Detection of Novel BEST1 Variations in Autosomal Recessive Bestrophinopathy Using Third-generation Sequencing

Objective:Autosomal recessive bestrophinopathy(ARB),a retinal degenerative disease,is characterized by central visual loss,yellowish multifocal diffuse subretinal deposits,and a dramatic decrease in the light peak on electrooculogram.The potential pathogenic mechanism involves mutations in the BEST1 gene,which encodes Ca2+-activated Cl−channels in the retinal pigment epithelium(RPE),resulting in degeneration of RPE and photoreceptor.In this study,the complete clinical characteristics of two Chinese ARB families were summarized.Methods:Pacific Biosciences(PacBio)single-molecule real-time(SMRT)sequencing was performed on the probands to screen for disease-causing gene mutations,and Sanger sequencing was applied to validate variants in the patients and their family members.Results:Two novel mutations,c.202T>C(chr11:61722628,p.Y68H)and c.867+97G>A,in the BEST1 gene were identified in the two Chinese ARB families.The novel missense mutation BEST1 c.202T>C(p.Y68H)resulted in the substitution of tyrosine with histidine in the N-terminal region of transmembrane domain 2 of bestrophin-1.Another novel variant,BEST1 c.867+97G>A(chr11:61725867),located in intron 7,might be considered a regulatory variant that changes allele-specific binding affinity based on motifs of important transcriptional regulators.Conclusion:Our findings represent the first use of third-generation sequencing(TGS)to identify novel BEST1 mutations in patients with ARB,indicating that TGS can be a more accurate and efficient tool for identifying mutations in specific genes.The novel variants identified further broaden the mutation spectrum of BEST1 in the Chinese population.

Innovation and development of the third-generation hybrid rice technology

The breeding and large-scale application of hybrid rice contribute significantly to the food supply worldwide.Currently,hybrid seed production uses cytoplasmic male sterile(CMS)lines or photoperiod/thermo-sensitive genic male sterile(PTGMS)lines as female parent.Despite huge successes,both systems have intrinsic problems.CMS systems are mainly restricted by the narrow restorer resources that make it difficult to breed superior hybrids,while PTGMS systems are limited by conditional sterility of the male sterile lines that makes the propagation of both PTGMS seeds and hybrid seeds vulnerable to unpredictable climate changes.Recessive nuclear male sterile(NMS)lines insensitive to environmental conditions are widely distributed and are ideal for hybrid rice breeding and production,but the lack of effective ways to propagate the pure NMS lines in a large scale renders it impossible to use them for hybrid rice production.The development of"the third-generation hybrid rice technology"enables efficient propagation of the pure NMS lines in commercial scale.This paper discusses the establishment of"the thirdgeneration hybrid rice technology"and further innovations.This new technology breaks the limitations of CMS and PTGMS systems and will bring a big leap forward in hybrid rice production.

Applications of sediment sudden deposition model based on the third-generation numerical model for shallow water wave

The existing numerical models for nearshore waves are briefly introduced, and the third-generation numerical model for shallow water wave, which makes use of the most advanced productions of wave research and has been adapted well to be used in the environment of seacoast, lake and estuary area, is particularly discussed. The applied model realizes the significant wave height distribution at different wind directions. To integrate the model into the coastal area sediment, sudden deposition mechanism, the distribution of average silt content and the change of sediment sudden deposition thickness over time in the nearshore area are simulated. The academic productions can give some theoretical guidance to the applications of sediment sudden deposition mechanism for stormy waves in the coastal area. And the advancing directions of sediment sudden deposition model are prospected.

Recent progress in third-generation low alloy steels developed under M^3 microstructure control

During the past thirty years, two generations of low alloy steels(ferrite/pearlite followed by bainite/martensite) have been developed and widely used in structural applications. The third-generation of low alloy steels is expected to achieve high strength and improved ductility and toughness, while satisfying the new demands for weight reduction, greenness, and safety. This paper reviews recent progress in the development of third-generation low alloy steels with an M^3 microstructure, namely, microstructures with multi-phase, meta-stable austenite, and multi-scale precipitates. The review summarizes the alloy designs and processing routes of microstructure control, and the mechanical properties of the alloys.The stabilization of retained austenite in low alloy steels is especially emphasized. Multi-scale nano-precipitates, including carbides of microalloying elements and Cu-rich precipitates obtained in third-generation low alloy steels, are then introduced. The structure–property relationships of third-generation alloys are also discussed. Finally, the promises and challenges to future applications are explored.

Taking early preventive interventions to manage the challenging issue of acquired resistance to third-generation EGFR inhibitors

Although the clinical efficacies of third-generation epidermal growth factor receptor(EGFR)-tyrosine kinase in-hibitors(TKIs)such as osimertinib in the treatment of non-small cell lung cancer(NSCLC)with EGFR-activating mutations are promising,drug-acquired resistance inevitably occurs whether they are used as first-line or second-line treatment.Therefore,managing the acquired resistance to third-generation EGFR-TKIs is crucial in the clinic for improving patient survival.Great efforts have been made to develop potentially effective strategies or regimens for the treatment of EGFR-mutant NSCLC patients after relapse following these TKIs therapies with the hope that patients will continue to benefit from treatment through overcoming acquired resistance.Although this approach,which aims to overcome drug-acquired resistance,is necessary and important,it is a passive practice.Taking pre-ventive action early before disease progression to manage the unavoidable development of acquired resistance offers an equally important and efficient approach.We strongly believe that early preventive interventions using effective and tolerable combination regimens that interfere with the process of developing acquired resistance may substantially improve the outcomes of EGFR-mutant NSCLC treatment with third-generation EGFR-TKIs.Thus,this review focuses on discussing the scientific rationale and mechanism-driven strategies for delaying and even preventing the emergence of acquired resistance to third-generation EGFR-TKIs,particularly osimertinib.

Development of the “Third-Generation” Hybrid Rice in China

Rice is a major cereal crop for China. The development of the ‘‘three-line" hybrid rice system based on cytoplasmic male sterility in the 1970 s(first-generation) and the ‘‘two-line" hybrid rice system based on photoperiod-and thermo-sensitive genic male-sterile lines(second-generation)in the 1980 s has contributed significantly to rice yield increase and food security in China. Here we describe the development and implementation of the ‘‘third-generation" hybrid rice breeding system that is based on a transgenic approach to propagate and utilize stable recessive nuclear male sterile lines, and as such, the male sterile line and hybrid rice produced using such a system is nontransgenic. Such a system should overcome the intrinsic problems of the ‘‘first-generation" and‘‘second-generation" hybrid rice systems and hold great promise to further boost production of hybrid rice and other crops.

The spatiotemporal evolution of EGFR C797S mutation in EGFR-mutant non-small cell lung cancer:opportunities for third-generation EGFR inhibitors re-challenge

Third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have demonstrated impressive activity in EGFR T790M-positive non-small cell lung cancer (NSCLC) progressed from prior EGFR-TKIs (1,2)However, resistance inevitably occurred after approximately 10 months of treatment.

Thoughts on the Development of Bridge Technology in China

In the history of bridge engineering, demand has always been the primary driving force for development. Driven by the huge demand for construction since China’s reform and opening-up, Chinese bridge has leapt forward both quantitatively and qualitatively in three major stages, by completing the transition from “follower” to “competitor,” and nally to “leader.” A new future is emerging for Chinese bridge engi- neering. As an important part of China’s transportation infrastructure, the bridge engineering industry is facing challenges in this new era on how to support the construction of a new form of transportation. This paper provides a summary of the status of bridge technology in China, based on a basic analysis of stock demand, incremental demand, and management demand. It is our belief that the Chinese bridge engi- neering industry must ful ll three outstanding requirements: construction ef ciency, management effec- tiveness, and long-term service. Intelligent technology based on information technology provides a new opportunity for innovation in bridge engineering. As a result, the development path of bridge engineering needs to be changed. This paper puts forward the idea of developing a third-generation bridge project that is characterized by intelligence, and discusses this project’s implications, development focus, and plan. In this way, this work provides a direction for the improvement of the core competitiveness of China’s bridge engineering industry.

Third Generation Horseradish Peroxidase Biosensor Based on Self-assembling Carbon Nanotubes to Gold Electrode Surface

A third-generation horseradish peroxidase (HRP) biosensor has been developed by adsorbing HRP on multi-wall carbon nanotube (MWNTs) monolayer modified gold electrode surface. The assembly process was investigated by electrochemical and spectroscopic techniques. Results showed that the immobilized HRP exhibited direct electrochemical behavior toward the reduction of H2O2. The resulting biosensor shows a fast amperometric response (<2 s) to H2O2. The linear response range was from 5.0×10-7～1.0×10-5 mol/L with a detection limit of 1.0×10-7mol/L. Moreover, the biosensor has a good reproducibility, and long-term stability.

Rapid identification of full-length genome and tracing variations of monkeypox virus in clinical specimens based on mNGS and amplicon sequencing

The monkeypox virus(MPXV)has triggered a current outbreak globally.Genome sequencing of MPXV and rapid tracing of genetic variants will benefit disease diagnosis and control.It is a significant challenge but necessary to optimize the strategy and application of rapid full-length genome identification and to track variations of MPXV in clinical specimens with low viral loads,as it is one of the DNA viruses with the largest genome and the most AT-biased,and has a significant number of tandem repeats.Here we evaluated the performance of metagenomic and amplicon sequencing techniques,and three sequencing platforms in MPXV genome sequencing based on multiple clinical specimens of five mpox cases in Chinese mainland.We rapidly identified the full-length genome of MPXV with the assembly of accurate tandem repeats in multiple clinical specimens.Amplicon sequencing enables cost-effective and rapid sequencing of clinical specimens to obtain high-quality MPXV genomes.Third-generation sequencing facilitates the assembly of the terminal tandem repeat regions in the monkeypox virus genome and corrects a common misassembly in published sequences.Besides,several intra-host single nucleotide variations were identified in the first imported mpox case.This study offers an evaluation of various strategies aimed at identifying the complete genome of MPXV in clinical specimens.The findings of this study will significantly enhance the surveillance of MPXV.

PacBio Sequencing and Its Applications

Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing （SGS） technologies, making it well-suited for unsolved problems in genome, transcriptome, and epigenetics research. The highly-contiguous de novo assemblies using PacBio sequencing can close gaps in current reference assemblies and characterize structural variation （SV） in personal genomes. With longer reads, we can sequence through extended repetitive regions and detect mutations, many of which are associated with dis- eases. Moreover, PacBio transcriptome sequencing is advantageous for the identification of gene isoforms and facilitates reliable discoveries of novel genes and novel isoforms of annotated genes, due to its ability to sequence full-length transcripts or fragments with significant lengths. Addition- ally, PacBio＇s sequencing technique provides information that is useful for the direct detection of base modifications, such as methylation. In addition to using PacBio sequencing alone, many hybrid sequencing strategies have been developed to make use of more accurate short reads in conjunction with PacBio long reads. In general, hybrid sequencing strategies are more affordable and scalable especially for small-size laboratories than using PacBio Sequencing alone. The advent of PacBio sequencing has made available much information that could not be obtained via SGS alone.

Oxford Nanopore MinION Sequencing and Genome Assembly

The revolution of genome sequencing is continuing after the successful secondgeneration sequencing （SGS） technology. The third-generation sequencing （TGS） technology, led by Pacific Biosciences （PacBio）, is progressing rapidly, moving from a technology once only capable of providing data for small genome analysis, or for performing targeted screening, to one that promises high quality de novo assembly and structural variation detection for human-sized genomes. In 2014, the MinION, the first commercial sequencer using nanopore technology, was released by Oxford Nanopore Technologies （ONT）. MiniON identifies DNA bases by measuring the changes in electrical conductivity generated as DNA strands pass through a biological pore. Its portability, affordability, and speed in data production makes it suitable for real-time applications, the release of the long read sequencer MiniON has thus generated much excitement and interest in the genomics community. While de novo genome assemblies can be cheaply produced from SGS data, assem- bly continuity is often relatively poor, due to the limited ability of short reads to handle long repeats. Assembly quality can be greatly improved by using TGS long reads, since repetitive regions can be easily expanded into using longer sequencing lengths, despite having higher error rates at the base level. The potential of nanopore sequencing has been demonstrated by various studies in genome surveillance at locations where rapid and reliable sequencing is needed, but where resources are limited.

Correlation analysis between underwater noise and Liutex for DTMB4119 propeller

Propeller is an important equipment of ocean structures.It has characteristics of complex geometry and periodic vortex shed,which means high research value.This paper takes DTMB419 propeller as the research object.A detailed study is conducted on the wake vortices in non-cavitation state and cavitation state.Three vortex identification methods are used to compare the vortices capture effects from downstream sections and three-dimensional perspectives.The results show that,the third-generation vortex identification technologyΩ_(R)shows obvious advantages.At the same time,FW-H equation is used to predict propeller noise,and the relationship between nonlinear sound source distribution and vortices distribution is analyzed.It is found that the distribution of Lighthill tensor is consistent withΩ_(R),which proves the advantages and potential of third-generation vortex identification technology from acoustic perspective.

Recent advances in single-cell sequencing technologies

Single-cell omics sequencingwas first achieved for the transcriptome in 2009,whichwas followed by fast development of technologies for profiling the genome,DNA methylome,3D genome architecture,chromatin accessibility,histone modifications,etc.,in an individual cell.In this review we mainly focus on the recent progress in four topics in the single-cell omics field:single-cell epigenome sequencing,single-cell genome sequencing for lineage tracing,spatially resolved single-cell transcriptomics and third-generation sequencing platform-based single-cell omics sequencing.We also discuss the potential applications and future directions of these single-cell omics sequencing technologies for different biomedical systems,especially for the human stem cell field.

Screening of probiotics with efficient α-glucosidase inhibitory ability and study on the structure and function of its extracellular polysaccharide

Inhibition ofα-glucosidase activity is an important strategy in lowering the concentration of blood sugar.In this paper,using domestic and foreign characteristic food-derived substances as the sources of lactobacillus,the performance of them were evaluated by measuring the strains’α-glucosidase inhibitory ability.Finally,the cell-free extracellular supernatants(CFS)of Lactobacillus rhamnosus LB1lac10 was determined to have the highest α-glucosidase inhibition ability.Based on the Nanopore third-generation sequencing technology platform,the genome of LB1lac10 was sequenced and functional gene annotation was performed.After that,the biological activity and structural composition of the exopolysaccharide produced by L.rhamnosus LB1lac10 were studied.The purified exopolysaccharide EPS1-1 also showed efficientα-glucosidase inhibitory ability.The structure and conformation characteristics of EPS1-1 were further analyzed.The EPS1-1 from L.rhamnosus LB1lac10 had a molecular weight of 88,650 Da,and it was mainly composed of mannose,glucuronic acid,glucose,xylose,galactose,and arabinose.From the FT-IR and NMR analyses,EPS1-1 had functional groups of a typical polysaccharide structure and contained two types of glycosidic bonds with α-configuration pyranose.The main glycosidic bond corresponded to→4)-α-D-Glcp-(1→,which might be an important reason why EPS1-1 could inhibit α-glycosidase.Thermodynamic studies showed that EPS1-1 had high heat resistance to meet the needs of food processing.The results suggested that L.rhamnosus LB1lac10 could be used as a potential probiotic to lower blood sugar,and the EPS1-1 has the potential to serve as a naturalα-glycosidase inhibitor to regulate the concentration of blood glucose.