MeCP2 inhibited the expressions of numerous miRNAs by binding to their upstream elements, including not only the promoter but also the distal enhancer. Among the affected miRNAs, miR-22 was identified to remarkably suppress gastric cancer (GC) cell proliferation, arrest G1–S cell cycle transition, and induce cell apoptosis by targeting MeCP2, MTHFD2, and MTHFR. Understanding GC metabolism characteristics is the key to developing novel therapies that target GC metabolic pathways. Our study revealed that the metabolic profiles in GC tissues were altered. SAM (S-adenosylmethionine), a universal methyl donor for histone and DNA methylation, which is specifically involved in the epigenetic maintenance of cancer cells, was found increased. The production of SAM is promoted by the folate cycle. Knockdown of MTHFD2 and MTHFR, two key enzymes in folate metabolism and methyl donor SAM production, significantly suppressed GC cell proliferation. MiR-22 overexpression reduced the level of endogenous SAM by suppressing MTHFD2 and MTHFR, inducing P16, PTEN, and RASSF1A hypomethylation. In conclusion, our study suggests that miR-22 was inhibited by MeCP2, resulting in deficiency of endogenous SAM, and ultimately leading to tumor suppressor dysregulation.This study investigated the effect of methyl-CpG-binding protein 2 (MeCP2) on miRNA transcription. Our results of miRNA chip assay and ChIP-seq showed that  DNA methylation participates in the regulation of gene expression by recruiting methyl-CpG-binding proteins, such as methylated DNA-binding domain (MBD) 1–4 and methyl-CpG-binding protein 2 (MeCP2). MeCP2 amplification and overexpression have been observed in several human cancer types4. MeCP2 is overexpressed in primary gastric cancer (GC) tissues and is involved in the regulation of GC cell proliferation and apoptosis5.Stomach cancer is the fourth most common malignancy in men and the fifth most common malignancy in women6. MiR-22, a tumor suppressor gene, is downregulated in a wide variety of tumors, such as colorectal cancer, hepatocellular carcinoma, breast cancer, and lung cancer9. MiR-22 is involved in tumorigenesis by targeting HIF-1α, SIRT1, CDK6, Sp1, HDAC4, MAX, Galectin-9, NET1, PAPST1, ESR1, TIAM1, cyclin A2/CDKN1A, and Erbb3, and its promoter is frequently hypermethylated22. Topologically associating domains (TADs) facilitate the formation of three-dimensional genomic architecture, providing physical contacts between the genes and the regulatory elements. Chromatin TADs affect the expression of cancer-associated genes by inhibiting or facilitating the interactions between enhancers and promoters26. We also performed a bioinformatics assay to specifically detect the physical interactions between the enhancer and the promoter of miR-22 in ESCs and cancer cells.Approximately 47% of the investigated human miRNAs have been associated with CpG islands, suggesting that miRNAs are subject to transcriptional regulation by DNA methylation27. Methionine is the substrate for S-adenosyl-methionine synthetase. In addition, in the mitochondria, 5,10-MTHF is regulated by MTHFD2, which generates formic acid through a complicated reaction and is transferred to the cytoplasm for folate metabolism. Folate metabolism is usually altered in cancer29. Targeted gene analysis indicated that the folate metabolism-related enzymes, MTHFR and MTHFD2, which participate in the formation of the methyl donor SAM, may be novel targets of miR-22, suggesting that miR-22 participates in methyl metabolism. Previous studies have shown that MTHFR mutation is closely related to tumor formation30, but the expression and molecular mechanism of MTHFR in cancer still need to be explored. Silencing the expression of MTHFD2 inhibited the proliferation of multiple tumors34 significantly, suggesting that MTHFD2 is an oncogene. It remains unclear whether MTHFR and MTHFD2 are involved in tumor progression by regulating SAM. SAM is a major sustainer of tumor suppressor genes and histone methylation and has a concentration-dependent effect on the proliferation of colorectal cancer cells29. A study on network regulation showing that folate metabolism contributes to SAM formation and influences the epigenetics and development of carcinomas is important for developing innovative treatment strategies.Folate metabolism, also known as one-carbon metabolism, involves a series of transformations and supports epigenetic maintenance. SAM, a reactive methyl carrier, plays a major role in epigenetics. Methylene tetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5, 10-methyleneTHF  to 5-methyTHF (5-MTHF) in the cytoplasm. 5-MTHF is the most important naturally occurring form of folate found in organisms. 5-MTHF is converted to tetrahydrofolate (THF) with the transfer of a methyl group to homocysteine to form methioninemiR-22 expression and evaluate the role of miR-22 in one-carbon metabolism by targeting MTHFR and MTHFD2.In this study, we aimed to investigate the molecular mechanism by which MeCP2 regulates MeCP2 on the transcription of miRNAs, we performed a microRNA chip assay. The results suggested that many miRNAs, such as miR-22, were markedly affected by MeCP2. MiR-22, a conserved microRNA, was negatively correlated to MeCP2 mRNA according to the TCGA data  Identifier). ChIP-PCR . As shown in Fig. miR-22 were improved by the methylation inhibitor. Furthermore, bisulfite sequencing showed that the methylation levels of the two CpG sites in the MeCP2-binding location remarkably decreased after treatment with 5-Aza (44.62%) compared with the methylation levels after treatment with DMSO (95.56%)  and control vector to GC cells, and performed immunoprecipitation and immunofluorescence assays with an anti-GFP or anti-MeCP2 antibody to confirm the exogenous gene expression phenotype and subcellular localization  Fig. . These rcer Fig. . In addicer Fig. . The ChIion Fig. . Subsequ-22 Fig. . In orde0Kb Fig. .miR-22 expression levels by performing qRT-PCR in 39 matched GC and adjacent tissue samples. Results showed that the expression of miR-22 appeared downregulated in GC tissues  that promoted cell proliferation and verified that they reversed the inhibitory effects of miR-22 overexpression and MTHFD2 and MTHFR knockdown on cell proliferation  rely on SAM biosynthesis. Methionine restriction inhibits CSCs, and SAM supplementation rescues the effects of methionine restriction, at least partly46. Based on several sets of data discussed, SAM is the principal biological methyl donor synthesized in cells, which maintains chromatin methylation. In this study, we demonstrated that miR-22 reduces P16, RASSF1A, and PTEN methylation by decreasing endogenous SAM  of SAM promote colon cancer cell line growth and inhibits proliferation at high doses (3 mM)49. At the same time, dietary intake of high concentrations of folic acid, an important molecule in carbon metabolism, can increase levels of serum folic acid, which can lead to DNA methylation of P16, MLH1, and MGMT50. Our study demonstrates that SAM reduces the expression of the tumor suppressor genes P16, P21, PTEN, and RASSF1A. Interestingly, P21 inhibition is mainly regulated by histone methylation52. However, this does not contradict our conclusion that SAM deficit induced P21 upregulation. As described in our introduction, MTHFR catalyzes the reduction of methionine and 5-MTHF to SAM. A recent study reported that the methionine cycle flux specifically influences the epigenetic state of cancer cells and drives tumor initiation. Histone methylation is upregulated in SAM-treated cells and downregulated in MTHFR knockdown cells45. Other research findings indicate that dietary methionine influences therapy in mouse cancer models through one-carbon metabolism53. To confirm the effect of miR-22 on one-carbon metabolism, we examined the changes in THF and SAM concentrations and found that miR-22 prevented the synthesis of both substances and induced the expression of P16, P21, PTEN, and RASSF1A. SAM reversed the inhibition of miR-22, MTHFD2, and MTHFR knockdowns on cell proliferation and antagonized the expression of P16, P21, PTEN, and RASSF1A induced by miR-22, suggesting that miR-22 is involved in the inhibition of SAM synthesis by targeting MTHFD2 and MTHFR.SAM, the most common methyl group donor, is involved in tumor progression by regulating multiple cell processes, such as proliferation, differentiation, cell cycle regulation, and apoptosis. SAM levels are related to different effects on the regulation of cell processesAM Figs. . These emiR-22 expression is inhibited by MeCP2. MiR-22 targets MTHFD2 and MTHFR to inhibit SAM synthesis and induces P16, P21, PTEN, and RASSF1A upregulation, which inhibits the proliferation of GC cells. This study suggests that the MeCP2 -miR-22-MTHFD2-MTHFR axis may be a therapeutic target for GC patients.In conclusion, 2 incubator at 37 °C. The cell lines were transfected with Lipofectamine 2000  following the manufacturer’s protocol.Paired GC and adjacent non-tumor gastric tissues were obtained from 36 patients who underwent surgical resection at the First Affiliated Hospital of Xi’an Jiaotong University and had not undergone previous surgery, radiotherapy, or chemotherapy. The tissue samples were immediately snap-frozen in liquid nitrogen until RNA extraction. Both tumor and non-tumor tissues were histologically confirmed. Informed consent was obtained from each patient, and the study was approved by the Institute Research Ethics Committee at Cancer Center, Xi’an Jiaotong University. Human GC AGS and MKN45 GC cell lines were obtained from the Cell Bank . The AGS and MKN45 cell lines were maintained in 1640 medium  supplemented with 10% fetal bovine serum (PAA Laboratories GmbH) and cultured in a humidified 5% CO6mir-22 or control lentivirus transfected with MKN45 cells were resuspended in 100 μL PBS and directly injected into the groin of each mouse. The tumor volumes were calculated using the following formula: V = L × (W × ½)2 . After 40 days, the GFP activity of xenografts in mice was detected using a small animal imaging system. After the mice were killed, the xenografts were removed, and the next step was performed.Five-week-old male BALB/c nude mice  were bred under specific pathogen-free conditions. All animal experiments were approved by the Institutional Animal Care and Use Committee of Xi’an Jiaotong University and were performed according to the institution’s guidelines for laboratory animals. A total of 1 × 10miR-22 precursor was subcloned into pcDNA6.2 GW/eGFP, which was purchased from Invitrogen. The synthetic primary transcript of miR-22 was designed into EcoRI and HindIII enzyme restriction sites to facilitate the cloning of vectors. The complementary sites in the 3′-UTR of MeCP2, MTHFD2, and MTHFR for miR-22 were cloned into a pmirGLO vector at the SacI and XhoI sites . The PEGFP-C1 vector containing cDNA of MeCP2 was purchased from GENEWIZ . The GFP-tagged MeCP2 vector was only used to identify the MeCP2 valid domain that interacts with miR-22. The MeCP2, MTHFD2, and MTHFR overexpression vector and control (OE control) were purchased from GeneChem . The inhibitor of miR-22-3p, a small interfering RNA (siRNA) targeting MeCP2, MTHFD2, and MTHFR, was purchased from GenePharma. The MiR-22 lentivirus overexpression vector was purchased from GeneChem .A human Equal numbers of cells were trypsinized, resuspended, and seeded at a density of 1000 cells/mL into six-well plates and incubated at 37 °C for 14 days. The colonies were stained with 0.1% crystal violet for 15 min and visualized by phase imaging.The cell cycle experiments were performed as follows: the cells were trypsinized, washed, and fixed with 70% ice-cold ethanol at 4 °C overnight. The cells were washed and suspended in 100 μl of 0.1 mg/mL RNase A and 100 μL of 0.05 mg/mL propidium iodide (PI) for 15 min at room temperature. The distribution of the cell cycle phase was examined by flow cytometry .Apoptosis analysis was performed following the manufacturer’s instructions . The percentage of cell phase was examined by flow cytometry .MeCP2ΔMBD) was amplified twice by performing an overlap PCR with the following primers: AF (5′-CAAGCTTCGATGGCTGCCGCTGCTGCCGCTGCTCC-3′), AR (5′-TCTGTTCCCTCCTGCTAGGGCTATCCCTGATGATGGACCTCCTT-3′), BF (5′-AAGGAGGTCCATCATCAGGGATAGCCCTAGCAGGAGGGAACAGA-3′), and BR (5′-CGGGATCCTCAGCTCACTCTCTCGGTGACAGGG-3′). The primers AF and BR contained HindIII and BamHI restriction sites, respectively, which were used to clone them into a PEGFP-C1 vector. The TRD deletion gene (MeCP2ΔTRD) was constructed using the same protocol, but different overhang primers: AR (5′-TCCTTGACTTCGATGCTGACGGTGCCCTCGCTGGTAGCGGCTTTG-3′) and BF (5′-CAAAGCCGCTACCAGCGAGGGCACCGTCAGCATCGAAGTCAAGGA-3′). The TRD partial deletion gene (MeCP2ΔTRD+NLS) was constructed based on the MeCP2ΔTRD vector using the following primers: AR (5′-TAGGAATGGCCTGGGGGTCGGCCTCAGCCTTCCTGCCCTCGCTGGTAGCGGCTT-3′) and BF (5′-CCCCCAGGCCATTCCTAAAAAGAGGGGCAGGAAGACCGTCAGCATCGAAGTCAA-3′).The MBD deletion gene  and antibodies against MeCP2, IgG, and GFP. The DNA was isolated, and 10% of the IP lysate was used as input. ChIP products were assayed by qPCR and PCR gel analysis. Two standard methods were utilized to normalize the ChIP-qPCR data: the percent input method and the fold enrichment method. The input and IP products were amplified by PCR, followed by electrophoresis analysis.ChIP was performed as previously describedCells were seeded on eight-well chamber slides. After 48 h, the cells were fixed with 4% paraformaldehyde for 15 min. After washing with PBS, the cells were permeabilized for 20 min with PBST (0.5% Triton X-100 in PBS) at room temperature and then washed with PBS. The cells were blocked with PBSB (0.1% Triton X-100 and 4% BSA in PBS) for 2 h at room temperature and then incubated with the primary antibody overnight at 4 °C. After washing three times with PBS, the cells were incubated with the secondary antibody for 2 h at room temperature in the dark. After washing three times with PBS, the slides were incubated with DAPI (1 µg/mL in 1× PBS) for 10 min at room temperature in the dark. After washing with PBS three times, the slides were observed under a fluorescence microscope.The levels of CpG methylation were measured with a BSP assay using a Bisulfite Conversion Kit  per the manufacturer’s instructions. In brief, genomic DNA was extracted using a genomic DNA extraction kit . The DNA was subjected to C/T conversion using a bisulfite reagent. We amplified the converted DNA with methylation-specific primers, designed by the MethPrimer and used TA cloning after a sequencing assay to measure the methylation level.miR-22 enhancers were amplified from the whole genome of a Hek-293 cell; enhancer1 and enhancer2 were constructed into a pGL3-Promoter Vector . The serial architecture enhancer was constructed by overlap PCR. The AGS and MKN45 cells were transfected with pGL3-Control Vector , pGL3-Promoter Vector, pGL3-Promoter Vector-E1, pGL3-Promoter Vector-E2, pGL3-Promoter Vector-E1+E2, and pGL3-Basic Vector . After 24 h, the luciferase activity was measured using a microplate system.The miR-22 and MeCP2, MTHFD2, and MTHFR. The Hek-293 cells were co-transfected with miR-22 expression vector and pmirGLO–MeCP2, -MTHFD2, and -MTHFR 3′UTR-WT or pmirGLO–MeCP2, -MTHFD2, and -MTHFR 3′UTR-MUT reporter vectors. After 48 h of transfection, the luciferase activity of each group was tested using the Dual-Glo Luciferase Assay kit (Promega) following the manufacturer’s protocol.A dual-luciferase assay was used to verify the relationship between MTHFD2 and miR-22 expression were downloaded from the UCSC Xena (https://xenabrowser.net)54. Type of data: gene expression RNAseq, Version: 2017-10-13, platform: IlluminaHiSeq_RNASeqV2. Type of data: miRNA mature strand expression RNAseq, version: 2017-09-08, platform: IlluminaHiSeq_miRNASeq. The Hi-C data, including A549 (GSE92819), Caki2 (GSE105465), H1-ESC (GSE52457), NCIH460 (GSE105725), T47D (GSE105697), and K562 (GSE63525) were downloaded from the GEO data set56. TAD borders were analyzed using the TADtool57.Data regarding Patient characteristics and clinico pathologicCondition and method of mobile phase in HPLC-MS assayAll primer sequence, antibody and siRNA informationDataset 1Dataset 2ADataset 2BDataset 3supplementary figure 1supplementary figure 2supplementary figure 3supplementary figure 4supplementary figure 5supplementary figure 6Supplementary figure legends


Squamous cell carcinoma of the head and neck (SCCHN) has a high recurrence rate after (chemo) radiation therapy [(C)RT]. The relationship between the changing levels of immune checkpoint molecules and immune cells in pre-(C)RT tissues and locally recurrent tissues in the irradiated field, after (C)RT completion, is not known. This study aimed to assess the changes in these immune parameters between pre-(C)RT tissue and the same area after local recurrence post-(C)RT. We retrospectively reviewed 30 (C)RT-treated patients with SCCHN. We performed immunohistochemical analyses on these immune parameters using paired tissue samples obtained pre-(C)RT and at local recurrence sites post-(C)RT. No significant changes in immune parameters were found between the pre-(C)RT and locally recurrent tissues. An increased density of CD8+ tumor-infiltrating lymphocytes (TILs) showed a significantly positive correlation with PD-L expression on tumor cells (TC-PD-L1). Patients with increased TC-PD-L1 expression and CD8+TIL density showed favourable prognosis, and one of them showed a favourable response to nivolumab therapy. Our study shows a positive association between TC-PD-L1 upregulation and increased CD8+TIL density, and demonstrates that patients with these changes have a favourable survival outcome. Programmed death 1 (PD-1) is a receptor expressed on the T cell membrane, which binds to programmed death-ligand 1 (PD-L1) on tumor cells, resulting in T cell anergy and inhibition of antitumor activity2. In the case of squamous cell carcinoma of the head and neck (SCCHN), the “CheckMate 141” phase 3 trial demonstrated that nivolumab therapy, which inhibits the PD-l/PD-L1 and PD-L2 axis, increases the survival periods of people with recurrent/metastatic SCCHN (RMSCCHN) compared to the therapy selected by investigators3. The expression levels of PD-L1 on tumor and immune cells is a known predictive biomarker that correlates with the efficacy of PD-1/PD-L1 inhibitors4. This correlation was confirmed in a recent analysis of 2-year long-term survival data from CheckMate 141, showing that nivolumab therapy results in favourable overall survival (OS) and OS benefits in patients with PD-L1 expression ≥ 1%4.Immune checkpoint inhibitors (ICI) are utilized in systemic therapies that induce T cells to specifically target and kill tumor cells5. Recent studies have shown that PD-L1 expression changed in response to chemotherapy (CT), RT, and molecular target therapy8. RT is known to induce cancer cell death and has positive immune-modulatory abilities, including causing the increased expression of MHC-class I molecules, releasing various tumor antigens, and accumulating tumor-infiltrating lymphocytes (TILs)11. Furthermore, a recent study showed that tumor cell PD-L1 expression is upregulated in response to IFN-gamma, produced by CD8+ T cells, through a process named adoptive immune resistance status. Thus, this kind of immune escape might indicate favourable responses to PD-1/PD-L1 inhibitors12.Radiation therapy (RT) or chemo-radiation therapy (CRT) is now a standard procedure for patients with head and neck cancer14. Leduc et al.15 reported that docetaxel, platinum, and fluorouracil (TPF) induction chemotherapy for head and neck cancer increased PD-L1 expression on tumor cells and immune cells. Lim et al.14 reported that patients with oesophageal cancer receiving neoadjuvant chemo-radiotherapy displayed a significant increase in PD-L1 expression between pre-CRT and post-CRT evaluations, in 12 paired tissue samples.Several studies have reported changes in the immune checkpoint molecule expression levels that correlate with immune cell levels between the tissue samples obtained at pre-treatment sites and sequentially surgically resected tissue after neoadjuvant CT or CRTHowever, to our knowledge, no information regarding the association of immune checkpoint molecules and immune cells between pre-(C)RT and local recurrence sites post-(C)RT is available. This study aimed to investigate the changes in immune parameters of paired-biopsy specimens obtained from pre-(C)RT and local recurrence tissue sites post-(C)RT in patients with head and neck cancer.The clinical characteristics of the 30 patients enrolled in this study are shown in Table The expression levels of PD-L1 on tumor cells (TC-PD-L1), PD-L1 on immune cells in the stroma (IC-PD-L1), PD-L2 on tumor cells (TC-PD-L2), PD-L2 on immune cells in the stroma (IC-PD-L2), HLA-class I expression, and CD8+TIL density, are shown in Table p = 0.418; IC-PD-L1, p = 0.422; TC-PD-L2, p = 0.057; IC-PD-L2, p = 0.190; HLA-class I, p = 0.476; CD8+TILs, p = 0.976).The correlations between changes in the density of TC-PD-L1, IC-PD-L1, TC-PD-L2, IC-PD-L2, HLA-class I, and CD8+TILs in pre-(C)RT and local recurrences in tissues are shown in Fig. p = 0.007), whereas no significant differences were observed for IC-PD-L1 (p = 0.260), TC-PD-L2 (p = 1.000), IC-PD-L2 (p = 1.000), and HLA-class I expression (p = 0.226). The correlations between increased TC-PD-L1 expression and CD8+TIL density or with other changes in immune parameters (TC-PD-L1 expression and CD8+TIL density) and patient characteristics are shown in Supplementary Table The correlations between changes in CD8+TIL density and TC-PD-L1, IC-PD-L1, TC-PD-L2, IC-PD-L2, or HLA-class I expression are shown in Table p = 0.048) and OS (p = 0.067).A Kaplan–Meier analysis was performed to evaluate DSS and OS for the patients with both increased TC-PD-L1 expression and increased CD8+TIL density, as well as for patients in the other groups  Fig. . PatientTwo patients, both of whom had p16-positive oropharyngeal cancer, received nivolumab therapy Fig. . Patient16. Herein, we investigated the association between changes in immune parameters, including PD-L1 using pre-(C)RT and locally recurrent paired tissue samples. Our results showed that there were no significant changes in the immune parameters between pre-(C)RT and local recurrence sites after (C)RT.A recent study reported that radiation-induced immune responses led to PD-L1 upregulation and increased dendritic and cytotoxic T cell activation and proliferation15. In studies involving patients with squamous oesophageal cancer or rectal cancer receiving CRT, CRT reportedly significantly increased PD-L1 expression14. Further, Dovedi et al.17 reported that in a pre-clinical study using a mouse model, radiation-induced upregulation of PD-L1 expression on tumor cells peaked at 72 h after RT and then began to decline. Additionally, Patel et al.13 reported that the analysis of PD-L1 expression at a median of 28 days after completion of RT might be suboptimal.Recent reports have shown a definite increase in PD-L1 expression levels and immune infiltrating cells induced by chemotherapy and CRT. In the patient’s cohort with SCCHN, induction chemotherapy increased PD-L1 expression on tumor cells and immune cells, including CD8+TILsAccordingly, changes in immune parameters including PD-L1 expression, were closely associated with chemotherapy, (C)RT, and the period after the completion of (C)RT. Thus, we investigated this using recurrent tissue samples, but not pre-(C)RT tissue samples, to exactly evaluate the current tumor immune microenvironment (TIM). This is because of TIM between pre-(C)RT tumor tissue sites and tumor recurrence sites after (C)RT might differ. To the best of our knowledge, this is the first investigation of changes in immune parameters between pre-(C)RT and post-(C)RT recurrence in patients with head and neck cancer.18. A TIM with both upregulated PD-L1 expression and an increased density of CD8+TILs was considered to be a type I TIM, in the conditions of immune escape induced by the inactivation of T cells19. Dovedi et al.17 found that PD-L1 expression in tumor cells induced by IFN-gamma, produced by CD8+T cells, may act as a biomarker for local antitumor response levels. Previous reports showed that, in several cancer types, type I TIMs in several cancer types had favourable prognoses compared to other types. In addition, type I TIM (adaptive immune resistance) may be the most likely to respond to PD-1/PD-L1 inhibitors21. Of the two patients undergoing nivolumab monotherapy, patient 1 had upregulated PD-L1 expression on tumor cells and an increased CD8+TIL density and showed a clinically complete response to nivolumab therapy. However, patient 2 showed no PD-L1 upregulation and no increase in CD8+TIL density, had a clinically defined progressive disease after nivolumab therapy, and died of cancer. Thus, these results suggest that investigating the changes in PD-L1 and CD8+TIL density might help evaluate an immunologic shift via interferon-gamma, induced by (C)RT; furthermore, this might be useful for predicting the responses to immune checkpoint inhibitor treatments in cases of recurrence after a definitive (C)RT.We examined the association of changes in immune checkpoint molecule expression and the number of immune cells and found a positive correlation between TC-PD-L1 expression and CD8+TIL density. Furthermore, patients with upregulated PD-L1 expression and an increase in CD8+TIL density had significantly more favourable prognoses. A recent report described the four types of TIM, type I (adaptive immune resistance), type II , type III (intrinsic induction), and type IV (tolerance)Our study had several limitations that should be taken into consideration. First, this study was retrospective and consisted of a small number of patients. Second, clinical characteristics at the primary and treatment sites (CRT or RT) were heterogeneous. Third, precise evaluation of TIL densities, PD-L1, PD-L2, and HLA-class I expression might be affected by intratumoral heterogeneity, due to biopsy tissue specimens.In summary, although we show no definite association between the changes in PD-L1 or PD-L2 expression (tumor and immune cells), TIL densities, and HLA-class I expression between pre-(C) RT tissue and the local recurrence sites after (C)RT, there was a positive association between the upregulation of PD-L1 expression on tumor cells and an increased CD8+TIL density. Furthermore, patients with both upregulation of PD-L1 expression on tumor cells and an increased CD8+TIL density had more favourable survival likelihoods compared with the other patients. Considering the limitations of this study, further large prospective studies are required to confirm our results.We retrospectively screened 30 patients diagnosed with SCCHN at Kurume University Hospital between 2004 and 2016. The selected patients had been diagnosed pathologically with SCCHN, had local recurrence after the completion of radiotherapy or chemo-radiotherapy, and had adequate frozen histological specimens containing tumor cells in paired tissue samples from both pre-(C)RT and local recurrence sites. Local recurrence in the radiated field was regularly examined using trans-nasal endoscopy, and tissue biopsies were performed if local recurrence was suspected. The malignant positivity of the biopsy confirmed local recurrence. Patient exclusion criteria included those with distant metastases from the local recurrence, those undergoing palliative treatments, or those with inoperable disease. The present study was conducted in accordance with the provisions outlined in the Declaration of Helsinki and was approved by the Institutional Review Boards of Kurume university. Informed consent was obtained from the patients. However, if the patients died, informed consent was obtained from the patient’s family.22, we used 4-μm-thick sections of formalin-fixed, paraffin-embedded tissues. The tissue sections were mounted on glass slides and incubated with an anti-PD-L1 rabbit monoclonal antibody  using BenchMark ULTRA . Briefly, each slide was heat-treated using Ventana’s cell conditioning solution (CC1) for 30 min and incubated with the PD-L1 antibody for 30 min. The automated system used the ultraVIEW DAB detection kit with 3,3′ diaminobenzidine (DAB) as the chromogen. Mouse monoclonal human PD-L2  antibody was used after treatment with proteinase K  for 5 min, after which the antibody was added and incubated for 30 min23. This automated system used a refined polymer detection system  with HRP (Horseradish peroxidase)-polymer bound to an anti-mouse secondary antibody, using 3,3′ diaminobenzidine (DAB) as the chromogen. Immunostaining for HLA-class I  and CD8  was performed on the fully automated Bond-III system. Briefly, each slide was treated using the heat-induced epitope retrieval solution 2 for 10 min at 99 °C, before being incubated with the primary antibody for 30 min at room temperature. This was followed by incubation with the secondary antibody for 30 min at room temperature.As previously reported24, the expression of PD-L1 (TC-PD-L1) and PD-L2 (TC-PD-L2) on tumor cells was evaluated using a defined tumor proportion score. The expression of PD-L1 (IC-PD-L1) and PD-L2 (IC-PD-L2) on immune cells (lymphocytes or macrophages) in the stroma occupying the tumor were assessed using the mononuclear immune cell density score. Furthermore, CD8+TILs were counted in five high-magnification fields of view (magnification: 40×), and these averages were assessed.All IHC findings were evaluated by two experienced pathologists (A.K. and J.A.) who were unaware of the patients’ conditions. Any disagreement between the pathologists was resolved by a joint review to obtain a single consensus category. As previously reportedp < 0.05. Statistical analyses were performed using JMP Pro 14 statistical software .Correlations between the changes to immune parameters in the local lesions before and after (C)RT were analyzed using a Wilcoxon signed-rank test. Disease-specific survival (DSS) and OS were calculated from the date of initial treatment after local recurrence to the date of death due to cancer or any cause, respectively. The Kaplan–Meier method was used to assess patient survival curves, and the log-rank test was used to evaluate the differences between groups. All tests were two-sided, and the differences were considered statistically significant at Supplementary table S1.


Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) proteins are a diverse and evolutionarily conserved family of cytidine deaminases that provide a variety of functions from tissue-specific gene expression and immunoglobulin diversity to control of viruses and retrotransposons. APOBEC family expansion has been documented among mammalian species, suggesting a powerful selection for their activity. Enzymes with a duplicated zinc-binding domain often have catalytically active and inactive domains, yet both have antiviral function. Although APOBEC antiviral function was discovered through hypermutation of HIV-1 genomes lacking an active Vif protein, much evidence indicates that APOBECs also inhibit virus replication through mechanisms other than mutagenesis. Multiple steps of the viral replication cycle may be affected, although nucleic acid replication is a primary target. Packaging of APOBECs into virions was first noted with HIV-1, yet is not a prerequisite for viral inhibition. APOBEC antagonism may occur in viral producer and recipient cells. Signatures of APOBEC activity include G-to-A and C-to-T mutations in a particular sequence context. The importance of APOBEC activity for viral inhibition is reflected in the identification of numerous viral factors, including HIV-1 Vif, which are dedicated to antagonism of these deaminases. Such viral antagonists often are only partially successful, leading to APOBEC selection for viral variants that enhance replication or avoid immune elimination. Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) proteins constitute a protein family that deaminates cytosines within nucleic acids. This review begins with a description of APOBEC proteins and their enzymatic activity, structure, and function. This description is followed by a discussion of the primarily inhibitory relationship between APOBECs and virus replication. This inhibitory relationship is exemplified by the multiple mechanisms used by viruses to thwart APOBEC function as well as the induction of APOBEC proteins by type I interferons (IFNs), which induce the antiviral state in infected and neighboring cells. Due to the large numbers of papers on APOBEC enzymes, we will focus on publications since our previous review with the Harris laboratory .APOBEC gene family, namely APOBEC1 (A1), APOBEC2 (A2), APOBEC3A (A3A), APOBEC3B (A3B), APOBEC3C (A3C), APOBEC3D (A3D), APOBEC3F (A3F), APOBEC3G (A3G), APOBEC3H (A3H), APOBEC4 (A4) and activation-induced cytidine deaminase (AICDA). Other animal species have fewer APOBEC genes . D. D156]. Although hypermutation of HIV-1 proviruses by A3 deaminases has been widely studied in cell lines, there is ample evidence of A3 activity in infected patients. Patient samples showed G-to-A hypermutation of proviruses recovered from HIV-1-infected peripheral blood mononuclear cells (PBMCs). Although many normal proviruses were recovered, 43% of the mutant proviruses had in-frame stop codons and non-synonymous nucleotide changes . High A3gag, pol, and nef sequences encoding immunodominant CD8+ T cell epitopes [The beneficial role of A3-mediated mutations for in vivo HIV-1 infection has been documented in multiple studies. Results showed that deaminase-generated variability of HLA-recognized viral epitopes allowed escape of virally infected cells from cytotoxic CD8+ T cells ,165,166.epitopes . Howeverepitopes . For HLAepitopes . A recenThe lethal effect of A3G-induced HIV-1 hypermutations generated in vivo was shown by analyzing viral polymerase sequences from 3000 chronically infected patients . In thisChronically infected HIV-1 patients are known to harbor numerous defective proviruses and a reservoir of virus-positive cells . Open quHIV-1 encodes the Vif protein to inhibit the function of A3s to prevent hypermutation and allow efficient virion replication ,179,180.Recent studies have provided additional details of Vif antagonism of E3 enzymes. A RING-finger E3 ubiquitin ligase (MDM2) downregulates Vif through proteasomal degradation ,190. Co-A second mechanism for Vif-mediated antagonism occurs through restricted translation of A3G mRNA. Vif inhibits the translation of A3G mRNA to further decrease its intracellular level in HIV-1-infected cells. Subsequently it prevent A3G packaging into virions ,196,197.Although Vif decreases the level of A3G by several means to prevent its antiviral activities, cells have developed several ways to counteract Vif and stabilize A3G. First, cellular proteins may directly interrupt A3G degradation by Vif ,199. A hcyclin-F E3 ligase complex, leading to proteasomal degradation. Consequently, cyclin F stabilizes A3G levels, which increases virion incorporation and decreases infectivity. However, in primary HIV-1-infected T cells, cyclin F levels are downregulated, although the mechanism is unknown [A3G levels also are maintained through recruitment of cellular proteins to reduce Vif levels ,202. Cyc unknown .Together, these studies suggest that HIV-1 maintains a fine balance between Vif-mediated destruction of A3 enzymes and the ability of the host to resist viral infection. A3G-induced mutations allow rapid virus selection, particularly in the face of high interferon levels or antiviral drugs. Although reverse transcription allows basal levels of mutation, mutagenesis by A3 enzymes provides viruses with the ability to respond immediately to changes in the host environment.Other lentiviruses counteract APOBECs by different mechanisms. The Vif protein from the poorly pathogenic feline immunodeficiency virus (FIV) subtype B only weakly antagonizes feline A3 proteins. Instead, the viral protease cleaves feline A3 proteins in released virions  Table 1Table 1. pol and tax genes, respectively) typical of A3G preferred motifs, although other deaminases, such as AID, were implicated. No significant correlation was observed between A3G expression levels and the number of G-to-A mutations in proviral genomes [HBZ gene lacks A3G-targeted sequence motifs or evidence of A3 mutation, consistent with selection for its function [Human T-Cell Leukemia Virus type 1 (HTLV-1) is a human deltaretrovirus that causes both Adult T-Cell Leukemia/Lymphoma (ATL) and HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) . Like HI genomes . Howeverfunction .Although HTLV-1 hypermutation is rare in vivo, a small percentage of genomes (0.1–0.5%) are edited extensively, and up to 97% of cytidines in the edited genomes are deaminated . Cell cucis to inhibit A3G packaging. Thus, A3G packaging efficiency into nascent virions is reduced compared to HIV-1 particles made in the absence of Vif [HBZ gene needed for proliferation of HTLV-1-infected cells [Two reasons have been proposed to explain the relative resistance of HTLV-1 to APOBEC-mediated hypermutation . First, e of Vif . Second,e of Vif ,216,217.e of Vif . A3 mutaed cells  (Table 1The first in vivo evidence indicating that A3 proteins protect their hosts from retrovirus infection was obtained using the betaretrovirus Mouse Mammary Tumor Virus (MMTV) . Unlike Mtv sequences [In contrast, our experiments with two different MMTV strains that lack expression of the Rem protein showed APOBEC-mediated deamination in vivo . Rem is equences , indicatenv gene from proviral DNA isolated from MMTV-SD and MMTV-WT-induced tumors. Both transition and transversion mutations were increased in MMTV-SD proviruses relative to MMTV-WT proviruses. We observed that G-to-A mutations on the plus strand were elevated in the absence of Rem expression, yet C-to-T mutations showed the largest increase on both proviral strands [To determine whether selection of proviruses occurs due to mutation by APOBEC deaminases, we performed sequence analysis of the  strands . Human A strands ,222. How strands . AID is  strands . TherefoC-3′ motif, a known “hotspot” for AID hypermutation [C-3′ and 5′-ATC-3′ motifs, which are associated with mA3 activity, in MMTV-SD proviruses [C-3′ mutations known as AID “coldspots” [Aicda−/− (AID-knockout) mice compared to wild-type BALB/c animals. Low-level proviral C-to-T mutations were not significantly different in tumors from AID-knockout and wild-type mice when the inoculated virus expressed Rem [C’3′ and 5′-TYC-3′ motifs  declined in Aicda−/− mice. These data suggest that MMTV-encoded Rem is a Vif-like antagonist of mutations induced by AID, and perhaps other APOBECs, during viral replication in vivo [Sequence motif analysis was performed to determine the identity of APOBEC enzymes targeting MMTV proviruses lacking Rem expression. Significant mutations of the 5′-WRmutation , were obmutation . Howeveroviruses ,224,225,ldspots” ,227. Furssed Rem . Surpris in vivo   expression from an internal viral promoter used in lymphocytes . MMTV Satagonist .To determine whether Rem functions similar to HIV-1 Vif by targeting A3 enzymes for degradation, we co-expressed either AID or mA3 in the presence or absence of Rem. Rem expression alone or in the context of an intact provirus led to proteasome-dependent degradation of AID, but not mA3. In addition, as previously shown, mA3 was packaged into virions . Our resmA3-knockout mice and infected with AKV also showed higher infectivity than virus-infected splenocytes from mA3+/+ or heterozygous mA3+/− mice [CAA-3′ [C-3′ or 5′-TC-3′) [Like the betaretrovirus MMTV, gammaretroviruses are restricted by APOBEC proteins. AKV, an endogenous, ecotropic murine leukemia virus (MLV) strain, which also replicates as an exogenous virus, has been shown to be hypermutated by mA3 ,229,230.+/− mice . More regag gene. This product, glyco-Gag (gGag), uses a CUG initiation codon upstream and in the same frame as the Gag polyprotein [gag reading frame results in 88 additional amino acids at the Gag N-terminus to give a signal peptide that allows ER insertion and gGag Pr85 production [Several studies have shown that gammaretroviruses make a glycosylated translation product of the yprotein ,232,233 yprotein ,235,236 oduction . Pr85 gGoduction ,239. Theoduction ,241. Theoduction . The numoduction . HoweverC context, whereas mA3 preferentially mutated in the CC dinucleotide context in vitro. The investigators also obtained F-MLV sequences from spleens of infected B6 wild-type mice. TC motif mutations in these transcripts were interpreted as mA1 mutagenic activity that restricts F-MLV in vivo [Although the role of mA3 has been the primary focus of research on murine retroviruses, other APOBEC proteins also have been studied. Mouse APOBEC1 (mA1) was first shown to restrict MLV, but a second study contradicted these results ,243. Com in vivo .mA3 gene demonstrated lower numbers of GA to AA mutations compared to wild-type or mA1-knockout mice, suggesting that mA3 targeted the F-MLV reverse transcripts in the TC, but not the CC context. Since mA1 could potentially edit retroviral RNA, this study also analyzed the percentage of F-MLV TC-TT and CC-CT mutations within viral transcripts. No differences in dinucleotide mutation frequencies were observed between viral RNA obtained from mice with or without a functional mA1 gene. Therefore, this study indicated that mA1 does not restrict F-MLV replication in vivo.Subsequently, several groups demonstrated that mA3 also edits cytidines in the TC motif ,224,225.Spumavirinae also are restricted by APOBECs. The foamy viruses use Bet protein to sequester A3 proteins away from the virus assembly site and prevent their incorporation into virions [gag gene of the SFV genome [The retroviral subfamily  virions ,248,249  virions . In cont virions . Unlike  virions ,251 and  virions ,251. Sim virions ,254,255.V genome . In humaV genome .APOBECs also restrict other RNA-containing viruses such as coronaviruses, measles viruses, and enteroviruses 71 (EV71) ,260,261.To test this idea, Milewask et al.  infectedTo determine whether A3 proteins cause hypermutation of the viral genome, recombinant GC-rich HCoV virus strains were modified with GC-rich potential “hot-spots” for cytidine deamination. No enrichment of G-to-A or C-to-T mutations was observed in coronaviruses containing these GC-rich regions after infection of cells expressing different A3s. Sequence heterogeneity of coronaviruses was detected in infected cells expressing A3F. Both wild-type and catalytic inactive mutants of A3C, A3F, and A3H interacted with the HCoV-NL63 nucleocapsid (N) protein, but not other A3 proteins . Colocalization also was observed between A3C and NL63 N proteins, suggesting that APOBEC proteins restrict coronavirus infections . FurtherEpstein-Barr Virus (EBV) belongs to the gammaherpesvirus subfamily and infects B cells to induce infectious mononucleosis and multiple tumors, such as Burkitt lymphoma . In EBV-In addition, all A3 family genes, except A3A, are significantly upregulated in EBV-positive gastric cancers compared to EBV-negative gastric cancers . Cheng eLike the gammaherpesviruses, EBV and KSHV, the alphaherpesvirus herpes simplex virus type 1 (HSV-1) encodes a large RNR subunit (ICP6), which binds A3A and A3B and relocalizes these deaminases to the cytosol. Therefore, both alpha- and gammaherpesviruses antagonize A3A and A3B by altering their subcellular localization, suggesting that A3 nuclear functions inhibit herpesvirus DNA replication ,275. HowHuman cytomegalovirus (HCMV), a betaherpesvirus, infects most individuals worldwide. Although HCMV infection in healthy individuals is mostly asymptomatic, HCMV commonly causes congenital infections, which may result in neurological defects, such as deafness or mental retardation in infants ,278. HCMIn support of this idea, HCMV infection has been shown to increase different A3 members in a cell type-specific manner. In HCMV-infected primary human foreskin fibroblasts (HFFs), A3G was induced by IFNβ, yet did not affect HCMV replication. A3G knockout or A3G expression from an adenovirus vector also had no effect . PautassHepadnaviridae family is a major cause of liver cirrhosis and hepatocellular carcinoma [Hepatitis B Virus (HBV) in the arcinoma ,281. As arcinoma ,283. A3Carcinoma . Since Aarcinoma . OverexpType I interferon (IFNα and β) or type III interferon (IFNλ1-3) treatment, which induced A3 enzymes with different kinetics, reduced HBV cccDNA and increased cytidine deamination. Type I and III IFNs increased A3A and A3G, but not A3B levels in hepatocytes . A3B hasAlthough studies of Vif-minus HIV-1 first focused attention on A3G-mediated hypermutation, APOBECs also inhibit HIV-1 replication through deamination-independent mechanisms ,291,292.A recent study examined A3G deamination-independent inhibition and the “roadblock” model. These experiments revealed that A3G binds directly to reverse transcriptase, rather than viral RNA, to inhibit HIV-1 DNA synthesis ,303. OthMost studies about APOBEC-mediated antagonism of HIV-1 have focused on A3s since these enzymes are expressed in target cells, such as T cells and macrophages. Human A4 did not cause cytidine deamination of HIV-1 cDNA and, in contrast to other APOBECs, even enhanced replication . NonetheB6 protein showed a higher selectivity at the -2 position for the canonical 5′-TTC-3′ substrate, whereas the mA3BALB enzyme preferred the 5′-ATC-3′ motif with a higher selectivity for the −1 position [Experiments in mice suggested that mA3 has a deamination-independent mechanism for MMTV inhibition . Additioposition . Thus, mposition ,91,312.Our results with the MMTV T-cell-tropic strain, TBLV, also suggest that tumor-derived proviruses from B6 mice have limited differences in their mutation profiles in the presence or absence of Rem compared with those from BALB/c mice   has been shown to exclude the host-derived APOBEC protein, rhesus A3G (rA3G), from virion particles . MPMV GaMouse APOBEC3 restricts infection by exogenous murine gammaretroviruses, such as Friend, Moloney, and AKV murine leukemia viruses  ,221,319.Co-immunoprecipitation of overexpressed M-MLV RT and mA3 in 293T cells revealed that both the wild-type mA3 and the E73Q/E253Q mutant bind reverse transcriptase. Binding was not dependent on RNA since RNase A treatment of the extracts prior to the co-immunoprecipitation did not affect the interaction. Overall, these results confirmed that mA3 restricts retroviral replication independent of deamination activity in vivo . RestricgGag) replicated more poorly in wild-type mice compared to replication observed in mA3-knockout mice. Cores of mutant virus were more unstable than wild-type virus cores, allowing mA3 access to the reverse transcription complex [gGag mutant virus reverted to yield wild-type gGag by 6 weeks post-infection in B6 mice that express mA3. Reversion did not occur in the mA3-knockout mice, but did occur in transgenic mice on the knockout background expressing the mA3 E73Q/E253Q mutant, which lacks catalytic activity and is not packaged into virions [How do MLVs counteract deaminase-independent mA3 inhibition? Similar to studies with AKV, the gGag mutant of M-MLV  ,328. GouA subsequent study  showed tPicornaviridae family), which causes hand, food, and mouth disease (HFMD) [Enterovirus 71 (EV71) is a single-stranded positive-sense RNA virus (e (HFMD) . In 2018e (HFMD) . EV71 vie (HFMD) .The activity of A3G against a number of different positive-stranded viruses has been used as the rationale for development of antiviral drugs. The drug IMB-26 previously shown to stabilize A3G levels during HIV-1 infection  was the Paramyxoviridae family  were inhibited in human A3G-overexpressing monkey cells as well as in T-cell lines expressing A3G at physiological levels [The negative-stranded RNA viruses in the l levels . The inhl levels . A subsel levels  showed tl levels . These dOrthomyxoviridae family, influenza A virus (IAV), was previously shown to upregulate A3G, but not A3F, transcription in infected cells. Upregulation was induced by the accumulation of viral RNA in infected cells [APOBEC genes may be informative for their effects on IAV replication.Another negative-stranded RNA virus in the ed cells . Increased cells . A more ed cells . TherefoVery few studies have investigated the effects of the APOBEC family members, A2 and A4. However, the A4 cytidine deaminase was shown to inhibit the paramyxovirus, Newcastle Disease Virus (NDV). A4 affected NDV replication in chickens by reducing viral RNA levels  Table 1Table 1. In vivo experiments using mA3-knockout mice with transgenic hA3 alleles suggested that A3s do not affect HSV-1 replication or genome deamination . The faiMost studies have reported that the HBV genome is edited by A3 proteins. However, HBV replication also appears to be inhibited by a deaminase-independent mechanism ,341,342.Polyomaviruses are small, non-enveloped DNA viruses that cause or are associated with various human diseases, including nephropathy and Merkel cell carcinoma . InfectiAPOBECs restrict several types of DNA elements within mammalian genomes that spread through reverse transcription. These elements include long terminal repeat (LTR)-containing retrotransposons, also known as endogenous retroviruses (ERVs), and non-LTR retrotransposons, such as long interspersed element 1 (LINE-1 or L1) or Alu ,354,355.Tlr7) knockout mice, which show emergence of ERVs as well as hA3G+/+mA3−/− mice [hA3G+/+mA3−/−Tlr7−/− mice on the B6 background to control mice lacking hA3G expression, hA3G, but not mA3, restricted ERV emergence [Tlr7−/− mice [ERVs compose approximately 8 to 10% of the human and mouse genomes ,357,358.−/− mice , the effmergence . In cont−/− mice . However−/− mice . TherefoRecent bioinformatic analyses have revealed enrichment of G-to-A editing in ERVs within genomes from different species ,364. TheAnother study of 160 mammalian species showed that ERVs drive the evolution of A3 genes . A stronLINE-1/L1 elements are retrotransposons that lack envelope genes and LTRs, but they move through reverse transcription and are subject to APOBEC-mediated antagonism. Non-LTR retrotransposons comprise approximately 30% of the human genome . Most anAID and its catalytically inactive mutant both have been shown to inhibit L1 retrotransposition, indicating that restriction does not occur through deamination and hypermutation . The DNAAll A3 family proteins have been demonstrated to inhibit L1 and/or Alu retrotransposition, ,374,376,hA3G+/+mA3−/−Tlr7−/− B6 transgenic mice (discussed above), human A3G does not suppress L1 in vivo [A3B is a nuclear cytidine deaminase expressed in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) ,352,368. in vivo , consist in vivo . Earlier in vivo ,378.A3H was shown to have weak anti-L1 activity ,376, butC-3′ motifs may have evolved from their capability for damaging L1 while minimizing host gene damage [Recent genomic analysis has provided more information about the roles of APOBECs in restricting L1 in vivo ,380. Bioe damage .In a retrotransposition assay based in cell culture, A1 was shown to reduce the mobility of L1 by a deaminase-independent mechanism . The A1 Viruses have provided many insights into host cell functions, and the APOBEC family proteins are no exception. Cytidine deaminases have evolved to serve multiple purposes, including host-specific functions, such as lipid metabolism, as well as defense against infectious viruses and retrotransposons. Evolutionary studies of the APOBEC cytidine deaminases have revealed the early appearance of A2 and AID, yet only AID has demonstrable deaminase activity. Interestingly, virus-mediated antagonism of APOBECs can be divided into deaminase-dependent and deaminase-independent mechanisms, even with respect to the same viral target and APOBEC family member. In fact, deaminase-independent mechanisms appear to have been the first to evolve to inhibit retrotransposons. Although the deamination-independent mechanisms appear to be prevalent, they have not been as widely studied as the mechanisms of deamination. The mechanistic choice may depend on host and/or cell-specific factors. APOBEC members often are inducible, particularly through interferons, which produce an antiviral response within the infected cells to provide an early warning system for their uninfected neighbors. Nevertheless, APOBECs have dual effects. Just as AID generates hypermutation of the immunoglobulin variable region for antibody affinity maturation and antiviral activity, APOBEC enzymes also generate viral diversification. RNA editing by A1 or other APOBECs with different cofactors could induce tissue-specific antiviral activities. From RNA-containing coronaviruses and retroviruses to DNA-containing herpesviruses and HBV, APOBECs provide a means for control of virus replication, but also harbor the potential for emergence of new pathogens and viral resistance to drugs or host immune responses.


BackgroundBeta (β)-thalassemia major patients frequently suffer from many vascular problems. Thrombophilia is a blood disorder that comprises imbalances in the blood coagulating factor due to ecological and hereditary components. Previous evidence shows that thrombosis is the commonest risk in beta-thalassemia patients. Several studies have examined that MTHFR C677T, prothrombin G20210A (PT G20210A), and Factor V Leiden G1691A (FVL G1691A) polymorphism play a crucial role in the development of β-thalassemia major, yet the result was questionable and uncertain. Therefore, in this study, we executed the correlation between these gene polymorphisms with β-thalassemia major patients.MethodsSuitable keywords were used to search related articles in PubMed, Google Scholar, and Web of Science. In this random-effects meta-analysis, we analyzed the odds ratio (OR) for the estimation of risk.Results2=61%) and allele C vs T (I2=71%) of MTHFR and genotype GG vs GA (I2=95%), GG vs GA+AA (I2=95%), GA vs GG+AA (I2=95%), and allele G vs A (I2=93%) of FVL G1691A.A total of nine research articles with 645 β-thalassemia major patients and 989 healthy controls were incorporated in this meta-analysis. The pooled OR was assessed in MTHFR C677T, PT G20210A, and FVL G1691A polymorphism. This random-effects meta-analysis demonstrated that MTHFR C677T, PT G20210A, and FVL G1691A gene polymorphism did not significantly associate with β-thalassemia major. Moreover, the heterogeneity was significantly found in genotype CC vs CT+TT C677T  gene polymorphism are not a risk factor for β-thalassemia major. Beta (β)-thalassemia major is a single genetic disease affected by mutations in β-globin genes. These mutation causes a complete deficiency or reduces the formation of β-globin chains . This diHighly thromboembolic events have been associated with β-thalassemia, which leads to the thrombosis of arterial or venous blood -6. VarioMTHFR C667T gene polymorphism altered the function of enzymes and increased the risk of thrombosis . A previVarious epidemiological studies have explored the associations of the MTHFR C677T, PT G20210A, and FVL G1691A gene polymorphism with β-thalassemia. Still, the outcomes of the studies were conflicting and indecisive. In light of the above evidence, we executed this meta-analysis of accessible articles connecting MTHFR C677T, PT G20210A, and FVL G1691A gene polymorphism to the risk of β- thalassemia.Setting and study designThis meta-analysis study was done in the Center for Advance Research (CFAR), Cytogenetics Lab, King George’s Medical University, Lucknow.Identification and methods: search and selection of studiesβ-thalassemia related articles were independently recognized by investigators from PubMed, Google, and Web of Science. The search terms were as follows:  AND . Moreover, the references of all articles and reviews were manually searched for extra appropriate studies.Results were restricted to MTHFR C677T, PT G20210A, and FVL G1691A gene polymorphism and β-thalassemia major in humans. A total of 780 articles were identified in PubMed, Google, and Web of Science. Inclusion criteria for the selection and inclusion of studies were: i) Case‑control study of β-thalassemia patients and controls ; (2) MTHFR C677T, PT G20210A, and FVL G1691A gene polymorphism; (3) Published articles; (4) Full article in English; (5) Studies with the expression of MTHFR C677T, PT G20210A, and FVL G1691A gene, and studies on animals or in the lab were excluded. The association of MTHFR (C677T), PT (G20210A), and FVL (G1691A) gene with β-thalassemia and control was the main concern for the selection of studies.Data collectionData were extracted by authors individually from articles text, figures, or tables in each published article. The above-mentioned details were collected from individual studies, i.e., types of SNP, the technique for polymorphism, and the number of patients and controls Table .Data synthesisThe data were obtained from every study: title of the article, name of the principal author’s, journal name, date of publication, name of nation, and strategy utilized for genotyping.Statistical analysisRevMan 5.3 (Review Manager version 5.3) of the Cochrane Collaboration, London, United Kingdom, was used for statistical interpretation. The odds ratio (OR) was used for risk assessment. In addition, the heterogeneity among studies was estimated by the Q test with p-values <0.05 and >50% I2 was considered significant. Initially, the associations-based studies were analyzed; after that, the subgroup examinations by SNP type were accomplished to find the SNP type-specific impacts.Search resultsThe search outcomes of published articles have been shown in Figure MTHFR C677T gene polymorphism and the risk of β-thalassemiaFor MTHFR C677T polymorphism, a total of eight studies, including 612 β-thalassemia patients and 871 healthy controls were examined Table .Prothrombin G20210A gene polymorphism and the risk of β-thalassemiaFor prothrombin G20210A polymorphism, a total of six studies, including 521 β-thalassemia patients and 863 healthy controls were examined Table .Prothrombin G20210A gene polymorphism and the risk of β-thalassemiaFor prothrombin G20210A polymorphism, a total of six studies, including 521 β-thalassemia patients and 863 healthy controls were examined Table .Factor V Leiden G1691A gene polymorphism and the risk of β-thalassemiaFor Factor V Leiden G1691A polymorphism, a total of four studies, including 484 β-thalassemia patients and 681 healthy controls, were examined Table . This raMeta-analysis is a potent analyzing tool for accumulative data with low and small power studies. In this study, we explore the correlation of MTHFR C677T, PT G20210A, and FVL G1691A gene polymorphism with β-thalassemia major patients. Here, we meta-analyzed the published information about the polymorphism of MTHFR C677T of 612 β-Thalassemia major patients from eight published studies and, particularly, the changes in allele and genotype occurrences of MTHFR C677T gene polymorphism in patients as compared with controls. We also meta-analyzed the published data about the polymorphism of PT G20210A and FVL G1691A of 521 and 484 β-thalassemia major patients from six and four studies, respectively, and compared the genotype and allele frequencies of PT G20210A and FVL G1691A polymorphism in patients as compared with controls.In this random-effects meta-analysis, we did not find an association between MTHFR C677T, PT G20210A, and FVL G1691A gene polymorphism with β-Thalassemia major patients as compared to controls. Overall, it can be concluded from general and subgroup analyses that the MTHFR C677T, PT G20210A, and FVL G1691A gene polymorphisms may not play an important role in the pathogenesis of a thromboembolic event. Still, there is not a sufficient amount of relevant studies to give a safe and good assumption. In the future, however, more well-designed studies with a bigger sample size and metacentric studies will be necessary to validate the current outcomes.In meta-analyses, heterogeneity is one of the significant factors. In this study, heterogeneity was significantly observed in genotype CC vs CT+TT (I2=61%) and allele C vs T (I2=71%) of MTHFR C677T polymorphism and genotype GG vs GA (I2=95%), GG vs GA+AA (I2=95%), GA vs GG+AA (I2=95%), and allele G vs A (I2=93%) FVL G1691A gene polymorphism. However, heterogeneity was not significantly found in other genotypes of MTHFR C677T and FVL G1691A and the genotype and alleles of PT G20210A gene polymorphism.A previous study reported that the T allele  frequencies of MTHFR C677T polymorphism were 21.5% and 21% in β-Thalassemia major and healthy controls, respectively. The frequency of MTHFR C677T mutations was marginally greater but not significantly different between β-thalassemia major patients and controls ,27. The The mutation of the MTHFR C677T gene was not statistically correlated with an increased risk of thrombophilia in patients with β-thalassemia major ,22. ContWhen interpreting the meta-analysis outcomes, various limitations should be considered. First of all, the appropriate studies were limited in number. To limit the conceivable outcomes of distribution bias, published articles were incorporated. Thereafter, the present results were based on unadjusted appraisals. Finally, our analyses did not reflect the possibility of linkage disequilibrium and interaction between gene-gene or SNP-SNP.The results of the present meta-analysis suggest that the genotype and allele frequencies of MTHFR C677T, prothrombin G20210A (PT G20210A), and Factor V Leiden G1691A (FVL G1691A) gene polymorphism were not significantly correlated with the risk for the development of β-thalassemia major. However, the outcomes of this meta-analysis were based on mutation (single gene polymorphism), and significant heterogeneity was likewise identified. Therefore, the outcomes of the current study should be interpreted and need further well-designed studies.


Background: Cardiac autophagic flux is impaired during myocardial ischemia/reperfusion (MI/R). Impaired autophagic flux may exacerbate MI/R injury. Charged multivesicular body protein 2B (CHMP2B) is a subunit of the endosomal sorting complex required for transport (ESCRT-III) complex that is required for autophagy. However, the reverse role of CHMP2B accumulation in autophagy and MI/R injury has not been established. The objective of this article is to elucidate the roles of AMP-activated protein kinase (AMPK)/atrogin-1 pathways in inhibiting CHMP2B accumulation in ischemia–reperfusion injury.Methods: Male C57BL/6 mice (3–4 months) and H9c2 cardiomyocytes were used to evaluate MI/R and hypoxia/reoxygenation (H/R) injury in vivo and in vitro, respectively. MI/R was built by a left lateral thoracotomy and occluded the left anterior descending artery. H9c2 cells were firstly treated in 95% N2 and 5% CO2 for 15 h and reoxygenation for 1 h. Metformin (100 mg/kg/d) and CHMP2B (Ad-CHMP2B) transfected adenoviruses were administered to the mice. The H9c2 cells were treated with metformin (2.5 mM), MG-132 (10 μM), bafilomycin A1 (10 nM), and compound C (20 μM).Results: Autophagic flux was found to be inhibited in H/R-treated cardiomyocytes and MI/R mice, with elevated cardiac CHMP2B accumulation. Upregulated CHMP2B levels in the in vivo and in vitro experiments were shown to inhibit autophagic flux leading to the deterioration of H/R-cardiomyocytes and MI/R injury. This finding implies that CHMP2B accumulation increases the risk of myocardial ischemia. Metformin suppressed CHMP2B accumulation and ameliorated H/R-induced autophagic dysfunction by activating AMPK. Activated AMPK upregulated the messenger RNA expression and protein levels of atrogin-1, a muscle-specific ubiquitin ligase, in the myocardium. Atrogin-1 significantly enhanced the interaction between atrogin-1 and CHMP2B, therefore, promoting CHMP2B degradation in the MI/R myocardium. Finally, this study revealed that metformin-inhibited CHMP2B accumulation induced autophagic impairment and ischemic susceptibility in vivo through the AMPK-regulated CHMP2B degradation by atrogin-1.Conclusion: Impaired CHMP2B clearance in vitro and in vivo inhibits autophagic flux and weakens the myocardial ischemic tolerance. Metformin treatment degrades CHMP2B through the AMPK-atrogin-1-dependent pathway to maintain the homeostasis of autophagic flux. This is a novel mechanism that enriches the understanding of cardioprotection. Globally, ischemic heart disease is among the leading causes of mortality and disability  and autophagy are two major cell quality control pathways. Parallel to the UPS, autophagy is a lysosome-dependent bulk degradation system that inhibits the abnormal accumulation of intracellular proteins and organelles. It is an important process for cellular self-renewal, dynamic energy equilibrium, the maintenance of functional homeostasis, and cardiomyocyte survival Chang, . AutophaThe endosomal sorting complex required for transport (ESCRT) protein complexes have been reported to exhibit an effect on lysosome–autophagosome fusion   was intraperitoneally administered to the mice daily, for 7 days as previously described  were purchased from the Experimental Animal Center of the Fourth Military Medical University. Ethical approval for the use of animals in this study was obtained from the Animal Ethical Experimentation Committee of the Fourth Military Medical University (IACUC-20200602). Animals were housed under a 12:12-h light/dark cycle with GSE71226. We reanalyzed these data using a volcano plot.Human data were obtained from the Open Access database of National Center for Biotechnology Information. Total RNA samples obtained from the peripheral blood of coronary heart disease (CAD) patients and healthy controls among the Chinese Han people and deposited in Gene Expression Omnibus (GEO) were assessed through the GEO Series accession number MI/R mice model was established as previously described   for 15 h and reoxygenated (95% air and 5% CO2) for 1 h , a subclone of the original clonal cell line derived from embryonic BD1X rat heart tissue, were cultured in Dulbecco's modified Eagle's medium (Gibco) that had been supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin mixture . The cell culture was incubated in a 5% CORabbit p-AMPKα (#2535), AMPKα (#5832), CHMP2B (#76173), α-tubulin (#9099), p62 (#16177), LC3B (#3868), and Atg 5 (#12994) antibodies were purchased from Cell Signaling Technology ; rabbit atrogin-1  and LAMP1 (ab24170) antibodies were purchased from Abcam ; sheep anti-CHMP2B (AF7509) for immunofluorescence was purchased from R&D System , whereas all primary MG-132 (10 μM)  assay was performed to quantitatively evaluate cell viability as previously described  concentrations were collected 24 h after MI/R and analyzed spectrophotometrically as previously described   as previously described  were used for immunoprecipitation purposes. The complete open reading frame of rattus CHMP2B was constructed by DNA synthesis containing Not I and Kpn I restriction sites within the 5′ and 3′ termini . Synthetic complementary DNA was inserted into the pFlag-CMV-2 vector. A green fluorescent protein (GFP)-mRFP-LC3 virus is an adenovirus tool for researching autophagy flux (20812860). Cells were treated with GFP-mRFP-LC3 adenovirus  with a multiplicity of infection as 100:1. Forty-eight hours after transfection, the cells were subjected to experimental administration of metformin , as previously described. When mRFP-GFP-LC3 fusion proteins fuse with acidic lysosomes, red fluorescence is observed, and GFP is quenched. A yellow fluorescence is observed when autophagosomal clearance is impaired.Cells were collected by tyrisin , washed thrice using phosphate-buffered saline, stained using the AnnexinV-FITC/PI apoptosis detection kit  and analyzed in a FACSCalibur flow cytometer .RNA extraction and real-time quantitative PCR were performed as previously described  as previously described . A p ≤ 0.05 was considered to be statistically significant.All data were presented as mean ± standard error of the mean. One-way analysis of variance, followed by Dunnett's test in vitro state of the autophagy/lysosomal system under H/R condition was evaluated. We found that the expression levels of Atg 5, LC3 II/I, LAMP1, and p62 were significantly elevated in H/R-treated H9c2 cells  in H/R-induced autophagy-impaired cardiomyocytes . MetformMoreover, we reanalyzed clinical data from CAD patients  or Ad-CHMP2B for 48 h. Adenovirus-infected cells are GFP-positive . AdenoviMetformin treatment significantly enhanced the phosphorylation of AMPK in H/R-treated cardiomyocytes, enhanced the expression of atrogin-1 (a muscle-specific ubiquitin ligase)  or an equivalent volume of vehicle was administered to C57 mice for 7 days . Written informed consent was not obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.Conceptualization, investigation, project administration, and supervision: HM, YL, and LY. Data curation: TL and YY. Formal analysis: TL, NM, and YW. Funding acquisition: YY, NM, YW, WJ, LY, and HM. Methodology: ML. Resources: MC. Software: WJ. Validation, visualization, and writing — original draft: TL. Writing — review and editing: HM. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Litopenaeus vannamei, we synthesized five dsRNAs targeting Liv-SGP-A, −B, −C, −F, and −G and injected them into subadults. The following treatments were employed: sgpG-dsRNA (targeting Liv-SGP-G), sgpC-dsRNA (targeting Liv-SGP-C), and mixed-dsRNA . The expression of Liv-SGP-G in eyestalks was significantly decreased at 10, 20, and 30 days after the injection of sgpG-dsRNA In addition, it was significantly decreased at 10 and 30 days after the injection of mixed-dsRNA. The expression of vitellogenin (Vg) gene expression in the ovaries, and concentrations of Vg protein in the hemolymph, were not changed by the administration of any dsRNA treatment . Although the administration of dsRNAs corresponding to multiple VIHs did not promote ovarian maturation, this is the first report of the co-transcriptional repression of Liv-SGP-G by the injection of dsRNA for homologous genes . These results indicate that subadults can respond to the techniques of transcriptional silencing.Vitellogenesis-inhibiting hormone (VIH) negatively regulates reproduction in shrimp and other decapod crustaceans. In order to assess the effects of transcriptional silencing by multiple VIH subtype I sinus gland peptides (SGPs) on ovarian maturation in female whiteleg shrimp,  Litopenaeus vannamei and other penaeid shrimp species, VIH comprises a set of peptide hormones that belong to the crustacean hyperglycemic hormone (CHH) family. CHH-family neuropeptides also include CHH, VIH, molt-inhibiting hormone (MIH), and mandibular organ-inhibiting hormone (MOIH). Most CHH-family peptides are synthesized at and secreted predominantly from the X-organ/sinus gland complex, but some are synthesized at other tissues  and investigated their expression levels in relation to molting and eyestalk ablation  gene expression in the ovary and levels of Vg in the hemolymph even 20 days after injection .Previously, we analyzed gene expression levels for VIH subtype I peptides in the eyestalks following unilateral eyestalk ablation in adult and subadult ablation . The queL. vannamei, subadult females can also be induced to undergo ovarian maturation using eyestalk ablation; therefore, such females are often used as experimental material in order to study the mechanisms of ovarian maturation  and were gradually acclimated to recirculated natural seawater  after which they were kept in 600-L tanks at 28°C under a 13-h light/11-h dark photoperiod for 2 weeks before use in experimentation. All shrimp were fed with a commercial diet  at a rate of 2–3% body weight per day until use. The treatment of all animals complied with institute regulations and Japanese policy on animal use  and the gene for green fluorescent protein (GFP) as a negative control were prepared as previously elaborated  and T7 promoter for Liv-SGP-B; T7-sgpC-L and T7-sgpC-R (5′-TAATACGACTCACTATAGGGAGACTATTTCCCGACCATCTGG-3′) for Liv-SGP-C; T7-sgpF-L (5′-TAATACGACTCACTATAGGGAGAAAGCGCTCCCTCTTCGACC-3′) and T7-sgpF-R (5′-TAATACGACTCACTATAGGGAGACTTTATTTGCCGACGGTCTGCAGG-3′) for Liv-SGP-F; and T7-VIH-L (5′-TAATACGACTCACTATAGGGAGAAAGCGAGCAAACTTCGAC-3′) and T7-VIH-R (5′-TAATACGACTCACTATAGGGAGACTACTTGCCCACCGTCTG-3′) for Liv-SGP-G. DNA fragments that encoded each mature peptide were purified and used to synthesize each dsRNA , following the protocol described in Kang et al. . This was diluted with TE buffer to yield 0.75 µg µl−1 total dsRNA. We next randomly selected 80 female shrimp having a body weight of above 15 g and injected 18 of them each with one of the above four dsRNA preparations at 3 µg g−1 body weight as in our previous report  to 0.75 µg µls report . In brieeviously . Body weLiv-SGP-G was quantified by two-step qPCR as reported previously  was performed with TaqMan Fast Universal PCR Master Mix  on a Model 7500 Fast Real-Time PCR System  as previously reported  . In order to maximize the quality of RNA for subsequent qPCR analysis, the purified eyestalk total RNA was purified once more using an RNeasy MinElute cleanup kit (Qiagen) according to the manufacturer’s protocol. The relative expression of eviously . In briereported , 18, usior actb) . ExpressLiv-SGP-A, -B, and -C was also quantified by two-step qPCR as reported previously  was performed using the same protocols of qPCR for Liv-SGP-G, but using the following gene-specific primers and TaqMan probes: SgpA-Fw, SgpA-Rv, and SgpA-Prb for Liv-SGP-A; SgpB-L2, SgpB-Rv, and SgpB-Prb for Liv-SGP-B; and LvsgpC-Fw, LvsgpC-Rv, and LvsgpC-Prb for Liv-SGP-C. The standard curves for Liv-SGP-A, -B, and -C were linear over seven orders of magnitude of serially diluted 4 ng of each plasmid  with purified total RNA as the template. The expression of vg and actb mRNAs was quantified on a Model 7500 Fast Real-Time PCR System as previously reported  to 50.1 to 0.10 ng per well for the TR-FIA Vg assay. For sample-coating, the 96-well plates were incubated at 4°C overnight with 100 μl of sample or standard, and then the wells were blocked with 1% BSA in assay buffer (Perkin Elmer). After blocking, vitellin antiserum from Penaeus japonicus (anti-PjVn) was diluted 1:10,000 in assay buffer for use as the primary antibody. Secondary antibody was Delfia Eu-N1-labeled anti-rabbit antibody (Perkin Elmer), diluted 1:2,000 in assay buffer. Plates were incubated following each step for 2 h at 24°C, and the wells were washed five times for 3 min at a time with 0.3 ml of washing buffer  in an ImmunoWash 1575 Microplate Washer (Bio-Rad) each time between steps. Finally, enhancement solution (Perkin Elmer) was added to the plate, and the fluorescence was measured at 615 nm on a time-resolved fluorometer .Hemolymph Vg was measured by time-resolved fluoroimmunoassay (TR-FIA) as previously reported . In brie−1; EtOH, 330 ml L−1 95%; acetic acid, 115 ml L−1) for 16–20 h and then held in 70% ethanol at room temperature before processing for histological analysis. Tissues were dehydrated through a graded series of ethanol and lemosol , and then embedded in paraffin. Tissues were sectioned to 5 µm, hydrated through a graded ethanol series, stained with hematoxylin and eosin, and again dehydrated through a graded ethanol series and xylene. Oocyte developmental stage, determined according to previous reports, was observed under a light microscope  and omitted. All results are expressed as the mean ± SEM. Significant differences were assessed by one-way ANOVA using SigmaPlot v. 11 software . The significance between vg expression levels and Vg concentrations in all groups was tested by multiple comparisons between the untreated  group and treatment groups by Dunnett’s method. The significance of expression levels of Liv-SGP-G among individuals was tested by multiple comparisons between the initial group and treatment groups by Kruskal–Wallis ANOVA on ranks using Dunn’s method.Significant outliers among each group were identified by Grubbs’s test for outliers (α = 0.01) on the GraphPad website  to 30 days (828.9 ± 393.6) after injection, with no significant difference from the initial group (664.0 ± 337.9). In the sgpG-dsRNA group, expression was significantly lower than that of the initial group at 10 days (3.011 ± 1.413) to 30 days (3.052 ± 0.943). In the sgpC-dsRNA group, expression was lower, but not significantly, than that in the initial group at 10 days (54.08 ± 13.14), and then increased gradually from 20 days (145.7 ± 51.31) to 30 days (229.0 ± 83.46). In the mixed-dsRNA group, expression was significantly lower than that in the initial group at 10 days (14.91 ± 2.379), lower (but not significantly) at 20 days (20.89 ± 5.447), and then significantly lower at 30 days (14.21 ± 2.830).vg mRNA expression in the ovary. In the initial group, expression was weak (four out of six shrimps) or easily detectable (two out of six shrimps) (vg mRNA levels 220.6 ± 147.8). In the GFP-dsRNA group, expression was weak at 10 days (15.16 ± 14.08) and 20 days (1.408 ± 0.578), and then strong at 30 days (343.1 ± 193.1), but with no significant difference compared to the initial group owing to high variation in measured values. In the other groups, vg mRNA expression was weak. Expression of vg mRNA in the hepatopancreas was weak or not detectable in all treatment groups, including the initial group (data not shown).−1). In the GFP-dsRNA group, levels were low at 10 days (3.76 ± 0.801 µg ml−1) and 20 days (7.51 ± 0.573 µg ml−1), with no significant difference observed from the initial group, but were significantly increased at 30 days (80.7 ± 40.6 µg ml−1). Levels in the other groups from 10 to 30 days were not significantly different from those of the initial group.GSI was low (<2%) from 10 to 30 days in all individuals Figure 3In the sgpG-dsRNA group, ovaries were mostly in the previtellogenic stage in all six shrimps at 10 days   gene expression levels using of Marsupenaeus japonicus ovary as the in vitro incubation system  is produced in the eyestalks and the removal of the source of VIH by eyestalk ablation allows ovarian maturation to occur. In previous work of this laboratory, several sinus gland peptides (SGP) were purified from the eyestalks of subadult n system . Thereafnstrated . Thus fonstrated . As showLiv-SGP-G) silenced the transcription of Liv-SGP-G in the eyestalks for at least 30 days. Although we did not include treatment groups for eyestalk ablation and a vehicle control, the observance of positive effects in eyestalk-ablated animals is a well-documented fact ; negligible effects can be inferred from the results of the GFP-dsRNA group, the treatment for which was prepared using the same vehicle buffer. As expected, eyestalk ablation induces ovarian maturation and vg mRNA expression in subadult females  also did not promote ovarian maturation. Interestingly, however, the administration of mixed-dsRNAs  significantly reduced the expression of Liv-SGP-G   . AlthougPenaeus monodon and L. vannamei  transcription for at least 30 days  at 3 µg g−1 body weight suppressed transcription, but did not promote ovarian maturation or increases in endogenous Vg gene expression or concentration even after 20 days  and analyzed their effects on ovarian maturation. These results will help improve the current understanding of the biological functioning of VIH subtype I peptides, and in the future may lead to the development of alternatives to eyestalk ablation.In conclusion, we investigated the dynamics of the expression of https://www.ncbi.nlm.nih.gov/genbank/, LC278950; https://www.ncbi.nlm.nih.gov/genbank/, LC278952; https://www.ncbi.nlm.nih.gov/genbank/, LC278953.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: BJK and MNW designed the experiments. BJK and ZS performed the experiments and collected raw data. All authors contributed to the article and approved the submitted version.This research was supported by in-house funds provided by the Japan International Research Center for Agricultural Sciences (JIRCAS).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


P=0.019, 0.01, 0.02). In pre-clinical models, we found that chronic exposure of cell lines to rituximab led to downregulation of ICAM-1 and acquirement of a rituximab resistant phenotype. In vitro exposure of rituximab resulted in rapid aggregation of B-cells regardless of the ICAM-1 expression levels. MTT assay showed knockdown of ICAM-1 could cause rituximab resistance. Neutralization of ICAM-1 did not affect rituximab activity in vitro and in vivo. Our data illustrated that in post-rituximab era, R-CHOP significantly improved the ORR, PFS and OS in ICAM-1 negative subset patients. Downregulation of ICAM-1 may contribute to rituximab resistance, and that rituximab, by promoting cell-cell aggregation, may sensitize cells to the cytotoxic effects of chemotherapy agents.Intercellular adhesion molecule-1 (ICAM-1) is a cell-surface receptor contributing to lymphocyte homing, adhesion and activation. The prognostic significance of the protein is unknown in diffuse large B-cell lymphoma (DLBCL) in post-rituximab era. We detected expression of ICAM-1 immunohistochemically in 102 DLBCL tissue samples. Overexpression of ICAM-1 was found in 28 (27.5%) cases. In patients with low ICAM-1 expression levels, the addition of rituximab to CHOP  chemotherapy resulted in an improved overall response rate, progression-free survival (PFS) and overall survival (OS) ( Lymphoma is the fifth most common cancer in China, among which diffuse large B-cell lymphoma (DLBCL) is the predominant subtype . The immIntercellular adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein of the immunoglobulin-like superfamily of adhesion molecules, which is constitutively expressed on lymphocytes and endothelial cells –8. It coThus, ICAM-1 seems a potential predictor of clinical outcome in pre-rituximab era. However, in rituximab era, the prognostic value of ICAM-1 has not been known in lymphoma. Here we studied the expression level of ICAM-1 in 102 diffuse large B-cell lymphoma patients and investigated its association with rituximab treated progression-free survival (PFS) and overall survival (OS). We also used rituximab-sensitive and resistant cell line models to further explore the mechanism of action.Among 102 patients who were all hospitalized, median age was 52 years  at the time of diagnosis and 52 (51%) were male. According to Ann Arbor staging criteria, 67 patients (65.7%) had stage I/II diseases, and 35 patients (34.3%) had stage III/IV diseases. 80 patients (78.4%) were IPI 0 or 1 and 22 patients (21.6%) were IPI 2 or more. Eastern Cooperative Oncology Group (ECOG) scores of patients were 0-1. Concerning of cell of origin, 41 patients were germinal center B-cell lymphoma (GCB) and 56 patients were non-germinal center B-cell lymphoma (non-GCB) according to Han’s algorithm. 59 patients (57.8%) received R-CHOP and 43 patients (42.2%) received CHOP regimen as the first-line treatment. Basic features of the patients are recorded concerning the clinical and pathological parameters .P=0.019) (ICAM-1 expression was observed on lymphoma cell membrane. The protein highly expressed in 28 (27.5%) cases. Image P=0.019) .P = 0.03), and a worse OS  compared to those with higher expression of ICAM-1 by Kaplan-Meier analysis   , 2D. Howctively) , 2F.P=0.409); however, in ICAM-1 negative group, an improved ORR was found in patients treated with R-CHOP compared to those with CHOP regimen   .To investigate the mechanism underlying that low ICAM-1 expression group had better ORR, PFS and OS treated by R-CHOP compared to CHOP regimen, we used RSCL and RRCL generated in our lab as previous described.We found the level of surface ICAM-1 protein decreased in RRCL by flow cytometry staining. However, ICAM-1 binding ligand CD11a did not have significant decrease level . WesternThe major cellular killing activity of rituximab is by CMC and ADCC, both of which depend on cellular interaction with complement and/or other cells. We observed that both RSCL and RRCL aggregated after rituximab treatment, regardless of ICAM-1 expression levels . FurtherP> 0.05, P values are shown in P> 0.05)  .DLBCL is a heterogeneous disease in both clinical and biological settings. Many factors identified have prognostic values, such as Ki67, BCL-2 and c-MYC etc , 24. ICAP=0.03). OS between the two group showed a strong tendency towards statistical significance (P =0.05), which is consistent with other reports [In our study, ICAM-1 was detected mainly on the membrane. In pre-rituximab era, Kaplan-Meier analysis indicated that patients with high expression of ICAM-1 had a better PFS compared to those with low expression of the protein in the CHOP group , the association between patients with GCB or non-GCB and their PFS and OS was not found statistical difference.Although higher ICAM-1 was detected in GCB subtype patients . In brief, formalin-fixed, paraffin-embedded tissue sections were incubated overnight at 4° C with mouse monoclonal antibody against ICAM-1  at 1:100 dilutions. Slides with no primary antibodies added served as negative controls.The expression level of ICAM-1 was assessed by percentage of positive cells. Tumors were considered positive when at least 75% of tumor cells expressed ICAM-1. Assessment was done by three pathologists without prior knowledge of the clinical features or follow-up data of the patients.Human lymphoma cell line Raji and RL were purchased from American Type Culture Collection . Rituximab resistant cell line Raji 4RH, RL 4RH were generated by exposure sensitive cells to rituximab at escalating dosages over times in the presence of human complement as previously described .All the cell lines were maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 g/ml streptomycin, maintained in 5% CO2 at 37° C.6 cells/ml were achieved by gentle pipetting before plating on a 6-well plate. Re-aggregation of cell cluster was then observed, and pictures were taken under microscope at different time intervals.Disruption of rituximab sensitive cell line (RSCL) clusters at the concentration of 1×106 cells were centrifuged and RPMI 1640-10%FBS media was removed, leaving 500ul in the tube with cells. Calculated volume of rituximab, or isotype controls was added to each tube of cells. Cells were resuspended briefly so that they mixed well with antibody, and then incubated for 1hr in 37° C incubator for binding of antibody to cell surface to occur. After 1hr incubation, 1ml of RPMI1640-10% FBS media was added back to each tube. Cells were again resuspended well by gentle pipetting before being transferred to a 6-well plate.1×10RSCL and rituximab resistant cell lines (RRCL) were treated with rituximab or Herceptin isotype control for 48h. Then the cells were stained with either FITC-conjugated mouse anti-human ICAM-1 (BD Invitrogen) or Isotype control (mouse IgG2α-FITC) (BD Invitrogen) for 30 minutes. Cells were then washed with PBS, fixed with 2% paraformaldehyde and analyzed by flow cytometry using FACSCCalibur (BD Biosciences). Cells were analyzed using the FCS express software .Cells were lysed in 200 μl lysis buffer . The protein extracts (20 μg) were separated by 10% SDS–PAGE and transferred to polyvinylidene fluoride membranes . The membrane was blocked for 1 hour by 5% non-fat milk and then incubated with primary antibodies overnight at 4 ° C. After washed 3 times with Tris-buffered saline/0.1% Tween 20, the membrane was incubated with horseradish peroxidase-linked secondary antibodies for 1 hour. Proteins were visualized using Tanon full-automatic light detecting system with the BeyoECL Star (Ultra hypersensitive ECL chemiluminescence kit). All the data were confirmed by three individual experiments.The forward sequences of shRNAs targeting ICAM1 were as follows: sh-1: CCGGCCGGTATGAGATTGTCATCATCTCGAGATGATGACAATCTCATACCGGTTTTTG;sh-2:CCGGGCCAACCAATGTGCTATTCAACTCGAGTTGAATAGCACATTGGTTGGCTTTTTG. The shRNA oligo sequences were cloned into a lentiviral shRNA expressing plasmid GV248 . A scramble sequence TTCTCCGAACGTGTCACGT constructed as stem-loop-stem structure were also cloned into the same plasmid and were used as control (SC). For the production of lentivirus, 293T cells were transfected with shRNA-expressing plasmids  and packaging plasmids (PSPAX2 and pMD2G) by Lipofectamine® 3000 (Invitrogen). Virus supernatants were harvested at 48h, 72h and 96h after transfection and were used to infect cultured cell lines. The infected cells were then isolated by puromycin selection.51Cr release assay. In brief, cells were labeled with 51Cr at 37° C, 5% CO2 for 2 hours and then were plated at a cell concentration of 1x105 cells/well (CMC assay) or 1x104 cells/well (ADCC assay). Cells were then exposed to rituximab (10μg/ml), ofatumumab, TG20, R603 and GA101 or isotype (10μg/ml) and human serum  or PBMCs  for six hours at 37° C and 5% CO2. 51Cr release was measured and percentage of cell-lysis was calculated as previously described.The ADCC and CDC were accessed by 5 cells/ml in 96 well plates and was exposed to 10ug/ml of rituximab for different time intervals. At the indicated time points, 20 μl of 0.5 mg/ml MTT  was added to each well and incubated at 37° C for 4 h. In the end, the culture was replaced with 150 μL DMSO  to stop the reaction. The absorbance values (OD 570 nm) were measured using a spectrophotometer.RSCL was plated at a cell density of 1 × 10Following 1hr incubation with the indicated antibodies , or isotype controls), Raji cells were inoculated into SCID mice through intraperitoneally. The growth of tumor was measured twice per week and mice were sacrificed when one dimension of the tumor reached 2cm, or when necrotic/ulcerated tumor developed. The average cumulative days of survival were calculated.2 test. Differences of the cell viability between control and sh-ICAM-1 cell lines treated with rituximab were analyzed using students t-test. Kaplan-Meier survival curves were constructed for survival analyses, and differences were tested by the log-rank test. OS was defined as the time between the date of surgery or biopsy and the date of death or the date of last contact. PFS refers to the period from the beginning of treatment to the observed progression of the disease or the occurrence of death for any reason. The data of patients alive at the end of the study were censored. All P values were two-sided, and the results were considered significant if P < 0.05.The analyses were performed using SPSS 23.0  or GraphPad Prism 8 software . Associations between protein expression and clinical/laboratory parameters or treatment response were assessed by χ