A novel workflow to improve genotyping of multigene families in wildlife species: An experimental set-up with a known model system.
- 作者列表："Gillingham MAF","Montero BK","Wihelm K","Grudzus K","Sommer S","Santos PSC
:Genotyping complex multigene families in novel systems is particularly challenging. Target primers frequently amplify simultaneously multiple loci leading to high PCR and sequencing artefacts such as chimeras and allele amplification bias. Most genotyping pipelines have been validated in nonmodel systems whereby the real genotype is unknown and the generation of artefacts may be highly repeatable. Further hindering accurate genotyping, the relationship between artefacts and genotype complexity (i.e. number of alleles per genotype) within a PCR remains poorly described. Here, we investigated the latter by experimentally combining multiple known major histocompatibility complex (MHC) haplotypes of a model organism (chicken, Gallus gallus, 43 artificial genotypes with 2-13 alleles per amplicon). In addition to well-defined 'optimal' primers, we simulated a nonmodel species situation by designing 'cross-species' primers based on sequence data from closely related Galliform species. We applied a novel open-source genotyping pipeline (ACACIA; https://gitlab.com/psc_santos/ACACIA), and compared its performance with another, previously published pipeline (AmpliSAS). Allele calling accuracy was higher when using ACACIA (98.5% versus 97% and 77.8% versus 75% for the 'optimal' and 'cross-species' data sets, respectively). Systematic allele dropout of three alleles owing to primer mismatch in the 'cross-species' data set explained high allele calling repeatability (100% when using ACACIA) despite low accuracy, demonstrating that repeatability can be misleading when evaluating genotyping workflows. Genotype complexity was positively associated with nonchimeric artefacts, chimeric artefacts (nonlinearly by levelling when amplifying more than 4-6 alleles) and allele amplification bias. Our study exemplifies and demonstrates pitfalls researchers should avoid to reliably genotype complex multigene families.
: 新系统中复杂多基因家族的基因分型尤其具有挑战性。靶引物经常同时扩增多个位点，导致高PCR和测序伪影，如嵌合体和等位基因扩增偏倚。大多数基因分型管道已经在非模型系统中得到验证，其中真正的基因型是未知的，并且伪影的产生可以是高度可重复的。进一步阻碍准确的基因分型，人工制品和基因型复杂性之间的关系 (即PCR中每个基因型的等位基因数) 仍然描述不足。在这里，我们通过实验结合模型生物的多个已知的主要组织相容性复合体 (MHC) 单倍型 (鸡，Gallus gallus，43个人工基因型，每个扩增子有2-13个等位基因) 来研究后者。除了定义明确的 “最佳” 引物之外，我们还通过基于来自密切相关的alli形物种的序列数据设计 “交叉物种” 引物来模拟非模型物种情况。我们应用了一个新的开源基因分型管道 (ACACIA; https://gitlab.com/psc_santos/ACACIA)，并将其性能与另一个先前发表的管道 (AmpliSAS) 进行了比较。当使用阿拉伯胶时，等位基因识别准确度更高 (对于 “最佳” 和 “杂交物种” 数据集，分别为98.5% 对97% 和77.8% 对75%)。由于 '交叉物种' 数据集中的引物错配导致的三个等位基因的系统性等位基因缺失解释了高等位基因调用可重复性 (当使用阿拉伯胶时为100%)，尽管准确性低，这表明可重复性在评估基因分型工作流程时可能具有误导性。基因型复杂性与非嵌合artefacts、嵌合artefacts (当扩增超过4-6个等位基因时，通过水平非线性) 和等位基因扩增偏倚呈正相关。我们的研究举例说明并证明了研究人员应该避免的陷阱，以可靠地对复杂的多基因家族进行基因型分型。
METHODS::The ATP binding-cassette superfamily corresponds the mostly transmembrane transporters family found in humans. These proteins actively transport endogenous and exogenous substrates through biological membranes in body tissues, so they have an important role in the regulation of many physiological functions necessary for human homeostasis, as well as in response regulation to several pharmacological substrates. The development of multidrug resistance has become one of the main troubles in conventional chemotherapy in different illnesses including cancer, being the increased efflux of antineoplastic drugs the main reason for this multidrug resistance, with a key role of the ABC superfamily. Likely, the interindividual variability in the pharmacological response among patients is well known, and may be due to intrinsically factors of the disease, genetic and environmental ones. Thus, the understanding of this variability, especially the genetic variability associated with the efficacy and toxicity of drugs, can provide a safer and more effective pharmacological treatment, so ABC genes are considered as important regulators due to their relationship with the reduction in pharmacological response. In this review, updated information about transporters belonging to this superfamily was collected, the possible role of these transporters in cancer, the role of genetic variability in their genes, as well as some therapeutic tools that have been tried to raise against main transporters associated with chemoresistance in cancer.
METHODS:BACKGROUND:Cholinergic neurotransmission regulates neuroinflammation in Parkinson disease (PD). RESEARCH DESIGN AND METHODS:The authors conducted a delayed-start study of donepezil for cognitive decline in non-demented PD patients. The study consisted of a 96-week randomized placebo-controlled double-blind phase 1, followed by a 24-week donepezil extension phase 2. The primary outcome measure was a change in the Mini-Mental State Examination (MMSE) at week 120. RESULTS:A total of 98 patients were randomly allocated to the early-start (donepezil-to-donepezil) and delayed-start (placebo-to-donepezil) groups. Mean (SD) of the baseline MMSE was 27.6 (2.0) and 28.0 (2.1), respectively. MMSE change at week 120 was better in the early-start group than in the delayed-start group, but the difference was not significant. The MMSE declined in apolipoprotein ε4 carriers, but not in non-carriers, and the factor interaction (intervention × ε4 genotype) was highly significant (P < 0.001). Analyzed with the interaction, the difference was significant (group difference 1.95 [0.33 to 3.57], P = 0.018). The MMSE decline slope in phase 1 was significantly better in the early-start group than in the delayed-start group (P = 0.048). CONCLUSIONS:Cognitive function deteriorated in ε4 carriers, but not in non-carriers, and early-start donepezil may postpone cognitive decline in the former.
METHODS::Since the discovery of dental pulp stem cells, a lot of teams have expressed an interest in dental pulp regeneration. Many approaches, experimental models and biological explorations have been developed, each including the use of stem cells and scaffolds with the final goal being clinical application in humans. In this review, the authors' objective was to compare the experimental models and strategies used for the development of biomaterials for tissue engineering of dental pulp with stem cells. Electronic queries were conducted on PubMed using the following terms: pulp regeneration, scaffold, stem cells, tissue engineering and biomaterial. The extracted data included the following information: the strategy envisaged, the type of stem cells, the experimental models, the exploration or analysis methods, the cytotoxicity or viability or proliferation cellular tests, the tests of scaffold antibacterial properties and take into account the vascularization of the regenerated dental pulp. From the 71 selected articles, 59% focused on the "cell-transplantation" strategy, 82% used in vitro experimentation, 58% in vivo animal models and only one described an in vivo in situ human clinical study. 87% used dental pulp stem cells. A majority of the studies reported histology (75%) and immunohistochemistry explorations (66%). 73% mentioned the use of cytotoxicity, proliferation or viability tests. 48% took vascularization into account but only 6% studied the antibacterial properties of the scaffolds. This article gives an overview of the methods used to regenerate dental pulp from stem cells and should help researchers create the best development strategies for research in this field.