Magnaporthe oryzae is one of the most destructive diseases of rice. An increasing number of microRNAs (miRNAs) have been reported to fine-tune rice immunity against M. oryzae and coordinate with growth and development.Rice blast caused by Osa-miR159a) played a positive role in rice resistance to M. oryzae. The expression of Osa-miR159a was suppressed in a susceptible accession at 12, 24, and 48 h post-inoculation (hpi); it was upregulated in a resistant accession of M. oryzae at 24 hpi. The transgenic rice lines overexpressing Osa-miR159a were highly resistant to M. oryzae. In contrast, the transgenic lines expressing a short tandem target mimic (STTM) to block Osa-miR159a showed enhanced susceptibility. Knockout mutations of the target genes of Osa-miR159a, including OsGAMYB, OsGAMYBL, and OsZF, led to resistance to M. oryzae. Alteration of the expression of Osa-miR159a impacted yield traits including pollen and grain development.Here, we showed that rice microRNA159a (Osa-miR159a positively regulated rice immunity against M. oryzae by downregulating its target genes. Proper expression of Osa-miR159a was critical for coordinating rice blast resistance with grain development.Our results indicated that The online version contains supplementary material available at 10.1186/s12284-021-00469-w. Among them, 757 mature miRNAs have been identified in rice.Plant microRNAs (miRNAs) act as fine-tuning regulators and play regulatory roles in gene expression via cleavage, translational inhibition, or DNA methylation of target sites with sequences complementary to the miRNAs  transcription factor genes, which control leaf shape , and function in flowering and male fertility  double mutant displays severe growth and developmental defects including curled rosette leaves and stunted plant height  miRNAs, Osa-miR159 genes generating five mature isoforms: Osa-miR159a, Osa-miR159b, Osa-miR159c, Osa-miR159d, Osa-miR159e, and Osa-miR159f. These isoforms mediate mRNA cleavage of three genes, GAMYB (LOC_Os01g59660), GAMYBL (LOC_Os06g40330), and ZF . GAMYB has been shown to function in rice development. For example, miR159-GAMYB modulates the expression of gibberellic acid (GA)/abscisic acid (ABA)-related genes to maintain the energy supply and enhance developmental processes in Wuxiang S, a photo-thermosensitive genic male sterile rice line  and an isotrichodermin C-15 hydroxylase (HiC-15), which are essential for fungal virulence  and the suppressed expression of Osa-miR159a (STTM159) through short tandem target mimic (STTM), which is an effective method to block mature miRNA binding to target sites of the target genes  showed a severe disease phenotype, whereas the accession (IRBLkm-Ts) that contains the gene Pikm, which confers M. oryzae resistance displayed resistance  in LTH  and introduced the construct into Nipponbare (NPB), generating 24 independent transgenic lines, out of which we chose two lines that showed high Osa-miR159a accumulation for further investigation  and introduced it into NPB, which may prevent Osa-miR159a from binding to its target sites   Fig. a. ConsisEV) Fig. b. In conEV) Fig. a, and thEV) Fig. b, c. TheOsa-miR159a delayed infection, whereas blocking Osa-miR159a facilitated M. oryzae infection.Next, we exploited the GFP-tagged strain GZ8 to observe the infection process in sheath cells using laser scanning confocal microscopy. Compared with the control, our observation found that the infection progress was delayed in OX159 Fig. c, d, butOsa-miR159a positively regulated resistance to M. oryzae, we used RT-qPCR to examine the expressions of some marker genes, including OsNAC4, OsPR10b (Pathogenesis-Related 1b) and OsJAMYB, acting in immune responses after infection with M. oryzae  and OsGAMYBL (LOC_Os06g40330)   of OsZF , especially in relation to pollen development. However, the phenotype of zf mutants was very similar to that of the wild type, except for the slightly reduced height of the zf mutants , but further studies are required. In addition, our results showed that knockout lines of OsGAMYB and OsGAMYBL exhibited increased resistance to M. oryzae  allele from the rice cultivar Digu, thus, inhibiting H2O2 degradation and enhanced disease resistance  enhances resistance to Rhizoctonia solani  and Zhong Hua 11 (ZH11) were used for transgenic analysis. The susceptible accession Lijiangxin Tuan Heigu (LTH) and the resistant accession International Rice Blast Line Pyricularia-Kanto51-m-Tsuyuake (IRBLkm-Ts) was used in this study. The rice plants were grown in a growth chamber maintained at 26 °C and 70% relative humidity under 12 h of light and 12 h of darkness. To assay yield traits, rice plants were grown in a paddy field in the Wenjiang district of Chengdu, China, from April to September.P < 0.01.Rice agronomic traits were measured from five plants in the paddy field at maturity. The 1000-grain weight, grain length, and grain width were measured using an SC-A grain analysis system  using the filled grains that were dried in an oven at 42 °C for 1 week. These data were analyzed by a one-way ANOVA followed by post hoc Tukey’s HSD analysis. Differences were considered significant at Osa-miR159a, we amplified the DNA sequence containing 321 bp upstream and 306 bp downstream of Osa-miR159a from NPB genomic DNA with the specific primers miR159a-KpnI-F and miR159a-SalI-R  of Osa-miR159a (STTM159), we inserted the amplified DNA fragments of STTM159 into the KpnI and SalI sites of the binary vector 35S-pCAMBIA1300, resulting in a target mimic of the miR159 construct. The entire sequences of STTM159 were 5′-ggtaccTGCAGCTCCTGATCGGGCATGCAAGTTGTTGTTGTTATGGTCTAGTTGTTGTTGTTATGGTCTAATTTAAATATGGTCTAAAGAAGAAGAATATGGTCTAAAGAAGAAGAATCAGAGCTCCCTCAGTCAATCCAAAgtcgac-3′. Both of the constructs were transformed into the NPB background via Agrobacterium-mediated transformation. To generate Osa-miR159a target gene knockout lines, we constructed the CRISPR/Cas9 plasmids as described previously  was used in this study. The strain was incubated on oatmeal and tomato agar media (OTA) at 28 °C under a 12-h light and 12-h dark cycle. After 10 days, the hyphae were scratched, and the fungus on the plates was further incubated with 24-h light treatment to promote sporulation. Three to 5 days later, the spores were collected for spray- or punch-inoculation. For spray-inoculation, seedlings at the three-leaf-stage were inoculated with a spore suspension (3 × 105 conidia/mL). Disease lesions were recorded at 5 days post-inoculation (dpi). For punch-inoculation, 5 μL of the spore suspension (5 × 105 spore/mL) was drop-inoculated at wound sites on the leaves of seedlings at the three-leaf-stage following previously described methods  were inoculated with a spore suspension (1 × 104 conidia/mL) as described previously  with M. oryzae strain GZ8. Scale bars, 10 mm. b Relative lesion area on the inoculated leaves from (a). Error bars indicate standard deviation (SD) (n = 3). Asterisks (**) above the bar indicates significant differences (P < 0.01) determined by the Student’s t-test. c Confocal images showing the infection status of eGFP-tagged M. oryzae strain GZ8 in the indicated lines at 24, 36, and 48 h post-inoculation (hpi). Scale bars, 25 μm. d Quantification analysis on the process of GZ8 infection in the indicated lines at the indicated time points. Over 200 conidia in each line were analyzed. The experiments were repeated two times with similar results. e-g The expression of defense-related genes in wild type and OX159 lines following the inoculation of M. oryzae strain GZ8. RNA was extracted at the indicated time points for the reverse transcription-quantitative PCR (RT-qPCR) assay. The amounts of collected mRNA were normalized to that in the wild type at 0 hpi. Error bars indicate SD (n = 3). Different letters above the bars indicate significant differences (P < 0.01) as determined by a one-way ANOVA followed by post hoc Tukey’s HSD analysis.Additional file 2 : Figure S2. Overexpression of STTM159 results in enhanced susceptibility to Magnaporthe oryzae. a Blast disease phenotype at 5 days post-inoculation (dpi) with M. oryzae strain GZ8. Scale bars, 10 mm. b, c Relative fungal growth and lesion area on the inoculated leaves from (a). Error bars indicate SD (n = 3). Asterisks (**) above the bar indicates significant differences (P < 0.01) determined by the Student’s t-test. d Confocal images showing the infection status of a GFP-tagged M. oryzae strain GZ8 in the indicated lines at 24, 36, and 48 h post-inoculation (hpi). Scale bars, 25 μm. e Quantification analysis of the process of GZ8 infection in the indicated lines at the indicated time points. Over 200 conidia in each line were analyzed. The experiments were repeated two times with similar results. f-h The expression of defense-related genes in wild type (WT) and STTM159 lines following the inoculation of M. oryzae strain GZ8. RNA was extracted at the indicated time points for the reverse transcription-quantitative PCR (RT-qPCR) assay. The amounts of collected mRNA were normalized to that in the WT at 0 hpi. Error bars indicate SD (n = 3). Different letters above the bars indicate significant differences (P < 0.01) as determined by a one-way ANOVA followed by post hoc Tukey’s HSD analysis.Additional file 3 : Figure S3.Osa-miR159 mature isoforms and the accumulation of Osa-miR159a target genes in the indicated lines. a The sequence alignments of Osa-miR159 mature isoforms and their positions on the chromosome in rice. b The structure of target genes and the sequence alignment of the target sites in the target genes. White boxes indicate the 5′-UTRs and 3′-UTRs. Black boxes indicate exons. Black lines indicate introns. Red lines indicate the target sites of Osa-miR159a. c Reverse transcription-quantitative PCR (RT-qPCR) data show the relative mRNA amount of target genes in OX159 and STTM159 in comparison with NPB containing the empty vector (EV). Data are shown as mean ± SD (n = 3). Different letters above bars indicate significant differences (P < 0.05) as determined by a one-way ANOVA followed by post hoc Tukey’s HSD analysis.Additional file 4 : Figure S4. The phenotype and 1000-grain weight (g) of the zf mutants. a The phenotype of zf mutants and EV control at the reproductive stage. The height of zf mutants slightly less than that of the wild type plants. b The 1000-grain weight (g) of the indicated plants showed no difference.Additional file 5 : Table S1. The primers used in this research.


Thermococcus kodakarensis using single-particle cryo-electron microscopy (EM).DNA polymerase D (PolD) is the representative member of the D family of DNA polymerases. It is an archaea-specific DNA polymerase required for replication and unrelated to other known DNA polymerases. PolD consists of a heterodimer of two subunits, DP1 and DP2, which contain catalytic sites for 3′-5′ editing exonuclease and DNA polymerase activities, respectively, with both proteins being mutually required for the full activities of each enzyme. However, the processivity of the replicase holoenzyme has additionally been shown to be enhanced by the clamp molecule proliferating cell nuclear antigen (PCNA), making it crucial to elucidate the interaction between PolD and PCNA on a structural level for a full understanding of its functional relevance. We present here the 3D structure of a PolD-PCNA-DNA complex from Pyrococcus abyssi onto our EM map showed the 3D atomic structural model of PolD-PCNA-DNA. In addition to the canonical interaction between PCNA and PolD via PIP (PCNA-interacting protein)-box motif, we found a new contact point consisting of a glutamate residue at position 171 in a β-hairpin of PCNA, which mediates interactions with DP1 and DP2. The DNA synthesis activity of a mutant PolD with disruption of the E171-mediated PCNA interaction was not stimulated by PCNA in vitro.Two distinct forms of the PolD-PCNA-DNA complex were identified by 3D classification analysis. Fitting the reported crystal structures of truncated forms of DP1 and DP2 from Based on our analyses, we propose that glutamate residues at position 171 in each subunit of the PCNA homotrimer ring can function as hooks to lock PolD conformation on PCNA for conversion of its activity. This hook function of the clamp molecule may be conserved in the three domains of life.The online version contains supplementary material available at 10.1186/s12915-020-00889-y. Pyrococcus furiosus PolB (PfuPolB) -dTTP , 20 nM PCNA, and 5 nM PolD, at 72 °C for 1, 2, and 4 min. The reaction mixture was pre-incubated for 3 min, and PolD was added to initiate the reaction. After incubation, aliquots (8 μl) were fractionated and spotted onto DE81 filters . The filters were washed with 5% Na2HPO4 solution thrice and dried. Incorporated radioactivity was measured with a scintillation counter Tri-Carb 3110TR (PerkinElmer).The primer extension ability of TkoPolD was measured by counting incorporated radioactivity into DNA strands using dNTP containing [methyl-4)2SO4, 1 mM DTT, 0.1 mg/ml BSA, 2 mM MgCl2, and 10 nM Cy5-labeled DNA substrates 2SO4, 1 mM DTT, 0.1 mg/ml BSA, 2 mM MgCl2, 2, 5 nM Cy5-labeled DNA  of the interactions were determined from the association and dissociation curves of the sensorgrams, using the BIAevaluation (ver. 4.1) software .The Biacore J system  was used to test the physical interactions of PCNA with various PolD mutants. PCNA was fixed on a CM5 Sensor Chip , according to the manufacturer’s recommendations. To measure the kinetic parameters, purified PolDs  in a running buffer , with six different concentrations , were applied for 120 s to the PCNA-immobilized chip, at a continuous flow rate of 30 μl/min at 25 °C. The bound analytes were removed by washing with regeneration buffer  at the end of each cycle. The apparent equilibrium constants . PolD , PCNA, and DNA  were mixed and incubated for 3 min at 60 °C. The protein solutions were applied to a Superose 6 PC 3.2/30 column  and were eluted with buffer containing 50 mM Tris–HCl, pH 8.0, and 0.3 M NaCl. Aliquots (5 μl) of applied solution and aliquots (8 μl) of each fraction from the eluates were subjected to 10% SDS-PAGE, followed by Coomassie Brilliant Blue staining. The standard marker proteins, including thyroglobulin , γ-globulin , ovalbumin , and myoglobin , were also subjected to gel filtration as controls.Additional file 1:Table S1. Model refinement statistics. Figure S1. The synthetic deoxyoligonucleotides used in this study. Bases with lower case letters are linked by phosphorothioate bond for protection from the 3′–5′ exonuclease activity. Figure S2. Sample preparations. (A) Gel filtration chromatography of the reconstituted PolD-PCNA-DNA (30/45) complex. The absorbances at 260 nm and 280 nm are indicated by red and blue lines, respectively. (B) The peak fractions in (A) were analyzed by a gradient (10–20%) SDS-PAGE. The lanes for the purified proteins used for complex reconstitution and the markers are indicated by the notations “PCNA,” “PolD,” and “Marker,” respectively. (C) Gel filtration chromatography of the reconstituted PolD-PCNA-DNA (25/35) complex. (D) The peak fraction indicated by the asterisk (*) in (C) was analyzed by a gradient (10–20%) SDS-PAGE. Figure S3. Electron microscopic images of PolD-PCNA-DNA complex. (A) Representative electron microscopic image of negatively stained PolD-PCNA-DNA (30/45) complex, recorded by a CCD camera. (B) Representative cryo-electron microscopic image of PolD-PCNA-DNA complex, recorded by a CCD camera, without Volta Phase Plate (VPP). The white and magenta broken circles indicate tetrameric and pentameric clusters of the complex, respectively. (C) Representative cryo-electron microscopic image of PolD-PCNA-DNA complex, recorded by a Falcon II direct electron detector, using VPP. The white and magenta broken circles indicate tetrameric and pentameric clusters of the complex, respectively. (D) Representative cryo-electron microscopic image of PolD-PCNA-DNA complex, recorded by a Falcon 3EC direct electron detector without phase plate. The scale bars indicate 50 nm. Figure S4. Flow charts of 3D classification and refinement procedures for the PolD-PCNA-DNA (30/45) complex. The details are described in the Methods section. Figure S5. Flow charts of 3D classification and refinement procedures for the PolD-PCNA-DNA (25/35) complex. The details are described in the Methods section. Figure S6. EM analysis. (A) Comparison between representative 2D class averages (avg) and projections from the 3D model (prj) of the Form A complex. (B) Comparison between representative 2D class averages (avg) and projections from the 3D model (prj) of the Form B complex. Gold standard Fourier shell correction (FSC) curves for the EM density maps of the Form A complex (C), and Form B complex (D). Resolutions are given for the FSC 0.143 criteria. Euler angle distributions of particles used for the final maps of, (E) Form A complex, and (F) Form B complex. Figure S7. 3D map and atomic model fitting of PolD-PCNA-DNA complex (Form A). (A) Top view. The arrows with the angles indicate rotations to see the bottom view (B) and the side views of (C), (D), and (E), respectively. The PCNA-interacting C-terminal loop of DP2, harboring the PIP-box, is indicated by the red arrow in (D). The crystal structures of T. kodakarensis PCNA and P. abyssi DP1 are shown in green and cyan ribbon. The N-terminal, catalytic, center, and C-terminal domains of P. abyssi DP2 are shown in red, purple, yellow, and orange ribbon, respectively. (G) Top view of the Form A map, with the crystal structures of DP1  and DP2  docked as rigid bodies. (H) Top view of the Form A map in which each domain of DP2 , catalytic (purple), center (yellow), and C-terminal (orange)) is docked independently into the map. Note that the PCNA region of the map is omitted for clarity. Figure S8. 3D map and atomic model fitting of PolD-PCNA-DNA complex (Form B). (A) Top view. The arrows with the notation of C, D, E indicates the view angle of side views of (C), (D), and (E), respectively. (B) Bottom view. (C) – (E) Side views. The PCNA-interacting C-terminal loop of DP2, harboring the PIP-box, is indicated by the red arrow. The crystal structures of PCNA and DP1 are shown in green and cyan ribbons, respectively. The N-terminal, catalytic, center, and C-terminal domains of DP2 are shown in red, purple, yellow, and orange ribbon, respectively. (F) Oblique view of the Form B map, in which PolD is aligned in the same direction as Supplementary Figure S7G. The crystal structures of DP1  and DP2  are docked as rigid bodies into head and body region, respectively. (G) The same view as shown in (F), in which each domain of DP2 , catalytic (purple), center (yellow), and C-terminal (orange)) is docked independently into the map. Note that the PCNA region of the map is omitted for clarity. Figure S9. Structural similarity between eukaryotic family B and archaeal family D DNA polymerases. (A) Structure-based sequence alignment of the C-terminal domains of p261 of Polε (p261C), p180 of DP2 (p180C) and DP2 of PolD (DP2C) were performed with reference to a previous report [T. kodakarensis PolD (right). Secondary structures of the C-terminal domains are shown in schematic representations. Zn1, Zn2, Zinc-finger; α1–3, alpha helix; β, beta sheet. Figure S10. Interactions between PCNA and various PolD mutants. SPR analyses were performed to detect the physical interactions of PCNA with various PolD mutants . Purified PCNA was immobilized on a sensor chip, and six different concentrations  of purified PolDs were analyzed. The apparent equilibrium constants (KD) are shown at the top of each sensorgram. Figure S11. DNA-protein interaction of Form A. (A) Enlarged view of the polymerase active site. The 3′-terminus of the DNA and the active site residues D965 and D967 in DP2 are colored gold. The positions of the side chains are those of crystal structures and should be regarded as approximate ones. (B) Side view cross section of Form A. The red colored DNA strand is obtained by extending the upstream of the DNA duplex of the P. abyssi PolD-DNA structure fitted to our Form A map. Note that the DNA orientation of the reported P. abyssi structure cause a collision with the inner wall of the PCNA channel. (C) The bottom view of atomic model shown in (B). Figure S12. Crystal structure of FEN1–PCNA (PDB: 1UL1), superimposed on the EM maps. (A) FEN1 in the X configuration (purple), placed on the free PCNA subunit of Form A map. (B-D) FEN1 (blue) in the X (B), Y (C), and Z (D) configurations placed on the free PCNA subunit of Form B map. Note that only one configuration of FEN1 molecule is displayed in each figure for clarity. (PDF 13043 kb)s report . Amino-a


For OERs and HERs, N-PC@Ni shows better overpotential values of 314 and 179 mV at 10 mA cm−2, and its corresponding Tafel slopes are 132 and 98 mV dec−1, respectively. The chronopotentiometry curve of N-PC@Ni reveals better stability toward OER and HER at 50 mA cm−2 for 8 h. These consequences provide new pathways to fabricate efficient electrocatalysts of metal-free heteroatom-doped porous carbon from bio-waste/biomass for energy application in water splitting and metal air batteries.Tremendous developments in energy storage and conversion technologies urges researchers to develop inexpensive, greatly efficient, durable and metal-free electrocatalysts for tri-functional electrochemical reactions, namely oxygen reduction reactions (ORRs), oxygen evolution reactions (OERs) and hydrogen evolution reactions (HERs). In these regards, this present study focuses upon the synthesis of porous carbon (PC) or N-doped porous carbon (N-PC) acquired from golden shower pods biomass (GSB) via solvent-free synthesis. Raman spectroscopy, X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) studies confirmed the doping of nitrogen in N-PC. In addition, morphological analysis via field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) provide evidence of the sheet-like porous structure of N-PC. ORR results from N-PC show the four-electron pathway (average  Unfortunately, the high electrocatalytic activity of Pt is accompanied with several disadvantages, including high costs, sluggish kinetics, over potential and poor operational durability. Similarly, catalysts for OERs (IrO2 and RuO2) suffer due to high cost and the lack of long-term stability [2O3, Co3O4/C and Mn3O4), metal phosphates  and dichalcogenides (MoS2 and CoS2), have been investigated as electrocatalysts in ORRs, OERs and HERs [The current global-energy crisis has provoked researchers to investigate alternative energy storage and conversion systems that should be environmentally benign, inexpensive and highly efficient . The eletability ,15. All tability . On the and HERs ,20,21,22and HERs . Howeverand HERs , banana and HERs , waste sand HERs , kidney and HERs , orange and HERs , soybeanand HERs  and pomeand HERs , via cosΔE = EOER 10 - EORR1/2) at alkali medium and nitrogen reduction reaction. Huang et al. [Typha orientalis biomass plant and studied its ORR activity in alkali and acid medium, and these NCS show better catalytic activity, stability and better methanol tolerance. However, synthesis of NCS has used multi-step processes such as the hydrothermal method and freeze drying followed by annealing with NH3. Again, the synthesis strategy reported in the aforementioned literatures is multi-step, and it should be reduced to a facile one step and a cost-effective method. In this regard, synthesis of N-doped porous carbon (N-PC) from biomass via solvents that have been free pyrolyzed with urea is more suitable method. In this study, we presented the cost-effective synthesis of PCs and N-PCs from relatively abundant, natural, renewable golden shower pod biomass (GSB) via a solvent-free method (pyrolysis with urea). The key factor is the doping of N in a porous carbon skeleton, which alters the electronic and chemical structure of the PC and improves the efficiency of the electrochemical properties. Heteroatoms, such as nitrogen (N), sulfur (S), phosphorous (P), fluorine (F) and boron (B), doped porous carbons receive pervasive attention owing to their accelerated electron transfer and electrocatalytic properties, resulting in improvement in ORRs, HERs and OERs ,31,32. Fg et al.  synthesig et al.  synthesin = 3.6) of ORR with a half-wave potential (E1/2) of 0.76 V and Tafel slope of 86 mV dec−1. The OER and HER of the N-PC@Ni catalyst shows better over potential values of 314 and 179 mV at 10 mA cm−2, and it shows better stability over 8 h at 50 mA cm−2.The synthesized PC and N-PC catalysts were characterized with various analytical methods include XRD analysis, Raman and morphology analysis. The catalytic activity of N-PCs for ORRs, OERs and HERs was examined in alkaline medium to determine the suitability of the catalyst for energy storage and conversion applications. The N-PC catalysts show robust tri-functional catalytic performance toward HERs, OERs and ORRs in alkaline electrolyte. The ORR results from N-PC show the four-electron pathway , potassium hydroxide  and polyvinylidene fluoride (PVDF) were purchased from Merck Chemicals . Golden shower pod biomass (GSB) was collected from local premises .Analytical grade concentrated hydrochloric acid , urea  water and then dried in an oven at 90 °C for 24 h. The dried GSB was pyrolyzed in a tubular furnace at 800 °C for 2 h in an Argon (Ar) atmosphere with a 20 mL minfor 12 h .4N2O and dried at 80 °C for 12 h. GSB [N-PC was prepared by treating the dried GSB powder with urea via a single step process. Dried GSB powder and urea (1:1 weight ratio) were grained mechanically for 1 h and pyrolyzed at 800 °C under the Ar atmosphere for 2 h. The resultant N-PC was washed by DI water to remove any unreacted CH2 h. GSB .The diffractograms were recorded using a PANanalytical X’pert Pro X-ray diffractometer (XRD) . Field emission scanning electron microscopy (FESEM) was performed on an TESCAN-MIRA3 LMH . Transmission electron microscopy (TEM) was captured with an FEI Tecnai T 20 U-Twin . Raman spectra were performed using a Lab RAM HR (Horiba Jobin Yvon)  with 632.8 nm laser excitation. X-ray photoelectron spectra (XPS) analysis was carried-out using a Kratos Axis Ultra . The surface area and pore diameters were analyzed with a Quantachrome ASIQ  win surface area analyzer.Electrochemical measurements were probed on CHI7001E instrumentation equipped with a three-electrode electrochemical cell. An N-PC-modified glassy rotating disk electrode (RRDE) was employed as a working electrode, and Hg/HgO and a graphitic rod were served as the reference and counter electrodes, respectively. For catalyst slurry preparation, 10 mg of N-PC, was dispersed in isopropyl alcohol (IPA) (0.25 mL) and DI water (0.75 mL) for 20 min. Then, 20 μL of Nafion (5 %) was added as a binder, and the materials were further sonicated for 30 min. −2). All the catalysts were underwent cyclic voltammetry (CV) tests at a scan rate of 10 mV s−1 from 0.2 to 1.2 V (vs the reversible hydrogen electrode (RHE)) in an O2-saturated 0.1 M KOH electrolyte. Linear sweep voltammetry (LSV) tests were carried out between the potential ranges from 0.2 to 1.2 V (vs RHE). Long-term stability tests for the N-PC and Pt-C chronoamperometry tests were carried out for 10 h, at −0.3 V (vs. Hg/HgO). All recorded potentials were converted to vs. RHE with 0.1 M KOH through the given equation (1).The as-prepared slurry (5 μL) was coated onto the RRDE electrode  was deposited onto the Ni foam (1 × 1 cm2) and dried at 80 °C for overnight, and was then used as a working electrode. RuO2, PC and Pt-C electrodes were also fabricated with a similar procedure, and their OER and HER activities were compared with the N-PC electrode. Three-electrode electrochemical cell setup was utilized to measure the electrocatalytic behavior of prepared electrodes. One out of N-PC@Ni, RuO2@Ni, PC@Ni, Pt-C@Ni or the bare Ni foam electrode was used as a working electrode, Ag/AgCl and a graphitic rod were served as reference and counter electrodes, respectively in 1 M KOH aqueous electrolyte solution. LSV measurements were recorded with a scan rate of 1 mV s−1 for OERs and HERs. The long-term durability measurements for OERs and HERs were evaluated by chronopotentiometry at a current density of 50 mA cm−2 and −50 mA cm−2 respectively, for 8 h. All recorded potentials were converted to vs. RHE with 1 M KOH through the given equation (2).The N-PC catalyst ink was prepared as follows. Firstly, carbon black, polyvinylidene fluoride (PVDF) and N-PC (1:1:8 weight ratio) were thoroughly grinded together with The morphologies of PC and N-PC are presented in It can be seen form −1 and at ~1352 cm−1, corresponding two G band D (defect A1) and other deconvoluted two peaks at ~1504cm−1 (D3 band) and at 1205 cm−1 (D4 band) are corresponding to presence of amorphous carbon and impurities or polyenes, respectively. Further additionally broad peaks were obtained at 2700–3000 cm−1, belong to the 2D+S3, which is the mode for out of plane vibration of single or few layer graphene sheet. Whereas, the calculated intensity ratios of ID1/IG for N-PC and PC are 1.16 and 0.94 respectively, which confirms the presence of amorous carbon with monocrystalline graphite [The XRD pattern a of PC agraphite ,40. 2 adsorption/desorption isotherm and type-IV adsorption isotherm for N-PC are provided in 0P/P < 0.15) is indicative of micropores, and the curve at the intermediate pressure (0P/P 0.5–0.9) indicates the existence of a highly mesoporous structure in N-PC. The Brunauer-Emmett-Teller (BET) surface area of N-PC as calculated from the isotherm is 839 m2 g−1. The BJH pore size distribution indicates that the pore diameters are in the range of 2 to 4 nm  equation [The electrochemical ORR performances of PC, N-PC and Pt-C were examined through CV in 0.1 M KOH saturated with Othe N-PC a and Pt-the N-PC  have diseduction . Table Sequation . −1) and the inverse of square root of rotation rate. −1 and with a potentials range of 0.3 to 0.6 V, showing similar electron transfer (closer to 4) at various potentials (− electron transfer process (during the O2 conversion to H2O). The influence of catalyst loading on variation in the number of electrons was also studied and the results given in −2 to 0.10 mg cm−2 on RRDE and the corresponding number of electrons transfer was evaluated. As the catalyst loading increases, the electron transfer number increases. The 0.025, 0.05, 0.075 and 0.1 mg cm−2 loading show ‘n’ values of 3.2, 3.6, 3.7 and 3.8, respectively. These results reveal that loading of the catalyst does not significantly influence the ‘n’ values [−1) is lower than that of PC (146 mV dec−1) or higher than commercial Pt-C (74 mV dec−1), indicating that N-PC catalyst has a faster electron transfer rate than PC during ORRs. The four electron pathway during O2 reduction on N-PC is described as follows. When incorporating heteroatoms in a porous carbon skeleton, the heteroatom  polarizes the adjacent carbon atom. Such carbon atoms emerge out of the skeleton and generate a chemical bond with O2. This is followed by a set of protons and electrons approaching the system to support the process of four electron reduction [Such a K–L plot derived from the plot between the inverse of the current density  and PC@Ni electrode (147 mV dec−1), but it is higher than that of the RuO2@Ni (88 mV dec−1). This is because polarized carbon sites provided by N doping accelerate the OER performance in N-PC@Ni. CV curves were reordered in the non-faradaic region at various scan rates (5–50 mV s−1) to quantify the electrochemical double-layer capacitance (Cdl) of catalysts (ΔJ = Janodic - Jcathodic) vs RHE and scan rate). As given in dl of 2.28 and 2.76 mF cm−2, respectively, suggesting the great number of active sites on the N-PC to support the catalytic activity.OER activities of PC@Ni, N-PC@Ni, RuOaterials  and N anel slope d. The Taatalysts . The dou−2. Afterward, potential decay was measured at each constant time interval of 300 s during applying the current density from 10 to 175 mA cm−2. By increasing current densities, the potentials of the N-PC@Ni electrode have risen gradually and are stable at each current density for 300 s. This confirms the superior mass transport property, conductivity and mechanical integrity of the N-PC@Ni electrode. When decreasing the current density by 10 mA cm−2 again, the 1.67 V was observed, which is almost similar to its initial potential. Such small variation in the potential is due to the peeling-off N-PC from Ni foam as a result of the quick generation/discharge of gas molecules at a higher current density. The multiple-current chronopotentiometric curve was recorded for the N-PC@Ni electrode in 1 M KOH aqueous solution, as represented in −2 for 8 h  and Ni foam (121 mV dec-1). Otherwise, it is higher than Pt-C@Ni (33 mV dec−1). Suggestively, efficient HER kinetics are provided by N-PC compared to PC@Ni and Ni. The stability of the N-PC@Ni electrode was also evaluated using multi-step chronopotentiometry in 1 M KOH. −2 with step size of −10 mA cm−2. For each current density, the holding time was 300 s. The obtained result ratifies that N-PC@Ni has a low tendency to peeling-off during HER.The HER electrocatalytic activity of PC@Ni N-PC@Ni, Pt-C@Ni and Ni electrodes was evaluated in 1 M KOH solution. −2 for 8 h in 1 M KOH. The chronopotentiometry curve , energy-dispersive analysis X-ray (EDAX), Raman and X-ray photoelectron spectroscopy (XPS) techniques. N-PC revealed robust, tri-functional, catalytic performance towards HERs, OERs and ORRs in alkaline electrolyte. The relatively large and self-organized porous structures of N-PC were observed by FE-SEM and BET. The N-PC displays ORR, OER and HER activities comparable to Pt-C and RuOThe mechanical integrity and mass-transfer properties of N-PC were also better during OER and HER processes. The strategy adopted in this present work and investigation demonstrated that the structure of N-PC can be further tuned with varying the heteroatom content and type. If efforts are put further, this strategy might open up new possibilities for the fabrication of efficient porous carbon architectures from bio-wastes for energy applications, such as water splitting and a metal–air battery.


Dugesia ryukyuensis by feeding asexual planarians with minced sexual planarians. We identified dl-tryptophan (Trp) as one of the sex-inducing substances. dl-Trp can induce ovarian development, the first and essential step of sexual induction. d-Trp must act as a principal bioactive compound in terms of ovarian development, because the ovary-inducing activity of d-Trp was 500 times more potent than that of l-Trp. However, how Trp controls sexual induction is still unknown.Many animals switch between asexual and sexual reproduction in nature. We previously established a system for the sexual induction of planarian Dr-SLC38A9 is highly expressed in sexual worms, especially in the yolk glands. In situ hybridization analyses showed that Dr-SLC38A9 is expressed in the ovarian primordia of asexual worms and in the mature ovaries, testes, and yolk glands of sexual worms. In addition, Dr-SLC38A9 RNA interference during sexual induction resulted in the suppression of the development of reproductive organs. These results suggest that Dr-SLC38A9 is involved in the development of these organs. Moreover, we demonstrated that the reproductive organ-specific expression of Dr-SLC38A9 is enhanced by the addition of d-Trp.In this study, qRT-PCR analyses suggested that the putative amino acid transporter gene d-Trp activates the expression of Dr-SLC38A9 to promote sexual induction in the planarian D. ryukyuensis.We propose that The online version contains supplementary material available at 10.1186/s40851-021-00173-z. Many animals switch between asexual and sexual reproduction, depending on environmental conditions and/or the phase of their life cycle –4. The sDugesia ryukyuensis (OH strain) by feeding with the minced bodies of sexually mature worms of the species Bdellocephala brunnea  was calculated for each sample, and then ∆∆Ct [where ∆∆Ct = ∆Ct (sample) − the average of ∆Ct ] was calculated. Calibrators were asexual worms Fig. , EGFP knR v3.2.2 . The ShaDr-SLC38A9. A probe for Dr-nanos was synthesized as previously described . The slides were dipped in 1 μg/mL Hoechst No. 33342  to counterstain the nuclei.In situ hybridization of whole-mount worms and sections was performed according to our previously published methods , 33. Sagescribed . Both prDr-SLC38A9 dsRNAs were a 529-bp fragment (399–927) and 521-bp fragment (1053–1573) of the ORF. The enhanced green fluorescent protein (EGFP) dsRNA was the full-length ORF (720 bp). These dsRNAs were purified using phenol/chloroform and then annealed. To prepare the test foods used for the knockdown experiments under sex-inducing conditions, dsRNAs were added to minced B. brunnea and stored at − 80 °C until use. The worms were fed twice at 2-day intervals with chicken liver supplemented with 200 ng of EGFP or Dr-SLC38A9 dsRNA. On 1 day after the second feeding, the posterior and anterior regions of the worms were amputated, and the trunk fragments were incubated without food for 7 days. The partially regenerated worms were then fed chicken liver supplemented with 200 ng of EGFP or Dr-SLC38A9 dsRNA twice at 2-day intervals to allow the worms to fully regenerate. The resultant asexual worms were then fed minced B. brunnea supplemented with 200 ng of the dsRNAs of the indicated genes daily for 6 weeks.Double-stranded RNA (dsRNA) was synthesized in vitro using the MEGAscript High Yield Transcription kit. The B. brunnea worms (1 g wet weight) as previously described , , and , respectively. Next, to compare the gene expression levels among the three groups, Tukey’s honestly significant difference (HSD) test was used. The expression levels in cocoons indicates those in yolk glands because cocoons are mostly filled with yolk gland cells. Considering that tryptophan is enriched in yolk glands ). We examined the development of ovaries and the copulatory apparatus in Dr-SLC38A9-knockdown worms externally using a stereomicroscope. After 4 weeks of treatment, the percentage of Dr-SLC38A9-knockdown worms that had developed ovaries and copulatory apparatuses was significantly lower than that of EGFP-knockdown (control) worms  = 1.00, mean (Dr-SLC38A9 KD) = 0.284, t(14) = 4.40, P = 0.00061]). Moreover, histological examination revealed that the testes of Dr-SLC38A9 knockdown worms were smaller  = 1.00, mean (Dr-SLC38A9 KD) = 0.0374, t(14) = 3.15, P = 0.0071]). In addition, histological examination revealed that Dr-SLC38A9 knockdown inhibited the development of yolk glands  and Dryker gene , and thrDr-SLC38A9 was related to the activity of sex-inducing substances. In this experiment, we chose sexual planarians as test worms because they contain many reproductive organs expressing Dr-SLC38A9. In the experimental sexual induction of asexual planarians, approximately 30 worms of the asexual planarians were fully sexualized by daily feeding with minced bodies of sexually mature B. brunnea worms (60 mg wet weight) for 4 weeks. We fed 15 sexual worms with approximately 120 mg wet weight of the sexually mature B. brunnea worms or chicken liver (control) over 3 days and carried out in situ hybridization analyses using a probe for Dr-SLC38A9 RNA. Stronger Dr-SLC38A9 expression was detected specifically in the ovaries  from B. brunnea . The resulting expression of Dr-SLC38A9 was significantly enhanced by the M0 + M10 fraction of B. brunnea  as one of the sex-inducing substances contained in the M0 + M10 fraction . As a result, worms fed with or without l-Trp did not show any statistically significant difference in the expression of Dr-SLC38A9, although the mean value was higher than that of the control  and acts as an Arg sensor [d-Trp sensor to control cell growth and proliferation during sexual induction. The current study suggests that d-Trp is taken directly into the ovaries and testis via the Dr-SLC38A9 transporter. Nevertheless, the biological function of d-Trp in the ovaries and testes remains unknown. A possibility is that it might act as a ligand for nuclear receptors. Additionally, qPCR analysis implied that TR33723 | c0_g1_i1 is also highly expressed in the yolk glands, and TR37685|c0_g2_i3 is expressed in the ovaries. We consider that in future works, it will be important to study whether TR33723|c0_g1_i1, TR37685|c0_g2_i3, and Dr-SLC38A9 proteins are capable of transporting d-Trp.Transporters play key roles in many biological processes that are regulated by amino acids . The solentation . As mentsic acid . This sut of Trp . Recentlg sensor , 37. SLCg sensor , 37, whig sensor . Therefod-Trp was the first substance to be identified as sex-inducing in the sexual induction of planarian D. ryukyuensis. In the present study, to investigate the mechanism of sexual induction of planaria by d-Trp, we isolated a putative amino transporter gene, Dr-SLC38A9, activated by d-Trp. Considering its expression and function, Dr-SLC38A9 could play a role in the early developmental stages of germline cells and yolk glands. Its function in yolk glands could be related to the enrichment of Trp therein. Thus, the functional analysis of Dr-SLC38A9 in this study is an important first step in clarifying the mechanism of planarian sexual induction by d-Trp.Additional file 1: Figure S1. Five stages of sexualization in the planarian D. ryukyuensis. The OH strain of the planarian D. ryukyuensis begins to develop reproductive organs upon switching from the asexual to the sexual state. The process can be roughly divided into five stages based on the development of reproductive organs [e organs . Brieflye organs  form, anAdditional file 2: Figure S2. Nucleotide sequence of the Dr-SLC38A9 gene and its predicted amino acid sequence. The isolated cDNA has a 78-bp 5′ untranslated region and an 84-bp 3′ untranslated region. A polyadenylation signal (AATAAA) is located 13 bp upstream of the beginning of the poly(A) tail. Dr-SLC38A9 contains an open reading frame encoding a 585-amino acid polypeptide.Additional file 3: Figure S3. Ectopic germline induction by d-Trp administration in the dorsal midline.  After we fed asexual worms food supplemented with d-Trp for 7 weeks, a serial sagittal section of a d-Trp-fed worm was prepared. (A) A section stained with hematoxylin and eosin. (B) In situ hybridization of Dr-nanos using an adjacent section. Red arrowheads represent a germ-like cell mass with a positive Dr-nanos signal. The dorsal side is at the top. In, intestine. Scale bar, 100 μm. (C–E) Whole-mount in situ hybridization of Dr-nanos. The test worms were fed (C) chicken liver homogenate (control) and (D) Trp for 7 weeks. (E) Stage 3 worms. Red arrowheads represent Dr-nanos expression in the dorsal midline at the prepharyngeal region, whereas blue arrowheads represent Dr-nanos expression in the presumptive region of the testes. Scale bars, 2 mm. Red and blue arrowheads represent Dr-nanos expression in the dorsal midline at the prepharyngeal region and presumptive region of the testes. These ectopic germlines were induced by administration of l-Trp, as well as d-Trp.Additional file 4: Supplementary Table S1. Primer sets used for qRT-PCR.Additional file 5: Supplementary Data Sheet S1. The raw data of qRT-PCR analyses for Fig. 1, 4 and 5.


Background: Esophageal adenocarcinoma (EAC) is a deadly disease with limited treatment options. STING is a transmembrane protein that activates transcription of interferon genes, resulting in stimulation of APCs and enhanced CD8+ T-cell infiltration. The present study evaluates STING agonists, alone and in combination with radiation to determine durable anticancer activity in solid tumors.Materials and Methods: Esophagojejunostomy was performed on rats to induce reflux leading to the development of EAC. At 32 weeks post operatively, rats received intratumorally either 50 μg STING (ADU-S100) or placebo (PBS), +/– 16Gy radiation. Drug activity was evaluated by pre- and post- treatment MRI, histology, immunofluorescence and RT-PCR.P < 0.0001). Downstream gene expression, pre- to on- and post- treatment, demonstrated significant upregulation of IFNβ, TNFα, IL-6, and CCL-2 in the treatment groups vs. placebo. On- or post- treatment, radiation alone, ADU-S100 alone, and ADU-S100 + radiation groups demonstrated enhanced PD-LI expression, induced by upregulation of CD8+ T-cells (p < 0.01).Results: Mean MRI tumor volume decreased by 30.1% and 50.8% in ADU-S100 and ADU-S100 + radiation animals and increased by 76.7% and 152.4% in placebo and placebo + radiation animals, respectively (de novo EAC.Conclusions: ADU-S100 +/– radiation exhibits potent antitumor activity and a promising immunomodulatory profile in a  Esophageal cancer is the sixth most common cause of cancer related deaths worldwide . CurrentTargeting the PD-1/PD-L1 axis has proven to be highly efficacious in patients with advanced solid tumors including melanoma, renal cell carcinoma, and non-small cell lung cancer , 5. DuraThe key regulator of the innate immune system is the stimulator of interferon genes (STING), an endoplasmic reticulum adaptor protein, which stimulates the production of type 1 interferons (IFN) from cancer cells and dendritic cells (DC) in the tumor microenvironment. Briefly, the tumor or pathogen derived cytosolic DNA in these cells is recognized by cytosolic enzyme cGAMP synthase leading to the generation of cyclic GMP-AMP, which in turn binds to and activates STING signaling. This initiates a cascade where STING in the cytoplasm binds to TANK-binding kinase 1 (TBK1) and IκB kinase (IKK), that activates a host of transcription factors: interferon regulatory factor 3 (IRF3), nuclear factor-κB (NF-κB), and signal transducer and activator of transcription (STAT6). Subsequently, nuclear translocation of these transcription factors leads to the induction of type I IFNs and other immune modulatory cytokines , 11. TypTherefore, as expected, in preclinical models STING agonists have demonstrated not only potent activity against the targeted primary tumors but also for distant metastasis and recurrence , 14. Addde novo EAC through identical physiological and molecular processes that occur in humans [In this study we evaluate the impact of a STING agonist, ADU-S100, a synthetic cyclic dinucleotide (CDN) agonist of STING, known to activate all human and mouse STINGs and induce the expression of cytokines and chemokines , in combn humans , 20. Addn humans . This prn = 20), placebo with radiation (P+R) (n = 16), STING agonist (S) (n = 29) and STING agonist with radiation (S+R) (n = 20). The overall mortality rate post randomization was 20% (n = 17), with the S and S+R treatment cohort representing 64.7% of the total mortality (n = 11). Causes of mortality within the control groups (P and P+R) included respiratory infection secondary to aspiration of reflux (n = 2), strictures at the anastomotic site (n = 2), perforated stomach ulcers (n = 1) and unknown etiology (n = 1).Within the treatment groups (S and S+R), causes of mortality included respiratory infection secondary to aspiration (n = 6), strictures at the anastomotic site (n = 3), perforated stomach ulcers (n = 1) and unknown etiology (n = 1). There was no significant increase in observed mortality in the STING agonist treated animals (22.5%) when compared to the controls (16.7%) (p = 0.59). Sixty-eight animals completed the study, including 30 control (P = 18 and P+R = 12) and 38 treatment (S = 23 and S+R = 15) animals.Thirty weeks after the Modified levrat surgery was performed, 85 rats were randomized to the four intervention groups: placebo (P) (p < 0.0001) – p =< 0.0001) – Overall, a comparison of MRIs in the study groups between 32 and 40 weeks demonstrated a mean increase in percentage tumor volume of 76.7% and 152.4% in the P and P+R arms respectively, and a decrease of 30.1% and 50.8% in the S and S+R arms, respectively (ANOVA test p < 0.01) – Analysis of STING pathway gene expression with 30-week pre-randomization biopsy RQ values set as the baseline demonstrated on- and post- S treatment a significant mean difference in RQ values. Similar but more moderate peak and trough changes in RQ values of downstream genes were observed in S+R group on- and post- treatment. On the contrary, P and P+R groups demonstrated minor difference in on- and post- treatment RQ values from baseline pre- randomization biopsy levels for all the individual genes. For the individual genes, Mixed model F-test demonstrated statistically significant difference for group effect, time effect and interaction effect between group and time, respectively , S  and S+R  compared to P . Additionally, in the drug intervention subgroups, post-hoc analysis utilizing Wilcoxon-Mann-Whitney test showed a significant difference in CD-8 positivity between pre- and both on- (p < .0001 (S); p = 0.0002 (S+R)) and post- treatment (p < .0001 (S); p = 0.0004 (S+R)) time points – p = 0.0001; mean post- = 35.3, p = 0.0149), S  and S+R , compared to P . Likewise, in the drug intervention subgroups, post-hoc analysis utilizing Wilcoxon-Mann-Whitney test showed a significant difference in PD-L1 positive cells between pre- and both on- (p < .0001 (S); p = 0.0021 (S+R)) and post- treatment (p = 0.0003 (S); p = 0.0003 (S+R)) time points – TIL infiltration was upregulated with higher mean CD-8+ T-cell densities on- and post- treatment in P+R  targets is needed. Data from CheckMate 032, has demonstrated nivolumab plus ipilimumab is superior to nivolumab alone, with ORR of 24% and 12%, respectively however it is unclear if this is a viable strategy moving forwards due to enhanced toxicity with this particular IO-IO combination .Through our current study, we provided strong evidence to suggest cross-link between the STING and PD-L1 pathways in EAC. A host of earlier preclinical cancer studies have also demonstrated enhanced activity of ICIs when combined with a STING agonist , 22, 34.The main limitation of our study was the inability to test a PD-1/PD-L1 inhibitor in combination with S and S+R due to unavailability of a rat cross reactive antibody. However, we did show PD-L1 upregulation on- or post- treatment with S and S+R hence substantially addressing this limitation. Another limitation may have been that we did not specifically study systemic immune response, for instance looking at peripheral T-cell trafficking. Yet previous preclinical data have well documented systemic immune modulation following a 50 μg dose of ADU-S100 .In conclusion, our findings suggest potent antitumor activity of ADU-S100 alone and in combination with radiation against EAC with evident molecular pathway activation and reasonable safety. Additionally, the attractive synergistic association between STING activation and PD-L1 expression may represent a new IO-IO concurrent combinatorial antitumor strategy well-suited for further clinical testing in gastroesophageal cancers, to provide broader and more durable responses.All animal research was performed with the approval of the Institutional Animal Care and Use Committee of Allegheny General Hospital in Pittsburgh, PA, USA, under Protocol #1057. Humane care was provided to all animals per the standards set forth in “The Guide for the Care and Use of Laboratory Animals.”Published data was utilized to select a dose strength and frequency for ADU-S100 that was deemed highly immunostimulatory . ADU-S10Modified Levrat surgery of end-to-side esophagojejunostomy was performed on 85, 8 week old male, Sprague-Dawley rats from Harlan Laboratories, to induce GDER and progression to EAC , as prevActivity of ADU-S100 was determined through the comparison of pre and post-treatment tumor volumes of each animal utilizing evaluable MRIs, quantitative real-time polymerase chain reaction (qRT-PCR) analysis of gene expression from serial tissue samples, and evaluation of immunofluorescence for CD8 and PDL1 proteins. MRI imaging response was scored according to the clinical gold standard of response evaluation criteria in solid tumors (RECIST version 1.1) .Pre-, on- and- post-treatment biopsy tissues were flash-frozen in Tissue-Tek Optimal Cutting Temperature (OCT) compound . Upon necropsy, the entire esophagus and jejunum, to a length approximately 1 cm distal to the anastomosis was harvested. After the specimen was cut open longitudinally, samples were rinsed in ice-cold phosphate buffered saline to remove debris, oriented to maximize exposure of suspicious areas, and flash-frozen in OCT. Next, frozen OCT blocks with adequate and evaluable tissue were cut into 5 μM sections using a cryostat  and stained with hematoxylin and eosin (H&E) for pathological assessment, gene expression analysis, and immunoassays.2 Primer Assays Endogenous controls run on each plate included SNORD-95 , and miR16 . All samples were normalized against pathologically confirmed normal esophagus and were run in technical duplicates.A total of 80 μM of tissue was macrodissected using a cryostat . Briefly, RNA was isolated, reverse transcribed, and RT-PCR was performed using rat primers; CCL2 , IFNβ , IL6 , IFNγ , and TNFα  . Raw datCD8 and PDL1 antigens were examined using an indirect double sequential immunofluorescence assay. Sample sections were fixated in 4% paraformaldehyde in PBS solution for 15 min at room temperature, then washed 3 times in 1xTBS and blocked with 1xTBS containing 5% goat serum and 1% BSA for 2 hours. Incubation with anti-CD8 antibody  was performed for 1 hour at 1:100 dilution followed by 3 TBS washes containing 0.025% Tween-20. A second incubation with anti-PDL1 antibody  at 1:100 dilution at 20 ug/mL for 1 hour was done followed by 3 TBS washes containing 0.025% Tween-20. Secondary antibody incubation was performed using 1:300 dilutions of goat anti-mouse IgG H&L Alexa Fluor 488  and of goat anti-rabbit Alexa 594  for anti-CD8 and PDL1, respectively. This was followed by 3 TBST washes. Slides were then rinsed in water and allowed to dry followed by the addition of one drop of Prolong Gold with 4′, 6-diamidino-2-phenylindole (DAPI) . After incubation for 24 h at room temperature in the dark, cells were observed on a fluorescence microscope with light intensity 50% and exposure time of 60 ms for DAPI channel, with light intensity 60% and exposure time of 120 ms for GFP channel, and with light intensity 70% and exposure time of 750 ms for RFP channel.For PD-L1 and CD8 staining each, a quantitative density analysis was performed for each sample using 3 selected microscopic fields and counting the number of stained cells per 100 tumor cells. Rat lymph node and PD-L1+ tumor control tissue served as positive controls for CD8 and PD-L1, respectively. Scoring was performed by three blinded trained research associates with collaborative consensus on cases with discrepant interpretation.t test or Wilcoxon-Mann-Whitney test was utilized for comparison of mean difference between any two treatment groups for CD8 cell density, PD-L1 cell density, and gene expression. ANOVA test was used to compare all groups for percent change of tumor volume. Fisher’s exact test was used for comparison of mortality status and change in MRI tumor volume among all treatment groups, respectively. A mixed model F test was performed to evaluate each gene and each protein expression between group effects (treatment group) as well as within subject effects . A p value < 0.05 was considered to be statistically significant. All statistical analyses were performed using SAS software .An independent two-tailed


Human leukocyte antigen G (HLA-G) is a non-classical human leukocyte antigen (HLA) class I protein that interacts with inhibitory receptors and is commonly overexpressed in various cancers, thereby establishing itself as an inhibitory checkpoint immune ligand. It is also expressed in trophoblast cells during pregnancy and protects the fetus from immune rejection. Despite its crucial role and its intriguing expression pattern, the regulation of HLA-G’s expression is only partially understood. HLA-G’s mRNA is expressed in many tissues but the protein expression is restricted only to the cells mentioned above. Therefore, we suggest that HLA-G is post-transcriptionally regulated. Here, we reveal a distinctive site present only in the 3′ Untranslated region (UTR) of HLA-G, which might explain its unique expression pattern. Consequently, we attempted to find binding factors such as RNA binding proteins (RBPs) and microRNAS (miRs) that regulate HLA-G expression by interacting with this distinct site present in its 3′ UTR. Our research indicates that this site should be further studied in order to reveal its significance. HLA-G belongs to the family of HLA class I  class I), which presents peptides to T cells and, thereby, activates cytotoxic T cells (CTLs) of the adaptive immune system. HLA class I proteins, including HLA-G, are also ligands of NK cell inhibitory receptors. Thus, HLA class I proteins have a dual role: they activate the adaptive immune response and inhibit innate cell activity . This coHLA-G is a crucial immune modulating molecule. It serves as an inhibitory checkpoint ligand by binding to inhibitory receptors  such as Despite the importance of HLA-G, its unique expression pattern regulation is not fully understood. The mRNA of HLA-G is expressed in several tissues however, protein expression in these tissues cannot be detected ,14. ThusSix miRs were already reported to regulate HLA-G expression by binding its 3’ UTR: miR-148a, miR-148b, miR-152, miR-133a, miR-628-5p, and miR-548q . HoweverTo identify factors that specifically affect HLA-G expression, we compared the 3′ UTR of HLA-G to those of HLA-A, -B, and -C  a His-tagged version of DDX47 in JEG-3 cells, which endogenously express HLA-G at the protein level  and veriTo further examine the regulatory role of DDX47, we screened for cells that do not express the HLA-G protein, but still express HLA-G mRNA, as detected by qRT-PCR A. We selZSWIM8, the second RBP candidate for HLA-G regulation, has a 5646 bp coding sequence (CDS) . Due to Next, we speculated that miRs can affect HLA-G expression by targeting its unique 26 bp region. In silico miR screening analysis identified miR-1301 to have a predicted binding site in this region A. miR-13Many genes are often controlled by a combination of several factors. Therefore, we hypothesized that the regulation of HLA-G might require a combination of effectors. Hence, JEG3 and 721.221-HLA-G cells that overexpressed miR-1301 A,B were The 3′ UTR of a given gene is an important regulatory site harboring many regulatory elements that control its post-transcription regulation . The 3′ In addition to miRs, we recently discovered and published our findings regarding the RBP HNRNPR, which binds the 3′ UTR of both HLA-G and the classical HLA class I and acts as their general positive regulator . During By performing an RNA purification assay, we identified RBPs that interact with the 26 bp region. However, overexpression or downregulation of the given RBPs did not ultimately influence the surface expression levels of HLA-G. The reason for this observation might be the construct used for these assays, which included an additional 45 bp downstream sequence, common to all 3′ UTRs of classical HLA class I mRNAs. An RNA affinity purification assay capable of precipitating RBPs bound only to the 26 bp sequence may yield more specific candidates that bind this region. This assay may be difficult to perform due to the short length of the RNA transcript; it might be too small to bind streptavidin beads and RBPs simultaneously. Alternatively, maybe other RBPs that may have been precipitated but were ruled out for low statistical significance score, were those that regulate HLA-G expression specifically. We also screened for miRs with a hypothetical binding site within the 26 bp region, and selected miR-1301. We found that this miR either alone or in combination with a given RBP did not affect HLA-G surface protein expression. To conclude, we identified a region in the 3′ UTR of HLA-G, which is distinctive to this HLA molecule. We used different methods to identify regulators that bind to this site and specifically control HLA-G protein expression. None of our candidates affected surface HLA-G expression levels. Nevertheless, we strongly suggest that the unique site present in the 3′ UTR of HLA-G is of value and may explain the unique expression pattern of HLA-G. Hence, this site should be further studied to reveal its significance. l-glutamine, and 1% sodium pyruvate . No datasets were generated or analyzed during the current study.JEG3 cells were maintained in DMEM medium . 721.221, C1R, RAJI, BJAB and JURKAT were maintained in RPMI medium (Sigma-Aldrich). All media were supplemented with 10% fetal bovine serum and 1% MEM EAGLE, 1% penicillin-streptomycin, 1% The binding of RBPs to RNA was analyzed by RNA affinity purification as previously described . In shorZSWIM8 knockdown vectors and scrambled vector cloned into pLKO.1-puro plasmids were purchased from Sigma Aldrich. Transduced JEG3 cells were grown in the presence of 2 μg/mL puromycin. DDX47 and HLA-G were cloned into the pHAGE-DsRED(−)-eGFP(+) lentiviral vector that also contains GFP. miRNA-1301 vectors were generated as previously described . The oliPrimer sequences: miR-130 forward: gatccccTTGCAGCTGCCTGGGAGTGACTTCttcaagagaGAAGTCACTCCCAGGCAGCTGCAAtttttggaaa, miR-1301 reverse: agcttttccaaaaaTTGCAGCTGCCTGGGAGTGACTTCtctcttgaaGAAGTCACTCCCAGGCAGCTGCAAggg, DDX47 forward (NotI): atGCGGCCGCAccatggcggcaccc DDX47 reverse (AgeI): TGACCGGTTTAATGGTGATGGTGATGGTGACGGCCTTTCCG, HLA-G forward (NotI): GCGGCGGCCGCGCCGCCACCATGGTGGTCATGGCG HLA-G 14 bp deletion reverse (AgeI): GCGACCGGTAAAGTTCTCATGTCTTCCATTTATotal RNA was extracted with TRI reagent , followed by Turbo-DNase  treatment. A poly(A) tail was added using the poly(A) kit (Ambion). cDNA libraries were generated from 1 μg of RNA using M-MLV reverse transcriptase , in the presence of an adapter primer. Detection of the various transcripts was performed with qRT-PCR.For qRT-PCR, cDNAs were labeled in SYBR Green based detection using a QuantStudio 12k Flex Real-time PCR cycler . The primers used for targeting HLA-G, GAPDH, and miR-1301 were: GAPDH forward: GAGTCAACGGATTTGGTCGT, GAPDH reverse: GATCTCGCTCCTGGAAGATG, HLA-G forward: AGTCAAAGACAGGGTGGTGG, HLA-G reverse: GGAGTGGCTCCACAGATACCThe reverse primer for miR-1301 was the 3′ adapter primer , The forward primer we used was: TTGCAGCTGCCTGGGAGTGACTTCLysates of JEG3, 721.221, JURKAT, C1R, RAJI, or BJAB cells were prepared using lysis buffer containing SDS, PMSF, and aprotinin, followed by SDS gel electrophoresis. Proteins were transferred onto a nitrocellulose membrane and specific protein bands were detected using antibodies detecting His-tag ), ZSWIM8 ) Vinculin ), and GAPDH ). All were diluted in 5% BSA in 1× PBS. Chemiluminescence was detected by secondary antibody-linked horse-radish peroxidase .® 647 catalog number 115-606-062). All stainings were analyzed by flow cytometry using the FCS Express software.The following antibodies were used throughout the manuscript: anti-HLA-G , anti-classical HLA class I proteins mAb . Staining was performed with 0.2 μg mAbs per 100,000 cells. Binding was detected by a secondary antibody used in a final dilution of 1:200 (Abcam, Cambridge, UK, Alexa Flour


However, their efficacy was limited by poor expansion, loss of function, and short persistence of the TCR T cells. Here, we studied whether overexpression of c-Jun, a transcription factor required for T cell activation, in the TCR T cells could enhance their expansion, function, and persistence in HCC tumor models.The overall 5-year survival rate of hepatocellular carcinoma (HCC), a major form of liver cancer, is merely 20%, underscoring the need for more effective therapies. We recently identified T cell receptors (TCR) specific for the HLA-A2/alpha fetoprotein amino acids 158-166 (AFP158-specific TCR or both the TCR and c-Jun (TCR-JUN), were constructed and used to transduce primary human T cells to generate the TCR or TCR-JUN T cells, respectively. We compared the expansion, effector function, and exhaustion status of the TCR and TCR-JUN T cells in vitro after HCC tumor stimulation. Additionally, we studied the persistence and antitumor effects of the TCR and TCR-JUN T cells using the HCC xenografts in NSG mice.Recombinant lentiviral vectors (lv), expressing either the HLA-A2/AFP158 TCR T cells, the TCR-JUN T cells have better expansion potential in culture, with enhanced functional capacity against HCC tumor cells. In addition, the TCR-JUN T cells were less apoptotic and more resistant to exhaustion after HepG2 tumor stimulation. In the HCC xenograft tumor model, c-Jun overexpression enhanced the TCR T cell expansion and increased the overall survival rate of the treated mice. Importantly, the TCR-JUN T cells were less exhausted in the tumor lesions and demonstrated enhanced tumor infiltration, functionality, and persistence.We could effectively transduce primary human T cells to express both TCR and c-Jun. Compared to the HLA-A2/AFP158 TCR engineered T cells. The c-Jun gene co-delivery has the potential to enhance the antitumor efficacy of AFP specific TCR T cells when treating patients with HCC.c-Jun overexpression can enhance the expansion, function, and persistence of the A2/AFP Hepatocellular carcinoma (HCC) accounts for 90% of primary liver cancer , the six158) and showed that human T cells engineered with such TCR genes could recognize and kill AFP+ HepG2 tumor cells, which resulted in regression of HCC xenografts in immunocompromised NSG mice   . Different (TME) , 18, armnt (TME) –22, and nt (TME) , 24. Addnt (TME) .158 specific TCR T cells in treating HCC xenografts. Our data showed that c-Jun overexpression enhanced the functionality and persistence of TCR T cells, with more resistance to exhaustion and activation-induced cell death. More TCR-JUN T cells were also found in the tumor lesions. The TCR-JUN T cells had better function compared to TCR T cells, which extended mouse survival. Thus, engineering T cells to overexpress c-Jun could be a potential approach to enhance TCR T cell cancer therapy.c-Jun is a member of the AP-1 family of transcription factors, which is required for T cell activation, and its activity is strongly augmented by TCR signaling through Jun kinase (JNK) , 27. DurThe Jurkat, HEK293T, and HepG2 cells were purchased from American Type Culture Collection (ATCC) and cultured according to recommendation. No more than 10 passages of the cells were used to assure authenticity. HepG2-Luc and HepG2-GFP cells were created in lab, as described (scid IL2Rgammanull (NSG) mice were purchased from Jackson Laboratory. Animal protocol is approved by IACUC of Augusta University. 8-10-week-old mice were inoculated subcutaneously (SC) with the indicated number of HepG2-Luc. Tumors were measured by bioluminescent intensity (BLI) using the Ami X spectral imaging system from Spectral Instruments. BLI values  were analyzed using AmiView software.The NOD-158-specific TCR construction were previously described  buffy coats were purchased from Shepeard Community Blood Center in Augusta, GA, and separated by Ficoll-Paque PREMIUM . Primary human T cells were isolated using the EasySep Human T Cell Isolation Kit  according to the manufacturer’s protocol. Human T cells were activated with Dynabeads™ Human T-Activator CD3/CD28  using 1.5:1 beads to cell ratio for 24 hours and were transduced by lvs at 30 Multiplicity of Infections (MOI) as previously described (4/well) were grown on a 96-well flat bottom plate in triplicate overnight, and TCR T cells were then added at indicated effector/target (E/T) ratios. The supernatants were collected after 20 hours and were analyzed for IL-2 and IFNγ by ELISA (BioLegend) and for cytotoxicity by LDH activity (Promega).HepG2 cells (1 × 10+ cells (1×104/well) were plated in triplicate on a 96-well flat bottom plate and grown overnight. TCR T cells were added at the indicated E/T ratio. Pictures were taken every 3 hours using the IncuCyte SX1 Live-Cell analysis system for 3 days. 5 images per well were collected at each time point. The total GFP area was assessed at each time point by IncuCyte analysis software.The cytotoxicity of TCR T cells was also analyzed by IncuCyte real-time quantitative live-cell imaging and analysis (Sartorius). HepG2-GFP6/well) were seeded into 6-well plate and grown overnight. After 24 hours, the medium was removed, and TCR T cells (day 7 after lv transduction) were added at 0.5:1 E/T ratio and co-cultured for up to 6 days in RPMI-1640 complete medium with and without 40 UI/mL of IL-2. Duplicate wells were plated for each condition. Mock non-transduced T cells were included as controls. TCR T cells’ expansion was calculated, and the expression of exhaustion markers and apoptosis of TCR T cells was assessed by flow cytometry. Cells were counted with Countess II™ Automated Cell Counter using trypan blue stain (0.4%)  in Countess™ Cell Counting Chamber Slides .HepG2 cells  or NovoCyte Quanteon (Agilent). For some staining, Zombie Green Fixable Viability Kit (BioLegend) was added to exclude dead cells. The antibodies included BioLegend CD45, CD8, CD4, CD3, TCRb, PD1, LAG-3, TIM-3, Annexin-V, Bcl-2, IL-2, and IFNγ. The c-Jun (60A8), rabbit (DA1E) mAb IgG XP isotype control, and anti-rabbit IgG (H+L), F(ab’)2 fragment were from Cell Signaling. Unless otherwise indicated, all antibodies were used at 1:200 dilution in PBS. The c-Jun (60A8) and rabbit (DA1E) mAb IgG XP isotype control were diluted in antibody dilution buffer (0.5% BSA in 1x PBS) and used according to manufacturer’s recommendation .+ T cells were monitored in the blood by staining with indicated antibodies anti-human CD45 and anti-mouse TCRbeta (the TCR specific for HLA-A2/AFP158 were identified from immunized mice (TCR or TCR-JUN T cells (10-12 days after transduction) of the indicated numbers were injected into tumor-bearing mice intravenously (IV). Human T cells and TCRzed mice . Mouse Cex vivo analysis of tumor infiltrating TCR T cells, tumor tissue was collected, washed in PBS, and mechanically dissociated and digested in RPMI media supplemented with 0.1% collagenase type V (Sigma C9263), 0.1% hyaluronidase (Sigma H6254), and 100 unit/mL DNase I (Sigma D4527)) for 30-45 minutes at 37°C. After RBCs lysis, the single cell suspension was ready for analysis by flow cytometry and for cytotoxicity by IncuCyte Live-Cell analysis system.For 5 HepG2 tumor cells for 20 hours in a 24-well plate according to the percentage of TCR+ T cells and the desired E/T ratios. Monensin (BioLegend) was added to the co-culture for the last 4 hours. Intracellular cytokine staining of IL2 and IFNγ was performed according to the manufacturer’s instruction (BioLegend).To study the cytokine production, TIL TCR T cells were prepared and co-cultured with 2.5×10Tumor tissues were collected and fixed in 10% Buffered Formalin Phosphate  for 24 hours and then replaced with 70% ethanol. The paraffin-embedded tissues were sectioned and stained by anti-CD8 antibody  and HRP conjugated secondary anti-Rabbit IgG, which was revealed by TMB substrate.in vitro stimulation with HepG2 tumor cells were isolated by cell sorting based on the hCD45 and TCRb markers. Total RNA was isolated by Quick-RNA Microprep Kit (Zymo Research) and then mRNA was isolated by oligo-dT. The RNA libraries were prepared from the purified mRNA and sequenced by Novogene. The paired-end 150bp raw sequencing reads were examined by FastQC v0.11.8. There were minimal 20 million sequence reads for each sample. Adaptor sequences and low-quality bases were trimmed using Trim Galore! v0.6.3. The cleaned reads were mapped to reference genome (mm10) using STAR aligner v2.7.8a. The QC and mapping steps were performed in Galaxy server . The sequence counts for each gene were collected by Rsubread featureCounts functions v1.22.2. The differential gene expression analysis was performed using DESeq2 v1.32.0. The heatmaps were generated using ComplexHeatmap v2.8.0 in R v4.1.The TCR T cells after t-test or 2-way ANOVA in the Prism software (GraphPad Inc.). Survival curves were compared using the log-rank Mantel–Cox test. Data points represent biological replicates and are shown as the mean ± standard error of mean (SEM) or mean ± standard deviation (SD) as indicated in the figure legends. Significance was assumed with *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001.Statistical analyses were performed using student two-tailed in vitro. To this end, we first constructed 2 lentiviral vectors (lvs) that expressed the TCR specific for HLA-A2/AFP158 and the c-Jun separately (158 TCR and c-Jun (TCR-JUN)  T cells after either TCR-JUN-lv or TCR-lv transduction; this level was also similar to that of the mock non-transduced T cells. In contrast, the c-Jun level in TCR+ T cells after TCR-JUN lv transduction was 3-folds higher than that in the TCR negative T cells  of TCR T cells. To this end, the TCR and TCR-JUN T cells were co-cultured with HepG2 tumor cells at different effector/target (E/T) ratios for 20 hours. Using LDH assay, we found that the TCR-JUN T cells maintained similar cytotoxicity against HepG2 tumor cells as the TCR T cells  in the peripheral blood of TCR-JUN T cells treated mice on day 38 after tumor injection (26 days after ACT), even though the % of total human T cells was only slightly higher  HCC xenograft tumor model in NSG mice to study whether the c-Jun overexpression would enhance the + T cells in the tumors treated with TCR-JUN T cells compared to TCR T cells. On day 13 after ACT, we found that there were significantly more TCR+ T cells in the tumor treated with TCR-JUN T cells, almost 5-folds, than in the TCR T cell-treated tumors  before ACT . We found that c-Jun overexpression enhanced T cell expansion driven by anti-CD3/CD28 magnetic beads, as well as TCR T cell expansion by tumor antigenic stimulation. The TCR-JUN T cells produced higher amounts of IL-2 and IFNγ, without significantly altering the cytotoxicity. Importantly, after ACT into tumor bearing mice, more TCR-JUN T cells accumulated in the tumor lesions, which were functional and associated with better overall survival of the treated mice. These findings are in agreement with previous reports that c-Jun overexpression provides CAR-T cells resistance to exhaustion , which may play an important role in suppressing the tumor infiltrating T cells and limiting their ability to exert their effector function. Thus, in addition to the provision of tumor specific T cells, creating a favorable TME is needed for TCR T cells to exert their function to achieve antitumor effects. Future work using immune-competent mice will help to study if c-Jun overexpression would enhance the ACT in the immunosuppressive TME in solid tumors.The persistence of T cells usually correlates with the efficacy of the ACT , 45, 46.During T cell exhaustion, continuous TCR signaling from persistent antigen exposure results in the induction of IRF4, BATF, and NFAT transcription factors, with continuous upregulation of co-inhibitory receptors such as PD-1, LAG-3, and TIM-3. The co-inhibitory signals disrupt c-Jun activation, resulting in diminished expression of effector cytokines, whereas NFAT activation remains undisrupted to form ternary complexes with IRF4 and BATF at the enhancer and promoters of exhaustion-associated genes , 30. CARc-Jun has been described as an oncogene linked to a range of cancers , 48. Befin vitro and in vivo, with more resistance to exhaustion and apoptosis and better effector function. Taken together, c-Jun can potentially enhance the efficacy of TCR T cells.In conclusion, our study demonstrated that the c-Jun overexpression improves the expansion and persistence of TCR-Ts https://www.uniprot.org/uniprotkb/P05412/entry). Readers can also contact corresponding authors to acquire these 2 sequences.The TCR sequence is included in the Patent title “Human alpha fetoprotein-specific murine T cell receptors and uses thereof”. The Patent Number 11041011. The c-Jun sequence is from Uniprot P05412 (The animal study was reviewed and approved by IACUC of Augusta University.in vitro and in vivo experiments. QL, RM, and YP were involved in constructing and producing lentiviral vectors, animal care, and animal studies. MH and YH wrote the manuscript. QL, RM, and YP all read and agree with the manuscript. All authors contributed to the article and approved the submitted version.YH and MH formulated the hypothesis and designed and conducted most of the


The clinically used androgen receptor (AR) antagonistsfor thetreatment of prostate cancer (PCa) are all targeting the AR ligandbinding pocket (LBP), resulting in various drug-resistant problems.Therefore, a new strategy to combat PCa is urgently needed. Enlightenedby the gain-of-function mutations of androgen insensitivity syndrome,we discovered for the first time small-molecule antagonists towarda prospective pocket on the AR dimer interface named the dimer interfacepocket (DIP) via molecular dynamics (MD) simulation, structure-basedvirtual screening, structure–activity relationship exploration,and bioassays. The first-in-class antagonist M17-B15 targeting theDIP is capable of effectively disrupting AR self-association, therebysuppressing AR signaling. Furthermore, M17-B15 exhibits extraordinaryanti-PCa efficacy in vitro and also in mouse xenograft tumor models,demonstrating that AR dimerization disruption by small molecules targetingthe DIP is a novel and valid strategy against PCa. Small molecules targeting the dimer interface pocket locatedat the ligand-binding domain of androgen receptor (AR) are capableof suppressing AR signaling and combating prostate cancer. AR is a member of the nuclear receptor(NR) family of ligand-dependent transcription factors, and it containsfour functional modules, including a large N-terminal domain (NTD),a DNA-binding domain (DBD), a short hinge region, and a C-terminalligand-binding domain (LBD).3 The bindingof testosterone or DHT to the ligand binding pocket (LBP) of the ARLBD triggers a conformational change of AR that in turn promotes thedissociation of AR from heat shock proteins, and AR dimerization,and transport of AR from the cytosol into the cell nucleus. Subsequently,the AR dimer binds to a specific region of DNA known as the androgenresponse element (ARE) to activate or repress specific gene transcription.5 Abnormal AR activity is associated with various diseases, such asprostate cancer (PCa), androgen insensitivity syndrome (AIS) and spinalbulbar muscular atrophy (SBMA).8Androgens are responsible for normal physiology,development, andmetabolism of reproductive and nonreproductive systems.11 Point mutations within the AR LBD are frequentlydescribed in thisdisease.12 The AR LBD possesses a three-layersandwich fold architecture .The two AR LBD monomers in the homodimer are arranged “head-to-head”and form a large dimer interface by centering the helix 5 (H5). Nearly40 different AIS-related mutations have been reported on the AR dimerinterface.13 However, so far, how the mutationson the AR dimer interface affect the biological functions of AR remainspoorly understood.AIS is the most common cause of the 46,XY disorder in sexdevelopment.14 At the beginning, we explored the structural ensemblesof the wild type AR LBD and two AIS-associated mutants with the mutations(W751R and F754V) located at the dimer interface by molecular dynamics(MD) simulations and small-angle X-ray scattering (SAXS). The simulationand experimental results illustrate that the AR LBD homodimer canbe easily disrupted by either W751R or F754V. Therefore, we speculatedthat blocking the AR dimerization by small molecule antagonists targetingthe interface is probably favorable to combat PCa. Interestingly,an AR LBD dimer interface pocket (DIP) was discovered by our theoreticalpredictions. To prove the druggability of the DIP, an integrativestructure-based virtual screening was performed and a novel hit compoundM17 was identified. Then through a four-step structure optimization,M17-B15 with >31-fold improved inhibitory activity on AR transcriptionalfunction was discovered. M17-B15 exhibited potent anti-PCa efficacywithout detectable toxicity, and it can specifically target to ARrather than its phylogenetically related nuclear receptors. Collectively,our findings demonstrate that AR dimerization disruption by smallmolecules targeting the DIP is a novel and valid strategy to developnew classes of AR antagonists against PCa.In this study, we report the discovery processof a series of novelthiadiazole-amide-based small molecules that block the AR LBD dimerizationas our granted patent “CN113444081B”.WT)and two AIS-associated mutants with mutations located in H5 , i.e., W751R (ARW751R withchanged hydrophobicity) and F754V (ARF754V with reducedhydrophobicity and bulkiness), by molecular dynamics (MD) simulations.16 Analysis of the time evolution of the root-mean square deviations(RMSDs) of the protein backbone atoms of the ARWT dimerconfirmed that the simulated system is equilibrated after ∼260ns  and the two monomers (monoA and mono B) are stable . However, the RMSDs for the ARW751R and ARF754V dimers are both dramatically fluctuant, though those for each monomerare stable . The alignmentof the initial structure (magenta) and the last snapshot (blue) extractedfrom the MD trajectory for the ARWT dimer shows a highstructural similarity with a RMSD of 2.24 Å (Movie S1), while those for the ARW751R and ARF754V dimers exhibit significant structural differences with RMSDs of11.94 and 14.86 Å, respectively (Movies S2–3). Based on the theoreticalpredictions, the mutations disrupt the AR LBD dimerization and leadto self-dissociation.We exploredthe structural ensembles of the wild type AR LBD  of the ARW751R or ARF754V mutant bound with DHT is much smaller than that of ARWT, suggesting a compaction of the protein system. As each AR monomeris rigid, the compaction can only be explained by the emergence ofthe AR LBD monomer. In addition, Dmax ofARW751R is almost identical to that of ARF754V, suggesting that neither ARW751R nor ARF754V stably exists in the form of a homodimer. Considering that the ARdimerization is essential for AR transcriptional activity, blockingthe AR dimerization by small-molecule antagonists targeting the dimerinterface can be a powerful way to combat PCa.The predictions were then confirmed by the small-angleX-ray scattering(SAXS) technique, which gives the overall dimension and shape silhouetteof the biomacromolecule. As shown in 17 Novel AR antagoniststoward the DIP are expected to offer a new approach to treat PCa.We thenanalyzed the dimer interface and successfully discovered a potentialsmall-molecule binding pocket located at each LBD monomer as illustratedin Figure S3A) and androgen-dependent cell line LNCaP viability . The serum level of prostate-specificantigen (PSA) is usually used for detecting and monitoring the PCaprogression and treatment response.18 Herein,the secreted PSA from LNCaP cells was evaluated, and the result showsthat M17 dose-dependently inhibits the secretion of PSA . Furthermore, the binding of M17to the conventional LBP was excluded by a competitive ligand-bindingassay . These results promptedus to investigate the cell viabilities of androgen-independent celllines  upon the treatment of M17.As shown in Figures S3E–H, M17 exhibitscomparable effects with the marketed second-generation AR antagonistenzalutamide (Enz) on the proliferation of the four cell lines. Inaddition, the cytotoxicity of M17 to a normal cell line murine embryonicfibroblast (NIH-3T3) was determined and both M17 and Enz show negligiblecytotoxicity against NIH-3T3 . These data support that M17 targets AR but not the LBP with highsafety profile, and it is worth chemical optimization.Next, the structure-based virtual screening (SBVS) procedure wasemployed toward the DIP 1F. ThrouTable S1. The AR antagonistic activities weredecreased or totally lost when methylfuran (A ring) was replaced bydifferent groups (M17-A2 and M17-A2). We also examined the role ofthe benzene ring (B ring) in 3,4-dichlorophenyl. The compound withphenyl (M17-B1) exhibits optimal AR antagonistic activity comparedwith the rest of the compounds (M17-B2 ∼ M17-B4). Subsequently,we designed compounds with various substituents at different positionsof phenyl (M17-B5 ∼ M17-B11) and found that the meta-substitutedcompound M17-B10 is more potent. We thus designed compounds with differentsubstituents at the meta-position of the phenyl (M17-B12 ∼M17-B23), which led to the discovery of the potent compound M17-B15(IC50 = 0.03 μM). This compound achieved >31-foldimprovement in AR transcriptional inhibition compared with the originalhit M17  of 1.32 × 10–5 M. Similar to M17, M17-B15 does not target the LBP as indicatedby the competitive ligand-binding assay .19 The ensemble refinement against the CXMS restraints indicatesthat 7 conformers in the ensemble can satisfy all the cross-linkingpairs (Biolayer interferometry(BLI) experiments were performed to determine the binding affinityof M17-B15 to the purified AR LBD. As shown in ng assay 3B. We th M17-B15 3C. Furthngpairs 3D.By trngpairs 3E. FurthTo explore whether M17-B15 can inhibit the AR dimerizationat thecellular level, the N-terminal yellow fluorescent protein (YFP)-labeledand C-terminal cyan fluorescent protein (CFP)-labeled AR-LBD plasmidswere constructed for the acceptor-bleaching fluorescence resonanceenergy transfer microscopy (FRET) assay. After treating with 10 nMDHT, the AR LBD dimerization occurred, which induced the YFP- andCFP-labeled AR LBDs to move closer to each other, thereby exhibitinghigh FRET efficiency. However, lower FRET efficiency was observedin the presence of 10 μM M17-B15 3F, indicFigures S4A–C), and M17-B15 does not show obvious inhibitionon the cell viability of 22RV1, PC3, and Du145 . The dose–response curves forM17-B15 and Enz on LNCaP cells are quite similar, which is consistentwith their anti-AR transcriptional activities , consistent with the data from thecell viability assays.Like Enz, M17-B15shows negligible cytotoxicity against NIH-3T3, human liver cells (Chang),and human gastric epithelial cell  and ARF876L/T877A (FL-ARF876L/T877A) on M17-B15 was evaluated using the dual-luciferasereporter assay. As a comparison, a recently approved AR antagonistdarolutamide, which targets the AR LBP and could significantly inhibitthe transcriptional activity of the AR F876L and F876L/T877A mutants,was also applied.21 As shown in 50 valuesof 0.16 μM and 0.10 μM for FL-ARF876L, and0.15 μM and 0.34 μM for FL- ARF876L/T877A,respectively. In addition, M17-B15 significantly inhibits the DHT-inducedtranscriptional and translational levels of PSA , Enz (2.5 mg/kg/week),and M17-B15 (2.5 mg/kg/week) were administered to the establishedsubcutaneous xenografts of LNCaP cells. As shown in Figures S5A–C). The Venndiagram displays that there are 14 shared differentially expressedgenes , which are down-regulatedin the antagonist-treated groups of Enz and M17-B15 while up-regulatedin the DHT-treated group. The corresponding heatmap shows their relativeexpressions . Among these DEGs,4 of them have been reported and their up-regulations are linked torecurrent PCa, including KLK3 (encoding PSA), CDC20, CENPF, and MKI67.22 Therefore, we validated the vitalgenes of CDC20, CENPF, and MKI67 by qPCR. It is shown that the treatment by Enz orM17-B15 significantly reduces the expressions of all these genes comparedwith those by DHT  for abbolactone is potent at the concentrationof 0.4–10 μM, while those for Enz and M17-B15 are notobserved even at a relatively high concentration of 10 μM. Thesecompounds do not show an obvious effect on the MR agonistic activities.We analyzed the LBDs of 48 human nuclear receptors(NRs), and theirphylogenetic tree was constructed. The LBDs of progesterone receptor(PR), glucocorticoid receptor (GR), and mineralocorticoid receptor(MR) show the highest evolutionary conservation 5C. That ectively 5D. It isectively 5E–F. Simtivities5G. TheseFigure S7, with the concentrationonly as high as 50 μM, mild to moderate inhibitory effects wereobserved for either Enz (except for U2OS) or M17-B15 (except for A549).Furthermore, both M17-B15 and Enz do not show an obvious impact onthe cell cycle and apoptosis of the LNCaP cell line . The above results highlight the potential highAR specificity of M17-B15 and good druggability of the DIP.In addition, the cell viabilityinhibition of various of humancancer cell lines for Enz and M17-B15 was evaluated, including osteosarcoma(U2OS), breast cancer (MCF-7), hepatocellular carcinoma (HepG2), leukemia(HL60), non-small cell lung cancer (H1299 and A549), colorectal cancer(SW480), glioma (U87), and cervical adenocarcinoma (Hela) cell lines.As shown in Investigation of the dynamic behavior ofAIS mutations at the ARhomodimer interface illuminated the strategy of small molecules targetingthe protein interface to disrupt AR homodimerization to treat PCaand AR-related diseases. Herein, we identified a potential pocketnamed the DIP located at the dimer interface near H5, and the druggabilityof the previously unexploited pocket was validated by a novel smallmolecule M17-B15. TM17-B15 exhibits potent anti-PCa efficacy in vitroand in vivo, and it specifically targets AR toward its homologuesPR, GR, and MR. However, structural optimization is still urgentlyrequired for M17-B15, especially to the chemical modification of thesolvent-exposed moiety of 3-(2-oxopropyl)-1,2,4-thiadiazol to improveits pharmacokinetic properties. In addition, we believe that the ARDIP will inspire interesting studies on the discovery of new classesof therapeutics including novel proteolysis-targeting chimeras (PROTACs)and molecular glues for treating PCa and AR-related diseases. Moreover,the DIP is as excellent as the conventional target of the LBP in ourstudy, providing a new option for the other NRs to develop variantNR modulators. Overall, this study provided a successful paradigmfrom gain-of-function mutations to a potential druggable pocket andnew therapeutic strategies.