Association between the organic cation transporter 3 methylation and hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the deadliest cancers in the world and has a high death rate in the world. This research while examining the expression of OCT3 at the mRNA level has also studied gene methylation profile in patients with HCC in comparison with people without HCC. The volunteers were: patients with HCC (n=81) and a healthy control group (n=90). The expression of OCT3was studied using the qRT-PCR method. The methylation profile was evaluated by genomic DNA using methylation specific PCR (MSP) method. The expression level of OCT3 marker mRNA in patients has decreased significantly compared to healthy individuals (0.58 ± 0.311 vs 1.20 ± 0.355, P <0.001). No significant statistical relationship was found between demographic data and OCT3 expression in participants (P >0.05). The amount of methylation (UM + MM) in cancer patients has raised vs controls (P <0.001) and has increased the risk of cancer (OR=0.379, 95% CI=1.171-2.839, P <0.001, and OR=2.727, 95% CI=1.251-5.945, P <0.001, respectively).Changes in OCT3 levels appear to be associated with HCC. Also, changing the methylation pattern of this gene can reveal HCC pathology.

Establishment and application of a quantitative real-time PCR detection system for Brucella S2 vaccine strain / 中华地方病学杂志

Objective:To establish a quantitative real-time PCR detection system for Brucella S2 vaccine strain. Methods:Based on the differences in the entire genome sequence between Brucella S2 vaccine strain and other reference strains of Brucella, primers and probes were designed to establish a quantitative real-time PCR detection system for Brucella S2 vaccine strain. The DNA of 22 reference strains of Brucella and 8 non- Brucella control strains were obtained from the National Institute for Infectious Disease Control and Prevention of the Chinese Center for Disease Control and Prevention. At the same time, environmental samples were obtained from the brucellosis vaccine manufacturers, and bacterial DNA from environmental samples was extracted using a blood/tissue genomic DNA extraction kit. The obtained DNA was pre-amplified by conventional PCR, and then subjected to quantitative real-time PCR secondary amplification (nested fluorescence quantitative PCR) using the amplified PCR product as a template. The specific fluorescence curve and corresponding number of cycles (Ct value) were observed, and the sensitivity was tested. Results:The quantitative real-time PCR detection system established did not detect specific fluorescence curves (without Ct values) for 21 reference strains of Brucella and 8 non- Brucella control strains, except for S2 vaccine strains. The established detection system had a minimum detection limit of 4.34 fg (genomic DNA) for detecting the DNA of Brucella S2 vaccine strain; DNA of Brucella S2 vaccine strain was detected in 3 of the 14 environmental samples collected. Conclusion:The quantitative real-time PCR detection system established can detect Brucella S2 vaccine strain in samples, with good sensitivity and specificity.

Mycoplasma pneumoniae infection and macrolide resistance of children hospitalized in Ningbo Area， Zhejiang Province in 2019‒2021 / 上海预防医学

ObjectiveTo investigate Mycoplasma pneumoniae （MP） infection and macrolide resistance of hospitalized children in Ningbo Area in 2019‒2021. MethodsA total of 6 782 respiratory throat swab specimens were collected from hospitalized pediatric patients with pneumoniae， admitted in Ningbo Women and Children's Hospital from January， 2019 to December 2021. MP and its mutations in 23S rRNA were detected by real-time polymerase chain reaction. ResultsAmong 6 782 respiratory throat swab specimens from 2019‒2021， 1 290 cases （19.02%） were MP positive， and the positive rate decreased year by year （P<0.05）. The positive rate in 2019 was 28.12%， higher than that in 2020 （7.16%） and 2021 （5.16%） （all P<0.017）. The mutation of 23S rRNA occurred in 947 cases， with a mutation rate of 73.41%. The mutation rate in 2020 was 84.04%， higher than that in 2019 （73.01%） and 2021 （66.23%）. The differences of positive rate and mutation rate in different seasons were significant （P<0.05） （all P<0.008）. The positive rate was the highest in summer （25.00%）， and the mutation rate was the highest in winter （78.89%）. The positive rate of female children was 20.52%， higher than that of male children （17.82%） （P<0.05）， and the mutation rates of female and male children were 74.93% and 71.77% （P>0.05）， respectively. The difference of positive rate among the different age was significant （P<0.05）. The positive rates in the 5‒ and 8‒ years groups were 27.24% and 26.38%， higher than those in the 1‒ and 2‒ years groups， respectively. The difference of mutation rate among the four groups in age was not significant （P>0.05）. ConclusionThe infection rate of MP in children decreases in Ningbo Area from 2019 to 2021. MP infection may be related with gender， seasonal distribution， age， and the resistance rate of MP to macrolide is high.

Development and verification of a quantitative real-time PCR method for determination of host cell DNA residues in inactivated SARS-CoV-2 vaccine(Vero cells) / 中国生物制品学杂志

@#Objective To develop and verify a quantitative real-time PCR method for determination of the content of host cell DNA residues in severe acute respiratory symptom coronavirus 2(SARS-CoV-2) inactivated vaccine(Vero cells),in order to better control the safety of products.Methods DNA was extracted from inactivated SARS-CoV-2 vaccine(Vero cells) bulk by magnetic bead separation method,and the DNA residues of host cells were quantitatively analyzed by probetype PCR.The linear range,repeatability,intermediate precision,quantitative limit,specificity,durability and accuracy of the developed method were verified,and the host cell DNA re sidues of 5 batches of inactivated SARS-CoV-2 vaccine(Vero cells)were determined by this method.Results DNA standard curve showed good linearity in the range of 300～0.003 pg/μL(each R~2> 0.99);Relative standard deviations(RSD) of repeatability and intermediate precision verification were less than 20%;The quantitative limit was 0.001 pg/μL;Sample dilution and purified liquid dilution had no interference to detection;The results of 60 min incubation at 53,55,57 ℃ and 56,60,64 min incubation at 55 ℃ showed no significant difference;The recoveries of accuracy verification were 79%～83%,RSD <5%.This method had good adaptability in detecting DNA residues in the bulk of inactivated SARS-CoV-2 vaccine(Vero cells).Conclusion The quantitative realtime PCR method for determination of host cell DNA residues in inactivated SARS-CoV-2 vaccine(Vero cells) has been successfully developed,of which the linearity and range,repeatability,intermediate precision,quantitative limit,specificity,durability and accuracy meet the acceptable standards,and are suitable for the detection and quality control of host cell DNA residues in inactivated SARS-CoV-2 vaccine(Vero cells).

Multicenter post-marketing clinical evaluation on the clinical performance of the five domestic fast nucleic acid detection reagents for 2019-nCoV / 中华检验医学杂志

Objective:This multicenter clinical evaluation analyzed the clinical performance of five fast nucleic acid detection systems for 2019-nCoV.Methods:Clinical performance of the five fast nucleic acid detection reagents approved in China was evaluated in the present study. Fifty-seven throat swabs samples from COVID-19 patients and fifteen throat swabs samples from healthy people were collected from the First Affiliated Hospital of Zhejiang University school of Medicine, Tongji Hospital of Tongji Medical College of HUST, and National Institute of Viral Disease Control and Prevention of CDC to evaluate the positive coincidence rate, negative coincidence rate, total coincidence rate, the detection time and retest rate as well as the relation between positive intensity and positive coincidence rate of the five fast nucleic acid detection systems in November 2020.Results:The positive coincidence rates of the five kits were 92.59% (50/54), 83.64% (46/55), 98.25% (56/57), 94.44% (51/54) and 98.18% (54/55); and the negative coincidence rates were 93.33% (14/15), 93.33% (14/15), 86.67% (13/15), 100% (14/14) and 93.33% (14/15); and the total coincidence rates were 92.75% (64/69), 85.71% (60/70), 95.83% (69/72), 94.20% (65/69) and 97.14% (68/70), respectively. The positive coincidence rate of the five kits reached 100% for the strong-positive (90/90) and medium-positive samples (84/84), but only 82.18% (83/101) for weak-positive samples (cycle threshold value>33), and the retest rate of two kits were 15.28% (11/72) and 12.50% (9/72), which were both higher than 10%. Total time from sample extraction to amplification was between 32.33-65.33 minutes for these five kits.Conclusion:The five fast nucleic acid detection reagents have good performance and can be used as a supplement to routine nucleic acid detection reagents.

Screening of reference genes for quantitative real-time PCR in Artemisia argyi / 中国中药杂志

Artemisia Argyi Folium, a traditional Chinese medicine of important medicinal and economic value, sees increasing demand in medicinal and moxibustion product market. Screening stable and reliable reference genes for quantitative real-time PCR(qRT-PCR) is a prerequisite for the analysis of gene expression in Artemisia argyi. In this study, eight commonly used reference genes, Actin, 18s, EF-1α, GAPDH, SAND, PAL, TUA, and TUB, from the transcriptome of A. argyi, were selected as candidate genes. The expression of each gene in different tissues(roots, stems, and leaves) of A. argyi and in leaves of A. argyi after treatment with methyl jasmonate(MeJA) for different time(0, 4, 8, 12 h) was detected by qRT-PCR. Then, geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder were employed to evaluate their expression stability. The results demonstrated that Actin was the most stable reference gene in different tissues and in leaves treated with MeJA, and coming in the second was SAND. Furthermore, the expression of DXS and MCT which are involved in terpenoid backbone biosynthesis was detected in different tissues and after MeJA treatment. The results showed that the expression patterns of DXS and MCT in different tissues and under MeJA treatment calculated with Actin and SAND as internal reference genes were consistent, which validated the screening results. In conclusion, Actin is the most suitable reference gene for the analysis of gene expression in different tissues of A. argyi and after MeJA treatment. This study provides valuable information for gene expression analysis in A. argyi and lays a foundation for further research on molecular mechanism of quality formation of Artemisia Argyi Folium.

Inclusion of molecular monitoring (BCR-ABL1) in the treatment of chronic myeloid leukemia in the Brazilian Public Health System (SUS): an urgent need for treatment management

ABSTRACT Introduction: Chronic Myeloid Leukemia (CML) is a myeloproliferative disease that affects mainly adults between 50 and 55 years. In Brazil, information from the Sistema Único de Saúde (SUS) Outpatient Information System indicates that 12,531 patients had the Autorização de Procedimento Ambulatorial (APAC) approved for the CML treatment in 2017. Disease monitoring through molecular response evaluation is critical to the care of CML patients. The quantitative PCR test (real-time polymerase chain reaction) provides adequate evaluation parameters that allow the health professional to intervene at the right moments in order to reduce the chance of progression of the disease, providing the best outcome to the patient, including the possibility of treatment discontinuation for eligible patients. Although the test is included in the Clinical Protocol and Therapeutic Guidelines (PCDT) of CML, it is not possible to monitor the molecular response within SUS since there is no reimbursement for this test. Objective: Obtain expert recommendations on the importance, financing, and reimbursement of molecular monitoring in SUS. Methods: Six CML experts with different perspectives participated in the panel. The discussion was based in the main publications about the quantitative PCR test in CML monitoring. Results: Experts' recommendations: Molecular monitoring should be part of the integral treatment of patients with CML to reduce the chances of disease progression and costs to the health system; The government should put into practice what is provided in the PCDT of Chronic Myeloid Leukemia in Brazil: performing the monitoring of the molecular response via quantitative PCR; The government should create a code with adequate nomenclature and reimbursement value in SIGTAP, so that the test is carried out and covered by the public health network, as it is contained in the PCDT of the disease and the existing APAC does not cover the operational costs for its performance; Patients with chronic phase CML should perform a quantitative PCR every 3 months and, after reaching the MMR, should perform the examination every 6 months, as recommended by international guidelines; Patients should be monitored in reference laboratories that are standardized according to the international scale; The laboratories that are within the reference public centers could absorb all the test demand in Brazil, and other centers could be qualified through an ABHH accreditation; Adequate molecular monitoring may allow some patients to stop taking drugs and selffinancing the molecular test for all SUS patients Conclusion: A solution for the molecular test (BCR-ABL1) funding is urgent to ensure the monitoring of CML patients in SUS. The savings that might be generated with patients that stop taking the medication when adequately monitored may finance the test.

Selection and Validation of Appropriate Reference Genes of Cinnamomum cassia and Cinnamomum cassia var. macrophyllum / 中国实验方剂学杂志

Objective:To screen out stable internal reference genes suitable for real-time quantitative polymerase chain reaction（Real-time PCR） analysis of different parts of<italic> Cinnamomum cassia</italic> and <italic>C. cassia</italic> var. <italic>macrophyllum</italic>，in order to provide stable internal reference genes for gene expression analysis of three different parts of and <italic>C. cassia</italic> var.<italic> macrophyllum</italic> branches and leaves. Method:With 6 different tissues and organs， such as bark，branches and leaves of two plants of <italic>C. cassia</italic> and <italic>C. cassia</italic> var. <italic>macrophyllum</italic> as experimental materials，Real-time PCR technology was used to detect the five internal reference genes， namely glyceraldehyde-3-phosphate dehydrogenase（GAPDH），actin，ubiquitin-ligase enzymes（UBE），histone and tubin（TUB）. The analysis of the expression of the data. Furthermore， three commonly used internal reference gene analysis software，namely geNorm，NormFinder and BestKeeper，was used to analyze and evaluate the stability of the candidate internal reference gene. Result:The internal five reference genes were expressed in the bark，branches and leaves of the two plants，but with differences in stability. Comprehensive analysis showed that the expression stability of candidate internal reference genes was in the order of GAPDH>actin>UBE>histone>TUB. The internal reference genes of the two plants were analyzed separately，and the optimal internal reference gene was still GAPDH，indicating that GAPDH was the most suitable internal reference gene. TUB and histone ranked low in the three software，and should be eliminated in the screening of reference genes. They were not suitable for gene expression analysis of <italic>C. cassia </italic>and <italic>C. cassia</italic> var. <italic>macrophyllum</italic>. Conclusion:The most suitable internal reference gene for different parts of cinnamon，branches，and leaves of <italic>C. cassia</italic> and <italic>C. cassia</italic> var. <italic>macrophyllum</italic> was GAPDH. In this study，a screening system for internal reference genes of Real-time PCR of <italic>C. cassia</italic> and <italic>C. cassia</italic> var. <italic>macrophyllum</italic> was established to provide theoretical basis for studying functional regulation and expression of genes during the accumulation of effective components in different parts.

Selection of optimal qRT-PCR reference genes for Aconitum vilmorinianum / 中国中药杂志

Screening suitable reference genes is the premise of quantitative Real-time PCR(qRT-PCR)for gene expression analysis. To provide stable reference genes for expression analysis of genes in Aconitum vilmorinianum, this study selected 19 candidate re-ference genes(ACT1, ACT2, ACT3, aTUB1, aTUB2, bTUB, 18S rRNA, UBQ, eIF2, eIF3, eIF4, eIF5, CYP, GAPDH1, GAPDH2, PP2A1, PP2A2, ACP, and EF1α) based on the transcriptome data of A. vilmorinianum. qRT-PCR was conducted to profile the expression of these genes in the root, stem, leaf, and flower of A. vilmorinianum. The Ct values showed that 18S rRNA with high expression level and GAPDH2 with large expression difference among organs were not suitable as the reference genes. NormFinder and geNorm showed similar results of the expression stability of the other candidate reference genes and demonstrated PP2A1, EF1α, and CYP as the highly stable ones. However, BestKeeper suggested EF1α, ACT3, and PP2A1 as the top stable genes. In view of the different results from different softwares, the geometric mean method was employed to analyze the expression stability of the candidate re-ference genes, the results of which indicated that PP2A1, EF1α, and ACT3 were the most stable. Based on the comprehensive analysis results of geNorm, NormFinder, BestKeeper, and geometric mean method, PP2A1 and EF1α presented the most stable expression in different organs of A. vilmorinianum. PP2A1 and EF1α were the superior reference genes for gene expression profiling in different organs of A. vilmorinianum.

Serum expression of microRNA-16 in a cohort of Egyptian patients with ulcerative colitis and its correlation with disease extent and severity / Expressão sérica do microRNA-16 em uma coorte de pacientes egípcios com colite ulcerosa e sua correlação com a extensão e gravidade da doença

Abstract Ulcerative colitis is one of the IBDs. Its etiology and pathogenesis remain undefined with an interaction between environmental, genetic and immunological factors is the most accepted explanation. Several recent studies have examined microRNA expression in the peripheral blood and tissues from IBD patients. The study aims at assessing the expression of serum miR-16 in ulcerative colitis patients and its correlation with disease extent, activity and severity. It included 30 treatment naïve ulcerative colitis patients of different presentations. Serum miR-16 expression was assessed using reverse transcriptase quantitative real time PCR (RT-qPCR), and then correlated with that of a group of 20 healthy subjects to assess its role in diagnosis of ulcerative colitis. Also, it was correlated with disease extent (proctitis, left sided colitis, extensive colitis) and disease activity and severity indices (Truelove and Witts criteria, fecal calprotectin and UCEIS). Thirty ulcerative colitis patients were enrolled, 53% had mild, 37% had moderate, while 10% had severe disease. Concerning endoscopic extent, 8 had proctitis, 14 had left sided colitis and 8 had extensive colitis. Serum expression of miR-16 in the 30 patients were compared to that of the healthy control subjects. The patients' group showed median serum miR-16 expression of 1.91, 1.13 for the control group with a significant difference between both groups. Correlation between serum miR-16 expression with disease extent, activity and severity showed no significant relation. From the current study we can conclude that increased serum expression of miR-16 is associated with ulcerative colitis despite no significant relation to disease activity extent or severity.

Resumo A colite ulcerativa é uma das DII. Sua etiologia e patogênese permanecem indefinidas; a interação entre fatores ambientais, genéticos e imunológicos é a explicação mais aceita. Vários estudos recentes avaliaram a expressão de microRNA no sangue e tecidos periféricos em pacientes com DII. O presente estudo teve como objetivo avaliar a expressão do miR-16 sérico em pacientes com colite ulcerativa e sua correlação com a extensão, atividade e gravidade da doença. Foram incluídos 30 pacientes de colite ulcerativa, com diferentes apresentações, que ainda não haviam sido submetidos a nenhum tipo de tratamento. A expressão sérica de miR-16 foi avaliada usando transcrição reversa seguida de reação em cadeia da polimerase quantitativa (RT-qPCR) e, em seguida, correlacionada com a de um grupo de 20 indivíduos saudáveis para avaliar seu papel no diagnóstico de colite ulcerativa. Além disso, foi feita uma correlação com a extensão da doença (proctite, colite do lado esquerdo, colite extensa) e com os índices de atividade e gravidade da doença (critérios de Truelove e Witts, calprotectina fecal e UCEIS). Trinta pacientes com colite ulcerativa foram incluídos no estudo, classificada como leve em 53%, moderada em 37% e grave em 10%. Quanto à extensão endoscópica, oito apresentavam proctite, 14 apresentavam colite do lado esquerdo e oito apresentavam colite extensa. A expressão sérica de miR-16 nos 30 pacientes foi comparada à dos indivíduos controle saudáveis. No, grupo de pacientes, a expressão sérica de miR-16 foi de 1,91 (grupo controle: 1,13), uma diferença estatisticamente significativa entre os dois grupos. Não foi observada relação significativa entre a expressão sérica de miR-16 e a extensão, atividade e gravidade da doença. A partir do presente estudo, pode-se concluir que o aumento da expressão sérica do miR-16 está associado à colite ulcerativa, apesar de não haver relação significativa com a extensão ou gravidade da atividade da doença.

Quantitative real-time PCR assay with immunohistochemical evaluation of HER2/neu oncogene in breast cancer patients and its correlation with clinicopathological findings

Human epidermal growth factor receptor HER2/neu status is an important prognostic factor for breast cancer as it is crucial in stimulating growth and cellular motility. Overexpression of HER2/neu is observed in 10%?35% of the human breast cancer and is associated with prognosis and response to treatment. The magnitude of amplification must be determined to facilitate better prognosis and personalized therapy in the affected patient. This study aims to investigate the HER2/neu status in breast cancer by concurrent HER2/neu protein overexpression immunohistochemically with HER2/neu DNA amplification by quantitative real-time polymerase chain reaction (PCR), allowing accurate and precise quantification of HER2/neu amplification after a follow-up period. A total of 54 paired tissue samples from formalin-fixed paraffin-embedded (FFPE) breast cancer patients enrolled in this study were collected to evaluate tumor and normal tissues. Only cases with 80% and more tumor cells were included. For confirmation of immunohistochemistry (IHC) results, qPCR was used to determine the HER2/neu amplification. The association between clinicopathological variables like age, tumor size, histological grade, stage, lymph node status, hormone receptor status, family history, recurrence rate, and vital status was evaluated. We observed that 11/54 (20.4%) of the tumor tissues are positive for HER2/neu protein overexpression by IHC. A total of 8 out of these 11 cases (72.7%), which presented a score of 3+, showed gene amplification of HER2/neu. The concordance rate between IHC and qPCR was 94.4%. HER2/neu gene amplification was found to be significantly associated with recurrence, increased risk of death, and progesterone receptor status, supporting a negative prognostic role of HER2/neu in breast cancer survival. In conclusion, IHC can be used as an initial screening test to detect HER2/neu protein overexpression, and the use of qPCR can verify the IHC results and establish HER2/neu status in routine clinical practice.

Integrative molecular characterization of Chinese prostate cancer specimens / 亚洲男科学杂志(英文版)

Prostate cancer (PCa) exhibits epidemiological and molecular heterogeneity. Despite extensive studies of its phenotypic and genetic properties in Western populations, its molecular basis is not clear in Chinese patients. To determine critical molecular characteristics and explore correlations between genomic markers and clinical parameters in Chinese populations, we applied an integrative genetic/transcriptomic assay that combines targeted next-generation sequencing and quantitative real-time PCR (qRT-PCR) on samples from 46 Chinese patients with PCa. Lysine (K)-specific methyltransferase 2D (KMT2D), zinc finger homeobox 3 (ZFHX3), A-kinase anchoring protein 9 (AKAP9), and GLI family zinc finger 1 (GLI1) were frequently mutated in our cohort. Moreover, a clinicopathological analysis showed that RB transcriptional corepressor 1 (RB1) deletion was common in patients with a high risk of disease progression. Remarkably, four genomic events, MYC proto-oncogene (MYC) amplification, RB1 deletion, APC regulator of WNT signaling pathway (APC) mutation or deletion, and cyclin-dependent kinase 12 (CDK12) mutation, were correlated with poor disease-free survival. In addition, a close link between KMT2D expression and the androgen receptor (AR) signaling pathway was observed both in our cohort and in The Cancer Genome Atlas Prostate Adenocarcinoma (TCGA-PRAD) data. In summary, our results demonstrate the feasibility and benefits of integrative molecular characterization of PCa samples in disease pathology research and personalized medicine.

Adaptive evolution and selection of stress-resistant Saccharomyces cerevisiae for very high-gravity bioethanol fermentation

Background: In industrial yeasts, selection and breeding for resistance to multiple stresses is a focus of current research. The objective of this study was to investigate the tolerance to multiple stresses of Saccharomyces cerevisiae obtained through an adaptive laboratory evolution strategy involving a repeated liquid nitrogen freeze­thaw process coupled with multi-stress shock selection. We also assessed the related resistance mechanisms and very high-gravity (VHG) bioethanol production of this strain. Results: Elite S. cerevisiae strain YF10-5, exhibiting improved VHG fermentation capacity and stress resistance to osmotic pressure and ethanol, was isolated following ten consecutive rounds of liquid nitrogen freeze­thaw treatment followed by plate screening under osmotic and ethanol stress. The ethanol yield of YF10-5 was 16% higher than that of the parent strain during 35% (w/v) glucose fermentation. Furthermore, there was upregulation of three genes (HSP26, HSP30, and HSP104) encoding heat-shock proteins involved in the stress response, one gene (TPS1) involved in the synthesis of trehalose, and three genes (ADH1, HXK1, and PFK1) involved in ethanol metabolism and intracellular trehalose accumulation in YF10-5 yeast cells, indicating increased stress tolerance and fermentative capacity. YF10-5 also showed excellent fermentation performance during the simultaneous saccharification and fermentation of VHG sweet potato mash, producing 13.40% (w/ v) ethanol, which corresponded to 93.95% of the theoretical ethanol yield. Conclusions: A multiple-stress-tolerant yeast clone was obtained using adaptive evolution by a freeze­thaw method coupled with stress shock selection. The selected robust yeast strain exhibits potential for bioethanol production through VHG fermentation.

Cloning and heterologous expression of a hydrophobin gene Ltr. hyd from the tiger milk mushroom Lentinus tuber-regium in yeast-like cells of Tremella fuciformis

Background: Hydrophobins are small proteins secreted by filamentous fungi, which show a highly surface activity. Because of the signally self-assembling abilities and surface activities, hydrophobins were considered as candidates in many aspects, for example, stabilizing foams and emulsions in food products. Lentinus tuber-regium, known as tiger milk mushroom, is both an edible and medicinal sclerotium-producing mushroom. Up to now, the hydrophobins of L. tuber-regium have not been identified. Results: In this paper, a Class I hydrophobin gene, Ltr.hyd, was cloned from L. tuber-regium and expressed in the yeast-like cells of Tremella fuciformis mediated by Agrobacterium tumefaciens. The expression vector pGEH-GH was under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter. The integration of Ltr.hyd into the genome of T. fuciformis was confirmed by PCR, Southern blot, fluorescence observation and quantitative real-time PCR (qRT-PCR). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that recombinant hydrophobin rLtr.HYD with an expected molecular mass of 13 kDa was extracted. The yield of rLtr.HYD was 0.66 mg/g dry weight. The emulsifying activity of rLtr.HYD was better than the typical food emulsifiers sodium caseinate and Tween 20. Conclusions: We evaluated the emulsifying property of hydrophobin Ltr.HYD, which can be potentially used as a food emulsifier.

Differential expression of circular RNA in oral squamous cell carcinoma / 口腔疾病防治

Objective@# To analyze circular RNA (circRNA) expression profiles in oral squamous cell carcinoma (OSCC) and its clinical significance. @*Methods @#The expression of circRNA was detected with circRNA microarray assay in three samples of OSCC tumor and matched adjacent tissues. Quantitative real-time PCR (RT-qPCR) was used to verify the expression of circRNA in 45 pair OSCC tissues and normal adjacent tissues. The relationship between the expression of circRNA and the clinicopathological characteristics of OSCC was analyzed. circRNAs/miRNAs interaction were predicted using Arraystar' s home-made miRNA target prediction software. @*Results @#155 circRNAs were differentially expressed between the OSCC tissues and matched adjacent tissues, of which 45 circRNAs were up-regulated and 110 circRNAs were down-regulated in OSCC tissues (fold changes ≥1.5 and P < 0.05). In the selected three circRNAs that were most significantly upregulated or downregulated in OSCC, the RT-qPCR results showed that hsa_circ_0001874, hsa_circ_0001971 and has_circ_0067934 were increased, while hsa_circ_0000520, hsa_circ_0023944 and hsa_circ_0000140 were decreased in OSCC tissues versus normal adjacent tissues (P < 0.001). The results were generally consistent with the microarray data. Among the circRNA expression profiles in OSCC, the up-regulation of hsa_circ_0001874 was the highest and the expression of hsa_circ_0001874 was significantly correlated with TNM stage and tumor grade. The result of Arraystar' s home-made miRNA target prediction software indicated that miR-103a-3p, miR-107, miR-593-5p, miR-661 and miR-662 may be potential target genes of hsa_circ_0001874.@*Conclusion @#The differentially expressed circRNAs in OSCC tissues and normal adjacent tissues were identified, and these dysregulated circRNAs and their potential target gents may play important roles in the development of OSCC.

Applicable Value of AMSS-PCR in Lung Cancer Gene Mutation Detection / 中国肺癌杂志

BACKGROUND@#The detection of driver oncogenes of lung cancer is of great importance. There are various gene detection techniques nowadays which are different from each other. We carried out this study to investigate the specificity and sensitivity of assay panels based on an Amplification Refractory Mutation System-polymerase chain reaction (ARMS-PCR) technique of Amplification Mutation Specific System (AMSS) in detection of lung cancer gene mutation. To estimate the applicable value of assay panels in clinical settings.@*METHODS@#We collected cancer tissue specimens or fluid specimens from 309 patients. Mutation results were presented for those samples previously detected by ARMS-PCR. In comparison, we carried out AMSS-PCR using (epidermal growth factor receptor, EGFR) assay panel and Six-Alliance assay panel as well as Sanger sequencing. Software SPSS 22.0 (SPSS IBM) was used for statistical analysis.@*RESULTS@#The rates of consistency between the results by assay panels and Sanger sequencing or ARMS-PCR were 97.41% and 97.73%, respectively. Besides, EGFR assay panel had higher consistency rates with other detection methods than Six-Alliance assay panel. As for consistency test, the Kappa values of assay panels with Sanger sequencing, assay panels with ARMS-PCR, and ARMS-PCR with Sanger sequencing were 0.946, 0.953, and 0.913, respectively. The receiver operating characteristic curve (ROC) area under curve (AUC) of assay panels was 0.976 referring to Sanger sequencing, and 0.975 as ARMS-PCR was referred to.@*CONCLUSIONS@#AMSS-PCR can make an optimal cancer gene mutation detection method for clinical settings.

Establishment of a quantitative real-time PCR for rapid detection of Panax quinquefolius / 中国中药杂志

In this study, the specific primers and probes of Panax quinquefolius were designed for a quantitative real-time PCR, and the rapid identification method of P. quinquefolius was established by optimizing conditions. The method was used to validate 43 samples of the traditional Chinese medicine,and the results showed that 22 samples of P. quinquefolius were identified accurately. The limit of detection of the method can be reach to 1×10⁻⁴ ng. The method is accurate, fast, sensitive and specifically.

Study of Bacteria Associated with Dental Caries Using a 3 Tone Disclosing Agent / 대한소아치과학회지

The aim of this study was to determine the efficacy of a 3 tone plaque disclosing gel in assessing the risk of caries related to the population of Streptococcus mutans, Streptococcus sobrinus, and Lactobacillus spp. quantified using a quantitative real-time polymerase chain reaction (qRT-PCR).15 healthy children of ages 9 – 12 years were randomly examined. The 3 tone plaque disclosing gel was applied on teeth surfaces, which changed the color to pink or red, blue or purple and light blue. Plaque was divided into 3 groups based on staining. Genomic DNA from each sample was subjected to a qRT-PCR assay for quantitative detection of target bacteria. The Kruskal-Wallis test was conducted for correlation between the color of plaque and the number of bacterial species.The levels of S. mutans, S. sobrinus, and Lactobacillus spp. were significantly different in the plaque samples of the 3 groups (p < 0.05). The proportion of S. sobrinus to S. mutans showed correlation to the color of plaque.The different color-dyed plaque was related to the number of acidogenic bacteria. The 3 tone plaque disclosing gel could be used as one of the indicators to assess the clinical risk of caries associated with the population of S. mutans, S. sobrinus, and Lactobacillus spp.

Cloning of BpTRX gene and promoter and expression pattern analysis under H2S treatment in Betula platyphylla / 中草药

Objective To study the full-length, promoter sequences and its coding structure and properties of thioredoxin (Trx) gene of Betula platyphylla (BpTRX), and reveal the expression pattern of BpTRX under H2S treatment. Methods The BpTRX gene was cloned by PCR, and the promoter region sequences of BpTRX was obtained by using chromosome walking technique. The BpTRX gene, the promoter and its encoded protein were analyzed by bioinformatics software. The phylogenetic tree of BpTRX was constructed. The expression patterns of BpTRX gene under H2S exogenous stress were analyzed by quantitative real-time PCR. Results The full-length of BpTRX gene is 351 bp, encoding 117 amino acids. The BpTRX gene was closely related to the TRX-H protein of castor bean, soybean, alfalfa and grape. The obtained BpTRX promoter region sequences are 873 bp, which contains the essential elements of transcription and a large number of stress response and hormone response elements. Conclusion The full length and partial promoter sequences of the BpTRX gene were obtained. The response trend of BpTRX gene to H2S treatment was in the form of bimodal, which was first rises, then decreases, then rises and then declines.

Cloning, bioinformatics, and expression analysis of squalene epoxidase in Antrodia cinnamomea / 中草药

Objective To clone the squalene epoxidase gene of Antrodia cinnamomea (AcSE) and analyze the bioinformatics and expression of the gene. Methods AcSE was cloned by rapid-amplification of cDNA ends (RACE) from cDNA of A. cinnamomea. The physical and chemical properties of AcSE protein were analyzed, and its secondary structure, tertiary structure, and function were predicted by using bioinformatics analysis. The expression of AcSE in mycelium and fruit body of A. cinnamomea at different culture time was detected by using quantitative real-time PCR (qRT-PCR). Results The full-length cDNA sequence of AcSE were 1 446 bp (Genbank: KT070558), encoding a 481-amino-acid polypeptide. The molecular weight of AcSE was 53 300 and pI was 6.36. Domain analysis results showed that AcSE had three transmembrane domains without coiled-coil structure, and hydrophobic and hydrophilic regions existed alternately. The gDNA sequence of AcSE was 1 607 bp, contained four exons and three introns. A gene expression analysis by relative qRT-PCR showed that the highest expression level of AcSE was in mycelia incubated for 7 d of A. cinnamomea, and it was 7.89 times than that in fruiting body, and a gradual decline was observed with the extension of the culture time. Conclusion Gene AcSE was firstly cloned from A. cinnamomea, and it would lay a foundation for exploring the mechanism of terpenoid biosynthesis in A. cinnamomea.