  . The combination therapy showed significantly prolonged survival compared to CD44-targeted NIR-PIT group, although IL-15 treatment group had no significant difference in survival compared to CD44-targeted NIR-PIT group  (Our previous study showed MOC1 tumor has low and more heterogeneous CD44 expression and CD44-targeted NIR-PIT was less effective against this type of tumor . The NIReaching. B. The tuer test) C. The conk test) D. This cThe combination of IL-15 administration and CD44-targeted NIR-PIT showed superiority in survival to IL-15 treatment alone in all three tumor types tested here. IL-15 binds and sends signals through a heterotrimeric receptor composed of the IL-15-specific IL-15Rα, shared IL-2R/IL-15Rβ (CD122) with the IL-2 receptor, and the common type I cytokine receptor γ-chain γc, CD132; ,26). IL-2; ,26. IIn all tumor types used in this study, MC38-luc, LL/2, and MOC1, infiltration of Tregs was observed within the TME . Tregs tIn addition to CD8 T cells, we observed increasing number of Gzmb-high non-T cytotoxic cells within a few days after IL-15 administration or the combination therapy. Since IL-15 is known to stimulate the cytotoxic function on both CD8 T and NK cells, these cells were most likely to be NK cells. Therefore, the combination therapy of CD44-targeted NIR-PIT and IL-15 administration demonstrate its therapeutic efficacy probably by stimulating both the NK-mediated innate immunity and T-cell mediated adoptive immunity.This report represents an extension of previous studies which attempt to augment the immune response initiated by NIR-PIT. For instance, a previous study demonstrated that the combination of CD44-targeted NIR-PIT and either immune checkpoint blockade or Treg-targeted NIR-PIT showed inconsistent therapeutic efficacy among these three tumor models ,14,24. CThis study had several limitations. First, we used subcutaneously implanted tumor models, whereas an orthotopic or transgenic mouse model would be considered as a superior clinically relevant animal model . However2; CELLTREAT Scientific Products, Pepperell, MA, USA) in a humidified incubator at 37 °C in an atmosphere of 95% air and 5% carbon dioxide. MOC1 cells were cultured in HyClone Iscove’s modified Dulbecco’s medium /HyClone Ham’s Nutrient Mixture F12  at a 2:1 mixture with 5% FBS, 1% penicillin/streptomycin, 3.5 ng/mL EGF , 40 ng/mL hydrocortisone , and 5 ng/mL insulin  in the tissue culture flasks in a humidified incubator at 37 °C in an atmosphere of 95% air and 5% carbon dioxide. Cells were authenticated via in vitro growth characteristics.MC38 cells (murine colon cancer), which were generously provided by Dr. Thomas Waldmann, NIH  stably eWater soluble, silica-phthalocyanine derivative, IRDye700DX NHS ester was obtained from LI-COR Bioscience . An anti-mouse/human CD44 mAb (IM7) was purchased from Bio X Cell . All other chemicals were of reagent grade.Briefly, anti-CD44-IgG  was incubated with IR700 NHS ester  and 0.1 mol/L Na2HPO4 (pH 8.5) at room temperature for 1 h. The mixture was purified with a gel filtration column . The protein concentration was measured with Coomassie Plus protein assay kit  by determining absorption at 595 nm with a UV-Vis spectroscopy system . We abbreviate IR700-conjugated anti-CD44 mAb as anti-CD44-mAb-IR700.3 in volume were used for the experiments. Tumor volumes were calculated from the greatest longitudinal diameter (length) and the greatest transverse diameter (width) using the following formula; tumor volume = length × width2 × 0.5, based on caliper measurements. Mice were monitored each day and tumor volumes were measured three times a week for MC38-luc and LL/2 tumors and twice a week for MOC1 tumors until the tumor volume reached 2000 mm3, whereupon the mice were euthanized by inhalation of carbon dioxide gas. All procedures were performed in compliance with the Guide for the Care and Use of Laboratory Animals and approved by the local Animal Care and Use Committee on 12-02-2019 (MIP-003). Six- to eight-week-old female C57BL/6 mice (strain #000664) were purchased from the Jackson laboratory . The lower part of the body of the mice was shaved as fur can interfere with light activation of the conjugate and bioluminescence imaging. Mice with tumors reaching approximately 150 mm3 mice were divided randomly into one of the following four experimental groups: (1) no treatment (control); (2) intraperitoneal injection of 5 μg murine IL-15  on days 0, 2, 4, and 6 following intravenous injection of 100 μg anti-CD44-mAb-IR700 (IL-15 treatment); (3) intravenous injection of 100 μg anti-CD44-mAb-IR700 followed by external NIR light irradiation at 50 J/cm2 on day 0 (CD44-targeted NIR-PIT); (4) intravenous injection of 100 μg anti-CD44-mAb-IR700 followed by external NIR light irradiation at 50 J/cm2 on day 0 and 100 J/cm2 on day 1 with intraperitoneal injection of 5 μg murine IL-15 on days 0, 2, 4, and 6 (combination). For the mice with MC38-luc tumor, LL/2 tumor, and MOC1 tumor in the NIR-PIT treated groups, intravenous injection of the anti-CD44-mAb-IR700 was performed 5, 5, and 28 days after tumor inoculation, respectively. NIR light was administered to tumor-bearing mice using a red-light emitting diode (LED), which emits light in the range of 670–710 nm wavelength  at a power density of 50 mW/cm2 as measured with an optical power meter . IR700 absorbs light at approximately 690 nm. IR700 fluorescence images were obtained before and after therapy.MC38-luc cells (8 million), LL/2 cells (8 million), and MOC1 cells (4 million) were subcutaneously injected in the dorsum of mice. Once tumors reached volumes of approximately 150 mm/(RLU before treatment) × 100 (%); . BiolumMulticolor immunofluorescence was performed using Opal 7-Color Automation immunohistochemistry staining Kit  and BOND RXm auto stainer . The following antibodies were used: anti-CD44 , anti-CD8 , anti-CD4 , anti-FOXP3 , anti-pan cytokeratin , anti-CD45 , and anti-Granzyme B . The staining was performed according to the Opal 7 color protocol provided by manufacturer with following modification: (i) antigen retrieval was performed using BOND ER2 solution  for 20 min and (ii) the ImmPRESS HRP anti-Rabbit IgG (Peroxidase) Polymer Detection Kit  was used instead of anti-mouse/human secondary antibody provided in the kit. Stained slides were mounted with VECTASHIELD Hardset Antifade Mounting Medium (Vector Laboratories) and then imaged using Mantra Quantitative Pathology Workstation (Akoya Biosciences). Images were analyzed with inForm software (Akoya Biosciences). inForm software was trained to detect tissues and cell phenotypes according to following criteria: areas with pan-cytokeratin expression = tumor, other areas = stroma, CD4+FOXP3+ cells = Tregs, CD4+FOXP3− = CD4 T cells, CD8+ = CD8+ T cells, respectively. At least five images were taken from each tumor sample, areas for each tissue phenotype and cell count of each phenotype were combined to calculate the cell density. As for Gzmb expression analysis, cell phenotyping  and Gzmb expression analysis (Gzmb + and other) were performed separately using inForm for the same set of images, then cell segmentation data were consolidated and analyzed using phenoptrReports and phenoptr (Akoya Biosciences) to calculate the density of Gzmb+CD8 T cells.p value of less than 0.05 was granted significant.Quantitative data were expressed as means ± SEM. The Mann–Whitney U test was used to compare differences between two groups. For multiple comparisons (≥3 groups), a one-way analysis of variance (ANOVA) followed by the Tukey–Kramer test or Dunnett’s multiple comparison test was used. The cumulative survival was analyzed by the Kaplan–Meier survival curve analysis, and the differences between tested groups were compared using the log-rank test followed by Bonferroni correction. Statistical analysis was performed with JMP 13 software . A CD44-targeted NIR-PIT combined with IL-15 treatment showed superior in vivo therapeutic efficacy to either CD44-targeted NIR-PIT or IL-15 treatment alone in colon, lung, and oral cancer models. IL-15 might have potential to become a versatile adjuvant after cancer NIR-PIT.


Scientific Reports 10.1038/s41598-017-09568-5, published online 25 August 2017Correction to: This Article contains errors.Figure 3A is mislabelled and the corresponding figure caption is incorrect. The correct version of Figure 3 appears below as Figure


In this work, we explore the feasibility of using 5c3c-derived rHBV for liver-specific delivery of IL-21 as treatment of chronic HBV infection.Chronic infection by hepatitis B virus (HBV) is associated with high risks of liver fibrosis, cirrhosis and hepatocellular carcinoma. In mouse models of HBV persistence, interleukin 21 (IL-21) has been identified as a potent inducer of viral clearance. Strict hepatotropism makes recombinant HBV (rHBV) vectors ideal for liver-targeting gene delivery. Previously, we established an rHBV vector termed 5c3c, which is highly replicative by itself, but requires HBV envelope proteins provided Methods: 5c3c-derived rHBV replicons harboring mouse or human IL-21 genes (termed 5c3c-mIL-21 and 5c3c-hIL-21 respectively) were constructed and then tested for the production of rHBV virions in vitro and in vivo. 5c3c-mIL-21's anti-HBV effects were determined in chronic HBV mouse model. Furthermore, superinfection by rHBV virions was analysed using HBV-infected HepG2/NTCP cells and human liver chimeric mice.Results: 5c3c-mIL-21 and 5c3c-hIL-21 were efficiently replicative and produced enveloped virions when provided with envelope proteins, both in vitro and in vivo. In mouse model of HBV persistence, IL-21 expressed from injected 5c3c-mIL-21 replicon induced complete viral clearance. 5c3c-mIL-21 and 5c3c-hIL-21 virions could infect HepG2/NTCP cells and engender sustained IL-21 expression. Most importantly, IL-21-expressing rHBV virions could superinfect HBV-infected HepG2/NTCP cells and human hepatocytes in human liver chimeric mice, and engender sustained IL-21 expression and rHBV production.Conclusion: These data suggest the high potential of 5c3c-derived IL-21-expressing rHBV as a novel therapeutic against chronic HBV infection. The complex is then packaged by core proteins (HBcAg), also translated from pgRNA, to form progeny nucleocapsid, wherein reverse transcription continues and rcDNA is eventually produced. Nucleocapsids could be enveloped by viral large/middle/small (L/M/S) surface or envelop proteins (HBsAg) to form mature virions, or recycled back to nucleus to form new nuclear cccDNA Hepatitis B virus (HBV) chronically infects ~250 million people worldwide Previously, by using multiple mouse models of HBV persistence, we identified and confirmed interleukin 21 (IL-21) as a potent inducer of HBV clearance in trans to be able to produce mature virions, but its genome replication is self-reliant. 5c3c-derived virions are infective for primary tupaia hepatocytes and engenders cargo gene expression in infected cells Despite many restrictions imposed by its complex and compact genome organization, HBV's strict hepatotropism makes it an ideal vector for liver- targeting gene delivery Figure S1). This is consistent with previously published data Previously, we demonstrated that IL-21 delivered by recombinant serotype 8 AAV virus elicited clearance of HBV persistence in mouse models In order to evaluate rHBV as vector for liver-specific delivery of IL-21, we used our previously described rHBV vector 5c3c null as IL-21-deficient control . 5c3c-mIL-21 and 5c3c-mIL-21null have the same genome size of 3287 bp, compared to 2846 bp of 5c3c and 3215 bp of wild type. When the respective replicon plasmids were transfected into Huh-7 cells, expectedly only 5c3c-mIL-21 produced measurable mIL-21 in culture supernatants , while both 5c3c-mIL-21 and 5c3c-mIL-21null displayed replication efficiencies comparable to wild type HBV . Similar results were obtained when human IL-21 (hIL-21) coding sequences (405 bp) were used . Furthermore, when and only when co-transfected with helper plasmid expressing HBV envelope proteins (pLMS), both 5c3c-mIL-21 and 5c3c-mIL-21null secreted enveloped virions into culture media . These data indicated that 5c3c- based rHBV replicon harboring mIL-21 cargo gene could support mIL-21 expression in transfected cells of liver origin, and package mature rHBV virions in the presence of HBV envelope proteins. Viral DNA was also detectable in supernatants of 5c3c-mIL-21- and 5c3c-mIL-21null-transfected cells in the absence of envelope proteins , indicating secretion of unenveloped viral nucleocapsids by replicon-transfected cells as confirmed by anti-HBcAg antibody pulldown followed by Southern blot analysis .Murine IL-21 coding sequences (441 bp) modified according to the above rule were inserted into 5c3c to create 5c3c-mIL-21, and premature termination was introduced in mIL-21 reading frame to create 5c3c-mIL-21in vivo after hydrodynamic injection (HDI) into BALB/c mice via tail vein with or without helper plasmid. As shown in Figure 2, HDI with 5c3c-mIL-21 replicon resulted in markedly elevated serum levels of IL-21. Interestingly, serum 5c3c-mIL-21 rHBV DNA was only detectable in mice co-injected with helper plasmid, but not in mice injected with replicon plasmid alone, despite the fact that Southern blot analysis of intracellular capsid-associated HBV DNA showed that both 5c3c-mIL-21 and 5c3c-mIL-21null replicons were efficiently replicative . Apparently, unenveloped nucleocapsids observed in culture media of replicon-transfected cells  were not present in serum from mice injected with the same replicon. In human infections, such unenveloped nucleocapsids were not detectable in serum either in vitro and in vivo HBV replication systems. Since rHBV virions produced in HDI mice are not contaminated with unenveloped nucleocapsids, further separation and purification are not required, which simplifies rHBV production.Genome replication and progeny virus production capabilities of 5c3c-derived IL-21-expressing rHBV replicons were then further tested null replicon plasmids. As shown in Figure 3, injection of 5c3c-mIL-21, but not 5c3c-mIL-21null, resulted in markedly accelerated decrease of serum HBsAg and HBeAg in all recipient mice, and eventual disappearance of both markers in 5 out of 6 mice by 10 weeks post first injection (w.p.i.) of rHBV replicon plasmid. Similarly, HBV DNA in pooled sera started to decrease rapidly in 5c3c-mIL-21-injected mice after the first injection and dropped close to detection limit by 10 w.p.i. . In contrast, decrease of serum HBsAg and HBeAg was comparably slow in 5c3c-mIL-21 null-injected and untreated mice, and none of these mice cleared any viral marker by 10 w.p.i., with serum HBV DNA levels remaining largely unchanged through the observation period .In our previous studies, CMV promoter-driven expression of IL-21 from plasmid or recombinant AAV induced viral clearance in BALB/c mice harboring HBV (genotype B strain BPS) persistence Figure , rHBV DNA was readily detectable in serum after 5c3c-mIL-21 or 5c3c-mIL-21null replicon injection and remained at nearly unchanged levels till 4 w.p.i.. Afterwards, rHBV DNA levels started to decrease and dropped to near detection limit by 10 w.p.i. in 5c3c-mIL-21-injected mice. In 5c3c-mIL-21 null-injected mice, however, rHBV DNA levels did not change markedly up till the end of observation period , similar to what was observed for total HBV DNA .Since BPS expresses both large and small envelope proteins that support packaging of mature, infective virions Figure S6, in 5c3c-mIL-21-injected BPS persistence mice that cleared serum HBsAg at 10 w.p.i., neither BPS nor 5c3c-mIL-21 replicon DNA was detectable in liver; whereas in 5c3c-mIL-21null-injected mice, which failed to clear serum HBsAg, both BPS and 5c3c-mIL-21null DNA persisted at considerable levels. These results are in agreement with serum viral DNA analysis results  and proved true and complete clearance of both wild type BPS and rHBV DNA in 5c3c-mIL-21-injected BPS persistence mice.In order to confirm the clearance of BPS in 5c3c-mIL-21-injected mice, mouse liver DNA was extracted at 10 w.p.i. and subjected to Southern blot analysis using BPS- and rHBV-specific probes. As shown in Figure ), but coincided with transient ALT elevations peaking at 4 w.p.i. and reverting to normal levels by 10 w.p.i. , which was slightly preceded by a transient serum IL-21 elevations peaking at 2 w.p.i. . When cellular immune response at 10 w.p.i. were analyzed, splenocytes from 5c3c-mIL-21-treated mice contained more HBsAg- and HBcAg-specific IFN-γ-producing cells compared to untreated and 5c3c-mIL-21null-treated mice . These data are consistent with our previous studies using plasmid and recombinant AAV that expressed IL-21 under the control of CMV promoter Mechanistically, 5c3c-mIL-21-induced HBV clearance was not associated with HBsAb development , which reconfirmed that both wild type and recombinant HBV virions enter susceptible cells through preS1-dependent mechanism(s). Similarly, Southern blot analysis showed that post-infection replication of wild type or recombinant HBV genomes were only detectable in cells infected with HepaAD38 supernatant, or 5c3c-mIL-21/pLMS or 5c3c-mIL-IL-21null/pLMS co-transfection supernatants .In order to qualify for therapeutic applications in human, infectivity of IL-21-expressing rHBV virions needs to be validated first. For this purpose, HepG2 cells stably transfected with HBV receptor NTCP (HepG2/NTCP) were incubated with 5c3c-mIL-21/pLMS or 5c3c-mIL-IL-21Figure , sustained and increasing mIL-21 expression and secretion by rHBV was only detected in supernatants from cells infected with 5c3c-mIL-21/ pLMS co-transfection supernatant, whereas sustained and increasing HBsAg secretion was only detectable in supernatants from cells infected with wild type HBV . These results are consistent with the expected IL-21 and HBsAg expression capacities of the respective virions . In addition, when HBV DNA in culture supernatants were analyzed at the end of observation period (8 days post infection), only cells infected with HepaAD38 supernatant produced enveloped HBV DNA, namely virions, into culture media, while cells infected with 5c3c-mIL-21/pLMS or 5c3c-mIL-IL-21null/pLMS co-transfection supernatants only produced unenveloped viral DNA in the form of unenveloped nucleocapsids  as observed in replicon transfection experiments . These data are also consistent with the inability of these 5c3c-derived rHBV replicons to produce virions by themselves due to lack of envelope expression .Culture supernatants of infected HepG2/NTCP cells were also serially sampled and analyzed. As shown in Figure S8). Moreover, 5c3c-mIL-21 and 5c3c-mIL-IL-21null rHBV virions produced in HDI mice  also displayed infectivity using this assay .Highly similar results were obtained when rHBV expressing human IL-21 was used in the same assay , while sustained and increasing HBsAg secretion was observed for both cells infected with wild type HBV, or superinfected with 5c3c-mIL- 21 or 5c3c-mIL-21null virions . Southern blot using HBV- and mIL-21-specific probes in parallel confirmed rHBV replication in superinfected cells . Moreover, both unenveloped and enveloped rHBV DNA was detectable in supernatants from cells superinfected with 5c3c-mIL-21 or 5c3c-mIL-21null virions, but not in supernatant from cells mono-transfected with 5c3c-mIL-21 only . These results demonstrated that superinfection of HBV-infected cells by 5c3c-mIL-21-derived rHBV virions not only resulted in continuous mIL-21 production, but also sustained progeny rHBV production with the help of envelope proteins produced by pre-existing wild type HBV.For therapeutic applications targeting chronic HBV infections, IL-21-expressing rHBV should not only be infective for hepatocytes, but also be capable of superinfecting hepatocytes already infected with wild type HBV, since virtually all hepatocytes are already HBV-infected in this context in vivo. Mice were first infected with wild-type HBV by injection via tail vein with sera from pCMV1.1HBV HDI mice, and 14 days later, injected again with sera form 5c3c-mIL-21/ pLMS co-injected mice, or left untreated. As shown in Figure , prominent and increasing production of mIL-21 into serum was detectable only in mice receiving 5c3c-mIL-21-derived rHBV virions. When serum HBsAg and wild type HBV DNA were measured using ELISA and quantitative real time PCR respectively, continuously increasing levels were observed in all mice . In contrast, serum rHBV DNA was only detected in HBV-infected mice receiving 5c3c-mIL-21 virions , and the levels gradually increased, in correlation with serum mIL-21 levels in the same mice . The identity of rHBV DNA was also confirmed in Southern blot using HBV- and mIL-21-specific probes in parallel . Collectively, these results corroborated the in vitro data and proved that 5c3c-mIL-21 rHBV virions are capable of superinfecting HBV-infected hepatocytes in vivo. Since unenveloped nucleocapsids are not observed in mice , increasing serum rHBV DNA levels in mice superinfected with 5c3c-mIL-21 rHBV represent marked and steady production of progeny rHBV virions by hepatocytes harboring both wild type and 5c3c-mIL-21 rHBV infection.Finally, human liver chimeric mice Figure S11).Tissue distribution of HBV and rHBV infection in human liver chimeric mice was evaluated after sacrifice, and as can be expected, HBV infection was only detected in liver samples and rHBV infection was only detected in liver samples from super-infected chimeric mice . Secondly, integration of AAV genome into host chromosome remains a possibility Figure S1 nevertheless would be troubling when applications in human are considered.The potency of IL-21 for inducing viral clearance in mouse models of HBV persistence has been repeatedly demonstrated in trans . In addition, Sp1-driven expression of IL-21 from rHBV replicon induced viral clearance in mouse model of HBV persistence . Furthermore, IL-21-expressing rHBV virions were shown to be infective using HepG2/NTCP cells . Collectively, these data suggest rHBV as a valid vector for liver-targeting delivery of IL-21.HBV boasts nearly exclusive hepatotropism, which even dwarfs other hepatitis viruses. Despite its small genome size and complex genome organization, our previous efforts successfully established a recombinant HBV vector system capable of delivering cargo gene expression controlled by liver-specific HBV Sp1 promoter into hepatocytes in vitro and in vivo . Such superinfection not only allows rHBV to engender expression of cargo gene, IL-21 in this case, in HBV-infected cells, but also enables production of rHBV progeny virions using envelope proteins expressed from preexisting wild type HBV genomes . In addition, the sustained production of rHBV virions and IL-21 in HBV-infected human liver chimeric mice super-infected with IL-21-expressing rHBV through a single injection  indicated that in vivo, rHBV would be able to persist as long as wild type HBV has not been cleared. In an immunocompetent HBV-infected host, such qualities of IL-21-expressing rHBV would allow continued expression of IL-21 until wild type HBV has been reduced to negligible levels as a result of the effects of IL-21 and/or other co-administered therapeutics. Since IL-21 acts through inducing HBV-specific CD8+ T cells in mice Figure 1), it could be envisioned that IL-21-activated HBV-specific cellular immunity  would attack and destroy hepatocytes infected with wild type HBV, rHBV, or both. This would effectively constitute an ideal self-removal mechanism that ensures clearance of IL-21-expressing rHBV upon eventual clearance of wild type HBV. Such a mechanism would also partially alleviate the risk associated with possible integration of rHBV sequences into hepatocyte chromosome by targeting hepatocytes with integrated rHBV sequences retaining HBcAg expression.In CHB patients, a predominant majority of hepatocytes were already infected with HBV In summary, results presented here demonstrated that rHBV is a powerful vector for delivering IL-21, and possibly also other HBV-targeting genes, specifically into hepatocytes, including previously HBV-infected hepatocytes. Therapeutic application of such rHBV virions in the context of human chronic HBV infections warrants further studies.Figure S2 and Figure S3) and inserted into 5c3c in frame with ATG-less preS1 to create 5c3c-mIL-21 and 5c3c-hIL-21, respectively. Mutants with premature termination codons (5c3c-mIL-21null) or obliterated start codons (5c3c-hIL-21null) were created through site-directed mutagenesis .Construction of most of the plasmids used in this work has been previously described. Briefly, replicon plasmid pCMV1.1HBV contains 1.1× over-length wild-type HBV genome under the control of cytomegalovirus (CMV) promoter in vitro. HepAD38, which is stably transfected with HBV replicon, was used for wild type HBV virion production. HepG2 cells stably transfected with HBV receptor NTCP (HepG2/NTCP) were used for infection. All cells were cultured and transfected using TurboFect  as previously described Human hepatoma cell line Huh-7 was used for replicon analysis and rHBV virion production 11 genome equivalents (geq) of recombinant serotype 8 AAV expressing EGFP  in 200 μL PBS via tail vein. At 1 week post injection (w.p.i.), tissues were collected after sacrifice by cervical dislocation and frozen sections prepared for fluorescent microscopy analysis. For evaluating therapeutic effects of IL-21-expressing rHBV replicon, mice were first injected with 10 μg BPS plasmid DNA via tail vein through hydrodynamic injection (HDI). Four weeks later, mice with persistently positive serum HBsAg were selected and injected with 25 μg 5c3c-mIL-21 or 5c3c-mIL-21null via HDI, or left untreated, and injected once again 3 weeks later. Serial serum samples and tissues were collected at indicated time points or after sacrifice as previously reported Male specific pathogen-free BALB/c mice aged 6-8 weeks were purchased from Experimental Animal Centre, Fudan University. For analyzing tissue- distribution of infection by AAV, BALB/c mice were injected with 2×10Mouse procedures were approved by the Animal Ethics Committee of School of Basic Medical Sciences, Fudan University.in vitro and in vivo, supernatants form HepAD38 cells and sera from BALB/c mice hydrodynamically injected with pCMV1.1HBV at 3 days post injection were concentrated using Amicon Ultra-15 centrifugal filters . To obtain rHBV virions, rHBV replicon plasmid and helper plasmid pLMS were transfected into Huh-7 cells or injected into BALB/c mice via HDI at 1:1 ratio, and supernatants or sera were concentrated as described above. Infection of HepG2/NTCP cells with HBV was performed using a multiplicity of infection (MOI) of 1000:1 (geq versus cell numbers) as previously reported null/pLMS co-injected mice were used to superinfect HepG2/NTCP cells at 1000:1 MOI.To obtain wild-type HBV virions produced 6 geq of wild type HBV concentrated from pCMV1.1HBV injected mice were injected via tail vein, and 2 weeks later, 2×106 geq of rHBV concentrated from 5c3c-mIL-21/pLMS co-injected mice were injected in the same way.For superinfection in chimeric mice, 2×10Table S1. Capsid-associated HBV DNA and nuclear HBV replicon DNA were extracted from transfected cells and mouse liver cells respectively, and analyzed in Southern blot as previously described HBsAg, HBeAg and HBsAb were measured using ELISA . Mouse and human IL-21 expressed by transfected or infected cells were measured using ELISA . Total, wild-type and rHBV DNA in culture medium and mouse serum were measured using quantitative real time PCR. Wild-type and rHBV DNA in tissue samples from sacrificed human liver chimeric mice were analysed using PCR. Primer sequences used are listed in HepG2/NTCP cells infected with wild-HBV and/or rHBV virions were subjected to immunofluorescence analysis to detect HBcAg, HBsAg, and mIL-21. Primary antibodies against HBcAg (1:500) and mIL-21 (1:1000) were purchased from Dako (Denmark) and R&D SYSTEMS (USA), respectively. Primary antibody against HBsAg has been previously reported Mouse splenocytes cultured in RPMI 1640 (Invitrogen) containing 2 mM L-glutamine, 50 U/mL penicillin, and 10% foetal bovine serum (Invitrogen) were stimulated with 15 μg/mL recombinant HBsAg as described t-test. A P-value <0.05 was considered to indicate statistical significance in all analyses. Group positivity percentages of serum HBV markers were expressed using Kaplan-Meier plot. GraphPad 6 was used for plotting and statistical tests.The levels of serum HBV markers were presented as the mean ± standard errors of the mean (SEM). Statistical significance was calculated using unpaired two-tailed


In silico analysis identified two potential heme docking sites on MD-2 near conserved amino acids W23/S33/Y34 and Y36/C37/I44. Heme-induced NF-κB activity was reduced by 39 and 78% in HEK293 cells transfected with MD-2 mutants W23A and Y34A, respectively, compared to WT-MD-2. NF-κB activation by LPS was not affected by the same mutants. Biotinyl-heme/streptavidin-agarose pulled down 68% less W23A and 80% less W23A/S33A/Y34A mutant MD-2 than WT-MD-2. In contrast, at the Y36/C37/I44 MD-2 site, heme-induced NF-κB activity was significantly increased by mutants Y36A (191% of WT-MD-2) and unchanged by mutants C37A and I44A . In conclusion, these data suggest that heme binds and activates TLR4 signaling at amino acids W23 and Y34 on MD-2.Myeloid differentiation factor-2 (MD-2) binds lipopolysaccharide (LPS) and initiates toll-like receptor-4 (TLR4) pro-inflammatory signaling. Heme also activates TLR4 signaling, but it is unknown if heme interacts with MD-2. Therefore, we examined MD-2 for a potential heme activation site. Heme-agarose and biotin-heme/streptavidin-agarose pulled down recombinant MD-2, which was inhibited by excess free heme. UV/visible spectroscopy confirmed MD-2-heme binding. To determine whether MD-2 was required for heme-mediated TLR4 signaling, HEK293 cells were transfected with MD-2, TLR4, CD14, and an NF-κB luciferase reporter, and then stimulated with heme or LPS. Heme or LPS treatment elicited robust reporter activity. Absence of MD-2, TLR4 or CD14 plasmid abolished NF-κB reporter responses to heme or LPS.  Toll-like receptors are central to vertebrate innate immune responses , 2. TheyThe MD-2/TLR4 complex also recognizes a diverse number of endogenous molecules released from injured cells called damage-associated molecular patterns or DAMPs. One such DAMP is heme , 9, 12. Large amounts of heme can be released intravascularly by trauma, sepsis, malaria and red blood cell disorders such as sickle cell disease (SCD). Recent studies underscore the importance of heme-mediated MD-2/TLR4 activation in inflammation, vaso-occlusion, lethality and pulmonary injury in SCD , 13. In Plasmid pFlag-CMV1–hMD2 was a gift from Doug Golenbock (Addgene plasmid #13028). Site-directed mutagenesis was performed using a QuickChange II XL site-directed mutagenesis kit (Agilent Technologies). Mutagenic primers were designed using web-based QuickChange primer design program, and synthesized by Integrated DNA Technologies (IDT) with polyacrylamide gel electrophoresis (PAGE) purification. Mutant strands were synthesized and transformed into XL10-gold ultracompetent cells (Agilent), the DNA from the colonies were checked with restriction digestion and confirmed with DNA sequencing.2 incubator at 37°C. Cells in T225 cm2 culture flasks were transiently transfected with polyethylenimine  (Polysciences) using a 4:1 ratio of PEI to DNA (w/w). After 18 h at 37°C, the cells were changed to ProCHO-AT protein-free media (Lonza). After 4 days in ProCHO-AT media, the conditioned media was collected, and cleared by centrifugation at 600× g for 30 min at 4°C and filtered using a 0.22 μm Stericup Vacuum Filtration System (EMD Millipore). In the UV/Vis heme-MD-2 binding assays described below, the recombinant proteins in the filtered media were purified using anti-Flag M2 affinity gel (Sigma-Aldrich) column chromatography following the manufacturer's instructions. The bound Flag fusion proteins were eluted by competition with Flag peptide, and further concentrated using 10k centrifugal filter units (Amicon). The purity and concentration of the protein was determined by a 4–15% SDS-PAGE and Coomassie R-250 stain (Bio-Rad) with BSA standards (0.2–5 μg) loaded on the same gel to estimate the Flag-MD-2 concentration by comparing the band intensities with BSA. The recombinant WT and mutant MD-2 proteins were confirmed by Western blots with a primary MD-2 antibody (Abcam).WT and mutant N-Flag-hMD-2 fragments from pFlag-CMV1–hMD-2 plasmids were subcloned into expression plasmids with a CAG promoter (pT2/Caggs-Flag-hMD2) that were used to produce WT and mutant MD-2 recombinant proteins in Chinese Hamster Ovary Cells (CHO) as described previously  with modPull-down assays were used to determine if there was a physical interaction between MD-2 and heme. Conditioned ProCHO-AT culture media (30 ml) from T225 tissue culture flasks containing CHO cells overexpressing recombinant wild-type (WT) Flag-MD2, mutants or recombinant Flag-hemopexin (Hpx) as a positive control as previously described  were conThe UV/Vis absorption spectra (250–600 nm) of heme, MD-2 and heme + MD-2 were measured using a Nanophotometer P330 (Implen) with an optical path length of 1 cm. Human recombinant Flag-Hpx was used as a positive heme-binding control.http://mleg.cse.sc.edu/hemeBIND/). HemeBind is a specialized algorithm that combines both structure- and sequence-based methods to identify potential heme-binding sites on heme proteins  residues on human and mouse MD-2 were identified using the HemeBind web server that is freely accessible online  or empty vector, along with 15 ng of Firefly luciferase NF-κB reporter vector , 15 ng of Renilla luciferase control vector , 10 ng of wt or mutant MD-2 expression vector (pFlag-CMV1-hMD2), and 10 ng of CD14 expression vector  per well using Lipofectamine Plus reagent (LifeTechnology). Four h after transfection, cells were incubated 24 h in media containing 10% fetal bovine serum (FBS). Twenty-four hours after transfection, cells were incubated with media with 1% FBS (control), or media with 1% FBS supplemented with heme (10 μM), LPS  or heme + LPS for 6 h, then cells were lysed and luciferase activity was measured.Human embryonic kidney 293 (HEK293) cells were seeded in 96-well plates at 2 × 10t-test. Statistical significance was considered to be p < 0.05.Results are presented as means ± standard deviation unless otherwise indicated. Analyses were performed with SigmaStat 3.5 for Windows . Comparisons of multiple treatment groups were made using One Way ANOVA with Holm-Sidac correction, Kruskal-Wallis One Way Analysis of Variance on Ranks, or a student's unpaired To determine if heme binds to MD-2, recombinant MD-2 was expressed by transfecting CHO cells with a plasmid encoding human MD-2 with a Flag tag at the N-terminus. After transfection, cells were washed and incubated for 3 or 4 days in protein-free ProCHO medium. Transfected, but not untransfected CHO cells secrete soluble recombinant human MD-2 into the media . Flag-MDThe recombinant human flag-MD-2 (rhMD-2) was highly purified as seen on an SDS PAGE gel stained with Coomassie Brilliant Blue R-250 . After 4To confirm heme binding to MD-2, additional pull-down assays were run using recombinant MD-2, biotin-heme and streptavidin-agarose . Flag-MDTo further confirm heme-MD-2 binding, UV/visible spectroscopy was used to detect heme binding to MD-2. Proteins such as Hpx when bound to heme have an increase in absorbance at ~414 nm . Recombiin silico analyses identified two potential heme docking sites on both human and murine MD-2 near conserved amino acids W23/S33/Y34 and Y36/C37/I44 highlighted in magenta and green, respectively , heme (10 μM), LPS (10 ng/ml), or heme + LPS for 6 h, followed by measurement of NF-κB luciferase reporter activity. Heme, LPS and heme + LPS treatment elicited robust luciferase activity in the presence of CD14, MD-2, and TLR4  contained <0.01 endotoxin units/ml. Our NF-κB reporter assays used 10 uM hemin. We have previously shown that anti-LPS IgG directed against the LPS core does not inhibit heme-induced TLR4 signaling , but sigSome of our MD-2 Western blots appear to have 2 bands. Ohnishi et al.  reportedThere could potentially be other heme-activation sites on MD-2 or TLR4 that were not identified. We used a specialized algorithm that combines both structure- and sequence-based methods to identify potential heme-binding sites on heme proteins . This alAnother possibility is that mutations of MD-2 at the proposed activation site might affect the folding and 3-dimensional structure of MD-2 and thereby indirectly affect MD-2 binding to heme and TLR4 signaling. However, nearby mutations Y36A, C37A, and I44A on MD-2 did not inhibit heme-mediated MD-2/TLR4 signaling. An important caveat of our data interpretation assumes the mutants were properly folded.Another limitation is the limited characterization of heme-MD-2 interactions. Future experiments will further characterize the interactions of heme with MD-2 using differential scanning fluorimetry to detect heme's binding affinity to MD-2 . Heme biFuture studies will use virtual screening to identify small molecules that might interact at the W23/Y34 site and inhibit heme-mediated MD-2/TLR4 signaling. Their interaction with MD-2 will be characterized by differential scanning fluorimetry, circular dichroism, and analytical centrifugation as described above for heme-MD-2 interactions. Candidate molecules will be screened for their ability to inhibit heme-mediated chemokine production in human monocyte/macrophages. Follow-up screens will include inhibition of heme activation of P-selectin and von Willebrand factor expression on the membrane of endothelial cells, vaso-occlusion in SCD mice, and heme binding to MD-2.We conclude that heme activates TLR4 signaling at residues W23 and Y34 on MD-2. This site on MD-2 appears to be a possible target for inhibition of heme-mediated TLR4 signaling. We speculate that targeted inhibition of heme-mediated TLR4 signaling would be beneficial in hemolytic diseases such as SCD without affecting innate immunity to gram negative bacteria expressing LPS.All datasets generated for this study are included in the article/JB and GV designed the research. JT and JH contributed computational tools. JB, PZ, KN, JT, and GV wrote and edited the manuscript. JB, PZ, and JT prepared the figures. PZ, JN, and ZK performed the biochemical analyses. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Scientific Reports 10.1038/s41598-020-59145-6, published online 10 February 2020Correction to: This Article contains an error in Figure 2 where the average length for Domesticated and Wild Larvae have been incorrectly plotted. The correct Figure 2 appears below as Fig.


C. elegans Hedgehog-related pathway: wrt-6 and wrt-10. The warthog (wrt) family member wrt-6 encodes a predicted secreted signalling molecule that contains an N-terminal Wart domain and a C-terminal autoprocessing domain (Hint or Hog domain) . wrt-10 encodes another member of this Wrt family that contains the Wart domain but lacks the Hog domain .We have generated new putative null alleles of two members of the wrt-6 or wrt-10 . We isolated the wrt-6 (aus41) allele that contains a 49 bp out-of-frame deletion, predicted to cause a premature stop codon after 7 amino acids . Wild-type (N2) animals were injected with a mix consisting of the sgRNA expression vector (250 ng/µL), Cas9 expression vector (peft-3::cas9::tbb-2) (50 ng/µL) (gift from the de Bono Lab), pCFJ90 (pmyo-2::mCherry::unc-54) (2.5 ng/µL) and pCFJ104 (pmyo-3::mCherry::unc-54) (5 ng/µL). We used a 3-primer method for screening for deletions in mCherry-positive animals . The external forward and reverse primers were used for Sanger sequencing.sgRNA target sequences were incorporated into a wrt-6(aus41)Oligonucleotides used to detect and genotype Expected PCR amplicon: Wild-type: 578 and 327 bp. Mutant: 529 bpExternal forward: GTGTCGTCGGATTTCTTCATTCInternal forward: GCGGATTCCATTCATGATGGExternal reverse: GAGCCACTTACAGTCCAAGGwrt-10(aus36) and (aus37)Oligonucleotides used to detect and genotype aus36), 537 bp (aus37)Expected PCR amplicon: Wild-type: 539 and 241 bp. Mutant: 534 bp  IIHRN666 wrt-10(aus37) IIHRN667 wrt-6(aus41) XHRN680 Strains have not been outcrossed. Will be available at the CGC.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of CoV disease 2019 (COVID-19) is a highly pathogenic and transmissible CoV that is presently plaguing the global human population and economy. No proven effective antiviral therapy or vaccine currently exists, and supportive care remains to be the cornerstone treatment. Through previous lessons learned from SARS-CoV-1 and MERS-CoV studies, scientific groups worldwide have rapidly expanded the knowledge pertaining to SARS-CoV-2 virology that includes in vitro and in vivo models for testing of antiviral therapies and randomized clinical trials. In the present narrative, we review SARS-CoV-2 virology, clinical features, pathophysiology, and animal models with a specific focus on the antiviral and adjunctive therapies currently being tested or that require testing in animal models and randomized clinical trials. MetagSARS-CoV-2 is a member of the Coronavirinae subfamily in the family Coronaviridae and the order Nidovirales. Based on genetic and phylogenetic relationships, this subfamily is classified into four genera: α-, β-, γ-, and δ-CoV. Members of the α- and β-CoV genera infect mammals . MembersSARS-CoV-2 virions are spherical, with a diameter of 60-100 nm conforming to the typical CoV diameter of 125 nm . SARS-CoLike SARS-CoV-1, SARS-CoV-2 also utilizes human angiotenisin-converting enzyme 2 (ACE2) as a receptor for cellular entry . ACE2 isFull-length ACE2 protein consists of an N-terminal peptidase domain (PD) and a C-terminal collectrin-like domain (CLD) followed by a single transmembrane helix and an ~40-residue intracellular segment . The cryUnlike SARS-CoV-1-S, SARS-CoV-2-S harbors a multi-basic furin cleavage site (RRAR*SV) before the canonical S1/S2 site (AY*TM) which allows efficient proteolytic processing during viral egress and prior to the subsequent round of entry . This prFollowing binding and endocytosis, efficient proteolytic priming of S2 at a site proximal to the fusion peptide is essential for exposing the fusion peptide . Like SAThe SARS-CoV-2 genomic RNA contains a 5′ cap and a 3′ poly(A) tail that allow immediate translation to produce two coterminal replicase polyproteins, pp1a and pp1ab, by utilizing a ribosomal frameshifting mechanism. Replicase polyproteins are encoded within two-thirds of the genome (as ORF1a and ORF1b)  [1]. TheThe nsps are assembled into the replicase–transcriptase complex which begins to generate anti-sense (-) genomic and sub-genomic RNAs that serve as templates for positive-sense genome (+) and sub-genomic mRNA synthesis, respectively. These sub-genomic mRNAs are 3′ co-terminal with the viral genome and form a nested set of RNAs due to discontinuous extension of the anti-sense RNA. The nsps also promote membrane rearrangement of rough endoplasmic reticulum (ER) membranes into double-membrane vesicles, in which the above replication and transcription processes occur .Structural proteins translated from sub-genomic mRNAs are inserted into the ER and pass along the secretory pathway to the ER-Golgi intermediate compartment (ERGIC) . Along tFollowing transmission by droplets, contact, and other modes, the virus replicates in cells of respiratory (upper and lower) and gastrointestinal tracts. The clinical spectrum of COVID-19 can vary from asymptomatic and mild symptomatic to an adverse clinical condition characterized by severe respiratory failure requiring mechanical ventilation and hospitalization and critically ill condition . CriticaPatients with moderate pneumonia display such symptoms as cough and shortness of breath (tachypnea). Patients clinically diagnosed with severe pneumonia display symptoms with or without fever, severe dyspnea, respiratory distress, tachypnea (> 30 breaths/min), and hypoxia (SpO2 < 90% on room air) . PatientClinical features of COVID-19 patients with sepsis are particularly critical with a wide range of signs and symptoms involving multiple organs . These s+ T-cell populations in COVID-19 patients are functionally exhausted (NKG2A+), whereas convalescing patients recover functionality , MERS [EC50: 0.15 μM], and SARS-CoV-1 [EC50: 0.14 μM] propagated in Calu-3 cell lines and primary human airway epithelial cultures  and MERS-CoV [EC50: 0.074 μM] at low micromolar concentrations  at low micromolar concentration in vitro with no cytotoxic effects  in vitro. Furthermore, LPV/RTV in combination with IFN-β displayed no synergistic antiviral activity against MERS-CoV compared to IFN-β alone  in Calu-3 cells. The same study also demonstrated near similar EC50 for LPV/RTV [EC50: 8.5 μM] and LPV [11.6 μM], suggesting similar activity as observed for SARS-CoV-1.Lopinavir (LPV) and ritonavir (RTV) are FDA-approved HIV-1 protease inhibitors that also appear to inhibit 3CLpro activity in vitro ,63. LopiDespite in vitro antiviral activity against MERS-CoV, prophylactic or therapeutic administration of LPV/RTV + IFN-β in a mouse model failed to reduce the virus titer, prevent body weight loss / pulmonary function / inflammatory lung disease. In contrast, in a common marmoset model of severe MERS-CoV disease, LPV/RTV treatment resulted in reduced lung / extrapulmonary tissue viral loads and improved the clinical, radiological, and pathological features compared to untreated and mycophenolate mofetil-treated marmosets. The MIRACLE trial (MERS-CoV Infection tReated with A Combination of Lopinavir/ritonavir and intErferon-β1b) is presently investigating the efficacy of combination therapy of LPV/RTV + IFN-β compared to placebo in MERS-confirmed hospitalized patients provided with standard supportive care .50 of SARS-CoV-2, LPV/RTV with or without ribavirin at 400 mg / 100 mg twice daily is part of the recommended treatment for COVID-19 patients in China  [Another second-generation HIV-1 protease inhibitor darunavir demonstrated in vitro antiviral activity against SARS-CoV-2 [EC 300 μM] . Darunav 300 μM] . An RCT  300 μM] .50: 1.13 μM] at low micromolar concentration in Vero E6 cells [50 range reported for SARS-CoV-1 [EC50: 1–8.8 μm] and MERS-CoV [EC50: 3.0 μM] in Vero E6 and Huh7 cell lines, respectively  exhibited a 7.6-fold greater potency than chloroquine [EC50: 5.47 μM] in SARS-CoV-2-infected Vero cells [50: 6.5 ± 3.2 μM] exhibited approximately a 5-fold greater potency compared to hydroxychloroquine [EC50: 34 ± 5 μM] in SARS-CoV-1-infected Vero cells   . A non-r0–30 μM] . There a50: 9.99 μM] towards MERS-CoV replication in Vero E6 cells [50: 16.33 μg/mL] compared to Vero cells [EC50: 41.45 μg/mL] [50: 50 μg/mL] reported to be effective in Vero cells  . Similarro cells . Howeverro cells .th day of admission resulted in fatal outcome in all patients   [50: 2.12 μM] [Nitazoxanide [2-acetyloxy-N-(5-nitro-2-thiazolyl) benzamide] is an FDA-approved broad-spectrum anti-parasitic compound effective against intestinal protozoal and helminthic infections, specifically, m parvum . Recentlm parvum , rotavirm parvum , astrovim parvum , norovirm parvum , Japanesm parvum , rubellam parvum , Zika vim parvum , hepatitm parvum , and hepm parvum . Nitazox3 µg/mL] . Similarctively] . Effecti2.12 μM] . Current2.12 μM] .Clinical trials have successfully demonstrated its effectiveness in treating influenza virus, norovirus and rotavirus, hepatitis B virus, and hepatitis C virus. In a phase 2b/3 study for the outpatient management of influenza, a treatment dose of 600 mg nitazoxanide by mouth BID was associated with a 17·3hr reduction in time to alleviation of symptoms when compared with placebo . In a ph50 2.10 μM] than remdesivir and LPV [Homoharringtonine is a plant alkaloid known to exhibit potent anti-viral effects against herpesviruses , coronaviruses , rhabdoviruses (vesicular stomatitis virus or VSV and rabies virus), and other viruses  . Omaceta and LPV . AntivirEmetine is an alkaloid derived from ipecac and is FDA-approved for the treatment of ameobiasis. Emetine was demonstrated to have anti-viral effects towards a wide range of DNA and RNA viruses including bovine herpes virus-2, HSV-2, human cytomegalovirus, Buffalo Poxvirus, ZIKV, EBoV, HIV-1, Newcastle’s disease virus or NDV, pestes des petits ruminants virus, rift valley fever virus, influenza and rabies virus ,101. In Corticosteroids are used to mitigate the host inflammatory response which contributes to acute lung injury and acute respiratory distress syndrome (ARDS) in severe pneumonia cases. However, the benefit of these agents outweighs adverse effects that include delayed viral clearance and enhanced risk of secondary infection . Presentth and 7th day) from two severe patients [Tocilizumab is an FDA-approved humanized monoclonal antibody that inhibits IL-6 receptor in membrane-bound or soluble form. It was originally approved for the treatment of rheumatoid arthritis (RA), but has been used in patient therapy of cytokine-release syndrome (CRS) following chimeric antigen receptor T cell (CAR-T) therapy . IL-6 ispatients . In monopatients . Like toThe use of convalescent plasma (CP) from patients recovered from viral infections has been in practice for more than 100 years and has been used successfully for SARS-CoV-1, MERS-CoV, EboV, and H1N1 influenza . TheoretIn summary, SARS-CoV-2 is a highly transmissible CoV that led to the current pandemic and disruption of human activity. There is presently a limited understanding of SARS-CoV-2 pathogenesis, mostly extrapolated from SARS-CoV-1 and MERS-CoV studies. While these studies are useful, development of an animal model that faithfully recapitulates SARS-CoV-2 pathogenesis in humans will allow identification of viral and host factors critical for COVID-19. Furthermore, animal model allows researchers to carry out the immediate task of developing and testing antiviral interventions that will eventually control COVID-19 in humans. In the present summary, we detailed the current understanding of SARS-CoV-2, a result of incredible efforts of researchers worldwide.


C. elegans. We provide evidence that the transcription factor ZIP-2, a well-known immune response effector in C. elegans, is activated in response to age-associated mitochondrial dysfunction. ZIP-2 mitigates multiple aging phenotypes, including mitochondrial disintegration and reduced motility of the pharynx and intestine. Importantly, our data suggest that ZIP-2 is activated during aging independently of bacterial infection and of the transcription factors ATFS-1 and CEBP-2. Thus, ZIP-2 is a key component of an endogenous pathway that delays aging phenotypes in C. elegans. Our data suggest that aging coopted a compensatory strategy for regulation of aging process as a guarded process rather than a simple passive deterioration process.Physiological stresses, such as pathogen infection, are detected by “cellular Surveillance Activated Detoxification and Defenses” (cSADD) systems that trigger host defense responses. Aging is associated with physiological stress, including impaired mitochondrial function. Here, we investigated whether an endogenous cSADD pathway is activated during aging in  C. elegans maintains cellular homeostasis during physiological stress, such as pathogen infection or toxic drug treatment  IV, PD4251 ccIs4251 [(pSAK2) myo-3p::GFP::LacZ::NLS + (pSAK4) myo-3p::mitochondrial GFP + dpy-20(+)]; dyp-20(e1282), ERT20 jyEx6 [zip-2p::GFP + myo-2p::mCherry], PE255 feIs5 [sur-5p::luciferase::GFP + rol-6(su1006)], zip-2(ok3730); ccIs4251 [(pSAK2) myo-3p::GFP:: LacZ::NLS + (pSAK4) myo-3p::mitochondrial GFP + dpy-20(+)], zip-2(ok3730); agIs17 [myo-2p::mCherry + irg-1p::GFP] IV, ZC376.7(gk3094); agIs17 [myo-2p::mCherry + irg-1p::GFP] IV, ZC376.7(gk3094); jyEx6 [zip-2::GFP + myo-2p::mCherry], ZC376.7 (gk3094); agIs17 [myo-2p::mCherry + irg-1p::GFP] IV, daf-2(e1370); agIs17 [myo-2p::mCherry + irg-1p::GFP] IV, and daf-2(e1370); ccIs4251 [(pSAK2) myo-3p::GFP::LacZ::NLS + (pSAK4) myo-3p::mitochondrial GFP + dpy-20(+)], VC3201(atfs-1(gk3094)).All strains were maintained at 20°C. The following strains were used in this study. N2 wild-type, The high MV or low MV worms were divided and harvested as described in https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE99020.Microarray expression data that reported in this paper have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession number GSE99020. To identify genes that are differentially expressed between high MV and low MV groups, the microarray data were analyzed with following procedure. The log2 value of the measured probe intensities from each microarray dataset was normalized using quantile normalization method . Next, aAll Gene Ontology Biological Processes (GOBPs) or Cellular Processes (GOCCs) enrichment analysis were performed using DAVID online software . For eachttp://www.phage.dk/plugins/). The locomotion velocity data were imported into an Excel spreadsheet. The peak locomotion velocity observed in the 30 second period was used as the MV.MV measurement was performed as previously described . Single Morphological categories were defined as previously described . MitochoC. elegans RNAi feeding libraries generated by Ahringer laboratory . RNA interference Escherichia coli strains were cultured as previously described [For RNAi experiments, we used the commercial escribed .in vivo used PE255 strains as described previously [The amount of ATP production was measured eviously . FluoresTM Real-time C1000 Touch Thermal cycler (Bio-Rad) and analyzed using ΔΔCt methods described in the manufacturer’s manual. Sequences of primers used for quantitative RT-PCR analysis; zip-2-Forward: GTTCTTTCCACAGCTTGTGC, zip-2-Reverse: GATGACGAATCGGACGATAC, irg-1-Forward: GCTGAAATTCACTTGTAGTGAG, irg-1-Reverse: GAGACCATAATTTCAATTGCTC, irg-2-Forward: CACCTCATTATTGCATTGTTTC, irg-2-Reverse: GTTGTAGACTTTTGAAAGGTTG, act-3-Forward: AAGTCATCACCGTCGGAAAC, and act-3-Reverse: TTCCTGGGTACATGGTGGTT.Total RNA was extracted by using miRNeasy mini kit . cDNA was generated by using a reverse transcription system  and was used for quantitative PCR. Quantitative real time PCR was performed with SYBR green dye  using CFX96Worms fed Fluoresbrite® YG Carboxylate Microspheres 0.50 μm diameters with heat-killed bacteria for a 1 hour and then the accumulation of fluorescent bead in the intestine was observed using an Eclipse Ni (Nikon).The number of contractions in the terminal bulb of pharynx was counted for 10 seconds. The pharyngeal pumping was observed by a SMZ 745T microscope (Nikon) equipped with a 2X lens .The concentration of mitochondrial inhibitors used for testing was defined as described previously . MitochoSupplementary FiguresSupplementary Table 1Supplementary Tables 2, 3


Scientific Reports 10.1038/s41598-020-59697-7, published online 20 February 2020Correction to: The original version of this Article contains an error where two supplementary files containing raw data, which were included with the initial submission, were omitted. These files appear below.Supplementary Data set 1.Supplementary Data set 2.


Correction to: BMC Musculoskelet Disord 21, 449 (2020)https://doi.org/10.1186/s12891-020-03479-9Following publication of the original article , the autThe correct title is shown below.“Bilobalide protects against ischemia/reperfusion-induced oxidative stress and inflammatory responses via the MAPK/NF-κB pathways in rats”The original article  has been


There are no specific drug therapies or vaccines for the pandemic of coronavirus disease 2019 (COVID-19), which is associated with substantial mortality. Attenuating or reversing the cytokine storm is critical for treating patients with severe COVID-19 pneumonia. Mesenchymal stem cells (MSCs) have been shown to have powerful immunoregulation and reparative properties in injured tissue with good safety [6 cells per kilogram of weight) were suspended in 100 ml normal saline. We report numbers (percentages) for categorical variables and the median (interquartile range [IQR]) or mean ± standard deviation (SD) for continuous variables. Intergroup comparisons were performed with paired t tests.We wish to report our experience using UC-MSCs for the treatment of severe COVID-19 pneumonia at Taikangtongji Hospital in Wuhan, China, from January 3, 2020, to April 4, 2020. Patient data, including demographics, clinical data, laboratory indices, treatment, and in-hospital outcomes, were collected. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polymerase chain reaction (PCR) results of all patients were positive before UC-MSCs infused. Patients were diagnosed and treated according to national guidelines. Before the intravenous drip was established, UC-MSCs , decreased C-reactive protein level , decreased procalcitonin level , decreased interleukin-6 level , decreased D-dimer level , and elevated PaO2/FiO2  ; 25 patients (80.6%) were male. The proportion of treatment with oxygen was the highest (31 [100%]), followed by antivirals (26 [83.9%]), antibiotics (23 [74.2%]), intravenous immunoglobulin (8 [25.8%]), intravenous albumin (8 [25.8%]), and methylprednisolone (6 [19.4%]). The median (IQR) volume of infused UC-MSCs was 200 mL (100–300 mL). No adverse events were attributable to intravenous transplantation of UC-MSCs. After the first infusion of UC-MSCs, the SARS-CoV-2 PCR results of 30 patients (96.8%) became negative after a mean time of 10.7 days  (Table Our experience showed that UC-MSC therapy may restore oxygenation and downregulate cytokine storms in patients hospitalized with severe COVID-19 without any infusion reaction. This approach is a promising candidate for the treatment of severe COVID-19 . During


Serological tests were positive for HIV infection; theCD4 count was 40 cells/mm3. A CT-guided needle biopsy and India ink stainingdetected Cryptococcus neoformans characterized by multiple, round,thin-walled, encapsulated yeast cells  revealed aparavertebral soft tissue mass with hypodense foci, extending between the T3-T5vertebrae on the left side. The mass occupied the T3-T4 and T4-T5 neural foraminabilaterally, with spinal cord compression at these levels  B. Multist cells E.Cultur,,Vertebral involvement in cryptococcal infection is extremely rare. The clinical symptomsand radiological findings of skeletal cryptococcosis are nonspecific. Histopathology anddetection of fungi in the lesions confirm the diagnosis


Scientific Reports 10.1038/s41598-018-30640-1, published online 14 August 2018Correction to:In Figure 2C, the merge image of immunofluorescent stainingof NMuMG (E9) at 7 days is incorrect. The correct Figure 2 appears below as Figure Additionally, the accession number under the ‘Data Availability’ section was incorrectly provided:“The datasets generated and/or analyzed during the current study are deposited at Gene Expression Omnibus  EMT RNA-Seq data); GSE117474 (NMuMG (E9) MET RNA-Seq data); GSE1145722 (PyMT-1099 EMT and MET RNA-Seq data).”should read:“The datasets generated and/or analyzed during the current study are deposited at Gene Expression Omnibus  EMT RNA-Seq data); GSE117474 (NMuMG (E9) MET RNA-Seq data); GSE114572 (PyMT-1099 EMT and MET RNA-Seq data).”


Early assessment of atherosclerosis (leading cause of death in West and soon world) remains an elusive clinical goal, which if realized could lead to significant improvements in mortality and morbidity.Gadolinium (Gd)-containing immunomicelles targeting macrophages improved magnetic resonance (MR) detection of murine atherosclerosis. We sought to determine if immunomicelles targeting the macrophage scavenger receptor-B (CD36) improved ex-vivo MR detection and characterization of human aortic atherosclerosis.Gd-containing micelles, anti-CD36 immunomicelles and Fc-micelles were created. Macrophages were incubated with fluorescent micelles and immunomielles to determine uptake via confocal microscopy and inductively coupled plasma mass spectroscopy (ICP-MS) was performed to quantify Gd uptake. Human aortic specimens with moderate to severe atherosclerosis were harvested at autopsy. Using a 1.5 T Siemens clinical scanner, T1, T2, and PDW 3-dimensional scans were performed and post-contrast scans were repeated after 24 h incubation. T1 analysis and cluster analysis were performed comparing immunohistopathology with MR images. P-values < 0.05 were considered significant.Micelles had a mean diameter of 125 nm, average of 14,900 Gd-ions, and mean relaxivity was 37 mM-1 s-1 at 1.5 T and 37°C. Confocal microscopy and ICP-MS demonstrated significant in vitro uptake of immunomicelles by macrophages while non-targeted micelles had minimal uptake. On T1 imaging, immunomicelles increased CNR by 52.5%  while Fc-micelles increased CNR by 17.2%  and micelles increased CNR by 18.7% . Please see Figure Macrophage-specific (CD36) immunomicelles bind to human macropages in vitro and improved MR detection and characterization of human aortic atherosclerosis. Thus, immunomicelles could help identify high-risk human plaque.