Hepatocellular carcinoma (HCC) is an invasive malignant neoplasm with a poor prognosis. The development of chemoresistance severely obstructs the chemotherapeutic efficiency of HCC treatment. Therefore, understanding the mechanisms of chemoresistance is important for improving the outcomes of patients with HCC. Eukaryotic translation initiation factor 5A2 (eIF5A2), which is considered to be an oncogene, has been reported to mediate chemoresistance in various types of cancer; however, its precise role in HCC remains unclear. Accumulating evidence has suggested that autophagy serves a dual role in cancer chemotherapy. The present study aimed to investigate the role of autophagy in eIF5A2-mediated doxorubicin resistance in HCC. High expression levels of eIF5A2 in human HCC tissues were observed by immunohistochemistry using a tissue microarray, which was consistent with the results of reverse transcription-quantitative PCR analysis in paired HCC and adjacent healthy tissues. HCC patient-derived tumor xenograft mouse model was used for the in vivo study, and knockdown of eIF5A2 effectively enhanced the efficacy of doxorubicin chemotherapy compared with that in the control group. Notably, eIF5A2 served as a repressor in regulating autophagy under chemotherapy. Silencing of eIF5A2 induced doxorubicin sensitivity in HCC cells by triggering lethal autophagy. In addition, 5-ethynyl-2 '-deoxyuridine, lactate dehydrogenase release assay and calcein-AM/PI staining were used to determine the enhanced autophagic cell death induced by the silencing of eIF5A2 under doxorubicin treatment. Suppression of autophagy attenuated the sensitivity of HCC cells to doxorubicin induced by eIF5A2 silencing. The results also demonstrated that knockdown of the Beclin 1 gene, which is an autophagy regulator, reversed the enhanced autophagic cell death and doxorubicin sensitivity induced by eIF5A2 silencing. Taken together, these results suggested eIF5A2 may mediate the chemoresistance of HCC cells by suppressing autophagic cell death under chemotherapy through a Beclin 1-dependent pathway, and that eIF5A2 may be a novel potential therapeutic target for HCC treatment.

本文从大陆与台湾地区医学检验本科教育的基本情况、课程设置、实践教学、英语教学、毕业论文几方面进行比较,分析两岸医学检验本科教育的共同点及各自特点,以供同行探讨和借鉴.

CRISPR/Cas9 has been confirmed as a distinctly efficient, simple-to-configure, highly specific genome-editing tool that has been used to treat monogenetic disorders. Epidermolytic palmoplantar keratoderma (EPPK) is a common autosomal dominant keratin disease resulting from dominant-negative mutation of the KRT9 gene, and it has no effective therapy. We performed CRISPR/Cas9-mediated treatment on a knockin (KI) transgenic mouse model that carried a small indel heterozygous mutation of Krt9, c.434delAinsGGCT (p.Tyr144delinsTrpLeu), which caused a humanized EPPK-like phenotype. The mutation within exon 1 of Krt9 generated a novel protospacer adjacent motif site, TGG, for Cas9 recognition and cutting. By delivering lentivirus vectors (LVs) encoding single-guide RNAs (sgRNAs) and Cas9 that targeted Krt9 sequence into HeLa cells engineered to constitutively express wild-type and mutant keratin 9 (K9), we found the sgRNA was highly effective in reducing expression of the mutant K9 protein in vitro. We injected the LV into the fore-paws of adult KI-Krt9 mice three times every 8 days and found that the expression of K9 decreased ∼14.6%. The phenotypic mitigation was revealed by restoration of the abnormal differentiation and aberrant proliferation of the epidermis. Our data are the first to show that CRISPR/Cas9 is a potentially powerful therapeutic option for EPPK and other PPK subtypes.

Epidermolytic palmoplantar keratoderma (EPPK) is a relatively common autosomal dominant skin genetic disorder caused by mutations in the keratin 9 gene (KRT9),with few therapeutic options for the affected so far.Here,the hot mutation sites of KRT9,KRT9 gene mutation as a reliable indicator applied in clinical prenatal molecular diagnosis of EPPK,the construction of Krt9 gene mutation knock-in mouse model,and the mutant-specific shRNA-mediated treatment of EPPK were summarized.

Epidermolytic palmoplantar keratoderma (EPPK) is a relatively common autosomal-dominant skin disorder caused by mutations in the keratin 9 gene (KRT9), with few therapeutic options for the affected so far. Here, we report a knock-in transgenic mouse model that carried a small insertion-deletion (indel) mutant of Krt9, c.434delAinsGGCT (p.Tyr144delinsTrpLeu), corresponding to the human mutation KRT9/c.500delAinsGGCT (p.Tyr167delinsTrpLeu), which resulted in a human EPPK-like phenotype in the weight-stress areas of the fore- and hind-paws of both Krt9(+/mut) and Krt9(mut/mut) mice. The phenotype confirmed that EPPK is a dominant-negative condition, such that mice heterozygotic for the K9-mutant allele (Krt9(+/mut)) showed a clear EPPK-like phenotype. Then, we developed a mutant-specific short hairpin RNA (shRNA) therapy for EPPK mice. Mutant-specific shRNAs were systematically identified in vitro using a luciferase reporter gene assay and delivered into Krt9(+/mut) mice. shRNA-mediated knockdown of mutant protein resulted in almost normal morphology and functions of the skin, whereas the same shRNA had a negligible effect in wild-type K9 mice. Our results suggest that EPPK can be treated by gene therapy, and this has significant implications for future clinical application.

为了解决理实一体化课程存在的问题,文章结合微信平台设计一种混合式教学模式,通过前期调研、平台建设、资源构建、教学实践、分析反馈等一系列步骤实施教学研究,结果表明混合式教学模式能有效联结课堂内外各个环节,学生运用微信相关功能进行预习、复习,能够迅速提高课堂教学效率,师生间打破时空的限制进行交流互动,及时解决教学难题.同时也对混合式教学中出现的问题提出解决办法,通过不断更新调整,为其他课程实施混合式教学提供了借鉴.

病原生物与免疫是高职护理专业职业能力素养课程的重要组成部分,针对教改中凸显的问题,通过优化整合与护理岗位紧密的实践项目,以微课(微视频)为辅助,调整并设计实践教学内容和过程,形成了适用于基础医学任务引领的实践情景教学,学生在逻辑分析能力培养和无菌基本技能获得等方面得到了明显的提升.

在基础医学课程整合的背景下,针对出现的过多基础医学课程群,基于专业群构建基础医学平台课程,实现与专业的有效对接.并在教学内容融合、应用型知识强化及教学模式改革等方面得到了提升,同时为进一步的改革深化提出了设想和建议.

Epidermolytic palmoplantar keratoderma (EPPK) is the most frequent form of such keratodermas. It is inherited in an autosomal dominant pattern and is clinically characterized by diffuse yellowish thickening of the skin on the palms and soles with erythematous borders during the first weeks or months after birth. EPPK is generally caused by mutations of the KRT9 gene. More than 26 KRT9 gene mutations responsible for EPPK have been described (Human Intermediate Filament Database, www.interfil.org), and many of these variants are located within the highly-conserved coil 1A region of the α-helical rod domain of keratin 9. Unfortunately, there is no satisfactory treatment for EPPK. Thus, prenatal molecular diagnosis or pre-pregnancy diagnosis is crucial and benefits those affected who seek healthy descendants. In the present study, we performed amniotic fluid-DNA-based prenatal testing for three at-risk pregnant EPPK women from three unrelated southern Chinese families who carried the KRT9 missense mutations p.Arg163Trp and p.Arg163Gln, and successfully helped two families to bear normal daughters. We suggest that before the successful application of preimplantation genetic diagnosis (PGD), and noninvasive prenatal diagnosis of EPPK that analyzes fetal cells or cell-free DNA in maternal blood, prenatal genetic diagnosis by amniocentesis or chorionic villus sampling (CVS) offers a quite acceptable option for EPPK couples-at-risk to avoid the birth of affected offspring, especially in low- and middle-income countries.

目的 了解宁波市老年居民的营养健康知识、态度、行为现状即KAP水平及其影响因素,为在社区针对性地开展营养科普宣教提供科学依据.方法 整群抽样问卷调查方式,选取东裕社区500名60岁及以上的老人进行入户访谈式调查.结果 调查对象K、A、P得分的合格率分别为38.2％、85.8％、23.8％.主要缺乏对中国居民膳食指南、营养素的主要食物来源、营养相关疾病等知识方面的了解;饮食习惯、食物选择、三餐能量安排等饮食行为存在不合理.营养知识的主要影响因素是文化程度、职业、常住户口;营养态度的主要影响因素是文化程度、职业、年龄、营养知识水平;营养行为的主要影响因素是营养知识水平、态度、年龄、文化程度.老年居民营养信息获取途径主要是电视广播、社区宣传窗、报刊杂志.结论 宁波市某社区老年居民的营养健康知识水平普通较低,饮食行为存在诸多不合理,但营养态度较好.应加强对社区老年居民的营养健康知识的宣教和健康饮食行为的指导.

根据高职护理教育培养高素质、高端技能型护理人才的目标,探索人体机能学实践教学改革。优化实践教学内容、整合课程,设计基于工作过程的人体机能学综合性实践项目,改进评价方式,突出对学生实践能力的培养,体现知识的应用性和实践教学的专业针对性。

目的 通过对社区老年居民营养知识、态度和行为的调查分析,了解其对营养健康科普知识的需求程度和相关内容,以便开展针对性的营养健康教育,提高老年居民的健康素质.方法 采取整群随机抽样法,选取东裕社区500名60岁及以上老年人进行入户访谈式问卷调查.结果 调查对象营养知识、态度和行为得分合格率分别为38.2％、85.8％和23.8％.主要缺乏对中国居民膳食指南、营养素的主要食物来源和疾病相关营养知识等方面的了解,饮食习惯、食物选择和三餐能量安排等饮食行为存在不合理现象.结论 宁波市某社区老年居民的营养健康知识水平普通较低,饮食行为存在诸多不合理现象,但营养态度较好.应加强对社区老年居民营养健康知识的宣教和健康饮食行为的指导.

基于基础医学课程特点和网络资源功能,构建针对多个专业的基础医学平台课程,体现各专业共同的基本核心内容,突出各专业针对性和职业性的特色内容;同时实践立体化教学模式,以提高教学效果。

Exome is the collection of all exons,i.e.the protein-coding sequencing,within the genomic DNA of an individual.Exome constitutes around 1% of the entire human genome or 30 megabases(Mb),split across approximately 180,000 exons.It estimated that about 85% variants located on these protein-coding regions.Traditionally,the family linkage analysis and positional cloning methods are used to identify the disease-causing gene for Mendelian disorders.However,because of the difficulty in collection of an adequate number of affected individuals and families for a statistically powerful study,or incomplete/non-penetrance of the disease,or de novo mutations for sporadic cases,these approaches are not applicable.Thus,the whole exome-capture sequencing(WES) represents a cost-effective,reproducible and robust strategy for the sensitive and specific identification of variants in protein-coding changes in individual human genomes.In addition,during the gene testing for larger gene of monogenic defects such as DMD,FBN1,PKD1,WES is more cost-effective than PCR followed by Sanger sequencing technique.

BACKGROUND:Mutations in the KRT6A or KRT16 gene cause pachyonychia congenita type 1 (PC-1), while mutations in KRT16 or KRT6C underlie focal palmoplantar keratoderma (FPPK). A new classification system of PC has been adopted based on the mutated gene. PC rarely presents the symptoms of diffuse plantar keratoderma. Mutation in the tail domain of keratins is rarely reported. PC combined with fissured tongue has never been described.OBJECTIVES:To investigate the genotype-phenotype correlations between clinical features and gene mutational sites in two unrelated southern Chinese PC pedigrees (one family presented with specific fissured tongue, the other with diffuse plantar keratoderma).MATERIALS & METHODS:The whole coding regions of the KRT6A/KRT16/KRT17/KRT6B genes were amplified and directly sequenced to detect the mutation. To confirm the effect of the IVS8-2A>C mutation in KRT6A at the mRNA level, total RNA from the plantar lesion of a patient was extracted and reverse-transcribed to cDNA for sequence analysis.RESULTS:Two novel de novo mutations, a splice acceptor site variant IVS8-2A>C (p.S487FfsX72) in KRT6A and a heterozygous substitution c.AA373_374GG (p.N125G) within exon 1 of KRT16, were found separately in the two PC families.CONCLUSION:Genotype-phenotype correlations among PC patients with codon-125 mutation in KRT16 were established, while the phenotypes caused by the IVS8-2A>C mutation in KRT6A need further studies to confirm the rare feature of fissured tongue.

BACKGROUND:Genetic screening for germline mutations in the RET proto-oncogene has been extensively exploited worldwide to optimize the diagnostic and clinical management of multiple endocrine neoplasia type 2 (MEN2) patients and their relatives. However, a distinct lag period exists not only in the recognition but also in the medical treatment of patients with MEN2. Here we present a comprehensive genetic and clinical analysis of MEN2 among Chinese families followed from 1975 to 2011. Our series comprises 36 index cases and 134 relatives from 11 independent families.METHODS:Genetic diagnosis was performed in all participants by direct sequencing all relevant RET exons. Thyroidectomy was performed in 50 patients with varying cervical neck dissection procedures. Patients with pheochromocytoma (PHEO) underwent specific surgery. Demographic, clinical profiles, mutation types, tumor histopathologic features, and follow-up records were systematically analyzed.RESULTS:The RET mutations p.C634Y (n=34), p.C634R (n=6), p.C618S (n=13), p.V292M/R67H/R982C (n=7), p.L790F (n=2), and p.C634Y/V292M/R67H/R982C (n=1) were confirmed in 31 index cases and then identified in 32 at-risk relatives (mutation carriers), with MEN2A as the most common clinical subtype. The overall penetrance of PHEO in patients with MEN2A was 46.7%. A total of 50 patients underwent thyroidectomy, and there was a significant lowering of their mean age at thyroidectomy and the tumor diameter of the mutation carriers that were detected and operated on compared with the index cases (age at first surgery: 29.3 vs. 39.3 years, p<0.05; maximum size: 1.1 vs. 3.3 cm, p<0.001). There was also a decrease in the TNM staging and the proportion of patients who underwent inappropriate initial thyroid surgery (pN1: 31.6% vs. 100%, p<0.001; inappropriate surgery: 0% vs. 29%). Meanwhile, disease-free survival (DFS) increased (DFS: 100% vs. 58.1%, p<0.05). Both medullary thyroid carcinoma-specific (n=1) and PHEO-specific (n=5) deaths were reported during the study period.CONCLUSIONS:Our results further substantiate that gene scanning of all relevant RET exons is a powerful tool in the management of MEN2 patients, especially in asymptomatic carriers, and has led to earlier diagnosis and more complete initial treatment of patients with MEN2 in China.

Danon disease is a rare X-linked lysosomal storage disease characterized by hypertrophic cardiomyopathy, myopathy and mental retardation, and is due to a primary defect in lysosome-associated membrane protein-2 (LAMP 2). More than 26 mutations in the LAMP2 gene have been described, including a small number of de novo mutations, some of which are suspected to be caused by germline mosaicism. Here, we describe the first molecularly documented evidence of somatic mosaicism for a LAMP2 mutation, identified in the asymptomatic mother of a boy with Danon disease caused by the frameshift mutation c.808dupG (p.A270Gfx3) within exon 6. In addition, in order to gain insight into the possible explanation for the mother's lack of phenotype, the level of somatic mosaicism and the X-chromosome inactivation pattern were investigated. This study provides new insight into the causes of phenotypic variability in female mutation-carriers and underlines the importance of parental molecular testing for accurate genetic counseling for Danon disease.

Epidermolytic palmoplantar keratoderma (EPPK) is generally associated with dominant‐negative mutations of the Keratin 9 gene ( KRT9 ), and rarely with the Keratin 1 gene ( KRT1 ). To date, a myriad of mutations has been reported with a high frequency of codon 163 mutations within the first exon of KRT9 in different populations. Notably, a distinct phenotypic heterogeneity, digital mutilation, was found recently in a 58‐year‐old female Japanese EPPK patient with p.R163W. Here, we report the most common mutation, c.C487T (p.R163W) of KRT9 , in two large EPPK pedigrees from southeast China. The arginine residue in peptide position 163 remains almost constant in at least 47 intermediate filament proteins ranging from snail to human. A substitution in arginine alters both the charge and shape of the 1A rod domain and disrupts the function of the helix initiation motif of keratins, finally compromising the integrity of filaments and weakening their stability in the epidermis of palms and soles. We summarize the clinical symptoms of EPPK in Chinese and show that knuckle pads are associated with KRT9 mutations. We suggest that the frequency of p.R163W in Chinese EPPK patients (31.03%) is consistent with that in the general population (29.33%), and that codon 163 is truly a hotspot mutational site of KRT9. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

Mutation-based molecular diagnostics of autosomal dominant polycystic kidney disease (ADPKD) is complicated by genetic and allelic heterogeneity, large multi-exon genes, and duplication sequences of PKD1. Recently, targeted resequencing by pooling long-range polymerase chain reaction (LR-PCR) amplicons has been used in the identification of mutations in ADPKD. Despite its high sensitivity, specificity and accuracy, LR-PCR is still complicated. We performed whole-exome sequencing on two unrelated typical Chinese ADPKD probands and evaluated the effectiveness of this approach compared with Sanger sequencing. Meanwhile, we performed targeted gene and next-generation sequencing (targeted DNA-HiSeq) on 8 individuals (1 patient from one family, 5 patients and 2 normal individuals from another family). Both whole-exome sequencing and targeted DNA-HiSeq confirmed c.11364delC (p.H3788QfsX37) within the unduplicated region of PKD1 in one proband; in the other family, targeted DNA-HiSeq identified a small insertion, c.401_402insG (p.V134VfsX79), in PKD2. These methods do not overcome the screening complexity of homology. However, the true positives of variants confirmed by targeted gene and next-generation sequencing were 69.4%, 50% and 100% without a false positive in the whole coding region and the duplicated and unduplicated regions, which indicated that the screening accuracy of PKD1 and PKD2 can be largely improved by using a greater sequencing depth and elaborate design of the capture probe.

Objective To investigate the dominant genotypes of H.pylori(Hp) and its relationship to the resis-tance of antibiotics in Ningbo district.Methods Specimens of gastric mucosa were collected by biopsy from 40 patients,26 with chronic gastritis(CG) and 14 with peptic ulcer(PU) .PCR was conducted to detect the cagA,vac-Am1,vacAm2,vacAs1a and babA2 genotypes of Hp isolates.Antibiotic susceptibility test was performed by disk diffusion technique.Results The positive rates of cagA,vacAm1,vacAm2,vacAs1a and babA2 genotypes in 40 Hp strains were 100%,52.5%,85.5%,92.5%and 57.5%,respectively.There were no significant differences in the detective rates of HP genotypes between chronic gastritis and peptic ulcer(>0.05) .Of 40 Hp strains,0 was re-sistant to amoxicillin,15%to azithromycin,7.5%to clarithromucin,22.5%to lavo-ofloxacin and 35%was resis-tant to metronidazole.Resistance rate of metronidazole was higher than that of azithromycin and significantly hig-her than that of amoxicillin and clarithromucin(<0.01) ;the distributions of cagA,vacAm1,vacAm2,vacAs1a and babA2 genotypes were not significantly different between resistant strains and susceptibile strains of Hp to metronidazole(>0.05) .Conclusion CagA,vacAm2,vacAs1a,babA2 and vacAs1a/m2 are the dominant geno-types of Hp in the CG and PU patients in Ningbo.The determination of subtypes of Hp gene is still not helpful to choose antibiotics in Hp eradication treatments.