Paenibacillus sp. str. FPU-7 effectively hydrolyzes chitin by using a number of chitinases. A unique chitinase with two catalytic domains, ChiW, is expressed on the cell surface of this bacterium and has high activity towards various chitins, even crystalline chitin. Here, the crystal structure of ChiW at 2.1 Å resolution is presented and describes how the enzyme degrades chitin on the bacterial cell surface. The crystal structure revealed a unique multi-modular architecture composed of six domains to function efficiently on the cell surface: a right-handed β-helix domain , a Gly-Ser-rich loop, 1st immunoglobulin-like (Ig-like) fold domain, 1st β/α-barrel catalytic domain , 2nd Ig-like fold domain and 2nd β/α-barrel catalytic domain (GH-18). The structure of the CBM-54, flexibly linked to the catalytic region of ChiW, is described here for the first time. It is similar to those of carbohydrate lyases but displayed no detectable carbohydrate degradation activities. The CBM-54 of ChiW bound to cell wall polysaccharides, such as chin, chitosan, β-1,3-glucan, xylan and cellulose. The structural and biochemical data obtained here also indicated that the enzyme has deep and short active site clefts with endo-acting character. The affinity of CBM-54 towards cell wall polysaccharides and the degradation pattern of the catalytic domains may help to efficiently decompose the cell wall chitin through the contact surface. Furthermore, we clarify that other Gram-positive bacteria possess similar cell-surface-expressed multi-modular enzymes for cell wall polysaccharide degradation.The Gram-positive bacterium  N-acetyl-D-glucosamine (GlcNAc) as a repeating unit with the β-1,4-linkage, and chitin-derived sugars, e.g., chitosan, oligo- and monosaccharides, have beneficial effects as elicitors and anti-tumor agents. Accordingly, chitin is of industrial, agricultural, cosmetic and medicinal interest . Th. Th11]. llenging . Thus, tllenging . Crystalllenging . HoweverChiA1CD)  as a refChiA1CD) , and theChiA1CD) , and refBased on the crystal structure, ChiW-SLHd was functionally divided into three regions: CBM-54, the GS (glycine-serine)-rich loop and a catalytic region Figs  and 2. TB-factor (93.2 Å2) than that of the full-length protein (33.2 Å2). These observations do not contradict the hypothesis that the loop is an intrinsically flexible region. ChiW is fastened on the bacterial cell surface with an SLH domain, which enables ChiW to readily collect chitin oligosaccharides into the cell. The flexible motion of the catalytic region via the GS-rich loop probably facilitates attachment of the enzyme to the molecular surface of the solid substrate chitin in an appropriate orientation.The GS-rich loop  connects the catalytic region and CBM-54 . Althoug8/α7-barrel as a core structure -insertion domain and insertion domain-2 3 (2 from (GlcNAc)3 with ChiW-CD was confirmed by HPLC analysis 5 or (GlcNAc)6, the electron density maps corresponding to the substrates were too weak and complicated to interpret because of their diversity and the heterogeneity in substrate binding modes within the active site. The structure of the ChiW-CD-product complex contained one (GlcNAc)2 molecule at the bottom of the deep cleft of Cat-1, indicating that it occupied two subsites, −1 and −2  with the β-anomer and the −2 subsite GlcNAc adopts a stable chair conformation (4C1). The ring distortion at the −1 subsite has been observed in the complex of other glycosidases ; YP_004027356: s-layer domain-containing protein [Caldicellulosiruptor kristjanssonii I77R1B]; YP_004797824: S-layer protein [Caldicellulosiruptor lactoaceticus 6A]; ZP_07387876: S-layer domain protein [Paenibacillus curdlanolyticus YK9]; YP_003008964: S-layer protein [Paenibacillus sp. JDR-2]; ZP_08511493: hypothetical protein HMPREF9413_5209 [Paenibacillus sp. HGF7]; EIJ83768: S-layer domain protein [Bacillus methanolicus MGA3]; ZP_09079447: S-layer domain-containing protein [Paenibacillus elgii B69]; YP_003009590: glycoside hydrolase family 16 [Paenibacillus sp. JDR-2]; ABJ15796: endo-beta-1,3-glucanase [Paenibacillus sp. CCRC 17245]; YP_005047467: beta-propeller domain-containing protein, methanol dehydrogenase [Clostridium clariflavum DSM 19732]; ZP_08278755: hypothetical protein HMPREF9412_0339 [Paenibacillus sp. HGF5]; YP_003240334: S-layer domain-containing protein [Paenibacillus sp. Y412MC10]; YP_006468477: cell wall-binding protein [Desulfosporosinus acidiphilus SJ4]; YP_006189237: mannan endo-1,4-beta-mannosidase [Paenibacillus mucilaginosus K02]; YP_004640549: mannan endo-1,4-beta-mannosidase [Paenibacillus mucilaginosus KNP414]; YP_005312442: mannan endo-1,4-beta-mannosidase [Paenibacillus mucilaginosus 3016]; YP_002506677: S-layer protein [Clostridium cellulolyticum H10]; YP_003850806: Mannan endo-1,4-beta-mannosidase [Thermoanaerobacterium thermosaccharolyticum DSM 571]; YP_004310855: glucan endo-1,3-beta-D-glucosidase [Clostridium lentocellum DSM 5427]; YP_006391314: glycoside hydrolase family 26 [Thermoanaerobacterium saccharolyticum JW/SL-YS485]; YP_004459776: S-layer domain-containing protein [Tepidanaerobacter acetatoxydans Re1]; ZP_08191088: S-layer domain-containing protein [Clostridium papyrosolvens DSM 2782]; ZP_09079445: hypothetical protein PelgB_33676 [Paenibacillus elgii B69]; ZP_08420347: putative S-layer homology domain protein [Ruminococcaceae bacterium D16]; EIC10715: glycoside hydrolase family 16, partial [Ruminiclostridium thermocellum AD2]; ZP_06250297: glycoside hydrolase family 16 [Ruminiclostridium thermocellum JW20]; CAC27412: endo-1,3(4)-beta-glucanase [Ruminiclostridium thermocellum YP_001039201: glycoside hydrolase family 16 [Ruminiclostridium thermocellum ATCC 27405]; ZP_05428042: glycoside hydrolase family 16 [Ruminiclostridium thermocellum DSM 2360]). Identical amino acid residues among the conserved proteins are indicated by red asterisks. The blue colons and green periods indicate conserved residues.The numbers in the left-hand column are the protein accession numbers (ZP_07902840: S-layer domain protein ; gi|312201661|gb|ADQ44988.1| Glucan endo-1,3-beta-D-glucosidase [Caldicellulosiruptor kronotskyensis 2002]; gi|302573661|gb|ADL41452.1| Glucan endo-1,3-beta-D-glucosidase [Caldicellulosiruptor obsidiansis OB47]; gi|311776336|gb|ADQ05822.1| Glucan endo-1,3-beta-D-glucosidase [Caldicellulosiruptor hydrothermalis 108]; gi|373943664|gb|AEY64585.1| Ig-like domain-containing protein, putative S-layer protein [Clostridium sp. BNL1100]; gi|219998061|gb|ACL74662.1| S-layer domain protein [Clostridium cellulolyticum H10]; gi|961447213|gb|ALS25414.1| SLH domain-containing protein [Paenibacillus naphthalenovorans]; gi|1022705355|gb|ANA79717.1| S-layer protein [Paenibacillus glucanolyticus]; gi|1031374757|gb|ANF98139.1| hypothetical protein AR543_20420 [Paenibacillus bovis]; gi|261280556|gb|ACX62527.1| S-layer domain protein [Paenibacillus sp. Y412MC10]; gi|1049752686|gb|ANY65940.1| hypothetical protein BBD42_05270 [Paenibacillus sp. BIHB4019]; gi|686546821|gb|AIQ57517.1| hypothetical protein PBOR_11680 [Paenibacillus borealis]; gi|686541165|gb|AIQ51862.1| hypothetical protein R70331_10275 [Paenibacillus sp. FSL R7-0331]; gi|686535581|gb|AIQ46279.1| hypothetical protein R70723_10605 [Paenibacillus sp. FSL R7-0273]; gi|1049752686|gb|ANY65940.1| hypothetical protein BBD42_05270 [Paenibacillus sp. BIHB4019]; gi|686541165|gb|AIQ51862.1| hypothetical protein R70331_10275 [Paenibacillus sp. FSL R7-0331]; gi|686535581|gb|AIQ46279.1| hypothetical protein R70723_10605 [Paenibacillus sp. FSL R7-0273]; gi|1049753427|gb|ANY66681.1| hypothetical protein BBD42_09565 [Paenibacillus sp. BIHB4019]; gi|662720239|gb|AIE61449.1| putative secreted protein [Bacillus methanolicus MGA3]; gi|375301816|gb|AFA47950.1| hypothetical protein Awo_c11660 [Acetobacterium woodii DSM 1030]; gi|375303290|gb|AFA49424.1| hypothetical protein Awo_c26710 [Acetobacterium woodii DSM 1030]; gi|375300765|gb|AFA46899.1| S-layer domain containing protein [Acetobacterium woodii DSM 1030]; gi|995956117|gb|AMJ42396.1| endoglucanase precursor [Clostridium propionicum DSM 1682]; gi|134052896|gb|ABO50867.1| S-layer domain protein [Desulfotomaculum reducens MI-1]; gi|974001378|dbj|BAU26536.1| Cellulosome-anchoring protein precursor [Aneurinibacillus soli]; gi|686555149|gb|AIQ65844.1| hypothetical protein PSTEL_24790 [Paenibacillus stellifer]; gi|686516405|gb|AIQ27106.1| hypothetical protein P40081_02015 [Paenibacillus sp. FSL P4-0081]; gi|1028462806|gb|ANE45729.1| hypothetical protein SY83_04795 [Paenibacillus swuensis]; gi|605573144|gb|AHV95811.1| S-layer domain protein [Paenibacillus sabinae T27]; gi|332696012|gb|AEE90469.1| S-layer domain-containing protein [Tepidanaerobacter acetatoxydans Re1]; gi|311777858|gb|ADQ07344.1| glycoside hydrolase family 43 [Caldicellulosiruptor hydrothermalis 108]; gi|312201710|gb|ADQ45037.1| glycoside hydrolase family 16 [Caldicellulosiruptor kronotskyensis 2002]; gi|312201700|gb|ADQ45027.1| glycoside hydrolase family 16 [Caldicellulosiruptor kronotskyensis 2002]; gi|1049073550|gb|ANW97748.1| hypothetical protein CSTERTH_01225 [Clostridium stercorarium subsp. thermolacticum DSM 2910]; gi|472398113|gb|AGI38312.1| xylan-binding protein [Clostridium stercorarium subsp. stercorarium DSM 8532]; gi|1049073550|gb|ANW97748.1| hypothetical protein CSTERTH_01225 [Clostridium stercorarium subsp. thermolacticum DSM 2910]; gi|343963700|gb|AEM72847.1| S-layer domain-containing protein [Caldicellulosiruptor lactoaceticus 6A]; gi|312181573|gb|ADQ41743.1| S-layer domain-containing protein [Caldicellulosiruptor kristjanssonii I77R1B]; gi|247542485|gb|ACS99503.1| glycoside hydrolase family 16 [Paenibacillus sp. JDR-2]; gi|806915496|emb|CQR58104.1| hypothetical protein PRIO_5717 [Paenibacillus riograndensis SBR5]; gi|247542612|gb|ACS99630.1| Licheninase [Paenibacillus sp. JDR-2]; gi|482514681|gb|AGK07609.1| PglB [Paenibacillus sp. S09]; gi|384088723|gb|AFH60159.1| BglA2 [Paenibacillus mucilaginosus K02]; gi|336299299|gb|AEI42402.1| BglA2 [Paenibacillus mucilaginosus KNP414]; gi|378567862|gb|AFC28172.1| BglA2 [Paenibacillus mucilaginosus 3016]; gi|686549654|gb|AIQ60350.1| hypothetical protein PBOR_27925 [Paenibacillus borealis]; gi|302777163|gb|ADL67722.1| Mannan endo-1,4-beta-mannosidase [Thermoanaerobacterium thermosaccharolyticum DSM 571]; gi|389569310|gb|AFK85715.1| glycoside hydrolase family 26 [Thermoanaerobacterium saccharolyticum JW/SL-YS485]; gi|806911027|emb|CQR53170.1| Mannan endo-1,4-beta-mannosidase [Paenibacillus riograndensis SBR5]; gi|686552417|gb|AIQ63112.1| hypothetical protein PSTEL_08405 [Paenibacillus stellifer]; gi|1049753193|gb|ANY66447.1| beta-mannosidase [Paenibacillus sp. BIHB4019]; gi|336297576|gb|AEI40679.1| Mannan endo-1,4-beta-mannosidase [Paenibacillus mucilaginosus KNP414]; gi|384090036|gb|AFH61472.1| mannan endo-1,4-beta-mannosidase [Paenibacillus mucilaginosus K02]; gi|378568983|gb|AFC29293.1| Mannan endo-1,4-beta-mannosidase [Paenibacillus mucilaginosus 3016]; gi|973198192|gb|ALX07409.1| Glucan endo-1,3-beta-D-glucosidase [Ruminiclostridium thermocellum AD2]; gi|1046311560|gb|ANV75148.1| Glucan endo-1,3-beta-D-glucosidase [Ruminiclostridium thermocellum DSM 2360]; gi|316939453|gb|ADU73487.1| Glucan endo-1,3-beta-D-glucosidase [Ruminiclostridium thermocellum DSM 1313]; gi|359826770|gb|AEV69543.1| beta-propeller domain-containing protein, methanol dehydrogenase [Clostridium clariflavum DSM 19732]; gi|1049078145|gb|ANX02323.1| S-layer protein [Clostridium stercorarium subsp. leptospartum DSM 9219]; gi|1049075499|gb|ANW99697.1| S-layer protein [Clostridium stercorarium subsp. thermolacticum DSM 2910]; gi|472400210|gb|AGI40409.1| xylan-binding protein [Clostridium stercorarium subsp. stercorarium DSM 8532]; gi|160428582|gb|ABX42145.1| S-layer domain protein [Lachnoclostridium phytofermentans ISDg]; gi|373946072|gb|AEY66993.1| Ig-like domain-containing surface protein [Clostridium sp. BNL1100].The amino acid sequences of 74 CBM-54 family members were taken from the CAZy database and aligned by ClustalW. The tree is constructed by the neighbor-joining method with 1,000 bootstrap replications. Numbers at branching points refer to bootstrap values. The characterized CBM-54 domains, (TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


More importantly, compared to other FLT3 inhibitors, A674563 is able to overcome FLT3 ligand-induced drug resistance through simultaneous inhibition of FLT3-ITD- and AKT-mediated signaling. Our findings suggest that A674563 might be a potential drug candidate for overcoming FLT3 ligand-mediated drug resistance in FLT3-ITD positive AML.The FLT3-ITD mutation is one of the most prevalent oncogenic mutations in AML. Several FLT3 kinase inhibitors have shown impressive activity in clinical evaluation, however clinical responses are usually transient and clinical effects are rapidly lost due to drug resistance. One of the resistance mechanisms in the AML refractory patients involves FLT3-ligand induced reactivation of AKT and/or ERK signaling via FLT3 wt kinase. Via a screen of numerous AKT kinase inhibitors, we identified the well-established orally available AKT inhibitor, A674563, as a dual suppressor of AKT and FLT3-ITD. A674563 suppressed FLT3-ITD positive AML both  The typ50: 0.06 μM), MOLM14 (GI50: 0.18 μM) and MV4-11 (GI50: 0.075 μM), versus the FLT3 wt-expressing cell lines (about 5-20 fold less potent). The well-characterized FLT3 kinase inhibitors, AC220 and TCS359, exhibited a similar trend. The clonogenic assay also confirmed the selective efficacy of A674563 against FLT3-ITD positive AML cell lines  compared to FLT3-wt expressing cell lines  (We screened a panel of AKT inhibitors using FLT3 inhibitors AC220  and TCS3.387 μM) .50: 0.088 μM) compared to parental BaF3 cells (GI50: 1.6 μM). (Table 50: 0.045 μM). It was less potent against drug-acquired secondary mutants such as FLT3-ITD-D835Y (GI50: 0.42 μM) and FLT3-ITD-F691L (GI50: 0.43 μM). In addition, all of the inhibitory activities could be substantially rescued after addition of IL-3, which suggests that inhibitory effects are on-target. (50: 1.0 μM). We also found that A674563 was most potent against auto-phosphorylation of FLT3-ITD (EC50: 0.085 μM) and FLT3-D835Y (EC50: 0.045 μM), but was least potent against auto-phosphorylation of FLT3-ITD-D835Y (EC50: 1.26 μM), FLT3-ITD-F691L (EC50: 0.4 μM) and FLT3 wt (EC50: 0.87 μM); this is consistent with the demonstrated anti-proliferative activity of A674563 . Table  Interest-target.  However,3 Figure .50: 134 nM) versus FLT3 wt (IC50: 2171 nM).  of TCS359 combined with 1/50 of the GI50 concentration of MK2206 (0.12 μM/5.2 μM) was able to potently and synergistically inhibit the cell proliferation   against these FLT3-ITD cells, which would leave little margin to observe synergy with an AKT inhibitor, we chose the less potent FLT3 inhibitor, TCS359, to observe the combination effect more clearly  in which width (W) is defined as the smaller of the two measurements and length (L) is defined as the larger of the two measurements.Five weeks old female Balb/c-nu mice were purchased from the Shanghai Experimental Center, Chinese Science Academy . All animals were housed in a specific pathogen-free facility and used according to the animal care regulations of Hefei Institutes of Physical Science Chinese Academy of Sciences. Prior to implantation, cells were harvested during exponential growth. 10 million MOLM-14 cells in 1640 were formulated as a 1:1 mixture with Matrigel (BD Biosciences) and injected into the subcutaneous space on the right flank of Balb/c-nu mice. Daily oral administration was initiated when MOLM-14 tumors had reached a size of 200 to 400 mmIn Situ Cell Death Detection Kit (POD)  according to the manufacturer's instructions.Tumor tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin. Six-micron tissue section were prepared, deparaffinized, dehydrated, and then stained with hematoxylin and eosin (H&E) using routine methods. Commercially available primary antibody to human Ki-67  was used for Ki-67 staining. After heat-induced antigen retrieval, formalin-fixed and paraffin-embedded tumor tissue sections were stained with primary antibody overnight at 4°C. The slides were subsequently incubated with ImmPRES anti-rabbit Ig or anti-mouse Ig  at room temperature for 30 min, stained with peroxidase substrate 3, 3ʹ-diaminobenzidine chromogen (Vector Laboratories), and finally counterstained with hematoxylin. TUNEL staining was assessed using


Notably, CD20 is strongly expressed by neoplastic B cells, enabling this cell surface protein to be exploited as a therapeutic target3. Targeting CD20 with anti-CD20 monoclonal antibodies such as rituximab has shown clinically meaningful outcomes7. To enhance antibody-mediated therapy, immunotoxins are utilized as an innovative cancer therapy tool that links an antibody or antibody fragment with a toxin, selectively localizing the toxin to the target cells to induce lethality8.Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma (NHL) subtype with aggressive clinical demonstration characterized by the expression of neoplastic B-cell markers such as CD5, CD19, CD20, CD79, and BSAP/PAX512. Although targeted therapy such as Bruton’s tyrosine kinase (BTK) inhibition by ibrutinib has achieved high response rates (68%) in relapsed/refractory MCL, therapeutic resistance has emerged as a barrier to improved patient outcomes and survival13. MT-3724 has the potential to bypass possible resistance mechanisms mediated via acquired BTK mutations or the activation of alternative survival signaling pathways by inhibiting tumor growth and survival through toxin-mediated activity15. To assess the anti-MCL effects of MT-3724, we tested its in vitro and in vivo efficacy in MCL cell lines and patient-derived xenograft (PDX) mouse models.MT-3724, an engineered toxin body (ETB) comprised of a modified cytotoxic Shiga-like toxin 1A (3F7) and a CD20-specific single-chain variable fragment (scFv), recognizes CD20-expressing cells and triggers protein synthesis inhibition and apoptosis50 value ranging from 78 to 1383 ng/mL  was treated with different MT-3724 doses for 24 h. As previously reported, Jeko-R is an acquired ibrutinib-resistant MCL cell line generated through chronic exposure to low ibrutinib concentrations19. MT-3724 induced apoptosis, and the percentage of apoptotic cells , and Ingenuity Pathway Analysis (QIAGEN Bioinformatics) was performed. Overall, 63 pathways comprising 307 proteins were analyzed Supplementary Tables(DOCX 22 kb)Supplementary Figure 1(JPG 64 kb)Supplementary Figure 2(JPG 46 kb)Supplementary Figure 3(JPG 110 kb)Supplementary Figure 4(JPG 72 kb)Supplementary Figure 5(JPG 78 kb)Supplementary Figure Legends(DOCX 15 kb)


Post-translational mechanisms regulating cell-matrix adhesion turnover during cell locomotion are not fully elucidated. In this study, we uncovered an essential role of Y118 site-specific tyrosine phosphorylation of paxillin, an adapter protein of focal adhesion complexes, in paxillin recruitment to autophagosomes to trigger turnover of peripheral focal adhesions in human breast cancer cells. We demonstrate that the Rab-7 GTPase is a key upstream regulator of late endosomal sorting of tyrosine118-phosphorylated paxillin, which is subsequently recruited to autophagosomes via the cargo receptor c-Cbl. Essentially, this recruitment involves a direct and selective interaction between Y118-phospho-paxillin, c-Cbl, and LC3 and is independent from c-Cbl E3 ubiquitin ligase activity. Interference with the Rab7-paxillin-autophagy regulatory network using genetic and pharmacological approaches greatly impacted focal adhesion stability, cell locomotion and progression to metastasis using a panel of human breast cancer cells. Together, these results provide novel insights into the requirement of phospho-site specific post-translational mechanism of paxillin for autophagy targeting to regulate cell-matrix adhesion turnover and cell locomotion in breast cancer cells. Cell migration is a physiological process fundamental for embryonic development, immune and inflammatory responses, wound healing, and tissue homeostasis , 2. AberIn general, motile cells exhibit finger-like protrusions  of the plasma membrane that are stabilized by the formation of focal adhesions (FAs). The synchronous cycle of FA assembly/disassembly are essential for cell locomotion  and requIn this study, we report a novel role for the late endosomal protein Rab7 GTPase in the regulation of the selective recruitment of tyrosine 118 (Y118)-phosphorylated paxillin, but not Y31-phospho-paxillin to autophagosomes via its interaction with c-Cbl, which serves as a cargo receptor independently of its E3 ubiquitin ligase activity, in human breast cancer cells. This novel Rab7-mediated turnover of 118Y-p-paxillin was inhibited upon Atg12 downregulation, thereby reinforcing the implication of the autophagic pathway. This regulatory axis was not seen with other FA proteins such as FAK and was not affected by Src manipulation. Although autophagy-mediated FA turnover has recently been identified in mouse mammary 4T1 cells , studiesTo understand the role of the Rab7 GTPase in cancer cell migration, we first investigated its impact on FA turnover using two human breast cancer cell lines with intrinsic invasive properties, MDA231-M2 and BT-20 cells, and their matched counterparts where Rab7 was stably knockdown by shRNA Figure . Time-laInvestigation of cell migration confirmed that in MDA231-M2 cells where Rab7 was downregulated, cell locomotion was significantly compromised compared to control cells Figure . Similarin vitro observations and cancer progression in vivo, we investigated the impact of Rab7 down-regulation on cancer cell progression to metastasis using MDA231-M2 cells expressing control shNT or Rab7 shRNA implanted orthotopically into the mammary fat pad of SCID mice. After 40 days observation, a significant inhibition in the number of lung metastases was seen with the Rab7-silenced cells compared to control cells , wild-type Rab7 (GFP-Rab7) or a Rab7-GTPase defective mutant (GFP-Rab7-T22N)  in contrRab7 plays an essential role in the maturation of late autophagic vacuoles , 19. TheConsidering that Rab7 can play a role in both endosomal and autophagic pathways which converge at late autophagic vacuoles, we seeked to identify which of these pathways is primarily responsible for 118Y-p-paxillin trafficking to autophagosomes. Therefore, we silenced either Rab5  or Atg12 (autophagic upstream regulator) using siRNA Figure  and thenTo establish the importance of paxillin post-translational tyrosine modifications necessary for Rab7- mediated autophagosomal targeting, we investigated the two tyrosine residues previously established to be phosphorylated in dynamic focal adhesions , namely In order to gain further insight into mechanisms of paxillin recruitment to autophagosomes, we co-transfected BT-20 cells with FLAG-paxillin and GFP-LC3, followed by immunoprecipitation of FLAG-paxillin. As shown in Figure The E3 ubiquitin ligase activity of c-Cbl, via its LIR (light chain 3 (LC3)-interacting region) domain, has been reported to recruit Src to LC3 . AdditioCell locomotion is controlled by actin cytoskeleton-associated adhesions, represented by a network of proteins among which the scaffolding protein paxillin plays a fundamental regulatory role in the formation of both nascent and mature focal adhesions at leading edges of motile cells –27. ThesOur study identified the striking impact of Rab7 (implicated in transport from early to late endosomes of late endocytic structures/lysosomes) in the regulation of paxillin tyrosine-118 phosphorylation turnover via the autophagy pathway, an evolutionary conserved catabolic process which delivers cytoplasmic cargo to lysosomes via double membrane vesicles called autophagosomes  but not The critical function of Rab7 to recruit autophagy effector FA proteins is of fundamental importance given that autophagy, an evolutionary conserved catabolic process which delivers cytoplasmic cargo to lysosomes via autophagosomes , is implPaxillin has a proline-rich motif that binds to the Src SH3 domain and Src-FAK kinases, which mediate tyrosine phosphorylation of paxillin . In turnDuring selective autophagy, recognition of protein targets involves direct interaction between the substrate and LC3 through LIR motifs, which include cargo receptor proteins such as c-Cbl, NBR1 and p62 . InteresIn summary, our data provides new mechanistic insights into the role of Rab7 in targeting Y118 site-specific phosphorylation of paxillin to the autophagy pathway to promote focal adhesion turnover ; anti-GFP, anti-β-actin, anti-Rab7, anti-p62 and anti-LC3B (Santa Cruz); anti-416Y-p-Src and anti-NBR1 , anti-GFP (Roche); anti-LAMP-1 and anti-HA (Abcam); anti-LC3B, anti-Atg12 and anti-c-Cbl (Novus); anti-Rab5a (BD transduction laboratory); anti-paxillin, anti-Src and anti-FAK (Millipore); anti-LAMP1 (R&D systems); anti-FLAG (Sigma). Anti-mouse and anti-rabbit IgG-peroxidase-conjugated secondary antibodies for Western blot assays were from Bio-Rad. Alexa Fluor594 and 488 conjugated secondary antibodies were from Life Technology. Alexa Fluor 647 conjugated secondary antibodies were from Millipore. Epidermal growth factor was from Gibco; Histodenz (Nycodenz), nocodazole and chloroquine (CQ) were from Sigma-Aldrich. When indicated CQ was used at a concentration of 20 μM, a non-toxic concentration found to induce optimal autophagy inhibition (based on changes in LC3I/II ratio), which is in accordance with previous studies .The breast cancer cell lines MDA-231, MCF-7, BT-20 and 4T1 were obtained from the American Type Culture Collection. MDA231-M2 is a highly metastatic variant of MDA-MB-231 cells we isolated from lung nodules induced by the parental MDA-MB-231 . All celEGFP-paxillin (plasmid #15233), FLAG-paxillin (plasmid #15212), GFP-LC3 (plasmid #22405), mCherry-LC3 (plasmid #40827), GFP-Rab7 (plasmid #12605), GFP-Rab7-T22N (plasmid #12662) were obtained from Addgene. FLAG-paxillin-Y118F was further modified by GeneScript Inc. Primers used for mutation of the EGFP-paxillin and HA-Cbl  using site-directed mutagenesis kit (Stratagene): EGFP-paxillin-Y31F: sense, 5′-CAGAGGAAACGCCTTT CTCCTACCAACTGG-3′; antisense, 5′-CCAGTT GGGTA GGAGAAAGGCGTTTCCTCTG-3′, EGFP-paxillin-Y118F: sense, 5′- GAGGAGGAA CACCTGTTCAGCTT CCCAAACAG-3′; antisense, 5′-CTTGTTTGGGAAGCT GAACACGTGTTCCTCCTC-3′ and HA-Cbl-C381A: sense, 5′-GATGGGCTCCACATTCCAACTAGCTAA AA TATGTGCTGAAAATGATA -3′; antisense, 5′-TATCA TTTTCAGCACATATTTTAGCTAGT TGGAATGTGG AGCCCATC-3′. All plasmids were transfected into BT-20 cells using Lipofectamine LTX and PLUS reagents (Invitrogen) according to the manufacturer′s instructions.Non-targeted siRNA sequence: sense, 5′-UUCUCC GAACGUGUCA CGUdTdT-3′; antisense, 5′-ACGGUG ACACGUUCGGAGAAdTdT-3′, c-Cbl siRNA sequences: sense, 5′-GGGAAGGCUUCUAUUUGUUdTdT-3′; antisense, 5′-AACAAAUAG AAGCCUUCCCdTdT-3′, p62 siRNA sequence: sense, 5′-GCAUUGAAGUUGAUAUCGAUd TdT-3′; antisense, 5′-AUCGAUAUCAACUUCAAUGC dTdT-3′, NBR1 siRNA sequence: sense, 5′-GGAGUGG AUUUACCAGUUAUUdTdT-3′;antisense, 5′-AAUAA CUGGUAAAUCCACUCCdand FAK siRNA sequence: sense, 5′-GCGAUUAUAUGUUAGAGAUAG dTdT-3′; antisense, 5′-CUAUCUCUAACAUAUAAUCGCdTdT-3′ were from Dharmacon. Src siRNA was from Sigma with the following sequences: sense, 5′-CUGAGAGGG CGGUGGGUA UdTdT-3′ (WD04736566); anti-sense, 5′-AUACCCACCGCCCUCUCAGdTdT-3′ (WD04736567). siRNA for Rab5a: sequence 1 (siGENOME siRNA D-004009-01); and sequence 2 (siGENOME siRNA D-004009-02); Atg12: sequence 1 (siGENOME siRNA D010212-03); and sequence 2 (siGENOME siRNA D010212-05) were from Thermo Fisher Scientific. Control siRNA  or target siRNA oligonucleotides were expressed in cells by incubated with INTERFERin (Polyplus transfection) in serum-free RPMI medium according to the manufacturer's instruction.Stable Rab7 knockdown cells were generated from a polyclonal population as described previously . The target shRNA sequence for Rab7  was 5′-CTGCTGCGTTCTGGTATTTGA-3′ (targeting nucleotide 478-498). This sequence was cloned as an inverted repeat into pSUPER.puro vector according to the manufacturer's instructions (OligoEngine).Sub-confluent cells were washed with PBS, lysed in RIPA buffer  supplemented with 1mM PMSF and protease inhibitor cocktail (Roche) for 10 min on ice and centrifuged  to separate cell lysate. Cell lysate  was then added with SDS sample buffer  and boiled for 5 min. Samples were then resolved through 13% SDS-PAGE gels, transferred to nitrocellulose membrane, blotted with primary antibodies at different dilution in cold room overnight, and then amplified with horseradish peroxidase-conjugated secondary antibodies for 1h in room temperature and enhanced by chemiluminescence detection system.Cells transfected with both FLAG-paxillin (or FLAG-paxillin-Y118F) and GFP-LC3 were washed with PBS and lysed in lysis buffer  supplemented with 1mM PMSF and protease inhibitor cocktail (Roche) for 10 min on ice and cell lysate isolated following centrifugation . Cell lysate  was immunoprecipitated with either 2 μg anti-FLAG or anti-GFP antibodies or IgG (sigma) overnight at 4°C, and then incubated with protein G magnetic beads (Millipore) for 1 h at 4°C. Beads were washed three times with IP buffer , then added with SDS sample buffer  and boiled for 5 min, then separated by 10% SDS-PAGE. For interaction between c-Cbl and paxillin, cells were transfected with HA-Cbl (or HA-Cbl-C381A) and FLAG-paxillin and pulled down using anti-FLAG antibody.8) were washed with PBS and resuspended in 1 ml cold buffer A  supplemented with 1mM PMSF and protease inhibitor cocktail (Roche). Cells were disrupted by repeated aspiration through 26 gauge needle and centrifuged  and 0.95 ml supernatant was diluted with 1.45 ml 85.6% Nycodenz (Sigma) solution to become 52%, then placed on the bottom of the 13 ml ultra-clear centrifuge tube (Beckman). A discontinuous Nycodenz gradient  was layered on the top of the 52% supernatant and centrifuged at 24,700 rpm at 4°C for 3 h in a SW41 rotor (Beckman). Fractions were washed with cold PBS and collected as indicated . Pellets were collected at 14,800 rpm, 4°C for 1 h and used for immunoblotting experiments.Cells  and incubated with primary antibodies overnight in 4°C . The cells were washed with PBS containing 0.2% TritonX-100 and subsequently incubated with Alexa Fluor 488-labelled, Alexa Fluor 594-labelled, or Alexa Fluor 647-labelled secondary antibodies  for 1 hr in room temperature. The nuclei were stained with DAPI (0.1 μg/ml) for 5 min before mounted with aqueous mounting medium. Cells were imaged using WaveFX spinning disk confocal microscope system (Quorum Technologies INC.). Images shown are representative of three independent experiments2 and stimulated with 20 ng/ml epidermal growth factor. Fluorescent images were captured every 5 min for 2 h using a heated 64 X NA 1.40 objective at WaveFX spinning disk confocal microscope system (Quorum Technologies INC.). Fluorescence intensities of individual adhesions were measured over time by Volocity imaging software (PerkinElmer) and quantified according to the previously described protocol . Measurements were made on at least 20 individual adhesions in four separated cells for both Rab7 silenced and control groups. For investigating colozalization, cells were transfected with both GFP-paxillin and mcherry-LC3, then plated in RPMI 1640 medium with 10% FBS on a multiwell chambered coverglass (LabTek). Fluorescent images were captured every 15 sec for 2 h using a heated 63 X NA 1.40 objective using a WaveFX spinning disk confocal microscope system.For the monitoring of FA turnover, cells were transfected with EGFP-paxillin and plated in RPMI 1640 serum-free medium on a multiwell chambered coverglass (LabTek) prepared by coating overnight in 4°C with 5 μg/ml fibronectin. The coverglass was directly placed on a heated stage with 5% COFA disassembly and reformation, cells were grown on glass-coverslips coated with 5 μg/ml fibronectin for 24 h in 4°C, serum starved for 4 h in RPMI1640 serum free medium and then treated with 2μM nocodazole in serum-free medium for 2 h to allow a complete depolymerization of microtubules. Nocodazole was washed-out with serum-free medium and cells were further incubated at 37°C to allow a reformation of FA structure. Subsequently, cells were fixed and processed for immunofluorescence staining at different time intervals. Cells were stained with Rab7, 118Y-p-paxillin and LC3B. Focal adhesion dynamics was analyzed as previously described  using Im2 and stimulated with 20 ng/ml epidermal growth factor before monitoring the cell movement using a heated 40 X NA 1.40 objective at WaveFX spinning disk confocal microscope system (Quorum Technologies INC.). Single cell speed was determined by manually tracing the cell periphery every 20 min for 2 h by using Volocity imaging software (PerkinElmer).Cells were seeded at very low density in serum-free medium on a multiwell chambered coverglass (LabTek) prepared by coating overnight in 4°C with 5 μg/ml fibronectin. The coverglass was placed on a heated stage with 5% COt test. Data were deemed to be statistically significant if P < 0.05. Error bars indicate SEM unless otherwise indicated.All experiments counting 118Y-p-paxillin and LC3 puncta were performed in triplicate with similar results. Quantitative data in single cell migration experiments and puncta quantification were performed in 15 separate cells for both Rab7 silenced and control cells. For FA disassembly and FA life time quantification, measurements were made on at least 40 focal adhesions in 10 separate Rab7 silenced and control cells. All data are presented as mean ± SEM using Prism (GraphPad Software). Statistical significance was analyzed using unpaired two-tailed Student's 6 cells/ each mouse) expressing scramble shRNA and their matched cells stably expressing Rab7 shRNA were transplanted into the mammary fat pad of SCID mice. Tumor size was measured using a caliper, and tumor volume was calculated as π/6 (length × width2). Primary tumors were excised once they had reached a mean size of 0.8 cm3. The wound was closed with a single layer of surgical clips. Mice were sacrificed 40 days after transplanting. The lungs were fixed in 10% Bouin's fixative and lung metastatic nodules on the surface were counted using a stereomicroscope . Statistical comparison between groups was performed using a computer-based statistical package from Statistical Product and Service Solution  as we described earlier [All experiments were carried out according to protocol number 4101 of the McGill University Animal Care Committee. MDA231-M2 cells (1 × 10 earlier .


PHASs) in the rice genome. However, the regulatory mechanism that determines spatiotemporal expression of these RNAs has remained elusive. ETERNAL TAPETUM1 (EAT1) is a basic-helix-loop-helix (bHLH) transcription factor indispensable for induction of programmed cell death (PCD) in postmeiotic anther tapetum, the somatic nursery for pollen production. In this study, EAT1-dependent non-cell-autonomous regulation of male meiosis was evidenced from microscopic observation of the eat1 mutant, in which meiosis with aberrantly decondensed chromosomes was retarded but accomplished somehow, eventually resulting in abortive microspores due to an aberrant tapetal PCD. EAT1 protein accumulated in tapetal-cell nuclei at early meiosis and postmeiotic microspore stages. Meiotic EAT1 promoted transcription of 24-PHAS RNAs at 101 loci, and importantly, also activated DICER-LIKE5  mRNA transcription that is required for processing of double-stranded 24-PHASs into 24-nt lengths. From the results of the chromatin-immunoprecipitation and transient expression analyses, another tapetum-expressing bHLH protein, TDR INTERACTING PROTEIN2 (TIP2), was suggested to be involved in meiotic small-RNA biogenesis. The transient assay also demonstrated that UNDEVELOPED TAPETUM1 (UDT1)/bHLH164 is a potential interacting partner of both EAT1 and TIP2 during early meiosis. This study indicates that EAT1 is one of key regulators triggering meiotic phasiRNA biogenesis in anther tapetum, and that other bHLH proteins, TIP2 and UDT1, also play some important roles in this process. Spatiotemporal expression control of these bHLH proteins is a clue to orchestrate precise meiosis progression and subsequent pollen production non-cell-autonomously.The 24-nucleotides (nt) phased secondary small interfering RNA (phasiRNA) is a unique class of plant small RNAs abundantly expressed in monocot anthers at early meiosis. Previously, 44 intergenic regions were identified as the loci for longer precursor RNAs of 24-nt phasiRNAs  promotes large-scale remodeling of meiotic chromosomes with dramatic increases of histone H3 lysine 9 dimethylation, and that loss of MEL1 resulted in early meiotic arrest with few crossovers present. In rice anthers, MEL1-associating small interfering RNAs (masiRNAs) were composed of large amounts of premeiotic 21-nt phasiRNAs, plus low levels of both 24-nt repeat-associated siRNA and meiotic 24-nt phasiRNAs. Production of 24-nt phasiRNA during the meiotic stage was largely EAT1-dependent. Collectively, our findings suggest a possibility that unknown small RNA-mediated signaling regulates male meiosis non-cell-autonomously, probably a downstream output involves large-scale chromosome remodeling promoted by Argonaute proteins, while a possibility of EAT1-dependent, but small RNA-independent signaling cannot be excluded. In any cases, the studies on MEL1 and tapetal bHLH proteins will be a clue to reveal small RNA-mediated processes determining meiotic epigenetic landscape.Meiotic crossover formation shuffles homologous genes between parental genomes, and enables transmission of new gene sets to the offspring. Frequency and positions of crossovers are determined by numerous genetic and epigenetic factors, and low nucleosome-density regions are associated with crossover hot spots in yeasts and  Small noncoding RNAs are 20–30 nucleotides (nt) long and associate with Argonaute family proteins to serve as guide molecules for RNA silencing in various biological processes, such as cell type specification, cell proliferation, cell death, metabolic control, transposon silencing and antiviral defense . Plant gtrans-acting siRNA (tasiRNA) and phased secondary siRNA (phasiRNA) are identified as plant-specific small RNA subgroups. In monocot model plants, rice and maize, phasiRNAs are abundantly expressed in the male reproductive organs, and in this study, the term "phasiRNA" will be used for monocot reproductive phasiRNAs derived from protein-noncoding regions. Both tasiRNA and phasiRNA are produced via miRNA-dependent primary processing, and characterized by phased alignment on both sense and antisense strands in genomic regions. However, they are distinct in several points. First, phasiRNAs are abundantly expressed in developing reproductive organs .–24.22–24In this study, we focused on the rice bHLH TFs, because they are key transcriptional regulators for differentiation and development of anther somatic layers. TDR INTERACTING PROTEIN2 (TIP2)/bHLH142 is expressed in several undifferentiated cell layers to form the middle layer and tapetum , 28. TAPPHAS precursor RNAs to produce 24-nt phasiRNAs in tapetum. This study demonstrates that EAT1 is one of key regulators triggering meiotic phasiRNA biogenesis in anther tapetum, and that other bHLH proteins, TIP2 and UDT1, also play important roles in this process.In addition to its role in tapetal PCD, we found that EAT1 is required earlier in tapetal development to support meiosis, while the loss of EAT1 function has little impact on the tapetum morphology. EAT1 shows a bimodal expression at both early meiosis and postmeiosis. Interestingly, EAT1 expressed during early meiosis promoted both transcription and processing of 24-bHLH genes: UDT1, TDR, TIP2 and EAT1, all of which are involved in tapetal cell-fate decision [UDT1, TDR and TIP2 were expressed as expected from previous reports  and postmeiosis (ST.5), whereas it was previously thought to function only in postmeiotic tapetal PCD [To determine the impact of bHLH proteins in communication between somatic tapetal cells and PMCs in rice anthers, we first performed quantitative reverse-transcription PCR (qRT-PCR) of four decision –32. In t reports , S1 Dataetal PCD , 31.EAT1pro-EAT1-GFP transcriptional fusion construct  and 69.4% at metaphase I (n = 36)  (eat1-4 PMCs exhibited interphase-like nuclei with fully decondensed chromosomes (n = 163), in contrast to wild-type PMCs  . In addi(n = 42) . Anothern = 192, . In addin = 192, , S1 Datan = 192, . These reat1-4 plants. The data were obtained from three different meiotic stages: premeiosis (ST.1), early meiosis (ST.2) and late meiosis (ST.4), each with three biological replicates. 142,048,793 reads from wild-type and 146,928,874 reads from eat1-4 anthers . Of 44 24-PHAS loci previously reported [PHAS loci and analyzed hereafter.mRNA-seq also identified 6,097 regions generating long intergenic noncoding RNAs (lincRNAs), and 248 showed ST.2-enriched and EAT1-dependent expression . Next, we genome . As a rereported , 24 werePHAS transcripts detected at the 101 loci in wild-type ST.2 anthers were 688-fold and 24-fold higher than those in ST.1 and ST.4 anthers, respectively. In addition, the values were 55-fold higher than in eat1-4 anthers at ST.2  using EAT1-GFP-expressing plants. Both sequences included E-box motifs, short CANNTG sequences potentially targeted by bHLH proteins [chr5-20-Ebox1 was enriched 5.4-fold and the chr6-97-Ebox2 was enriched 6.1-fold in ChIP of EAT1-GFP-expressing anthers (PHAS loci.Chromatin-immunoprecipitation (ChIP)-qPCR analysis was performed to examine EAT1-binding to the upstream proteins  Fig 5A)cis seque anthers , S1 DataDCL5 was 2.1-fold downregulated in eat1-4 ST.2 anthers in mRNA-seq analysis  genes. Indeed, analysis , S1 Dataanalysis , S1 Dataectively , S1 Dataynthesis , 41, andoduction  -Luciferase fusion reporter into rice protoplasts (PHASs was significantly 4.46 (chr5-20) and 3.99-fold (chr6-97) elevated in EAT1-UDT1 cotransfection, compared to the no effector control  and pDCL5 (1.95 fold)  . Interes.61 fold , while id  in rice protoplasts. EAT1 fused with the C-terminal split of YFP (EAT1-cYFP and cYFP-EAT1) gave positive BiFC signals when coexpressed with UDT1-nYFP , while tPHAS precursors and the EAT1 gene itself by interacting with UDT1 at the molecular level. EAT1 also promotes the DCL5 transcription, but likely with an unknown bHLH partner.The above results demonstrate that the meiotic EAT1 TF promotes the transcription of 24-tip2-2 loss-of-function allele newly identified in this study had a T-DNA insertion in the third intron  was 4.3-fold enriched in ChIP of YFP-TIP2  and pDCL5 activities (4.91 fold) (PHAS activity (2.52 and 2.67 fold)  . TIP2-TD67 fold) . Both TIectively , consistectively  and to tectively .PHASs and DCL5 by interacting with UDT1 at the molecular level in early meiosis.Collectively, these results suggest that TIP2 has the potential to activate transcription of both 24-Small RNAs are sorted to confer association with specific Argonaute family proteins . MEL1 iseat1-4 and mel1-1 flowers, respectively  were defined as canonical 24-nt MEL1-associating siRNAs (masiRNAs)  and protein coding regions  (PHAS loci were detected in ST.2 and ST.4 (5.2 and 9.0%), but hardly detected in ST.1 anthers (<0.1%)  and ST.4 (<0.5%) in addition to ST.1 (<0.1%)  . Throughd 10.7%) . In cont (<0.1%) . This re (<0.1%)  right. I (<0.1%) , S1 Datacytosine , consistch stage , S1 DataPHAS loci for example  were mapped at the third phase of the sense strand in ST.2 and ST.4 anthers, respectively  bHLH122, the EAT1 ortholog, also shows bimodal expression , and MALhasiRNAs . A posithasiRNAs , whereasnd pEAT1 . Furthermel1 mutant anthers with few meiotic DSBs in male meiocytes [eat1-4 anthers (multiple sporocytes1 (msp1) and tpd1-like gene in rice1a (tdl1a), in which a subset of inner anther-wall cells turn into PMCs [ms23 anther lacking the tapetum fails to produce 24-nt phasiRNAs, but the ocl4 anther developing the tapetum succeeds [The observation that a subset of tapetum-originating phasiRNAs was sorted to MEL1 Argonaute, which is abundantly expressed in PMCs but not in tapetal cells . Though eiocytes  produce  anthers . A recennto PMCs , 47. In succeeds . These rPHAS transcripts or their processed intermediates are transferred from tapetum, and processed into mature 24-nt phasiRNAs by 24-PBR components in PMCs. TIP2 and EAT1 are detectable in somatic companions, but hardly in PMCs  of all protein-coding transcripts expressed in ST.2 anthers , but also non-C-terminal ones are supposed to move together in the intercellular movement, because the enrichment of C-terminal phasiRNAs in MEL1-RIPseq in this study is simply due to the selectivity of MEL1 . The anaArabidopsis AGO4 plays important roles in chromosome condensation and segregation during the first meiotic division [Arabidopsis AGO9, is also required for meiotic chromosome condensation [Functions of other Argonaute proteins in plant meiosis still remain to be debated. Rice flowers highly express many Argonaute proteins in addition to MEL1/AGO5  , whose mdivision , comparaensation . In eithPHAS precursor loci showed higher DNA methylation in all cytosine contexts  in isolated maize PMCs. The highest context was CHH methylation, implying that reproductive phasiRNAs are involved in RNA-directed DNA methylation (RdDM) in PMCs. RdDM includes both de novo DNA methylation and histone H3 lysine-9 (H3K9) methylation in plants [Dukowic-Schulze et al.  unveiledn plants –59. Suppn plants .Meiosis is a special type of cell division to transmit new haplotypes to the next generation, and additionally, to survey incompatibilities in ploidy levels and chromosomal structures between both parents. This process must be strictly regulated by complicated mechanisms genetically and epigenetically. Recent genome-wide studies have revealed that small RNA-mediated and non-cell-autonomous regulation is likely general in reproduction of eukaryotic species. Further analyses of tapetum-expressing bHLH TFs and meiotic phasiRNAs in anthers will bring new epigenetic insights into plant reproduction systems.eat1-4 mutant is a Tos17 insertion line produced from the rice variety, cv. Nipponbare [mel1-1 mutant [Tos17 line with the Nipponbare background, was kindly provided by the National Bioresource Project (NBRP) Rice, conducted by the Japan Agency for Medical Research and Development (AMED). The tip2-2 mutant is a T-DNA tag line with the genetic background of cv. Dongjin [Tos17-internal primers  containing 4% paraformaldehyde (PFA) for 3 h and washed six times in PMEG buffer for 2 hours. After dehydration using ethanol series, they were embedded in Technovit7100 resin (Heraeus Kulzer), sectioned in 2 μm thick slices using a LM2255 microtome (Leica Microsystems), stained with 0.1% toluidine blue O  and photographed using a BX50 light microscope (Olympus) and a DP50 camera system (Olympus). Cellulosic cell wall staining was conducted according to the method described previously [Rice spikelets were fixed in PMEG buffer -SK(-) vector. The 1.2 kbp XhoI-EcoRV fragment including the 3’ downstrem region of the EAT1 gene was also subcloned into another pBSII-SK(-), and from this plasmid, the 2.0 kbp XhoI-EcoRV fragment harboring a sGFP sequence just in the front of the EAT1 stop codon was constructed using EAT1-specific primers, bHLH141stop-BamHI/bHLH141XhoI-BamHI and bHLH141stop-NotI/M13-Rev, and a CaMV35S-sGFP(S65T)-nos3ʹ vector [EAT1pro-EAT1-GFP.EAT1-GFP  was consʹ vector , kindly 35S-sGFPST-nos3ʹ vTIP2pro-YFP-TIP2  vector. From the 6.6 kbp fragment, the 1.8 kbp HindIII-SalI fragment including the translational initiation site (TIS) was subcloned into pBSII-SK(-). From this plasmid, the YFP sequence was inserted just in front of TIS by using TIP2-specific primers bHLH142start-NcoI/M13-Rev and bHLH142start-BsrGI/T7-EcoRI and a pEYFP vector (a cloning vector with EYFP sequence in pUC18 backbone). Then, the 1.8 kbp fragment with the YFP sequence was inserted back into the original 6.6 kbp genomic fragment/pBSII-SK(-) plasmid. The resultant 7.4-kbp insert was digested, blunt-ended, and reinserted into pGWB601 binary vector [TIP2pro-TIP2-YFP (TIP2 stop codon in the above 6.6 kbp genomic fragment/pBSII-SK(-) was replaced by YFP sequence by using TIP2-specific primers, bHLH142stop-NcoI/M13-20 and bHLH142stop-BsrGI/M13-Rev, and a pEYFP. Finally, the 7.4 kbp of TIP2pro-TIP2-YFP insert was assembled in the pPZP2H-lac. In above constructions, KOD-FX DNA polymerase (TOYOBO) was used for PCR.YFP-TIP2  was consy vector , kindly TIP2-YFP , the TIPMEL1pro-GFP-MEL1 construction, the GFP sequence was inserted just in front of MEL1 TIS in pKN16, a binary vector containing the 18 kbp MEL1 genomic fragment [MEL1 TIS, were amplified from pKN16 with primer pairs up_nf/up_nr and up_atgf/up_r, respectively. Linker-attached sGFP coding sequence was amplified from CaMV35S-sGFP(S65T)-nos3ʹ with ngfp_f/ngfp_r primers. The PCRs were conducted using a PrimeSTAR Max DNA polymerase (TaKaRa). The three amplified DNA fragments were mixed with the NruI-AscI-digested pKN16 and incubated with an In-Fusion HD enzyme premix (TaKaRa) to assemble MEL1pro-GFP-MEL1, following manufacturer’s instructions. All the primer sequences for the construction were listed in In fragment . Two DNAThe constructs were transformed into rice calli using agrobacterium-mediated transformation , in whicAnthers embedded in 6% SeaKem GTG agarose (Lonza) were sliced into 50 μm thickness by MicroSlicer DTK-ZERO1 (D.S.K.), and mounted on slide grasses with VECTASHIELD (Vector Laboratories) containing DAPI. Fluorescent images were captured using Fluoview FV300 CLSM system (Olympus).Spikelet (lemma) and anther lengths were measured under SMZ645 stereo microscopy (Nikon). 0.8–1.2 mm anthers were fixed with 4% PFA/PMEG and provided for chromosome observations as previously described . Fluores12-18 primer (Life Technologies) and SuperscriptIII reverse transcriptase (Life Technologies). The products were 20-fold diluted and supplied for real-time qPCR using gene-specific primers . Eighteen libraries differentially indexed by FastGene Adapter kit (Nippon Genetics) were multiplexed (9 per lane) and sequenced by HiSeq2500 (Illumina) with SR50 (single ended). Adapter sequences were removed in silico using R package QuasR [Total RNAs were extracted from ST.1, ST.2 and ST.4 anthers of wild-type and ge QuasR .eat1-4 ST.2 anthers, and (3) genes with each standard deviation less than a half of the FPKM mean value of three replicates in wild-type ST.2 anthers. The lincRNAs were determined by Cuffdiff2 (merged.gtf), in which protein-coding genes were removed as referring to MSU7.0 annotation, and unannotated but transcribed genomic regions larger than 200 bp were extracted. FPKM values of lincRNAs were calculated by BEDtools [mRNA-seq reads were mapped on the rice genome IRGSP1.0 using Tophat (v2.0.14) . DiffereBEDtools . FurtherPHAS loci. Regional abundance of mRNA-seq and 24-nt sRNA-seq reads mapped on the rice genome . The libraries were 9-plexed per lane and sequenced by HiSeq2500 (illumina) with SR52, a 2-bp extended version of SR50, for higher-quality sequencing. After trimming by QuasR, 24-nt long sRNA-seq reads were extracted by ShortRead , and mapshown in  was calc program . Phased  program .indica rice variety, cv. 93–11, was obtained from Sequence Read Archive of DNA Data Bank of Japan (DDBJ-SRA) under the accession code SRR034102 [http://hannonlab.cshl.edu/fastx_toolkit/) and the reads retaining 20- or 21-nt length were mapped onto rice IRGSP1.0 genome using Bowtie 2 [PHAS loci identified in this study , the standard 5ʹ rapid amplification cDNA end (5ʹ RACE) method was applied using a GeneRacer kit (Thermo Fisher Scientific), total RNA from ST.2 wild-type anther, and gene specific primers  were supplied for ChIP as described previously . The ant24-PHAS , DCL5, EAT1 and DCL3a genes, all originated from the japonica rice cv. Nipponbare, were inserted in the upstream of the firefly Luciferase CDS and the nopaline synthase (nos) terminator. This reporter construct was cloned into pBSII-SK(-) plasmid    . For norII-SK(-) , and cotEAT1, TIP2 and UDT1 cDNAs, originated from Nipponbare ST.2 anthers, was inserted into the either of upstream or downstream of both pBS-35S-nYFP and pBS-35S-cYFP vectors. For the nuclear marker, the rice Histone 2B (H2B) cDNA were fused with the maize Ubiquitine promoter and in-frame with the tagRFP gene (Evrogen), cloned into pPZP2H-lac binary vector [A pair of split YFP vectors (pBS-35S-nYFP and pBS-35S-cYFP) were kindly provided by Drs. D. Tsugama  and T. Takano  . Each ofy vector , and cotmel1-1 and eat1-4 flowers at ST.1, ST.2 and ST.4, each of which included two biological replicates, were obtained using anti-MEL1 antibody as described previously [mel1-1 and eat1-4 fractions. In this process, 24-nt masiRNAs were defined in 24-nt RNA sequences as having ≥15 RPM detected in wild-type ST.1, ST.2 or ST.4 flowers, and ≥RPM 4-fold enriched in wild-type compared to mel1-1.RIP fractions from wild-type, eviously . LibraryEAT1; Os04g0599300, TIP2; Os01g0293100, TDR; Os02g0120500, UDT1; Os07g0549600, MEL1, Os03g0800200, DCL5; Os10g0485600, DCL3a; Os01g0909200, DCL4; Os04g0509300, DCL1; Os03g0121800, RDR6; Os01g0527600, AP25; Os03g0186900. (Rice Annotation Project Database (RAP-DB) (http://rapdb.dna.affrc.go.jp)).S1 FigbHLH genes; UDT1, TDR, TIP2 and EAT1, in wild-type (cv. Nipponbare) anther development. The bottom numbers of the developmental stage correspond to Expression patterns of four tapetum-related (TIF)Click here for additional data file.S2 FigA( and G) Genomic structure of EAT1 and Tos17 insertion of eat1-4 (A) and TIP2 and T-DNA insertion of tip2-2 (G).B( and H) qRT-PCR results of underlined regions of EAT1 transcript in wild-type and eat1-4 flowers (B) and of TIP2 transcript in wild-type and tip2-2 flowers (H). In (B) and (H), total RNAs from early meiotic flowers (around 2.0 mm) long were used.C( and I) Flower morphology of eat1-4 (C) and tip2-2 (I). Bars, 1 mm.D, E, J( and K) I2KI staining of mature pollen in the anther of EAT1 wild type D)EAT1 mRNA expression during anther development in wild type (cv. Nipponbare) and eat1-4 plants (F) and TIP2 mRNA expression in wild type (cv. Dongjin) and tip2-2 anthers (L). In (F), expression data of EAT1 transcripts in wild-type anthers were identical to those of In qRT-PCR analyses, relative expression values and standard errors were calculated by three biological replicates.(TIF)Click here for additional data file.S3 FigAP25 expression during anther development in eat1-4 (left), tip2-2 (right) and their respective wild-type siblings. Relative expression values and standard errors were calculated by three biological replicates.(TIF)Click here for additional data file.S4 FigA( to J) Anther cross-sections of wild-type (EAT1) A( to E) and eat1-4F( to J). The cross-sections of ST.4 and their magnified views are shown in K( to X) Anther cross-sections of wild-type (TIP2) K( to Q) and tip2-2 (R to X).A: ST.1; B: ST.2; C: ST.3; N( and U): ST.4; D: ST.5; E: ST.6; Q( and X): magnified view of ST.4.Bars, 20 μm.(TIF)Click here for additional data file.S5 FigA) Box plots of spikelet lengths in each meiosis I stage in wild-type and eat1-4 mutant anthers. Zyg.; early zygotene, Pac.; pachytene, Dip.; diplotene, Div; the stage including diakinesis, metaphase I, anaphase I, dyad, and second division. n.s. and *** indicate no significance and significance at P = 0.001 (Student's t-test), respectively, between the wild-type and eat1-4. Arrowheads indicate average values.(B) Column charts showing the spectrum of meiotic stages in single wild-type and eat1-4 anthers.((TIF)Click here for additional data file.S6 FigPHAS loci identified in this study. Then, the frequency of in-phase reads to total reads were box-plotted. The sRNA-seq reads from three replicates were combined in each stage and plotted. The median values were 0.814 and 0.820 in wild-type ST.2 and ST.4 samples, respectively.Of 254,163 and 877,203 reads of 24-nt small RNAs from ST.2 and ST. 4 anthers, 329,112 (ST.2) and 1,138,234 reads (ST.4) were defined as in-phase reads for the 24-nt phased interval that starts from the predicted miR2275 cleavage site  on 93 24(TIF)Click here for additional data file.S7 FigA) ChIP-qPCR results of DCL3a and DCL4 promoter region using transgenic (TG) plants expressing EAT1-GFP B)( and YFP-TIP2 C)Click here for additional data file.S8 FigA) qRT-PCR results of 24-nt phasiRNA biogenesis-related genes, DCL1, RDR6, and two pri-miR2275 transcripts in wild-type and eat1-4 anthers.(B) qRT-PCR results of five 24-PHAS transcripts, DCL5, DCL1, RDR6, and two pri-miR2275 transcripts in wild-type and tip2-2 anthers. In qRT-PCR analyses, relative expression values and standard errors were calculated by three biological replicates.(C) Schematic illustration of genomic compositions of the 5ʹ upstream regions of pri-miR2275b locus. The diagrams are equivalent to (D( and E) ChIP-qPCR results of pri-miR2275b promoters using TG plants expressing EAT1-GFP E and YFP-TIP2 (E). In ChIP-qPCR analyses, relative abundance and standard errors were calculated by two or three biological replicates each subjected to three PCR replications. n.s.; not significant. * and **; significant at P = 0.05 and P = 0.01 in Student's t-test, respectively, less than the leftmost positive ChIP result in each graph.(TIF)Click here for additional data file.S9 FigA) and an empty vector. Bars, 1 mm.C) in developing anther sections from ST.1 to ST.5. in a transgenic plant harboring TIP2pro-YFP-TIP2. YFP-TIP2 signals were intensified in tapetal nuclei (arrowhead) and also detected in middle layer nuclei (arrow) in ST.2 and ST. 3 anthers, and not detected in the ST.2 anther from the negative control . Bars, 20 μm.D and E) TIP2-YFP expression and localization in wild-type and eat1-4 ST.2 anthers (D) and EAT1-GFP expression and localization in wild-type and tip2-2 ST.2 anthers (E). Bars, 20 μm.(F and G) Expression pattern of TIP2 mRNA in wild-type and eat1-4 anthers (F), and EAT1 mRNA in wild-type and tip2-2 anthers (G). Relative expression values and standard errors were calculated by three biological replicates.((TIF)Click here for additional data file.S10 FigA), DCL5, EAT1 or DCL3a fused with the firefly Luciferase. CaMV35S; cauliflower mosaic virus 35S promoter, nos; nopaline synthase terminator.B)( The results of the transient expression assay. Any one or two effector plasmids encoding EAT1 (E1), TIP2 (T2), UDT1 (U1) and TDR (TD) proteins were cotransfected with the reporter constructs into rice protoplasts. The number above each bar is the fold change of the Luciferase activity compared to the negative control without the effector (leftmost bars). *, ** and ***; the significant fold changes at P = 0.05, 0.01 and 0.001 in Student’s t-test, respectively, compared to the negative control. Error bars indicated standard deviation of three biological replicates. The significant >2 fold changes were in bold.(TIF)Click here for additional data file.S11 Fig(A) BiFC results of EAT1-cYFP and TIP2-cYFP constructs combined with a nYFP-UDT1 sonstruct and those of negative control combinations. (B) BiFC results of cYFP-EAT1 and cYFP-TIP2 constructs combined with a UDT1-nYFP construct and those of negative control combinations. (C) BiFC results of cYFP-EAT1 and cYFP-TIP2 constructs combined with a nYFP-UDT1 construct. Some negative control results common in (TIF)Click here for additional data file.S12 FigPHAS loci, protein-coding genes, intergenic regions except for 24-PHAS loci and repetitive regions, in eat1-4 anthers at ST.1, ST.2 and ST.4 stages. The numbers with parentheses indicated the read counts of 24-nt masiRNAs extracted from MEL1-IPseq results.(A) Pie-charts representing the ratios of 24-nt MEL1-associating siRNAs (masiRNAs) originated from 24-PHAS loci (top), and from all 2,110 species of 24-nt masiRNAs mapped on the rice genome (bottom).(B) The sequence logos generated from 68 species of 24-nt masiRNAs mapped onto 24-(TIF)Click here for additional data file.S13 FigMEL1 included was indicated by an arrowhead.A histogram representing the distribution of FPKM values of all 38,311 rice genes in wild-type ST.2 anthers. The area where (TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(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 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.


Mastadenovirus; family Adenoviridae) serotype 55 is a reemerging pathogen associated with acute respiratory disease. Here, we report the complete genome sequence of HAdV-55 strain AFMC 16-0011, isolated from a military recruit, using next-generation sequencing technology.Human adenovirus (HAdV) (genus  Adenoviridae family, which comprises five genera. Human adenoviruses (HAdVs) are classified in the Mastadenovirus genus. There are more than 68 HAdV genotypes, characterized by neutralization assay, which are grouped within seven species (A to G) (–6–Adenoviruses are double-stranded DNA viruses that belong to the (A to G) –5. HAdV A to G) – are assoA to G) –, 6–9. ReThe whole-genome sequence of HAdV-55 strain AFMC 16-0011 was sequenced using next-generation sequencing (NGS) technology. DNA was sheared using an M220 focused-ultrasonicator . The library was prepared using the TruSeq Nano DNA sample preparation kit . Deep sequencing was performed on a MiSeq benchtop sequencer (Illumina) using a MiSeq reagent kit version 2 (Illumina) with 2 × 150-bp paired-end reads. All reads with quality scores >Q20 score were trimmed for reference mapping and consensus sequence extraction using CLC Genomics Workbench version 7.5.2 . NGS data of the HAdV-55 strain AFMC 16-0011 generated 19,831 reads with an average read length of 150 bp. Gaps at the 5′ and 3′ termini were filled by designing specific primers, using the Sanger sequencing method. The full-length genome of HAdV-55 strain AFMC 16-0011 was 34,776 nucleotides with 48.77% G+C content. The inverted termini repeat sequences were 137 bp in length. A comparison of the whole-genome sequences of HAdV-55 strain AFMC 16-0011 and HAdV-55-B/CHN/BJ01/2011/55 (P14H11F14) (GenBank accession no. JX491639) from China showed 99.93% sequence similarity at the nucleotide level. The whole-genome sequencing of HAdVs will accelerate the acquisition of new knowledge about their phylogenetic, phylogeographic, and evolutionary origin.KX494979.The whole-genome sequence of HAdV-55 strain AFMC 16-0011 was deposited in GenBank under the accession number


AMP-activated protein kinase (AMPK) activation could protect osteoblasts from dexamethasone (Dex). This study aims to provoke AMPK activation via microRNA downregulation of its negative regulator protein kinase C ζ (PKCζ). Results show that microRNA-25-5p (miR-25-5p) targets PKCζ's 3’ untranslated regions (UTRs). Forced-expression of miR-25 downregulated PKCζ and activated AMPK in human osteoblastic cells (OB-6 and hFOB1.19 lines), which thereafter protected cells from Dex. Reversely, expression of antagomiR-25, the miR-25 inhibitor, upregulated PKCζ and inhibited AMPK activation, exacerbating Dex damages. Notably, PKCζ shRNA knockdown similarly activated AMPK and protected osteoblastic cells from Dex. AMPK activation was required for miR-25-induced osteoblastic cell protection. AMPKα shRNA or dominant negative mutation almost completely blocked miR-25-induced cytoprotection against Dex. Further studies showed that miR-25 expression increased NADPH activity and suppressed Dex-induced oxidative stress in osteoblastic cells. Such effects by miR-25 were abolished with AMPKα knockdown or mutation. Significantly, miR-25-5p level was increased in patients’ necrotic femoral head tissues, which was correlated with PKCζ downregulation and AMPK hyper-activation. These results suggest that miR-25-5p targets PKCζ and protects osteoblastic cells from Dex possibly via activating AMPK signaling. Long-term and/or excessive medication of dexamethasone (Dex) or other glucocorticoids (GC) will lead to different degrees of bone damages, including osteoporosis and osteonecrosis . Osteobli.e. acetyl-CoA carboxylase (ACC) -2,5 diphenyltetrazolium bromide (MTT) assay described in our previous studies , 11.As described , the HisThe two lentiviral shRNAs (GV248-puromycin vector) against human PKCζ were designed, synthesized and verified by Genepharm Co. . The AMPKα shRNA was described in our previous study . OsteoblThe dominant-negative mutant of AMPKα  construct was a gift from Dr. Lu's group , which wNADPH activity assay was described in previous studies , 41. BriROS production was measured via a DCFH-DA fluorescent dye (Invitrogen). After treatment, cells were incubated with 1 μM of DCFH-DA (Invitrogen) for 30 min. Cells were then washed and analyzed for fluorescence using the flow cytometer . The ROS intensity in the treatment group was normalized to that of control group.Surgery-isolated fresh necrotic femoral head tissues and their surrounding normal femoral head tissues were collected from Dex-taking patients. Fresh tissue specimens were dissolved in tissue lysis buffer  and were subjected to qRT-PCR assay and Western blot assay. The experiment protocols requiring human samples were approved by the institutional review board and ethics committee of all authors’ institutions, and written informed consent was obtained from each patient. A total of 9 patients were included. All studies were conducted according to the principles expressed in the Declaration of Helsinki.p < 0.05 were considered statistically significant.The data presented were mean ± standard deviation (SD). Statistical differences were analyzed by one-way ANOVA followed by multiple comparisons performed with post hoc Bonferroni test (SPSS version 18.0). Values of


Phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 1 (P-Rex1) has been implicated in cancer growth, metastasis, and response to phosphatidylinositol 3-kinase (PI3K) inhibitor therapy. The aim of this study was to determine whether P-Rex1 expression differs between primary and metastatic human breast tumors and between breast cancer subtypes. P-Rex1 expression was measured in 133 specimens by immunohistochemistry: 40 and 42 primary breast tumors from patients who did versus did not develop metastasis, respectively, and 51 breast-derived tumors from metastatic sites . Primary breast tumors showed significant differences in P-Rex1 expression based on receptor subtype. ER+ and HER2+ primary tumors showed higher P-Rex1 expression than primary triple-negative tumors. HER2+ metastases from all sites showed significantly higher P-Rex1 expression compared to other metastatic receptor subtypes. Solid organ  metastases showed higher P-Rex1 expression compared to bone metastases. P-Rex1 expression is increased in ER+ and HER2+ breast cancers compared to triple-negative tumors. P-Rex1 may be differentially expressed in metastatic tumors based on site and receptor status. The role of P-Rex1 in the development of breast cancer metastases and as a predictive biomarker of therapeutic response warrants further investigation. By using estrogen receptor subtypes , 2. Thessubtypes .The development of distant metastases in breast cancer remains the main cause of mortality: among women with de novo distant metastatic disease, the overall 5-year survival rate is 26% . HoweverWe and others recently proposed that the protein phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 1 (P-Rex1) is a potential breast cancer biomarker to predict sensitivity to therapeutic inhibitors of phosphatidylinositol 3-kinase (PI3K) . P-Rex1 This retrospective study was approved by the Dartmouth Committee for the Protection of Human Subjects. The Dartmouth-Hitchcock Medical Center pathology database was retrospectively searched from 2003 to 2015 to identify primary invasive breast cancers and matched metastases. Pathology reports were reviewed to determine ER, PR, and HER2 status. Cancers were categorized as luminal A (ER+ and/or PR+ but HER2−), luminal B (ER+ and/or PR+ and HER2+), HER2+ (ER− and PR− but HER2+), or triple-negative . H&E-stained histologic sections from primary and metastatic tumors were reviewed, and one representative formalin-fixed paraffin-embedded (FFPE) tissue block was selected for immunohistochemistry (IHC) analysis.μm, mounted on slides, and air-dried at room temperature. An automated protocol was performed using the Leica Biosystems BOND RX including bake/dewax and antigen retrieval . Primary antibody binding utilized the Leica Biosystems BOND Refine Detection kit (DS98000) with a 15-minute incubation and anti-P-Rex1 antibody . Visualization was performed using the Leica Refine kit with DAB chromogen and hematoxylin counterstain. Slides were dehydrated, cleared, and mounted with Tissue-Tek Glass Mounting Medium (Sakura). For positive and negative P-Rex1 IHC controls, we used the following: (1) MCF-7 breast cancer cell line- (ATCC-) derived xenografts expressing P-Rex1 and (2) ZR75-1 breast cancer cells (ATCC) transiently transfected with siRNA targeting P-Rex1 (GE Dharmacon) or a nonsilencing control (Qiagen) . The generation of xenografts and transfection of cells was described previously [Tumor tissue sections were cut at 4  t-test and ANOVA with Tukey's honest significant difference (HSD) were used to compare histoscores between groups. Associations between P-Rex1 histoscore and clinical/pathologic features  were analyzed using the multivariate linear model. p ≤ 0.05 was considered significant.Correlation of P-Rex1 histoscores between the two pathologists was estimated using Pearson correlation. Unpairedn = 24), brain (n = 19), liver (n = 4), and lung (n = 4). Clinical and pathologic features of the primary breast tumors are detailed in P-Rex1 expression was evaluated in 133 cases by IHC from 101 patients: 42 primary breast tumors from patients who did not develop distant metastasis within 3–11 years of follow-up (mean = 9.7 years); 40 primary breast tumors from patients who later developed distant metastasis; and 51 breast-derived metastatic tumors, of which 36 had a matching primary breast tumor available for analysis. Tumors from metastatic sites included bone (R2 = 0.73). Overall, there was a significant difference in P-Rex1 expression in primary breast cancers based on receptor status (ANOVA p = 0.019). Primary ER+   and HER2+ (131 ± 66) breast tumors showed higher P-Rex1 expression than primary TNBCs (75 ± 62) . HER2+ metastases generally had higher P-Rex1 expression (187 ± 55) compared to luminal A (131 ± 68), luminal B (123 ± 75), and triple-negative tumors (100 ± 49). There was a nonsignificant difference in P-Rex1 expression between metastatic sites when combining all receptor subtypes (ANOVA p = 0.07), with a trend toward decreased expression in bone. Analysis of combined brain and visceral  metastases demonstrated higher P-Rex1 expression compared to bone metastases , which may be related to the enrichment of HER2+ metastases in the brain and luminal A/B metastases in bone.P-Rex1 expression in tumors from different metastatic sites is shown in n = 18) and decreased expression (n = 15) in the metastases  primary breast tumors. For those patients with matched primary cancers and metastases, there was essentially an equal number of cases that demonstrated increased P-Rex1 expression (tastases . An incrp = 0.04) and P-Rex1 and tumor grade (p = 0.006) remained statistically significant.In a multivariate analysis including primary tumor subtype, grade, initial stage, initial lymph node status, lymphovascular invasion in primary tumor, and whether or not a patient eventually developed metastatic disease , the associations between P-Rex1 expression and molecular subtype  and HER2+ breast cancers had the highest P-Rex1 expression, while triple-negative tumors had lower levels. These findings are in alignment with the few prior studies that have examined P-Rex1 expression in human breast cancers, including our prior report that demonstrated a correlation between P-Rex1 and ER in primary breast tumors and breast cancer cell lines  Sosa et  cancers . Sosa et cancers . In our P-Rex1 is a mediator of tumor progression in models of prostate cancer and melanoma. Utilizing prostate cancer cell lines and a mouse xenograft model, Qin et al. demonstrated that P-Rex1 acts as detector of chemotactic signals with the subsequent activation of Rac to promote metastasis . In melaOur study uniquely examined multiple distant metastatic sites, many of which also had a matched primary breast tumor available for analysis. HER2+ breast cancer metastases, which were from either brain or lung, had the highest levels of P-Rex1 expression. These observations lend further support to the role of P-Rex1 in HER2-driven oncogenic signaling , 15.Bone metastases had the lowest levels of P-Rex1 expression, despite the majority of bone metastases being of the luminal (ER+) subtypes. Given that high P-Rex1 levels have been associated with poorer outcomes, the lower P-Rex1 expression in bone metastases potentially correlates with the prognosis related to metastatic site; patients with visceral metastases tend to have worse survival. It should be noted that many of the bone metastases were sampled via core biopsy and underwent decalcification, typically with ethylenediaminetetraacetic acid (EDTA). The impact of decalcification on P-Rex1 IHC expression is largely unknown and might have affected interpretation of expression levels in bone specimens.The definition of the luminal B subtype continues to evolve, with recent evidence suggesting the existence of both luminal B/HER2+ and luminal B/HER2− subtypes [This study independently corroborates the P-Rex1 expression patterns that have been reported in breast cancer cell lines and at the mRNA level in human tumors. Using a commercially available antibody on a cohort of routine clinical cases, we confirmed that P-Rex1 is increased in ER+ and HER2+ breast cancers compared to triple-negative tumors. Furthermore, our data suggest that P-Rex1 may be differentially expressed in metastatic tumors based on site and receptor status. Provided that increased P-Rex1 expression might predict sensitivity to PI3K inhibitors in breast cancer , 6, the p = 0.04) and grade (p = 0.006) remained significantly associated with P-Rex1 histoscore in the multivariate analysis .Figure S1: P-REX1 immunohistochemical controls. A) Positive control; MCF-7 breast cancer xenograft that expresses P-Rex1 (x155). B) Positive control; ZR75-1 breast cancer cells transfected with non-silencing control siRNA (x145). C) Negative control; ZR75-1 breast cancer cells transfected with siRNA targeting P-Rex1 (x145). Figure S2: Univariate and multivariate analyses of associations between clinical and pathologic features and P-Rex1 histoscore. Based on univariate analyses (shown below), we found that primary tumor subtype and grade were significantly associated with P-Rex1 histoscore; these covariates were included in a multivariate linear model as follows: Subtype: TN vs. other, Grade: Intermediate vs. other. Primary tumor subtype (


Antibody-dependent cell-mediated cytotoxity (ADCC) can eliminate HIV-1 infected cells, and may help reduce the reservoir of latent virus in infected patients. Sera of HIV-1 positive individuals include a number of antibodies that recognize epitopes usually occluded on HIV-1 envelope glycoprotein (Env) trimers. We have recently described eCD4-Ig, a potent and exceptionally broad inhibitor of HIV-1 entry that can be used to protect rhesus macaques from multiple high-dose challenges with simian-human immunodeficiency virus AD8 (SHIV-AD8). Here we show that eCD4-Ig bearing an IgG1 Fc domain (eCD4-IgG1) can mediate efficient ADCC activity against HIV-1 isolates with differing tropisms, and that it does so at least 10-fold more efficiently than CD4-Ig, even when more CD4-Ig molecules bound cell surface-expressed Env. An ADCC-inactive IgG2 form of eCD4-Ig (eCD4-IgG2) exposes V3-loop and CD4-induced epitopes on cell-expressed trimers, and renders HIV-1-infected cells susceptible to ADCC mediated by antibodies of these classes. Moreover, eCD4-IgG2, but not IgG2 forms of the broadly neutralizing antibodies VRC01 and 10–1074, enhances the ADCC activities of serum antibodies from patients by 100-fold, and significantly enhanced killing of two latently infected T-cell lines reactivated by vorinostat or TNFα. Thus eCD4-Ig is qualitatively different from CD4-Ig or neutralizing antibodies in its ability to mediate ADCC, and it may be uniquely useful in treating HIV-1 infection or reducing the reservoir of latently infected cells. Antibodies can bind HIV-1-infected cells by recognizing the viral envelope glycoprotein (Env) on the cell surface. Antibody-bound cells then recruit natural killer cells to eliminate these infected cells. Here we demonstrate the unique properties of eCD4-Ig, a potent and exceptionally broad antibody-like HIV-1 entry inhibitor. Like antibodies, eCD4-Ig can efficiently mediate killing of infected cells. However, unlike most antibodies, eCD4-Ig can promote Env conformational changes that then allow abundant but otherwise inert antibodies in patient sera to mediate killing of infected cells. This property may be especially useful in efforts to cure HIV-1 by reactivating virus in latently infected cells and then killing these virus-producing cells. Natural killer (NK) cells and other Fc-gamma receptor (FcγR)-expressing cells can eliminate HIV-1 infected cells through antibody-dependent cell-mediated cytotoxicity (ADCC) . These A80s) less than 10 μg/ml /(%p24+ cells in targets + effectors − inhibitor) × 100.TZM-bl neutralization assays were performed as previously described ,16,43. BHEK293T cells were transfected with plasmids expressing HIV-1 envelope glycoprotein variants lacking cytoplasmic residues 732–876 (HXBc2 numbering). Cells were collected 48 hours post transfection with non-enzymatic dissociation buffer . Cells were washed with flow cytometry buffer  before incubation with eCD4-mouse-Ig for 1 hour on ice. Cells were subsequently incubated with human IgG1-containing antibodies. Antibody binding was determined with FITC-conjugated goat anti-mouse, and APC-conjugated goat anti-human secondary antibodies . Between each antibody incubation, cells were washed twice with flow cytometry buffer. After incubation with secondary antibody, cells were washed once with flow cytometry buffer, once with PBS, and then resuspended in 1% paraformaldehyde in PBS. Binding was analyzed with an Accuri C6 Flow Cytometer and data analyzed with the C6 Software .S1 FigA-B) HEK293T cells were transfected to express the BG505 Env with a deletion in its cytoplasmic tail to increase expression on the cell surface. Cells were pre-incubated with varying concentrations of eCD4-Ig (solid lines) or CD4-Ig (dotted lines) with mouse Fc domains , as indicated. Cells were washed and then incubated with 0.4 μg/mL of the indicated antibodies, and analyzed by flow cytometry. Mean fluorescence intensity values (MFI) are normalized to the value of antibody binding in the absence of eCD4-Ig or CD4-Ig. Error bars represent range (n = 2). Data are representative of at least three independent experiments.((TIF)Click here for additional data file.S2 FigHIV-1 isolates 89.6, NL4-3, or YU2 were incubated for 1 hour with 5-fold serial dilutions of eCD4-IgG1 or IgG2 or the indicated antibodies. TZM-bl cells were then added to virus-inhibitor combinations and incubated for 40 hours. Infection is represented as the percentage of luciferase activity in the absence of inhibitor. Values represent means +/- S.E.M. (n = 3). Data are representative of at least three independent experiments.(TIF)Click here for additional data file.S3 FigA) ADCC assays similar to those described in B) An in vitro neutralization assay performed as in ((TIF)Click here for additional data file.


Drosophila larval motor neurons. The number of synaptic boutons and the amount of neurotransmitter release at the Khc-73 mutant larval neuromuscular junction (NMJ) are normal, but we find a significant decrease in the number of presynaptic release sites. This defect in Khc-73 mutant larvae can be genetically enhanced by a partial genetic loss of Bone Morphogenic Protein (BMP) signaling or suppressed by activation of BMP signaling in motoneurons. Consistently, activation of BMP signaling that normally enhances the accumulation of phosphorylated form of BMP transcription factor Mad in the nuclei, can be suppressed by genetic removal of Khc-73. Using a number of assays including live imaging in larval motor neurons, we show that loss of Khc-73 curbs the ability of retrograde-bound endosomes to leave the synaptic area and join the retrograde axonal pathway. Our findings identify Khc-73 as a regulator of endosomal traffic at the synapse and modulator of retrograde BMP signaling in motoneurons.Retrograde signaling is essential for neuronal growth, function and survival; however, we know little about how signaling endosomes might be directed from synaptic terminals onto retrograde axonal pathways. We have identified Khc-73, a plus-end directed microtubule motor protein, as a regulator of sorting of endosomes in  Drosophila neuromuscular junction, receptors for the Bone Morphogenic Protein signaling pathway are transported from the synapse to the neuron cell body for the proper establishment of synaptic growth and function of motoneurons. Using this system we demonstrate that a kinesin motor protein, Khc-73, is necessary for the efficient sorting of retrograde bound vesicles to the retrograde transport route.Retrograde axonal transport is essential for normal synaptic function and neuronal survival. How endosomes are specifically sorted from the synaptic terminal for retrograde axonal transport is currently not known. At the  Drosophila larval neuromuscular junction (NMJ). In particular the Bone Morphogenic Protein signaling pathway (BMP) has been identified as a major regulator of synaptic growth and function. As such, many regulators of synaptic endosomal sorting have been identified in the regulation of BMP signals at synaptic terminals. Nevertheless, how activated receptors are preferentially sorted to travel to the nucleus is currently unknown.Bidirectional communication between the neuronal cell body and distant synaptic terminals is essential for synapse formation, plasticity and neuronal survival , 2. Thisretrograde sorting of signaling vesicles at the Drosophila larval NMJ.The movement of endosomes within the cytoplasm is directed through the actions of microtubule binding proteins such as minus end dynein motors, and plus end directed kinesins. Co-ordination and competition between these opposing motors for endosome cargoes regulates the transport of proteins to their correct targets , 14–18. Drosophila NMJ, plays an important role in the regulation of synaptic function by influencing presynaptic neurotransmitter release  (RRID:BDSC_22058), UAS-Rab5:YFP (RRID:BDSC_9775), UAS-Rab7:GFP(RRID:BDSC_42706), UAS-Rab11:GFP (RRID:BDSC_50782), VGlut-Gal4 (RRID:BDSC_24635), MadK00237 (RRID:BDSC_10474), UAS-Mito-HA-GFP (RRID:BDSC_8442), nSyb-Gal4 (RRID:BDSC_51635). UAS-luciferase (RRID:BDSC_35788). puckered LacZ insertion pucE69 ; P{y[+m8] = Mae-UAS.6.11}Khc-73[DP00530]. Virgin y[w[67c23]; P{y[+m8] = Mae-UAS.6.11}Khc-73[DP00530] female flies were mated to Cyo/+; Δ2–3, Sb/TM6b males. Male progenies of y[w[67c23]; P{y[+m8] = Mae-UAS.6.11}Khc-73[DP00530]/Cyo; Δ2–3, Sb/+ were mated to virgin y, w; CyoGFP/Adv females. Yellow, non Sb, yellow eyed progeny were singly mated to y, w; Adv/CyoGFP virgins and individual stocks were established. P-element excisions were screened with the following primers: OED91: CTGACGGCGCTGTTGCTTG and OED96: GATCTAGAGATGATTCTGCATCACTAG TAAAAATT.nt from y w[67c23]Khc-73 promoter GAL4 construct was generated by cloning a 4kb fragment upstream of the translational start site with primers OED453: CAG GTA CCG CCG AGG AAC CGC TAA CG and OED452:CAG GTA CCC GCG GAT GTG GAT GCA GC. Vector pW+SN attB was modified with a GAL4 sequence cloned as a KpnI/NotI fragment.Khc-73 promoter was subsequently inserted into the unique KpnI site. Genomic Khc-73 is from BACPAC clone CH321-36I16 (BACPAC Resources Center).CH321-36I16 was made by standard embryo injection of BACPAC clone CH321-36I16 (BACPAC Resources Center) with ΦC31 –mediated integration into attP site at position 86F of chromosome III.Transgenic fly Wandering third instar larvae were dissected, prepared and embedded as described in . Ultra-tWandering third instar larvae were dissected as previously described . Third IWestern blots were performed as previously described . Muscle Synapses were imaged using a ConfoCor LSM710 and Zeiss LSM 780 on an Axiovert 200M inverted microscope  with 63x/1.4 oil objective. Image analysis was performed with ImageJ 1.46j (NIH) , Imaris Wandering third instar larvae were dissected in room temperature HL3 to remove the guts and fat bodies. The larval filet was then inverted and stretched onto a coverslip using magnetic dissection pins inside a chamber consisting of a coverslip surrounded by magnet strips. Larval prep was maintained at room temperature in an HL3 bath during imaging. NMJs at hemisegment A3 and A4, muscles 6/7 and 4 were imaged. Axons were imaged at hemisegment A3 to A4. Larvae were imaged for a maximum of 30 minutes after dissection. Axons and NMJs were imaged with 63x 1.4NA oil objective on Axiovert 200 inverted microscope with Zeiss LSM780 confocal .The nerve crush assay was performed as previously described . BrieflyWandering third instar larvae were dissected in cold HL3 solution following standard protocol . The spoThe amplitudes of mEJC and EJC were measured using Mini Analysis 6.0.3 software (Synaptosoft) and verified by eye. QC was calculated by dividing the mean EJC amplitude by mean mEJC amplitude. The recording traces were generated with Origin 7.5 software (Origin Lab).Spontaneous and evoked potentials were measured as previously described . StandarS1 FigD. melanogaster and H. sapiens. K (Kinesin motor), FHA (Forkhead Associated), CC (coiled coil) and CAP-GLY  domains. (B) Khc-73 gene structure and location of DP00530 insert. Untranslated regions (UTR) are in solid black. Exons in grey. Deletions Khc-73193 and Khc-73149 are indicated by solid lines. (C) Western blot of Control (Khc-73100), Khc-73 heterozygotes (Khc-73149/+), Khc-73 mutants (Khc-73149) (left) and Control (Khc-73100), Khc-73193 (right) with anti-Khc-73 (top) and actin loading control (bottom).(A) Khc-73 protein domain structure in BG380-Gal4/+; UAS-HA-Khc-73). Scale bar is 5μm.(D) Localization of HA-Khc-73 in axons . Scale bar is 5μm. (F) Expression of Khc-73-GAL4 visualized with mCD8:GFP in ventral nerve cord . Scale bar is 100μm. (G) Expression of Khc-73-GAL4 visualized with mCD8:GFP in muscle 4 NMJ . Scale bar is 5μm. (H) Expression pattern of Khc-73-GAL4 visualized with mCD8:GFP in the adult CNS and VNC. (I) Quantification of bouton number in Control (Khc-73100) and Khc-73 (Khc-73149) mutants at muscle 6/7. N = 17, 18 NMJs. Error Bars are SEM. (J) Muscle surface area of muscle 6/7 in Control (Khc-73100) and Khc-73 (Khc-73149). Muscle 6/7 normalized to control. n = 17, 18. Error Bars are SEM. Student’s t-test. ns-no statistical significance. (K) High Frequency Stimulation trace for Control (Khc-73100) and Khc-73 (Khc-73193). N = 11, 13 NMJs. (L) Quantification of EJP amplitudes for genotypes in (K). Recording duration was 600 seconds at 10Hz. EJPs were binned per 30 seconds and amplitudes were averaged per bin. Relative EJP amplitudes were normalized to the average amplitude for the first 15seconds of recording for each genotype. Error bars are SEM. N = 9,8.(E) Localization of HA-Khc-73 in muscle 4 NMJ terminal boutons ((TIF)Click here for additional data file.S2 FigKhc-73193/+) and Khc-73 (Khc-73193/Khc-73193). N = 10, 8 NMJs. (C) Quantification of Cysteine String Protein (CSP) mean fluorescence intensity, normalized to HRP intensity and expressed as a percentage of control, of muscle 4 NMJs. Control (Khc-73100) and Khc-73 (Khc-73193), N = 9, 10. (D) Quantification of EPS-15 staining in control (w1118) and Khc-73 (Khc-73149) larval NMJs. N = 6, 6. (E) Muscle 4 terminal boutons stained with Dlg in control (Khc-73100) (top) and Khc-73 (Khc-73193) (bottom) third instar larvae. Scale bar is 5μm. (F) Quantification of Dlg fluorescence intensity normalized to HRP intensity and expressed as a percentage of control.(A) Muscle 4 terminal boutons stained with anti-synaptotagmin (SYT). Scale is 5μm. (B) Quantification of SYT mean fluorescence intensity normalized to HRP signal and expressed as a percentage of control for muscle 4 boutons in control (Khc-73100) and Khc-73 (Khc-73149) mutants. Error Bars are SEM. Student’s t-test. ***P<0.001, ns-no statistical significance.(G) Quantification of GluRIIA fluorescence intensity normalized to HRP intensity and expressed as a percentage of control for Control ((TIF)Click here for additional data file.S3 FigKhc-73100/+), Medea (MedeaC246/MedeaG112) and Khc-73; Medea  larvae. (B) Quantification of BRP puncta number for genotypes in (A). N = 19,20,20 NMJs. (C) Quantification of synaptic area identified with HRP staining for genotypes in (A). N = 19,20,20 NMJs. (D) Quantification of bouton number per NMJ for genotypes in (A). N = 19,20,20 NMJs. (E) Brp puncta in axons of Control (Khc-73100/+), Medea (MedeaC246/MedeaG112) and Khc-73; Medea  larvae. Green channel–Brp, Red channel–HRP. (F) Quantification of Brp puncta axon density for genotypes in (E) normalized to control. N = 10,10 and 10 Larvae. (G) Acetylated tubulin staining in muscle 4 NMJs of Control (Khc-73100/+) and Khc-73 (Khc-73149), Dashed line–Quantified region. Acetylated-tubulin (green) and HRP (red). Scale bar is 10μm. (H) Quantification of Acetylated-tubulin intensity in axon region indicated by dashed line in (G) N = 9,8. Error Bars are SEM. Student’s t-test. *P<0.05, **P<0.01, ***P<0.001. ns-no statistical significance. Scale bar is 5μm.(A) Brp puncta in terminal boutons of muscle 4 NMJs in Control ((TIF)Click here for additional data file.S4 FigKhc-73100), Khc-73149 and Khc-73193. N = 10, 12 and 15 NMJs. (B) Quantification of pMad intensity in VNC neurons for control (w1118) and Khc-73 mutants (Khc-73149). N = 6(217), 6(234), VNCs(nuclei). (C) Quantification of pMAD intensity at the NMJ in larvae overexpressing Khc-73 in motoneurons. Control  and Khc-73 OE . N = 20, 19. (D) Quantification of pMAD intensity in the ventral nerve cord in larvae overexpressing Khc-73 in motoneurons. Control  and Khc-73 OE  N = 4(100), 3(143), larvae (nuclei).Error Bars are SEM. Student’s t-test. ns-no statistical significance.(A) Quantification of pMad intensity at muscle 4 NMJs for control ((TIF)Click here for additional data file.S5 FigBG380-Gal4/+; OK371-Gal4/+; UAS-TKV-YFP/+) and Khc-73 . Scale bar is 10μm. (B) Quantification of mean fluorescence intensity as percentage of control for genotypes in (A). N = 14, 12 NMJs. (C) Live image of muscle 6/7 NMJs in live, unfixed larvae for Control  and Khc-73 . Scale bar is 10μm. (D) Quantification of mean fluorescence intensity as percentage of control for genotypes in (C). N = 15, 15 NMJs.(A) Image of muscle 4 NMJs in live, unfixed larvae for Control  Kymograph of TKV-YFP live imaging of axons in third instar larvae in control  and Khc-73 mutants  larvae. Scale bar is 10μm. Error Bars are SEM. Student’s t-test. ***P<0.001. ns-not statistically significant.(E) Quantitative PCR analysis of (TIF)Click here for additional data file.S6 FigOK371-Gal4, Khc-73149/UAS-Wit) and Wit OE, Khc-73 larvae  stained for Hrs (white) and Hrp (red). Scale bar is 5μm. (B) Quantification of Hrs staining in (A). N = 20, 20 NMJs. Error Bars are SEM. *P<0.05. Student’s t-test.(A) Muscle 4 terminal bouton in Wit OE Control ((TIF)Click here for additional data file.S7 FignSyb-Gal4, UAS-TKV-YFP/UAS-Rab7:GFP) stained with Wit (Green) and GFP (red) antibodies. Scale bar is 5μm. (B) Muscle 4 terminal boutons in motoneurons overexpressing Wit and TKV-YFP  stained with Wit (Green) and GFP (red) antibodies. Scale bar is 5μm.(A) Muscle 4 terminal boutons in motoneurons overexpressing Wit and Rab7:GFP ((TIF)Click here for additional data file.S8 FigBG380-Gal4/+; UAS-RAB7:GFP/+) and Khc-73 mutants . Scale bar is 5 μm. (B) Kymographs from live imaging of RAB7:GFP in axons of Control  and Khc-73 mutants . (C) Histogram of Retrograde velocities of RAB7:GFP puncta in Control  and Khc-73 mutants . n = 5, 8 axons and n = 64, 49 puncta respectively.(A) RAB7:GFP expressed in live third instar larval axons of Control  LacZ staining in control  and Khc-73 mutant  larval motor neuron nuclei in the ventral nerve cord. Scale bar is 10μm. (F) Quantification of mean LacZ fluorescence staining in nuclei for genotypes in (E). N = 279, (6) for control  and 535, (11) for Khc-73 mutant , Nuclei, , respectively. (G) LacZ staining in control  and Khc-73 mutant  larval motor neuron nuclei in the ventral nerve cord after nerve crush assay. Scale bar is 10μm. (H) Quantification of Anterior and Posterior mean fluorescence of nuclei in (G) expressed as Posterior/Anterior ratio. N = 95, 113, (4) for Khc-73100; pucE69 /+. 219, 300, (10) for Khc-73149; pucE69 /+. Anterior nuclei, posterior nuclei, , respectively. Error Bars are SEM. Student’s t-test. ns-no statistical significance.(D) Histogram of anterograde velocities of RAB7:GFP puncta in Control and (TIF)Click here for additional data file.S1 MovieOK371-GAL4/+; UAS-RAB7:GFP/+). Retrograde direction (toward motoneuron cell body) is to the left. Scale bar is 5 μm.Live imaging of Rab7:GFP in third instar larva, muscle 4 NMJ in control ((GIF)Click here for additional data file.S2 MovieKhc-73 mutant . Retrograde direction (toward motoneuron cell body) is to the left. Scale bar is 5 μm.Live imaging of Rab7:GFP in third instar larva, muscle 4 NMJ in (GIF)Click here for additional data file.S3 MovieOK371-GAL4/+; UAS-RAB7:GFP/+). Retrograde direction (toward motoneuron cell body) is to the left. Scale bar is 10 μm.Live imaging of Rab7:GFP in third instar larva axon in control ((GIF)Click here for additional data file.S4 MovieKhc-73 mutant . Retrograde direction (toward motoneuron cell body) is to the left. Scale bar is 10 μm.Live imaging of Rab7:GFP in third instar larva axon in (GIF)Click here for additional data file.S5 MovieBG380-Gal4/+; OK371-Gal4/+; UAS-TKV-YFP/+). Retrograde direction (toward motoneuron cell body) is to the left. Scale bar is 5 μm.Live imaging of TKV:YFP in third instar larva, muscle 4 NMJ in control ((GIF)Click here for additional data file.S6 MovieKhc-73 mutant . Retrograde direction (toward motoneuron cell body) is to the left. Scale bar is 5 μm.Live imaging of TKV:YFP in third instar larva, muscle 4 NMJ in (GIF)Click here for additional data file.S7 MovieOK371-GAL4/UAS-mito:GFP). Retrograde direction is to the left. Scale bar is 10μm.Live imaging of axons in third instar larva of control ((GIF)Click here for additional data file.S8 MovieKhc-73 mutant . Retrograde direction is to the left. Scale bar is 10μm.Live imaging of axons in third instar larva of (GIF)Click here for additional data file.