Proteome analysis of hepatocellular carcinoma by two-dimensional difference gel electrophoresis: novel protein markers in hepatocellular carcinoma tissues

Hepatocellular carcinoma (HCC) is a highly malignant tumor, and chronic infection with hepatitis B virus is one of its major risk factors. To identify the proteins involved in HCC carcinogenesis, we used two-dimensional fluorescence DIGE to study the differentially expressed proteins in tumor and adjacent nontumor tissue samples. Samples from 12 hepatitis B virus-associated HCC patients were analyzed. A total of 61 spots were significantly up-regulated (ratio >/= 2, p 相似文献   

Computational fluid dynamics endpoints for assessment of adenotonsillectomy outcome in obese children with obstructive sleep apnea syndrome

Background

Improvements in obstructive sleep apnea syndrome (OSAS) severity may be associated with improved pharyngeal fluid mechanics following adenotonsillectomy (AT). The study objective is to use image-based computational fluid dynamics (CFD) to model changes in pharyngeal pressures after AT, in obese children with OSAS and adenotonsillar hypertrophy.

Methods

Three-dimensional models of the upper airway from nares to trachea, before and after AT, were derived from magnetic resonance images obtained during wakefulness, in a cohort of 10 obese children with OSAS. Velocity, pressure, and turbulence fields during peak tidal inspiratory flow were computed using commercial software. CFD endpoints were correlated with polysomnography endpoints before and after AT using Spearman?s rank correlation (r_s).

Results

Apnea hypopnea index (AHI) decreases after AT was strongly correlated with reduction in maximum pressure drop (dP_TAmax) in the region where tonsils and adenoid constrict the pharynx (r_s=0.78, P=0.011), and with decrease of the ratio of dP_TAmax to flow rate (r_s=0.82, P=0.006). Correlations of AHI decrease to anatomy, negative pressure in the overlap region (including nasal flow resistance), or pressure drop through the entire pharynx, were not significant. In a subgroup of subjects with more than 10% improvement in AHI, correlations between flow variables and AHI decrease were stronger than in all subjects.

Conclusions

The correlation between change in dP_TAmax and improved AHI suggests that dP_TAmax may be a useful index for internal airway loading due to anatomical narrowing, and may be better correlated with AHI than direct airway anatomic measurements.     

基于微滴式数字PCR检测肠癌病人游离环状DNA KRAS突变的新方法

基于微滴式数字聚合酶链式反应(Droplet digital polymerase chain reaction,dd PCR)设计一种检测肠癌游离循环DNA(Circulating cell free DNA,cf DNA)中KRAS(V-Ki-ras2 Kirsten ratsarcoma viral oncogene homolog)基因突变的新方法并评估其灵敏度和准确性。根据肠癌病人KRAS基因的突变类型设计并合成,采用dd PCR扩增并评估其灵敏度和准确性;根据AMRS-PCR引物设计原理设计KRAS基因的实时定量PCR扩增引物并评估其准确性,进而比较dd PCR和q PCR二者之间的优缺点;最后针对52例肠癌病人的cf DNA采用dd PCR进行检测,研究dd PCR在cf DNA KRAS基因突变检测的应用。成功使用dd PCR和q PCR两种方法对KRAS野生型及7种突变型建立检测方法,使用质粒标准品及实际样品验证该两种方法可行并对其假阳性率、线性范围及检测下限等性能进行了评价,最后成功对52例临床患者和20例正常人的血浆cf DNA样本进行检测,临床灵敏度为97.64%,临床特异性为81.43%。dd PCR的检测性能优于q PCR,LOD达到个位数DNA拷贝,最低可确认突变浓度达到0.01%–0.04%。样本提取效率在方法学建立中也十分重要,直接影响到灵敏度和Cut Off值的判定。临床患者检测结果显示其KRAS突变率接近报道水平。     

思茅松HDR基因全长cDNA克隆与序列分析

1-羟基-2-甲基-2-E-丁烯基-4-焦磷酸还原酶(HDR)是甲基-D-赤藓醇-4-磷酸(MEP)途径中的最后一个酶,在植物萜类生物合成中起主控作用。该研究根据思茅松(Pinus kesiya var.langbianensis)树皮转录组数据分析结果,首先获得了思茅松HDR基因片段,然后根据所获得的基因片段设计特异引物,提取受伤后的思茅松树皮的RNA,并运用RT-PCR和RACE技术从思茅松树皮中克隆得到完整的HDR基因(Pk HDR)。生物信息学分析表明:克隆获得的Pk HDR1基因c DNA全长序列为1 876 bp,含有1个1 464 bp的开放阅读框(ORF),编码487个氨基酸。同源性分析结果表明:思茅松HDR蛋白与赤松(Pinus densiflora)HDR蛋白的相似性高达99%。亚细胞定位及结构域分析结果表明:思茅松Pk HDR氨基酸序列中包含转运肽序列(A1-A61)及植物HDR蛋白多个保守的功能位点(A143,A234,A288,A371)。系统进化分析结果表明:Pk HDR蛋白与赤松HDR蛋白的亲缘关系最为接近。半定量PCR检测结果表明:树皮的创伤促进思茅松HDR基因的表达。该研究成功克隆获得HDR基因,并确定其与松脂代谢密切相关,为阐明思茅松松脂生物合成机制和分子育种提供了参考。     

Field Studies of the Wheat Stomata Resistance Influenced by Soil Water Content

Concurrent observations of soil water potential and leaf stomata diffusion resistance were made on two, plots of wheat grown at Datun Agro-ecological Experimental Station in Beijing under two different soil water conditions. These data were further complemented by weather and physiological observation. In this paper, we mainly analysed the influence of soil water potential on the status of wheat leaf stomatal resistance. The results indicate that: (1) there is a obvious influence of soil water potential on the status of wheat leaf stomata under normal conditions and (2) there is a different upper and lower epidermis stomata of wheat leaf respond to the soil water potential. The lower epidermis stomata are more sensitive to soil water potential than upper epidermis one. (3) There is a linear relationship between the ratio of lower and upper epidermis stomata resistance and soil water potential in root layer, according to this we can diagnose the degree of wheat water deficit.     

Hepatitis C virus inhibits AKT-tuberous sclerosis complex (TSC), the mechanistic target of rapamycin (MTOR) pathway,through endoplasmic reticulum stress to induce autophagy

Hepatitis C virus (HCV) is able to induce autophagy via endoplasmic reticulum (ER) stress, but the exact molecular signaling pathway is not well understood. We found that the activity of the mechanistic target of rapamycin complex 1 (MTORC1) was inhibited in Huh7 cells either harboring HCV-N (genotype 1b) full-genomic replicon or infected with JFH1 (genotype 2a) virus, which led to the activation of UNC-51-like kinase 1 (ULK1) and thus to autophagy. We then analyzed activity upstream of MTORC1, and found that both protein kinase, AMP-activated, α (PRKAA, including PRKAA1 and PRKAA2, also known as AMP-activated protein kinase, AMPKα) and AKT (refers to pan AKT, including three isoforms of AKT1-3, also known as protein kinase B, PKB) were inhibited by HCV infection. The inhibition of the AKT-TSC-MTORC1 pathway contributed to upregulating autophagy, but inhibition of PRKAA downregulated autophagy. The net effect on autophagy was from AKT, which overrode the inhibition effect from PRKAA. It was further found that HCV-induced ER stress was responsible for the inhibition of the AKT pathway. Metformin, a PRKAA agonist, inhibited HCV replication not only by activating PRKAA as previously reported, but also by activating AKT independently of the autophagy pathway. Taken together, our data suggested HCV inhibited the AKT-TSC-MTORC1 pathway via ER stress, resulting in autophagy, which may contribute to the establishment of the HCV-induced autophagy.     

Countrywide Survey for MERS-Coronavirus Antibodies in Dromedaries and Humans in Pakistan

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a zoonotic pathogen capable of causing severe respiratory disease in humans. Although dromedary camels are considered as a major reservoir host, the MERS-CoV infection dynamics in camels are not fully understood. Through surveillance in Pakistan, nasal (n = 776) and serum (n = 1050)samples were collected from camels between November 2015 and February 2018. Samples were collected from animal markets, free-roaming herds and abattoirs. An in-house ELISA was developed to detect IgG against MERS-CoV. A total of 794 camels were found seropositive for MERS-CoV. Prevalence increased with the age and the highest seroprevalence was recorded in camels aged [ 10 years (81.37%) followed by those aged 3.1–10 years (78.65%) and B 3 years (58.19%).Higher prevalence was observed in female (78.13%) as compared to male (70.70%). Of the camel nasal swabs, 22 were found to be positive by RT-qPCR though with high Ct values. Moreover, 2,409 human serum samples were also collected from four provinces of Pakistan during 2016–2017. Among the sampled population, 840 humans were camel herders.Although we found a high rate of MERS-CoV antibody positive dromedaries (75.62%) in Pakistan, no neutralizing antibodies were detected in humans with and without contact to camels.     

<Emphasis Type="Italic">Sphingomonas humi</Emphasis> sp. nov., isolated from soil

A Gram-negative, non-motile, non-spore-forming, small, orange, rod-shaped bacterium was isolated from soil in South Korea and characterized to determine its taxonomic position. Phylogenetic analysis based on 16S rRNA gene sequence examination revealed that strain PB323^T belongs to the family Sphingomonadaceae. The highest degree of sequence similarity was found with Sphingomonas kaistensis PB56^T (98.9%), followed by Sphingomonas astaxanthinifaciens TDMA-17^T (98.3%). Chemotaxonomic characteristics (the G+C content of the genomic DNA 69.0 mol%, Q-10 quinone system, C_18:1 ω7c/ω9t/ω12t, C_16:1 ω7c/C_15:0 iso 2OH, C_17:1 ω6c, and C16:0 as the major fatty acids) corroborated assignment of strain PB323^T to the genus Sphingomonas. Results of physiological and biochemical tests clearly demonstrate that strain PB323^T represents a distinct species and support its affiliation with the genus Sphingomonas. Based on these data, PB323^T (=KCTC 12341^T =JCM 16603T =KEMB 9004-003^T) should be classified as a type strain of a novel species, for which the name Sphingomonas humi sp. nov. is proposed.