Caenorhabditis elegans, ingestion of double stranded RNA causes strong, systemic knockdown of target genes. Further insight into gene function can be revealed by tissue-specific RNAi techniques. Currently available tissue-specific C. elegans strains rely on rescue of RNAi function in a desired tissue or cell in an otherwise RNAi deficient genetic background. We attempted to assess the contribution of specific tissues to polyunsaturated fatty acid (PUFA) synthesis using currently available tissue-specific RNAi strains. We discovered that rde-1 (ne219), a commonly used RNAi-resistant mutant strain, retains considerable RNAi capacity against RNAi directed at PUFA synthesis genes. By measuring changes in the fatty acid products of the desaturase enzymes that synthesize PUFAs, we found that the before mentioned strain, rde-1 (ne219) and the reported germline only RNAi strain, rrf-1 (pk1417) are not appropriate genetic backgrounds for tissue-specific RNAi experiments. However, the knockout mutant rde-1 (ne300) was strongly resistant to dsRNA induced RNAi, and thus is more appropriate for construction of a robust tissue-specific RNAi strains. Using newly constructed strains in the rde-1(null) background, we found considerable desaturase activity in intestinal, epidermal, and germline tissues, but not in muscle. The RNAi-specific strains reported in this study will be useful tools for C. elegans researchers studying a variety of biological processes.RNA interference is a powerful tool for dissecting gene function. In  Caenorhabditis elegans has been a powerful system for discovering the molecular mechanisms underlying the RNAi phenomena and its role in gene regulation (C. elegans (RNA interference (RNAi) is an evolutionarily ancient defense mechanism against viruses and transposable elements . gulation . The disC. elegans because of two membrane proteins called SID-1 and SID-2 that facilitate the uptake of double stranded RNA into cells  , which degrades the passenger strand of the siRNA and uses the remaining strand to target mRNA for use as a template for synthesis of secondary siRNAs , including RRF-1, and a large family of Argonaute proteins  to monitor the flux of fatty acid desaturation reactions from substrates to products as a quantitative approach to measuring the RNAi silencing capacity in previously characterized C. elegans mutant strains. Our quantitative method revealed that some C. elegans strains deemed “RNAi deficient” retained substantial RNAi silencing capacity when exposed to the feeding method of RNAi delivery, and therefore the available strains used for tissue-specific RNAi are not useful for determining tissue-specificity of gene function. We present here a collection of new strains for tissue-specific RNAi and report that intestine and epidermal tissues produce the highest amounts of desaturated fatty acids, while the germline also shows activity of omega-3 and delta-5 fatty acid desaturases.Our objective was to determine various tissue contributions to the overall degree of fatty acid desaturation in the nematode HT115 RNAi bacteria. RNAi constructs for fat-1, fat-4, and empty vector control were obtained from the Ahringer RNAi library and sequence verified (act-5 and dpy-7 experiments) until worms reached young adult stage.NGM growth media was supplemented with 100 μg/mL ampicillin, 2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and seeded with the appropriate N2, wild type; WM27, rde-1(ne219) V; NL2098, rrf-1(pk1417) I; NL3511, ppw-1(pk1425) I; VS26, rde-10(hj20) I; VS27, rde-11(hj37) IV; WM30, rde-3(ne298) I; WM119, sago-2(tm894) ppw-1(tm914) I; C06A1.4(tm887) F58G1.1(tm1019) II; H10D12.2(tm1144) IV; sago-1(tm1195) V; neIs10 X; WM49, rde-4(ne301) III, HC196, sid-1(qt9) V; WM118, rde-1(ne300) V, neIs9 [myo-3::HA::RDE-1 + rol-6(su1006)]. The WM45, rde-1(ne300) V strain was provided by Craig Mello and the DCL569 strain was provided by Di Chen : frSi17 and frSi21 are single copy insertions on chromosome II (ttTi5605 location) of pNP160 (mtl-2p::RDE-1_3′rde-1ttTi5605] and pSO21(col-62p::RDE-1_3′rde-1ttTi5605) respectively by CRISPR using a self-excising cassette (SEC)  (rde-1(ne300) at 10 ng/µl together with pDD122 (eft-3p::Cas9) at 40 ng/µl, pCFJ90 (myo-2p::mCherry) at 2.5 ng/µl, pCFJ104 (myo-3p::mCherry) at 5 ng/µl, and #46168 (eef-1A.1p::CAS9-SV40_NLS::3′tbb-2 (rde-1(ne300) V; frSi17[pNP160 V; frSi21[pSO21 and confirmed by PCR or sequencing.The transgenes To measure fatty acid composition, approximately 1000 young adult stage worms (containing 0-8 embryos) were washed from feeding plates with water on ice and washed once to remove residual bacteria. After settling on ice again, as much water as possible was removed (∼90%). Fatty acids were converted to methyl esters for analysis as previously described (nlp-29p::gfp reporter was quantified with the COPAS Biosort (Union Biometrica). The ratio of GFP/size was then calculated to normalize the fluorescence . The results shown are representative of at least 3 independent experiments.Size was measured as time of flight = TOF as arbitrary units and expression of the t-tests, which were corrected for multiple comparisons using the Holm-Sidak method with an alpha value of 0.05.At least 3 biological replicates were used for analysis. Within each biological experiment, samples were collected in triplicate. P values shown in Table S1 were determined by comparing the control empty vector to the RNAi knockdown using Desaturation index for each strain was determined by comparing the relative peak areas of individual fatty acid methyl esters using the following formulas.DI = (20:4n-3 +20:5n-3)/(20:3n-6 + 20:4n-6)DI = (20:4n-6+20:5n-3)/(20:3n-6 + 20:4n-3)https://doi.org/10.25387/g3.12968096.Worm strains, data and reagents are available upon request. Accompanying the manuscript is one supplemental table, Table S1. Supplemental material available at figshare: C. elegans obtains fatty acids from its bacterial diet, and also has the capacity to generate fatty acids de novo from acetyl-CoA , whereas the FAT-4 ∆5 desaturase adds double bonds between the fifth and sixth carbon counting from the carboxyl end of the fatty acid. FAT-1 . In fat-1 or fat-4 null mutants, the corresponding desaturation indices drop to zero because there are no omega-3 or delta-5 unsaturated PUFAs formed in the mutant strains , as well as transgenic strains in which the rde-1 gene was expressed under control of tissue-specific promotors in the rde-1(ne219) mutant background (rde-1 mutant strain. We found that the rde-1(ne219) strain, which contains a single glutamate to lysine substitution at a conserved residue, retained considerable RNAi silencing activity. The FAT-1 desaturation index fell from 6.0 to 2.6 during fat-1 RNAi feeding treatment, while the FAT-4 desaturation index fell from 2.5 to 0.8 during fat-4 RNAi treatment (rde-1(ne219) mutant strain.We sought to determine the extent of fatty acid desaturation in various tissues by using the RNAi-deficient strain reatment , Table 1rde-1 mutation, rde-1(ne300), which is predicted to contain a premature stop codon within the protein’s PIWI domain (rde-1(ne219), we found the rde-1(ne300) strain to be strongly RNAi deficient. Treatment with fat-1 or fat-4 RNAi did not significantly change desaturation index compared to treatment with empty vector control. Previous reports concluded that rde-1 (ne219) was highly resistant to RNAi against several genes when using lethality and fluorescence as indicators  mutation retained RNAi activity capable of knocking down fat-1 and fat-4 nearly to the same extent as wild type (rrf-1 (pk1417) maintained RNAi capacity in the intestine and in the hypodermis  mutants because this strain is not truly deficient in RNAi activity in intestinal and hypodermal tissues, supporting the findings of Kumsta and Hansen  strain and the rrf-1 strain were not RNAi-deficient as described led us to use our biochemical GC-MS method to quantitatively assess the degree of RNAi deficiency in other strains reported to be RNAi deficient. We performed the RNAi and GC-MS analysis of various RNAi-deficient mutants using empty vector, fat-1, and fat-4 RNAi and calculated the desaturation indices. We found that sid-1 mutants were resistant to RNAi of fat-1 and fat-4 induced by the feeding method, as expected  strain, which carries mutations in several Argonaute proteins, including SAGO-1 and SAGO-2 that are known to interact with secondary siRNA (fat-1 and fat-4 RNAi. Neither rde-10 (hj20), nor rde-11 (hj37) were significantly RNAi resistant in our study (C. elegans mutant strains.Our findings that the expected . The RDEur study . The ranrde-1(null) background. For intestine-specific RNAi, we used the mtl-2 promotor to rescue rde-1 in the rde-1(ne300) strain  strain  background. The germline-specific strain DCL569 uses a single-copy insertion of a transgene expressing rde-1 under control of the sun-1 promoter , and the tissue-specific RNAi strains and determined the desaturase indices using GC-MS. We found that RNAi activity in intestinal, epidermal and germline tissues partially reduces the desaturation index compared to RNAi performed in rde-1(null) animals, while muscle-specific RNAi showed no detectable change in the desaturation index compared to the rde-1(null) control , rde-1 (ne300), ne298)rde-3 , sid-1 (qt9), and the multiple secondary Argonaute mutant strain (MAGO) were resistant to RNAi. And of those, only the null mutant rde-1 (ne300) and sid-1(qt9) were fully resistant to fat-1 and fat-4 RNAi.This study used tissue-specific RNAi paired with a highly quantitative method to determine the tissues in d mutant . This alrde-1. The rde-1 has been reported to be essential to the success of exogenous RNAi. The primary evidence being that the rde-1 (ne219) allele was completely resistant to pos-1 RNAi, which normally causes lethality  is, in contrast, completely resistant to RNAi against fatty acid desaturase genes, which suggests that RDE-1 function is indispensable. The ne219 allele was used as the RNAi deficient genetic background for tissue specific rescue of RNAi function (rde-1(ne219) background, because this strain is not completely RNAi deficient.We analyzed two mutant alleles of the Argonaute encoding gene, fat-1 and fat-4 genes are expressed in multiple somatic tissues, including epidermis and intestine , to nearly wild-type levels. The rrf-1 (pk1417) contains a large deletion that eliminates a large conserved region of the protein  mutant background that rescue sid-1 using neuronal, muscle, and intestine-specific promoters (rde-1(null) background. With these strains we were able to determine the extent of omega-3 and ∆5 desaturation in various tissues in C. elegans. The lack of RNAi activity in the rde-1(ne300) treated with fat-1RNAi and fat-4RNAi indicates that the rde-1(null) background is appropriate for tissue-specific RNAi experiments.Several tissue-specific RNAi strains have been constructed by others that do not rely on the tissue-specific rescue of fat-1 and fat-4 transcripts in epidermal and germ cells  and rrf-1 strains. Similarly, researchers should also be aware that the tissue-specific promotors may also exhibit a low level of expression in other tissues, which may explain the slight leakiness in the DCL569 strain. Expression in unexpected tissues was observed with several neuronal promotors expressed from multicopy arrays (C. elegans (Our findings emphasize that caution must be used in interpreting previous tissue-specific RNAi studies using the  elegans .


Mycobacterium tuberculosis-infected zebrafish and human macrophages, suggesting its utility in tuberculosis therapy.Tumor necrosis factor (TNF) is a critical host resistance factor against tuberculosis. However, excess TNF produces susceptibility by increasing mitochondrial reactive oxygen species (mROS), which initiate a signaling cascade to cause pathogenic necrosis of mycobacterium-infected macrophages. Here, using the zebrafish, we identify the mechanism of TNF-induced mROS in tuberculosis. Excess TNF in mycobacterium-infected macrophages elevates mROS production by reverse electron transport (RET) through complex I. TNF-activated cellular glutamine uptake increases the Krebs cycle intermediate succinate. Oxidation of this elevated succinate by complex II drives RET, thereby generating the mROS superoxide at complex I. The complex I inhibitor, metformin, a widely used anti-diabetic drug, prevents TNF-induced mROS and necrosis of  Mycobacterium marinum (Mm) have revealed the mechanisms behind this dual effect  is both a host resistance and susceptibility factor in tuberculosis (TB) –3. Findil effect –6. Althol effect , 7–9. Thl effect , 9. LTA4rm of TB . In cohorm of TB . These frm of TB . Moreoverm of TB .To gain mechanistic understanding of TNF-mediated pathogenic macrophage necrosis, we returned to the zebrafish larva. We found that excess TNF, acting through the kinase RIP3 and one of its substrates, PGAM5, increases mitochondrial reactive oxygen species (mROS) such as superoxide and hydrogen peroxide in infected macrophages  5, 6). . 6). Thehi animals, mROS in infected macrophages were further increased to 3.6-6.6-fold over uninfected macrophages  bacterial determinant is needed (hi mROS originated from superoxide production by the electron transport chain (ETC), we asked if it was inhibited by compounds that disrupt mitochondrial electron transport. mROS were inhibited by four compounds that disrupt mitochondrial electron transport through distinct mechanisms . TNF. TNF4). rophages . TNF didrophages . Moreoves needed . To confchanisms . Thus, T2•−) through single electron donation to O2 (2•− production at complex I is generated by two distinct mechanisms (2•− (2•− which dismutates to H2O2) , generating in the process a small amount of the mROS superoxide . Th. Th2•−) to H2O2) . These tto H2O2)  12, 14), 142•−) to H2O2) . By cont animals . Two oth in TNFhi larvae through injection of its mRNA. AOXs, which are present in plants, fungi, and some invertebrates but are absent in vertebrates, catalyze the transfer of electrons from the CoQH2 pool directly to O2, bypassing Complexes III and IV . AO. AOhi mRI and IV  (15). Thmplex IV  15, 16)hi mROS, CoQ pool .Mycobacterium tuberculosis (Mtb), the agent of human TB, using a leucine and pantothenic acid Mtb auxotroph that can be used in containment level 2 facilities , 1713, 1trations —increasetrations . Diethyltrations —performetrations . Thus, ihi macrophages without affecting mROS in wild-type macrophages (hi mROS. Two different inhibitors of glutaminase 1 (GLS1), BPTES and CB-839 (telaglenastat), performed as expected, inhibiting mROS in TNFhi but not wild-type macrophages  removed mROS in both wild-type and TNFhi animals , as previously proposed  increased succinate levels over baseline . Moreoveproposed , 21. Accproposed . Finallyproposed  and for proposed  5, 6). , 6. hi sWe dissected the interactions between TNF, mROS, and mycobacteria and what roles they play at distinct steps of the pathway. We had shown that both TNF and mycobacteria are required to increase mitochondrial succinate, which is required to induce mROS. Because exogenous succinate could induce mROS in wild-type animals in both infected and uninfected macrophages , we concWe have previously shown that the mROS are required for macrophage necrosis . We now To determine if mycobacteria were required for necrosis downstream of mROS induction, we examined if diethyl succinate and DEBM could also kill uninfected macrophages by enumerating macrophages in infected and uninfected animals . DiethylSimilar results were observed in human macrophages derived from the monocytic cell line THP-1. We had previously shown that TNF induces necrosis in Mtb-infected THP-1 cells through the same interorganellar pathway downstream of mROS as in Mm-infected zebrafish  6). We . We 6). We had previously shown that blocking mROS using scavengers such as N-acetyl cysteine inhibited TNF-induced macrophage necrosis and restored resistance . Four ofThough long thought to be an in vitro artifact, moderate levels of RET and resultant increases in mROS have important homeostatic roles in cell differentiation and oxygen sensing . HoweverWe also considered our findings in the light of work using cultured macrophages, which has shown that succinate is responsible for generating proinflammatory responses to lipopolysaccharide (LPS), a key virulence determinant of Gram-negative bacteria . LPS cauLTA4H genotype, and, given the association of TNF with necrotic lung granulomas  (with yellow fluorescent macrophages) (ZFIN ID: ZDB-FISH-150901-6828)  (ZFIN ID: ZDB-GENO-960809-7) and the transgenic line 01-6828) . Sample 26206 ΔleuD ΔpanCD  expressing tdTomato, mWasabi, or EBFP2 were grown as previously described (Mm M strain (ATCC #BAA-535) and Mtb H37Rv strain, mcescribed , 61. Forhi animals were created by injecting recombinant zebrafish soluble TNF (2-Xros injection. TTFA (thenoyltrifluoroacetone) (1 μM) (Cambridge Bioscience), atpenin A5 (2.5 nM) (Insight Biotechnology), diethyl succinate (500 nM) (reagent plus 99% Sigma), and DEBM  (1 μM) (Sigma) were administered 2 hours before MitoTracker Red CM-H2-Xros injection. DM-Glutamate (dimethyl glutamate) (60 μM) (Cambridge Bioscience) was administered 3 hours before MitoTracker Red CM-H2-Xros injection. DNP (2.4-dinitrophenol) (100 nM) (Agilent Technologies) was administered 3.5 hours before MitoTracker Red CM-H2-Xros injection. Rotenone (6.25 nM) (Sigma), piericidin A (50 nM) (Stratech Scientific), strobilurin B (100nM) (Insight Biotechnology), metformin (20 μM) , phenformin (20 μM) (Sigma), nigericin (5 μM) (Sigma), diazoxide (50 nM) (Cambridge Bioscience), UK5099 (10 μM) (Cambridge Bioscience), and M-pyruvate (methyl pyruvate) (50 nM) (Fisher Scientific) were administered 4 hours before MitoTracker Red CM-H2-Xros injection. DM-malonate  (10 μM) (Sigma), perhexiline (10 μM) (Stratech Scientific), 4-BrCA (4-bromocrotonic acid) (10 μM) (Insight Biotechnology), GPNA (10 μM) (Cambridge Bioscience), BPTES (5 μM) (Cambridge Bioscience), telaglenastat (5 μM) (Cambridge Bioscience), and R-162 (1 μM) (Cambridge Bioscience) were administered 5 hours before MitoTracker Red CM-H2-Xros injection. In experiments to assess cording, perhexiline was removed 5 hours after TNF administration, diethyl succinate and DEBM were administered for 10 hours and then removed, and metformin, phenformin, DM-malonate, diazoxide, and telaglenastat were added 1 dpi and removed 2 dpi. After drug removal, the larvae were maintained in fresh E2/2 medium for the rest of the experiment. In experiments to assess macrophage numbers, diethyl succinate and DEBM were administered 1 dpi for 24 hours until macrophage number was assessed 2 dpi. For experiments quantifying mitochondrial ROS production, drugs were added before MitoTracker Red CM-H2-Xros injection as indicated above and maintained during imaging.TNFuble TNF  as previuble TNF . To asseuble TNF . FCCP . The T7 promoter (5′-TAATACGACTCACTATAGG-3′) followed by the zebrafish Kozak sequence 5′-GCCGCCACC-3′ were inserted before the start codon by PCR. mRNA was synthesized using the mMessage mMachine kit (Ambion) and the polyA Tailing kit (Ambion). Approximately 2-4 nl of injection solution  from RIP3 e2/i2-splice-blocking (5′-TTTTAGAAATCACCTTGGCATCCAG-3′) and PGAM5-translation-blocking morpholino (5′-AGCGCCCTCCGAAAAGACATGCTTC-3′) (Gene Tools) were diluted to 0.15 mM in injection solution . ApproxiAOX-expressing 2 dpf larvae were treated with different concentrations of KCN for an hour. Heart rate (beats per minute) was assessed as a readout of cyanide poisoning of complex IV of the electron transport chain  in absenFluorescence microscopy was performed as described . Mycobac2-Xros, a cell-permeable fluorogenic probe for ROS which is targeted to the mitochondrion and produces red fluorescence upon oxidation by diverse ROS (Fisher Scientific) (w200Tg(mpeg1:YFP) larvae were infected 2 dpf. For all experiments where TNFhi animals are used, larvae were microinjected 1 dpi via CV with phosphate buffered saline (PBS) containing TNF and 50 mM MitoTracker Red CM-H2-Xros or PBS containing vehicle for TNF and MitoTracker Red CM-H2-Xros , larvae were prepared for confocal imaging and maintained at 28.5°C within a heated incubation chamber attached to the confocal microscope. Images of the CHT of each larva were taken starting 30-60 min after MitoTracker Red CM-H2-Xros administration. Mitochondrial ROS production was quantified using maximum projection images as MitoTracker Red CM-H2-Xros maximum fluorescence intensity per macrophage using NIS-Elements. When not otherwise stated in the figure legend, the mean of maximum MitoTracker Red CM-H2-Xros fluorescence was quantified only in Mm- or Mtb- infected macrophages.Mitochondrial ROS production was assayed by fluorescence intensity of MitoTracker Red CM-Hentific) , 6. Tg was added to culture medium 30 min before image acquisition. Macrophages were imaged using a Nikon Ti-E inverted microscope fitted with a 20X objective  and 2-5 arbitrary images per well acquired with NIS Elements (Nikon). Cell necrosis was quantified using a previously described method (THP-1 cells (ATCC TIB-202) were differentiated into macrophages and infected with single-cell suspensions of mCherry- or tdTomato-expressing Mtb mcP-values were defined as follows: Not significant, P>0.05; *P<0.05; ** P<0.01; ***P<0.001; and ****P<0.0001. The statistical tests used for each figure can be found in the corresponding figure legend. Where the n value is given and not represented graphically in the figure, n represents the number of zebrafish used for each experimental group.The following statistical analyses were performed using Prism 7 (GraphPad): two-way ANOVA or one-way ANOVA with Dunn’s or Tukey’s post-test and Fisher’s exact test. Error bars represent the standard error of mean. Post-test https://fiji.sc/) for image analysis of macrophage death, GraphPad Prism 7.0  for data graphing and statistical analyses, and CorelDRAW (CorelDRAW Graphics Suite x5) for figure preparation.The following software was used: NIS-Elements for image acquisition in wide-field and confocal microscopy, ImageJ (Supplementary Materials


Caenorhabditis elegans, the IL2 neurons arborize to cover the anterior body wall. In contrast, the FLP neurons arborize to cover an identical receptive field during reproductive development. Using time-course imaging, we show that branching between these two neuron types is highly coordinated. Furthermore, we find that the IL2 and FLP arbors have a similar dendritic architecture and use an identical downstream effector complex to control branching; however, regulation of this complex differs between stress-induced IL2 branching and FLP branching during reproductive development. We demonstrate that the unfolded protein response (UPR) sensor IRE-1, required for localization of the complex in FLP branching, is dispensable for IL2 branching at standard cultivation temperatures. Exposure of ire-1 mutants to elevated temperatures results in defective IL2 branching, thereby demonstrating a previously unknown genotype by environment interaction within the UPR. We find that the FOXO homolog, DAF-16, is required cell-autonomously to control arborization during stress-induced arborization. Likewise, several aspects of the dauer formation pathway are necessary for the neuron to remodel, including the phosphatase PTEN/DAF-18 and Cytochrome P450/DAF-9. Finally, we find that the TOR associated protein, RAPTOR/DAF-15 regulates mutually exclusive branching of the IL2 and FLP dendrites. DAF-15 promotes IL2 branching during dauer and inhibits precocious FLP growth. Together, our results shed light on molecular processes that regulate stress-mediated remodeling of dendrites across neuron classes.Stress-induced changes to the dendritic architecture of neurons have been demonstrated in numerous mammalian and invertebrate systems. Remodeling of dendrites varies tremendously among neuron types. During the stress-induced dauer stage of  C. elegans has a defined and relatively simple nervous system amenable for study. The head of C. elegans contains two classes of neurons with highly arborized dendritic arbors. The FLP neurons form dendritic arbors as part of healthy reproductive development. In contrast, the IL2 neurons undergo dendritic arborization only during a stress-induced developmental stage. When fully arborized, FLP and IL2 dendrites occupy the same area, although during distinct developmental stages. Using forward and reverse genetics, we find that branching in both neuron types is controlled by a similar protein complex, but that regulation of this complex differs between cell types. Our data demonstrate how molecular cues drive the transition between stressed and unstressed states in the nervous system.Neurons have processes called dendrites that receive information. Dendrites are often elaborately shaped and highly branched. Adverse stress in the mammalian brain can reduce dendritic branching in some neuron types, while increasing it in others. How stress can cause opposite branching responses in different neurons is unclear. The transparent nematode  Dendrite morphology is crucial for efficient neural signaling. Recent years have seen increased attention given to the molecular basis of dendritic arborization using both cell-culture studies and model organisms. Experience-driven remodeling of a dendrite can contribute to both the size and complexity of dendritic arbors in a diverse range of species. Depending on neuron class, these changes can include both increasing and decreasing dendritic volume –3. For eaenorhabditis elegans is one of several leading models for studying dendritic arborization  (3/3); [dpy-7p::sax-7] (2/2); [dpy-7p::mnr-1] (3/3); [klp-6p::daf-16] (1/1). Successful rescue was determined by a quantifiable change in body wall branch numbers, with ANOVA followed by Tukey’s multiple comparison. For DMA-1 overexpression, we crossed a [klp-6p::dma-1] rescuing array into the WT background. TV8545 wyEx3282[dma-1p::dma-1::gfp::SL2:: mcherry(fosmid)]; pha-1(e2123 ts) was used for all [dma-1::gfp] localization analyses. A complete list of plasmids, and assembly primers can be found in Some plasmids were generously donated from other labs . Plasmiddma-1(cbc1) was generated using a standard EMS mutagenesis protocol [daf-7(e1372) myIs13[klp-6p::gfp] daf-c background. Newly generated mutant strains were backcrossed to wild type at least twice before usage and assigned to a linkage group based on two-factor mapping with EG1020 bli-6(sc16) IV; dpy-11(e224) V; lon-2(e678) X and EG1000 dpy-5(e61)I; rol-6(e187)II; lon-1(e1820)III. Complementation testing for dma-1(cbc1) was performed on dauer pheromone-containing media with dma-1(tm5159)I. The dma-1(cbc1) lesion was identified with Sanger Sequencing.protocol  in a dafwormweb.org. Functional domains for dma-1 were determined using LRRfinder.com.Gene schematics were constructed using muIs32[mec-7p::gfp] to visualize FLP cell morphology. To generate synchronous cultures, eggs were collected from gravid hermaphrodites by bleaching with 5% bleach and 5N NaOH in M9 buffer  one-day-old adults were transferred from 15°C to 25°C. Twenty animals were examined for IL2 morphology every 24 hours for three consecutive days.S1 Figdma-1(tm5159)/dma-1(cbc1) heterozygotes show a reduction of branches. (A’) Schematic drawings traced from the accompanying micrographs indicate the IL2 dendrites that are visible in the images. Dorsal midline branches (green) and body wall branches (red). (B) Quantification of IL2-specific DMA-1 rescue. Replicate #2, WT (n = 20), dma-1(cbc1) (n = 18), and dma-1; klp-6p::dma-1 (n = 20). Replicate #3, WT (n = 22), dma-1(cbc1) (n = 21), and dma-1; klp-6p::dma-1 (n = 23). (C) Expression of sax-7 in the epidermis using the dpy-7 promoter rescues the sax-7 mutant branching phenotype. WT (n = 23), sax-7 (n = 19), and sax-7; dpy-7p::sax-7 (n = 19). (D) Expression of mnr-1 in the epidermis using the dpy-7 promoter rescues the mnr-1 mutant branching phenotype. Replicate #2, WT (n = 19), mnr-1 (n = 21), and mnr-1; dpy-7p::mnr-1 (n = 18). Replicate #3, WT (n = 19), mnr-1 (n = 22), and mnr-1; dpy-7p::mnr-1 (n = 20). Data was analyzed with ANOVA followed by Tukey’s test for multiple comparisons to determine significance (B-D). ***p ≤0.001 and ****p ≤0.0001. Error bars are the standard error of the mean. (E) Overexpression of DMA-1 in the IL2s using the klp-6 promoter does not induce ectopic branching during adult; WT (n = 10) and klp-6p::dma-1 (n = 17). Representative, epifluorescent images of unbranched adult IL2 dendrites, wild type (top) and DMA-1 overexpression (bottom). IL2 neurons are visualized by klp-6p::gfp (A and E). Scale bars, 10 μm.(A) (TIF)Click here for additional data file.S2 Figklp-6p::gfp. (B) Epifluorescent micrographs of adults showing body wall branches. Adults grown on replete food (top) and adults following L1 arrest (bottom). We observed no noticeable differences in branching regardless of the L1 arrest. FLP arbors (red arrows) and ALM neurite (yellow arrow). FLP neurons are labeled with mec-7p::gfp. Scale bars, 10μm.(A) Epifluorescent micrographs of dauers showing the IL2 body wall branches. Dauers isolated from pheromone plates not having gone through L1 arrest (top) and dauers that arrested at L1 and then grew to dauer in the presence of pheromone (bottom). We observed no noticeable differences in branching regardless of the L1 arrest. IL2 neurons are labeled with (TIF)Click here for additional data file.S3 Figrab-10 (n = 16) does not affect the number of IL2 body wall branches. The exoc-8 mutant (n = 21) has a slight increase in the number of body wall branches compared to wild-type (n = 17). (B) Quantification of IL2 arborization in rab-10 paralogs. Mutations in rab-8 (n = 19) and rab-35 (n = 21) do not affect IL2 branch number compared to wild-type (n = 22). (C) Quantification of IL2 arborization in unfolded protein response mutants. xbp-1 (n = 19) has a slight increase in branch number, while skr-5 (n = 21), F47F2.1 (n = 21), and tps-1 (n = 21) do not differ from wild-type in the number of body wall branches (n = 18). (D) Localization of dma-1p::dma-1::gfp in the rab-10 and ire-1 mutants. In the wild type (left), rab-10 mutant (middle), and ire-1 mutant dauers (right), dma-1p::dma-1::gfp is localized to the IL2 dendrite (red arrows). For statistical comparisons between more than two groups (A and C), we used ANOVA followed by Tukey’s test for multiple comparisons to determine significance. For statistical comparisons between two groups (B and C) we used t-tests to determine statistical significance. “ns” p >0.05, *p ≤0.05, **p ≤0.01. Error bars are the standard error of the mean. Scale bars, 10μm.(A) Mutation in (TIF)Click here for additional data file.S4 Figdaf-16(m27); daf-7(e1372) adult FLP neurons shows a fully arborized FLP dendrite. (A’) Schematic of daf-16(m27); daf-7(e1372) FLP dendrites. (B) Z-projection confocal micrograph lateral view of a daf-18(e1375); daf-7(e1372) adult with extensive FLP arbor. (B’) Schematic of daf-18(e1375); daf-7(e1372) FLP dendrites. (C) Z-projection confocal micrograph of FLP neurons in daf-9(m540) mutant adults. (C’) Schematic of daf-9(m540) FLP dendrites. FLP neurons are visualized by mec-7p::gfp. FLP cell body (red asterisks), ALM/AVM neuronal processes (yellow arrows). Scale bars, 10 μm.(A) Z-projection confocal micrograph of (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


The genome harbors a Panton-Valentine leukocidin phage, a type V staphylococcal cassette chromosome mec element, the delta-hemolysin-converting Newman phage ΦNM3, and the pathogenicity island SaPI3, encoding the superantigen enterotoxin B.We report a  de novo-assembled draft genome sequence of the Indian Staphylococcus aureus sequence type 88 (ST88) strain LVP-7, isolated from an ocular infection. The genome harbors a Panton-Valentine leukocidin phage, a type V staphylococcal cassette chromosome mec element, the delta-hemolysin-converting Newman phage ΦNM3, and the pathogenicity island SaPI3, encoding the superantigen enterotoxin B.We report a  Staphylococcus aureus is among the most common pathogens causing ocular infection. Previous reports suggest that both hospital-associated (HA) and community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) can cause eye infections (–Staphylococcus aureus (MSSA) strains belong to distinct clonal lineages carrying either type IV or V staphylococcal cassette chromosome mec (SCCmec) elements  extraction, LVP-7 glycerol stock stored at −80°C was streaked onto chromogenic agar medium . A single colony was picked and grown overnight in brain heart infusion (BHI) broth under aerobic conditions. gDNA was prepared using the phenol-chloroform method . Sequenc v3.14.1 . Gap fil v3.14.1 , with theference . The reseference , and dowA finder –21. The  finder – was used finder –, 24. Pro finder –, 26. Defspa (Staphylococcus aureus protein A) type t2526 and carries an SCCmec type V (5C2) cassette. This ST88 strain encodes Panton-Valentine leukocidin (PVL) phage Φ2958PVL, gamma-hemolysin components (hlgABC), and several super antigens such as sea, sep, sek, and selX (hlb)-converting S. aureus Newman phage (ΦNM3) was also identified, as depicted in seb enterotoxin, which may contribute to systemic S. aureus infection  and are available under the accession number PRJNA679674.This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number


Mice with treatment-induced tumor eradication developed immunological memory, enabling tumor rejection upon challenge and cancer-recurrence-free survival. The depletion of CD4 or CD8 abrogated the antitumor activity of combined therapy. PD-1 blockade or CCL21-DC tumor lysate vaccine monotherapy reduced tumor burden without tumor eradication. (4) Conclusion: Immune checkpoint blockade promotes the activity of the therapeutic cancer vaccine. PD-1 blockade plus CCL21-DC tumor lysate vaccine therapy could benefit lung cancer patients.(1) Background: Targeting inhibitory immune checkpoint molecules has highlighted the need to find approaches enabling the activation of immune responses against cancer. Therapeutic vaccination, which induces specific immune responses against tumor antigens (Ags), is an attractive option. (2) Methods: Utilizing a K-RasG12Dp53null murine lung cancer model we determined tumor burden, tumor-infiltrating T cell (TIL) cytolysis, immunohistochemistry, flow cytometry, and CD4 and CD8 depletion to evaluate the efficacy of PD-1 blockade combined with CCL21-DC tumor lysate vaccine. (3) Results: Anti-PD-1 plus CCL21-DC tumor lysate vaccine administered to mice bearing established tumors (150 mm The goal of this study is to determine the mechanisms of the PD-1 blockade and the therapeutic cancer vaccine in the modulation of immune activity in K-RasG12Dp53null lung adenocarcinoma, which is common in smokers and non-smokers. We hypothesize that the CCL21-DC tumor Ag vaccine mediated specific systemic immunity will benefit patients who have limited CD8 T cell infiltration of their tumors and or limited expression of tumor PD-L1. We report that PD-1 immune checkpoint blockade combined with CCL21-DC tumor Ag vaccine eradicates tumors and has the potential to augment therapy in lung cancer patients who have low baseline tumor T cell infiltration and do not respond to PD-1 therapy.Lung cancer is the leading cause of cancer death among men and women in the United States and worldwide; more than 1.9 million people died from lung cancer in 2017 . As receThe murine K-RasG12Dp53null lung adenocarcinoma was isolated from lung tumors of the K-RasG12Dp53null transgenic mice obtained from J. Kurie . The culture medium (CM) was RPMI 1640 supplemented with 10% fetal bovine serum , 100 units/mL penicillin, 0.1 mg/mL streptomycin, and 2 mM glutamine . Fluorescein isothiocyanate-, phycoerythrin-, allophycocyanin-, PerCP-, or APC-Cy7-conjugated anti-mouse Abs to CD4 (RM4-5) and CD8a (53-6.7) were from BD Biosciences or eBiosciences. For in vivo experiments, anti-PD-1 (BE0146), anti-CD4 (L3T4), and anti-CD8 (YTS169.4) were from BioXCell. Isotype control antibody (Ab) was from Sigma. The Abs used for T cell depletion were different for monitoring T cell levels. Bradford protein quantification dye was obtained from Sigma. Tissue digestion buffer was obtained from Miltenyi and used in Miltenyi tissue homogenizer according to the manufacturer’s instructions. T cell purification columns were from R&D.2 in air in CM. K-RasG12Dp53null and DCs were mycoplasma and murine viral pathogen free. The K-RasG12Dp53null cell line was used up to the 10th passage. DCs were cultured for 7 days in GM-CSF (20 ng/mL) and IL-4 (4 ng/mL) containing CM. The DCs were plated in flasks coated with 2% gelatin (Sigma). Non-adherent DCs were harvested on day 7 for experiments. The cultured DCs had increased levels (70–90%) of MHC class I and MHC class II expression, as evaluated by staining/flow cytometry (data not shown).K-RasG12Dp53null lung cancer cells and bone-marrow-derived DCs from femurs of syngeneic 129-E mice were routinely cultured in Corning T75 tissue culture flask in humidified atmosphere containing 5% COg for 2 h. CCL21-transduced DCs produced 10–16 ng CCL21/106 cells/24 h, as evaluated by CCL21-specific ELISA. The transduced DCs were pulsed with K-RasG12Dp53null tumor lysates prepared by digesting K-RasG12Dp53null tumors (day 15–20) with a Miltenyi Tissue dissociation kit in the gentleMACS Octo Dissociator at 37 °C for 40 min. Following digestion, tumor cells were heated for 5 h at 42 °C and recovered at 37 °C for 24 h in CM containing 10% mouse serum (MS). The cells were washed three times with PBS and suspended in RPMI followed by disruption by five freeze (liquid nitrogen) and thaw (37 °C) cycles. After freeze–thaw the lysates were passed through a 28-gauge needle 10 times. Tumor lysates were filtered through a 0.2 µm syringe filter. The protein content was determined by Bradford assay, and aliquots  were stored at −80 °C. Lysates were tested for bacterial endotoxin with the Limulus Amoebocyte lysate assay  according to instructions, and preparations routinely contained less than 0.01 EU/µg protein. DCs (50 × 106) were pulsed with tumor lysates (200 µg/mL) for 24 h in RPMI. Then, a 5 × 106 CCL21-DC tumor Ag vaccine/injection was utilized. The cells were washed three times with PBS and reconstituted in sterile PBS for injection.DC were transduced with a replication-deficient adenovirus expressing murine CCL21 or control virus without CCL21 insert at a MOI of 100:1 and centrifugation at 2000× 2, where a = large diameter and b = small diameter. K-RasG12Dp53null tumor cells (106) were injected on right supra scapular region of 129-E mice. The mice with 150 mm3 tumors, were injected with CCL21-DC lysate Ag pulsed vaccine (5 × 106) once per week for 3 weeks by s.c. administration on the left contra-lateral flank of the tumor. Mice were injected i.p. with anti-PD-1 (200 µg/dose) or isotype IgG2b Ab (200 µg/dose) every 48 h for 3 weeks. The anti-PD-1 was non-depleting neutralizing Ab. To determine the induction of immunological memory, mice that eradicated tumors in response to therapy were challenged by s.c. injection on the left flank with 106 K-RasG12Dp53null tumor cells. One week following the 2nd week of therapy, tumors were harvested to conduct the experiments described below. All animal work was conducted in accord with the Veterans Affairs Institutional Animal Care and Use Committee guidelines (id D16-00002). The Animal Care and Use Committee review board approved all the studies involving animals. Male and female mice were used in same proportion in all the experiments. Mice were re-challenged one month following tumor eradication with 106 tumor cells. The depleting anti-CD4 (L3T4) and anti-CD8 (YTS169.4) were from BioXCell. The monitoring anti-CD4 monoclonal antibody (GK1.5) was from eBioscience. The monitoring anti-CD8 monoclonal antibody (5H10) was from Invitrogen.Tumorigenesis was conducted to test whether anti-PD-1 would augment CCL21-DC tumor lysate vaccine over monotherapy. Here, 6–8-week-old pathogen-free 129-E mice (Charles River Lab) were used in the experiments, and tumor volume was determined using the following formula: Tumor Volume = 0.4ab6 single-cell suspension of tumor digest according to manufacturer’s instructions.Tumors were digested in the gentleMACS Octo Dissociator for evaluation by specific staining/flow cytometry. Flow cytometry analysis was conducted to determine CD8 T cells (FITC conjugated anti- mouse CD8) expressing granzyme B (PE conjugated anti-mouse granzyme B) in the TME following diluent, anti-PD-1, CCL21-DC lysate vaccine, and anti-PD-1 plus CCL21-DC lysate vaccine therapy. Fluorimetry staining followed by flow cytometry analyses were performed on single-cell suspensions of tumor digest. A total of 10,000 events were acquired on the Cytoflex flow cytometer , and the data were analyzed by CytExpert software. Cell staining was performed with fluorochrome-labeled specific Ab on 10H&E staining was conducted on tumor sections.2 grid subdivided into one hundred 1-mm2 squares). A grid square with specific staining occupying >50% of its area was scored as positive and the total number of positive squares was determined. Ten separate fields from four IHC sections from four mice per group were examined under high power (20× objective)IHC staining was conducted on tumor sections. CD3 T cells, apoptotic tumor cells, perforin, and granzyme B were assessed by microscopic examination of IHC-stained sections (brown) with a calibrated graticule  or isotype control via or cells . Abs useTumor-infiltrating T cells (TILs) from 12-day treatment groups were purified by Miltenyi beads and co-cultured with tumor cells in CM at a ratio of 5:1 for 24 h. Almar blue (20 µL) was added for 4 h, and the fluorescence was read at excitation/emission (530nm/595nm) in a Wallac Fluorescence reader.CD4 and CD8 T cells were purified with Miltenyi beads from the spleens of mice one month following vaccination (once per week for 3 weeks) with DC tumor lysate vaccine. Purified DCs from the TME of (i) diluent control, (ii) anti-PD-1, (iii) CCL21-DC tumor lysate vaccine, and (iv) anti-PD-1 plus CCL21-DC tumor lysate vaccine were pulsed with MHC Class I K-Ras peptide (LVVVGADGV) or MHC Class II (MTEYKLVVVGADGVG) and co-cultured with splenic CD8 or CD4 T cells of the vaccinated mice at a ratio of 1:5 for 24 h. IFNγ secreted by T cells in the co-culture was determined by IFNγ-specific ELISA (eBioscience).t-test for dual comparison. p-values < 0.05 were considered significant.All data are presented as mean ± SE. Statistical analysis was performed using Prism . We used analysis of variance for data with multiple groups, and we used unpaired Student’s We tested the antitumor efficacy of (1) diluent, (2) anti-PD-1, (3) CCL21-DC tumor lysate vaccine, and (4) CCL21-DC tumor lysate vaccine plus anti-PD-1 in the K-RasG12Dp53null tumor model. The treatments induced the following fold reductions in tumor burden compared to diluent control: (1) anti-PD-1 induced a 2-fold reduction, (2) CCL21 DC tumor lysate vaccine induced a 2-fold reduction, and (3) Anti-PD-1 plus CCL21-DC tumor lysate vaccine induced a 17-fold reduction. The anti-PD-1, CCL21-DC tumor lysate vaccine, and anti-PD-1 plus CCL21-DC tumor lysate vaccine treatments resulted in 3-, 3-, and 19-fold weight changes of tumors at the end of therapy in comparison to control, respectively. In comparison to the monotherapy that reduced tumor burden without causing tumor eradication, treatment groups receiving CCL21-DC tumor lysate vaccine plus anti-PD-1 led to 80% tumor eradication . The curT cells from the TME of the combined therapy had increased cytolytic activity (8-fold) against parental K-RasG12Dp53null tumor cells in vitro in comparison to diluent control, whereas monotherapy had a 2-fold increase A. Flow cWe conducted H&E and IHC staining to evaluate tumor immune infiltrates following therapy. In comparison to controls and monotherapy, H&E staining of tumor sections revealed enhanced immune infiltrates with reduced tumor burden in the anti-PD-1 plus CCL21-DC tumor lysate vaccine treatment group A. IHC ofCD4 and CD8 T cells from the spleens of mice were purified with Miltenyi beads following vaccination with DC tumor lysate vaccine. Purified DCs from the TME of (i) diluent control, (ii) anti-PD-1, (iii) CCL21-DC tumor lysate vaccine, and (iv) anti-PD-1 plus CCL21-DC tumor lysate vaccine were pulsed with MHC Class I K-Ras peptide (LVVVGADGV) or MHC Class II (MTEYKLVVVGADGVG) and co-cultured with splenic CD8 or CD4 T cells of the vaccinated mice at a ratio of 1:5 for 24 h. IFNγ secreted by T cells in the co-culture was determined by IFNγ-specific ELISA. In comparison to diluent control or monotherapy, anti-PD-1 plus CCL21-DC tumor lysate Ag induced the highest DC activity of presenting MHC Class I (27-fold) and MHC Class II (36-fold) K-RasG12D tumor peptides to tumor-vaccine-specific T cells. DC activity following monotherapy with anti-PD-1 presented MHC Class I (4-fold) and MHC Class II (4-fold) to tumor vaccine specific T cells. DC activity following monotherapy with CCL21-DC tumor lysate presented MHC Class I (3-fold) and MHC Class II (3-fold) to tumor vaccine specific T cells. Control peptide (FECNTAQAC)-pulsed DCs did not stimulate T cell IFNγ production .n = 4 mice/group).Purified NK cells were plated with tumor cells at the ratio of 5:1 for 24 h. Almar blue (20 µL) was added for 4 h after 24 h, and the fluorescence was read. Bars represent SE. Results are representative of a single experiment B (n = 4 n = 10 mice/group).Anti-CD4 and anti-CD8 led to >95% depletion of the respective T cell populations. Results are representative of two experiments (Targeting inhibitory immune checkpoint molecules has highlighted the need to find approaches enabling the activation of immune responses against cancer. Therapeutic vaccination, which induces specific immune responses against tumor Ags, is an attractive option. Although this strategy has shown minimum clinical efficacy when combined with other treatment modalities, therapeutic cancer vaccine strategies have a high probability of success when combined with immune checkpoint blockade therapy with the potential for cancer-free survival. This could be achieved by evaluating therapeutic vaccines combined with immune checkpoint blockade protocols in preclinical models prior to clinical translation. As effective therapeutic vaccines and combinations with biological therapy are defined in preclinical models, the understanding of the interactions in the TME that modulate antitumor activities can be utilized to develop effective technologies for long-term cancer-free survival.s.c. injections. DCs are highly specialized professional APCs with the capacity to capture, process, and present Ags to T cells, resulting in T cell activation. In the current study, the data demonstrate that PD-1 blockade augments T cell and DC activity of CCL21-DC tumor Ag vaccine in the TME to eradicate K-RasG12Dp53null lung adenocarcinoma. This suggests that a combination of PD-1 blockade therapy with therapeutic vaccination could prove essential to improve the management and clinical outcome of patients who do not respond or for those whose disease eventually progresses.We evaluated PD-1 blockade combined with CCL21-DC tumor Ag pulsed vaccine in the K-RasG12Dp53null murine model of lung adenocarcinoma. We are currently conducting a phase I clinical trial of CCL21-DC administered intratumorally in patients with advanced-stage lung cancer . This aps.c. administration of CCL21-DC tumor lysate vaccine results in tumor growth inhibition, but the vaccine therapy alone is not optimal. Immunomodulatory monoclonal Abs that target immune evasion pathways in cancer have shown efficacy in clinical trials [The results of this study present a unique translational opportunity because they provide a means for the activation of T cells to effectively target and eradicate the aggressive K-RasG12Dp53null mutant lung cancer. p53 normally serves as a tumor suppressor, the function of which is lost upon allelic loss or mutation. Lung cancers have a high p53-specific mutation in adenocarcinoma and in squamous cells. In the K-RasG12Dp53null tumor model, tumors develop in an immune-competent host. Our results demonstrate that this model responds to individual therapeutic vaccination or immune checkpoint blockade therapy, although the therapies administered individually are sub-optimal. This model has low baseline activated T cell infiltrates, similar to NSCLC patients who have low responses to PD-1 blockade. ,10. The l trials ,12. The l trials ,14. T ceCurrently, up to 50–60% of patients being treated with PD-1 checkpoint inhibitors do not have T cell infiltration and have reduced antitumor response. Our data demonstrate that the CCL21-DC tumor Ag vaccine has the means to overcome this limitation for long-term cancer-free survival and provide justification for evaluation for clinical translation.


PIK3CA mutations in kinase domain (KD), helical domain (HD) and other domain (OD), were 36 (18.7%), 26 (13.5%), 10 (5.2%), respectively. 21 (10.9%) patients had mutations in PI3K/AKT pathway genes other than PIK3CA (P/A). Compared to PI3K/AKT-wild type (WT) patients, PIK3CA-HD patients had a significantly shorter progression-free survival (PFS) . PIK3CA-KD, PIK3CA-OD and other P/A mutations showed similar PFS to WT patients . PIK3CA-HD patients had a distinct ctDNA mutation profile to patients with other PI3K/AKT mutations. PIK3CA-HD patients had a higher rate of FGFR and NF1 aberrations. In addition, more PIK3CA-HD carriers were TMB-high. Cox regression analyses suggested that PIK3CA-HD mutations, FGFR aberrations and high TMB were all significant risk factors for poor PFS. In conclusion, future research needs to focus more on the treatment strategies targeting PIK3CA-HD mutations.Nearly half of metastatic breast cancers (MBC) have genetic aberrations in the PI3K/AKT pathway. To investigate the distinct effect of these aberrations on MBC, 193 MBC patients who progressed after the early line (≤2) salvage treatment voluntarily received next generation sequencing (NGS) for a panel of 1,021 genes. 93 (48%) patients had genetic aberrations in the PI3K/AKT pathway. The number of patients with  PIK3CA mutations to be a major mediator of therapy resistance in breast cancer [PIK3CA gene encodes the PI3K catalytic subunit p110α [PIK3CA missense mutations; 10-30% of metastatic triple-negative breast cancers (TNBC) and HER2-positive breast cancers have PIK3CA missense mutations [PIK3CA mutations occur in helix domain (HD) exon 9 and kinase domain (KD) exon 20 [PIK3CA mutations are in exon 9 (hotspots: E545K and E542K), and 50% of PIK3CA mutations are in exon 20 (hotspot: H1047R) [PIK3CA-HD mutations are less potent in inducing mammary tumors [PIK3CA-KD mutations are associated with lymph node infiltration and are much more aggressive in carcinogenesis compared to PIK3CA-HD mutations [PIK3CA-KD mutations are associated with optimal prognosis [A recent study suggested t cancer . More tht cancer . PI3K isit p110α . Accordiutations . About 8 exon 20 . 26% of PIK3CA-HD mutations are more frequent in older age-onset patients, and they are not associated with lymph node infiltration as PIK3CA-KD mutations [PIK3CA-HD mutant tumors is significantly poorer than tumors with PIK3CA-KD mutations [PIK3CA-HD mutation-encoded protein PIK3CA(E545K) interacts with Ras-GTP [(H1047R), PIK3CA(E545K) is less efficient in activating the downstream Akt signaling [utations –11. Impoutations . Mechani Ras-GTP . Compareignaling .PIK3CA-HD and PIK3CA-KD mutations, to determine the different effects of PI3K/AKT pathway mutations on the prognosis of MBC patients with late-line treatment.Most MBC patients receive chemotherapy coupled with/without endocrine therapy or anti-HER2 therapy. The activation of PI3K/AKT/mTOR pathway, however, would promote tumor progression and induce drug resistance to endocrine therapy and chemotherapy –16. In tst or 2nd salvage treatment received ctDNA testing and then late-line therapy between December 2016 and December 2018. Ninety three patients (48%) had ctDNA mutations of the PI3K/AKT pathway molecules. Among these ninety three patients, thirty six patients (38.7%) had PIK3CA–KD missense mutations, including thirty one p.H1047R, one p.H1047L, two p.G1049R, and one p.M1043V missense mutations  mutations were 7.7 months (95% CI: 5.4-9.6), 5.1 months (95% CI: 3.5-13.0), 3.2 months (95% CI: 2.1-4.2), 11.2 months (95% CI: 3.0-NE), and 4.6 months (95% CI: 3.3-14.8), respectively . PIK3CA-HD mutant patients had a significantly lower PFS than WT patients (Logrank p<0.0001), PIK3CA-KD mutant patients (Logrank p=0.02), PIK3CA-OD mutant patients (Logrank p=0.0006) and P/A aberrant patients (Logrank p=0.01).All included patients failed and progressed in the early–line (≤2) salvage therapy. Then, the later line regimen was given by TPC (treatment physician choice). The median PFS for patients with WT, ectively . Apart fTP53, ERBB2, FAT1, and FGFR aberrations , a higher NF1 mutation rate , and a lower DNMT3A mutation rate   .PIK3CA-HD mutant MBC patients had a significantly higher TMB level than patients with PIK3CA-KD mutations (p=0.006), PIK3CA-OD mutations (p=0.045), or other P/A pathway mutations (p=0.01) (Tumor mutation burden (TMB) has been considered as a marker for immunotherapy. The more mutations the tumor has, the higher possibility of production and subsequent presentation of tumor-associated antigens (TAA) on MHC molecules, which leads to a higher tumor cell cytotoxicity after the inhibition of immune checkpoint signals , 18. BCs(p=0.01) .PIK3CA-HD mutations, FGFR aberrations, and high TMB levels, were significant risk factors for poor PFS for late-line therapy in MBCs , 2.22 , 2.17 , and 1.62 , respectively.Cox regression analyses suggested that none of the clinical indicators had significant impacts on PFS for late-line therapies, including the age of diagnosis, age of BC metastasis, TTM, primary BC laterality, ER, PR, HER2 status and the sites of metastatic lesions . However in MBCs . MultivaTP53 mutations, FGFR aberrations and TMB levels were imbalanced between PIK3CA-HD mutant and PIK3CA-KD mutant MBC patients, we performed a 1:1 propensity score matching (PSM) strategy to avoid the potential bias. Kaplan-Merier (KM) curves after PSM still showed a marginally significant difference between PIK3CA-HD mutant and PIK3CA-KD mutant MBCs  therapies, there is no consensus on the late-line therapy. Potential choices for hormone receptor (HR)-positive MBCs include mTOR inhibitor, FGFR inhibitor, Estradiol and Progestin , 24. CanPIK3CA, ESR1 and GATA3 mutations increased in progressed patients after chemotherapy [ESR1 mutations increased after AI treatment [RB1 mutation could be detected by ctDNA testing [After early-line therapies, MBC tumors become more heterogenous and have more somatic gene aberrations. otherapy . The ratreatment . After t testing . Based oPIK3CA-HD mutations were concentrated in HR-positive patients, and more than half of these patients were TMB-H  for novel treatment strategies, such as PD-1 inhibitors, FGFR inhibitors, etc.In this study, we found that in MBC patients with PI3K/AKT pathway aberration, ficantly . In partPIK3CA-HD mutations and FGFR aberration seemed to concentrate in HR-positive MBC patients. Is there any potential molecular mechanism underlying the coincidence? However, there is no report about the interaction between PIK3CA-HD mutations and FGFR aberration. In FGFR2mutant endometrial cancer, the sub-therapeutic doses of PI3K inhibitors could enhance the efficacy of anti-FGFR therapies [PIK3CA mutation and FGFR aberration might be independent to each other. If they have interaction, inhibiting each of them might also affect the other one. Only when they were independent, PI3K inhibitors and anti-FGFR therapies have synergic effects.Here, we found that both herapies . Such a PIK3CA-HD mutant MBC patients showed resistance to mTOR inhibitor Everolimus. More than 70% of patients who received Everolimus in late-line therapy progressed within six months. Due to the limited sample number, we did not demonstrate a concrete result here. However, we still suggested that for PIK3CA-HD mutant MBC patients, mTOR inhibitor Everolimus might be not as effective as in other PI3K/AKT pathway aberrant patients. PD-1 inhibitor plus FGFR inhibitor might be a promising option.This study involved 193 MBC patients who received late-line therapies from three affiliated hospitals in Central South University. The limitation was the relatively small sample size for each PI3K/AKT pathway aberrant category. The sample size is even smaller after PSM. In the foreseeable future, we would accumulate many more samples to further validate our hypothesis. For now, we raised our hypothesis and provided clues for reasonable treatment strategies, which might be beneficial for MBC patients who have no choice.PIK3CA mutations in helical domain had a specific ctDNA profile with high TMB and high FGFR aberration rate, which might lead to poor PFS for late-line therapy. PD-1/PD-L1 inhibitor and FGFR inhibitor could be promising as a late-line option for MBC patients with this specific genotype.MBC patients with This study included MBC patients who were within 2 weeks after progression of the early-line (≤2) salvage treatment. All patients had a confirmed pathology diagnosis . According to RECIST 1.1 standards, patients had at least one measurable distant disease lesion. All patients had an Eastern Cooperative Oncology Group/World Health Organization Performance Status (ECOG/WHO PS) of 0 or 1. Patients with inflammatory BC, multiple primary malignancies, immunodeficiency or organ transplantation history were excluded. Patients who have received mTOR inhibitor treatment were also not eligible for the study.This multicenter study was conducted across three local cancer centers. MBC patients who failed in early-line (≤2 lines) standard chemotherapies or standard chemotherapies combined with anti-HER2 therapies or endocrine therapies received big-panel NGS for ctDNA testing. After ctDNA testing, patients received late-line therapy.etc) [The primary endpoint was PFS, defined as the number of days from the beginning date of late-line therapy to progressive disease, defined as: 1) >20% growth of measurable target lesions and the absolute increase of target lesions >5mm, or 2) presence of new lesions . There wetc) .Peripheral blood samples were collected within 7 days before treatment. We performed ctDNA testing according to protocol described previously , 35, 36.To search for significant ctDNA aberrations, the R package “ComplexHeatmap” was used to rank the hot genetic aberrations in PI3K/AKT pathway aberrant subgroups. A two-sided log-rank test was used to test the influence of PI3K/AKT pathway aberrations in terms of late-line treatment PFS in MBCs. To reduce the potential bias between subgroups, we also performed 1:1 propensity score matching (PSM) analysis as described previously . Both thThe study protocol and informed consent form were censored by the independent ethics committee for each center. In our study, each participant filled and signed an approved written informed consent provided by the independent ethics committee.PIK3CA mutations in the helical domain exon 9 were associated with significantly worse prognoses in breast cancer. That study used frozen samples from 163 surgery patients. Here, we demonstrate the effect of PIK3CA helic domain mutations in metastatic breast cancer with late-line therapy by using plasma ctDNA. MBC patients with PIK3CA mutations in helical domain had a specific ctDNA profile with high TMB and high FGFR aberration rate, leading to poor PFS for late-line therapy. Our findings directly reflect the effect of genetic aberrations on treatment outcome and suggest potential strategies for MBC patients. PD-1/PD-L1 inhibitor and FGFR inhibitor could be promising as a late-line option for MBC patients with this specific genotype.12 years ago, Barbareschi M's team reported Supplementary Figure 1Supplementary Table 1


Inflammatory smooth muscle cells (iSMCs) generated from adventitial stem/progenitor cells (AdSPCs) have been recognised as a new player in cardiovascular disease, and microRNA-214-3p (miR-214-3p) has been implicated in mature vascular SMC functions and neointimal hyperplasia. Here, we attempted to elucidate the functional involvements of miR-214-3p in iSMC differentiation from AdSPCs and unravel the therapeutic potential of miR-214-3p signalling in AdSPCs for injury-induced neointimal hyperplasia.The role of miR-214-3p in iSMC differentiation from AdSPCs was evaluated by multiple biochemistry assays. The target of miR-214-3p was identified through binding site mutation and reporter activity analysis. A murine model of injury-induced arterial remodelling and stem cell transplantation was conducted to study the therapeutic potential of miR-214-3p. RT-qPCR analysis was performed to examine the gene expression in healthy and diseased human arteries.miR-214-3p prevented iSMC differentiation/generation from AdSPCs by restoring sonic hedgehog-glioma-associated oncogene 1 (Shh-GLI1) signalling. Suppressor of fused (Sufu) was identified as a functional target of miR-214-3p during iSMC generation from AdSPCs. Mechanistic studies revealed that miR-214-3p over-expression or Sufu inhibition can promote nuclear accumulation of GLI1 protein in AdSPCs, and the consensus sequence (GACCACCCA) for GLI1 binding within smooth muscle alpha-actin (SMαA) and serum response factor (SRF) gene promoters is required for their respective regulation by miR-214-3p and Sufu. Additionally, Sufu upregulates multiple inflammatory gene expression  in iSMCs. In vivo, transfection of miR-214-3p into the injured vessels resulted in the decreased expression level of Sufu, reduced iSMC generation and inhibited neointimal hyperplasia. Importantly, perivascular transplantation of AdSPCs increased neointimal hyperplasia, whereas transplantation of AdSPCs over-expressing miR-214-3p prevented this. Finally, decreased expression of miR-214-3p but increased expression of Sufu was observed in diseased human arteries.We present a previously unexplored role for miR-214-3p in iSMC differentiation and neointima iSMC hyperplasia and provide new insights into the therapeutic effects of miR-214-3p in vascular disease.Supplementary information accompanies this paper at 10.1186/s13287-020-01989-w. Smooth muscle cell (SMC) accumulation and phenotypic transition are critical steps for vascular remodelling in response to vascular injury. Although recent ground-breaking efforts, using SMC-restricted lineage-tracking techniques, have provided definitive evidence to support the conventional concept that medium SMCs are the main cellular origins of (neo)intimal SMCs upon injury –3, otherMicroRNAs (miRNAs) are endogenous, highly conserved single short strands of non-coding RNA. They have been reported as important regulators for stem cell differentiation, cardiovascular development and cardiac regeneration and are emerging as novel therapeutics as well as clinical biomarkers for cardiovascular diseases, including restenosis after vascular injury or stent implantation  and atheIn particular, miR-214-3p has been reported to be highly expressed at early murine embryonic stages (E10.5~E12.5) in the heart , vasculaThe procedures for mouse aortic AdSPC isolation and culture are described in our previous study . UndiffeThe femoral artery denudation injury murine model has been widely used in preclinical studies to replicate the pathological processes of human percutaneous transluminal angioplasty , 35. AneProcedures for cell isolation from the injured femoral arteries were similar to the protocols described in the previous studies –38. Two 6 AdSPCs transfected with control AgomiR (AdSPCs/Cel-miR-67) or miR-214-3p AgomiR (AdSPCs/miR-214), was randomly applied perivascularly to the injured femoral arteries. The injured arteries (~ 5.0 mm) were harvested at days 7 and 28 post-injury for gene expression, immunofluorescence staining and morphometric analysis.Eight-week-old male C57BL/6 mice (20~22 g) purchased from Charles River Laboratories were subjected to arterial injury and AdSPC transplantation. Briefly, arterial injury was induced by an endothelial denudation procedure as previously described , 21, 32.Additional materials and methods used in this study are described in detail in the t test was used for comparisons between 2 groups, or one-way analysis of variance with a post hoc test of Tukey’s analysis was applied when more than two groups were compared if the data displayed a normal distribution. Conversely, non-parametric Mann-Whitney U test was used for comparing two groups, if the data did not display normal distribution or if the number of observations from each group was smaller than 5 (n < 5). Spearman’s rank correlation analyses were conducted to characterise the relationships between the gene expression levels of miR-214-3p and Sufu in human arterial specimens. Alpha = 0.05 was chosen as the significance level, and a value of P < 0.05 was considered as statistically significant.Animals were randomly allocated to their experimental groups. Data collection and evaluation of all experiments were performed blinded to the group identity. The results are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism . The Shapiro-Wilk normality test was used for checking the normality of the data. Two-tailed unpaired Student’s Some data may not be made available because of privacy or ethical restrictions. All remaining data are contained within the article. Other detailed materials and methods were provided in the Isolated AdSPCs grew into colonies with a uniform morphology Figure SA and werVascular SMCs can switch to a ‘pro-inflammatory’ phenotype, or inflammatory SMC (iSMC), in response to vascular injury, which promotes monocyte/macrophage recruitment into the vascular wall and triggers the pathological processes of atherosclerosis and arterial remodelling . To mimiMultiple functional assays were conducted in mature SMCs with different phenotypes  and iSMCs to further characterise the iSMC phenotype. Compared to ‘synthetic’ SMCs, iSMCs displayed a trend of increased SMC gene expression with a significant drop in the expression of extracellular matrix genes Figure SA. Among Importantly, we observed an increased expression level of miR-214-3p induced by TGFβ1 alone, and this was inhibited once TNFα was added into the differentiating AdSPCs  signalling has been extensively implicated in adventitial smooth muscle progenitor cell self-renewal and differentiation , 42 and Suppressor of fused (Sufu) negatively regulates hedgehog signalling through direct binding of GLI1 and by retaining GLI1 in the cytoplasm , 46. IntCompared to the respective control cells, Sufu knockdown significantly upregulated SMC gene expression at both the mRNA Fig. a and proLuciferase activity assays showed that Sufu knockdown in the differentiating AdSPCs significantly activates SMC gene promoter activities Figure SA & S7B aThe Sufu-GLI1 signal axis has been reported to play a critical role in the intestinal inflammatory response, and decreased GLI1 activity predisposes to a heightened myeloid response to inflammatory stimuli in inflammatory bowel diseases . We wondSimilar to our previous studies , 21, 32,Triple immunofluorescence staining of the injured segments of the femoral arteries transplanted with GFP-labelled AdSPCs with antibodies against GFP, SMαA and S100A4 showed that the transplanted AdSPCs could differentiate into iSMCs  within the injured arterial walls Figure S. To furtTo further validate and translate our findings into a clinical setting, the gene expression levels of miR-214-3p and Sufu and their possible relationship were examined in healthy and diseased femoral arterial (FA) specimens collected in our previous study . RT-qPCRThe potential contributions of residential AdSPC-derived iSMCs to neointimal SMC hyperplasia and the underlying molecular mechanisms of iSMC differentiation/generation from the AdSPCs have yet to be fully recognised and elucidated and require further investigation. In the current study, we attempted to address this unexplored avenue by uncovering a novel role for miR-214-3p in modulating SMC differentiation and iSMC generation from AdSPCs in vitro and in vivo Fig. c. ImportmiR-214-3p belongs to the miR-199a-214 cluster, which is encoded by DNM3os that is transcribed from the opposite strand of the DNM3 gene . DifferiOne of the new aspects of this study is the establishment of a reliable cellular model to study iSMC differentiation/generation from AdSPCs. Apart from the classical ‘contractile’ to ‘synthetic’ phenotypic modulation model, extensive evidence also suggests that vascular SMCs can be primed to another distinct phenotype, namely the ‘pro-inflammatory’ phenotype or iSMCs, in response to vascular injury or inflammatory stimuli. These iSMCs contribute to vascular disease progression and are a pervasive feature of atherosclerotic lesion formation  and arteAnother new finding from this study is the identification of Sufu as a functional target gene of miR-214-3p in the context of iSMC differentiation from AdSPCs. Although multiple miR-214-3p target genes have been reported in various cellular contexts and diseases , SMC transcriptional factor SRF and inflammatory genes  through a transcriptional mechanism. Our data shows that Sufu exerts transcriptional regulation through modulation of GLI1 nuclear translocation. Data generated from luciferase activity Figs. g and S7GIn the current study, we have successfully uncovered the functional involvements of miR-214-3p in iSMC differentiation/generation from residential AdSPCs in the context of vascular remodelling after injury Fig. c. MoreovAdditional file 1: Table S1 and Supporting Methods.Additional file 2: Figure S1. Characterization of adventitia stem/progenitor cells (AdSPCs). (A) Phase-contrast image showing the primary culture of AdSPCs (P0). (B)AdSPCs at passage 3~8 were subjected to immunofluorescence staining with antibodies against AdSPC and other cell lineage markers, namely Sox10/FSP1 (fibroblast marker), Sox17/CD31 , and Nestin/SM-NHC (SMC marker), respectively. (C) Flow cytometry analysis of AdSPC markers (Sox10 and Nestin) expression in AdSPCs at passage 3. (D) Gene expression profiles in AdSPCs at the indicated passage number (P). The data presented here are representative (A-C) or mean±S.E.M. (D) of five independent experiments. *P<0.05 .Additional file 3: Figure S2. SMC differentiation from AdSPCs. Passage 3-8 AdSPCs were cultured in SMC differentiation medium (DMEM supplemented with 5% FBS and 5ng/ml TGFβ1) for the indicated time to allow for SMC differentiation. Total RNAs and proteins from undifferentiated AdSPCs (0d) or differentiating AdSPCs at day 2, 4, 6 and 8 were harvested and subjected to RT-qPCR (A) and Western blot (B) analyses, respectively. (C) Mature SMC marker expression in AdSPC-derived SMCs. Day 8 differentiated SMCs were stained with antibodies against SMαA and SM-MHC. The data presented here are representative images  or mean±S.E.M.  of five independent experiments. *P<0.05 .Additional file 4: Figure S3. miR-214 promotes SMC differentiation from AdSPCs. (A) Increased expression of miR-214 in AdSPCs in response to TGFβ1. (B-E) miR-214 modulates SMC marker expressions. Day 2 differentiating AdSPCs were transfected with miR-214 mimics, inhibitor or respective negative control (miR ctrl), and cultured for 2~3 days, followed by RT-qPCR (B & C) and western blot (D & E) analyses, respectively. The data presented here are representative (up panels in D & E) or mean±S.E.M.  of five independent experiments. *P<0.05. One-way ANOVA with a post hoc test of Tukey’s analysis for A & C, unpaired t-test for B. miR-214 indicates miR-214-3p.Additional file 5: Figure S4. Characterisation of iSMC phenotype. Mature SMCs were subjected to serum starvation for 48 hours, followed by the indicated treatments for additional 24 hours. Total RNAs and conditioned culture medium harvested from SMCs treated with vehicle , 5ng/ml TGFβ1 , 10ng/ml PDGF-BB , and iSMCs differentiated from AdSPCs (iSMCs) were subjected to RT-qPCR (A) and ELISA analyses (B), respectively. (C) Luciferase activity assays detected NFκB activation in cells with the indicated treatments. (D) BrdU incorporation assays to detect cell proliferation. (E) Trans-well migration assays detected cell migration under 30ng/ml PDGF-BB stimulation. The data presented here are representative images (left panels in E) or mean±S.E.M.  of five independent experiments (n=5). *P<0.05 (versus ssSMCs), #<0.05 (versus synSMCs) .Additional file 6: Figure S5. GLI1 cellular location was modulated by miR-214-3p. AdSPCs were differentiated into iSMCs (DMEM containing 5ng/ml TGFβ1 and 50ng/ml TNFα) for 4 days, then transfected with miR-214-3p mimics (miR-214) or negative control (miR ctrl), and cultured for 48 hours in the same culture medium. Cells were fixed and subjected to immunofluorescence staining (A). Fifty cells were randomly selected for quantification by Image J free software  from each treatment. Mean fluorescence intensity (MFI) of nuclear (B) and cytoplasmic (C) GLI1 (green), as well as DAPI (blue) from the same cells were quantified, and the ratio of MFI of GLI1 over DAPI were calculated accordingly. Veh indicates vehicle control for TNFα. The data presented here are representative (A) or mean±S.E.M. (B & C) of fifty cells (n=50). *P<0.05 (vs veh/miR ctrl); #P<0.05 (TNFα/miR-214 vs TNFα/miR ctrl) . miR-214 indicates miR-214-3p.Additional file 7: Figure S6. Sufu controls SMC gene expression and GLI1 cellular location. (A) SMC gene expression is negatively regulated by Sufu. AdSPCs (p3~p8) were infected with control (pCDH) or Sufu over-expression (pCDH-Sufu) lentivirus and cultured in iSMC differentiation induction medium for 2~3 days. The expression levels of a panel of SMC specific genes was examined by RT-qPCR analysis. (B) Sufu over-expression inhibited SMC gene promoter activity. AdSPCs infected with pCDH or pCDH-Sufu were transfected with SMC gene promoter reporters (pGL3-SMαA or pGL3-SM22α) as indicated. Luciferase activity assays were conducted at 48 hours post-transfection. (C) GLI1 gene expression was not affected by Sufu over-expression. (D) Sufu knockdown promotes GLI1 protein accumulation in nuclei. AdSPCs were infected with non-target (sh-NT) or Sufu (sh-Sufu) shRNA lentivirus, followed by iSMC differentiation. Cells were fixed and subjected to immunofluorescence staining with GLI1 antibody. Data presented here are representative images (D) or Mean±S.E.M of five or six independent experiments .Additional file 8: Figure S7. Sufu controls SMC gene expression through regulating SRF. (A-B) Serum response factor (SRF) binding sites within SMC gene promoters are required for Sufu-mediated SMC gene repression. AdSPCs infected with non-target (sh-NT) or Sufu (sh-Sufu) shRNA lentivirus were transfected with wild type (WT) SMC gene promoter reporters (pGL3-SMαA or pGL3-SM22α), or SRF binding site mutants [SRFmut], respectively. Luciferase activity assay were measured at 48 hours post-transfection. (C-D) Sufu over-expression inhibited SRF gene expression, while Sufu knockdown promoted its expression. (E) SRF gene promoter activity is increased by Sufu inhibition. (F) GLI1 binding site within SRF gene promoter region is necessary for SRF gene up-regulation by Sufu knockdown in AdSPCs. The potential wild type binding site (pGL3-SRF-WT) of GLI1 within SRF gene promoter and its mutant pGL3-SRF-GLI1mut) are depicted in this illustration (Insert). AdSPCs infected with sh-NT or sh-Sufu lentivirus were transfected with respective SRF gene promoter reporters as indicated. Luciferase activity assay were measured at 48 hours post-transfection. (G) Sufu inhibition increases GLI1 enrichment within SRF gene promoter. ChIP assays were performed using antibody against GLI1 or normal IgG, respectively. PCR amplifications of the adjacent regions were included as additional control for specific promoter DNA enrichment. Data presented here are Mean±S.E.M of five or six independent experiments .Additional file 9:Figure S8. miR-214 decreases iSMCs during arterial remodelling. After wire-induced femoral arterial injury, 100 μl of 30% pluronic gel containing 2.5 nmol control AgomiR (Cel-miR-67) or miR-214 AgomiR (miR-214) per artery per mouse was immediately applied and packed around the injured femoral arteries. Two weeks later, injured segments of femoral arteries were harvested for H&E staining analyses. Mice underwent a sham arterial injury procedure serve as control (Sham). SMCs and neointima (NI) cells were isolated from media of sham mice (SMCs/Sham) or media/neointima layers of injured mice received control (NI cells/Cel-miR-67) or miR-214 (NI cells/miR-214) AgomiR, respectively. Isolated cells were cultured in SMC medium for 5-7 days and subjected to various analyses as indicated. (B) Phase-contrast images were also taken prior to other analyses and included here. (C & D) Immunofluorescence staining assays with antibodies against SMαA and IL-6. Representative images (C) and percentage of iSMCs from each group ((D) were presented here. (E) RT-qPCR detection of gene expression. Data presented here are representative images or Mean±S.E.M of five independent experiments (n=5). *P<0.05 (versus sham), #P<0.05 (miR-214 versus Cel-miR-67) . miR-214 indicates miR-214-3p.Additional file 10: Figure S9. Transplanted AdSPCs differentiate toward iSMCs during arterial remodelling. After injury, 100μl of Geltrex contained 1x106 AdSPCs infected with Lenti-GFP per vessel per mice was immediately applied and packed around injured vessel. One week later, injured segments of femoral arteries were harvested and prepared, followed by triple immunofluorescence staining with antibodies against GFP, SMαA and S100A4. White arrows within merged image indicate iSMCs derived from transplanted AdSPCs. Data presented here are representative images from three mice.


Berberine is an isoquinoline alkaloid isolated from various Chinese herbs that has potential of anti-inflammatory, anti-lipidemic, anti-neoplastic, and anti-diabetic activity. In this study, we evaluated the anti-inflammatory efficacy of berberine on allergic airway inflammation by targeting epithelial cells. Allergic airway inflammation driven by T helper 2 (Th2)-type immunity is characterized by airway hyperresponsiveness, elevated IgE production, and eosinophilic infiltration. For eosinophil recruitment, major chemoattractant CCL11 (eotaxin-1) was secreted by lung epithelial cells. BEAS-2B cells, a human bronchial epithelial cell line, were pre-treated with berberine and then activated by IL-4 plus TNF-α. The viability of BEAS-2B cells was assessed. Expression levels of IL-6 and CCL11 were determined using ELISA and real-time PCR. The signaling pathways of MAP kinases, NF-κB, and STAT6 were analyzed by western blot. Berberine treatment (≤1 μM) didn't significantly affect the viability of BEAS-2B cells with or without IL-4 plus TNF-stimulation. Berberine significantly inhibited the secretion of IL-6 and CCL11 from pro-inflammatory cytokine-activated BEAS-2B cells. NF-κB and MAP kinase pathways were seemingly unaffected in BEAS-2B cells with berberine treatment. Significant reduction of nuclear STAT6 protein expression in activated BEAS-2B cells with berberine treatment was observed. Current study reveals that berberine has inhibitory effect in pro-inflammatory cytokine-activated BEAS-2B cells through reducing IL-6 and CCL11 production, which is possibly modulated by suppressing STAT6 signaling pathway. Asthma is one of the major public health concerns worldwide, with high prevalence and economic burden + T helper 2 (Th2) cells, mast cells, and eosinophils Airway epithelium acts as the first defense barrier against environmental stimuli by secreting numerous chemokines, cytokines and growth factors to recruit and activate a variety of immune cells including CD4The level of pro-inflammatory cytokine IL-6 was elevated in asthma patients Previous papers showed that IL-4 and tumor necrosis factor (TNF)-α can stimulate the fibroblasts of nasal polyps to produce eotaxin-1 Previous studies have reported that berberine, an alkaloid extracted from various traditional Chinese herbs, has pharmacological effects on intestinal bacteria, parasites, cardiovascular and cerebrovascular diseases, lowering blood sugar, and cancer in vitro model to examine the anti-inflammatory efficacy of berberine on pro-inflammatory cytokine-stimulated epithelial cells. NF-κB, STAT6 and MAP kinases signaling pathways involved in modulating eotaxin gene expression are assessed.In present study, we applied BEAS-2B cells, a human bronchial epithelial cell line, as an Figure 6 cells) or 24-well plates (4x105 cells) at 37 °C in a humidified 5% CO2 atmosphere. Berberine chloride (Sigma-Aldrich) was dissolved in DMSO (Sigma-Aldrich), where same amount of DMSO for dissolving berberine was used as control for comparison. BEAS-2B cells were treated with or without different concentrations of berberine for 16-18 hours. After pre-treatment, BEAS-2B cells were stimulated with IL-4 (20 ng/ml) (Peprotech) plus TNF-α (5 ng/ml) (Peprotech) and culture supernatants were harvested after 6 hours and 24 hours to detect IL-6 and CCL11 levels by ELISA assay. For inhibition assays, 10 μM of JNK inhibitor-SP600125 (Enzo) and ERK inhibitor-PD-98059 (Enzo) were added one hour before cytokine stimulation and culture supernatants were harvested after 24 hours for IL-6 and CCL11 detection.Human bronchial epithelial cells  were cultured in Dulbecco's Modified Eagle Medium/Nutrient mixture F-12 (DMEM/F12) medium (Gibco) containing 10% fetal bovine serum  and 1% penicillin/streptomycin (Gibco). Cells were seeded in 6-well plates (2 × 105 cells) were seeded in a 48-well plate overnight and then were pre-treated with various concentrations of berberine or DMSO for 16-18 hours. After pre-treatment, cells were stimulated without or with IL-4 and TNF-α for 24 hours and the viability was analyzed by Cell Counting-kit 8 (CCK-8) (Sigma-Aldrich). Briefly, after the supernatants were harvested, cells were washed with 1 ml PBS and then CCK-8 was added into each well to incubate at 37 °C in a humidified 5% CO2 atmosphere for 4 hours. Supernatants were transferred into a 96-well plate after incubation. Absorbance was measured on an ELISA reader at 450 nm (SpectraMax).BEAS-2B cells  were determined by ELISA according to the manufacturer's instructions.TM SYBR green supermix (Bio-rad). The relative expression of each gene was calculated by normalizing to the expression of GAPDH.RNA samples were collected at 3, 6, 12 and 24 hours after cytokine stimulation. Total RNA was extracted using TRIzol reagent (Invitrogen). Reverse transcription was performed by oligo-dT primers and M-MLV reverse transcriptase (Invitrogen). The mRNA expression of GAPDH, IL-6, and CCL11 were amplified by specific primers: 5'-GCAAATTCCATGGCACCG-3' for GAPDH forward primer and 5'-TCGCCCCACTGATTTTGG-3' for GAPDH reverse primer; 5'-CCAATCTGGATTCAATGAGGAG-3' for IL-6 forward primer and 5'-GGTCAGGGGTGGTTATTGCATC-3' for IL-6 reverse primer; 5'-AAAGCTCACACCTTCAGCCT-3' for CCL11 forward primer and 5'-TTTCTGGGGACATTTGCCAC-3' for CCL11 reverse primer. For real-time PCR, the reaction was performed by Lightcycler® 96 system (Roche) with iQ3VO4, 1 mM Tetranisole, 25X diluted protease inhibitor, 5 μM MgCl2, 20 μM Tris-HCl, 50 μM NaCl, 20% NP-40 on ice for 30 min. Cell lysates were centrifuged at 12,000 g for 30 min at 4 °C and the supernatants were harvested for western blot analysis. For cytoplasm and nucleus protein extraction, cell extracts were collected using Nuclear and Cytoplasmic Extraction Reagent kit (NE-PER kit) (Pierce Biotechnology) according to the manufacturer's instructions. Protein concentrations were determined using a BCA protein assay (Pierce Biotechnology) for western blot analysis.For total cell protein extraction, protein samples were collected at indicated time point after cytokine stimulation. Cells were lysed in cell lysis buffer containing 1 mM NaProtein samples (20 μg) were separated on sodium dodecyl sulfate-polyacrylamide gel, transferred to nitrocellulose membrane (Millipore) at 50 mA for 30 min and blocked in Tris-buffered saline with 0.1% Tween 20 (TBST) containing 5% BSA at room temperature for 1 hour. The blots were incubated with primary antibodies (rabbit anti-human antibody) to JNK, phosphorylated JNK, ERK, phosphorylated ERK, p38, phosphorylated p38, IκB, phosphorylated IκB, p65, STAT6, and phosphorylated STAT6, primary antibodies (mouse anti-human antibody) for β-actin and p38, and primary antibodies (goat anti-human antibody) for PCNA  overnight at 4 °C. Secondary antibodies conjugated with horseradish peroxidase were incubated for 2 hours. Finally, the blots were developed by using the Immobilon Western chemiluminescent HRP substrate (Millipore).P-value calculated by the Mann-Whitney U test. P<0.05 is considered significant. All graphs were generated and statistical analyses performed using GraphPad Prism 8.0 software.Results are presented as mean ± SEM. Significance was assessed using a two-tailed Before examining anti-inflammatory efficacy of berberine, we evaluated whether berberine has cytotoxicity on BEAS-2B cell line. Cells were pre-treated with different concentrations of berberine 0.1 μM-10 μM) or DMSO as control. Cells were stimulated with or without IL-4 plus TNF-α for activation and cell viability was assayed. The data indicated berberine at 1 μM or lower concentrations didn't affect the viability of BEAS-2B cells with or without pro-inflammatory cytokine stimulation compared with DMSO control  can be secreted from airway epithelial cells and fibroblasts after IL-4 plus TNF-α stimulation To investigate how berberine suppressed IL-6 production in activated BEAS-2B cells, the expression and activity of MAP kinases and NF-kB were examined. The expression of p-JNK1/JNK1, p-JNK2/JNK2, p-ERK1/ERK1, and p-ERK2/ERK2 were detected in BEAS-2B cells with IL-4 plus TNF-α stimulation Figure A and 6A.Allergic asthma is a chronic inflammatory disease of the airways that has become a global public health issue to result in a considerable burden on health services and expensive costs. The bronchial epithelium is recognized as a regulator of the initiation and maintenance of allergic airway inflammation Ephedra spp., which contains ephedrine, causes excitement and unwanted side effects Sophora flavescens, Glycyrrhiza uralensis, and Ganoderma lucidum, suppresses AHR, airway eosinophilia, and Th2 cell-secreted cytokines in asthmatic mice Nigella sativa or purple passion fruit peel were also reported to reduce asthmatic symptoms like cough, wheeze, and shortness of breath in asthmatic patients Scutellaria baicalensis, was reported to suppress STAT3 expression and promotes FoxP3 expression to alleviate asthmatic symptoms in mice Corticosteroids and bronchodilators are the most common treatment of asthma Coptis chinensis, Hydrastis canadensis, Berberis aristata and others, has potential of anti-inflammatory, anti-lipidemic, anti-neoplastic, and anti-diabetic activity in vivo is needed to further investigate. In conclusion, berberine perhaps is able to relieve airway inflammation by suppressing cytokine and chemokine production of epithelial cells.Alkaloid-containing plants have been used as medicine for animal and human starting from 4000 years ago. Alkaloids and derived species have been widely used to treat a variety of illnesses Supplementary figures and tables.Click here for additional data file.


All transgenes were inserted into the ttTi5605 transposon insertion site in chromosome II of wild-type C. elegans animals (N2) using CRISPR/Cas9-mediated genome editing, equalizing any potential positional effects. We observed very faint BFP signal in live worms from the signal copy transgene insertions. Additionally, the signal was further masked by bright autofluorescence from the intestine. To observe the cells expressing these transgenes we performed immunofluorescence experiments using primary antibody against BFP. Using this assay, we studied the expression from touch receptor neuron (TRN)-specific promoters, mec-4p (A) and mec-17p (B) and pan-neuronal promoters, cmk-1p (C) and unc-119p (D). As expected, BFP expression driven by the mec-4promoter is observed exclusively in the six TRN nuclei (A). However, under mec-17 promoter, BFP expression is observed in two additional neuronal nuclei  besides the six TRN nuclei (B). These two neurons are likely to be PVD or PDE neurons based upon the location of their nuclei relative to the PVM nucleus. In case of the pan-neuronal promoters, expression under cmk-1 and unc-119 promoters is observed in most, if not all neuronal nuclei, including in many head neurons (C and D top) and ventral nerve cord (VNC) neurons (C and D bottom). However, in a subpopulation of animals expressing BFP under the unc-119 promoter, intense fluorescence was observed in several additional larger nuclei along the lateral sides , which we identify as hypodermal cells and faint signals in even larger nuclei , which we identify as intestinal cells. We did not observe any fluorescence in our control animals (N2) which were treated similarly (data not shown). While this experiment confirmed expected expression patterns for mec-4p and cmk-1p, additional expression in other neurons for mec-17p and other tissue types in unc-119p promoters could reflect the specific promoter sequences used in this study or that low levels of expression in these unexpected cells were not readily detected from direct observation of fluorescent protein expression analyzed previously.Four transgenes designed to drive expression of Cre recombinase and a nuclear-localized blue fluorescent protein (All worm strains were grown at 20 °C on NGM plates containing OP50-1. Well-fed adult hermaphrodites were fixed according to the protocol described by Michael Koelle (1), with the following modifications:All steps after the initial washing off worms from the NGM plates were performed in 48-well plates instead of microcentrifuge tubes.Instead of centrifugation, worms were allowed to settle by gravity to reduce shear-induced rupturing of worms.Primary antibody against BFP  was used at 1:5000 dilution.F(ab’)2-Goat anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor® 568 conjugate , was used at 1:4000 dilution.(pgSi101[mec-4p::Cre::F2A::BFP::AID::NLS::tbb-2 3’UTR]) IIGN707 (pgSi102[mec-17p::Cre::F2A::BFP::AID::NLS::tbb-2 3’UTR]) IIGN751 (pgSi114[cmk-1p::Cre::F2A::BFP::AID::NLS::tbb-2 3’UTR]) IIGN730 (pgSi110[Cbr-unc-119p::Cre::F2A::BFP::AID::NLS::tbb-2 3’UTR]) IIThe following strains are used in this study.GN706 mec-4p: 1020 bp region immediately upstream of mec-4 start codon (2)mec-17p: 868 bp region immediately upstream of mec-17 start codoncmk-1p: 2000 bp region immediately upstream of cmk-1 start codon (3)Cbr-unc-119p: 988 bp region immediately upstream of Cbr-unc-119 start codon (4)The following promoter sequences was used in this study.Full sequences of all reported alleles are available upon request.