Gestational diabetes mellitus (GDM) decreases butyrylcholinesterase (BChE) activity and changes its relationship with lipids

Many conditions interfere with butyrylcholinesterase (BChE) activity, e.g., pregnancy or presence of the BCHE gene variant −116A can decrease activity whereas obesity and types I and II diabetes mellitus can increase activity. In this study, we examined BChE activity, −116A and 1615A BCHE gene variants, and anthropometric and biochemical variables associated with diabetes in patients with gestational diabetes mellitus (GDM) and in healthy pregnant women. BChE activity was measured spectrophotometrically using propionylthiocholine as substrate and genotyping of the −116 and 1615 sites of the BCHE gene was done with a TaqMan SNP genotyping assay. Three groups were studied: 150 patients with GDM, 295 healthy pregnant women and 156 non-pregnant healthy women. Mean BChE activity was significantly lower in healthy pregnant women than in women from the general population and was further reduced in GDM patients. BChE activity was significantly reduced in carriers of −116A in GDM patients and healthy pregnant women. Although GDM patients had a significantly higher mean body mass index (BMI) and triglycerides than healthy pregnant women, they had lower mean BChE activity, suggesting that the lowering effect of GDM on BChE activity was stronger than the characteristic enhancing effect of increased BMI and triglycerides.     

Performance comparison of four exome capture systems for deep sequencing

Background

Recent developments in deep (next-generation) sequencing technologies are significantly impacting medical research. The global analysis of protein coding regions in genomes of interest by whole exome sequencing is a widely used application. Many technologies for exome capture are commercially available; here we compare the performance of four of them: NimbleGen’s SeqCap EZ v3.0, Agilent’s SureSelect v4.0, Illumina’s TruSeq Exome, and Illumina’s Nextera Exome, all applied to the same human tumor DNA sample.

Results

Each capture technology was evaluated for its coverage of different exome databases, target coverage efficiency, GC bias, sensitivity in single nucleotide variant detection, sensitivity in small indel detection, and technical reproducibility. In general, all technologies performed well; however, our data demonstrated small, but consistent differences between the four capture technologies. Illumina technologies cover more bases in coding and untranslated regions. Furthermore, whereas most of the technologies provide reduced coverage in regions with low or high GC content, the Nextera technology tends to bias towards target regions with high GC content.

Conclusions

We show key differences in performance between the four technologies. Our data should help researchers who are planning exome sequencing to select appropriate exome capture technology for their particular application.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-449) contains supplementary material, which is available to authorized users.     

Dynamic expression of combinatorial replication-dependent histone variant genes during mouse spermatogenesis

Nucleosomes are basic chromatin structural units that are formed by DNA sequences wrapping around histones. Global chromatin states in different cell types are specified by combinatorial effects of post-translational modifications of histones and the expression of histone variants. During mouse spermatogenesis, spermatogonial stem cells (SSCs) self-renew while undergo differentiation, events that occur in the company of constant re-modeling of chromatin structures. Previous studies have shown that testes contain highly expressed or specific histone variants to facilitate these epigenetic modifications. However, mechanisms of regulating the epigenetic changes and the specific histone compositions of spermatogenic cells are not fully understood. Using real time quantitative RT-PCR, we examined the dynamic expression of replication-dependent histone genes in post-natal mouse testes. It was found that distinct sets of histone genes are expressed in various spermatogenic cells at different stages during spermatogenesis. While gonocyte-enriched testes from mice at 2-dpp (days post partum) express pre-dominantly thirteen histone variant genes, SSC-stage testes at 9-dpp highly express a different set of eight histone genes. During differentiation stage when testes are occupied mostly by spermatocytes and spermatids, another twenty-two histone genes are expressed much higher than the rest, including previously known testis-specific hist1h1t, hist1h2ba and hist1h4c. In addition, histone genes that are pre-dominantly expressed in gonocytes and SSCs are also highly expressed in embryonic stem cells. Several of them were changed when embryoid bodies were formed from ES cells, suggesting their roles in regulating pluripotency of the cells. Further more, differentially expressed histone genes are specifically localized in either SSCs or spermatocytes and spermatids, as demonstrated by in situ hybridization using gene specific probes. Taken together, results presented here revealed that different combinations of histone variant genes are expressed in distinct spermatogenic cell types accompanying the progression of self-renewal and differentiation of SSCs, suggesting a systematic regulatory role histone variants play during spermatogenesis.     

Variant CJD

It is now 18 years since the first identification of a case of vCJD in the UK. Since that time, there has been much speculation over how vCJD might impact human health. To date there have been 177 case reports in the UK and a further 51 cases worldwide in 11 different countries. Since establishing that BSE and vCJD are of the same strain of agent, we have also shown that there is broad similarity between UK and non-UK vCJD cases on first passage to mice. Transgenic mouse studies have indicated that all codon 129 genotypes are susceptible to vCJD and that genotype may influence whether disease appears in a clinical or asymptomatic form, supported by the appearance of the first case of potential asymptomatic vCJD infection in a PRNP 129MV patient. Following evidence of blood transfusion as a route of transmission, we have ascertained that all blood components and leucoreduced blood in a sheep model of vCJD have the ability to transmit disease. Importantly, we recently established that a PRNP 129MV patient blood recipient with an asymptomatic infection and limited PrP^Sc deposition in the spleen could readily transmit disease into mice, demonstrating the potential for peripheral infection in the absence of clinical disease. This, along with the recent appendix survey which identified 16 positive appendices in a study of 32 441 cases, underlines the importance of continued CJD surveillance and maintaining control measures already in place to protect human health.     

A new tool for monoclonal antibody analysis: Application of IdeS proteolysis in IgG domain-specific characterization

Monoclonal antibody (mAb) products are extraordinarily heterogeneous due to the presence of a variety of enzymatic and chemical modifications, such as deamidation, isomerization, oxidation, glycosylation, glycation, and terminal cyclization. The modifications in different domains of the antibody molecule can result in different biological consequences. Therefore, characterization and routine monitoring of domain-specific modifications are essential to ensure the quality of the therapeutic antibody products. For this purpose, a rapid and informative methodology was developed to examine the heterogeneity of individual domains in mAb products. A recently discovered endopeptidase, IdeS, cleaves heavy chains below the hinge region, producing F(ab')₂ and Fc fragments. Following reduction of disulfide bonds, three antibody domains (LC, Fd, and Fc/2) can be released for further characterization. Subsequent analyses by liquid chromatography/mass spectrometry, capillary isoelectric focusing, and glycan mapping enable domain-specific profiling of oxidation, charge heterogeneity, and glycoform distribution. When coupled with reversed phase chromatography, the unique chromatographic profile of each molecule offers a simple strategy for an identity test, which is an important formal test for biopharmaceutical quality control purposes. This methodology is demonstrated for a number of IgGs of different subclasses (IgG1, IgG2, IgG4), as well as an Fc fusion protein. The presented technique provides a convenient platform approach for scientific and formal therapeutic mAb product characterization. It can also be applied in regulated drug substance batch release and stability testing of antibody and Fc fusion protein products, in particular for identity and routine monitoring of domain-specific modifications.     

耐受偏二甲肼的芦苇变异株系的筛选

四氧化二氮/偏二甲肼(Unsymmetrical dimethyl-hybrazine, 简称UDMH)为常用的航天器双组元液体推进剂。偏二甲肼沸点低,具有“三致”毒性,可在使用过程中释放到环境中,对污染地的生物多样性和人类健康构成威胁,因此迫切需要清除环境中的偏二甲肼。该文采用细胞工程的技术手段,以芦苇幼苗的下胚轴为材料诱导愈伤组织,继而通过逐步提高筛选压力选育出可耐受偏二甲肼的变异细胞系,再诱导变异细胞系分化,为可治理含偏二甲肼的废水的人工湿地处理系统的构建提供理想的工程植物。结果表明:芦苇的愈伤组织在含有0.5 mg·L^-1 2,4-D的MS培养基上生长良好;偏二甲肼对该愈伤组织生长的半致死剂量为16.3 mmol·L^-1;在分别含有1.63、3.26和8.15 mmol·L^-1偏二甲肼的筛选培养基上进行3~6次继代培养后,可得到较稳定的抗性细胞系,培养23 d后的相对生长量分别为对照的90.4%、84.3%和43.4%,培养43 d后的相对生长量分别为对照的95.6%、91.7%和46.8%;但是只有前两类抗性细胞系可在含0.1 g·L^-1 KT、0.01 g·L^-1 NAA和相应浓度的偏二甲肼的MS培养基中诱导分化;将绿色再生苗转移到不含激素但含偏二甲肼的培养基上强化根的生长,再经过35 d左右的适应性驯化,70%以上的再生苗可成功地转移至温室中培养,为日后人工湿地系统的构建奠定了基础。     

芦苇耐盐变异体与野生型植株某些特性的比较

地通过细胞工程方法得到的耐盐芦苇（Ｐｈｒａｇｍｉｔｅｓ ｃｏｍｍｕｎｉｓ Ｔｒｉｎ．）变异系Ｒ５００２－１２与野生型植株进行了分子生物学和生理生化特性比较分析。筛选到５个ＲＡＰＤ引物对变异体和野生型植株的ＤＮＡＦ的随机扩增表现出不同的多态性,表明该变异体在分子水平上发生了变化。生理生化分析的结果表明,平行生长状态下的两种株系,其可溶性蛋白和同工酶的表达水平和种类不一样,变异体在ＮａＣｌ胁迫下,表达     

卫星搭载亚麻后代中PEG和NaCl抗性系的初步筛选

把空间生物学和细胞工程相结合,通过组织培养技术对其离体筛选,得到抗１．２％ＮａＣｌ和３５％ＰＥＧ的愈伤组织,将所得抗性系愈伤组织在２．０ｍｇ／Ｌ６－苄基氨基嘌呤、０．５ｍｇ／Ｌ吲哚乙酸的ＭＳ培养基上分化得到完整的植株。抗性系能在胁迫条件下保持高的生长速度和高效的脯氨酸合成能力,表明空间诱变与组织培养相结合有望可成为筛选抗胁迫变异系的有效途径。     

Comparison of Some Characteristics Between Phragmites communis and Its Salt Tolerant Variant

R5002-12, a salt tolerant line of Phragmites communis Trin., which was obtained from ethyl methane sulfonate (EMS) treated callus selected under saline stress, was compared with its wild line in respect to their molecular biological, physiological and biochemical characterizations. Five arbitrary primers were screened which showed differences in DNA amplified polymorphism between the variant and its wild line. Some new proteins appeared in the salt tolerant plant under salt stress. Electrophoresis of peroxidase and esterase also showed some differences in isozyme expression between them. The chlorophyll content of the variant was higher than that of the original variety, whether the plants were under salt stress or not.     

Predicting potentially pathogenic effects of hRPE65 missense mutations: a computational strategy based on molecular dynamics simulations

The human retinal pigment epithelium-specific 65-kDa protein (hRPE65) plays a crucial role within the retinoid visual cycle and several mutations affecting either its expression level or its enzymatic function are associated with inherited retinal diseases such as Retinitis Pigmentosa. The gene therapy product voretigene neparvovec (Luxturna) has been recently approved for treating hereditary retinal dystrophies; however, the treatment is currently accessible only to patients presenting confirmed biallelic mutations that severely impair hRPE65 function, and many reported hRPE65 missense mutations lack sufficient evidences for proving their pathogenicity. In this context, we developed a computational approach aimed at evaluating the potential pathogenic effect of hRPE65 missense variants located on the dimerisation domain of the protein. The protocol evaluates how mutations may affect folding and conformation stability of this protein region, potentially helping clinicians to evaluate the eligibility for gene therapy of patients diagnosed with this type of hRPE65 variant of uncertain significance.