In contrast to the well-established genomic 5-methylcytosine (5mC), the existence of N⁶-methyladenine (6 mA) in eukaryotic genomes was discovered only recently. Initial studies found that it was actively regulated in cancer cells, suggesting its involvement in the process of carcinogenesis. However, the contribution of 6 mA in tongue squamous cell carcinoma (TSCC) still remains uncharacterized. In this study, a pan-cancer type analysis was first performed, which revealed enhanced 6 mA metabolism in diverse cancer types. The study was then focused on the regulation of 6 mA metabolism, as well as its effects on TSCC cells. To these aspects, genome 6 mA level was found greatly increased in TSCC tissues and cultured cells. By knocking down 6 mA methylases N6AMT1 and METTL4, the level of genomic 6 mA was decreased in TSCC cells. This led to suppressed colony formation and cell migration. By contrast, knockdown of 6 mA demethylase ALKBH1 resulted in an increased 6 mA level, enhanced colony formation, and cell migration. Further study suggested that regulation of the NF-κB pathway might contribute to the enhanced migration of TSCC cells. Therefore, in the case of TSCC, we have shown that genomic 6 mA modification is involved in the proliferation and migration of cancer cells.
AlkB Homolog 1, Histone H2a Dioxygenase/metabolism* ; Carcinoma, Squamous Cell/pathology* ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Humans ; Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism* ; Tongue Neoplasms/metabolism*

The oral cavity of each person is home to hundreds of bacterial species.While taxa for oral diseases have been studied using culture-based characterization as well as amplicon sequencing,metagenomic and genomic information remains scarce compared to the fecal microbiome.Here,using metagenomic shotgun data for 3346 oral metagenomic samples together with 808 published samples,we obtain 56,213 metagenome-assembled genomes(MAGs),and more than 64％of the 3589 species-level genome bins(SGBs)contain no publicly available genomes.The resulting genome collection is representative of samples around the world and contains many genomes from candi-date phyla radiation(CPR)that lack monoculture.Also,it enables the discovery of new taxa such as a genus Candidatus Bgiplasma within the family Acholeplasmataceae.Large-scale metagenomic data from massive samples also allow the assembly of strains from important oral taxa such as Por-phyromonas and Neisseria.The oral microbes encode genes that could potentially metabolize drugs.Apart from these findings,a strongly male-enriched Campylobacter species was identified.Oral sam-ples would be more user-friendly collected than fecal samples and have the potential for disease diagnosis.Thus,these data lay down a genomic framework for future inquiries of the human oral microbiome.

Objective:To establish and validate the prediction model for postoperative sleep disturbance (PSD) in patients undergoing non-cardiac surgery.Methods:A total of 454 patients of both sexes, aged≥18 yr, of American Society of Anesthesiologists physical statusⅠ-Ⅲ, underwent non-cardiac surgery under general anesthesia from November 2019 to September 2020 were selected.The perioperative data were collected.The patients were divided into training set and validation set with a ratio of 7∶3 by using a simple random sampling method.The characteristic variables of PSD were selected using LASSO regression analysis and the independent risk factors were identified using multivariate logistic regression analysis in training set.Akaike′s information criterion was used to evaluate the quality of fit of the model.The nomogram of PSD in non-cardiac surgery patients was constructed based on the identified factors.The discrimination of the model was evaluated using receiver operating characteristic (ROC) curve, and the agreement of the model was evaluated using Hosmer-Lemeshow goodness-of-fit test and Brier score.Results:Seven risk factors (gender, preoperative anxiety, satisfaction with the ward environment, anesthesia time, the intraoperative consumption of midazolam and sufentanil and numerical rating scale (NRS) score at 3 days after operation) and two related factors (preoperative NRS score and general anesthesia combined with nerve block) were used to establish and verify the PSD nomogram.The area under the ROC curve was 0.805 (95% confidence interval [CI] 0.721-0.848) in training set.The area under the ROC curve was 0.773 (95% CI 0.684-0.876) in validation set.In training and validation sets, the calibration curves were tested by Hosmer-Lemeshow good of fit test, the P values were 0.590 and 0.950, respectively, and the Brier scores were 0.154 and 0.156, respectively.The nomogram predicated that the sensitivity (95% CI) and specificity (95%CI) were 81.83% (60.32%-95.14%) and 78.15% (71.83%-83.25%), respectively, in training set, and the sensitivity (95% CI) and specificity (95%CI) were 77.86% (39.84%-97.25%) and 78.15% 77.86% (68.74%-85.48%), respectively, in validation set.The optimal cut-off value of nomogram score was 113. Conclusion:In this study, the nomogram prediction model for PSD in patients undergoing non-cardiac surgery has been successfully established, which can visually and individually predict the risk of PSD.

Objective To construct lentiviral vector of late endosomal／lysosomal adaptor, mitogen-activated protein kinase and mammalian target of rapamycin activator 2 ( lamtor2) gene, and to explore its regulatory role on inflammatory response of macrophages after Klebsiella pneumoniae infection.Methods Two pairs of mouse lamtor2 short hairpin RNA (shRNA) were designed and sub-cloned into PLKO.1-puro to construct lentiviral vector, and were transfected into the murine RAW264.7 macrophage.There were two experimental groups including pLKO.1-puro shlamtor 2-1(sh1 group) and pLKO.1-puro shlamtor 2-2 (sh2 group), and the RAW264.7 cells transfected with non-treated pLKO.1-puro was set as control.The expession of lamtor2 were detected by real-time quantitative polymerase chain reaction ( RT-qPCR ) and Western blot.The levels of inflammatory factors including interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-αsecreted by the cells were detected by RT-qPCR.T test was used for comparison between groups.Results The recombinant lentiviral vector PLKO.1-shlamtor 2 transfected RAW264.7 cells successfully.The relative expressions of lamtor2 mRNA in the control group, the sh1 group and the sh2 group were 1.000 ±0.000, 0.596 ±0.125 and 0.120 ±0.080, respectively.The expression of lamtor2 in the sh2 group was significantly lower than that in the sh 1 group (t＝3.399, P＝0.015), and they were both significantly lower than the control group ( t ＝3.333 and 9.734, respectively, both P ＜0.05).After infection with Klebsiella pneumoniae, expression levels of IL-1β( t ＝15.20), IL-6 (t＝43.30) and TNF-α(t＝12.67) were significantly higher than those in the control group (all P＜0.01).Conclusion The lentiviral vector of lamtor2 can stably down-regulate the expression of lamtor2 gene in macrophages through RNA interference mechanism , which has a significant effect on the secretion of inflammatory factors of macrophages that are infected with Klebsiella pneumoniae.

Objective: To construct lentiviral vector of late endosomal/lysosomal adaptor, mitogen-activated protein kinase and mammalian target of rapamycin activator 2 (lamtor2) gene, and to explore its regulatory role on inflammatory response of macrophages after Klebsiella pneumoniae infection. Methods: Two pairs of mouse lamtor2 short hairpin RNA (shRNA) were designed and sub-cloned into PLKO.1-puro to construct lentiviral vector, and were transfected into the murine RAW264.7 macrophage. There were two experimental groups including pLKO.1-puro shlamtor 2-1(sh1 group) and pLKO.1-puro shlamtor 2-2 (sh2 group), and the RAW264.7 cells transfected with non-treated pLKO.1-puro was set as control. The expession of lamtor2 were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. The levels of inflammatory factors including interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α secreted by the cells were detected by RT-qPCR. T test was used for comparison between groups. Results: The recombinant lentiviral vector PLKO.1-shlamtor 2 transfected RAW264.7 cells successfully. The relative expressions of lamtor2 mRNA in the control group, the sh1 group and the sh2 group were 1.000±0.000, 0.596±0.125 and 0.120±0.080, respectively. The expression of lamtor2 in the sh2 group was significantly lower than that in the sh1 group (t=3.399, P=0.015), and they were both significantly lower than the control group (t=3.333 and 9.734, respectively, both P< 0.05). After infection with Klebsiella pneumoniae, expression levels of IL-1β (t=15.20), IL-6 (t=43.30) and TNF-α (t=12.67) were significantly higher than those in the control group (all P<0.01). Conclusion The lentiviral vector of lamtor2 can stably down-regulate the expression of lamtor2 gene in macrophages through RNA interference mechanism, which has a significant effect on the secretion of inflammatory factors of macrophages that are infected with Klebsiella pneumoniae.

Objective To analyze the differences of clinical manifestations and organ damage between patients with severe fever with thrombocytopenia syndrome(SFTS)and patients with tsutsugamushi disease,and to investigate the prognostic factors of SFTS.Methods The research was performed on 49 patients with SFTS and 16 patients with tsutsugamushi disease who visited the First Affiliated Hospital of Anhui Medical University from October 2014 to June 2017.The general information of patients including region,age,gender and clinical manifestations were evaluated.Blood routine,liver and kidney function,myocardial enzyme levels,lipase,amylase,electrolytes,C-reactive protein,procalcitonin,prothrombin time(PT)and activated partial thromboplastin time(APTT)were continuously monitored during the course of disease.T test was used for continuous variables of normal distribution,and non-parametric test was used for variables of non-normal distribution.Chi-square test was used for categorical variables.Results The mean age of SFTS patients was 62.1±15.5(ranging from 17 to 87 years)and the mean age of tsutsugamushi patients was 56.1±9.2(ranging from 47 to 73 years).There was no significant difference between the two groups(t=1.47,P=0.147).There were 25 males(51%)in SFTS patients and 8 males(50%)in tsutsugamushi disease patients.There was no significant difference between the two groups(x2=0.005,P=0.943).The incidences of headache,vomiting,superficial lymphadenectasis,disturbance of consciousness,proteinuria,hematuria,pulmonary infection,multiple organ dysfunction and acute pancreatitis in SFTS patients were all significantly higher than those in tsutsugamushi disease patients(x2=8.82,4.38,8.71,11.17,7.88,5.56,4.35,9.43,and 8.13,respectively,P <0.05 or 0.01).The counts of leukocytes(Z=2.73),neutrophils(Z=2.46),lymphocytes(Z=3.15),platelets(Z=4.25),albumin(Z=2.65)and sodium ion(t=2.10)in SFTS patients were all significantly lower than those in patients with tsutsugamushi disease(P <0.05 or 0.01).The levels of aspartate aminotransferase(Z=2.94),lactate dehydrogenase(Z=3.42),creatine kinase(CK)(Z=2.88),amylase(Z=2.11),lipase(Z=2.82),creatinine(Z=2.07)and urea nitrogen(Z=2.50)in fatal SFTS patients were all significantly higher than those in patients with tsutsugamushi disease(P <0.05 or 0.01).Among 49 SFTS patients,16 patients died and 33 patients recovered finally.The age(t=3.33),platelet count(Z=2.55),alanine aminotransferase(ALT)(Z=2.10),aspartate aminotransferase(AST)(Z=2.22),lactate dehydrogenase(Z=2.26),CK(Z=3.50),CK-MB(Z=3.10),creatinine(Z=2.17),urea nitrogen(Z=2.36),and sodium(t=2.65)between the two subgroups had significant differences(P <0.05 or 0.01).Conclusions SFTS is more severe and has high mortality,while tsutsugamushi disease has a better prognosis.Early differential diagnosis and early rational treatment are important to reduce the mortality of patients with SFTS.

Objective To investigate the clinical characteristics,diagnosis and treatment strategies of breast neuroendocrine carcinoma.Methods 20 cases with breast neuroendocrine carcinoma,who were admitted in Department of Breast Surgery,the Second Affiliated Hospital of Dalian Medical University from Mar.2005 to Dec.2017,were analyzed retrospectively.Results The average age of the 20 patients was(54.35±13.35) years.In aspect of surgery,18 patients received modified radical mastectomy,1 patient received total glandectomy and sentinel lymph node biopsy and stage I silicone implant breast reconstruction,and 1 patient received radical mastec tomy.In terms of pathological types,there were 5 cases (25.0％) of highly differentiated neuroendocrine carcinoma,4 cases (20.0％) of poorly differentiated neuroendocrine carcinoma (small cell carcinoma),and 11 cases (55.0％)of invasive breast cancer with neuroendocrine differentiation.In molecular typing,there were 7 cases (35.0％) of Luminal A,7 cases (35.0％) of Luminal B (HER2 negative),4 cases (20.0％) of Luminal B (HER2 positive),and one case(5.0％) of HER2 type and one case(5.0％) of Basal-like type.The positive rates of ER,PR and HER2 in this group were 90.0％,60.0％ and 25.0％ respectively.20 patients were followed up for 5 to 119 months,with an average follow-up of (59.85±24.51) months.One patient developed bone metastases in the 6th year after surgery and survived for 119 months.One patient developed pulmonary metastasis at the 20th month after surgery and died at the 28th month after surgery.So far,the remaining postoperative patients still survived and no sign of recurrence or metastasis was found.Conclusion The diagnosis of breast neuroendocrine carcinoma relies on histopathological and immunohistochemical detection.Its ER/PR positive rate is high,its molecular typing is mostly Luminal type,and neoadjuvant treatment can be performed when necessary.For specific patients whose ER or PR are positive,neoadjuvant endocrine therapy is also a well-established therapy,even the optimal results can be achieved.However,more cases are still needed for research.

Objective To study the clinical efficacy of respiratory allergic diseases in children treated by nasal atomization of budesonide combined with 3% hypertonic saline. Methods Children diagnosed as upper air-way cough syndrome or mild to moderate asthma without being controlled by anti-asthma treatment were included in the study.They contracted with complications of nasal congestion and/or running noseand other symptoms,and na-sal CT confirmed the nasal lesions.Thirty children undergoing conventional treatment(conventional drug therapy+keeping away from allergen)were defined as the control group. 89 children treated with nasal spray therapy(con-ventional drug therapy + keeping away from allergen + nasal spray treatment)were defined as the therapy group. The treatment group was subdivided into IgE-mediated group and non-IgE-mediated group according to IgE level. The treatment course was 7 days.The clinical symptoms score,nasal symptom visual scale(VAS),peak expirato-ry flow(PEF)index were observed and analyzed. Results The clinical symptom score and nasal VAS showed a decreasing trend and the percentage of PEF showed an increasing trend in the two groups within 1 week after treat-ment.The clinical symptom score and nasal VAS score of the treatment group were significantly lower than those of the control group,while the PEF%value was significantly higher.The difference in treatment method was interact-ed with the treatment time(P<0.05).The percentage of PEF in the non-IgE-mediated group was significantly high-er than that of IgE-mediated group 1 week after treatment(P < 0.05). Conclusion The nasal atomization of budesonide combined with hypertonic saline in the treatment of children with respiratory allergic diseases is effec-tive and of a good clinical value.

Objective To compare the differences in the intestinal microflora of WHBE rabbit and JW rabbit models of diarrhea-predominant irritable bowel syndrome(IBS). Methods 16 WHBE rabbits and 16 JW rabbits were randomly divided into normal control(NC)group and IBS model group, respectively(n=8). The diarrhea-predominant IBS model was established by wet-heat stress combined with intragastric gavage of senna decoction. The abdominal circumference index,water content of feces and colonic transit function were observed. After sacrifice,colon tissue samples were taken for histopathological examination and colon contents for intestinal flora diversity analysis. Results Compared with the NC group,the IBS model rabbits showed an increased abdominal circumference index and fecal water content,and a shortened colon transit time, but no obvious pathological changes were observed in the colon tissues. Meanwhile, the Shannon index and Chao1 index of IBS model rabbits were significantly decreased(P<0.05). According to the result of OTU classification analysis,Firmicutes and Bacteroidetes are the dominant bacteria in the intestinal microflora of rabbits. Compared with the NC group, the Firmicutes, Verrucomicrobia, Chloroflexi, Akkermansia, and Streptococcus in the WHBE rabbit IBS model group were significantly reduced(P < 0.05, P < 0.01), while Bacteroidetes and rc4-4 significantly increased(P < 0.05, P < 0.01). However, in the JW rabbit IBS model group, Eubacterium and Subdoligranulum were significantly increased(P< 0.05),while Lactobacillus,Coprobacter,Veillonella and Streptococcus were markedly decreased(P<0.05). Compared with the JW rabbit NC group,the abundance of Firmicutes,Odoribacter, Veillonella,Streptococcus,Oscillospira and Pseudoflavonifractor were significantly decreased(P<0.05, P<0.01), but Bacteroidetes,Verrucomicrobia,Eubacterium,Akkermansia and Coprobacter were significantly increased(P<0.05,P<0.01)in the WHBE rabbit NC group. Compared with the JW rabbit IBS model group, the abundance of rc4-4, Bacteroidetes,Coprobacter and Clostridium were significantly higher(P < 0.05, P < 0.01), while the Firmicutes, Dorea, Coprococcus and Subdoligranulum were significantly lower(P <0.05)in the WHBE rabbit IBS model group. Conclusions There is an intestinal microflora imbalance in rabbits with IBS, resulting in a decrease of microflora diversity. The changes of intestinal microflora in the WHBE rabbits and JW rabbits with IBS have their own characteristics, and have apparent differences.