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.

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.

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.

The Influences of American Deep Image on the Third-Generation Poets in China

The Third-Generation Poetry of China （namely Post-misty Poetry too） initiated with the introduction of Western modernist poetry, especially sorts of American Post-modernist poetry schools into China. ＂The relation between American poetry and Chinese poetry has a long history, which lies in the influences on the creation of the Third-Generation poets. This influence is probably unprecedented in its depth and breadth.＂ ＂Irrational association＂ and ＂leaping images＂ proposed by American Deep Image poets influenced by Freudian and Jungian unconscious perception gained an extraordinary appreciation among the Third-Generation poets who were in pursuit constantly of the experiments on poetic form and language. This paper mainly discusses the influences of American Deep Image on the Third-Generation poets of China through a case study of WANG Yin and CHEN Dongdong＇s poems.

Development of the Mechanical System on a Third-Generation Industrial Computed Tomography Scanner in Brazil

The development of measurement geometry for medical X-ray computed tomography （CT） scanners was carried out from the first to the fourth-generation. This concept has also been applied for imaging of industrial processes such as pipe flows or for improving design, operation, optimization and troubleshooting. Nowadays, gamma CT permits to visualize failure equipment points in three-dimensional analysis and in sections of chemical and petrochemical industries. The aim of this work is the development of the mechanical system on a third-generation industrial CT scanner to analyze laboratorial process columns which perform highly efficient separation, turning the ^6oCo, ^75Se, ^137Cs and/or ^192Ir sealed gamma-ray source（s） and the NaI（Tl） multidetector array. It also has a translation movement along the column axis to obtain as many slices of the process flow as needed. The mechanical assembly for this third-generation industrial CT scanner is comprised by strength and rigidity structural frame in stainless and carbon steels, rotating table, source shield and collimator with pneumatic exposure system, spur gear system, translator, rotary stage, drives and stepper motors. The use of suitable spur gears has given a good repeatability and high accuracy in the degree of veracity. The data acquisition boards, mechanical control interfaces, software for movement control and image reconstruction were specially development. A multiphase phantom capable to be setting with solid, liquid and gas was testing. The scanner was setting for 90 views and 19 projections for each detector totalizing 11,970 projections. Experiments to determine the linear attenuation coefficients of the phantom were carried out which applied the Lambert-Beer principle. Results showed that it was possible to distinguish between the phases even the polymethylmethacrylate and the water have very similar density and linear attenuation coefficients. It was established that the newly developed third-generation fan-beam arrangement gamma scanner unit has a good spatial resolution acceptable given the size of the used phantom in this study. The tomografic reconstruction algorithm in used 60 ~ 60 pixels images was the Alternative Minimization （AM） technique and was implemented in MATLAB and VB platforms. The mechanical system presented a good performance in terms of strength, rigidity, accuracy and repeatability with great potential to be used for education or program which dedicated to training chemical and petrochemical industry professionals and for industrial process optimization in Brazil.

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.

Diagnosis and treatment of refractory infectious diseases using nanopore sequencing technology:Three case reports

BACKGROUND Infectious diseases are still one of the greatest threats to human health,and the etiology of 20%of cases of clinical fever is unknown;therefore,rapid identification of pathogens is highly important.Traditional culture methods are only able to detect a limited number of pathogens and are time-consuming;serologic detection has window periods,false-positive and false-negative problems;and nucleic acid molecular detection methods can detect several known pathogens only once.Three-generation nanopore sequencing technology provides new options for identifying pathogens.CASE SUMMARY Case 1:The patient was admitted to the hospital with abdominal pain for three days and cessation of defecation for five days,accompanied by cough and sputum.Nanopore sequencing of the drainage fluid revealed the presence of orallike bacteria,leading to a clinical diagnosis of bronchopleural fistula.Cefoperazone sodium sulbactam treatment was effective.Case 2:The patient was admitted to the hospital with fever and headache,and CT revealed lung inflammation.Antibiotic treatment for Streptococcus pneumoniae,identified through nanopore sequencing of cerebrospinal fluid,was effective.Case 3:The patient was admitted to our hospital with intermittent fever and an enlarged neck mass that had persisted for more than six months.Despite antibacterial treatment,her symptoms worsened.The nanopore sequencing results indicate that voriconazole treatment is effective for Aspergillus brookii.The patient was diagnosed with mixed cell type classical Hodgkin's lymphoma with infection.CONCLUSION Three-generation nanopore sequencing technology allows for rapid and accurate detection of pathogens in human infectious diseases.

Effects of Compound Probiotics on Growth Performance and Intestinal Flora of Kunming Mice

[Objectives]This study was conducted to investigate the effects of adding compound probiotics on the growth performance and intestinal flora of Kunming mice.[Methods]Twelve healthy 2-week-old Kunming male mice with body weight of(11.09±0.43)g were selected.They were randomly divided into two treatment groups,namely blank control group(NC)and compound probiotics group(CB+LR+BS),with six mice in each group.The two groups were fed with commercial basal diet,and the compound probiotic experimental group was fed with basal diet supplemented with compound probiotics,in which the contents of Clostridium butyricum spores,Lactobacillus reuteri and Bacillus subtilis spores were 1×1010,1×1011 and 1×1010 CUF/kg,respectively.The body weight,feed intake and water intake of mice were counted every 4 d,and the experimental period was 13 d.On the 13 th day,the cecal contents of the mice were collected for analysis.[Results]There was no significant change in body weight and feed intake when compound probiotics were added to the diet.However,the addition of compound probiotics reduced the abundance of harmful bacteria such as Escherichia coli,urease-negative Helicobacter typhlonius and Salmonella enterica,while increasing the abundance of beneficial bacteria such as Anaerostipes hadrus,and the contents of IgG and IgM increased significantly(P<0.05).[Conclusions]In summary,the addition of compound probiotics could significantly improve the structure of intestinal microbial flora,increase the quantity of beneficial bacteria,reduce the quantity of harmful bacteria,and improve the immune function of mice.

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.

Generating barcodes for nanopore sequencing data with PRO

DNA barcodes,short and unique DNA sequences,play a crucial role in sample identification when processing many samples simultaneously,which helps reduce experimental costs.Nevertheless,the low quality of long-read sequencing makes it difficult to identify barcodes accurately,which poses significant challenges for the design of barcodes for large numbers of samples in a single sequencing run.Here,we present a comprehensive study of the generation of barcodes and develop a tool,PRO,that can be used for selecting optimal barcode sets and demultiplexing.We formulate the barcode design problem as a combinatorial problem and prove that finding the optimal largest barcode set in a given DNA sequence space in which all sequences have the same length is theoretically NP-complete.For practical applications,we developed the novel method PRO by introducing the probability divergence between two DNA sequences to expand the capacity of barcode kits while ensuring demultiplexing accuracy.Specifically,the maximum size of the barcode kits designed by PRO is 2,292,which keeps the length of barcodes the same as that of the official ones used by Oxford Nanopore Technologies(ONT).We validated the performance of PRO on a simulated nanopore dataset with high error rates.The demultiplexing accuracy of PRO reached 98.29%for a barcode kit of size 2,922,4.31%higher than that of Guppy,the official demultiplexing tool.When the size of the barcode kit generated by PRO is the same as the official size provided by ONT,both tools show superior and comparable demultiplexing accuracy.

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.

Structural Optimization of the Inlet Header of Supercritical Carbon Dioxide Printed Circuit Board Heat Exchanger

Supercritical carbon dioxide printed circuit board heat exchangers are expected to be applied in third-generation solar thermal power generation.However,the uniformity of supercritical carbon dioxide entering the heat exchanger has a significant impact on the overall performance of the heat exchanger.In order to improve the uniformity of flow distribution in the inlet header,this article studies and optimizes the inlet header of a printed circuit board heat exchanger through numerical simulation.The results indicate that when supercritical carbon dioxide flows through the header cavity,eddy currents will be generated,which will increase the uneven distribution of flow rate,while reducing the generation of eddy currents will improve the uniform distribution of flow rate.When the dimensionless factor of the inlet header is 6,the hyperbolic configuration is the optimal structure.We also reduced the eddy current region by adding transition segments,and the results showed that the structure was the best when the dilation angle was 10°,which reduced the non-uniformity by 21%compared to the hyperbolic configuration,providing guidance for engineering practice.

Central nervous system efficacy of aumolertinib versus gefitinib in patients with untreated,EGFR-mutated,advanced non-small cell lung cancer:data froma randomized phase III trial(AENEAS)

Background:The initial randomized,double-blinded,actively controlled,phase III ANEAS study(NCT03849768)demonstrated that aumolertinib showed superior efficacy relative to gefitinib as first-line therapy in epidermal growth factor receptor(EGFR)-mutated advanced non-small cell lung cancer(NSCLC).Metastatic disease in the central nervous system(CNS)remains a challenge in the management of NSCLC.This study aimed to compare the efficacy of aumolertinib versus gefitinib among patients with baseline CNS metastases in the ANEAS study.Methods:Eligible patients were enrolled and randomly assigned in a 1:1 ratio to orally receive either aumolertinib or gefitinib in a double-blinded fashion.Patients with asymptomatic,stable CNS metastases were included.Follow-up imaging of the same modality as the initial CNS imaging was performed every 6 weeks for 15 months,then every 12weeks.CNS responsewas assessed by a neuroradiological blinded,independent central review(neuroradiological-BICR).The primary endpoint for this subgroup analysis was CNS progression-free survival(PFS).Results:Of the 429 patients enrolled and randomized in the ANEAS study,106 patients were found to have CNS metastases(CNS Full Analysis Set,cFAS)at baseline by neuroradiological-BICR,and 60 of them had CNS target lesions(CNS Evaluable for Response,cEFR).Treatment with aumolertinib significantly prolonged median CNS PFS compared with gefitinib in both cFAS(29.0 vs.8.3 months;hazard ratio[HR]=0.31;95%confidence interval[CI],0.17-0.56;P<0.001)and cEFR(29.0 vs.8.3 months;HR=0.26;95%CI,0.11-0.57;P<0.001).The confirmed CNS overall response rate in cEFRwas 85.7%and 75.0%in patients treated with aumolertinib and gefitinib,respectively.Competing risk analysis showed that the estimated probability of CNS progression without prior non-CNS progression or death was consistently lower with aumolertinib than with gefitinib in patients with and without CNSmetastases at baseline.No new safety findings were observed.Conclusions:These results indicate a potential advantage of aumolertinib over gefitinib in terms of CNS PFS and the risk of CNS progression in patients with EGFR-mutated advanced NSCLC with baseline CNS metastases.

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.