Trp53 (but not Tgfbr2) deletion in endothelial cells (ECs) inhibited radiation-induced EndMT, reducing tumour regrowth and metastases with a high CD44v6+ cancer-stem-cell (CSC) content after radiotherapy. Osteopontin, an EndMT-related angiocrine factor suppressed by EC-Trp53 deletion, stimulated proliferation in dormant CD44v6+ cells in severely hypoxic regions after radiation. Radiation-induced EndMT significantly regulated tumour-associated macrophage (TAM) polarization. CXCR4 upregulation in radioresistant tumour ECs was highly associated with SDF-1+ TAM recruitment and M2 polarization of TAMs, which was suppressed by Trp53 deletion. These EndMT-related phenomena were also observed in irradiated human lung cancer tissues. Our findings suggest that targeting tumour EndMT might enhance radiotherapy efficacy by inhibiting the re-activation of dormant hypoxic CSCs and promoting anti-tumour immune responses.It remains controversial whether targeting tumour vasculature can improve radiotherapeutic efficacy. We report that radiation-induced endothelial-to-mesenchymal transition (EndMT) leads to tumour vasculature with abnormal SMA Radiotherapy is the main treatment for most cancer, but it is unclear if targeting tumour vasculature can enhance tumour radiosensitivity. Here, the authors show that tumour endothelial-mesenchymal transition after radiotherapy leads to proliferation of radioresistant CSCs and tumour associated macrophages polarization. Multiple strategies for targeting cancer cells, cancer stem cells (CSCs), tumour stroma, and tumour endothelial cells (ECs), as well as improving anti-tumour immune responses to increase tumour radiosensitivity, are being developed4. Several anti-angiogenics have been clinically evaluated in combination with radiotherapy6; however, their benefits are controversial. Bone marrow-derived cell (BMDC) recruitment to irradiated tumours may contribute to tumour relapse via vasculogenesis8. Although tumour-vasculature development after radiotherapy is not well characterized, targeting tumour ECs enhances radiotherapeutic efficacy; ceramide, sphingomyelinase, and Bax regulate EC apoptosis after irradiation10. Vascular damage may affect tumour responses to high radiation doses, e.g., during stereotactic radiosurgery/radiotherapy12. ECs lacking ataxia-telangiectasia mutated showed increased radiosensitivity13. However, it remains debatable whether EC targeting can improve radiotherapy efficacy.Anti-angiogenic or vascular-destructive agents potentially enhance tumour responses to radiotherapy14. CSCs are often quiescent after radiation or chemotherapy and their awakening causes tumour relapse and escape16. Understanding the mechanism regulating the dormant or proliferative status better is important for targeting CSCs.Cancer cells that acquire radioresistance exhibit CSC-like characteristics17. Radiotherapy can enhance immunosuppressive responses, including chemotactic signals that recruit several myeloid cell types17. Radio-immunomodulation studies have revealed crucial strategies for effectively combining immunotherapy and radiotherapy.Radiotherapy can stimulate anti-tumour immune responses. Immunomodulation using antibodies against programmed death 1 and programmed death-ligand 1 in combination with radiotherapy has been assessed in clinical trials18, affects the endothelium to enable tumour-cell extravasation19, and may give rise to pericyte-like cells within tumours20. Pericytes play critical roles in blood-vessel maturation and blood-barrier maintenance and regulate vessel integrity and function by interacting with ECs22. Tumour vessels harbouring less pericytes are more sensitive to radiation and chemotherapy23.Endothelial-to-mesenchymal transition (EndMT) promotes cancer-associated fibroblast formation in tumoursHere, we studied tumour EndMT and pericyte-derived tumour vasculature during tumour regrowth after radiotherapy. We analysed the effects of EndMT-regulated vasculature on the irradiated tumour microenvironment, especially, hypoxic dormant CSCs and tumour-associated macrophage (TAM) polarization of bone marrow-derived monocytes (BMDMs).26. Trp53 is a key regulator of radiation responses in ECs, and tansforming growth factor-β (TGFβ)-related signalling potentially is a key regulator of EndMT28. Thus, we explored the effects of small interfering RNA (siRNA)-mediated knockdown of Trp53 and Tgfbr2 on radiation-induced EndMT in human umbilical vein ECs (HUVECs). At 48 h post irradiation (hpi), Trp53 silencing in HUVECs markedly inhibited irradiation-induced messenger RNA (mRNA) expression of Snail1, Snail2, and Zeb2, which encode transcription factors implicated in EndMT29, compared to control siRNA-treated cells, whereas Tgfbr2 knockdown increased their expression , while reversing irradiation inhibited CD31 levels  and vessels were more dilated than in non-irradiated tumours . The changes in tumour size are shown in Supplementary Fig. Trp53-knockdown (EC-p53KD) and -knockout (EC-p53KO) mice . A syngeneic tumour model was established by implanting tumour cells (referred to as KP cells) isolated from a spontaneous lung adenocarcinoma in conditional KrasG12D;Trp53flox/flox mice30. Primary KP cells were implanted at passage 4 or less to maintain the cellular characteristics of spontaneous lung tumour. Trp53 mRNA–VE-cadherin+ cells were dominant in EC-p53KO, but not wild-type (WT) tumours, indicating that p53 was successfully knocked out in tumour ECs of EC-p53KO mice  than in non-irradiated WT (day 15), but not EC-p53KO mice   at 7 dpi in tumours of EC-tdTomato, but not EC-tdTomato-p53KO mice  cells in WT tumours were less TUNEL– or γH2AX– at 3 dpi, but highly Ki67+ (indicative of cell proliferation) during tumour regrowth (23 dpi), in contrast to SMA− ECs in EC-p53KO mice  mice . Supplementary Figure Tgfbr2 knockdown. Post-irradiation p-SMAD2/3 levels of CD31+ vessels were significantly higher in EC-TGFβR2KD than in WT mice -labelled BMDCs  during tumour regrowth after radiotherapy -overexpressing CT26 cells, we verified that mainly CD44v6 was increased in GFP+ cell membranes during tumour regression and regrowth when compared to non-irradiated tumours , in contrast to CD44v6+ cancer cells in non-severely hypoxic areas of irradiated WT tumours  lung tumour colonies (>100 μm tumour diameter) were CD44v6+ vs. 25% in non-irradiated mice  analysis. Various EndMT progression-associated molecules were upregulated at 48 hpi  and upregulated fibroblast-specific genes  in KP ECs isolated from WT, but not EC-p53KO mice  and hypoxic (1%/0.5% O2) conditions with/without OPN, in vitro. Hypoxia increased CD44v6 expression, with non-proliferative (Ki67–) KP cells surviving after irradiation  and proliferative CD44v6+ cells in EC-TGFβR2KD tumours at 21 dpi, and it induced similar effects in WT tumours +F4/80+ TAMs (M2-type) were highly accumulated in irradiated WT, but not EC-p53KO tumour vessels, compared to non-irradiated control vessels  in WT tumours  M2 subtype vs. macrophages cocultured with non-irradiated tumour ECs . When cocultured with irradiated WT, but not EC-p53KO tumour ECs showing EndMT, F4/80ECs Fig. . In contECs Fig. . Cocultuold Fig. .+ cells were highly recruited to both irradiated WT and EC-p53KO tumours  is highly expressed on irradiated ECs, which is inhibited by Trp53 deficiency Fig. . Indeed,urs Fig. . SDF-1+ urs Fig. . Becauseurs Fig. . These M-1+ Fig. .Fig. 6EC+ monocyte differentiation to M1/M2 TAMs, irradiated tumour ECs were cocultured for 48 h with macrophages differentiated from BMDMs by M-CSF. In accordance with the in vivo results, most F4/80+SDF-1+ cells were CD206+ (M2) after coculture with WT tumour ECs, but iNOS+ (M1) after coculture with EC-p53KO tumour ECs  when differentiated with cytokines interleukin-4 (IL-4) and IL-10 from M-CSF-treated BMDMs, whereas after coculture with EC-p53KO tumour ECs, iNOS+SDF-1+F4/80+ cells were flattened (like M1 macrophages) when differentiated with lipopolysaccharides (LPS) plus interferon-γ (IFN- γ) from M-CSF-treated BMDMs  of SDF-1+F4/80+ cells, which was inhibited by AMD3100 + CSCs and M2 macrophages in lung tissues of patients with lung cancer who received radiotherapy (Supplementary Table n = 10), which were compared to non-irradiated human lung tissues (n = 10) , although they were detected more in irradiated than in non-irradiated tissues  therapy or chemotherapy feature perivascular cells with significantly increased SMA expression38. Thus, radiotherapy with anti-VEGF or anti-angiogenic therapy may be more efficient when combined with a strategy for inhibiting tumour EndMT.The findings of this study support a model for TRP53-regulated radiation-induced EndMT and tumour vasculature Fig. . EndMT i8. In our study, during early tumour regrowth, BMDCs recruited to EndMT vasculature after irradiation colocalized with SMA+ cells, but not with ECs. We hypothesize that the recruited BMDCs support SMA+ vessel maturation, as the origin of tumour-vascular pericytes was traced to mesenchymal progenitor cells or BMDCs39. A single, high dose (20 Gy) of non-curative radiation used to examine vasculature development during the initial-response, early-regrowth, and late-regrowth tumour phases was insufficient to eradicate all intratumour and peripheral tumour vessels or angiogenic vessels surrounding the tumours. In contrast, tumour EndMT was rarely detected in recurrent KP tumours after radiotherapy with 2 times 25 Gy  CSCs may become awakened by angiocrine factors (such as OPN) secreted during tumour EndMT-related revascularization around hypoxic regions. Furthermore, we consider CD44v6+ CSCs more important than other CSCs in radiotherapy, as CD44v6+ populations were induced by radiation and might be mainly responsible for post-radiotherapy tumour regrowth and metastasis.We found that EC-p53KO tumours regulated OPN, an angiocrine factor acting on radioresistant CSCs, and EC receptors, which in turn controlled TAMs, in the radioresistant tumour microenvironment, supporting the observed role of tumour angiocrine factors in the crosstalk between CSCs and tumour vessels3. High-dose radiation induces antigen production via tumour cell death, tumour-specific immunity, and tumour-vascular damage49. Radiation can attract immunosuppressive cells to the tumour microenvironment17. TAM modulation is considered important in cancer immunotherapy because of the tumour-promoting effects of enhancing an immunosuppressive microenvironment and tumour angiogenesis51. Klug et al.52 reported that low-dose irradiation stimulates immunostimulatory macrophages, enabling cytotoxic T cells to infiltrate tumours, thus boosting tumour immunity. In the current study, we discovered that tumour vascularization via EndMT enhanced TAM polarization toward M2-like macrophages and proliferation after radiotherapy. M0 macrophages differentiated from BMDMs prominently showed Arg1+ M2 polarization in coculture with radiation-EndMT-derived tumour ECs. However, BMDMs mainly polarized to the iNOS+ M1 subtype during coculture with ECs from EC-p53KO mice, in which radiation-induced EndMT was inhibited. Accordingly, in vivo, at 1 dpi, tumours recruited monocytes and F4/80+ macrophages. At 7 dpi, M2 macrophages were mainly seen in WT tumours, while M1 macrophages were predominantly observed in irradiated EC-p53KO tumours, indicating that the tumour-vascular niche regulates TAM polarization after radiotherapy. Detailed effects of radiation-induced EndMT on other immune cells, including cytotoxic T and antigen-presenting cells, remain to be explored.Irradiation seems to modulate tumour immunogenicity in conventional radiotherapy and stereotactic ablative radiotherapy50. Interestingly, strongly SDF-1+ monocytes and subsequently SDF-1+CD206+ M2 macrophages were recruited to KP tumours after radiotherapy. Furthermore, EndMT tumour-derived ECs highly expressing CXCR4 induced M2 polarization and proliferation of SDF-1+ macrophages, which was inhibited by Trp53 silencing. AMD3100 significantly inhibited M2 SDF-1+ macrophage polarization in coculture with irradiated tumour ECs. Relevant to our findings with SDF-1+ macrophages, SDF-1 produced in monocytes contributes to macrophage differentiation36. In accordance with previous findings53, SDF-1+ tumour cells were observed after radiation, but in this study, SDF-1 expression was higher in monocytes than in tumour stromal cells; this discrepancy may reflect a requirement for SDF-1 cleavage for cellular activity, as monocytic SDF-1 was not strongly detected with antibodies targeting the C terminus of SDF-1. We are currently studying SDF-1 cleavage in relation to monocyte differentiation.High SDF-1/CXCL12 expression in tumours aids in the capture of CXCR4-expressing monocytes11. In this study, targeting EndMT more efficiently inhibited tumour regrowth after a single high dose of radiation than after fractionated radiotherapy. In fractionated radiotherapy, tumour EndMT was significantly inhibited by EC-p53KO; however, CD44v6+ CSC proliferation and tumour growth were not inhibited. Nevertheless, we cautiously suggest that in fractionated radiotherapy, targeting EndMT may enhance therapeutic efficacy when combined with inhibition of CSC proliferation.The effect of radiation-induced tumour-vascular damage on TAM polarization, which was increased by EndMT, suggests that high-dose radiation (~10 Gy) can evoke stronger tumour immune responses than low-dose radiation, because it indirectly causes tumour cell death via vascular damage+CD44v6+ CSCs and SDF-1+CD206+ macrophages, even though the patients had received fractioned radiotherapy, indicating that radiation-induced CD44v6+ tumour cells and SDF-1+ M2 macrophages might be targeted for overcoming tumour radioresistance.In accordance with our findings in vivo, tumour EndMT was prominently seen in lung cancer tissues of patients who received irradiation, with large populations of OPNIn conclusion, this study provides evidence that targeting radiation-induced EndMT is a new, promising strategy for enhancing tumour radiosensitivity by inhibiting radiation-induced abnormal tumour vasculature. Specifically, targeting vascular EndMT may be effective in regulating radioresistant CSC proliferation and maximizing the anti-tumour immunity of radiotherapy.54. Specific pathogen-free C57BL/6 Tie2-Cre, Trp53flox/flox, Tgfbr2flox/flox, and LSL-KrasG12D mice were purchased from the Jackson Laboratory. Tie2-GFP mice were a kind gift from Dr. Gou Young Koh (Korea Advanced Institute of Science and Technology). Male Tie2-Cre mice were crossed with female Trp53flox/flox, Tgfbr2flox/flox, or Trp53flox/floxTgfbr2flox/flox (generated by crossing Trp53flox/flox and Tgfbr2flox/flox) mice to generate Tie2-Cre;Trp53flox/+, Tie2-Cre;Trp53flox/flox, Tie2-Cre;Tgfbr2flox/+, Tie2-Cre;Trp53flox/+Tgfbr2flox/+, or Tie2-Cre;Trp53flox/floxTgfbr2flox/+ mice. Tie2-Cre;Trp53+/+, Tie2-Cre;Tgfbr2+/+, Tie2-Cre;Trp53+/+Tgfbr2+/+, or Tie2-Cre–/– littermates were used as controls. All animal experimental data shown are representative of three independent experiments. All experiments were conducted with 6–8-week-old mice. The mice had access to a standard diet and water ad libitum. All mice were anaesthetized with a combination of anaesthetics before being killed. For in vivo proliferation assays, mice received an intraperitoneal injection of 5-bromo-2-deoxyuridine (BrdU) (2 mg/mouse) (Sigma-Aldrich) 4 h before tumour harvesting. BrdU+ cells were detected using an anti-BrdU mouse monoclonal antibody .All animal experiments were approved by the Institutional Animal Care and Use Committee of the Korea Institute of Radiological & Medical Sciences and are reported in accordance with the ARRIVE  guidelinesn = 7; Origene tissues, n = 3) who received radiotherapy and 10 patients  who did not receive radiotherapy are shown in Supplementary Table The analysis of lung cancer patient tissues was approved by the ethics committee of Severance Hospital, Yonsei University (Korea). In addition, a lung cancer tissue with information related to radiotherapy was purchased from Origene (cat. no. CT565899). Clinicopathological characteristics of 10 patients  were injected subcutaneously into the right thigh of 8-week-old male BALB/c mice. When the tumour volume reached 150–200 mm3, tumours or whole mice were irradiated with a sublethal dose (8 Gy). Bone marrow cells were harvested from the tibias and femurs of male BALB/c donor mice and were fluorescently labelled with CM-Dil dye , per the manufacturer’s instructions. CM-Dil-labelled cells (2 × 107) were suspended in 200 μl of Opti-MEM (Gibco) and intravenously injected into whole body-irradiated mice at 6 hpi. Tumours from control and transplanted mice were harvested at 7 dpi. KP tumour-bearing recipient mice were sub-lethally irradiated (9 Gy), and 9 h after irradiation, they received Tie2-GFP mouse-derived bone marrow cells (1 × 107 cells) intravenously and were allowed to recover for 3 weeks.CT26 cells .Tumour tissues were covered with optimal cutting temperature (OCT) compound  and snap-frozen on dry ice or fixed in 10% (v/v) neutral-buffered formalin, embedded in paraffin, and sectioned. The sections were deparaffinized and stained as previously described5), differentiated by treatment with murine M-CSF every other day for 7 days, were placed on the tumour ECs. The cocultures were harvested at 48 h. Tumour ECs and BMDMs cultured alone were used as controls.For coculture experiments, tumour ECs were plated in 6-well plates, with or without radiation. BMDMs (1.2 × 105) were injected subcutaneously into the right thighs of C57BL/6 mice. When the tumours reached 150–200 mm3, a single dose of radiation (20 Gy) was delivered using the X-RAD 320 platform (Precision X-ray). AMD3100 (Sigma-Aldrich) was dissolved in distilled water, diluted in phosphate-buffered saline, and administrated daily intraperitoneally (5 mg/kg) for 7 days.KP tumour cells (2 × 105) were injected subcutaneously into the right thighs of male BALB/c mice. KP tumour cells (2 × 105) were injected subcutaneously into the right thighs of Tie2-Cre;Trp53flox/+, Tie2-Cre;Trp53flox/flox, Tie2-Cre;Tgfbr2flox/+, Tie2-Cre;Trp53flox/+Tgfbr2flox/+, Tie2-Cre;Trp53flox/floxTgfbr2flox/+, Tie2-Cre;R26RtdTomato;Trp53flox/flox, and VE-cadherin-CreERT2;Trp53flox/flox mice and control littermates. Tumour volumes were determined according to the formula (L × W × H)/2, using the calliper-measured tumour length (L), width (W), and height (H). When tumour volumes reached 150–200 mm3, radiation was delivered, using the X-RAD 320 platform (Precision X-ray). For OPN blocking experiments, mice were intraperitoneally injected with an anti-mouse OPN neutralizing antibody  four times, every 2 days.CT26 cells  1 min prior to killing. For hypoxia assessment, pimonidazole (60 mg/kg) was administered intraperitoneally. After 45 min, the mice were killed and tumours were harvested, fixed, and immunostained using the Hypoxyprobe Plus Kit (Hypoxyprobe) per the manufacturer’s instructions. To evaluate tumour metastasis, metastatic nodules on lung surfaces observed by the naked eye were counted. To visualize tumour vessel leakage, mice were intravenously injected with fluorescein isothiocyanate–dextran  5 min before killing.At day 7, tumour samples were harvested and digested into single-cell suspensions. Cells were stained with macrophage-specific antibodies. The cells were fixed and permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences) and stained with an anti-SDF1 antibody. Then, the cells were analysed on a FACSCalibur flow cytometer . Gating strategies for flow cytometry analysis are shown in Supplementary Figure 137Cs] source (Atomic Energy of Canada) at 3.81 Gy/min. To establish hypoxia, cells were incubated in a Forma 1025/1029 Anaerobic Chamber (Thermo Fisher Scientific) flushed with 0.5% or 1% O2 for severe or mild hypoxia, respectively. Cells were incubated with 10 μM EdU for 2 h, trypsinized, and surface-stained with an anti-CD44v6 antibody . The cells were labelled using the Click-iT Alexa Fluor 488 Assay Kit  and analysed on a FACSCalibur .For in vitro proliferation assays, KP cells were irradiated with gamma rays from a  source (Atomic Energy of Canada) at 3.81 Gy/min. For the analysis of secreted proteins, conditioned media were collected between days 6 and 7 of HPMEC culture after irradiation and assayed using Proteome Profiler Human Soluble Receptor Array, Hematopoietic Kit , according to the manufacturer’s instructions. For the analysis of cellular proteins, immunocytochemistry was performed as previously described26. OPN released by HUVECs into the culture medium was measured using a human OPN Quantikine ELISA Kit (R&D Systems), following the manufacturer’s instructions.HUVECs, human pulmonary microvascular endothelial cells (HPMECs), and human pericytes were obtained from PromoCell  and were cultured in Endothelial Cell Growth Medium 2, Endothelial Cell Growth Medium MV2, and Pericyte Growth Medium, respectively (Promocell) under 5% CO55. Fragments-per-kilobase-of-transcript values were calculated with Cufflinks55. Genes with a maximal log2-transformed read count <5 across samples were excluded from the analysis. To identify differentially expressed genes, we compared the direction of fold changes (>1.2) against irradiation (IR) alone. Reverse-regulated genes (>1.2-fold) between TGFBR2 knockdown+IR and p53 knockdown (with and without TGFBR2 knockdown)+IR compared with IR alone were selected. To analyse angiocrine factors and phenotypic changes in endothelial cells, matrisome gene lists and surfactome gene lists were used. The matrisome comprises core matrisome-related proteins, including extracellular matrix (ECM) glycoproteins, collagens, and proteoglycans, as well as ECM-associated proteins, including ECM-affiliated proteins, ECM regulators, and secreted factors56. The surfactome represents proteins at the plasma membrane, including G protein-coupled receptors, receptor tyrosine kinases, and integrins57. Among the differentially expressed genes, matrisome and surfactome genes were displayed in R using the ComplexHeatmap package58 and were used as input for the GeneMANIA 3.4.1 plugin59 within Cytoscape 3.4.060 for functional enrichment analysis. RNA-seq data generated in this study are available on Gene Expression Omnibus Total RNA was isolated from HUVECs, and RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA-seq libraries were constructed using the SENSE mRNA-Seq Library Prep Kit (Lexogen), according to the manufacturer’s instructions, and were sequenced as 100 bp paired-end runs on the HiSeq 2000 platform (Illumina). RNA-seq reads were mapped to UCSC hp19 using TopHat softwareGAPDH. Primers are listed in Supplementary Table RNA was isolated using TRI reagent , and 1 μg of RNA was used to synthesize complementary DNA with an Omniscript RT Kit . PCR was conducted in triplicate on the CFX96 TM Real-Time system , using qPCR SYBR Green master mix (Invitrogen). For each sample, target gene expression was normalized against the geometric mean of the reference gene t-test (Figs. p value < 0.05 was regarded significant. Experimenters were blinded to group assignments and outcome assessments.Student’s st Figs. , 7a–d anSupplementary InformationPeer Review File


Salmonella genomic island 1 (SGI1) is an integrative mobilizable element integrated into the chromosome of bacteria, which plays an important role in the dissemination of antimicrobial resistance genes. Lots of SGI1 variants are found mainly in Salmonella enterica and Proteus mirabilis. In this study, a total of 157 S. enterica and 132 P. mirabilis strains were collected from food-producing animals in Sichuan Province of China between December 2016 and November 2017. Detection of the SGI1 integrase gene showed that three S. enterica and five P. mirabilis strains were positive for SGI1, which displayed different multidrug resistance profiles. Five different SGI1 variants, including two novel variants (SGI1-PmBC1123 and SGI1-PmSC1111), were characterized by whole genome sequencing and PCR linkage. In two novel SGI1 variants, IS26-mediated rearrangements resulted in large sequence inversions of the MDR regions extending outside the SGI1 backbone. The sul3-type III class 1 integron (5′CS-sat-psp-aadA2-cmlA1-aadA1-qacH-IS440-sul3) and gene cassettes aac(6′)-Ib-cr-blaOXA–1-catB3-arr-3 are found in SGI1-PmSC1111. Mobilization experiments indicated that three known variants were conjugally mobilized in trans to Escherichia coli with the help of a conjugative IncC plasmid. However, the two novel variants seemed to lose the mobilization, which might result from the sequence inversion of partial SGI1 backbone. The identification of the two novel SGI1 variants in this study suggested that IS26-mediated rearrangements promote the diversity of SGI1. Salmonella genomic island 1 (SGI1) is an IME initially identified in the multidrug resistance (MDR) Salmonella Typhimurium DT104 clone , such as integrative and conjugative elements (ICEs) and integrative mobilizable elements (IMEs), are distinct regions integrated into the chromosome of bacteria and acquired via horizontal transfer . GIs oft04 clone . SGI1  have been reported (trmE gene) as SGI1. SGI1 and related islands often harbor various antimicrobial resistance genes including carbapenems resistance gene blaNDM–1  and 132 P. mirabilis strains (74 from swine and 58 from chicken) isolated from diseased tissues or anal swabs of animals among 30 poultry and 30 swine farms in Sichuan Province between December 2016 and November 2017. All isolates were identified using an automated system . The presence of SGI/PGI/AGI/GIPmi1 was screened by PCR targeting the integrase gene , amoxicillin-clavulanic acid (AMC), cefoxitin (FOX), cefotaxime (CTX), ceftriaxone (CRO), chloramphenicol (CHL), florfenicol (FFC), nalidixic acid (NAL), ciprofloxacin (CIP), streptomycin (STR), spectinomycin (SPT), apramycin (APR), doxycycline (DOX), trimethoprim (TMP), sulfizoxazole (SUL), trimethoprim-sulfamethoxazole (SXT) and polymyxin B (PB). S. enterica and acquired antimicrobial resistance genes were identified by MLST 2.01 and ResFinder 3.12, respectively. The complete nucleotide sequences of SGI1 variants were analyzed using the BLAST programs3. SNPs from genomes of the strains positive for SGI/PGI were called and a phylogeny based on the concatenated alignment of the high quality SNPs was inferred using CSI Phylogeny 1.44 with parameters as defaults.All SGI/PGI-positive strains were sequenced using Illumina HiSeq platform (400-bp paired-end reads with about 200-fold average coverage). The draft genomes were assembled using software SPAdes_3.12.0. The gaps among contigs that carried SGI1 fragments were filled in by PCR linkage. Because the complete genetic structures of SGI1 in strains PmBC1123 and PmSC1111 could not be assembled by PCR linkage, whole genomes of those two strains were further sequenced using PacBio RS II sequencing instrument (100-fold average read depth). The chromosomes were assembled into one scaffold using software SMRT portal v.3.2.0. The MDR regions were confirmed by PCR linkage between regions belonging to non-repeated genetic elements. Multi-locus sequence type of in trans to other bacteria with the help of the conjugative IncA/C plasmid /streptomycin (30 mg/L). The transfer frequency of SGI1was determined by dividing the number of E. coli SGI1 transconjugants by the number of P. mirabilis or S. enterica donor cells , MH998665 (SGI1-PmSC1111), CP034091 (PmBC1123) and CP034090 (PmSC1111), respectively. The Whole Genome Shotgun projects have been deposited at DDBJ/ENA/GenBank under the accession RQSE00000000 (SCMYP1), RQJQ00000000 (SC10), RQSD00000000 (SC968), RQSF00000000 (PmBC55), RQSG00000000 (PmSN55) and RQSH00000000 (PmDJ107).The complete nucleotide sequences of two novel SGI1 variants and the genomes of Pmi1 integrase gene showed that 3 S. enterica and 5 P. mirabilis strains were positive for SGI1. Antimicrobial susceptibility testing indicated those 8 strains displayed different MDR profiles . S. Albany strain SCMYP1 of swine origin exhibited resistance to the third generation cephalosporins and polymyxin B. Two P. mirabilis strains (PmSN55 and PmDJ107) of chicken origin were resistance to amoxicillin-clavulanic acid, cefoxitin and the third generation cephalosporins. Three SGI1-containg S. enterica strains belonged to different STs  and S. Infantis strain SC10 (ST32) harbors SGI1 variant SGI1-F that carries dfrA1-orfC and blaCARB–2 (ampicillin) gene cassettes, as well as resistance genes sul1 (sulfonamides), tetA(G) (tetracycline) and floR (chloramphenicol and florfenicol). S. Derby strain SC968 (ST40) harbors SGI1 variant SGI1-I that carries gene cassettes aadA2 (streptomycin and spectinomycin) and dfrA1-orfC. Three P. mirabilis strains harbor SGI1 variant SGI1-W carrying aadA2-lnuF gene cassettes. SGI1-W was firstly detected in P. mirabilis of poultry origin in China , hph (hygromycin) and aacC4 (apramycin) shows 99.9% nucleotide identity to the corresponding region of Tn6450  that carries gene cassettes n SGI1-Z  and SGI1n SGI1-Z , respect in PGI1 . The 20.f Tn6450 , indicatalg firstly described in Vibrio cholerae serogroup O103  being in inverse orientation and flanked by 8-bp inverted repeats (AAATGTGT)  belonging to sul3-type III (res gene (S027) carries gene cassettes dfrA1-orfC. This region contains five copies of IS26 in the same orientation. However, the 8-bp target site duplications could not be observed around these IS26, suggesting this complicated MDR region might be formed via the incorporation of several IS26-mediated translocatable units successively (aac(6′)-Ib-cr (fluoroquinolones and aminoglycosides), blaOXA–1 (ampicillin), catB3 (chloramphenicol) and arr-3 (rifampicin) never reported to date in SGI1.SGI1-PmSC1111 is 82,352 bp in size . A 1,258 bp insertion sequence, encoding transposase OrfAB subunits A and B, was inserted into the backbone gene S007 and flanked by 3-bp target site duplication (AAG). This IS element shows 98.6% nucleotide identity to ISoup O103 , and 86%e SGI1-K . An IS26AATGTGT) . The dowtype III . The claessively , 2016. TtrmE gene  in  species . HoweverS. enterica and P. mirabilis isolates are listed in S. Albany strain SCMYP1 harbors blaCTX–M–55 and mcr-1, explaining the resistance to third generation cephalosporins and polymyxin B, respectively. P. mirabilis strains PmSN55 and PmDJ107 harbors AmpC cephalosporinase gene blaCMY–2 carried by an 11.7-kb contig that is identical to the corresponding region of SXT/R391 ICE ICEPmiJpn1 . Very recently we reported a novel SXT/R391 ICE that carried cfr, blaCTX–M–65, fosA3, and aac(6′)-Ib-cr in P. mirabilis and could be transferred to E. coli  and gene cassettes aac(6′)-Ib-cr-blaOXA–1-catB3-arr-3 are reported in SGI1 for the first time. Our study highlights that IS26-mediated rearrangements promote the diversity of SGI1.In this study, we characterized SGI1 in The datasets generated for this study can be found in the GeneBank, MH998664, MH998665, CP034091 and CP034090.This study was carried out in accordance with the recommendation of ethical guidelines of Sichuan University. The protocol was approved by the Sichuan University Animal Ethics Committee. Individual informed consent for the use of samples was obtained from all the animal owners.X-CW, C-WL, Z-ZK, and YZ performed the experiments. C-WL analyzed the data and conceived of the study. X-CW, C-WL, and Z-ZK wrote the manuscript. All authors contributed to manuscript revision and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Activating TRPML1 channels with ML-SA1 acidified endolysosomes and restricted Tat-mediated HIV-1 LTR transactivation. These effects of ML-SA1 appeared to be mediated through activation of BK channels, because the effects of ML-SA1 on Tat-mediated HIV-1 LTR transactivation were blocked using pharmacological inhibitors or shRNA knock-down of BK channels. On the other hand, activating TRPML1 and BK channels enhanced cellular degradation of exogenous Tat. These results suggest that acidifying endolysosomes by activating TRPML1 or BK channels may provide therapeutic benefit against latent HIV-1 infection, HIV-1 associated neurocognitive disorders, and other HIV-1 comorbidities.HIV-1 Tat is essential for HIV-1 replication and plays an important role in latent HIV-1 infection, HIV-1 associated neurological complication, and other HIV-1 comorbidities. Secreted from HIV-1 infected or transfected cells, Tat can be up-taken into cells by receptor-mediated endocytosis and internalized into endolysosomes. To reach nucleus where it can facilitate HIV-1 viral replication, exogenous Tat has to escape the degradation by endolysosomes. Because of findings that endolysosome de-acidification with, for example, the weak-base anti-malarial drug chloroquine prevents exogenous Tat degradation and enhances the amount of Tat available to activate HIV-1 LTR, we hypothesize that acidifying endolysosomes may enhance Tat degradation in endolysosomes and restrict LTR transactivation. Here, we determined the involvement of endolysosome-resident transient receptor potential mucolipin 1 channel (TRPML1) and the big conductance Ca Although combined antiretroviral therapy (ART) effectively suppresses HIV-1 replication, it does not completely eliminate the virus. In this ART era, reservoirs of HIV-1 exist centrally and peripherally6, low levels of neuroinflammation persist, and the prevalence of HIV associated neurocognitive disorders (HAND) remains high (30–50%)8. The existence of viral reservoirs makes complete eradication of HIV-1 extremely challenging11. Therefore, additional strategies are needed to block viral reactivation in sanctuary sites and to prevent disease progression including HAND. Because ART does not block Tat secretion from HIV-1 infected cells12, and brain levels of Tat remain elevated even when HIV-1 levels are below detectable levels with ART13, one strategy might be to prevent Tat from activating HIV-1 replication through elongation of the HIV-1 long terminal repeat (LTR)16.Infecting 37 million people globally20 and extracellular Tat crosses plasma membranes by various mechanisms including endocytosis; a major pathway for Tat entry22 following interactions with specific cell surface proteins and receptors26. Once internalized into endolysosomes, Tat has to escape from endolysosomes into the cytosol before it transits to the nucleus and activates the HIV-1 LTR promoter29. Typically, robust HIV-1 LTR transactivation requires high concentrations of exogenous Tat and disruption of plasma membranes using, for example, scrape-loading methods30. When endogenously expressed in cytosol, Tat can be imported directly into nucleus and activate HIV-1 LTR transactivation using importin β-dependent nuclear localization signals31. In contrast, secreted Tat or exogenously added Tat has to first enter the endolysosome system via endocytosis. Thus, avoiding endolysosome degradation is critical for exogenous Tat to first escape endolysosomes and then enter nucleus to activate HIV-1 Tat LTR transactivation. Consistent with findings of others33, we found that the lysosomotropic agent chloroquine enhanced extracellular Tat-mediated HIV-1 LTR transactivation34. Given that chloroquine does not increase22, or even decrease35, HIV-1 LTR transactivation under conditions when Tat is expressed intracellularly, it is generally thought that chloroquine, a weak base, neutralizes the acidic pH of endolysosomes and prevents exogenous HIV-1 Tat degradation, thus increasing the amount of Tat available to activate HIV-1 LTR in nucleus.Two-thirds of cellular Tat can be secreted from HIV-1 infected or transfected cells37. Others and we have found that activating endolysosome-resident transient receptor potential mucolipin 1 channel (TRPML1) channels with the agonist ML-SA1 resulted in endolysosome acidification39. In the present studies, we determined the involvement of TRPML1 and the big conductance Ca2+-activated potassium (BK) channel in regulating endolysosome pH, as well as extracellular Tat-mediated HIV-1 LTR transactivation and cellular degradation of extracellularly added Tat. TRPML1 and BK channel activation acidified endolysosomes, enhanced cellular degradation of extracellularly added Tat, and restricted Tat-mediated HIV-1 LTR transactivation. Thus, TRPML1 and BK channels might be targeted therapeutically to prevent re-activation of latent HIV-1 infection, and to decrease the prevalence and severity of HIV-1 associated neurocognitive disorders and other HIV-1 comorbidities.Thus, acidifying endolysosomes could enhance HIV-1 Tat degradation in endolysosomes, preventing Tat escape from endolysosomes and blocking subsequent activation of HIV-1 LTR in the nucleus. The acidic endolysosome luminal pH is maintained by the electrogenic pumping of protons by vacuolar-ATPase (v-ATPase) in conjunction with chloride and other ions37. Recent studies have shown that ML-SA1 activation of TRPML1, a cation channel residing on endolysosomes, decreases endolysosome pH40. Accordingly, it was important for us to first confirm using U87MG cells the extent to which ML-SA1 affects endolysosome pH. The concentration of 20 μM was used because at this concentration ML-SA1 protects against lysosomal lipid accumulation in Niemann-Pick type C40 and clears intraneuronal accumulation of amyloid beta39. We found that ML-SA1 (20 μM) decreased significantly (p < 0.001) endolysosome pH by about 0.15 pH units  decreased protein levels of TRPML1 Fig. . We next42, we determined next the involvement of BK channels in ML-SA1-induced acidification of endolysosomes and reduction in Tat-mediated LTR transactivation. Using immunostaining methods, we demonstrated that BK channels co-localized with LAMP1 and TRPML-1 with similar Pearson’s correlation coefficients  decreased protein expression levels of BK channels Fig. . FunctioNext, we determined potential functional interactions between TRPML1 and BK channels. BK channel knockdown decreased significantly (p < 0.01) the ability of ML-SA1 to acidify endolysosomes Fig.  and prev33. As such, acidifying endolysosomes with ML-SA1 and NS1619 could enhance cellular degradation of exogenously added Tat and decrease the amount of Tat available to activate HIV-1 LTR in nucleus. Here, we tested this hypothesis by directly measuring cellular protein levels of Tat using immunoblotting methods. Cells were co-treated with 5.0 μg/ml of Tat and DMSO as a control, ML-SA1 (20 μM), or NS1619 (20 μM) for 24 h in the presence of chloroquine (100 μM). ML-SA1 and NS1619 significantly (p < 0.001) decreased cellular protein levels of Tat /H+ exchanger73 or endolysosome membrane hyperpolarization following BK channel activation74 may enhance the activity or assembly of v-ATPase.TRPML1 channels can form macromolecular complexes with BK channels and BK channels are activated by TRPML1-mediated calcium release20, Tat can bind to cell surface receptors including LRP1, heparin sulfate proteoglycan, CD26, and CXCR4 that all participate in receptor-mediated endocytosis25. Following its endocytosis, Tat enters endolysosomes before escaping into the cytosol, where it can transit to the nucleus and activate the HIV-1 LTR promoter29. Consistent with others’ findings33, we demonstrated that chloroquine, a well-known anti-malarial drug that affects morphological features and functions of endolysosomes, increased exogenous Tat-mediated HIV-1 LTR transactivation. A consequence of enhanced HIV-1 LTR transactivation is increased HIV-1 replication and enhanced HIV-1 infectivity. Indeed, not only does chloroquine enhance HIV-1 LTR transactivation, it also enhanced HIV-1 infectivity in cells that require endocytosis for HIV-1 virus entry76. However, chloroquine did not increase HIV-1 LTR transactivation when Tat was transiently expressed intracellularly35, under which condition Tat can be imported directly into nucleus and activate HIV-1 LTR transactivation31. Thus, chloroquine-induced endolysosome de-acidification likely prevents the degradation of exogenous HIV-1 Tat in endolysosomes and increases the amount of Tat available to activate LTR in the nucleus33. Furthermore, we found that endolysosome de-acidification by the chloroquine derivative LYS01 and the v-ATPase inhibitor bafilomycin enhanced exogenous Tat-mediated LTR transactivation34. Moreover, lipofectamine-based transfection reagents, which avoid lysosome degradation, enhanced exogenous Tat-induced LTR transactivation77. Thus, although it remains unclear as to how Tat escapes endolysosomes to enhance LTR transactivation, it is clear that endolysosome pH plays an important role in regulating the cellular levels of Tat and its actions.Secreted from HIV-1 infected cellsBecause of our findings that activating TRPML1 with ML-SA1 acidified endolysosome and prevented chloroquine-induced increases in endolysosome pH, we determined whether activating TRPML1 affected Tat-mediated HIV-1 LTR transactivation. ML-SA1 attenuated Tat-mediated HIV-1 LTR transactivation and when TRPML1 was knocked down, ML-SA1-induced restriction in Tat-mediated HIV-1 LTR transactivation was completely prevented. We speculate that endolysosome pH is critical for degradation of Tat degradation via pH-sensitive hydrolysis in endolysosomes. Given that optimum pH is require for efficient enzymatic hydrolysis in endolysosomes, a critical level of endolysosome pH may be required for optimum activities of endolysosomal enzymes that degrade Tat protein efficiently. As such, even though TRPML1 protein was only partially knock-down, ML-SA1 might fail to decrease the endolysosome pH to this critical level, and Tat cannot be degraded efficiently, leaving enough Tat available to mediate HIV-1 LTR transactivation.Because we found that activating BK channels acidified endolysosomes and that pharmacological blocking of BK channels and knocking down BK channels attenuated ML-SA1-induced endolysosome acidification, we next determined the involvement of BK channels in Tat-mediated HIV-1 LTR transactivation. Similar to ML-SA1, activating BK channels restricted Tat-mediated HIV-1 LTR transactivation. In addition, BK channel knockdown prevented the restrictive effect of NS1619 on Tat-mediated HIV-1 LTR transactivation. Furthermore, blocking BK channels with penitrem A and knocking down BK channels prevented ML-SA1-induced restriction in Tat-mediated HIV-1 LTR transactivation, whereas knocking down TRPML1 did not prevent NS1619-induced restriction in Tat-medicated HIV-1 LTR transactivation. In addition, ML-SA1 and NS1619 significantly decreased cellular protein levels of exogenous Tat. Together our data suggests that activating TRPML1 channels and subsequent activation of BK channels acidifies endolysosomes, enhances endolysosome degradation of exogenous Tat, and reduces cellular amount of Tat available to mediate HIV-1 LTR transactivation Fig. .Figure 8In summary, our findings indicate that activating TRPML1 channels and subsequent activation of BK channels acidifies endolysosomes, enhances cellular degradation of exogenous Tat, and restricts Tat-mediated LTR transactivation. Our findings suggest that TRPML-1 and BK channels may represent therapeutic targets against HIV-1 infection, HIV-1 associated neurocognitive disorders, and other HIV-1 comorbidities.2 95% O2 incubator at 37 °C temperature. U87MG cells were stably transfected with an integrated luciferase gene under the control of an HIV-1 Tat-driven LTR promoter following neomycin antibiotic (Sigma-Aldrich) selection pressure. Cells were grown to 50% confluence in 35 mm2 dishes and 96 well plates, and cells were not used past their tenth passage. These cells were provided generously by Dr. Lena Al-Harthi .U87MG cells were grown in 1X DMEM (Invitrogen) supplemented with 10% fetal calf serum and penicillin/streptomycin (Invitrogen) in a 5% COU87MG cells were seeded at a confluency level of about 30–40%  on 96 well plates one-day prior to being taken for experimentation. Cells were incubated with 2 μg/ml HIV-1 Tat (ABL Inc. and NIH AIDS program) in the presence of 100 μM of chloroquine (Sigma-Aldrich). In some experiments, cells were co-treated with TRPML1 agonist ML-SA1 (Millipore), BK channel blocker Penitrem A (Tocris), or BK channel activator NS1619 (Tocris). 48 hrs post-incubation, luciferase activity assays (Promega) were performed and relative luminescence units were quantified using a fluorometer/luminometer plate-reader (Spectra MAX GEMINI EM).In U87MG cells, expression levels of TRPML1 and BK channels were knocked down using 1 μg/ml shRNA (Santa Cruz) against TRPML1 (Sc-44519) and BK channels (Sc-42511); control shRNA (Sc-108060) was used as a control. Jet prime reagent (2 μl) was used for transfection. Following 36 h incubation, 25 μg/ml puromycin (Invitrogen) was added to activate the shRNA promoter. After incubation for 2 to 3 days, cells were passaged to remove the dead and dying cells, and the remaining living cells were cultured for an additional 36 h prior to being taken for experimentation. Knockdown efficiency was confirmed by immunoblotting using antibodies against TRPML-1  and BK-channel .64. Cells were loaded with LysoSensor (2 µM) for 5 minutes at 37 °C. Light emitted at 510 nm in response to excitation at 340 nm and 380 nm was measured for 20 ms every 5 seconds using a filter-based imaging system (Zeiss) and data were analyzed using Slidebook 6 software (3i). The ratio of light excited at 340/380 nm and emitted at 510 nm was converted to pH using a calibration curve established using 10 µM of the H+/Na+ ionophore monensin, and 20 µM of the H+/K+ ionophore nigericin dissolved in 20 mM 2-(N-morpholino) ethane sulfonic acid (MES), 110 mM KCl, and 20 mM NaCl; pH was adjusted between 3.0 to 7.0 with HCl/NaOH.Endolysosome pH was measured ratiometrically with LysoSensor ; a dual excitation dye for pH measurements in acidic organelles78. Cells were incubated with 2 µM of Fura-2/AM for 1 hour at 37 °C. After incubation, cells were washed twice with calcium-free media (with 0.2 mM EGTA). Cells were excited at 340 and 380 nm and light emission was captured at 510 nm using our filter-based imaging microscope . Images were taken using Slidebook 6 (3i) software every 2 seconds; baseline levels and peak increases in free intracellular calcium were determined. The levels of free intracellular calcium levels were represented as a ratio of 340/380 nm.Free intracellular calcium levels were measured ratiometrically using Fura-2/AM (Invitrogen). The protocol used was basically as described in a previous study of ours3VO4, and protease inhibitor cocktail (Sigma). After centrifugation , supernatants were collected, and protein concentrations were determined with a DC protein assay (Bio-Rad). Proteins (10 μg) were separated by SDS-PAGE (12% gel) and transferred to PVDF membranes accordingly too Iblot2 methods (Invitrogen). The PVDF membranes were incubated overnight at 4 °C with antibodies against GAPDH , TRPML1 , BK channels , and HIV-1 Tat . The blots were developed with enhanced chemiluminescence, and bands were visualized and analyzed by our LI-COR Odyssey Fc Imaging System.Cells were harvested and lysed in 1X RIPA lysis buffer (Thermofisher) containing 10 mM NaF, 1 mM NaCells were fixed with 1% paraformaldehyde for 10 min followed by cold methanol (−20 °C) for 10 min. The cells were then washed with PBS, blocked with 5% goat serum, and incubated overnight at 4 °C with primary antibodies (1:100) against TRPML1 , BK channels  and LAMP1 . After washing with PBS, cells were incubated with corresponding Alexa 488- or Alexa 546-conjugated secondary antibodies a(Invitrogen). Cells were examined by confocal microscopy (Zeiss LSM800). Controls for immunostaining specificity included staining neurons with primary antibodies without fluorescence-conjugated secondary antibodies (background controls), and staining neurons with only secondary antibodies; these controls helped eliminate auto-fluorescence in each channel and bleed-through (crossover) between channels.All experiments were repeated 2–3 times using at least 2 different passages of cells. All data were presented as means and standard deviations. Statistical significance between two groups was analyzed with a Student’s t-test. Statistical significance among multiple groups was analyzed with a one-way ANOVA plus Tukey’s post-hoc test. And, statistical significance among multiple groups involving two factors was analyzed with two-way ANOVA. p < 0.05 was accepted to be statistically significant.No live vertebrates (or higher invertebrates), humans, or human samples were used in any experiments conducted for this manuscript.Supplementary information


Drosophila. Knock-down of one of the genes identified in this screen, the Ras-ERK-ETS pathway transcription factor Aop, alleviates the damaging effects of mitochondrial dysfunction in the nervous system. Inhibition of Ras-ERK-ETS signaling also restores function in Drosophila models of human diseases associated with mitochondrial dysfunction. Importantly, Ras-ERK-ETS pathway inhibition partially reverses the mitochondrial retrograde transcriptional response. Therefore, mitochondrial retrograde signaling likely contributes to neuronal dysfunction through mis-regulation of gene expression.Mitochondrial dysfunction activates the mitochondrial retrograde signaling pathway, resulting in large scale changes in gene expression. Mitochondrial retrograde signaling in neurons is poorly understood and whether retrograde signaling contributes to cellular dysfunction or is protective is unknown. We show that inhibition of Ras-ERK-ETS signaling partially reverses the retrograde transcriptional response to alleviate neuronal mitochondrial dysfunction. We have developed a novel genetic screen to identify genes that modify mitochondrial dysfunction in  Drosophila nervous system. Inhibition of Ras-ERK-ETS signaling improves neuronal function in Drosophila models of mitochondrial disease. Targeting Ras-ERK-ETS signaling may therefore have therapeutic potential in mitochondrial disease patients. Using a transcriptomic approach, we find that inhibition of Ras-ERK-ETS signaling partially reverses the mitochondrial retrograde transcriptional response. Surprisingly therefore, the mitochondrial retrograde transcriptional response contributes to neuronal dysfunction.Loss of mitochondrial function activates the mitochondrial retrograde signaling pathway resulting in large scale changes in nuclear gene transcription. Very little is known about retrograde signaling in the nervous system and how the transcriptional changes affect neuronal function. Here we identify Ras-ERK-ETS signaling as a novel mitochondrial retrograde signaling pathway in the  ATP is generated either by glycolysis in the cytosol, or through the action of the tricarboxylic acid (TCA) cycle and β-oxidation of fatty acids coupled to oxidative phosphorylation (OXPHOS) in mitochondria. The mitochondrial electron transport chain (ETC) couples the transfer of electrons to the pumping of protons into the inter-membrane space . This crMitochondria are abundant in almost every cell type and are particularly important in the nervous system. During nervous system development neural stem cell progenitors use mainly glycolytic metabolism, but upon differentiation into neurons switch to become dependent on OXPHOS for ATP , 4. MatuPINK1 and Parkin, cause familial Parkinson’s disease . TF. TFD42-Gnflation  and TFAMnflation ) suppresnflation . Furthernflation , confirmRas85D loss-of-function allele Ras85DΔC40B, consistent with Aop acting downstream of Ras.To test whether increasing Aop affects neuronal function we overexpressed Aop alone in motor neurons, or together with TFAM. Overexpression of Aop in neurons causes a strong climbing deficit and complete failure of wing inflation . MoreoveDrosophila. To test whether reducing Pnt expression also modifies neuronal mitochondrial dysfunction we used both a validated RNAi targeting Pnt (pnt (pntΔ88). Knock-down of Pnt in motor neurons, or heterozygosity for pnt, suppresses the TFAM overexpression climbing and wing inflation phenotypes  is a second target of Ras-ERK signaling in ting Pnt  and heteenotypes . Combinion alone . However85DΔC40B . Simulta85DΔC40B . PntP2 o85DΔC40B , which iV12. Expression of Ras85DV12 in motor neurons causes lethality with D42-Gal4 and with OK371-Gal4. Knock-down of either Aop or Pnt suppresses the Ras85DV12 lethality phenotype  form of ERK (dpERK). dpERK expression is significantly increased in larval motor neurons overexpressing TFAM . Clonal Similar to mitochondrial dysfunction in late larval sensory neurons, which does not affect ATP levels due to a compensatory increase in glycolysis , overexpDrosophila models of human disease associated with mitochondrial dysfunction. To test this we used pan-neuronal  knock-down of the OXPHOS complex IV subunit Surf1, a model for the primary mitochondrial childhood encephalomyelopathy Leigh syndrome, and park25 homozygous mutant flies, a model for familial Parkinson’s disease  and AT[RK] experiments, embryos were laid over a 24 hour period at 25°C, incubated for a further 24 hours at 25°C, then incubated at 29°C for three days prior to analysis. For AT[NL] and AT[RK] experiments embryos were laid at 25°C and larvae maintained at 25°C until dissection.UAS-TFAM3M [UAS-TFAM10M [park25 [UAS-Surf123.4 RNAi [Ras85DΔC40B [UAS-mito-roGFP2-Grx1 [UAS-ATeam1.03NL (AT[NL]) and UAS-ATeam1.03RK (AT[RK]) flies ) flies  were froe Center  and the  et. al. . Gene na et. al. .TM6B,tub-Gal80  were crossed to males carrying RNAi transgenes. 1–2 days after eclosion of the progeny wings were observed and scored in males. RNAi lines were only classed as enhancers if most flies had a ≥90ᵒ wing curve. RNAi lines were classed as suppressors if most flies had a <45ᵒ wing curve , rabbit anti-Dcp1 , mouse anti-Wingless , chicken anti-β galactosidase , mouse anti-Aop , mouse anti-brp , rabbit anti-dpErk1/2 , HRP-Cy3 , rat anti-PntP2 (1/500)  ATP biosensor expressing and AT[RK] ATP insensitive expressing control wing imaginal discs or larval CNS tissues were dissected in Schneider’s medium (Thermo Scientific) and imaged immediately at 21°C using a 458nm excitation laser and detecting emitted light between 460-499nm (CFP) and 535-650nm (FRET) using a Zeiss LSM 710 confocal microscope. For control experiments wing imaginal discs were incubated in Schneider’s medium with 100μM oligomycin (VWR)/50 mM 2-deoxyglucose (SLS) for 40 minutes at 21°C, then imaged immediately in the same medium. CFP and FRET channel signal intensity at the same three randomly selected points in each wing disc, or three cell bodies in each VNC, was determined using the ImageJ Point and Measure tools and used to calculate the FRET/CFP ratio as a measure of ATP levels . Imaging20 wing imaginal discs per genotype were dissected in PBS, homogenized in 100μl extraction buffer  and incubated at 70°C for 5 mins. ATP levels were measured using the ATP Determination kit (Molecular Probes) according to the manufacturer’s instructions.Drosophila anti-TFAM , mouse anti-ATP5A , mouse anti-MTCO1  and rabbit anti-Actin . After three ten minute washes in TBS-T, the membranes were incubated for 90 minutes with fluorescently labelled secondary antibodies  diluted in TBS-T, then washed three times for ten minutes in TBS-T. The membranes were then scanned and analysed using an Odyssey infrared scanner (LI-COR). Odyssey infrared imaging systems application software version 3.0.25 was used to quantify the intensity of the bands on the blots. Normalised expression level was calculated by determining the band intensity relative to Actin.Western blot analysis was performed as previously described . PrimaryqRT-PCR and qPCR of mtDNA were performed as previously described . PrimersCNS tissue from 20 late third instar larvae per genotype were dissected in cold PBS and transferred directly into 100μl lysis buffer containing β-mercaptoethanol . The lysis buffer was kept on ice while all the brains were dissected. RNA was prepared following manufacturer’s instructions, including DNase treatment and stored at -80°C. Samples were prepared in triplicate.Drosophila genome v2 GeneChips for 20 hours at 45°C. They were then washed, stained (GeneChip Fluidics Station 450) and scanned (GeneChip Scanner 3000 7G) according to the manufacturer’s instructions (Nugen Technologies Inc & Affymetrix).RNA was measured for quantity and integrity on an RNA Pico Chip (Agilent Technologies). 10ng of RNA per genotype was converted into labelled cDNA with the Nugen Ovation System V2 (NuGEN Technologies Inc.). 7mg of labelled cDNA was hybridised to Affymetrix Microarray data was processed using the MAS5.0 algorithm using the Transcriptome Analysis Console (ThermoFisher). Means were calculated using Tukey's Bi-weight average algorithm and differential expression between groups was calculated using un-paired one way analysis of variance (ANOVA). A statistical cutoff of p<0.05 and a fold change cutoff of ±1.5 fold were used. Correlations between datasets were analysed using GraphPad Prism (GraphPad Software Inc.). The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE114054. GO analysis was performed using DAVID  bioinformatics resources .GraphPad Prism (GraphPad Software Inc.) was used to create graphs and for statistical analysis. Data with a p-value less than or equal to 0.05 was considered significant. Comparisons of two samples of continuous data were analysed with an unpaired, two-tailed student’s t-test, where appropriate. Data were analysed for normality using the D’Agostino & Pearson omnibus normality test. Data that did not pass the normality test were analysed with the Mann Whitney test. Variance of the samples was assessed with an F test. If the variances of the two samples were significantly different then the Welch’s correction was applied to the t-test. In order to compare more than two samples of continuous data, one-way ANOVA was used with Tukey’s post hoc test. If data did not pass the D’Agostino & Pearson omnibus normality test, the Kruskal-Wallis, followed by Dunn’s post hoc test were utilised. Categorical data were analysed using chi-squared.S1 Figc01716  have strongly reduced levels of TFAM expression . (I-L) TFAM overexpression , or knock-down of TFAM using two independent RNAi lines (TFAMJF02307 and TFAMHMC04965) in the wing using MS1096-Gal4 cause a curved wing phenotype. (M-P) The curved wing phenotype caused by knock-down of TFAM using MS1096-Gal4 and heterozygosity for TFAMc01716  is almost completely rescued by co-expression of TFAM in both males (N) and females (P).(A) qPCR of mtDNA copy number from late third instar larvae with ubiquitous knock-down of TFAM (4217R-1) using tub-Gal4. Controls are tub-Gal4 hemizygotes. (B) Western analysis of coxI, ATP synthase α and TFAM expression from late third instar larvae control , or with ubiquitous knock-down of TFAM using tub-Gal4 (lanes 4–6). (C-E) Quantification of coxI, ATP synthase α and TFAM expression.  TFAM staining in wing imaginal discs with TFAM knock-down (4217R-1) (F) or TFAM overexpression (G) using MS1096-Gal4. Arrows mark the dorso-ventral compartment boundary . Scale bar: 10μm. (H) A MARCM clone in the late third instar larval wing imaginal disc showing that cells that are homozygous of TFAM(TIF)Click here for additional data file.S2 Fig(A-C) The FRET/CFP fluorescence emission ratio of the AT[NL] FRET–based ATP biosensor expressed in the wing imaginal disc using MS1096-Gal4 is decreased when the tissue is incubated with oligomycin (OM)/2-deoxyglucose. (D-F) The FRET/CFP fluorescence emission ratio of the AT[RK] control protein, which does not bind ATP, is unchanged when the tissue is incubated with oligomycin (OM). (G-J) Knock-down (4217R-1) or overexpression of TFAM do not alter the FRET/CFP fluorescence emission ratio of the AT[NL] FRET–based ATP biosensor in the wing disc. Images show a merge of the CFP (green) and FRET (red) channels. (K) ATP luciferase assay of wing discs with TFAM RNAi and overexpression using MS1096-Gal4. (L-N) Ratio images show no change in mitochondrial glutathione redox potential reporter mito-roGFP2-Grx1 fluorescence after excitation at 405nm (red) and 488nm (green) in wing discs with TFAM knock-down and overexpression using MS1096-Gal4. (O) Quantification of mito-roGFP2-Grx1 fluorescence ratio. (P-R) DHE staining in MS1096-Gal4 hemizygous control (P), TFAM knock-down (Q) and overexpression (R) wing discs. (S) Quantification of DHE staining in the dorsal compartment of the wing disc. (T-V) Mitochondrial morphology with TFAM knock-down (U) and TFAM overexpression (V) in the wing imaginal disc using MS1096-Gal4, compared to control (T). Mito-GFP is used to label mitochondria. Scale bar: 10 μm.  Quantification of mitochondrial number (W) and volume (X) in wing imaginal discs.  qRT-PCR of Thor (Y) and Hsp22 (Z) mRNA expression in wing imaginal discs with TFAM knock-down and TFAM overexpression using MS1096-Gal4. Data are represented as mean +/- SEM, n.s. not significant, *p≤0.05, **≤0.01, ***p≤0.001, a.u. arbitrary units.(TIF)Click here for additional data file.S3 Fig1118 showing the 45° curve at the wing tip. (C-H) Male progeny from crosses of MitoMod with DJ-1α RNAi (HMJ21180) (C), DJ-1α overexpression (D), DJ-1β RNAi (HMS01915) (E), DJ-1β overexpression (F), Lrrk RNAi (HMS00456) (G), Pink1 overexpression (H).(A) MS1096-Gal4, + control male. (B) Male progeny from MitoMod fly crossed to w(TIF)Click here for additional data file.S4 Fig1118.  Wing discs from the progeny of MitoMod crossed to Ino80 RNAi (E) and Aop RNAi (F).  Quantification of Dcp1 expression. Dcp1 expression is shown in green in (A)-(F) and white in (A’)-(F’) and DAPI staining shown in blue. Dotted line marks the dorso-ventral compartment boundary . (I) Quantification of Dcp1 expression in MitoMod wing discs combined with knock-down of Chrac-14, Ing3 and MTA1-like. (J) Quantification of Dcp1 expression in wing discs overexpressing TFAM combined with knock-down of Ino80 or Aop. Data are represented as mean +/- SEM, n.s. not significant, *p≤0.05, ***p≤0.001.(A) A wing disc from a MS1096-Gal4, + larva stained for Dcp1 expression and DAPI.  Wing discs from larvae with knock-down of Ino80 and Aop using MS1096-Gal4. (D) A wing disc from the progeny of MitoMod crossed to w(TIF)Click here for additional data file.S5 Fig(A-C) Segment A3, muscle 4 NMJ in late third instar larvae from control (A), with TFAM RNAi (4217R-1) (B), or TFAM overexpression (C) in motor neurons using OK371-Gal4. Motor neuron specific expression of mitoGFP (green in (A-C) and white in (A’-C’)) was used to visualise mitochondria and staining for horse radish peroxidase (red) to visualise neuronal membranes.  Quantification of mitochondrial number (D) and volume (E).  Knock-down of TFAM (4217R-1) using either OK371-Gal4 (F) or D42-Gal4 (G) causes reduced climbing ability in adults, but this phenotype is weaker than with TFAM overexpression.  An eye imaginal disc from a control GMR-Gal4/+ larva (H), or a larva expressing Aop RNAi (3166R-1) using GMR-Gal4 (I), which shows almost complete loss of Aop expression in photoreceptor neurons posterior to the morphogenetic furrow (arrow), where GMR-Gal4 is expressed (posterior is to the right).  Adult eyes from GMR-Gal4/+ control (J), or GMR-Gal4/Aop RNAi (3166R-1) (K) flies showing a rough eye phenotype caused by Aop knock-down. (L-N) Overexpression of TFAM with D42-Gal4 causes wings to either inflate normally (L), semi-inflate (M) or fail to inflate (N) in around 50% of flies. (O-R) Knock-down of Dsor using independent RNAi lines (HMS00037 and JF01697) suppresses the climbing  and wing inflation phenotypes  caused by TFAM overexpression with D42-Gal4. (S-V) Knock-down of Rl using independent RNAi lines (JF1080 and HMS00173) suppresses the climbing  and wing inflation phenotypes  caused by TFAM overexpression with D42-Gal4. The numbers of flies counted for each genotype are shown in white. Data are represented as mean +/- SEM, n.s. not significant, *p≤0.05, **≤0.01, ***p≤0.001. Controls are Gal4 hemizygotes.(TIF)Click here for additional data file.S6 FigΔ88 heterozygous background. (F) The wing inflation phenotype caused by overexpression of TFAM with D42-Gal4 is suppressed in a pntΔ88 heterozygous background. The numbers of flies counted for each genotype are shown in white.  Knock-down of Pnt combined with heterozygosity for Ras85D suppresses the climbing (G) and wing inflation phenotypes (H) caused by TFAM overexpression with D42-Gal4 compared to either condition alone.  Knock-down of Aop combined with heterozygosity for Ras85D does not affect the climbing (I) and wing inflation phenotypes (J) caused by TFAM overexpression with D42-Gal4 compared to either condition alone.  Knock-down of Pnt and Aop together does not affect the climbing (K), but suppresses the wing inflation phenotype (L) caused by TFAM overexpression with D42-Gal4 compared to Aop knock-down alone. (M) Overexpression of PntP2 in motor neurons with D42-Gal4 causes reduced climbing and is lethal when combined with TFAM overexpression.  Knock-down of Aop (N) or Pnt (O) rescues the lethality caused by expression of Ras85DV12 with OK371-Gal4. Controls are D42-Gal4 hemizygotes. Data are represented as mean +/- SEM, n.s. not significant, *p≤0.05, ** p≤0.01 ***p≤0.001.  Mosaic analysis with a repressible cell marker (MARCM) control (P) or TFAM overexpression (Q) clones (green) stained for dpERK expression (red). An eye imaginal disc from a control GMR-Gal4/+ larva (A), or a larva expressing Pnt RNAi (JF02227) using GMR-Gal4 (B), which shows almost complete loss of PntP2 expression in photoreceptor neurons posterior to the morphogenetic furrow (arrow). Posterior is to the right.  Adult eyes from GMR-Gal4/+ control (C), or GMR-Gal4/Pnt RNAi (JF02227) (D) flies showing a rough eye phenotype caused by Pnt knock-down. (E) The reduced climbing ability of flies with TFAM overexpression using D42-Gal4, is suppressed in a pnt(TIF)Click here for additional data file.S7 Fig25 larvae compared to w1118 controls. Data are represented as mean +/- SEM, n.s. not significant, *p≤0.05, ** p≤0.01, ***p≤0.001.(A-C) Overexpression of TFAM does not alter the FRET/CFP fluorescence emission ratio of the AT[NL] FRET–based ATP biosensor expressed in motor neurons with OK371-Gal4. Images show a merge of the CFP (green) and FRET (red) channels. Data are represented as mean +/- SEM, a.u. arbitrary units.  Quantification of mitochondrial number (D) and volume (E) from segment A3, muscle 4 NMJ in late third instar larvae from control, or with Aop RNAi (3166R-1), TFAM overexpression, or Aop RNAi (3166R-1) and TFAM overexpression together in motor neurons using OK371-Gal4. (F) Quantification of active zone number from the same genotypes as in . Controls are OK371-Gal4 hemizygotes. (G) dpERK expression is increased in the VNC by pan-neuronal knock down of Surf1 with nSyb-Gal4, compared to hemizygous nSyb-Gal4 controls. (H) dpERK expression is unchanged in the VNC in park(TIF)Click here for additional data file.S8 Fig25 male flies is not improved by ubiquitous knock-down of Aop (3166R-1) using Da-Gal4. Controls are w1118. Data are represented as mean +/- SEM, n.s not significant,** p≤0.01***, p≤0.001. Knock down of Pnt rescues the climbing (A) and wing inflation (B) phenotypes causes by knock-down of Surf1 with nSyb-Gal4. (C) The climbing defect in park(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableThe outcome of the screen is in the Result column. Any lines that had a phenotype with MS1096-Gal4 alone were excluded from the screen, so the result reads ‘Excluded’. The confirmed column refers to confirmation of the result with an independent RNAi for the same gene, Y: confirmed, N: not confirmed, see (XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 TableAny lines that had a phenotype with MS1096-Gal4 alone read ‘Excluded’ in the Result column.(XLSX)Click here for additional data file.S6 TableAny lines that had a phenotype with MS1096-Gal4 alone read ‘Excluded’ in the Result column.(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.S10 Table(XLSX)Click here for additional data file.S11 Table(XLSX)Click here for additional data file.S12 Table(XLSX)Click here for additional data file.S13 Table(XLSX)Click here for additional data file.S14 Table(XLSX)Click here for additional data file.


N81-STTM vector expresses a short tandem target mimic (STTM) containing two target-mimic sequences separated by a short spacer sequence. Our results showed that ZMBJ-CMV-2bN81-STTM can be used to investigate miRNA function in Nicotiana benthamiana and maize seedlings. The ZMBJ-CMV-2bN81-STTM-based downregulation of Nbe-miR165/166 or Nbe-miR159 induced specific and strong miRNA-sequestering phenotypes, and increased the expressions of their predicted target genes. For maize, the ZMBJ-CMV-2bN81-STTM based downregulation of zma-miR167 or zma-miR482 caused a decrease of lateral roots growth and a plant stunting phenotypes, respectively. In both cases, the target genes of zma-miR167- or zma-miR482 were increased significantly. Thus, we consider ZMBJ-CMV based VbMS system as a useful tool for high-throughput investigations of miRNA functions in maize.Endogenous microRNAs (miRNAs) play pivotal roles in plant development and responses to various biotic or abiotic stresses. Up to now, more than 500 maize miRNAs have been identified. However, functions of these identified miRNAs remained largely unknown due mainly to the lack of rapid and reliable tools. We previously reported a cucumber mosaic virus strain ZMBJ (ZMBJ-CMV)-based gene silencing vector for rapid and efficient gene function studies in maize lines with agronomical importance. Because ZMBJ-CMV induces very mild disease symptoms but strong gene silencing in maize, we decided to further modify this vector to suppress miRNA expressions in maize. The newly developed ZMBJ-CMV-2b Zea mays) is one of the most important cereal crops worldwide. It can be used for human consumption, animal feed and biofuel production . However, only four identified maize miRNAs, including miR166, miR156, miR172 and miR528 have been functionally characterized (Maize (oduction . To improduction . microRNoduction . miRNAs oduction . To datecterized .Corngrass1 (Cg1) mutant was found to overexpress two tandem miR156 in meristem tissues and in lateral organs (tasselseed4 (ts4) mutant was determined to express mir172 that can target APETALA2, a floral homeotic transcription factor. Maize mutant carrying a ts4 mutation was shown to promote carpel development on tassels and more meristem branching  or short tandem target mimics (STTM), through stable transformation, or through using virus-based miRNA silencing (VbMS) vector . For insl organs . Maize tranching . Analysienotypes . A more enotypes .IPS1 (INDUCED BY PHOSPHATE STARVATION1)-derived structure containing a three-nucleotide interruption linker in the miRNA cleavage site. This structure could be used as a target mimic to sequester miRNAs of interest (Earlier studies on miRNA functions were done through expressing target mimics (TM) using an interest . Unfortuinterest . Recentlinterest . With beinterest .Nicotiana benthamiana and/or tomato, respectively  systems have been developed to express STTM in plant. These VbMS systems were shown to have efficient miRNA suppression abilities and are simple to use. For example, four plant viruses: tobacco rattle virus (TRV), cotton leaf crumple virus (CLCrV), cucumber mosaic virus LS strain (LS-CMV) and potato virus X (PVX) had been modified to express STTMs to silence miRNAs. These systems were shown to be effective in Arabidopsis, cotton, ectively . For monectively . We specN. benthamiana and two endogenous miRNAs in B73 maize plants. We consider that this VbMS system is useful for routine and fast analyses of miRNA functions in maize.We previously reported ZMBJ-CMV as a maize-infecting strain of CMV . We thenN81 were described previously (Pfu polymerase (TransGen Biotech). The resulting STTM fragments were digested with restriction enzyme KpnI and XbaI, and cloned individually into the KpnI/XbaI site in pCMV201-2bN81 using the In-Fusion HD Cloning kit as instructed (Takara Bio).ZMBJ-CMV plasmid pCMV101, pCMV301 and pCMV201-2beviously . A 48 nteviously . All theN. benthamiana plants were grown in pots inside growth chambers set at 24/22°C (day/night), 16/8 h (light/dark) and 60% relative humidity.Maize seeds (inbred line B73) were obtained from the National Maize Improvement Center, China Agricultural University, Beijing, China. All seeds were germinated on wet blotting papers inside petri dishes at 25°C and in the dark. The germinated seeds were transplanted into pots and then grown inside growth chambers set at 20/18°C (day/night) and 16/8 h (light/dark).4, 2 mM NH4NO3, 2 mM CaCl2, 0.75 mM K2SO4, 0.1 mM KCl, 0.25 mM KH2PO4, 0.2 mM Fe-EDTA, 1 × 10−3 mM MnSO4, 1 × 10−3 mM ZnSO4, 1 × 10−4 mM CuSO4, 5 × 10−6 mM (NH4)6Mo7O24, and 1 × 10−3 mM H3BO3, pH 5.8–6.0.In other experiments, the germinated seeds were grown in hydroponic culture media with continuous aeration as described . The nutN81-STTM and its derivatives, were introduced individually into Agrobacterium tumefaciens strain C58C1. Preparations of A. tumefaciens cultures and infiltration of N. benthamiana leaves were same as described by For ZMBJ-CMV-based VbMS, plasmid pCMV101, pCMV301, pCMV201-2bN. benthamiana leaves were harvested at 3 dpi and then ground in 0.1 M phosphate buffer, pH 7.0. After 3 min centrifugation at 4,000 rpm and at 4 °C, supernatant of each sample was used to inoculate maize seeds using the vascular puncture method . After RNase-free DNase I (Takara Bio) treatment, 2 μg total RNA was used in each first-strand complementary DNA synthesis together an oligo(dT) primer or CMV-specific primers, and the Moloney murine leukemia virus reverse transcriptase (Promega Corporation).The 2EIF4a and ZmUbi were used as internal controls for N. benthamiana and maize, respectively. Stem-loop RT-qPCR was performed as described (t test. Each experiment was replicated at least three times.RT-qPCR was performed using a FastSYBR mixture (Beijing ComWin Biotech). Expressions of escribed . The miRhttps://www.ncbi.nlm.nih.gov/) under the following accession numbers: ZmARF3 (GRMZM2G078274); ZmARF9 (GRMZM2G073750); ZmARF16 (GRMZM2G028980); ZmARF18 (GRMZM2G035405); ZmARF22 (GRMZM2G089640); ZmARF30 (GRMZM2G475882); ZmARF34 (GRMZM2G081158); ZmIAR3 (GRMZM2G090779); Z. mays polyubiquitin (XM_008647047.3); ZmTPS2 (GRMZM2G079928).Sequences of miRNAs described in this article were retrieved from the GenBank . In this study, we cloned various STTMs at the multiple cloning sites (MCS) in pCMV201-2bN81  transcription factors important for plant shoot apical dominance and leaf polarity . Downregormation Figure 2MYB-like transcription factor genes in N. benthamiana , a predicted target of miR159, was increased by approximately 2.4 folds compared with the ZMBJ-CMV-infected plants  genes (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi). The function of miR167 in maize root development remains unknown.Arabidopsis and soybean miR167 was reported to play critical roles in lateral root development and architecture . In maizauxin response factors (ZmARF3 and ZmARF30) and INDOLE-3-ACETIC ACIDALANINE RESISTANT3 (ZmIAR3), were up-regulated significantly in the ZMBJ-CMV-STTM167-infected maize plants compared with the ZMBJ-CMV-infected control plants  as a novel and wounding-responsive microRNA  was significantly up-regulated , leading to a delayed growth of maize seedling (N. tabacum), expression of Escherichia coli TPS (OTS A) results in an increase of photosynthetic activity, stunted plant growth and formation of lancet-shaped leaves , the Ministry of Agriculture and Rural Affairs of China , and the Ministry of Education of China (the 111 Project B13006).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conﬂict of interest.


Background: Endothelial dysfunction and subsequent inflammation contribute to the development of vascular cognitive impairment (VCI). Soluble intercellular adhesion molecule-1 (sICAM-1) is upregulated in endothelial dysfunction and promotes an inflammatory response; however, the relationship between sICAM-1 and VCI remains equivocal. Objective: To determine whether sICAM-1 contributes to the prediction of VCI. Methods: Community-dwelling older adults (n=172) from the “Cohort of Obesity, Sarcopenia and Frailty of Older Mexican Adults” (COSFOMA) study were identified as VCI or controls using standard neuropsychological evaluations and neuroimaging. sICAM-1 was quantified using ELISA, and multivariate logistic regression determined the association between sICAM-1 and VCI. Results: 31 VCI cases were identified. sICAM-1 was higher in VCI [VCI: 450.7 (241.6) ng/ml vs. Control: 296.9 (140.9) ng/ml]. sICAM-1 concentrations above the 90th percentile (464.1 ng/mL) was associated with VCI group membership in all models [OR = 6.9 (95% CI: 1.1- 42.2)]. The final saturated model explained 64% of the variance in VCI group membership. Conclusion: High concentrations of sICAM-1 are independently associated with VCI group membership. Efforts to further characterize the relationship between indices of endothelial dysfunction and pathological changes to the aging brain should be further pursued.