HPV prevalence and genotype distribution in 2,306 patients with cervical squamous cell carcinoma in central and eastern China.

Background: To explore the positivity rate and genotype distribution of human papillomavirus (HPV) in cervical squamous cell carcinoma (CSCC) tissues in central and eastern China and to provide theoretical basis for cervical cancer screening and prophylactic HPV vaccine development in China. Methods: DNA was extracted from paraffin-embedded tissues of CSCC samples and exfoliated cervical cells of cervical cancer screening populations. 23 HPV genotypes were detected by combining polymerase chain reaction (PCR) and reverse dot hybridized gene chip detection technology in 2,306 CSCC tissues and 10,245 cervical cancer screening populations. The genotype distribution of HPV infection was analyzed. Results: The overall infection rate of HPVs in 2,306 CSCC patients was 92.71%. The frequency of single-type HPV infection and multiple-type HPV infection were 86.48% and 13.51%, respectively. The most common HPV genotypes detected in Chinese CSCC tissues were HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52, HPV-58, and HPV-59. The overall positivity rate of these eight high-risk HPV (HR-HPV) genotypes in HPV-positive CSCC was as high as 96.91%. Of which the positivity rate of seven HR-HPV genotypes related to nine-valent HPV vaccines in HPV-positive CSCC was 95.09%. Meanwhile, the overall infection rates of HR-HPV and low-risk HPV (LR-HPV) in female aged 35-64 years who underwent cervical cancer screening were 13.16% and 1.32%, respectively. The high-frequency HR-HPV genotypes in cervical cancer screening women were HPV-52, HPV-58, HPV-16, HPV-53, HPV-68, HPV-39, HPV-51, and HPV-56, with positivity rates of 2.25%, 1.60%, 1.31%, 1.22%, 0.93%, 0.92%, 0.78%, and 0.74%, respectively. Conclusion: Among women screened for cervical cancer in China, detecting the 8 high-frequency HR-HPV genotypes can reduce technical difficulty and reagent costs, while also improving the efficiency and effectiveness of cervical cancer screening. HPV genotyping assists gynecologists in assessing the risk of HR-HPV-positive cervical intraepithelial neoplasia and guiding them in implementing appropriate interventions. Furthermore, HPV genotyping is helpful for doctors to follow up HR-HPV-positive women and to evaluate the protective effect of HPV vaccine.

Cuproptosis-related gene SERPINE1 is a prognostic biomarker and correlated with immune infiltrates in gastric cancer.

PURPOSE: The serine protease inhibitor clade E member 1 (SERPINE1) has been studied as a potential biomarker in a variety of cancers, but poorly studied in gastric cancer (GC). The purpose of this study was to explore the prognostic value of SERPINE1 in GC and primarily analyze its functions. METHODS: We analyzed the the prognostic value of SERPINE1 and studied the relationship with clinicopathologic biomarkers in gastric cancer. The expression of SERPINE1 was analyzed by GEO and TCGA databases. Moreover, we validated the results by immunohistochemistry. Next, the correlation analysis between SERPINE1 and the cuproptosis-related genes was analyzed by the "Spearman" method. CIBERSORT and TIMER algorithms were used to analyze the correlation of SERPINE1 with immune infiltration. Furthermore, GO and KEGG gene enrichment analyses were used to study the functions and pathways that SERPINE1 might be involved in. Then, drug sensitivity analysis was performed using CellMiner database. Finally, a cuproptosis-immune-related prognostic model was constructed using genes related to immune and cuproptosis, and verified against external datasets. RESULTS: SERPINE1 was up-regulated in gastric cancer tissues, which tends toward poor prognosis. Using immunohistochemistry experiment, the expression and prognostic value of SERPINE1 were verified. Then, we found that SERPINE1 was negatively correlated with cuproptosis-related genes FDX1, LIAS, LIPT1, and PDHA1. On the contrary, SERPINE1 was positively correlated with APOE. This indicates the effect of SERPINE1 on the cuproptosis process. Furthermore, by conducting immune-related analyses, it was revealed that SERPINE1 may promote the inhibitory immune microenvironment. The infiltration level of resting NK cells, neutrophils, activated mast cells, and macrophages M2 was positively correlated with SERPINE1. However, B cell memory and plasma cells were negatively correlated with SERPINE1. Functional analysis showed that SERPINE1 was closely related to angiogenesis, apoptosis, and ECM degradation. The KEGG pathway analysis showed that SERPINE1 may be associated with P53, Pi3k/Akt, TGF-ß, and other signaling pathways. Drug sensitivity analysis showed that SERPINE1 could be also seen as a potential treatment target. The risk model based on SERPINE1 co-expression genes could better predict the survival of GC patients than SERPINE1 alone. We also verified the prognostic value of the risk score by GEO external datasets. CONCLUSION: SERPINE1 is highly expressed in gastric cancer and related to poor prognosis. SERPINE1 may regulate cuproptosis and the immune microenvironment by a series of pathways. Therefore, SERPINE1 as a prognostic biomarker and potential therapeutic target deserves further study.

Kainic acid induced hyperexcitability in thalamic reticular nucleus that initiates an inflammatory response through the HMGB1/TLR4 pathway.

OBJECTIVE: To explore the relationship between the proinflammatory factor high-mobility group box 1 (HMGB1) and glutamatergic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the development of epilepsy. METHODS: Thalamic reticular nucleus (TRN) slices were treated with kainic acid (KA) to simulate seizures. Action potentials and spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded within TRN slices using whole-cell patch clamp techniques. The translocation of HMGB1 was detected by immunofluorescence. The HMGB1/TLR4 signaling pathway and its downstream inflammatory factors (IL-1ß and NF-κB) were detected by RTPCR, Western blot and ELISA. RESULTS: KA-evoked spikings were observed in TRN slices and blocked by perampanel. sIPSCs in the TRN were enhanced by KA and reduced by perampanel. The translocation of HMGB1 in the TRN was promoted by KA and inhibited by perampanel. The expression of the HMGB1/TLR4 signaling pathway was promoted by KA and suppressed by perampanel. CONCLUSION: KA induced hyperexcitability activates the HMGB1/TLR4 pathway, which potentially leading to neuroinflammation in epilepsy.

Transcriptomic and metabolomic data reveal key genes that are involved in the phenylpropanoid pathway and regulate the floral fragrance of Rhododendron fortunei.

BACKGROUND: To reveal the key genes involved in the phenylpropanoid pathway, which ultimately governs the fragrance of Rhododendron fortunei, we performed a comprehensive transcriptome and metabolomic analysis of the petals of two different varieties of two alpine rhododendrons: the scented R. fortunei and the unscented Rhododendron 'Nova Zembla'. RESULTS: Our transcriptomic and qRT-PCR data showed that nine candidate genes were highly expressed in R. fortunei but were downregulated in Rhododendron 'Nova Zembla'. Among these genes, EGS expression was significantly positively correlated with various volatile benzene/phenylpropanoid compounds and significantly negatively correlated with the contents of various nonvolatile compounds, whereas CCoAOMT, PAL, C4H, and BALDH expression was significantly negatively correlated with the contents of various volatile benzene/phenylpropanoid compounds and significantly positively correlated with the contents of various nonvolatile compounds. CCR, CAD, 4CL, and SAMT expression was significantly negatively correlated with the contents of various benzene/phenylpropanoid compounds. The validation of RfSAMT showed that the RfSAMT gene regulates the synthesis of aromatic metabolites in R. fortunei. CONCLUSION: The findings of this study indicated that key candidate genes and metabolites involved in the phenylpropanoid biosynthesis pathway may govern the fragrance of R. fortunei. This lays a foundation for further research on the molecular mechanism underlying fragrance in the genus Rhododendron.

Rehabilitation of knee injury by chinese boxing / Reabilitação em lesão de joelho pelo boxe chinês / Rehabilitación de lesión de rodilla mediante el boxeo chino

ABSTRACT Introduction: Chinese boxing is a highly combative sport and injuries are inevitable. Rehabilitation of knee joint injuries can not only save the athlete's professional career but also improve their prevention ability. Objective: Study the situation of knee joint injuries in Chinese boxing and explore the strategies of sports rehabilitation. Methods: This experiment lasted 8 weeks. The knee joint activity and daily exercise ability of the control and experimental groups were tested. Results: In the experimental group, the knee joint extension range was increased to - 1.61° (± 2.228), the knee joint flexion range was increased to 97.14° (± 6.430), the walking ability score was increased to 32.25 (± 5.751) points, and the stair climbing and descending ability score was increased to 35.12 (± 8.764) points. The control group's scores were also optimized, but the range of optimization was smaller than that of the experimental group. Conclusion: Compared with traditional physical therapy and massage, sports rehabilitation training has a more significant effect on improving the range of motion and prevention ability on athletes' knee joints and should be promoted. Level of evidence II; Therapeutic studies - investigation of treatment outcomes.

RESUMO Introdução: O boxe chinês é um esporte altamente combativo e suas lesões são inevitáveis. A reabilitação de lesões nas articulações do joelho pode não apenas salvar a carreira profissional do atleta como também melhorar a sua capacidade de prevenção. Objetivo: Estudar a situação das lesões nas articulações do joelho pelo boxe chinês e explorar as estratégias de reabilitação esportiva. Métodos: Esta experiência teve duração de 8 semanas. A atividade articular do joelho e a capacidade de exercício diário dos grupos controle e experimental foram testadas. Resultados: No grupo experimental, o alcance da extensão da articulação do joelho foi aumentado para - 1,61° (± 2,228), o alcance da flexão da articulação do joelho foi aumentado para 97,14° (± 6,430), a pontuação da capacidade de caminhar foi aumentada para 32,25 (± 5,751) pontos, e a pontuação da capacidade de subir e descer escadas foi aumentada para 35,12 (± 8,764) pontos. Os índices do grupo de controle também foram otimizados, mas a faixa de otimização foi menor do que a do grupo experimental. Conclusão: Em comparação com a fisioterapia e massagem tradicionais, o treinamento de reabilitação esportiva tem um efeito mais significativo na melhoria da amplitude de movimento e na capacidade de prevenção sobre as articulações dos joelhos dos atletas, devendo ser promovido. Nível de evidência II; Estudos terapêuticos - investigação dos resultados do tratamento.

RESUMEN Introducción: El boxeo chino es un deporte altamente combativo y sus lesiones son inevitables. La rehabilitación de las lesiones de la articulación de la rodilla no sólo puede salvar la carrera profesional del deportista, sino también mejorar su capacidad de prevención. Objetivo: Estudiar la situación de las lesiones de la articulación de la rodilla por el boxeo chino y explorar las estrategias de rehabilitación deportiva. Métodos: Este experimento duró 8 semanas. Se comprobó la actividad de la articulación de la rodilla y la capacidad de ejercicio diario de los grupos de control y experimental. Resultados: En el grupo experimental, el rango de extensión de la articulación de la rodilla aumentó a - 1,61° (± 2,228), el rango de flexión de la articulación de la rodilla aumentó a 97,14° (± 6,430), la puntuación de la capacidad de caminar aumentó a 32,25 (± 5,751) puntos, y la puntuación de la capacidad de subir y bajar escaleras aumentó a 35,12 (± 8,764) puntos. Las puntuaciones del grupo de control también se optimizaron, pero el rango de optimización fue menor que el del grupo experimental. Conclusión: En comparación con la fisioterapia tradicional y el masaje, el entrenamiento de rehabilitación deportiva tiene un efecto más significativo en la mejora de la amplitud de movimiento y la capacidad de prevención en las articulaciones de la rodilla de los atletas y debería promoverse. Nivel de evidencia II; Estudios terapéuticos - investigación de los resultados del tratamiento.

[Identification of terpene synthase gene family members in Rhododendron and its relationship with terpenoid metabolism].

Terpene synthase (TPS) plays important roles in the synthesis of terpenoids which are the main fragrances in Rhododendron flowers. To understand the function of TPS genes in terpenoid metabolism in relation to flower aroma formation, we identified all TPS gene family members in Rhododendron by analyzing its genome database. We then used a transcriptomic approach to analyze the differential gene expression patterns of TPS gene family members in the scented flower Rhododendron fortunei compared to the non-scented flower Rhododendron 'Nova Zembla'. The contents of terpenoid compounds in petals of the above two Rhododendron species at different developmental stages were also measured by using qRT-PCR and head space-solid phase micro-extraction combined with gas chromatography-mass spectrometry. Our results showed that a total of 47 RsTPS members, with individual lengths ranged from 591 to 2 634 bp, were identified in the Rhododendron genome. The number of exons in RsTPS gene ranged from 3 to 12, while the length of each protein encoded ranged from 196 to 877 amino acids. Members of the RsTPS family are mainly distributed in the chloroplast and cytoplasm. Phylogenetic analysis showed that RsTPS genes can be clustered into 5 subgroups. Seven gene family members can be functionally annotated as TPS gene family since they were temporally and spatially expressed as shown in the transcriptome data. Notably, TPS1, TPS10, TPS12 and TPS13 in Rhododendron fortunei were expressed highly in flower buds reached the peak in the full blossoming. Correlation analysis between gene expression levels and terpenoid content indicates that the expression levels of TPS1, TPS4, TPS9, TPS10, TPS12 and TPS13 were positively correlated with the content of terpenoids in the petals of R. fortunei at all flower developmental stages, suggesting that these six genes might be involved in the aroma formation in R. fortunei.

Abnormal Ventral Somatomotor Network Homogeneity in Patients With Temporal Lobe Epilepsy.

Background: Abnormalities of functional connectivity in the somatomotor network have been thought to play an essential role in the pathophysiology of epilepsy. However, there has been no network homogeneity (NH) study about the ventral somatomotor network (VSN) in patients with temporal lobe epilepsy (TLE). Therefore, we explored the NH of the VSN in TLE patients in this study. Methods: The sample included 52 patients with left temporal lobe epilepsy, 83 patients with right temporal lobe epilepsy, and 68 healthy controls. The NH method was utilized to analyze the resting-state functional magnetic resonance imaging data. Results: Compared to the controls, rTLE patients had significantly higher NH in the bilateral postcentral gyrus, and significantly lower NH in the bilateral Rolandic operculum and the right superior temporal gyrus (STG). The NH values of the left postcentral gyrus were significantly higher in lTLE patients than in the healthy controls, and lTLE patients had lower NH in the right Rolandic operculum. The altered NH in the postcentral gyrus was negatively correlated with the illness duration, and the decreased NH in the left Rolandic operculum was negatively correlated with the executive control reaction time (ECRT). Conclusion: Our findings suggest that altered NH of the postcentral gyrus, Rolandic operculum and STG might be associated with the pathophysiology of TLE, and thus, highlight the contribution of the VSN to the pathophysiology of TLE.

[Effects of aridity on carbon, nitrogen, and phosphorus contents in soils and plants of mountain swamps, Zhejiang, China]. / 旱化对浙江山地沼泽湿地土壤与植物碳氮磷含量的影响.

Mountain swamps in Zhejiang Province have been suffered from severe drouhgt threats because of climate change and artificial interruption. Sphagnum bogs and swamps were gradually degraded into arid swamps. However, the effects of arid processes on the C, N, P contents of soils and their stoichiometry in mountain swamps are still unclear. We measured C, N and P concentrations, pH values, and bulk density in the upper 0-60 cm soil layers in the stands of five mountain swamps with the different arid levels. Moreover, the aboveground biomass and the C, N, P concentrations in the crushed plant mixture were also measured. The results showed that the soils of mountain swamps in Zhejiang Province were rich in soil organic carbon (SOC), total nitrogen (TN), but infertile in phosphorus (TP). Aboveground biomass, soil pH, bulk density increased, while SOC, TN, TP, C:N, C:P, N:P decreased with aridity. Soil pH and bulk density had significant negative correlations with SOC, TN, and TP in soils, respectively. The differences in the C, N, P accumulation in the soils were probably associated with litterfall production, the oxygen condition of wetlands, and the degree of plant decomposition at the different types of mountain swamps. In all, arid trends were obvious at the mountain swamps in Zhejiang Province. Soil nutrients, such as C, N, P, deceased, while plant community succeeded from the wet swamp to the mesophyte vegetation with the arid processes. The contents of C, N and P in the plants varied across species, and were not coupled with those in the soils.

Abnormal Degree Centrality as a Potential Imaging Biomarker for Right Temporal Lobe Epilepsy: A Resting-state Functional Magnetic Resonance Imaging Study and Support Vector Machine Analysis.

Previous studies have reported altered neuroimaging features in right temporal lobe epilepsy (rTLE). However, the alterations in degree centrality (DC) as a diagnostic method for rTLE have not been reported. Therefore, we aimed to explore abnormalities in the DC of the rTLE and whether such alterations could be applied to the diagnosis of rTLE. Resting-state functional magnetic resonance imaging (fMRI) was used to scan 82 patients with rTLE and 69 healthy controls. The DC and support vector machine (SVM) methods were used for an analysis of the imaging data. Compared to the control group, the rTLE patients exhibited lower DC values in the right hippocampus, right superior temporal gyrus, and right caudate. Compared to the control group, the rTLE patients showed higher DC values in the right medial superior frontal gyrus (SFGmed), left dorsolateral superior frontal gyrus (SFGdor), right inferior parietal lobule (IPL), and the left postcentral. The highest diagnostic accuracy of 99.34% (150/151), based on SVM analysis, was demonstrated for the combination of abnormal DC in the right IPL and the left SFGdor, along with a sensitivity of 100% (82/82), and a specificity of 98.55% (68/69) for the differentiation of rTLE patients from healthy controls. The study demonstrated abnormal functional connectivity in rTLE patients. Thus, a distinctive DC pattern may serve as an imaging marker for the diagnosis of rTLE patients.

Abnormal Fractional Amplitude of Low-Frequency Fluctuation as a Potential Imaging Biomarker for First-Episode Major Depressive Disorder: A Resting-State fMRI Study and Support Vector Machine Analysis.

Objective: Major depressive disorder (MDD) is a psychiatric disorder with serious negative health outcomes; however, there is no reliable method of diagnosis. This study explored the clinical diagnostic value of the fractional amplitude of low-frequency fluctuation (fALFF) based on the support vector machine (SVM) method for the diagnosis of MDD. Methods: A total of 198 first-episode MDD patients and 234 healthy controls were involved in this study, and all participants underwent resting-state functional magnetic resonance imaging (fMRI) scanning. Imaging data were analyzed with the fALFF and SVM methods. Results: Compared with the healthy controls, the first-episode MDD patients showed higher fALFF in the left mid cingulum, right precuneus, and left superior frontal gyrus (SFG). The increased fALFF in these three brain regions was positively correlated with the executive control reaction time (ECRT), and the increased fALFF in the left mid cingulum and left SFG was positively correlated with the 17-item Hamilton Rating Scale for Depression (HRSD-17) scores. The SVM results showed that increased fALFF in the left mid cingulum, right precuneus, and left SFG exhibited high diagnostic accuracy of 72.92% (315/432), 71.76% (310/432), and 73.84% (319/432), respectively. The highest diagnostic accuracy of 76.39% (330/432) was demonstrated for the combination of increased fALFF in the right precuneus and left SFG, along with a sensitivity of 84.34% (167/198), and a specificity of 70.51% (165/234). Conclusion: Increased fALFF in the left mid cingulum, right precuneus, and left SFG may serve as a neuroimaging marker for first-episode MDD. The use of the increased fALFF in the right precuneus and left SFG in combination showed the best diagnostic value.

[Quality evaluation of Polygonatum cyrtonema based on HPLC fingerprint and multi-component quantitative analysis].

The medicinal and edible Polygonatum cyrtonema is one of the original species of Polygonati Rhizoma. In this study,HPLC fingerprints for 25 batches of P. cyrtonema from 6 provinces were established. A total of 14 common peaks were identified and the similarities of the fingerprints were in the range of 0. 939-0. 999. In additon, the partial least squares-discriminant analysis(PLSDA) demonstrated that the samples had low discriminability except for JX-1 and most components of them had no significant correlation with environmental factors such as longitude, latitude, and altitude. Thus, chemical composition specificity of P. cyrtonema in natural distribution areas had no obvious regularity and their variation might be induced by the local environment. This conclusion explained the lack of records about Dao-di area of Polygonati Rhizoma. However, JX-1 boasted significantly higher content of 5-hydroxymethylfurfural(HMF) and 4',5,7-trihydroxy-6,8-dimethylhomoisoflavone( HIF), thick and long inflorescence and rhizome, and extremely high yield. Therefore, excellent variety of P. cyrtonema might have great potential to improve the quality and yield of Polygonati Rhizoma. Moreover, three components of HMF, polygonalline A(PA), and HIF were identified in the fingerprint. Among them, HMF has the activities of blood rheology improvement, antioxidation, and anti-myocardial ischemia and PA is an indolizine alkaloid with potential anti-inflammatory activity. HIF, the characteristic homoisoflavone in Polygonatum, has the pharmacological activities of regulating blood glucose and anti-tumor. A quantitative analysis method can provide a theoretical basis for the improvement of the quality evaluation of Polygonati Rhizoma.

MALAT1 Regulated mTOR-Mediated Tau Hyperphosphorylation by Acting as a ceRNA of miR144 in Hippocampus Cells Exposed to High Glucose.

AIM: High glucose (HG)-induced activation of mTOR promotes tau phosphorylation and leads to diabetes-associated dementia. This study aimed to explore the role of metastasis associated in lung adenocarcinoma transcript 1 (MALAT1) in HG-induced neuronal cell injury. METHODS: Hippocampus cells were isolated from C57BL/6J mice. After 6 days of culture, the cells were incubated with 5.5 mM glucose in normal medium or 75 mM glucose for 4 days. Cells were transfected with miR-144 mimic, miR-144 inhibitor, siRNA for MALAT1 or corresponding controls. Gene expression was detected by PCR and Western blot analysis. RESULTS: HG increased the levels of MALAT1 and p-tau in hippocampal cells. Knockdown of MALAT1 partially reversed the effects of HG on mTOR activity and p-tau protein levels. MALAT1 functioned as competing endogenous RNA (ceRNA) for miR-144, and pre-treatment with MALAT1 siRNA decreased mTOR activity and p-tau protein level in HG-treated hippocampal cells, which was significantly attenuated by miR-144 mimics. Moreover, miR-144 negatively regulated the expression of mTOR and knockdown of MALAT1 suppressed mTOR, while overexpression of mTOR abrogated protective effects of MALAT1 knockdown in HG-treated hippocampal cells. CONCLUSION: MALAT1 knockdown prevented HG-induced mTOR activation and inhibited tau phosphorylation. MALAT1 may be a therapy target for diabetes associated dementia.

Prevalence and Related Factors of Insomnia Among Chinese Medical Staff in the Middle and Late Stage of COVID-19.

Background: The outbreak of novel coronavirus disease (COVID-19) has brought serious psychological pressure to people, especially medical health staff. At present, there are few studies on insomnia and related factors of medical health staff in the middle and late stage of the epidemic of COVID-19. Therefore, the purpose of this study was to investigate the prevalence of insomnia and its related risk factors among medical workers in China in the middle and later stage of COVID-19 epidemic, as well as the relationship between insomnia and psychological resilience. Methods: From February 14 to March 29, 2020, a cross-sectional survey was conducted among 606 medical staff in China through Ranxing Technology's "SurveyStar" network platform. All subjects were assessed with the Insomnia Severity Index (ISI) and simplified Chinese version of Connor-Davidson Resilience scale (CD-RISC-10). Results: In the middle and later stages of the COVID-19 outbreak, the incidence of insomnia among medical staff was 32.0%. Compared with non-insomnia group, the insomnia group had younger age, lower education level, longer daily working hours and less psychological resilience. In addition, the prevalence of insomnia was higher in medical staff with a history of somatic diseases. The severity of insomnia of Chinese medical staff was associated with age, education level, daily working hours, psychological resilience and somatic diseases. Conclusions: Our study shows that nearly 1/3 of Chinese medical workers suffer from insomnia nearly a month after the COVID-19 outbreak. Compared with the general population, medical staff who are working with COVID are more prone to insomnia. Risk factors for insomnia include younger age, lower education level, longer working hours per day, and physical illness. The tenacious dimension of psychological resilience is a protective factor for insomnia.

Expression of Long Noncoding RNA LIPCAR Promotes Cell Proliferation, Cell Migration, and Change in Phenotype of Vascular Smooth Muscle Cells.

BACKGROUND The long noncoding RNA LIPCAR is a type of transcription product (>200 nucleotides long). Recent studies demonstrated that LIPCAR is a potential biomarker in cardiovascular disease and can predict survival in patients with cardiovascular disease. Therefore, the present study explored the role of LIPCAR in the regulation of proliferation, migration, and change in phenotype of vascular smooth muscle cells. MATERIAL AND METHODS Human vascular smooth muscle cells (VSMCs) were treated with 20 g/mL oxidatively modified low-density lipoprotein (ox-LDL) or 20 ng/ml platelet-derived growth factor BB (PDGF-BB) for 24 h, then the expression levels of LIPCAR were detected using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay. LIPCAR-overexpressing plasmids were transfected into VSMCs. After transfection, cell proliferation and migration were measured using the Cell Counting Kit-8 (CCK-8) and Transwell assays, respectively. The levels of a-smooth muscle actin (a-SMA) a molecular marker of the contractile VSMC phenotype, were measured using Western blot and immunofluorescence assays. Protein levels of cyclin-dependent kinase-2 (CDK2), proliferating cell nuclear antigen (PCNA), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor A (VEGF-A), and angiopoietin-2 (Ang-2) were assessed by Western blot. The level of tissue factor (TF) was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS Treatment with PDGF-BB or ox-LDL significantly increased levels of LIPCAR in VSMCs. Overexpression of LIPCAR markedly promoted cell proliferation and migration. Further, upregulation of LIPCAR increased CDK2, p21, PCNA, MMP2, MMP9, VEGF-A, Ang-2, and TF expression and decreased p21 expression. In addition, LIPCAR significantly decreased a-SAM expression. CONCLUSIONS Together, our data suggest that overexpression of LIPCAR promotes cell proliferation, migration, and phenotypic switch of vascular smooth muscle cells.

Plasma Exosomal miR-450b-5p as a Possible Biomarker and Therapeutic Target for Transient Ischaemic Attacks in Rats.

Transient ischaemic attack (TIA) and cerebral infarction are difficult to identify within the thrombolytic time window. Blood markers are efficient, economical and noninvasive and can be beneficial in the diagnosis of many diseases. Plasma exosomal biomarkers are rarely reported in TIA. Exosomal microRNAs (miRNAs) were extracted from plasma and cerebrospinal fluid after middle cerebral artery occlusion (MCAo) in rats (0 min, 5 min, 10 min, 2 h). Deep sequencing was used to detect exosomal miRNAs in rat plasma and confirm significant differentially expressed miRNAs. Polymerase chain reaction (PCR) was used to detect the differentially expressed miRNAs in plasma and cerebrospinal fluid. Exosomal miRNAs with the same expression trends in plasma and cerebrospinal fluid were selected, and bioinformatics analysis was then carried out. Finally, the area under the curve (AUC) of the receiver operating characteristic (ROC) curve was determined to assess the diagnostic accuracy of miRNAs for TIA in rats. First, high-throughput sequencing was used to detect the expression level of plasma exosome miRNA, and rno-miR-450b-5p with a decreasing expression level was screened. Second, the expression levels of exosomal miRNAs were verified in cerebrospinal fluid and plasma samples by PCR, and the results indicated that exosomal rno-miR-450b-5p was similarly expressed in cerebrospinal fluid and plasma. ROC analysis showed high AUC values for rno-miR-450b-5p (0.880) in the 10 min ischaemia rats compared with the control rats. Finally, bioinformatic analysis indicated that exosomal rno-miR-450b-5p may be involved in cerebral ischaemia. Plasma exosomal rno-miR-450b-5p has a high diagnostic value and may become a therapeutic target for rat TIA.

MicroRNA-125a inhibits tumorigenesis by targeting Smurf1 in colorectal carcinoma.

Aberrant expression of microRNAs (miRNAs) may contribute to the initiation and development of multiple types of human cancer. Several miRNAs have been found to be strongly correlated with the diagnosis, progression, and prognosis of colorectal carcinoma (CRC), but the role of miR-125a in CRC remains unclear. In the present study, the function of miR-125a on the expression of Smad ubiquitin regulatory factor 1 (Smurf1) was investigated in vitro and in vivo. We verified that Smurf1 is a downstream target gene of miR-125a and is involved in miR-125a-mediated regulation of CT26 cell (colon cancer cell) proliferation and migration. Overexpression of miR-125a suppresses CT26 cell growth by inhibiting cell proliferation. Additionally, wound healing assays were performed to show that overexpression of miR-125a significantly reduced CT26 cell migration, which was reversed by overexpression of Smurf1. In vivo, miR-125a overexpression downregulated the expression of Ki67 and Smurf1, thus leading to a marked reduction in tumor growth. These results revealed that miR-125a plays a critical role in CRC by directly targeting Smurf1, a finding that may facilitate the development of improved diagnostic and therapeutic techniques for CRC.

Disruption of CTLA-4 expression on peripheral blood CD8 + T cell enhances anti-tumor efficacy in bladder cancer.

Activation of programmed death-1 (PD-1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) on T cells leads to T cell exhaustion and ultimately facilitates tumor progression. Recent success of using immune cell checkpoint inhibitors offers a great promise to treat various cancers, including bladder cancer. However, the expression pattern and therapeutic value of PD-1 and CTLA-4 in peripheral blood T cells remain largely unexplored. In this study, we presume that disruption of the potential dysregulated checkpoint molecules in peripheral blood T cells may improve the anti-tumor efficacy of cytotoxic T cells in bladder cancer. We showed that both PD-1 and CTLA-4 expression were specifically elevated on CD8 + T cells but not CD4 + T cells in peripheral blood of patients with bladder cancer compared with that in healthy donors. Notably, CTLA-4 expression was significantly higher in muscle-invasive bladder cancer (MIBC) and correlated with tumor size. By blocking CTLA-4 with anti-CTLA-4 antibody and CRISPR-Cas9-mediated CTLA-4 disruption, we revealed that CTLA-4-disrupted CTLs had enhanced cellular immune response and superior cytotoxicity to the CD80/CD86-positive bladder cancer cells in vitro. Moreover, the CTLA-4-disrupted CTLs exhibited a pronounced anti-tumor effect in vivo as demonstrated by prophylactic assay and therapeutic assay in the subcutaneous xenograft model. Collectively, our findings confirm improved therapeutic efficacy of CTLA-4-disrupted CTLs and provides the potential strategy for targeting immune checkpoints to enhance the promising immunotherapy.

Diagnosis of Hyperacute and Acute Ischaemic Stroke: The Potential Utility of Exosomal MicroRNA-21-5p and MicroRNA-30a-5p.

BACKGROUND: Early and accurate diagnosis of ischaemic stroke (IS) requires the use of an optimized biomarker. Exosomal microRNAs have the potential to serve as biomarkers owing to their stability and specificity. We investigated the expression levels of plasma-derived exosomal microRNA-21-5p and microRNA-30a-5p in the different phases of IS. METHODS: One hundred forty-three patients with IS and 24 non-stroke controls were enrolled. The patients were divided into the following 5 groups: 1 group for the hyperacute phase IS (HIS, within 6 h); two for the acute phase IS (AIS, including days 1-3 and days 4-7); one for the subacute phase IS (SIS, days 8-14); and one for the recovery phase IS (RIS, days >14). Plasma exosomes were isolated using a QIAGEN exoRNeasy kit and examined by transmission electron -microscopy, nanoparticle tracking, and flow cytometry. The expression levels of miRNA-21-5p and miRNA-30a-5p were detected by quantitative real-time polymerase chain reaction. RESULTS: The plasma exosomal miR-21-5p levels in SIS and RIS were significantly higher than that in controls (p < 0.05 and p < 0.01 respectively). The levels of miR-30a-5p in HIS were significantly higher (p < 0.05) and in AIS (days 1-3) were lower than that in controls (p < 0.05). In AIS (days 1-3), both miRNAs were decreased compared with the HIS group (p = 0.053 and 0.001, respectively). The area under the curve (AUC) of the miR-21-5p was 0.714 for SIS (95% CI 0.570-0.859, p = 0.007), 0.734 for RIS (95% CI 0.596-0.871, p = 0.003); the AUC of the miR-30a-5p was 0.826 for HIS (95% CI 0.665-0.988, p = 0.001), 0.438 for AIS (days 1-3; 95% CI 0.240-0.635, p = 0.516). CONCLUSIONS: The plasma-derived exosomal miR-21-5p and miRNA-30a-5p in combination are promising biomarkers for diagnosing IS and distinguishing among HIS, SIS, and RIS, especially miRNA-30a-5p for the diagnosis of the HIS phase. Our results provide a new reference for clinicians to apply in early-stage diagnosis and identifies the possible value of biomarkers for IS thrombolysis therapy.

Plasma Exosomal miRNA-122-5p and miR-300-3p as Potential Markers for Transient Ischaemic Attack in Rats.

Background: Differentiation of transient ischaemic attack (TIA) from ischaemic stroke within the thrombolysis time window is difficult. Although TIA may be diagnosed within this window, the latest imaging technologies are complex and costly. Serum markers, which are non-invasive, rapid and economic, are used for diagnosis and prognosis of various diseases. Exosome-derived miRNA markers for TIA are unknown. Methods: We examined focal brain ischaemia produced by occlusion of the middle cerebral artery (MCAo) for 5 min, 10 min, and 2 h in rats. Exosomal miRNAs with consistent trends in cerebrospinal fluid (CSF) and plasma were identified by deep sequencing and quantitative real-time polymerase chain reaction (qRT-PCR). The areas under the curve (AUC) of the receiver operating characteristic (ROC) curve were used to evaluate the diagnostic accuracy of these miRNAs for TIA in rats. Results: Rno-miR-122-5p and rno-miR-300-3p were selected. Plasma exosomal rno-miR-122-5p was significantly downregulated in 10 min ischaemic rats compared with control and 5 min plasma. Plasma exosomal rno-miR-300-3p was significantly upregulated in 5 min ischaemic rats compared with control, 10 min and 2 h rats. Plasma and CSF levels of these miRNAs were correlated. ROC analysis showed high AUC values for rno-miR-122-5p (0.960) and rno-miR-300-3p (0.970) in the 10 and 5 min rats, respectively, compared with controls. Conclusions: Plasma exosomal rno-miR-122-5p and rno-miR-300-3p may be blood-based TIA biomarkers.

Plasma Exosomal miR-422a and miR-125b-2-3p Serve as Biomarkers for Ischemic Stroke.

BACKGROUND: MircroRNA (MiRNA) levels are associated with disease pathophysiology and are high in plasma exosomes. Plasma exosomal miRNAs serve as potential therapeutic targets and diagnosis biomarkers in some diseases but few studies have examined them in Ischemic Stroke (IS). Therefore, we explored the potential predictive value of plasma exosomal miR-422a and miR-125b-2-3p in different IS phases (acute and subacute phases). METHODS: Fifty-five IS patients and 25 age and sex matched healthy controls were recruited. Patients were classified into two groups: 27 patients in acute phase (days 1-3) and 28 patients in subacute phase (days 4-14). The plasma exosomal levels of miR-422a and miR-125b-2-3p were examined via quantitative real-time polymerase chain reaction (qRT-PCR). The Areas Under the Curve (AUC) of the Receiver Operating Characteristic (ROC) curve were constructed to evaluate the diagnostic accuracy of these miRNAs in IS. RESULTS: The expression levels of plasma exosomal miR-422a and miR-125b-2-3p were significantly decreased in the subacute phase group (P<0.001, P<0.001, respectively), and the miR-422a levels were increased in the acute phase group (P<0.005) as compared to the controls. Additionally, the expression levels of plasma exosomal miR-422a and miR-125b-2-3p were significantly decreased in the subacute phase group than in the acute phase group (P<0.001, P<0.005, respectively). ROC analysis showed high AUC values for miR-422a and miR-125b-2-3p in the subacute phase group as compared to those in healthy controls: 0.971 and 0.889, respectively, and miR-422a in the acute phase group as compared to healthy controls were 0.769. CONCLUSION: Plasma exosomal miR-422a and miR-125b-2-3p may serve as blood-based biomarkers for monitoring and diagnosing in IS patients, with plasma exosomal miR-422a showing the best diagnostic value. The use of these two plasma exosomal miRNAs in combination may be powerful for determining IS stage.