Methionine addiction is a fundamental and general hallmark of cancer cells, which require exogenous methionine, despite their ability to synthesize normal amounts of methionine from homocysteine. In contrast, methionine-independent normal cells do not require exogenous methionine in the presence of a methionine precursor. The methionine addiction of cancer cells is due to excess transmethylation reactions. We have previously shown that histone H3 lysine marks are over-methylated in cancer cells and the over-methylation is unstable when the cancer cells are restricted of methionine. In the present study, we show that methionine-addicted osteosarcoma cells are sensitive to both methotrexate (MTX) and recombinant methioninase (rMETase), but they affect histone H3 lysine-methylation in the opposite direction. Concentrations of MTX and rMETase, which inhibit osteosarcoma cells viability to 20%, had opposing effects on the status of histone methylation of H3K9me3 and H3K27me3. rMETase significantly decreased the amount of H3K9me3 and H3K27me3. In contrast, MTX significantly increased the amount of H3K9me and H3K27me3. The results suggest that increase or decrease in these methylated histone lysine marks is associated with proliferation arrest of methionine-addicted osteosarcoma. •Osteosarcoma cells are sensitive to both methotrexate and recombinant methioninase.•MTX increased the amount of H3K9me and H3K27me3.•RMETase decreased the amount of H3K9me3 and H3K27me3.•Increase/decrease in H3K9me3 and H3K27me3 is associated with proliferation arrest. Hoffman) . Methionl-methionine α-deamino-γ-mercapto-methane lyase (rMETase) [In addition, we previously have reported that the levels of histone H3 lysine-trimethylation were unstable in methionine-addicted cancer cells under methionine-restriction (MR) conditions [rMETase) . In contde novo purine synthesis, and 5,10-methylente-tetrahydrofolate, which is used for pyrimidine synthesis, causing inhibition of DNA synthesis.Methotrexate (MTX) is a first line therapy for osteosarcoma, which has been used for decades ,6. MTX iThe global status of the histone lysine-methylation may alter the expression of a vast number of genes . HistoneIn the present report, we show that rMETase and MTX have opposite effects on histone H3 lysine-methylation, at concentrations which inhibit proliferation of methionine-addicted osteosarcoma cells.22.1The 143B human osteosarcoma cell line was obtained from the American Type Culture Collection . Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1 IU/ml penicillin/streptomycin.2.2MTX was obtained from MedChemExpress  and dissolved in dimethyl sulfoxide (DMSO). Recombinant methioninase (rMETase)  is a homotetrameric enzyme, with a 172-kDa molecular mass. Production of rMETase was as previously reported [2.32 cells/well) in DMEM (100 μl/well) and incubated at 37 °C overnight. Cells were treated with different concentrations of MTX, between 1.25 μM and 160 μM; or rMETase, between 0.025 U/ml and 1.6 U/ml for 72h. At the end of the culture period, 10 μl of the WST-8 solution was added to each well and the plate was additionally incubated for 1 h at 37 °C. Absorption was measured with a microplate reader  at 450 nm. Drug sensitivity curves were obtained with Microsoft Excel for Mac 2016 ver. 15.52  and half-maximal inhibitory concentration (IC50) values were calculated with ImageJ ver. 1.53k . Experiments were performed twice, each in triplicate.Cell viability was assessed using the WST-8 reagent . Cells were cultured in 96-well plates  one time, and medium was changed to normal medium or medium with MTX (0.02 μM) or with METase (0.5 U/ml) and further incubated in 37 °C for 72h. The cells were lysed, and histones were extracted using a Epiquik Total Histone-Extraction Kit . Immunoblotting for these histones was performed as follows: Histone extract samples were loaded onto 12% SDS-PAGE gels and transferred to 0.2 μm polyvinylidene difluoride (PVDF) membranes. Blocking of the membranes was performed with the Bullet Blocking One for Western Blotting . Anti-H3K9me3 antibody ; anti-H3K27me3 antibody ; and anti-H3 antibody  were used. Total histone H3 was used as an internal loading control. Horseradish-peroxidase-conjugated anti-rabbit IgG  was used as a second antibody. Immunoreactivity was visualized with Clarity Western ECL Substrate . The signals were detected with the UVP ChemStudio  [The cells were cultured in 100 mm2.5All statistical analyses were performed with JMP pro ver. 15.0.0 . Tukey-Kramer HSD was performed to compare the means between 3 groups. The Dunnett test was applied to compare each of the means with the control group. Bar graphs were constructed to show the mean and error bars show standard deviation or standard error of the mean. A probability value ≤ 0.05 was defined as statistically significant.33.150: MTX (0.016 μM), and rMETase (0.28 U/ml) (Both MTX and rMETase inhibited the 143B osteosarcoma cells with the following IC28 U/ml) A and B, 28 U/ml) C.Fig. 1M3.2We evaluated the methylation status of histone H3K9me3 and H3K27me3 in osteosarcoma cells by immunoblotting, at the 20% inhibitory concentrations of rMETase or MTX. Compared to untreated control cells, rMETase decreased the amount of H3K9me3 (P = 0.04) and H3K27me3 (P < 0.001). In contrast, MTX increased the amount of H3K9me3 (P = 0.002) and H3K27me3 (P < 0.001) .Fig. 2 mouse models , and hasrMETase reduces the cell's external source of methionine. MTX reduces the cell's internal source of methionine by inhibiting dihydrofolate reductase (DHFR) and methylenetetrahydrofolate reductase (MTHFR), thereby inhibiting methionine biosynthesis from homocysteine. Our previous studies have indicated that cancer cells use external methionine and biosynthesized methionine differently ,38, whicOur current hypothesis is that the inhibition of internally-biosynthesized methionine by MTX may disrupt the methionine balance in the cancer cell, possibly allowing more external methionine to be used for histone methylation via S-adenosylmethionine (SAM). It is possible that both over-methylation and under-methylation of the two histone markers, H3K9me3 and H3K27me3, are detrimental to the proliferation of osteosarcoma cells.Future studies will focus on the mechanism of cell proliferation inhibition by rMETase and MTX, involving alteration of histone H3 lysine-methylation, including studies at the atomic/molecular level ,40.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Dictyostelium discoideum. The abundant amount of inositol hexakisphosphate  present in the amoeba allowed the discovery of the more polar inositol pyrophosphates, IP7 and IP8, possessing one or two high energy phosphoanhydride bonds, respectively. Considering the contemporary growing interest in inositol pyrophosphates, it is surprising that in recent years D. discoideum, has contributed little to our understanding of their metabolism and function. This work fulfils this lacuna, by analysing the ip6k, ppip5k and ip6k-ppip5K amoeba null strains using PAGE, 13C-NMR and CE-MS analysis. Our study reveals an inositol pyrophosphate metabolism more complex than previously thought. The amoeba Ip6k synthesizes the 4/6-IP7 in contrast to the 5-IP7 isomer synthesized by the mammalian homologue. The amoeba Ppip5k synthesizes the same 1/3-IP7 as the mammalian enzyme. In D. discoideum, the ip6k strain possesses residual amounts of IP7. The residual IP7 is also present in the ip6k-ppip5K strain, while the ppip5k single mutant shows a decrease in both IP7 and IP8 levels. This phenotype is in contrast to the increase in IP7 observable in the yeast vip1Δ strain. The presence of IP8 in ppip5k and the presence of IP7 in ip6k-ppip5K indicate the existence of an additional inositol pyrophosphate synthesizing enzyme. Additionally, we investigated the existence of a metabolic relationship between inositol pyrophosphate synthesis and inorganic polyphosphate (polyP) metabolism as observed in yeast. These studies reveal that contrary to the yeast, Ip6k and Ppip5k do not control polyP cellular level in amoeba.Initial studies on the inositol phosphates metabolism were enabled by the social amoeba  The previously characterized ip6k amoeba (Saccharomyces cerevisiae gene Kcs1 (kcs1Δ)  .6 to generate the inositol pyrophosphate IP7  that, as the name suggests, primarily phosphorylates IP7, also abbreviated to PP-IP5, IP8 or (PP)2-IP4 2-IP4 .5 (hereafter 5-IP7)   , while m1,5-IP8) .D. discoideum possesses one Ip6k gene and a Ppip5k homologous gene (see below). The similar enzymology between human, yeast and amoeba suggests a similar inositol pyrophosphate metabolism. However, this appears not to be the case since 1H,31P-NMR spectroscopy and enzymology studies 2-IP4 . studies , 1997 suin vivo   , led to  (polyP) . In thisynthesis . The SPXynthesis . Due to ynthesis . Howeveron yeast . TherefoS. cerevisiae kcs1Δ has been highly influential. In fact, D. discoideum ip6k knockout has been utilized as a proxy, using unrefined supporting data, which demonstrates that the amoeba has low polyP level  and polyP synthesis is an evolutionarily conserved feature.Like inositol pyrophosphates, polyP is also attracting a growing interest. PolyP, has been described as an important primordial chaperone able to regulate the aggregation of proteins which form in neurodegenerative disorders . HoweverD. discoideum offers the unique opportunity to address these issues since it is an excellent experimental model for both inositol pyrophosphate and polyP studies  searches using all inositol phosphate kinases found in escribed .2.23H-inositol-labeling Sax-HPLC analysis . Restriction sites were added at the 5′ SalI and 3’ NotI to cloned Ppip5K in a pADH-GST plasmid . The list of the yeast strains used in this study is given in D. discoideum lines were isogenic to the axenic strain AX2. Amoeba were cultivated at 22 °C in HL5 media, either in Petri dishes or in flasks at 120 rpm. Cells were diluted every 1–2 days to avoid confluence of dishes or when cell densities exceeded 5 × 106 cells/ml.Yeast were grown in rich  or synthetic complete medium  in the absence or presence of uracil to select for auxotrophy. For [2.5Logarithmic growing yeast grown in Sc-Ura media carrying pADH-Ppip5k or empty vector were washed and shifted in media with or without phosphate 10 μM for 3 h. Before FACS measurement, 50 μl of the yeast culture was diluted into 950 μl of TBS and immediately analysed using an LSRII flow cytometer (BD Biosciences).2.6D. discoideum AX2 and ppip5k were grown for 5–7 days in SIH media (Formedium) supplemented with 13C6-inositol (400 μM) synthesized as described  and washed once with KK2 (20 mM potassium phosphate pH 6.8). Pellets were resuspended in 40–100 μl perchloric acid (1M), incubated in ice and vortexed for 10 s every 2 min for a total period of 10 min. The extracts were centrifuged at  and the supernatants were neutralised with 1M potassium carbonate supplemented with 3 mM EDTA at 4 °C for 2 h and subsequently centrifuged. For polyP analysis cell were extracted using acidic phenol procedure . The filters were soaked in the specified buffer. Following the removal of excess liquid, cells were resuspended in 500 μl of buffer  and allowed to flow into the filter by delivery with a pipette in an outward spiral movement. Cells were harvested with a plastic scraper. Fruiting body images were then taken on an Olympus camera mounted on a dissecting microscope.Amoebas were starved by transferring cells from rapidly dividing vegetative cultures onto KK2 2% agar plates. 1 × 102.11D. discoideum extract for CE-MS analysis were prepared from vegetative growing strains in HL5 medium. Cultures (20–30 ml of 1–3 X 106 cell/ml) were spun at 500 g for 5 min; the cell pellet was washed in 1 ml KK2 and the inositol phosphate were extracted with 500 μl of perchloric acid 1M in the presence of 5 mM EDTA. The inositol phosphates in the perchloric acid extract were purified using TiO2  strain  strain . Two oth) strain . As oppo) strain , the sevynthesis . The amo3.27 and IP8 purified from amoeba were performed using two-dimensional 1H,31P-NMR  one-bond couplings is more efficient  homologue in D. discoideum encodes a 56 kDa protein, compared to the 130 kDa yeast protein and ∼150 kDa in mammalian cells  contains six poly-asparagine and one poly-glutamine repeats, which have also prevented us obtaining recombinant protein. Therefore, to test the amoeba Ppip5k activity, we cloned the gene into a yeast expression vector and transformed it into an array of S. cerevisiae mutants  developed by Thomas and O'Shea that expresses GFP under the control of the PHO84 promoter ,,D. disco1,5-IP8  reduction in IP8 levels while a 14.3 ± 6.7% (n = 4) reduction in the level of IP7 is recorded  isogenic to our AX2 background. Identically to the previously generated ip6k mutant, the new strain has no detectable level of IP8 and an almost completely depleted level of IP7  was excised by overexpressing a recombinant Cre  analysis . This se7 and 4/6,5-IP8 to be the major inositol pyrophosphates species, confirming the 13C-NMR studies  responsible for 5-IP7 synthesis and that together with the Ip6k would generate 4/6,5-IP8. D. discoideum inositol pyrophosphate metabolism revealed by these analyses where Dpk3 represents an as-yet uncharacterised kinase capable of producing inositol pyrophosphates. The presence of three IP7 isomers suggests the possibility for amoeba to synthesize three IP8 species however only 4/6,5-IP8 is detectable in our current CE-MS-qTOF experimental setup.Surprisingly, the analysis of  species A. Thus, 3.7ip6k, ppip5k, and ip6k-ppip5k could influences directly or indirectly phosphate homeostasis in D. discoidem. The social amoeba possess sub-millimolar concentration of the phosphate-rich IP6, IP7, and IP8 thus changing their concentration influences a large pool of cellular phosphate. Alternatively, inositol pyrophosphate could influence phosphate availability by regulating polyP metabolism, particularly as the primary function of polyP is to buffer cellular free phosphate concentration. We previously demonstrated that in S. cerevisiae polyP synthesis is under Kcs1 (the IP6K) control (S. pombe it is Asp1/Vip1 (the PPIP5K) that regulates polyP metabolism  grant MR/T028904/1, and by the European Union's Horizon 2020 research and innovation program under the 10.13039/100010665Marie Skłodowska-Curie Grant agreement PHEMDD 752903. This study was supported by the 10.13039/501100001659Deutsche Forschungsgemeinschaft (DFG) under Germany's Excellence Strategy . D.Q. gratefully acknowledges financial support from the Brigitte-Schlieben-Lange-Programm. R.K.H. gratefully acknowledges funding from the 10.13039/501100001664Leibniz-Gemeinschaft (SAW-2017-FMP-1).This work was supported by the The authors declare none conflict of interest. The funding bodies do not have any role in the study design, and in data collection and analysis.


Magnolia sieboldii K. Koch is endemic to China and has high medicinal and ornamental values. However, its seed exhibits morphophysiological dormancy, and the molecular mechanisms of which are not clearly understood. To reveal the regulation mechanism of the ABA signal in seed dormancy, the M. sieboldii ABA receptor Pyrabactin Resistance 1 (PYR1) gene was cloned and analyzed. Analysis of the MsPYR1 sequence analysis showed that the full-length cDNA contained a complete open reading frame of 987 bp and encoded a predicted protein of 204 amino acid residues. The protein had a relative molecular weight of 22.661 kDa and theoretical isoelectric point of 5.01. The transcript levels of MsPYR1 were immediately upregulated at 16 DAI and then decreased at 40 DAI. The highest transcript level of MsPYR1 was found in the dry seeds, indicating that the MsPYR1 gene may play an important role in the regulation of dormancy. The MsPYR1 gene cDNA was successfully expressed in E. coli Rosetta (DE3), and the protein bands were consistent with the prediction. The Anti-MsPYR1antibody could detect the expression of MsPYR1 in M. sieboldii. The results provided a foundation for further study of the function of the MsPYR1 gene.ABBREVIATIONSABA: Abscisic acid; MPD: morphophysiological; PYR1: Pyrabactin Resistance1; PYL: Pyr1-Like; RCAR: Regulatory Components of Aba Receptors; PP2C: protein phosphatases 2C; SnRK2: sucrose non-fermenting1-related protein kinase2; DAI: day after imbibition; NCBI: National Center for Biotechnology Information; BCA: Bicinchoninic acid; CDD: Conserved Domains.  Magnolia sieboldii K. Koch is widely distributed in China, Korea, and Japan. M. sieboldii has high ornamental and medicinal values ) for 15 min and rinsed thrice with sterilized distilled water. The seeds were allowed to imbibe different treatments, i.e., 10 mM ABA and water, in Petri dishes at 20°C for 48 h. Each treatment had three biological replicates of 100 seeds per Petri dish. After imbibition, the seeds were cold stratified in the dark at 4°C. Seed samples were collected at 0 h imbibition , 0 day after imbibition (DAI) and every 8th DAI then stored at −80°C for RNA extraction.TM Reverse Transcription System (Promega).The five key time points for seed germination were as follows: dry seed, 0 DAI, 16 DAI, 40 DAI, and 56 DAI. The seeds were collected at these time points and used for total RNA extraction. The RNA was extracted using the RNA Simple Total RNA Kit  [PYR1 (PYR1 clone was amplified using PrimeSTAR® HS DNA Polymerase (TaKaRa). RT-PCR reaction system was used in a 50 μL of reaction mixture including PrimeSTAR HS Taq (25 μL), primers (2 μL), cDNA template (1 μL), and ddH2O. The RT-PCR reaction conditions were as follows: 30 s at 98°C; 30 cycles of 10 s at 98°C, 15 s at 58°C, 1 min at 72°C; and a 10 min extension at 72°C. The PCR fragments were connected into the pMD18-T vector (TaKaRa) and sequenced by TaKaRa Co., Ltd.The full length of the primers of PYR1  was desi2O (6.4 μL). The qRT-PCR reaction conditions were as follows: 30 s at 95°C; and 40 cycles of 5 s at 95°C, 20 s at 60°C. The data were analyzed using the 2−ΔΔCt method described by Zhang et al [The qRT-PCR procedure was established using the qTOWER 2.0/2.2 Real Time PCR Systems (USA). All primers are listed in ng et al .http://www.cbs.dtu.dk/services/TMHMM/). The structural regions were predicted using the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The protein motif was conducted by using the Simple Modular Architecture Research Tool (SMART) (http://smart.embl-heidelberg.de/). The phylogenetic tree was constructed using Clustalx2.1 and MEGA7.0 [The open reading frame (ORF) and sequence alignment were analyzed using DNAMAN version 6.0. The transmembrane regions were predicted using TMHMM 2.0 ( MEGA7.0 .E. coli Rosetta (DE3). The positive clones were screened by PCR with pET28a-F and pET28a-R as primers  protein was determined, two New Zealand rabbits (2–2.5 kg) were injected subcutaneously with 400 µg, immunized once at 1.5 weeks and the process was performed four times. Blood samples were collected for detection, and the titer of the antiserum against PYR1 was determined by indirect ELISA. The titer was greater than 1:500,000, and the final blood samples were collected to prepare the antiserum. Western blot analysis was performed as described . After 1PYR1 cDNA was obtained from M. sieboldii seed by RT-PCR. The length of the PCR product contained a complete ORF of 615 bp and encoded a predicted protein of 204 amino acid residues , and the lowest content was Trp (0.5%) .Table 2.53th to Glu194th (The conserved domain of the MsPYR1 protein was predicted by Conserved Domains (CDD) in the NCBI. The MsPYR1 protein had a structural domain of the PYR/PYL/RACR protein family, which in turn belonged to the START/RHO_alpha_C/PITP/Bet_v1/CoxG/CalC superfamily and contained putative hydrophobic ligand binding sites, protein interfaces, and gate structures . The SMAGlu194th . The thrGlu194th .Figure 4MsPYR1 at different stratification stages  cell. After induction with 0.5 mM IPTG at 37°C for 2 h, 12% SDS-PAGE electrophoresis was performed (E. coli Rosetta (DE3) is frequently used for the high expression of eukaryotic or prokaryotic proteins [The recombinant plasmid PET28A-MSPYR1 was transformed into the erformed . The resproteins ,27.FigurE. coli Rosetta (DE3) strain had a low expression level of the recombinant proteins as inclusion bodies (The protein was loaded into a Ni-IDA affinity column to bind the polyhistidine-tagged recombinant MsPYR1. The purity of MsPYR1 eluted from the first Ni-IDA affinity column with 20 mM imidazole was approximately 20%. After purification of the second Ni-IDA affinity column with 250 mM imidazole, MsPYR1 was approximately 80%, which was exhibited as a single band on the SDS–PAGE with the molecular weight of 26 kDa . The expn bodies .Figure 7Anti-MsPYR1 antibodies are used as a convenient detection tool to bind/recognize the target proteins with higher stability and affinity. The presence of a cross-reacting protein appeared at the approximate size of 26 kDa in the purification of recombinant proteins and seed protein during the immunoblot analysis . When thMsPYR1 was cloned and successfully expressed in E. coli. The expression pattern of MsPYR1 at different stratification stages was analyzed using qPCR. The highest transcript level of MsPYR1 was found in the dry seeds. The pure recombinant MsPYR1 protein was successfully obtained. The Anti-MsPYR1 antibody could detect the expression of MsPYR1 in M. sieboldii. The results provided a foundation for further study of the function of the MsPYR1 gene. The results of present study provide valuable information for further functional studies of MsPYR1 gene.In the present study, an ABA receptor gene,


Chryseobacterium aquifrigidense FANN1 (NCBI: MW169027) was used to produce keratinase, and its biochemical properties were determined. The optimization of physicochemical parameters and analysis of the free amino acid constituents of the feather hydrolysate were also carried out. FANN1 showed a maximum keratinase yield of 1,664.55 ± 42.43 U/mL after 72 h, at optimal process conditions that included initial medium pH, incubation temperature, inoculum size, and chicken feather concentration of 8, 30°C, 4% (v/v), and 15 (g/L), respectively. Analysis of degradation product showed 50.32% and 23.25% as the protein value and total free amino acids, respectively, with a relatively high abundance of arginine (2.25%) and serine (2.03%). FANN1 keratinase was optimally active at pH 8.0 and relatively moderate to high temperature (40–50°C). EDTA and 1,10-phenanthroline inhibited the keratinase activity, and that suggests a metallo-keratinase. The enzyme showed remarkable stability in the presence of chemical agents, with residual activity 141 ± 10.38%, 98 ± 0.43%, 111 ± 1.73%, 124 ± 0.87%, 104 ± 3.89%, 107 ± 7.79%, and 112 ± 0.86% against DTT, H2O2, DMSO, acetonitrile, triton X-100, tween-80, and SDS, respectively. The residual activity of FANN1 keratinase was enhanced by Sunlight (129%), Ariel (116%), MAQ (151%), and Surf (143%) compared to the control after 60 min preincubation. Likewise, the enzyme was remarkably stable in the presence Fe3+ (120 ± 5.06%), Ca2+ (100 ± 10.33%), Na+ (122 ± 2.95%), Al3+ (106 ± 10.33%); while Co2+ (68 ± 8.22%) and Fe2+ (51 ± 8.43%) elicited the most repressive effect on keratinase activity. The findings suggest that C. aquifrigidense FANN1 is a potential candidate for keratinous wastes bio-recycling, and the associated keratinase has a good prospect for application in detergent formulation.Microbial keratinases’ versatility in the beneficiation of keratinous waste biomass into high-value products prompts their application in diverse spheres hence, advancing green technology and the bioeconomy. Consequently, a feather-degrading  In recent years, investment increase in the agro-industrial sector is evident for food security sustenance for the teeming population. Consequently, the vast tons of agro-wastes generated from this economic sector have been a global concern due to their associated environmental challenges . Non-fooThe chicken feather beneficiation process consequent to the rich keratin content includes conversion into feather meals for animal feed supplementation . HoweverBacillus sp., Streptomyces sp., Arthrobacter sp., and Chryseobacterium sp. from diverse ecological niches  based on 16S rRNA gene sequencing with a set of universal oligonucleotides - 27f (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492r (5′-CGGTTACCTTGTTACGACTT-3′) (The chicken feather-degrading bacterium used for the study was isolated from a soil sample collected from a poultry dumpsite (Unpublished). The isolate identity was confirmed as CTT-3′)  and phyl2HPO4, 0.4 g KH2PO4, 0.2 g MgCl2, 0.22 g CaCl2 in 1 L of distilled water. BSM (50 ml) was dispensed in 250 ml Erlenmeyer flasks and was supplemented with 15 g/L of pulverized chicken feathers (PCF). The keratinous substrate–PCF was prepared as described previously , containing 0.3 g Keviously . The flaThe keratinase activity assay followed the method described by The total protein content of the crude extract was quantified following Bradford protocols using bovine serum albumin as the standard protein . In addiThe significant process variables were studied using one variable at a time (OVAT) method. The initial medium pH and incubation temperature were varied from 3.0–9.0 and 25–50°C at intervals of 1 unit and 5°C, respectively. The effect of inoculum size on keratinase production was determined using the standardized bacterial suspension at a range of 1–7% , while the chicken feather concentration was varied from 5 g/L to 35 g/L  to establish the best concentration that would enhance extracellular keratinase production.C. aquifrigidense FANN1 was studied. The fermentation was carried out in 500 ml Erlenmeyer flasks with a working volume of 100 ml BSM at the optimal process variables. Aliquots were aseptically withdrawn at intervals (24 h) to determine various parameters tested.After establishing the optimal process variables, the time course profile of keratinolytic activity of The feather hydrolysate extracted from the culture broth by centrifugation was freeze-dried in a vacuum concentrator . The lyophilized powder was used to determine the free amino acid composition by pre-column derivatization, high-performance liquid chromatography (HPLC) separation, and quantification as previously described .Keratinase activity was determined over a pH range of 5.0–11.0 with the following buffer solutions (100 mM): sodium citrate; pH 5.0, potassium phosphate; pH 6.0–7.0, Tris-HCl; pH 8.0–9.0 and glycine-NaOH; pH 10.0–11.0. After that, the enzyme’s pH stability was evaluated by pre-incubating the enzyme with buffer solutions with pH 8.0 and pH 9.0 at 30°C for 4 h. Aliquots were withdrawn at 30 min intervals, and the residual activity was determined under the standard assay conditions. The enzyme activity determined without preincubation served as the control.The effect of temperature on keratinase activity was determined by carrying out standard keratinase activity assays at different temperatures ranging from 30°C to 80°C. In addition, the thermostability of the keratinolytic enzyme was evaluated by preheating the enzyme solution at 40°C and 50°C for 2 h. Then, aliquots were withdrawn at intervals of 30 min to determine the residual enzyme activity under the standard assay conditions.The enzyme solution was pre-incubated with protease inhibitors, including phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline at 5 mM. Similarly, 5 mM of dithiothreitol (DTT), sodium dodecyl sulfate (SDS), and 1% (v/v) of hydrogen peroxide, acetonitrile, dimethyl sulfoxide (DMSO), triton X-100, tween-80 were preincubated with an admixture of enzyme solution for 1 h at 30°C. Post-residual activity determination was carried out under the standard assay protocols.2+, Co2+, Fe3+, K+, Ca2+, Mn2+, Fe2+, Cu2+, Zn2+, Ba2+, and Al3+. The chloride salts of these metal ions were pre-incubated with the keratinolytic protease at 5 mM for 1 h. After incubation, the residual activity was determined under the standard assay conditions. The enzyme solution preincubated with distilled water served as the control.A group of metal ions used to evaluate the enzyme stability included MgThe effect of selected laundry detergents on keratinolytic protease stability was studied using the following commercial laundry detergent, including Sunlight, Omo, Ariel, MAQ, Surf, Sky, and Pro wash. The detergents were solubilized in tap water and mixed with the enzyme solution with a final concentration of 0.7% (w/v). The enzyme contents of the detergents were inactivated by heating at 100°C for 30 min prior to mixing with keratinolytic protease. The detergent-enzyme solutions were pre-incubated for 1 h at 30°C, and aliquots were withdrawn at 30 min intervals; then, residual activity was determined under the standard assay conditions. The enzyme solution preincubated with tap water along with the test solutions served as the control experiment.p < 0.05.The analysis of datasets generated from triplicate experiments was conducted in the Statistical Package for the Social Science (SPSS) version 23, and the results were presented as the mean and standard deviation. The statistical difference was compared at C. aquifrigidense FANN1 in BSM. The pH optimization study showed that keratinase was expressed by FANN1 at pH 3.0 (606.36 ± 29.56 U/mL), reaching optimum at pH 6.0, with the keratinase activity of 898.18 ± 59.14 U/mL , with a relatively higher concentration of arginine (2.25%) and serine (2.03%). Other amino acids in considerable concentration include glutamic acid (1.65%), glycine (1.93%), proline (1.69%), valine (1.67%), and leucine (1.71%), as shown in The analysis of the hydrolysate  generated from chicken feathers degradation by keratinolytic C. aquifrigidense FANN1 displayed the highest catalytic efficiency at pH 8  and Al3+ (106 ± 10.33%). Conversely, it showed varying degrees of activity loss in the presence of other metal ions evaluated (2+ (51 ± 8.43%).Enzyme stability studied in the presence of metal ions  showed that keratinase from ectively . The enzvaluated , with thThe impact of selected solid laundry detergents on keratinase stability was investigated. The results showed that keratinolytic protease displayed varying degrees of catalytic stability after 30 min of preincubation with the detergents . The enzC. aquifrigidense FANN1 that displayed efficient degradation of intact chicken feathers was used for this study. The construction of optimal fermentation conditions identified weakly acidic conditions as the best pH for maximum keratinase production by C. aquifrigidense FANN1. The effect of medium pH on the biosynthetic pathway of microbial secretory proteins has not been fully elucidated. However, it has been established that the interaction between microbial cells and their microenvironment fundamentally regulates various subcompartments of the secretory pathways , including Chryseobacterium sp. kr6  ion caused more inhibition of the catalytic performance of the enzyme than other heavy and transition metal ions used. A comparable pattern of enzyme activity inhibition at a similar concentration of the metal ions has been reported for metallo-keratinases in other studies (The keratinolytic protease displayed residual activity that varied among the respective metal ions tested, with the most stimulatory effect was obtained for Fe3+ and Na+, while the repressive effect was recorded against Znproperty . Therefo studies .C. aquifrigidense FANN1 keratinase generally exhibited a high degree of tolerance to the various detergents tested. The endogenous protease of detergents shows inconsistent stability due to variations in ingredients and concentrations among different detergent brands (Arthrobacter sp. KFS-1 keratinase in a similar detergent panel (C. aquifrigidense FANN1 keratinase is worthy of further investigation for potential applicability in the detergent formulation.Keratinolytic peptidase inclusion in the detergent formulation has significantly improved the washing performance of detergents than other classical proteases as it promotes the destaining of both soluble and insoluble proteinaceous stains while withstanding the influences of harsh washing conditions and detergent ingredients . The C. t brands . The resnt panel . The detC. aquifrigidense FANN1 isolated from a local poultry dumpsite was used to produce keratinase in a medium constituted with chicken feathers as the sole carbon and nitrogen source. FANN1 showed maximal keratinase titre after 72 h at optimized physicochemical conditions. The feather hydrolysate analysis showed that free amino acids were liberated from feather keratin, which underpins the isolate’s prowess in the valorization of keratin-rich agroindustrial residues. The enzyme was optimally active at pH 8.0 and 40–50°C. The sensitivity of the enzyme to protease inhibitors suggested a metallo-keratinase, with a remarkable tolerance of surfactants, organic solvents, reducing and oxidizing agents. Also, FANN1 keratinolytic protease showed excellent residual activity post-incubation with various commercially available laundry detergents, which indicates its significance as a potential bio-additive for detergent formulation. Thus, the keratinolytic propensity of C. aquifrigidense FANN1 is promising for bio-innovative developments. Prospectively, whole-genome sequencing and mining of keratinolytic determinants would be crucial to exploiting the bacterium further. Therefore, cloning, heterologous expression, and molecular optimization of the keratinolytic determinants are promising to enhance the suitability and versatility of the keratinolytic protease in a vast array of industrial and biotechnological developments.In conclusion, keratinolytic


ARID1A-deficient cancer cells exhibited elevated PDL1 expression. However, the aberrations in these molecules have not been studied in this population especially in Gastric Cancer (GC). In this backdrop we aimed to investigate the role of the ARID1A mutation and expression of ARID1A, TP53 and PDL1 genes in the etiopathogenesis of Gastric Cancer (GC) in the ethnic Kashmiri population (North India). The study included 103 histologically confirmed GC cases. The mutations, if any, in exon-9 of ARID1A gene was analysed by Polymerase Chain Reaction (PCR) followed by Sanger sequencing. The mRNA expression of the ARID1A, TP53 and PDL1 genes was analysed by Quantitative real time-PCR (qRT-PCR). We identified a nonsense mutation  in exon-9 among two GC patients (∼2.0%), which introduces a premature stop codon at protein position 1073. The mRNA expression of the ARID1A, TP53 and PDL1 gene was significantly reduced in 25.3% and elevated in 47.6 and 39.8% of GC cases respectively with a mean fold change of 0.63, 2.93 and 2.43. The data revealed that reduced mRNA expression of ARID1A and elevated mRNA expression of TP53 and PDL1 was significantly associated with the high-grade and advanced stage of cancer. Our study proposes that ARAD1A under-expression and overexpression of TP53 and PDL1 might be crucial for tumor progression with TP53 and PDL1 acting synergistically.AT-rich interactive domain-containing protein 1A (ARID1A), TP53 and programmed cell death-ligand 1 (PDL1) are involved in several protein interactions that regulate the expression of various cancer-related genes involved in the progression of the cell cycle, cell proliferation, DNA repair, and apoptosis. In addition, gene expression analysis identified some common downstream targets of ARID1A and TP53. It has been established that tumors formed by  Gastric cancer (GC) is prevalent and account for a large number of cancer deaths globally. Although there are considerable advances in cancer diagnosis and therapy, GC remains an important cancer worldwide and is responsible for over one million new cases in 2020 and an estimated 769,000 deaths, ranking fifth for incidence and fourth for mortality globally . In KashARID1A gene encodes a component of the switch/sucrose non-fermentable (SWI-SNF) chromatin remodelling complex [ARID1A gene expression is down-regulated in S and G2/M phases and is up-regulated in the G0/G1 phase, which supports the role of ARID1A at the G1 checkpoint for the proper arrest of cell cycle progression [via regulating the downstream transcriptional activity of several proto-oncogenes and tumor suppressor genes (TSGs) [ARID1A gene possesses a high frequency of somatic mutation in several types of malignancies leading to reduced or loss of expression, which in turn exhibits a positive correlation with tumorigenicity [ARID1A so far. Loss of ARID1A expression was reported in 11–51.3% of GCs and related to poor clinical parameters and shorter survival of GC patients [Multiple genetic and epigenetic alterations in oncogenes and tumor suppressor genes are involved in the process of gastric carcinogenesis . The ARI complex . ARID1A  complex . ARID1A gression . ARID1A s (TSGs) . ARID1A genicity , 8. Limipatients -10.TP53 is a well-studied tumor suppressor gene that plays a key role in regulating the cell cycle. It is a principal mediator of growth arrest, senescence and apoptosis in response to a broad array of cellular damage [TP53, thereby suggesting that ARID1A plays important roles in tumor suppression [TP53 are negatively associated with ARID1A loss [r damage . Interespression , 12. SomD1A loss .PDL1 was overexpressed in various cancers including GC [PDL1, a molecule able to down-regulate immune response, is thought to play an important role in the persistence of chronic infections and evasion of immune destruction by tumor cells . PD1 actuding GC -18. Howeuding GC . It was uding GC -18.ARID1A along with expression of TP53 and PDL1 genes in GC. Furthermore, we also correlate the expression with various clinicopathological parameters to have an idea about the role of ARID1A in the genesis of GC.At present, the outcome for patients with advanced GC is still bleak . EffortsThis was a cross-sectional study conducted by the Department of Biochemistry and General Surgery, Government Medical College Srinagar and Associated Shri Maharaja Hari Singh (SMHS) and Superspeciality Hospital, Srinagar, Kashmir, J&K, India.RNA-later  at 4°C overnight, to allow the solution to thoroughly penetrate the tissue before stored at −80°C to prevent any degradation until RNA isolation.The study included histopathologically confirmed 103 Gastric tumor tissue samples along with their adjacent normal tissue collected from the Department of Surgery, Shri Maharaja Hari Singh (SMHS) Hospital, Srinagar from March 2017 to March 2020. Adjacent normal tissue contained normal gastric mucosa present near the margins of resection, away from the tumor. The clinicopathological information of the patients was obtained from the Medical Records Department of hospital. All the GC cases were newly diagnosed intestinal type adenocarcinomas and did not receive any chemo or radiotherapy. The included GC cases were not having any other type of tumour and were free from any genetic disorder. One aliquot of tissue sample was snap-frozen immediately and stored at −80°C till further processing for DNA analysis. Another aliquot of tissue sample was immediately stored in QIAamp DNA Mini kit  according to the given protocol. The quality of the DNA was verified using 1% agarose gel electrophoresis. The concentration and purity of DNA was measured using NanoDrop 2000c Spectrophotometer . The DNA samples of high molecular weight, without any fragmentation/shearing, with OD (260/280) ratio between 1.8 and 1.9 were processed for further molecular analysis.DNA was extracted from tissue samples using ARID1A gene was amplified using primers: forward: 5′CACAGCACTATTTGGCTCCAG-3′; reverse: 5′-ATCATCTCTGGGCTGGCTG-3′ . The PCR amplification was carried out in a 50 µl volume containing 12.5 µl of 2X PCR master mix , 0.2 µM of each forward and reverse primers, 50–150 ng of genomic DNA. After initial denaturation at 94°C for 7 min, 35 cycles of denaturation at 94°C for 20 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s were performed. The final extension was given at 72°C for 7 min. The 343 bp amplified product was verified on 2.5% agarose gel and visualized on Omega Lum G Gel Documentation centre .Exon-9 of ABI prism 310 automated DNA sequencer  via Sanger dideoxy method.The amplified samples were sequenced, using NanoDrop 2000c Spectrophotometer . RNA having A260/A280 between 1.8 and 2.0 was considered as “Uncontaminated.” Furthermore, RNA integrity was also verified by the presence of 28S, 18S and 5S rRNA bands on 1.5% agarose gel. The first strand cDNA was synthesized with DNase-treated RNA; 1–2 µg RNA was reverse transcribed into cDNA using RevertAid First Strand cDNA Synthesis Kit , according to the manufacturer’s instructions. The reactions were incubated for 60 min at 37°C followed by 95°C for 10 min.Total RNA was extracted from tissue samples using TRIzol reagent . The concentration and purity of RNA was measured using 7500 Real-Time PCR system . The primers sequences used were; ARID1A forward 5′-CTTCAACCTCAGTCAGCTCCCA-3′, ARID1A reverse 5′-GGTCACCCACCTCATACTCCTTT-3′; TP53 forward 5′-TGCGTGTGGAGTATTTGGATG-3′, TP53 reverse 5′-TGGTACAGTCAGAGCCAACCTC-3′; PDL1 forward 5′-ACTGGCATTTGCTGAACGCA-3′, PDL1 reverse 5′- AGACAATTAGTGCAGCCAGGTCT-3′; GAPDH forward 5′- CTCCTCCTGTTCGACAGTCAGC-3; GAPDH reverse, 5′- CCCAATACGACCAAATCCGTT-3′ [GAPDH (housekeeping gene) was used as an internal control. The PCR reaction mixture contained 10 µl of KAPA SYBR® FAST master mix , 0.5 µl of cDNA of each sample, 0.2 µM of each forward and reverse primers in a final volume of 20 µl adjusted with Milli-Q water. The reaction mix was preheated at 95°C for 10 min and then amplified with 40 cycles at 95°C for 30 s, X°C for 1 min  and 72°C for 35 s. Specificity of the PCR products was determined by Melting curve analysis. All the samples were run in triplicates. The mRNA expression of ARID1A was defined on the basis of Ct (cycle threshold) value of each sample. The relative expression was calculated by the 2-∆∆Ct method [qRT-PCR was performed using CGTT-3′ , 21–22. t method .ARID1A, TP53, PDL1 alterations and various sociodemographic and clinicopathological characteristics was evaluated by Pearson’s χ2 test or Fisher’s exact test for discrete variables; paired t-test for continuous variables using multiple logistic regression analysis. The odds ratios (ORs) with 95% confidence intervals (CIs) was calculated. Two-sided p ≤ 0.05 was considered statistically significant.Data analysis was performed using SPSS software V 23.0 . The association between 2 of cases was 24.85 ± 4.45, while as the mean CEA levels of GC cases was 6.07 ± 2.04 ng/ml 57.3% of GC cases were non-smokers and 42.7% were smokers. H. Pylori status was positive in 36.9% (38 of 103) of GC patients. The detailed socio-demographic and clinicopathological parameters of GC patients are given in Out of all GC patients taken for the study 59.3% (61 of 103) were males whereas 40.7% (42 of 103) were females. The mean age (in years) of GC cases was 56.6 ± 12.1. The mean BMI in kg/mARID1A gene, we detected a nonsense mutation (c. 3219 C > T) at amino acid position 1073, among 02 out of 103 (∼2.0%) GC tumor tissues samples leading to formation of stop codon (CAG to TAG). Mutation was not found in any of the adjacent normal tissue samples. ARID1A gene.In the present study, Gastric tumor tissue samples were screened along with their adjacent non-tumor tissue samples for the presence of mutations, if any. On DNA sequencing of amplified exon-9 of ARID1A, TP53 and PDL1 mRNA expression in 103 Gastric tumor tissues and their adjacent normal tissue samples. The melt curve analysis showed zero formation of any non-specific products. ARID1A, TP53 and PDL1 in GC cases in terms of their ΔCt values. There was a significant decrease in the mRNA expression of ARID1A in Gastric tumor tissues compared to adjacent normal tissues  of GC patients (TP53 and PDL1 was increased in 47.6% (49 of 103) and 39.8% (41 of 103) of GC patients respectively . A higher percentage (37.9%) of GC patients with poorly differentiated disease were having reduced ARID1A mRNA expression compared to GC patients with well differentiated disease (18.2%) .The stratification of ARID1A mRNA levels with respect to various socio-demographic and clinicopathological parameters of GC patients is given in TP53 mRNA levels compared to GC patients having normal BMI and elevated TP53 mRNA levels . Among GC cases without H. Pylori infection 35.4% (23/65) were having TP53 mRNA overexpression compared to GC patients with H. Pylori infection among which 68.4% (26/38) were having TP53 overexpression . Among GC cases having stage I & II disease only 38.0% were having reduced TP53 mRNA expression compared to 68.8% patients with stage III&IV disease and elevated TP53 mRNA expression . A higher percentage of GC patients with poorly differentiated disease were having elevated TP53 mRNA expression compared to GC patients with well differentiated disease .The stratification of TP53 mRNA levels with respect to various socio-demographic and clinicopathological parameters of GC patients is given in p = 0.00). Among GC cases with well differentiated disease only 21.9% (14/66) were having PDL1 mRNA overexpression compared to GC patients with poorly differentiated disease among which 69.2% (27/37) were having PDL1 overexpression .The stratification of PDL1 mRNA levels with respect to various socio-demographic and clinicopathological parameters of GC patients is given in ARID1A mRNA underexpression and mRNA overexpression of TP53 and PDL1. Only TP53 overexpression and PDL1 overexpression were significantly associated with each other and the correlation was positive  among two (∼2.0%) GC patients that introduces a premature stop codon leading to the abortive termination of the ARID1A protein, thereby resulting in its complete or partial inactivation and reduced or loss of protein function. Mamo et al. previously reported nonsense mutation in exon-9, which introduces the premature stop codon into coding sequence at position W1073 [ARID1A gene varies between 8 and 39% in gastrointestinal cancers [ARID1A mutations were more frequent in Gastric tumors, especially with microsatellite instability and Epstein-Barr virus infection [ARID1A, resulting in loss of its functional protein, consequently leads to the activation of the PI3K/AKT pathway that promotes several mechanisms responsible for carcinogenesis, including cell proliferation, inhibition of apoptosis, cell adhesion, and transformation [ARID1A increases phosphorylations of AKT and promotes cell division and metastasis [ cancers , 10, 26.on W1073 . Furtheron W1073 , 28. How cancers  and betwnfection . The nonormation , 30. Sevtastasis , 30.ARID1A was under-expressed at mRNA level in 25.6% of GC cases with an average fold change of 0.63. Consistent with our study, Yang et al. found that the expressional loss of ARID1A was frequent in 30% of GC patients and has a significant correlation with poor survival and prognosis [et al. revealed that ARID1A was significantly lower in 65.15 and 52% of GC patients at mRNA and protein level respectively [ARID1A expression in clear cell and endometrioid ovarian carcinoma [ARID1A expression in ovarian cancer (29 vs. 18%) [ARID1A has been shown to trigger the initiation and progression of carcinogenesis in numerous types of cancers, including ovarian, breast, endometrial and cervical, breast, gastric, and colorectal cancers through several mechanisms that have not been fully elucidated [in vitro successfully inhibits the uncontrolled cell division [ARID1A expression in patients with the high-grade and advanced stage of the GC. Consistent with our findings, some investigations also reported that the reduced expression of the ARID1A has a significant association with higher grading and staging of GC [ARID1A has a significant association with age, gender, tumor location, and tumor size [ARID1A were low in the two GC patients with ARID1A c.3219 C > T mutation. Consistent with our study, Mamo et al. also reported that RNA levels of ARID1A were found very low in samples with nonsense mutations [Our result revealed that rognosis . Howeverectively . Previouarcinoma . Additiovs. 18%) . The expucidated . Studiesdivision . Chan-ondivision . We furtng of GC . Howeverng of GC . Althougmor size . Our stuutations . The intutations .TP53 in suppressing tumour growth is primarily due to its induction of cell cycle arrest and DNA repair or apoptosis, following genotoxic stress [TP53 relative mRNA expression in 47.6% of GC cases. The mean level of TP53 mRNA expression was almost 3 fold higher in GC tumours than in adjacent normal mucosa. In our study, some GC tumours showed high TP53 mRNA levels, while others showed a slight increase. So, TP53 mRNA may either be weakly expressed in all tumour cells or highly expressed in a few tumour cells owing to tumour heterogeneity. Our results clearly demonstrate that TP53 regulation may occur at a pretranslational step, involving either an increase in TP53 gene expression and/or stabilization of its mRNA which might lead to elevated content of TP53 protein in the cell. Although there is a particular paucity of studies that have analysed the relative expression of TP53 mRNA in cancer, TP53 has been shown to express at a high frequency in gastric adenocarcinomas [TP53 mRNA expression was significantly higher in triple-negative breast cancer (TNBC) [TP53 transcripts in 70% [TP53 mRNA has recently been shown to increase the amount of endogenous TP53 and to increase apoptosis in human melanoma cells, in part, by modulating the transcription of downstream target genes including downregulation of p21 and upregulation of TP53-induced death domain protein, to favour apoptosis rather than cell cycle arrest [The role of c stress . We obsercinomas . In liner (TNBC) . Our fins in 70%  and 66% s in 70%  of testee arrest . Reportse arrest . Thus, rTP53 mRNA levels with higher stage and higher grade of GC. In tune with our observations, Fenoglio-Preiser et al. has reported TP53 overexpression in almost 90% of invasive Gastric tumors [TP53 mRNA in ovarian and renal cell carcinoma has been associated with worse prognosis and higher tumour grade [TP53 expression to be more common in poorly differentiated tumors than in well differentiated lesions [TP53 mRNA overexpression was associated with lower recurrence rates and higher overall survival rates in breast cancer and gastric cancer [TP53 mRNA, tumour stage and disease prognosis in CRC and GC [TP53 mRNA also harbours information that helps control TP53 protein turnover rate [On stratification, we observed a significant association of elevated c tumors . Increasur grade . As per ur grade  and ther lesions . Kakeji  lesions  showed t lesions . In contc cancer , 48. In C and GC , 50. SimC and GC . It has ver rate .H. Pylori infection had significantly increased TP53 mRNA levels. Shiao et al. observed overexpression of TP53 in 15% of H. Pylori–positive chronic gastritis patients but 38% of H. Pylori–positive metaplastic gastritis patients [H. Pylori is a major cause of chronic inflammation of the human gastric antral mucosa leading to development of atrophic chronic gastritis and Gastric carcinoma (CG) [H. Pylori infection increased TP53 expression and the apoptosis rate in GC [TP53 levels before H. Pylori infection were low or undetectable and were elevated on exposure to H. Pylori infection accompanied with intense inflammation [H. Pylori were found to be in phase G1 of the cell cycle with TP53 overexpression suggesting that cell cycle arrest in G1 is associated with a reduction in cyclin E levels and an increase in TP53 and p21 expression showing that H. Pylori can induce cell stress, reduce the ability to repair damaged cells, and can increase the number of changes in the genome, leading to genetic instability and finally to GC [In our study, most of the GC patients with oma (CG) . In conste in GC . Wei et ammation . Accordily to GC .PDL1 relative mRNA expression was significantly higher in 39.8% of GC cases with an average fold change of 2.43 in tumor tissues compared to adjacent normal tissues. Wu et al. showed that immunohistochemical PDL1 expression was strongly positive in 42.2% of 102 human gastric carcinomas, weakly positive in adenoma samples and totally negative in normal gastric tissue [+ T cells upregulate PD1 expression and secrete IFN-γ when they encounter tumor antigens, resulting in the upregulation of PDL1 expression on tumor cells and immune cells and the ligation of PDL1 with PD1 will decrease T cell function and create a negative feedback mechanism that decreases antitumor immunity leading to tumorigenesis [PD1 acts as a T-cells inhibitor mainly by limiting T-cells activity within neoplastic tissues and its ligand, PDL1, is often overexpressed on tumor cells . In our c tissue . PDL1 exc tissue , 59. It igenesis . In contigenesis .PDL1 mRNA levels with higher stage and higher grade of GC. PDL1 overexpression has been associated to higher number of lymph node metastasis, larger tumor size, increased depth of invasion and poorer overall survival in various cancers [PD1, PDL1 and CD8 mRNA levels were significantly higher in undifferentiated GC [On stratification, we observed a significant association of elevated  cancers , 61. Acciated GC . More reiated GC .TP53 and PDL1 mRNA expression, suggesting that there is a synergistic effect between PDL1 and TP53 in the occurrence and development of tumors, which has also been demonstrated in NSCLC wherein TP53 has been shown to regulate PDL1 expression via miR-34 that binds PDL1 3′-untranslated region in NSCLC models [PDL1 and TP53 has previously been positively correlated [In our study, we observed a positive correlation between C models , 63. Morrrelated . All therrelated .ARID1A mRNA expression and upregulation of TP53 and PDL1 mRNA expression in GC which was in turn significantly associated high-grade and advanced stage of tumor suggesting that lower ARID1A expression and higher expression of TP53 and PDL1 might play a definite role in the initiation and progression of GC. Furthermore, a positive correlation was found between TP53 and PDL1 mRNA expression. This should be useful for future anti-tumour research and for the design of therapeutic agents specific to the inactivation process. However, further largescale and comprehensive researches are needed to support our results and conclusion.In summary, we observed down regulation of


To optimize the first layer with PbBr2 and SnI2, we performed different annealing treatments. Further, MA3 thin film was synthesized on the optimized first layer by evaporating MAI and post-annealing. The formed hybrid perovskite thin film exhibited absorptions at 1.0 and 1.7 THz with small absorbance (<10%). Moreover, no chemical and structural defect-incorporated absorption was found. In this study, the possibility of changing terahertz absorption frequency through the mixture of metal cations (Sn+ and Pb+) and halogen anions (Br− and I−) was verified.All mixed hybrid perovskite  Recently, organic–inorganic hybrid perovskite (OHP) materials, because of their universal properties such as controllable bandgap, weak exciton binding energy, and high-carrier mobility, are attracting great attention for solar-cell and light-emitting diode applications . Many re3NH3+ (MA+) ion, which were confirmed by powder neutron diffraction, were found to be ordered, whereas the 2D and 3D disorder was dependent on each structure being controlled by temperature (3NH3PbI3 (MAPbI3) and α-HC(NH2)2PbI3 (α-FAPbI3) thin films  method, which incorporated CH3NH2 molecular defects in the synthesized thin film  bromide (PbBr2) and tin (II) iodide (SnI2) using SVE method. Next, MA3 thin film was formed by evaporating MAI in the same method. In the THz time domain spectroscopy (THz-TDS), the formed hybrid perovskite thin film exhibited 1.0 and 1.7 THz absorptions, and there was no chemical and structural defect-incorporated absorption.In this study, we fabricated and characterized an all mixed hybrid perovskite, MA2 and PbBr2. The SVE chamber had two heaters for separate evaporations of SnI2 and PbBr2. The base pressure was 8.0 × 10–3 Pa. First, SnI2  was evaporated onto the substrates at room temperature with a fixed deposition rate and thickness of 10 Å/s and 50 nm, respectively,  onto the substrates with same deposition rate and thickness  flexible substrate  was cleaned by sonication in acetone for 10 min, rinsed in heated acetone for 1 min, and then treated in a UV-ozone chamber for 30 min before finally loading it into a vacuum chamber. To fabricate all mixed organic–inorganic hybrid perovskite thin film using SVE, first, we optimized the first mixed layers with SnIctively, . Next, whickness . Finallyhin film .2/PbBr2 was further deposited with CH3NH3I  with a fixed deposition rate and thickness of 2 Å/s and 300 nm, respectively,  solution containing 5 mg PTAA and 4 ml chlorobenzene. The PTAA solution was sonicated for 1 h .Kα source constructed by Rigaku. All XPS measurements were performed using Versa ProbeII with a monochromated AlKα (ULVAC-PHI) to obtain Pb 4f, I 4d, Sn 3d, Br 3d core-levels, and valence spectra. Binding energies were calibrated with respect to the Au 4f7/2 core-level (84.0 eV) , X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). SEM characterization was conducted at an acceleration voltage of 5 keV and an emission current of 10 μA using HITACHI SU9000. The XRD instrument was RINT-TTRIII/NM with a Cu 84.0 eV) .Transmission type THz-TDS was utilized to characterize the samples and elucidate their THz-wave absorption properties. . Transmi2/SnI2, we performed the post-annealing with different annealing times. Drastic changes in the surface morphologies and chemical states after the annealing treatments are shown.  Initially formed thin film structure had physical separation between SnI2 and PbBr2.2) The post-annealing process induced a physically mixed layer of SnITo find an optimized mixed thin film with PbBre shown. . From the shown. . Interesncrease, , whereasecrease, , indicat0+ and Pb0+ were observed for the no-annealing sample. However, they soon disappeared as the post-annealing process was initiated. Although there was still a small trace of Pb0+ after annealing, the annealing process almost induced a depletion of non-stoichiometry elements in the thin film. For the next deposition step, the optimized thin film is required with a rough stacking structure  and MAPbBr3 (5.95 Å). Furthermore, we have confirmed that the THz-wave absorptions of MA3 are different in comparison with those of MAPbI3 and MAPbBr3. MAPbBr3. . Unfortu3 by SVE in this study. Although the formed thin films were subjected to different annealing treatments, further optimizations are required in the thin film fabrication process. However, we verified the possibility of THz-wave absorption changes due to the mixture of metal cations (Sn+ and Pb+) and halogen anions (Br− and I−). To be a candidate material for THz-based application such as sensing and modulating devices, we believe this confirmation is a first step for the frequency modulating by the element mixtures.We fabricated MA


The results showed that changes in the chemical composition of FVB have a positive effect on the contact angle and increase the cellulose crystallinity index. Furthermore, the mechanical properties of the oriented board showed that 1% NaOH + 0.2% Na2SO3 with 60 min immersion has a higher value compared to other treatments. The best dimension stability was on a board with the modified FVB of 1% NaOH + 0.2% Na2SO3 with 30 and 60 min immersion. The bonding mechanism evaluated by FTIR spectra also showed that there is a reaction between the hydroxyl group in the modified FVB and the carboxyl group in citric acid. This showed that the modified combination treatment of NaOH+Na2SO3 succeeded in increasing the mechanical properties and dimensional stability of the orientation board from the FVB salacca frond.The fibrovascular bundle (FVB) in palm plants consists of fiber and vascular tissue. Geometrically, it is a long fiber that can be used as an oriented board raw material. This research aimed to examine the performance of citric acid-bonded orientation boards from modified FVB salacca frond under NaOH + Na The fibrovascular bundle (FVB) is a tissue in monocot plants, specifically palm plants, that consists of xylem, phloem, sclerenchyma fibers, and parenchyma tissue. According to Hakim et al. , the FVB2SO3 as a chemical for modification has not been widely reported. Therefore, the use of NaOH+Na2SO3 as a chemical modification exposes more –OH groups on the fiber surface and increases compatibility with adhesives since the surface structure facilitates higher interface bonding as raw material for composite boards [2SO3.Several types of research studies have been carried out on natural fibers using a chemical treatment to modify the surface structure and improve the compatibility between adhesive and raw material. This includes the use of alkali modification in natural fibers due to the convenience and simplicity of its application ,5. Previe boards ,7. AlkalThe use of modified FVB as a raw material for composite boards is more suitable when it is developed into an orientation board product . Geometr2SO3 combination and the citric acid adhesive has not been reported. Therefore, this research aims to evaluate the performance of citric acid-bonded oriented boards from modified FVB S. zalacca under NaOH+Na2SO3 treatment.Natural adhesives such as citric acid are used as an alternative in the manufacture of environmentally friendly composite boards ,17,18,19S. zalacca frond was used as a raw material for the manufacture of oriented board. The extraction was based on Hakim et al.’s study [2SO3). Meanwhile, the citric acid (anhydrous) used as adhesive without further purification was supplied by Rudang Chemical Company (Indonesia).The fibrovascular bundle extracted from the ’s study , and the2SO3 and their combination, as shown in Before the board was manufactured, the fibrovascular bundle was chemically modified with NaOH and NaThe contact angle was measured using a method by Schellbach et al. ; meanwhiMeanwhile, the mechanical properties of the modified fibrovascular bundle were evaluated based on the ASTM D-3379-75 standard. The FVB with 8–12% moisture content was cut to a length of about 90 mm + 0.1 mm and fixed on a 30 mm long paper frame using epoxy adhesive . The measurements were conducted in 50 replicates for each chemical treatment. The mechanical properties were determined using a universal testing machine , with a 1 mm/min crosshead speed. Before the test, the supporting paper’s middle part was cut out, and the test mounting was carried out according to the method proposed by Hakim et al.  Figure . In addiI002) (maximum intensity between 22.5° and 23°), while amorphous cellulose (Iam) is the minimum-intensity position (between 18° and 19°). Furthermore, the diffraction spectra were obtained at room temperature (20–22 °C) from radiation generated by Maximax X-ray Diffractometer-7000 . The measurements were carried out at 40 kV and 20 mA with a detector placed on the range of 2θ from 5° to 70° at a scan speed of 2°/min. Subsequently, the percentage crystallinity index (%CI) of the cellulose was calculated using the formula provided by Segal et al. [I002 is the maximal peak intensity at a 2θ angle of approximately 22°–23° and Iam is the minimum peak intensity (amorphous region) at a 2θ angle of about 18°–19°.The crystallinity index (CrI) was determined by considering the regions of crystalline and amorphous cellulose. Crystalline cellulose is determined at a 2θ peak in the reflection plane position  of the board were prepared for each fibrovascular bundle, each in three layers, namely face:core:back = 30%:40%:30% weight ratio  before being used as a raw material for orientation boards. The board was to be manufactured with dimensions of 250 × 250 × 8 mmht ratio . The thr3 were treated by the three-point bending method under dry conditions with a span distance of 150 mm and a crosshead speed of 10 mm/min. The IB specimens of 50 × 50 × 8 mm3 were randomly measured from both surfaces of each specimen. Furthermore, the SHP specimen of 75 × 50 × 8 mm3 was drawn two positions from the board of each sample with a speed of 2 mm/min, while the TS and WA of 50 × 50 × 8 mm3 were evaluated by water immersion for 24 h at 20 °C.All of the samples were conditioned at environmental conditions for approximately a week to reach a moisture content of ±6% before the measurement of the oriented board properties according to the Japanese Industrial Standard for particleboard (JIS A 5908). The mechanical properties of the oriented board were measured for modulus of rupture (MOR), modulus of elasticity (MOE), the strength of the internal bond (IB), and screw holding power (SHP) using a universal testing machine , while the physical properties were tested for thickness swelling (TS) and water absorption (WA). The static bending samples (MOR and MOE) of 200 × 50 × 8 mm−1 of resolution to determine the assignment of absorbance bands to specific functional groups.The fibrovascular bundle-oriented board samples were previously soaked in boiling water for 2 h to remove unreacted citric acid, conditioned in room temperature water for 1 h, dried at 35 ± 5 °C for 12 h, and powdered through a 100-mesh screen. FTIR spectroscopy was performed at room temperature (approximately 25 °C) using the FTIR-4200 spectrophotometer  and the KBr disk method with 12 cm2SO3 concentration, which has the capacity to destroy amorphous cellulose at high concentrations and longtime immersion. Similar to the previous research, 3% NaOH treatment + steaming on S. zalacca FVB frond yielded 54.53% cellulose [Dypsis lutescens) raised the cellulose content to 63.45% [The increase in cellulose from A0-30 to C12-30 treatment does not imply an increase in cellulose content, but rather that the other components, hemicellulose and lignin, decreased. Meanwhile, the α-cellulose fraction reduces during treatments C12-60, C14-30, and C14-60 because the α-cellulose component dissolves. This reduction was caused by the NaOH + Naellulose , and a 1o 63.45% .2SO3 and 60 min immersion. In contrast to previous studies, the alkaline treatment of 3% on the FVB of the frond of S. zalacca became 74.09% [The hemicellulose content of modified FVB decreased from 32.24% with the 30 min water immersion treatment to 21.91% with the treatment modified with 1%NaOH + 0.4% Nae 74.09% . Hemicele 74.09% ,28.S. zalacca frond FVB decreased from 28.1% (treatment A0-30) to 18.7% (treatment C14-60). This pattern of decline is relatively the same as that in previous studies using modified alkali [2SO3 treatment was more effective in maintaining α-cellulose, hemicellulose, and lignin content compared to previous studies by Darmanto et al. [S. zalacca as raw material with higher concentration and high-energy steam.The lignin component of the modified d alkali ,26,29,30o et al.  using S.3 under the water treatment with 30 min immersion. Meanwhile, the lowest was 0.35 g/cm3 under 1% NaOH + 0.4% Na2SO3 with 60 min immersion. This decreased density value is due to the degradation factor of the chemical components during the modification treatment, which causes the weight of FVB decrease.The modified FVB had the highest density value of 0.40 g/cm2SO3 decreased the contact angle compared to the water immersion treatment. Furthermore, it was affected by the surface roughness due to changes in the structure of the chemical composition of the surface [2SO3 treatment led to the removal of low molecular weight material such as hemicellulose and lignin, while the cellulose content increased. Based on previous research, the alkali modification reduced some impurities and wax in the FVB surface [S. zalacca [Tridax procumbens fiber [The contact angle was measured to determine when the modified FVB is easily wetted by a liquid; meanwhile, the value obtained is 24.02° with treatment H , which i surface . Surface surface . The res zalacca , 15% NaO zalacca , 5% NaOHns fiber , 5% NaOHns fiber , and 5% ns fiber .S. zalacca frond. Compared to the result of Darmanto’s research, this study was more effective and efficient because high temperatures and pressures were not used. Young’s modulus has the same pattern as the tensile strength of the modified FVB. The higher Young’s modulus of modified FVB increased from water treatment to C12-30 treatment but slightly decreased after C12-60, C14-30, and C14-60 treatment, respectively.Darmanto et al.  reported2SO3 treatment with 60 min immersion increased to 58.33%, whereas for the counterparts treated with 1% NaOH + 0.4% Na2SO3 with 60 min immersion, it decreased to 47.43%. The mechanical properties of fiber are affected by the crystallinity index. The higher the crystallinity index of a fiber, the higher is the predicted fiber strength [2SO3 with 60 min immersion resulted in an optimal increase in the crystallinity index of modified FVB. This occurs due to the degradation of amorphous components, which include hemicellulose, lignin, and wax. These chemical components are sensitive to alkali, so they are easily removed by alkaline modification treatment [2SO3 treatment with 60 min immersion decreased the crystallinity index because cellulose polymer is penetrated by sulfite ions (SO3−), so some of the cellulose and lignin components begin to dissolve [The mechanical properties of modified FVB are influenced by the material’s crystallinity index. strength . The trereatment . The 1% dissolve .2SO3 and decreased with the combined treatment with 1% NaOH + 0.4% Na2SO3.The MOR and MOE values of the oriented board were observed in two positions, namely perpendicular (ꓕ) and parallel (//). Generally, their values for an oriented board with 0° orientation angle were higher than those of the board with orientation angle 45° and 90°. The MOR value in the perpendicular position was higher than the MOR value in the parallel position for all orientation angles , which is similar to the previous research conducted by Munawar et al. and Baharin et al. ,40. Ther2SO3 with 60 min immersion, whereas the lowest values were achieved in the 30 min water immersion treatment, and they were 11.8 MPa (ꓕ) and 4.9 MPa (//) with orientation angle 0°, 7.9 MPa (ꓕ) and 7.3 MPa (//) with orientation angle 45°, and 7.6 MPa (ꓕ) and 7.5 MPa (//) with orientation angle 90°, respectively.As shown in 2SO3 for 60 min. Meanwhile, the lowest MOE values were obtained with the water immersion treatment for 30 min at 3.3 GPa (ꓕ) and 1.2 GPa (//) for 0°, 2.1 GPa (ꓕ) and 2.0 GPa (//) for 45°, and 2.5 GPa (ꓕ) and 2.2 GPa (//) for 90°, respectively.The highest MOE values were obtained at 7.4 GPa (ꓕ) and 4.0 GPa (//) for 0° orientation angle, 4.7 GPa (ꓕ) and 4.1 GPa (//) for 45°, and 5.2 GPa (ꓕ) and 5.1 GPa (//) for 90°, respectively, in the combination treatment of 1% NaOH + 0.2% NaSansevieria using phenol–formaldehyde (PF) adhesive also increases the MOR value by 403 MPa [Umemura et al.  reported 403 MPa . In this 403 MPa . This sh 403 MPa ,45. Mean 403 MPa ,47.Nypa fruticans frond that bonded citric acid and maltodextrin adhesives give an MOE of 0.5 GPa. Meanwhile, Widyorini et al. [The research by Santoso et al.  on the mi et al.  found thi et al.  using so2SO3 and then decreased again at the 1% NaOH + 0.4% Na2SO3 treatment for 30 min. The orientation board with an angle of 0° has a greater IB value than boards with angles of 45° and 90°. The IB value of the orientation board made from the modified FVB frond of S. zalacca is shown in The IB increased from water treatment for 30 min to the combination treatment of 1% NaOH + 0.2% Na2SO3 with 30 min immersion and 1% NaOH + 0.2% Na2SO3 for 60 min immersion, and it was 0.46 MPa (0°) and 0.35 MPa (90°), respectively, while the lowest was 0.29 MPa (0°) and 0.25 MPa (90°), respectively, with 1% NaOH + 0.4% Na2SO3 treatment with 60 min immersion.The highest IB value of the board was found in the combination treatment of 1% NaOH + 0.2% NaMoreover, the IB values of 90° and 45° orientation boards are lower than 0° because the 90° orientation boards are arranged crosswise, consisting of three layers of the face, core, and back for the bonding area and contact between the fibers on the surface of one layer with another layer to reduce. Some of the values in this research were still higher—0.37 MPa  and 0.15Sansevieria fiber bonded using phenol–formaldehyde (PF) had an IB value of 1.33 MPa, which is much higher compared to that of this study. Walther et al. [According to Munawar et al. , the orir et al.  also sta2SO3 with 30 min immersion; however, it decreased again on the combined treatment of 1% NaOH + 0.4% Na2SO3 with 30 min immersion. The results showed that the oriented board with 0° and 90° angles of orientation have relatively similar SHP values. The highest SHP values were 519 N (0°), achieved in the combined treatment of 1% NaOH + 0.2% Na2SO3 with 60 min immersion, and 504 N (45°) and 495 N (90°), achieved in the combined treatment of 1% NaOH + 0.2% Na2SO3 with 30 min immersion, respectively. The lowest values of HPS were 318 N (0°), 307 N (45°), and 293.5 N (90°), achieved in the combined treatment of 1% NaOH + 0.4% Na2SO3 with 60 min immersion. The histogram of the SHP of the oriented board is shown in The value of SHP increased from control to the combination treatment of 1% NaOH + 0.2% Na2SO3 treatment with 60 min immersion was the best in influencing the screw holding strength. According to Hung et al. [This research showed that the modified 1% NaOH + 0.2% Nag et al. , the adhg et al. . Compareg et al. . Finer pThe single-layer orientation board has a higher SHP value than the three-layer orientation board, while the 0° orientation angle is slightly higher than 45° and 90°. This showed that the orientation angle of 0° is a single-layer board, while 45° and 90° are a three-layer board.2SO3 for 60 min. In addition, the orientation board has the highest TS value of 19.64% (0°), 21.65% (45°), and 22.65% (90°) in the 30 min water immersion treatment, while the lowest value is 14.99% (0°) and 16.20% (45°) in the 1% NaOH + 0.2% Na2SO3 with 60 min immersion, and 17% (90°) in the 1% NaOH + 0.2% Na2SO3 with 30 min immersion. Generally, the WA and TS values decreased from treatment A (30 min water immersion) to treatment F (1% NaOH + 0.2% Na2SO3 with 60 min immersion) and increased again in treatment G (1% NaOH + 0.4% Na2SO3 with 30 min immersion). The WA and TS values of the modified orientation board are shown in The orientation board has the highest WA value in the water immersion treatment with 30 min immersion, which is 51.18% (0°), 54.87% (45°), and 55.26% (90°). By contrast, the lowest WA value is 43.50% (0°) in the 1% NaOH treatment with 30 min immersion, and 16.20% (45°) and 47.80% (90°) in the combination treatment of 1% NaOH + 0.2% Na2SO3 with 60 min immersion succeeded in reducing water absorption to 43.0%.The results have lower WA and TS values compared to the previous research conducted by Kemalasari and Widyorini , which s2SO3 on the chemical structure of the FVB orientation board of the salacca frond bonded with citric acid is shown in −1, which shows the C=O stretching group, indicating the formation of carbonyl group or C=O group [2SO3 for 30 min) and C12-60 (1% NaOH + 0.2% Na2SO3 for 60 min), while the modified treatment with a higher concentration of Na2SO3 (treatment C14-30 and C14-60) showed a decrease in intensity. This showed the mechanical properties of the orientation board, which has the best mechanical value in treatment C12-30 and C12-60.The effect of modified NaOH and the combination of NaOH + Na=O group ,19,42. T−1, but there was a significant peak on the modified orientation board (board+C12-30 and board+C12-60) at a wavelength of 1733 cm−1, which indicates the formation of a carbonyl group (C=O stretching) and an ester group C=O). This shows that there is a reaction between the hydroxyl group in the modified FVB and the carboxyl group in citric acid [ric acid . Furtherric acid  describe2SO3 treatment influences the modulus of rupture, modulus of elasticity, internal bond, as well as screw holding power of the orientation board. The 1% NaOH + 0.2% Na2SO3 treatment with 30 and 60 min immersion increased the mechanical properties. As a structural panel, orientation boards made of modified FVB bonded with citric acid as a natural adhesive still rank below the wood-based composite with conventional adhesives. These orientation boards can be recommended as nonstructural panels such as partitions and insulation boards. In terms of the use of structural panels, FVB can be improved as a potential new raw material for the development of composite boards by combining it with other raw materials such as wood and veneer.As expected, orientation boards were manufactured from FVB modified with citric acid adhesive and their mechanical properties were tested. It was found that the combination of NaOH+NaS. zalacca frond for raw material of the orientation board. The results showed that the combined treatment of 1% NaOH + 0.2% Na2SO3 with 30 and 60 min immersion has a positive effect on increasing the mechanical properties of the orientation board. The 0° orientation angle also had a better effect on the MOR and MOE tests for the position perpendicular to the fiber. Furthermore, the bonding mechanism of FVB modified by the combination of 1% NaOH + 0.2% Na2SO3 by immersion for 30 and 60 min bonded citric acid adhesive showed good bonding quality. The FTIR spectra also showed the peak intensity of the wavelength at 1733 cm−1 with the detection of C–O stretching the carbonyl and ester groups, which indicated that there is a reaction between the hydroxyl group in the modified FVB and the carboxyl group in citric acid. Based on these results, the combination of 1% NaOH + 0.2% Na2SO3 treatment for 30 and 60 min immersion is successful in reducing water absorption and thickness swelling of the orientation board.The research was conducted using the alkali modification of the fibrovascular bundle extracted from an


E2F family of transcription factors modulates multiple cellular functions associated with cell cycle and apoptosis. Here, we focused on the relevance of E2F1 to esophageal squamous cell carcinoma (ESCC) and identification of E2F1-mediated network in this study. Query of Gene Expression Omnibus database revealed that E2F1 was the core gene that was upregulated in ESCC. E2F1 downregulation inhibited ESCC cell activity. microRNA (miR)-375 was confirmed to be a downstream target of E2F1. E2F1 bound to miR-375 promoter and inhibited miR-375 transcription. Moreover, miR-375 inhibitor mitigated the repressive impacts of si-E2F1 on ESCC cells in part. Further study showed that sestrin 3 (SESN3) could interact with miR-375, and its knockdown annulled the stimulative effect of miR-375 inhibitor on ESCC development. Finally, E2F1 and SESN3 downregulation inhibited the phosphatidylinositol 3 kinase (PI3K)/AKT pathway activity in cells, while miR-375 inhibitor promoted PI3K/AKT pathway activation. These findings suggest that E2F1 inhibited miR-375 expression and promoted SESN3 expression to activate the PI3K/AKT pathway in ESCC. Esophageal cancer remains a commonly diagnosed and fatal cancer in China in 2015 , despiteE2F family of transcription factors modulates a vast network of cellular functions associated with cell cycle and apoptosis in cancers . Overexphttp://www.ncbi.nlm.nih.gov/geo). Among them, GSE20347 contains the tumor and normal adjacent tissues of 17 ESCC patients, and GSE17351 includes 5 pairs of primary ESCC and adjacent normal esophageal mucosa. Differentially expressed genes (DEG) between ESCC and normal tissues were screened out by GEO2R (http://www.ncbi.nlm.nih.gov/geo/geo2r/) with parameters set to p-value < 0.01 and log2fold change > 2, followed by heatmap plotting.Two data series consisting of 22 ESCC tissues and their matched normal tissue pairs were acquired from the GEO database (http://string-db.org/) database was utilized to determine related genes of E2F1. Data of the KEGG pathway enrichment analysis were obtained from KEGG (https://www.kegg.jp/kegg/rest/keggapi.html). TransmiR (http://www.cuilab.cn/transmir) was used to identify miRNAs interacting with E2F1. Pan-cancer analysis of E2F1 was conducted using Oncomine database. Cox analysis of E2F1 expression with ESCC patients’ characteristics was performed using the forestplot package in R . The promoter sequence of miR-375 was obtained from UCSC website (https://genome.ucsc.edu/index.html), and the binding site between miR-375 and E2F1 was predicted by ALGEEN . The target mRNAs of miR-375 and the binding site between miR-375 and SESN3 were acquired from the StarBase database (http://starbase.sysu.edu.cn/).STRING  from Cancer Hospital of China Medical University, Liaoning Cancer Hospital and Institute. The diagnosis was performed by three specialized pathologists. None of the patients received chemo-, immune- or radio-therapies before sample collection. After collection, the samples were instantaneously placed in liquid nitrogen and transferred to a − 80°C refrigerator until further analysis. Signed informed consent forms were acquired from patients. This item was endorsed by the ethics committee of Cancer Hospital of China Medical University, Liaoning Cancer Hospital and Institute.After conventional paraffin embedding, the human and mouse tumor tissues were cut into 4-μm histological sections, dewaxed with xylene, and hydrated with gradient density alcohol . Antigenin-situ hybridization assay kit . The tissues were fixed in 4% paraformaldehyde for 0.5 h, and cultured with 0.25% acetic anhydride for 10 min before hybridization, followed by permeabilization with Triton X-100 for 10 min and proteinase K treatment to expose target nucleic acids. The sections were incubated with probe-free hybridization buffer for 120 min at ambient temperature to block nonspecific binding. Hybridization was conducted using a synthetic miR-375 probe. The sections were treated with 10 μL hybridization solution overnight at 42°C, washed with gradient concentrations of saline citrate (20 min each) at 37°C, and sealed with 3% BSA for 0.5 h at 37°C. Next, the sections were cultured with anti-digoxigenin anti-serum alkaline phosphatase complex overnight at 4°C. Finally, diaminobenzidine color development solution was added, and the section was placed in Tris-ethylenediamine tetraacetic acid solution for 10–30 min to terminate the reaction. After gradient density alcohol dehydration, xylene dewaxing, neutral resin (Merck Millipore) sealing, and the staining results were observed under a microscope (Nikon).The miR-375 distribution in tissues was examined using an −ΔΔCq method [Total RNA was isolated from the cells with the help of TRIzol  as per the protocol. cDNA was produced with oligonucleotide primers in a 20 μL reaction system using iScript . Real-time PCR of cell line samples was conducted using the Universal Probe Library monochrome probe from the Roche LightCycler 480 system  on 5 μL diluted cDNA. The change in expression was measured with the help of the 2q method . The men2 (Thermo Fisher Scientific). The medium was refreshed at an interval of 24 h. When the cell confluence reached 80%, the cells were detached with 0.25% of trypsin. The miR-375 inhibitor (miR-375 inh) and its corresponding miR-375 control (miR-375 con), siRNA against E2F1 and SESN3 were from RiboBio . ESCC cells were plated overnight and transiently transfected using Lipofectamine 2000 (Thermo Fisher scientific). The cells were cultivated for 24 or 48 h prior to following experiments.An ESCC cell line KY150 and human esophageal epithelial cells HET1A were from EK Bioscience Biotechnology . EC109, EC9706, and YES2 from BeNa Culture Collection  were plated in RPMI-1640 plus 10% FBS at 37°C and 5% CO3 cells/well [2, CCK-8  was supplemented to each well and cultured at 37°C. After 3 d, the OD value was read at 450 nm under a microplate reader . Median inhibition concentration (IC50) values were analyzed from the dose response curves of GraphPad Prism 8.0 software.The cells were resuspended in PBS and plated in a 96-well plate at 2 × 10lls/well . Followi4 cells/well) were suspended in serum-free DMEM and supplemented to the apical chamber. For the invasion assay, 1 × 104 cells were plated into Matrigel-coated chambers. DMEM plus 10% FBS (500 μL) was plated to the basolateral chamber, and allowed to culture for 24 h. The cells in the basolateral chamber were stained with 0.1% crystal violet. The number of migratory or invasive cells was measured under a 200x magnification microscope  in three random fields of view.Cell motility was studied by cell invasion and migration . For theAnnexin V-FITC Apoptosis Detection kits (Abcam) were utilized for apoptosis examination. The cell samples were stained with 5 μL Annexin V-FITC and 5 μL PI for 0.5 h at ambient temperature in darkness and loaded onto the BD FACSCalibur Flow Cytometry System .7 ESCC cells were cross-linked with 1% formaldehyde and resuspended in lysis buffer [Briefly, the 1 × 10s buffer . The celThe SESN3 3ʹuntranslated region (3ʹUTR) sequence containing the miR-375 binding site was subcloned into pmirGLO luciferase reporter vectors  to produce a wild-type (WT) vector, while the corresponding mutant (MT) was produced by mutating the binding site. The luciferase reporter vector was co-transfected into EC109 cells with miR-375 con or miR-375 inh, and the luciferase reporter system (Promega) was utilized for luciferase activity analysis 24 h after transfection.6 cells) stably transfected with low expression plasmids were used for subcutaneous injection in nude mice. The length and width of tumors in nude mice were assessed every week to determine tumor volume as volume (mm3) = width2 × length/2. Euthanasia was performed using intraperitoneal injection of 1% pentobarbital sodium at 4 weeks after injection, and tumors were harvested for weighing and molecular analysis.Our research was permitted by the Ethical Committee of Cancer Hospital of China Medical University, Liaoning Cancer Hospital and Institute. Animal studies were executed in compliance with the ARRIVE guidelines. BALB/c nude mice  from the Shanghai Lab Animal Research Center  were acclimatized for 7 d and randomly allocated into six groups (n = 5). EC109 cells (2 × 10RIPA buffer (pH = 8.0) including 150 mM NaCl, 0.1% SDS, 50 mM Tris-HCl and 1% NP-40 was added to the proteinase inhibitor  and used to obtain whole cell lysates. The proteins were examined using a BCA kit (Thermo Fisher) and separated by SDS-PAGE. The proteins were transferred onto PVDF membranes , sealed with 5% BSA for 60 min at 25°C, and hybridized at 4°C with primary antibodies to MMP2 , MMP9 , Bax , PI3K , p-AKT , AKT , and GAPDH  for one night. The blots were incubated with secondary antibodies for 60 min at 25°C. Finally, the protein bands were detected using an ECL kit (Thermo Fisher Scientific). Densitometric analysis was performed using IPP 6.0 (Image-Pro Plus 6.0).t-test was carried out for two-group comparison, and ANOVA followed by Tukey’s post hoc test was utilized to compare differences when the number of groups was greater than two. Correlations between genes were calculated using Person’s correlation analysis. All differences were considered statistically significant when p < 0.05.All analyses were implemented using SPSS . The data were recorded as means ± SD. Paired In our results, we found that expressions of E2F1 was upregulated in ESCC, and silencing of E2F1 reduced the expression of MMP-associated proteins, cell proliferation, invasion and migration in ESCC cells. We also found that miR-375 was the downstream target of E2F1, and SESN3 was the putative target of miR-375 in ESCC. E2F1 promoted the SESN3 expression and the PI3K/AKT pathway through decreasing the transcription of miR-375.To identify candidate genes in ESCC, we implemented bioinformatics analyses using published datasets . BrieflyPan-cancer analysis of E2F1 revealed that E2F1 was commonly overexpressed in cancerous tissues . We asseTo study the specific function of E2F1 in ESCC progression, EC109 and YES2 cells with the relative high expression of E2F1 were delivered with si-NC or si-E2F1. After transfection, the mRNA and protein expression of E2F1 was remarkably reduced . In termBased on the results in The miR-375 target genes with the most enrichment pathways and biological processes were overlapped with the genes enriched in the PI3K/AKT pathway, and SESN3 was observed to be the only result . LuciferEC109 and YES2 cells were co-transfected with si-E2F1 + miR-375 inh, miR-375 inh + si-SESN3 or their respective controls (si-E2F1 + miR-375 con and miR-375 inh + si-NC). The downregulation of miR-375 contributed to a significant rise in cell proliferation in the presence of si-E2F1, while the downregulation of SESN3 similarly inhibited the effect of miR-375 inhibitor, resulting in a significant decline in cell proliferation activity . Migrate+ proliferating cells, which was mitigated by miR-375 inhibitor. In contrast, SESN3 downregulation reversed the effect of miR-375 downregulation and decreased tumor proliferation  . It is barcinoma . Furtherarcinoma . The wesTaken together, our results demonstrate a regulation mechanism between the transcription factor E2F1 and miR-375 in ESCC progression . ClinicaClick here for additional data file.