Functional Allele Validation by Gene Editing to Leverage the Wealth of Genetic Resources for Crop Improvement

Advances in molecular technologies over the past few decades, such as high-throughput DNA marker genotyping, have provided more powerful plant breeding approaches, including marker-assisted selection and genomic selection. At the same time, massive investments in plant genetics and genomics, led by whole genome sequencing, have led to greater knowledge of genes and genetic pathways across plant genomes. However, there remains a gap between approaches focused on forward genetics, which start with a phenotype to map a mutant locus or QTL with the goal of cloning the causal gene, and approaches using reverse genetics, which start with large-scale sequence data and work back to the gene function. The recent establishment of efficient CRISPR-Cas-based gene editing promises to bridge this gap and provide a rapid method to functionally validate genes and alleles identified through studies of natural variation. CRISPR-Cas techniques can be used to knock out single or multiple genes, precisely modify genes through base and prime editing, and replace alleles. Moreover, technologies such as protoplast isolation, in planta transformation, and the use of developmental regulatory genes promise to enable high-throughput gene editing to accelerate crop improvement.     

Nanopore Sequencing for De Novo Bacterial Genome Assembly and Search for Single-Nucleotide Polymorphism

Nanopore sequencing (ONT) is a new and rapidly developing method for determining nucleotide sequences in DNA and RNA. It serves the ability to obtain long reads of thousands of nucleotides without assembly and amplification during sequencing compared to next-generation sequencing. Nanopore sequencing can help for determination of genetic changes leading to antibiotics resistance. This study presents the application of ONT technology in the assembly of an E. coli genome characterized by a deletion of the tolC gene and known single-nucleotide variations leading to antibiotic resistance, in the absence of a reference genome. We performed benchmark studies to determine minimum coverage depth to obtain a complete genome, depending on the quality of the ONT data. A comparison of existing programs was carried out. It was shown that the Flye program demonstrates plausible assembly results relative to others (Shasta, Canu, and Necat). The required coverage depth for successful assembly strongly depends on the size of reads. When using high-quality samples with an average read length of 8 Kbp or more, the coverage depth of 30× is sufficient to assemble the complete genome de novo and reliably determine single-nucleotide variations in it. For samples with shorter reads with mean lengths of 2 Kbp, a higher coverage depth of 50× is required. Avoiding of mechanical mixing is obligatory for samples preparation. Nanopore sequencing can be used alone to determine antibiotics-resistant genetic features of bacterial strains.     

Camenisch群签名方案的改进和成员废除

如何缩短群签名的长度以及如何安全有效废除群成员是阻碍群签名走向实用的两个主要问题．该文对Camenisch群签名方案进行改进，通过缩短其所用知识签名的长度，达到缩短签名长度的目的，所提出的改进方案使签名由原来的2n 4元组缩短为n 5元组，长度缩短了近一半．同时利用公钥状态列表和可信时戳提出一个前向安全的高效群成员废除方案，这个方案的提出纠正了不能用证书撤销列表废除群成员的观点．该文还考虑了后加入成员的超前签名问题，所提出的成员废除方案能防止超前签名．     

优先队列的并行插入和删除

优先队列广泛地使用在许多并行算法中(例如,多处理机调度和某些组合优化算法)。在这些算法中,共享优先队列的存取冲突限制了加速比的提高。本文提出一种链表优先队列的并行插入和删除方法,具有较小并行开销和较大的并行度,并且保证和串行存取算法的优先顺序完全一致,即删除操作返回已经插入和正在插入的所有元素中的最佳元素。同时,我们还介绍了目前性能最好的堆的并行插入和删除算法,并对准和链表结构并行插入和删除算法的性能和适用范围进行了比较,进一步提出了散列结构的优先队列。在ENCORE Multimax520多处理机上的实验结果验证了我们的理论分析结果:使用链表结构的并行分枝限界算法性能上可获得很大提高。     

Linear-time algorithms for eliminating claws in graphs

Since many -complete graph problems are polynomial-time solvable when restricted to claw-free graphs, we study the problem of determining the distance of a given graph to a claw-free graph, considering vertex elimination a measure. Claw-free Vertex Deletion (CFVD) consists of determining the minimum number of vertices to be removed from a graph such that the resulting graph is claw-free. Although CFVD is -hard in general and recognizing claw-free graphs is still a challenge, where the current best deterministic algorithm for a graph G consists of performing executions of the best algorithm for matrix multiplication, we present linear-time algorithms for CFVD on weighted block graphs and weighted graphs with bounded treewidth. Furthermore, we show that this problem on forests can be solved in linear time by a simpler algorithm, and we determine the exact values for full k-ary trees. On the other hand, we show that CFVD is -hard even when the input graph is a split graph. We also show that the problem is hard to be approximated within any constant factor better than 2, assuming the unique games conjecture.     

Membrane Anchorage-Induced (MAGIC) Knockdown of Non-synonymous Point Mutations**

The promise of personalized medicine for monogenic and complex polygenic diseases depends on the availability of strategies for targeted inhibition of disease-associated polymorphic protein variants. Loss of function variants, including non-synonymous single nucleotide variants (nsSNVs) and insertion/deletion producing a frameshift, account for the vast majority of disease-related genetic changes. Because it is challenging to interpret the functional consequences of nsSNVs, they are considered a big barrier for personalized medicine. A method for inhibiting the specific expression of nsSNVs without editing the human genome will facilitate the elucidation of the biology of nsSNVs, but such a method is currently lacking. Here, I describe the phenomenon of membrane anchorage-induced (MAGIC) knockdown of allele-specific inhibition of protein and mRNA expression upon inner membrane tethering of point mutation-specific monoclonal antibodies (mAb). This phenomenon is likely mediated by a mechanism distinct from the protein degradation pathways, as the epitope-specific knockdown is replicated upon intracellular expression of a membrane-anchored single domain intrabody that lacks the Fc domain of the mAb. By harnessing the MAGIC knockdown of epitope-containing protein targets, I report a novel approach for inhibiting the expression of amino-acid-altering germline and somatic nsSNVs. As a proof-of-concept, I show the inhibition of human disease-associated variants namely, FGFR4 p.G388R, KRAS p.G12D and BRAF p.V600E protein variants. This method opens up a new avenue for not just therapeutic suppression of undruggable protein variants, but also for functional interrogation of the nsSNVs of unknown significance.     

基于高通量显微成像及分析技术的 DNA 重排研究

直接的重复序列广泛地存在于真核和原核细胞基因组中，并且与多种疾病（如遗传性神经肌 肉神经退行性疾病等）相关，因此定量重复序列的删除变得非常重要。结合高通量显微成像和分析技术，该文设计了基于三色荧光报告系统的方法来定量重复序列删除的发生。结果显示，在铜绿假单胞菌中，重复序列的删除频率在 recA 基因缺失突变株中明显降低，而 RadA 蛋白和 UvrD 蛋白的缺失则会提高重复序列的删除频率，并且重复序列的删除与细菌的生长率和启动子等因素无关。该研究有助于加深对直接重复序列相关问题的理解，并为直接重复序列删除定量提供了新的方法。     

Deletion and Recovery Scheme of Electronic Health Records Based on Medical Certificate Blockchain

The trusted sharing of Electronic Health Records (EHRs) can realize the efficient use of medical data resources. Generally speaking, EHRs are widely used in blockchain-based medical data platforms. EHRs are valuable private assets of patients, and the ownership belongs to patients. While recent research has shown that patients can freely and effectively delete the EHRs stored in hospitals, it does not address the challenge of record sharing when patients revisit doctors. In order to solve this problem, this paper proposes a deletion and recovery scheme of EHRs based on Medical Certificate Blockchain. This paper uses cross-chain technology to connect the Medical Certificate Blockchain and the Hospital Blockchain to realize the recovery of deleted EHRs. At the same time, this paper uses the Medical Certificate Blockchain and the InterPlanetary File System (IPFS) to store Personal Health Records, which are generated by patients visiting different medical institutions. In addition, this paper also combines digital watermarking technology to ensure the authenticity of the restored electronic medical records. Under the combined effect of blockchain technology and digital watermarking, our proposal will not be affected by any other rights throughout the process. System analysis and security analysis illustrate the completeness and feasibility of the scheme.     

Relation between αS1-casein,genotype, and quality traits of milk from Swedish dairy goats

Locally produced food is becoming popular among Swedish consumers. One product that has increased in popularity is artisan-manufactured goat cheese, and although the dairy goat industry in Sweden is small-scale, production is gradually increasing. In goats, the CSN1S1 gene regulates expression of the protein α_S1-casein (α_S1-CN), which has been found to be important for cheese yield. Over the years, breeding animals have been imported to Sweden from Norway. Historically, a high frequency of the Norwegian goat population carried a polymorphism at the CSN1S1 gene. This polymorphism, called the Norwegian null allele (D), leads to zero or significantly reduced expression of α_S1-CN. Using milk samples from 75 goats, this study investigated associations between expression of α_S1-CN and genotype at the CSN1S1 gene on milk quality traits from Swedish Landrace goats. Milk samples were grouped according to relative level of α_S1-CN (low: 0–6.9% of total protein; medium-high: 7–25% of total protein) and genotype (DD, DG, DA/AG/AA). While the D allele leads to extremely low expression of α_S1-CN, the G allele is low expressing and the A allele is highly expressing for this protein. Principal component analysis was used to explore the total variation in milk quality traits. To evaluate the effect of different allele groups on milk quality attributes, 1-way ANOVA and Tukey pairwise comparison tests were used. The majority (72%) of all goat milk samples investigated showed relative α_S1-CN content of 0% to 6.82% of total protein. The frequency of individuals homozygous for the Norwegian null allele (DD) was 59% in the population of sampled goats, and only 15% carried at least one A allele. A low relative concentration of α_S1-CN was associated with lower total protein, higher pH, and higher relative concentration of β-casein and levels of free fatty acids. Milk from goats homozygous for the null allele (DD) showed a similar pattern as milk with low relative concentration of α_S1-CN, but total protein was only numerically lower, and somatic cell count and α_S2-CN were higher than for the other genotypes. The associations between levels of α_S1-CN and the investigated genotype at the CSN1S1 gene indicate a need for a national breeding program for Swedish dairy goats.