Effect of Xiangsha Liu Junzitang Combined with Phlegm-removing and Detoxifying Traditional Chinese Medicine on Immune Escape in Lewis Lung Cancer Mice / 中国实验方剂学杂志

ObjectiveTo study the effect and mechanism of Xiangsha Liu Junzitang combined with phlegm-removing and detoxifying traditional Chinese medicine on immune escape in Lewis lung cancer mice. MethodA total of 60 specific-pathogen-free （SPF）-grade C57BL/6J male mice were injected subcutaneously with 0.2 mL of Lewis cell suspension （containing 2×106 cells·mL-1） in the right mid-axillary line. After 7 days， the mice that had been successfully modeled were randomly divided into six groups： the model group， the cisplatin group， the Xiangsha Liu Junzitang low-， medium-， and high-dose groups， and the combined group， with 10 mice in each group. The Xiangsha Liu Junzitang low-， medium- and high-dose groups were gavaged with 17.88， 35.75， 71.50 g·kg-1 Xiangsha Liu Junzitang solution once a day， respectively， and the dosage of cisplatin intraperitoneally injected into the mice was converted to 5 mg·kg-1 twice a week， and the tumour volumes of each group were measured every two days. The intervention lasted for 14 consecutive days. At the end of treatment， the tumour mass of mice in each group was weighed and the tumour inhibition rate was calculated. The morphological characteristics of tumours in each group were observed by hematoxylin-eosin （HE） staining. Fluorescent quantitative real-time polymerase chain reaction （Real-time PCR） assay was used to detect messenger ribonucleic acid （mRNA） contents of the natural killer group 2 member D （NKG2D） receptor， ribonucleic acid export-1 （RAE-1）， and γ interferon （IFN-γ） in the tumour tissues of each group. NKG2D， RAE-1， and IFN-γ mRNA in tumour tissues of each group. Immunohistochemistry （IHC） and Western blot were applied to detect the expressions of RAE-1， NKG2D， and IFN-γ in tumour tissues of each group， and Western blot was used to detect the expressions of interleukin-6 （IL-6）， Janus kinase 2 （JAK2）， p-JAK2， signal transducer and activator of transcription 3 （STAT3）， and p-STAT3 in tumour tissues of each group， as well as the protein levels of NKG2D， and RAE-1 in spleen tissues of each group. ResultCompared with that in the model group， the tumour mass decreased in all dose groups of Xiangsha Liu Junzitang， with no statistically significant difference. The tumour volume was reduced （P<0.05， P <0.01）. The pathological morphology was improved. The mRNA contents of NKG2D， RAE-1 and IFN-γ were increased in the medium-dose group （P<0.05， P<0.01）， and the protein expressions of NKG2D， RAE-1， and IFN-γ in tumour tissues were elevated （P<0.05， P<0.01）， and p-JAK2 and p-STAT3 protein expressions were decreased （P<0.05， P<0.01）. In spleen tissues， the protein expressions of NKG2D and RAE-1 in all dose groups of Xiangsha Liu Junzitang were significantly elevated （P<0.01）. Compared with those in the cisplatin group， NKG2D， RAE-1 and IFN-γ mRNA contents were elevated in the middle-dose group of Xiangsha Liu Junzitang， and the difference was not statistically significant. IHC showed that the protein expressions of NKG2D and IFN-γ in the combined group were significantly elevated （P<0.01）， and Western blot results showed that the protein expressions of RAE-1， NKG2D and IFN-γ were elevated （P<0.05， P<0.01）. p-JAK2 and p-STAT3 protein expressions were decreased in the combined group （P<0.05， P<0.01）. NKG2D and RAE-1 protein expressions were significantly increased in spleen tissues of the medium-dose groups and the combined group （P<0.01）. ConclusionXiangsha Liu Junzitang combined with phlegm-removing and detoxifying traditional Chinese medicine can inhibit the growth of tumours in Lewis lung cancer mice by up-regulating the expressions of RAE-1/NKG2D， promoting the activation of NK cells， and inhibiting immune escape， the mechanism of which may be related to down-regulation of the JAK2/STAT3 pathway.

Analysis of Mechanism of Yishen Huashi Granules for Improving Diabetic Nephropathy Based on Metabonomics and Transcriptomics / 中国实验方剂学杂志

ObjectiveIn this study， based on ultra-high performance liquid chromatography-mass spectrometry（UHPLC-MS/MS） and high-throughput transcriptome sequencing technology（RNA-seq）， we investigated the mechanism of Yishen Huashi granules in regulating serum metabolites and renal messenger ribonucleic acid（mRNA） expression to improve diabetic kidney disease（DKD）. MethodSD rats were randomly divided into normal group ， model group and Yishen Huashi granules group， with 8 rats in each group. The rat model of DKD was established by intraperitoneal injection of streptozotocin. Yishen Huashi granules group was given 5.54 g·kg-1·d-1 of Yishen Huashi granules by gavage， and the normal group and the model group were given the same amount of normal saline for 6 weeks. During the experiment， the body weight and blood glucose of rats were monitored， and the rats were anesthetized 24 hours after the last administration， blood was collected from the inferior vena cava， serum was separated， and renal function， blood lipid， and inflammatory indicators were detected. Kidney tissue of rats was fixed in neutral paraformaldehyde， and stained with hematoxylin-eosin（HE）， Masson and periodic acid-Schiff（PAS） to observe the renal pathological changes. UHPLC-MS/MS and RNA-seq were used to identify the changes of serum metabolism and the differences of renal mRNA expression， and real time fluorescence quantitative polymerase chain reaction（Real-time PCR） and Western blot were used to detect the differential mRNA and protein expression in renal tissue to explore the common expression mechanism. ResultCompared with the normal group， rats in the model group showed a decrease in body weight， a significant increase in blood glucose， urinary microalbumin to urinary creatinine ratio（UACR）， blood urea nitrogen（BUN）， cystatin-C（Cys-C）， β2-microglobulin（β2-MG）， interleukin-6（IL-6）， triglyceride（TG） and total cholesterol（TC）， and a significant decrease in total superoxide dismutase（T-SOD）（P<0.01）. After the intervention of Yishen Huashi granules， all the indexes were improved to different degrees in rats（P<0.05， P<0.01）. Compared with the normal group， the model group showed renal mesangial stromal hyperplasia， fibrous tissue hyperplasia and tubular vacuolar degeneration. Compared with the model group， the renal pathology of rats in Yishen Huashi granules group was improved to a certain extent. A total of 14 target metabolites and 96 target mRNAs were identified， the target metabolites were mainly enriched in 20 metabolic pathways， including sphingolipid metabolism， glycerophospholipid metabolism， and the biosynthesis of phenylalanine， tyrosine and tryptophan. The target mRNAs were enriched to obtain a total of 21 differential mRNAs involved in the TOP20 pathways closely related to glycolipid metabolism. A total of 6 pathways， glycerophospholipid metabolism， arachidonic acid metabolism， purine metabolism， primary bile acid biosynthesis， ascorbic acid and uronic acid metabolism， and galactose metabolism， were enriched by serum differential metabolites and renal differential mRNAs， among them， there were 7 differential metabolites such as phosphatidylethanolamine（PE） and 7 differential mRNAs such as recombinant adenylate cyclase 3（ADCY3）. Seven differential metabolites had high predictive accuracy as verified by receiver operating characteristic（ROC） curve， and the results of Real-time PCR and Western blot were highly consistent with the sequencing results. ConclusionYishen Huashi granules can reduce UACR， BUN and other biochemical indexes， correct the disorder of glucose and lipid metabolism， and improve renal function of DKD rats. And its mechanism may be related to the regulation of the level of PE and other blood metabolites， and expression of Phospho1 and other mRNAs in the kidney， of which six pathways， including glycerophospholipid metabolism， may play an important role.

High expression of VARS promotes the growth of multiple myeloma cells by causing imbalance in valine metabolism / 中南大学学报(医学版)

OBJECTIVES@#Multiple myeloma (MM) is a plasma cell malignancy occurring in middle and old age. MM is still an incurable disease due to its frequent recurrence and drug resistance. However, its pathogenesis is still unclear. Abnormal amino acid metabolism is one of the important characteristics of MM, and the important metabolic pathway of amino acids participates in protein synthesis as basic raw materials. Aminoacyl transfer ribonucleic acid synthetase (ARS) gene is a key regulatory gene in protein synthesis. This study aims to explore the molecular mechanism for ARS, a key factor of amino acid metabolism, in regulating amino acid metabolism in MM and affecting MM growth.@*METHODS@#The corresponding gene number was combined with the gene expression profile GSE5900 dataset and GSE2658 dataset in Gene Expression Omnibus (GEO) database to standardize the gene expression data of ARS. GSEA_4.2.0 software was used to analyze the difference of gene enrichment between healthy donors (HD) and MM patients in GEO database. GraphPad Prism 7 was used to draw heat maps and perform data analysis. Kaplan-Meier and Cox regression model were used to analyze the expression of ARS gene and the prognosis of MM patients, respectively. Bone marrow samples from 7 newly diagnosed MM patients were collected, CD138+ and CD138- cells were obtained by using CD138 antibody magnetic beads, and the expression of ARS in MM clinical samples was analyzed by real-time RT-PCR. Human B lymphocyte GM12878 cells and human MM cell lines ARP1, NCI-H929, OCI-MY5, U266, RPMI 8266, OPM-2, JJN-3, KMS11, MM1.s cells were selected as the study objects. The expression of ARS in MM cell lines was analyzed by real-time RT-PCR and Western blotting. Short hairpin RNA (shRNA) lentiviruses were used to construct gene knock-out plasmids (VARS-sh group). No-load plasmids (scramble group) and gene knock-out plasmids (VARS-sh group) were transfected into HEK 293T cells with for virus packaging, respectively. Stable expression cell lines were established by infecting ARP1 and OCI-MY5 cells, and the effects of knockout valyl-tRNA synthetase (VARS) gene on proliferation and apoptosis of MM cells were detected by cell counting and flow cytometry, respectively. GEO data were divided into a high expression group and a low expression group according to the expression of VARS. Bioinformatics analysis was performed to explore the downstream pathways affected by VARS. Gas chromatography time-of-flight mass spectrometry (GC-TOF/MS) and high performance liquid chromatography (HPLC) were used to detect the valine content in CD138+ cells and ARP1, OCI-MY5 cells and supernatant of knockdown VARS gene in bone marrow samples from patients, respectively.@*RESULTS@#Gene enrichment analysis showed that tRNA processing related genes were significantly enriched in MM compared with HD (P<0.0001). Further screening of tRNA processing-pathway related subsets revealed that cytoplasmic aminoacyl tRNA synthetase family genes were significantly enriched in MM (P<0.0001). The results of gene expression heat map showed that the ARS family genes except alanyl-tRNA synthetase (AARS), arginyl-tRNA synthetase (RARS), seryl-tRNA synthetase (SARS) in GEO data were highly expressed in MM (all P<0.01). With the development of monoclonal gammopathy of undetermined significance (MGUS) to MM, the gene expression level was increased gradually. Kaplan-Meier univariate analysis of survival results showed that there were significant differences in the prognosis of MM patients in methionyl-tRNA synthetase (MARS), asparaginyl-tRNA synthetase (NARS) and VARS between the high expression group and the low expression group (all P<0.05). Cox regression model multivariate analysis showed that the high expression of VARS was associated with abnormal overall survival time of MM (HR=1.83, 95% CI 1.10 to 3.06, P=0.021). The high expression of NARS (HR=0.90, 95% CI 0.34 to 2.38) and MARS (HR=1.59, 95% CI 0.73 to 3.50) had no effect on the overall survival time of MM patients (both P>0.05). Real-time RT-PCR and Western blotting showed that VARS, MARS and NARS were highly expressed in CD138+ MM cells and MM cell lines of clinical patients (all P<0.05). Cell counting and flow cytometry results showed that the proliferation of MM cells by knockout VARS was significantly inhibited (P<0.01), the proportion of apoptosis was significantly increased (P<0.05). Bioinformatics analysis showed that in addition to several pathways including the cell cycle regulated by VARS, the valine, leucine and isoleucine catabolic pathways were upregulated. Non-targeted metabolomics data showed reduced valine content in CD138+ tumor cells in MM patients compared to HD (P<0.05). HPLC results showed that compared with the scramble group, the intracellular and medium supernatant content of ARP1 cells and the medium supernatant of OCI-MY5 in the VARS-shRNA group was increased (all P<0.05).@*CONCLUSIONS@#MM patients with abnormal high expression of VARS have a poor prognosis. VARS promotes the malignant growth of MM cells by affecting the regulation of valine metabolism.

Diagnostic efficacy of joint detection of Xpert MTBRIF, TB-RNA and TB-DNA in bronchoalveolar lavage fluid for smear-negative pulmonary tuberculosis / 中国热带医学

@#Abstract: Objective　To investigate the diagnostic value of joint detection of Mycobacterium tuberculosis rifampicin resistance gene (Xpert MTB/RIF), Mycobacterium tuberculosis ribonucleic acid (TB-RNA) and Mycobacterium tuberculosis deoxyribonucleic acid (TB-DNA) in bronchoalveolar lavage fluid for smear-negative pulmonary tuberculosis. Methods A total of 806 patients with suspected smear-negative pulmonary tuberculosis admitted to our hospital from May 2020 to July 2022 were selected, 506 patients diagnosed as bacterial negative pulmonary tuberculosis by clinical, X-ray and sputum samples were classified as bacterial negative pulmonary tuberculosis group, and the other 300 patients with non-tuberculous pulmonary disease were classified as non-tuberculous pulmonary disease group. XpertMTB/RIF, TB-RNA and TB-DNA in bronchoalveolar lavage fluid of all patients were detected. With clinical, X-ray and sputum specimen examination of mycobacterium tuberculosis as the gold standard, the diagnostic efficacy of alveolar lavage solution Xpert MTB/RIF, TB-RNA and TB-DNA alone and in combination was analyzed. Results The positive detection rates of Xpert MTB/RIF, TB-RNA and TB-DNA in bronchoalveolar lavage fluid of the smear-negative pulmonary tuberculosis group and the non-tuberculosis pulmonary disease group were 69.96% (354/506) and 2.67% (8/300), 61.46% (311/506) and 5.00% (15/300), and 63.64% (322/506) and 8.00% (24/300), respectively. The rates in the smear-negative pulmonary tuberculosis group were higher than those in the non-tuberculosis lung disease group, and the differences were statistically significant (χ2=342.005, 246.930, 235.687, P<0.01). Compared with the gold standard, the sensitivity, specificity, accuracy, positive predictive value and negative predictive value of Xpert MTB/RIF in the diagnosis of smear-negative pulmonary tuberculosis were 69.96%, 97.33%, 80.15%, 97.79% and 65.77%, respectively; those values of TB-RNA were 61.46%, 95.00%, 73.95%, 95.40% and 59.38%, respectively; those values of TB-DNA were 63.64%, 92.00%, 74.19%, 93.06% and 60.00%, respectively; those values of combined diagnosis with Xpert MTB/RIF, TB-RNA and TB-DNA were 61.26%, 100.00%, 75.68%, 100.00% and 60.48%, respectively; the specificity and positive predictive value of combined detection were higher than those of single detection (P<0.05). Conclusions The joint detection of Xpert MTB/RIF, TB-RNA and TB-DNA in bronchoalveolar lavage fluid can improve the diagnostic efficacy of smear-negative pulmonary tuberculosis and is worthy of clinical promotion and application.

Prevalence and genotyping of Cryptosporidium spp. and Giardia lamblia in dogs and cats from a pet hospital in Shanghai Municipality / 中国血吸虫病防治杂志

Objective To investigate the prevalence and genotypes of Cryptosporidium spp. and Giardia lamblia in dogs and cats from a pet hospital in Shanghai Municipality. Methods A total of 145 fresh fecal samples were collected from pet dogs and cats in a pet hospital in Shanghai Municipality during the period from November 2021 to June 2022, including 99 dog fecal samples and 46 cat fecal samples. The small subunit ribosomal ribonucleic acid (SSU rRNA) gene of Cryptosporidium and the triose phosphate isomerase (TPI) gene of G. lamblia were amplified using nested PCR assay, and the positive amplification products were sequenced from both directions. The sequence assembly was performed using the software Clustal X 2.1, and sequence alignment was conducted using BLAST. A phylogenetic tree was created with the Neighbor-Joining method using MEGA 11.0 to identify parasite species or genotype. Results The overall prevalence of Cryptosporidium and G. lamblia was 20.00% (29/145) in 145 pet dog and cat fecal samples, with the prevalence of 0.69% (1/145) and 19.31% (28/145) in Cryptosporidium and G. lamblia, respectively. G. lamblia was only detected in dog fecal samples, with prevalence of 18.18% (18/99), while the detection rates of Cryptosporidium and G. lamblia were 2.17% (1/46) and 21.74% (10/46) in cat fecal samples. Nucleotide sequence analysis showed that one Cryptosporidium positive sample was characterized as C. felis, and 28 G. lamblia positive samples were all characterized as Giardia assemblage A, which showed 100% sequence homology with human isolates of Giardia. Phylogenetic analysis revealed that the sequences obtained in this study belonged to the same branch with the reported Giardia assemblage A. Conclusions Cryptosporidium and G. lamblia infection was prevalent in pet dogs and cats from the study pet hospital in Shanghai Municipality, and there is a zoonotic risk for the species and genotype. Intensified surveillance of Cryptosporidium and Giardia infection is recommended in pets and their owners, and improved management of pet keeping is required.

Application and Prospect of RNA Profiling Analysis in Forensic Body Fluid Identification / 法医学杂志

In forensic physical evidence identification, the accurate identification of the individual origin and their body fluid composition of the biological samples obtained from the crime scene play a critical role in determining the nature of a crime. In recent years, RNA profiling has become one of the fastest developing methods for body fluids identification. Due to the characteristics of tissue or body fluid specific expression, various types of RNA markers have been proven to be promising candidate markers for body fluids identification in previous studies. This review summarizes the research progress of RNA markers in body fluids identification, including the RNA markers that have been effectively verified in current research and their advantages and disadvantages. Meanwhile, this review prospects the application of RNA markers in forensic medicine.

Relationship between serum miR-199a-5p and miR-378a-3p levels and recurrence of infants with proliferative facial hemangioma after propranolol withdrawal / 口腔疾病防治

Objective@# To investigate the relationship between serum miR-199a-5p and miR-378a-3p levels and the recurrence of infants with proliferative facial hemangioma relapsed after propranolol withdrawal in infants.@*Methods@#Ninety-three infants with proliferative facial hemangioma were selected, all of whom received propranolol treatment. The recurrence of the hemangioma after treatment was followed up, and the children were divided into a recurrence group (23 patients) and a nonrecurrence group (70 patients). Venous blood was collected before and after treatment, and the serum levels of miR-199a-5p and miR-378a-3p were detected by qRT-PCR, and the relationship between the serum levels of miR-199a-5p and miR-378a-3p and the recurrence of proliferative facial hemangioma relapsed after propranolol withdrawal in infants was analyzed.@*Results @# The serum expressions levels of miR-199a-5p and miR-378a-3p in non-relapsed group were increased after treatment compared with before treatment (P < 0.05), and there was no significant difference in the levels of miR-199a-5p and miR-378a-3p in the serum of the recurrence group after treatment compared with before treatment (P > 0.05). After treatment, the levels of miR-199a-5p and miR-378a-3p in the serum of the recurrence group were lower than those of the nonrecurrence group (P < 0.05). After treatment, the serum levels of miR-199a-5p and miR-378a-3p in patients with Ⅲ ~Ⅳ Norm grade hemangioma were higher than those in patients with Ⅰ~Ⅱ Norm grade hemangioma (P < 0.05). Logistic regression analysis showed that the low expression of miR-199a-5p, low expression of miR-378a-3p and tumor grade Ⅰ~Ⅱ after treatment were risk factors for the recurrence of proliferative facial hemangioma after propranolol withdrawal in infants (P < 0.05).@*Conclusion@# Low serum levels of miR-199a-5p and miR-378a-3p are associated with the recurrence of proliferative facial hemangioma after propranolol withdrawal in infants.

Effects of ribonucleic acid for injection Ⅱ combined with cyclophosphamide on the tumor inhibition and survival of sarcoma cell S180 tumor-bearing mice / 肿瘤研究与临床

Objective:To investigate the effects of ribonucleic acid for injection Ⅱ, often called RNA Ⅱ for short, combined with chemotherapeutic drug cyclophosphamide (CTX) on the tumor inhibition and survival of sarcoma cell S180 tumor-bearing mice.Methods:The solid transplanted tumor mouse model of sarcoma cell S180 and peritoneal fluid tumor mouse model were established respectively. CTX (25 mg/kg, once for 2 days) alone or combined with low-dose (25 mg/kg, once a day) and medium-dose (50 mg/kg, once a day) RNA Ⅱ were injected intraperitoneally into solid transplanted tumor mice for 10 d. CTX (25 mg/kg, once for 2 days) alone, medium-dose (50 mg/kg, once a day) or high-dose (100 mg/kg, once a day) RNA Ⅱ alone or combined with CTX were injected intraperitoneally into peritoneal effusion tumor mice until all mice died. The two models were set up for modeling groups without drug treatment, 8 mice in each group. The body mass of solid transplanted tumor mice after administration was weighed, the tumor tissue in vivo was taken out and weighed after the mice were executed, and the tumor inhibition rate was calculated. The body mass of peritoneal effusion tumor mice after administration was weighed, the growth rate of body mass was calculated, the survival curve of each group was drawn, and the life extension rate was calculated.Results:(1) Solid transplanted tumor mice: the body mass of mice in each administration group was lower than that in the modeling group after administration. During the administration period, the tumor volume in the modeling group was much higher than that in each administration group. From the 8th day of administration, the tumor volume in vivo in the CTX group began to be larger compared with that in the two combined administration groups. After stopping the administration and killing the mice, the weighing showed that the tumor mass of each administration group was lower than that in the modeling group (all P < 0.01), the tumor mass of CTX + RNA Ⅱ low-dose group and CTX + RNA Ⅱ medium-dose group was lower than that of CTX group (all P < 0.05), and the tumor inhibition rate of the two groups was higher than that of CTX group (83.6%, 77.2% vs. 58.5%). (2) Peritoneal effusion tumor mice: after administration for 12 d, the body mass growth rate of mice in CTX group was increased rapidly and reached the highest, and the body mass growth rate of mice in the two combined administration groups was lower than that in other groups. The life prolongation rates of RNA Ⅱ high-dose group and CTX group were 48.2% and 53.2% respectively, which had the same effect on life prolongation. The life prolongation rate in RNA Ⅱ medium-dose group was 20.9%. The life prolongation rates of CTX + RNA Ⅱ medium-dose group and CTX + RNA Ⅱ high-dose group were 94.2% and 105.0% respectively. Conclusions:RNA Ⅱ combined with CTX can significantly prolong the survival time of sarcoma cell S180 tumor-bearing mice, increase the tumor inhibition rate and improve the quality of life of the mice. Both of them have a synergistic effect.

Tumor microenvironment responsive liposomes blocking CXCL12/CXCR4 pathway and synergistically enhancing immune efficacy of anti-PD-L1 / 药学学报

Blocking immune checkpoint programmed cell death receptor 1 (PD-1) or programmed death receptor-ligand 1 (PD-L1) can enhance anti-tumor activity of effector T cells. However, the lack of response in many patients to PD-1/PD-L1 therapy remains a question. Improving the immunosuppressive tumor microenvironment (TME) to enhance the efficacy of immune checkpoint inhibitors has become a promising cancer treatment strategy. We constructed a liposome system (PD-L1/siCXCL12-Lp) of CXCL12 siRNA and anti-PD-L1 peptide with matrix metalloproteinases (MMPs) responsiveness, which combined the TME regulation of siCXCL12 and the immune regulation of anti-PD-L1 peptide. All animal experiments were approved by the Biomedical Ethics Committee of Peking University. The authors found that PD-L1/siCXCL12-Lp directly down-regulated the expression of CXCL12 in vitro (33.8%) and in vivo (15.5%). It also effectively increased the ratio of CD8+/Treg by 20.0%, which helped the anti-PD-L1 peptide to better exert its immune effect. The combination therapy significantly inhibited tumor growth (52.08%) with great safety, which explored a new idea for cancer immunotherapy.

The Role of MicroRNA in Force-Related Periodontal Inflammatory Response and Tissue Remodeling / 医用生物力学

Micro-ribonucleic acid(miRNA) is a kind of non-coding single-stranded RNA, which can regulate the expression of genetic information by inhibiting mRNA translation of the target gene and participate in the occurrence and development of a variety of biological processes in vivo. miRNAs also take part in inflammatory diseases and tissue remodeling induced by mechanical forces. Mechanosensitive cells in periodontal tissue can lead to pathological/physiological changes such as periodontal inflammatory response and periodontal remodeling. miRNAs might have played important roles in the occurrence and development of force-related periodontal inflammatory diseases and tissue remodeling, by inhibiting the translation of specific genes in these cells. This article reviews the roles of miRNAs in force-related inflammatory response and tissue remodeling, especially in periodontal inflammatory response and tissue remodeling.

Effects of Peripheral Blood Different Pretreatment Methods and Preservation Time on RNA Quality / 法医学杂志

OBJECTIVES@#To evaluate the effects of different pretreatment methods and preservation time on RNA quality of peripheral blood samples, and to optimize the preservation method of peripheral blood samples.@*METHODS@#Eight pretreatment methods were used to preprocess the peripheral blood from 3 healthy unrelated individuals and the treated samples were stored at -80 ℃. Total RNA of samples was extracted using Quick-RNATM Miniprep Plus kit. DNA/RNA ShieldTM was added to peripheral blood and total RNA was extracted after preservation at -80 ℃ for 0, 5, 10, 15, 30 and 60 days, respectively. The concentration, purity and integrity of RNA were determined. Statistical analyses were performed by SPSS 22.0 software to compare the differences in RNA yield, purity and integrity among the eight pretreatment methods.@*RESULTS@#In terms of purity, leukocyte pretreated with RNAlaterTM and directly cryopreservation peripheral blood showed the worst purity. The other six methods showed better purity. In terms of yield, blood cells with DNA/RNA ShieldTM came out with the highest yield, followed by peripheral blood with DNA/RNA ShieldTM. In terms of integrity, peripheral blood preserved in PAXgene Blood RNA tube method had the best integrity. Except for peripheral blood pretreated with DNA/RNA ShieldTM and blood cells pretreated with DNA/RNA shieldTM, the other five methods had statistical differences when compared to the method by keeping peripheral blood in PAXgene Blood RNA tube. The purity of RNA stored at six-time gradients ranged from 1.815 to 1.952. With the increase of storage time, RNA yield decreased from 4.516 ng to 1.039 ng, and RNA integrity decreased from 8.533 to 7.150.@*CONCLUSIONS@#According to the results of total RNA's yield, purity and integrity, peripheral blood pretreated with DNA/RNA ShieldTM was the best pretreatment method. After the pretreatment, samples can be preserved for up to 60 days in low temperature.

Quality control analysis of brain tissue samples from Shanghai Brain Bank / 解剖学报

Objective To evaluate the feasibility of using brain tissues from Shanghai Brain Bank for the applications on biological research through the analysis of pH value of cerebrospinal fluids, RNA integrity number (RIN), transcriptome, proteome and morphology of brain tissues. Methods The pH value of fresh cerebrospinal fluid was detected by pH test paper; the RNA integrity of cryopreserved brain tissues was examined by Agilent RNA 6000 Nano chip and Agilent 2100 bioanalyzer; the transcriptome sequencing of superior temporal gyrus or caudate was performed using BGIseq-500 sequencer; the proteome of cryopreserved brain tissues was analyzed by Q Exactive HF mass spectrometer; at last, the morphology of the superior temporal gyrus was observed by HE staining. Results The pH value of the cerebrospinal fluid on average was about 6.5. The RIN values of more than 65% of brain tissues were more than 6, indicating good RNA quality. The clean reads ratio after transcriptome sequencing filtering was basically above 80%, indicating that the quality of sequencing library was high. The mass spectrometry analysis of frozen brain samples yielded more than 4000 protein groups and 30 000 peptides, indicating high quality of proteomic data. The morphology of brain tissues was relatively normal, with clearly visible neurons. Conclusion The quality of RNA and protein of brain tissues from Shanghai Brain Bank meets the basic needs for molecular and biological research, and the fixed brain samples can be used for morphological observations.

Las formulaciones anti-COVID-19 con ácido ribonucleico mensajero en la clasificación general de las vacunas antivirales

RESUMEN Las vacunas representan un hito fundamental para la prevención y el control de las enfermedades infectocontagiosas, con repercusión excepcional en la salud mundial. Su valor es incuestionable para evitar la aparición de numerosos padecimientos y muertes cada año. Existen numerosas clasificaciones de las vacunas, según se atienda a diferentes aspectos de su composición, síntesis o naturaleza. En este artículo se presenta una clasificación de los diseños actuales en que se sustentan las diversas plataformas tecnológicas de las vacunas antivirales. Se hace especial énfasis en las basadas en genes, entre ellas las vacunas de ácido ribonucleico mensajero que han recibido un impulso especial en su desarrollo desde el comienzo de la pandemia de la COVID-19. Las implicaciones de la respuesta satisfactoria de las vacunas de ácido ribonucleico mensajero podrían ir más allá de la actual pandemia de la COVID-19. Su éxito podría allanar el camino para el uso generalizado de esta plataforma tecnológica tanto para los patógenos emergentes como para los ya establecidos.

ABSTRACT Although the use of vaccines for disease prevention and control is a relatively recent social and health event, it has no doubt become one of the main tools of modern medicine to fight infectious diseases. The paper presents a classification of current designs substantiating the various technological platforms of antiviral vaccines, with special emphasis on those based on genes, among them messenger ribonucleic acid vaccines, which have experienced considerable development since the start of the COVID-19 pandemic. The implications of the successful response of messenger ribonucleic acid vaccines could go beyond the current COVID-19 pandemic. Its success could pave the way for the widespread use of this technology platform for both emerging and established pathogens.

Efficacy and safety of ribonucleic acid for injection Ⅱ combined with chemotherapy in treatment of advanced non-small cell lung cancer: a multicenter, retrospective and controlled study / 肿瘤研究与临床

Objective:To evaluate the efficacy and safety of ribonucleic acid for injection Ⅱ combined with chemotherapy in the treatment of advanced non-small cell lung cancer (NSCLC).Methods:Based on the LinkDoc database, 2 111 patients who were diagnosed with stage Ⅲ B and Ⅳ NSCLC in 8 research centers such as Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology from January 2014 to December 2017 were included. Patients were divided into observation group (1 039 cases) and control group (1 072 cases) according to whether or not they had used ribonucleic acid for injection Ⅱ during chemotherapy. Inverse probability of treatment weighting was used to correct the confounding factors of patients, and there were 1 078 cases in the control group and 1 033 cases in the observation group; the overall survival (OS), progression-free survival (PFS) and the occurrence of adverse events and chemotherapy-related adverse reactions were compared between the two groups. Results:The median OS time of the observation group and the control group was 18.51 months and 15.65 months, and the median PFS time was 7.00 months and 5.49 months, and the differences were statistically significant ( P values ??were 0.001 and 0.003). The incidence of adverse events in the observation group was slightly higher than that in the control group [75.6% (781/1 033) vs. 74.1% (799/1 078)], and the incidence of chemotherapy-related adverse reactions in the observation group was slightly higher than that in the control group [43.9% (453/1 033) vs. 40.7% (439/1 078)], but the differences were not statistically significant (both P > 0.05). Conclusions:Ribonucleic acid for injection Ⅱ can prolong the OS and PFS time of patients with stage Ⅲ B and Ⅳ NSCLC receiving chemotherapy. It is safe and can increase the clinical benefit of patients to a certain extent.

Quantitative Expression of RNA from Frozen Organs and Formaldehyde-fixed and Paraffin-embedded Tissues / 法医学杂志

Objective Quantitative analysis and comparison of the expression of ribonucleic acid （RNA） from frozen organs and formaldehyde-fixed and paraffin-embedded （FFPE） tissues. Methods Frozen specimens of human brain, myocardium and liver tissues as well as FFPE samples at different postmortem intervals were collected and mass concentration of RNA was extracted and detected. Reverse transcription-quantitative polymerase chain reaction （RT-qPCR） technology was used to analyze the amplification efficiency and relative expression of each RNA marker. Results The mass concentration and integrity of RNA extracted from FFPE samples were relatively low compared with frozen specimens. The amplification efficiency of RNA markers was related with RNA species and the length of amplification products. Among them, glyceraldehyde-3-phosphate dehydrogenase （GAPDH） and β-actin （ACTB） with relatively long amplification products failed to achieve optimal amplification efficiency, whereas 5S ribosomal RNA （5S rRNA） achieved ideal amplification efficiency and showed quite stable expression across various tissues, therefore it was chosen as internal reference marker. The expression quantity of GAPDH and ACTB in frozen specimens with longer postmortem intervals and in FFPE samples with relatively long amplification products was decreased. The expressions of tissue-specific microRNAs （miRNAs）, GAPDH and ACTB with relatively short amplification products had consistency in the same tissues and FFPE samples. Conclusion Through standardizing the RT-qPCR experiment, selecting the appropriate RNA marker and designing primers of appropriate product length, RNA expression levels of FFPE samples can be accurately quantified.

Combination of mRNA of Repair-related Genes in Rat Skeletal Muscles for Wound Age Estimation / 法医学杂志

Objective To investigate the estimation of early and mid-term wound age by a combination of four mRNAs, the DNA polymerase delta-interacting protein 3 （POLDIP3） mRNA, regulator of chromosome condensation 1 like （RCC1L） mRNA, proline-rich 5 （PRR5） mRNA, and ribonucleic acid export 1 （RAE1） mRNA in rats skeletal muscles. Methods The model of rat skeletal muscle contusion was established, and then contusion area muscle tissue was extracted 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 and 48 h after injury. Histomorphological changes during the repair process after rat skeletal muscle contusion were observed. The relative expressions of Poldip3, Rcc1l, Prr5 and Rae1 mRNAs were detected by reverse transcription real-time quantitative polymerase chain reaction （RT-qPCR）. Different stages of wound age were classified by using the expression patterns of four genes at various time points after injury. The accuracy of the results was verified by Fisher discriminant analysis. Results Histomorphological results showed that the repair process after skeletal muscle contusion occurred with the prolonging of time. Through combination of the expression trends of the four kinds of mRNAs, the 48 h after injury could be divided into three periods, 4-12 h, 16-28 h and 32-48 h. The Fisher discrimination method showed that the classification accuracy rates of the three periods were 83.3%, 75.0% and 73.3%, respectively. Conclusion The classification discrimination based on the relative expression of every gene has a higher accuracy, and the accuracy of wound age estimation with combination of mRNA relative expressions is higher than that with a single indicator. By combining with Fisher discrimination method, this method can be used for early and mid-term wound age estimation.

Differential expression of lncRNA in the serum of ankylosing spondylitis patients / 中华骨科杂志

Objective@#To investigate the differential expression of lncRNA in the serum of ankylosing spondylitis (AS) patients, with the goal of findingnew potential biomarkers for the diagnosis and targeted treatment of AS.@*Methods@#A total of 19 AS patients and 19 age-matched healthy controls treated at Nanjing Drum Tower Hospitalfrom January 2017 to September 2017 were recruited. Average age were 38.74±7.42 (range, 25-51) and 37.00±6.86 (range, 26-50). High-throughput lncRNA sequencing technology was used to detect differently expressed lncRNAs in the serum of 3 AS patients and 3 healthy controls. Target lncRNAs for further validation were selected according to the P values and fold-changes. In the rest of the serum samples (16 AS patients and 16 healthy controls), Trizol-based technique was used to extract total RNA, and after reverse transcription to obtain cDNA, RT-qPCR was preformed to confirm the sequencing results.@*Results@#Using high-throughput lncRNA sequencing, a total of 41 up-regulated and 2 down-regulated lncRNAs were detected in the serum of AS patients. After sorted by the P values, 4 lncRNAswith a fold-change larger than 2 were chosen as the target genes for RT-qPCR (ENST00000365494.1, P=2.6×10-277, fold-change: 2.05; ENST00000364938.1, P=2.49×10-77, fold-change: 2.19; ENST 00000363046.1, P=2.67×10-29, fold-change: 2.51; ENST00000384756.1, P=6.17×10-21, fold-change: 2.28). RT-qPCR results showed the relative expression of lncRNA ENST00000365494.1 was 1.80±0.22 (P=0.304), lncRNA ENST00000364938.1was 0.78±0.07 (P=0.417), lncRNA ENST00000363046.1was 1.28±0.24 (P=0.793), lncRNA ENST00000384756.1 was 1.52±0.25 (P=0.611)and tendency of up-regulation was found in 3 of them, which was consistent with the sequencing results. However, the difference did not achieve statistical significance.@*Conclusion@#Sequencing result could not be confirmed by RT-qPCR with a larger sample size, which implied the differential expression of lncRNA might not exist in the peripheral blood of AS patients, and further studies regarding lncRNA in AS could focus more on its differential expression and function in the focal tissue.

Ribonucleic acid II induces apoptosis of human leukemia kgla cells through pi3k/akt activation mediated by reactive oxygen / 中国药理学通报

Aim To investigate the correlation between ribonucleic acid ? on human leukemia cell lines KG1a and PI3K/Akt signaling pathway by reactive oxygen.Methods The cell cycle and the ROS level of ribonucleic acid ? in human leukemia cell lines were determined by flow cytometry.The levels of p-PI3K, p-Akt, p-MDM2, p21, cyclin D1 and CDK4 were detected by Western blot.The expression levels of protein p-PI3K, p-Akt and p53 after treated with ribonucleic acid ? and NAC were also detected by Western blot.Results The results of FCM revealed that the cell cycle was arrested at G0/G1 phase and ROS pre-vented the arrest of cell cycle.The results also showed that ROS level increased with the concentration of ribonucleic acid ? in KG1a cells.Western blot results showed after treated with ribonucleic acid ?, the expression levels of p-PI3K, p-Akt, p-MDM2, cyclin D1 and CDK4 decreased, while p21 increased in KG1a cells.After treated with ribonucleic acid ? and NAC, the expression levels of p-PI3K and p-Akt increased, while p53 decreased.Conclusion Ribonucleic acid ? can induce the apoptosis of leukemia KG1a cells through ROS-mediated PI3K/Akt signaling pathway.

Advances in the research of effects of competing endogenous RNAs and their regulatory networks in pathological scars of skin / 中华烧伤杂志

The skin pathologic scar is a skin fibrous proliferative disease characterized by abnormal proliferation of fibroblasts and overdeposition of extracellular matrix. Unclarity of genesis and development mechanism is the main reason that restricts its diagnosis and treatment. In recent years, it has been found that microRNAs play important roles in the regulation mechanism of pathological scars. The competing endogenous RNAs (ceRNAs) have microRNA response elements which can be competitively combined with microRNAs through sponge adsorption. Through the mutual regulation of RNAs, ceRNAs regulate the expression of target gene and participate in the development of disease. Based on the ceRNA hypothesis, this paper systematically reviews the biological functions and clinical significance of ceRNAs in pathological scars of skin, and discusses the role of ceRNAs and " RNA-microRNA-RNA" regulation network in pathologic scars. The ceRNA therapy may become a new model therapy for skin scars in the future.

Differential expression of lncRNA in the serum of ankylosing spondylitis patients / 中华骨科杂志

To investigate the differential expression of lncRNA in the serum of ankylosing spondylitis (AS) patients, with the goal of findingnew potential biomarkers for the diagnosis and targeted treatment of AS. Methods A total of 19 AS patients and 19 age?matched healthy controls treated at Nanjing Drum Tower Hospitalfrom January 2017 to September 2017 were recruited. Average age were 38.74±7.42 (range, 25-51) and 37.00±6.86 (range, 26-50). High?throughput lncRNA sequenc?ing technology was used to detect differently expressed lncRNAs in the serum of 3 AS patients and 3 healthy controls. Target ln?cRNAs for further validation were selected according to the P values and fold?changes. In the rest of the serum samples (16 AS pa?tients and 16 healthy controls), Trizol?based technique was used to extract total RNA, and after reverse transcription to obtain cD?NA, RT?qPCR was preformed to confirm the sequencing results. Results Using high?throughput lncRNA sequencing, a total of 41 up?regulated and 2 down?regulated lncRNAs were detected in the serum of AS patients. After sorted by the P values, 4 ln?cRNAswith a fold?change larger than 2 were chosen as the target genes for RT?qPCR (ENST00000365494.1, P=2.6×10-277, fold?change: 2.05; ENST00000364938.1, P=2.49×10-77, fold?change: 2.19; ENST 00000363046.1, P=2.67×10-29, fold?change: 2.51;ENST00000384756.1, P=6.17×10- 21, fold?change: 2.28). RT?qPCR results showed the relative expression of lncRNA ENST00000365494.1 was 1.80 ± 0.22 (P=0.304), lncRNA ENST00000364938.1was 0.78 ± 0.07 (P=0.417), lncRNA ENST00000363046.1was 1.28±0.24 (P=0.793), lncRNA ENST00000384756.1 was 1.52±0.25 (P=0.611)and tendency of up?regu?lation was found in 3 of them, which was consistent with the sequencing results. However, the difference did not achieve statistical significance. Conclusion Sequencing result could not be confirmed by RT?qPCR with a larger sample size, which implied the differential expression of lncRNA might not exist in the peripheral blood of AS patients, and further studies regarding lncRNA in AS could focus more on its differential expression and function in the focal tissue.