Genetic mutation of Tas2r104/Tas2r105/Tas2r114 cluster leads to a loss of taste perception to denatonium benzoate and cucurbitacin B

Background:Bitter taste receptors(Tas2rs)are generally considered to sense various bitter compounds to escape the intake of toxic substances.Bitter taste receptors have been found to widely express in extraoral tissues and have important physiological functions outside the gustatory system in vivo.Methods:To investigate the physiological functions of the bitter taste receptor cluster Tas2r106/Tas2r104/Tas2r105/Tas2r114 in lingual and extraoral tissues,multiple Tas2rs mutant mice and Gnat3 were produced using CRISPR/Cas9 gene-editing technique.A mixture containing Cas9 and sgRNA mRNAs for Tas2rs and Gnat3 gene was microinjected into the cytoplasm of the zygotes.Then,T7EN1 assays and sequencing were used to screen genetic mutation at the target sites in founder mice.Quantitative real-time polymerase chain reaction(qRT-PCR)and immunostaining were used to study the expression level of taste signaling cascade and bitter taste receptor in taste buds.Perception to taste substance was also studied using twobottle preference tests.Results:We successfully produced several Tas2rs and Gnat3 mutant mice using the CRISPR/Cas9 technique.Immunostaining results showed that the expression of GNAT3 and PLCB2 was not altered in Tas2rs mutant mice.But qRT-PCR results revealed the changed expression profile of m Tas2rs gene in taste buds of these mutant mice.With two-bottle preference tests,these mutant mice eliminate responses to cycloheximide due to genetic mutation of Tas2r105.In addition,these mutant mice showed a loss of taste perception to quinine dihydrochloride,denatonium benzoate,and cucurbitacin B(CuB).Gnat3-mediated taste receptor and its signal pathway contribute to CuB perception.Conclusions:These findings implied that these mutant mice would be a valuable means to understand the biological functions of TAS2Rs in extraoral tissues and investigate bitter compound-induced responses mediated by these TAS2Rs in many extraoral tissues.

Novel technique for cost reduction in mutation testing

Aimed at the problem of expensive costs in mutation testing which has hampered its wide use,a technique of introducing a test case selection into the process of mutation testing is proposed.For each mutant,a fixed number of test cases are selected to constrain the maximum allowable executions so as to reduce useless work.Test case selection largely depends on the degree of mutation.The mutation distance is an index describing the semantic difference between the original program and the mutated program.It represents the percentage of effective test cases in a test set,so it can be used to guide the selection of test cases.The bigger the mutation distance is,the easier it is that the mutant will be killed,so the corresponding number of effective test cases for this mutant is greater.Experimental results suggest that the technique can remarkably reduce execution costs without a significant loss of test effectiveness.

Towards Semantic Mutation Testing of Aspect-Oriented Programs

Aspect-oriented programs have received much attention from software testing researchers. Various testing techniques and approaches have been proposed to tackle issues and challenges when testing aspect-oriented programs including traditional mutation testing. In traditional mutation testing of aspect-oriented programs, mutants are generated by making small changes to the syntax of the aspect-oriented language. Recently, a new approach known as semantic mutation testing has been proposed. This approach mutates the semantics of the language in which the program is written. The mutants generated misunderstandings of the language which are different classes of faults. Aspect-oriented programming presents itself with different properties that can be further explored with respect to semantic mutation testing. This paper describes various possible scenarios that semantic mutation testing strategy might have particular value in testing aspect-oriented programs.

Implementation of Hybrid Particle Swarm Optimization for Optimized Regression Testing

Software test case optimization improves the efficiency of the software by proper structure and reduces the fault in the software.The existing research applies various optimization methods such as Genetic Algorithm,Crow Search Algorithm,Ant Colony Optimization,etc.,for test case optimization.The existing methods have limitations of lower efficiency in fault diagnosis,higher computa-tional time,and high memory requirement.The existing methods have lower effi-ciency in software test case optimization when the number of test cases is high.This research proposes the Tournament Winner Genetic Algorithm(TW-GA)method to improve the efficiency of software test case optimization.Hospital Information System(HIS)software was used to evaluate TW-GA model perfor-mance in test case optimization.The tournament Winner in the proposed method selects the instances with the best fitness values and increases the exploitation of the search to find the optimal solution.The TW-GA method has higher exploita-tion that helps to find the mutant and equivalent mutation that significantly increases fault diagnosis in the software.The TW-GA method discards the infor-mation with a lower fitness value that reduces the computational time and mem-ory requirement.The TW-GA method requires 5.47 s and the MOCSFO method requires 30 s for software test case optimization.

Novel Metrics for Mutation Analysis

A measure of the“goodness”or efficiency of the test suite is used to determine the proficiency of a test suite.The appropriateness of the test suite is determined through mutation analysis.Several Finite State Machine(FSM)mutants are produced in mutation analysis by injecting errors against hypotheses.These mutants serve as test subjects for the test suite(TS).The effectiveness of the test suite is proportional to the number of eliminated mutants.The most effective test suite is the one that removes the most significant number of mutants at the optimal time.It is difficult to determine the fault detection ratio of the system.Because it is difficult to identify the system’s potential flaws precisely.In mutation testing,the Fault Detection Ratio(FDR)metric is currently used to express the adequacy of a test suite.However,there are some issues with this metric.If both test suites have the same defect detection rate,the smaller of the two tests is preferred.The test case(TC)is affected by the same issue.The smaller two test cases with identical performance are assumed to have superior performance.Another difficulty involves time.The performance of numerous vehicles claiming to have a perfect mutant capture time is problematic.Our study developed three metrics to address these issues:FDR/|TS|,FDR/|TC|,and FDR/|Time|;In this context,most used test generation tools were examined and tested using the developed metrics.Thanks to the metrics we have developed,the research contributes to eliminating the problems related to performance measurement by integrating the missing parameters into the system.

Novel mutation of SPG4 gene in a Chinese family with hereditary spastic paraplegia:A case report

BACKGROUND Hereditary spastic paraplegia(HSP)is a group of neurogenetic diseases of the corticospinal tract,accompanied by distinct spasticity and weakness of the lower extremities.Mutations in the spastic paraplegia type 4(SPG4)gene,encoding the spastin protein,are the major cause of the disease.This study reported a Chinese family with HSP caused by a novel mutation of the SPG4 gene.CASE SUMMARY A 44-year-old male was admitted to our hospital for long-term right lower limb weakness,leg stiffness,and unstable walking.His symptoms gradually worsened,while no obvious muscle atrophy in the lower limbs was found.Neurological examinations revealed that the muscle strength of the lower limbs was normal,and knee reflex hyperreflexia and bilateral positive Babinski signs were detected.Members of his family also had the same symptoms.Using mutation analysis,a novel heterozygous duplication mutation,c.1053dupA,p.(Gln352Thrfs*15),was identified in the SPG4 gene in this family.CONCLUSION A Chinese family with HSP had a novel mutation of the SPG4 gene,which is autosomal dominant and inherited as pure HSP.The age of onset,sex distribution,and clinical manifestations of all existing living patients in this family were analyzed.The findings may extend the current knowledge on the existing mutations in the SPG4 gene.

Study on MCM Interconnect Test Generation Based on Ant Algorithm with Mutation Operator

A novel multi-chip module(MCM) interconnect test generation scheme based on ant algorithm(AA) with mutation operator was presented.By combing the characteristics of MCM interconnect test generation,the pheromone updating rule and state transition rule of AA is designed.Using mutation operator,this scheme overcomes ordinary AA’s defects of slow convergence speed,easy to get stagnate,and low ability of full search.The international standard MCM benchmark circuit provided by the MCNC group was used to verify the approach.The results of simulation experiments,which compare to the results of standard ant algorithm,genetic algorithm(GA) and other deterministic interconnecting algorithms,show that the proposed scheme can achieve high fault coverage,compact test set and short CPU time,that it is a newer optimized method deserving research.

Optimal Multiperiodic Control for Inventory Coupled Systems: A Multifrequency Second-Order Test

A complex autonomous inventory coupled system is considered. It can take, for example, the form of a network of chemical or biochemical reactors, where the inventory interactions perform the recycling of by-products between the subsystems. Because of the flexible subsystems interactions, each of them can be operated with their own periods utilizing advantageously their dynamic properties. A multifrequency second-order test generalizing the p-test for single systems is described. It can be used to decide which kind of the operation (the static one, the periodic one or the multiperiodic one) will intensify the productivity of a complex system. An illustrative example of the multiperiodic optimization of a complex chemical production system is presented.

Temperature Variation and Mutation Analysis over the Past 59 Years in Shenyang

In this paper,based on the observation data of air temperature during 1951-2009 in Shenyang,the interannual and interdecadal variation of annual average temperature,maximum and minimum temperature in Shenyang were conducted the statistical analysis by means of linear trend estimation and mutation detection by using Mann-Kendall method.As was demonstrated in the results,the annual average temperature,maximum and minimum temperature in Shenyang showed an upward trend,whose linear tendency rate was 0.231,0.181 and 0.218 respectively.The increment trend of annual average temperature,maximum and minimum temperature was extremely clear.The increase in minimum temperature was more significant than that in mean temperature and maximum temperature.The abrupt change point of annual mean temperature in Shenyang appeared in 1981;the abrupt change point of annual mean maximum temperature appeared in 1994;the annual mean minimum temperature underwent mutation in 1978.

Relationship between Mutation of IR in the mtr System of Neisseria Gonorrhoeae and Multiple Antibiotic Resistance

To study the relationship between mutation of the inverted repeat sequence （IR） in the multiple transferable resistant system （mtr） of Neisseria gonorrhoeae （NG） and its multiple antibiotic resistance, minimal inhibitory concentrations （MICs） for the clinically isolated strains were tested by agar-dilution-method. The mtr system＇s IR gene of NG was sequenced after amplification by polymerase chain reaction （PCR）. Either two susceptive or five penicillin-resistant strains had no base mutation in IR gene, while all of the 13 strains with multiple-antibiotic-resistance had a singlebase deletion （A/T）. The result suggests that a single-base deletion of the thirteen-base IR sequence in mtr system of NG might result in multiple antibiotic resistance but is not associated with single antibiotic resistance.

Establishment and application of a multiplex genetic mutation-detection method of lung cancer based on MassARRAY platform

Objective: This study aims to establish a method for highly parallel multiplexed detection of genetic mutations in Chinese lung cancer samples through Agena i PLEX chemistry and matrix-assisted laser desorption ionization time-of-flight analysis on Mass ARRAY mass spectrometry platform.Methods: We reviewed the related literature and data on lung cancer treatments. We also identified 99 mutation hot spots in 13 target genes closely related to the pathogenesis, drug resistance, and metastasis of lung cancer. A total of 297 primers, composed of99 paired forward and reverse amplification primers and 99 matched extension primers, were designed using Assay Design software. The detection method was established by analyzing eight cell lines and six lung cancer specimens. The proposed method was then validated through comparisons by using a Lung Carta^(TM) kit. The sensitivity and specificity of the proposed method were evaluated by directly sequencing EGFR and KRAS genes in 100 lung cancer cases.Results: The proposed method was able to detect multiplex genetic mutations in lung cancer cell lines. This finding was consistent with the observations on previously reported mutations. The proposed method can also detect such mutations in clinical lung cancer specimens. This result was consistent with the observations with Lung Carta^(TM) kit. However, an FGFR2 mutation was detected only through the proposed method. The measured sensitivity and specificity were 100% and 96.3%, respectively.Conclusions: The proposed Mass ARRAY technology-based multiplex method can detect genetic mutations in Chinese lung cancer patients. Therefore, the proposed method can be applied to detect mutations in other cancer tissues.

ACE gene missense mutation in a case with early-onset, rapid progressing dementia

The population of early-onset Alzheimer's disease(EOAD)accounts for 1%-2%of the total population of Alzheimer's disease,and genetic mutations are more common in EOAD.The first symptom of the patient in the present case report was the decline in memories of recent events,and the disease progressed rapidly in the following 2 years.Genetic testing has revealed the presence of genetic mutations(c.A479G,p.N160S)of ACE,which causes the 160th codon of the ACE protein to change from aspartic acid to serine,and at the same time genotype of apolipoprotein E(APOE)is ε3/ε4.We think that this patient carries the mutation type of the sensitive gene ACE and the risk gene APOE of Alzheimer's disease,and this is the reason why the disease progressed rapidly.Moreover,we discussed ACE genetic mutation's meaning in EOAD progression.

Mutation Analysis of STR Locus on 23 Autosomes in Hainan Population

[Objective] To analyze the mutation signature and regularity of STR locus on 23 autosomes in paternity testing cases in Hainan. [Methods] A total of 2715 paternity testing cases accepted by the Forensic Medical Identification Centre of our hospital from 2017 to 2020 derived from counties and cities in Hainan Province were collected, the cases containing gene mutations were selected, the mutation rate and details of each locus were counted, and the mutation regu-larity of 23 STR loci was analyzed. [Results] Of the 2715 cases identified as “support”, 1487 were triplet cases and 1640 were dyad cases, totaling 4614 meioses;There were 50 gene mutation events (including 17 triplet mutations and 33 dyad mutations), with an average mutation rate of 0.0047% and a cumulative mutation rate of 1.0837%. A total of 19 of the 23 STR loci were mutated, with a mutation rate of 0.1301% at the D12S391 locus and 0.0217% at five loci, TPOX, D1S1656, D2S441, D22S1045, and PentaD, while no muta-tion events were found at four loci, D19S433, TH01, D13S317, and D7S820. Of the 50 mutation events, 47 were one-step mutations, 1 was two-step, and 2 were three-step. There were 35 paternal mutations (13 triplets and 22 dyads), 6 maternal mutations (4 triplets and 2 dyads), and 9 indeterminate pater-nal/maternal mutations, with a paternal to maternal mutation ratio of 5.83:1. [Conclusion] The mutation rate of D12S391 locus is the highest, and the muta-tion rate of TPOX, D1S1656, D2S441, D22S1045 and PentaD loci is the lowest in Hainan population, and paternal mutations are more than maternal muta-tions. In the paternity test, if 1 - 3 STR loci do not conform to the genetic law, especially when the mutant locus is homozygous or the next of kin is identi-fied, it is necessary to use other kits to review and increase the number of loci or use the second-generation sequencing technology to confirm, carefully de-termine the mutation and ensure the accuracy of the identification conclusion.

New real-time-PCR method to identify single point mutations in hepatitis C virus

AIM To develop a fast, low-cost diagnostic strategy to identify single point mutations in highly variable genomes such as hepatitis C virus(HCV).METHODS In patients with HCV infection, resistance-associated amino acid substitutions within the viral quasispecies prior to therapy can confer decreased susceptibility to direct-acting antiviral agents and lead to treatment failure and virological relapse. One such naturally occurring mutation is the Q80 K substitution in the HCV-NS3 protease gene, which confers resistance to PI inhibitors, particularly simeprevir. Low-cost, highly sensitive techniques enabling routine detection of these single point mutations would be useful to identify patients at a risk of treatment failure. Light Cycler methods, based on real-time PCR with sequencespecific probe hybridization, have been implemented in most diagnostic laboratories. However, this technique cannot identify single point mutations in highly variable genetic environments, such as the HCV genome. To circumvent this problem, we developed a new method to homogenize all nucleotides present in a region except the point mutation of interest. RESULTS Using nucleotide-specific probes Q, K, and R substitutions at position 80 were clearly identified at a sensitivity of 10%(mutations present at a frequency of at least 10% were detected). The technique was successfully applied to identify the Q80 K substitution in 240 HCV G1 serum samples, with performance comparable to that of direct Sanger sequencing, the current standard procedure for this purpose. The new method was then validated in a Catalonian population of 202 HCV G1-infected individuals. Q80 K was detected in 14.6% of G1 a patients and 0% of G1 b in our setting. CONCLUSION A fast, low-cost diagnostic strategy based on real-time PCR and fluorescence resonance energy transfer probe melting curve analysis has been successfully developed to identify single point mutations in highly variable genomes such as hepatitis C virus. This technique can be adapted to detect any single point mutation in highly variable genomes.

A Novel Gene Mutation of Runx2 in Cleidocranial Dysplasia

Haploinsufficiency of the runt-related transcription factor 2（Runx2） gene is widely known to be responsible for cleidocranial dysplasia（CCD）. To date, more than 190 mutations in Runx2 gene have been reported to be related to CCD. In this study, a novel mutation of Runx2 gene was observed in a female with CCD. Genomic DNA was extracted from peripheral venous blood of the proband and eleven members of her family. Genetic testing on these twelve people identified a novel missense mutation（c.895 T〉C, Y299 H） in exon 5 of the RUNX2 gene in the proband. This mutation results in an amino acid change at codon 895（P.Tyr 299 His.） from a tryptophan codon（TAT） to a histidine codon（CAT）. Our finding may further extend the known mutation spectrum of the RUNX2 gene, and facilitate prenatal genetic diagnosis of CCD in the future.

2+0 CYP21A2 deletion carrier—a limitation of the genetic testing and counseling:A case report

BACKGROUND CYP21A2 gene mutations may all cause reduction or loss of 21-hydroxylase activity,leading to development of congenital adrenal hyperplasia(CAH)with different clinical phenotypes.For families with CAH children,genetic testing of the parents and genetic counseling are recommended to assess the risk of recurrence.CASE SUMMARY We report a case of CAH with a high suspicion before delivery.The risk of the child suffering from CAH during the pregnancy had been underestimated due to the deviation of genetic counseling and genetic testing results.Our report confirmed a CYP21A2 homozygous deletion in this case,CYP21A2 heterozygous deletion in the mother,and a rare 2+0 CYP21A2 deletion in the father.CONCLUSION It is important to analyze the distribution of CYP21A2 gene in the two alleles of parents of children with CAH.

A Fault-Based Testing Approach in Safety Critical Medical Systems

The advent of technology has opened unprecedented opportunities in health care delivery system as the demand for intelligent and knowledge-based systems has increased as modern medical practices become more knowledge-intensive. As a result of this, there is greater need to investigate the pervasiveness of software faults in Safety critical medical systems for proper diagnosis. The sheer volume of code in these systems creates significant concerns about the quality of the software. The rate of untimely deaths nowadays is alarming partly due to the medical device used to carry out the diagnosis process. A safety-critical medical (SCM) system is a complex system in which the malfunctioning of software could result in death, injury of the patient or damage to the environment. The malfunctioning of the software could be as a result of the inadequacy in software testing due to test suit problem or oracle problem. Testing a SCM system poses great challenges to software testers. One of these challenges is the need to generate a limited number of test cases of a given regression test suite in a manner that does not compromise its defect detection ability. This paper presents a novel five-stage fault-based testing procedure for SCM, a model-based approach to generate test cases for differential diagnosis of Tuberculosis. We used Prime Path Coverage and Edge-Pair Coverage as coverage criteria to ensure maximum coverage to identify feasible paths. We analyzed the proposed testing procedure with the help of three metrics consisting of Fault Detection Density, Fault Detection Effectiveness and Mutation Adequacy Score. We evaluated the effectiveness of our testing procedure by running the suggested test cases on a sample historical data of tuberculosis patients. The experimental results show that our developed testing procedure has some advantages such as creating mutant graphs and Fuzzy Cognitive Map Engine while resolving the problem of eliminating infeasible test cases for effective decision making.

基于变异的正则表达式反例测试串生成算法

正则表达式在计算机科学的许多领域具有广泛应用.然而,由于正则表达式语法比较复杂,并且允许使用大量元字符,导致开发人员在定义和使用时容易出错.测试是保证正则表达式语义正确性的实用和有效手段,常用的方法是根据被测表达式生成一些字符串,并检查它们是否符合预期.现有的测试数据生成大多只关注正例串,而研究表明,实际开发中存在的错误大部分在于定义的语言比预期语言小,这类错误只能通过反例串才能发现.研究基于变异的正则表达式反例测试串生成.首先通过变异向被测表达式中注入缺陷得到一组变异体,然后在被测表达式所定义语言的补集中选取反例字符串揭示相应变异体所模拟的错误.为了能够模拟复杂缺陷类型,以及避免出现变异体特化而无法获得反例串的问题,引入二阶变异机制.同时采取冗余变异体消除、变异算子选择等优化技术对变异体进行约简,从而控制最终生成的测试集规模.实验结果表明,与已有工具相比,所提算法生成的反例测试串规模适中,并且具有较强的揭示错误能力.

急性早幼粒细胞白血病细胞形态分类体系及其与实验室检查和FLT3-ITD突变的相关性分析

目的:建立一套用于表征急性早幼粒细胞白血病(APL)患者细胞形态特点的分类体系,并分析不同APL细胞形态特点与常规检验指标和基因变异的相关性。方法:根据APL白血病细胞的形态特征,建立一套14类的分类体系用以表征患者个体间和个体内的细胞形态异质性。将该分类体系用于40例APL患者的形态学分析,并将分类结果与患者的常规检验指标和基因变异特点进行统计学分析,以分析不同APL细胞形态特征与常规检验指标和基因变异的相关性。结果:FLT3-ITD突变阳性的APL病例组中核形规则、粗颗粒且不见Auer小体(1类)的细胞显著少于FLT3突变阴性病例组(P<0.05)。核形规则组相比于核形不规则组活化部分凝血活酶时间(APTT)明显较长(P<0.05);细颗粒组相比于粗颗粒组APTT明显较长(P<0.01)、骨髓白血病细胞比例相对更低(P<0.05);Auer小体阴性组的外周血白细胞计数、D-二聚体、乳酸脱氢酶和骨髓白血病细胞比例均显著高于Auer小体增多组(均P<0.05)。结论:本研究建立的形态学分类体系可以客观表征不同类型的APL白血病细胞,有助于更好地评估APL白血病细胞的个体内和个体间异质性和进一步用于精确分析APL的形态表型与生物学特性的相关性。

基于Mann-Kendall的广东省长效避孕服务利用和人工流产的趋势与突变分析

目的了解广东省长效避孕服务利用和人工流产变化趋势,为制定有效的生殖保健服务策略,提高妇女生殖健康提供依据。方法采用Mann-Kendall检验法对2008—2021年广东省长效避孕服务利用总例数、各种长效避孕方法例数与占比、人工流产例数、人工流产活产比进行趋势和突变分析。结果2008—2021年广东省长效避孕服务利用总例数呈现下降趋势,长效不可逆避孕方法例数呈先增后降的趋势、长效可逆避孕方法例数呈现下降趋势;这三个指标突变点分别出现在2016、2018、2014年。各种长效避孕方法服务利用上,放置宫内节育器术占67.3%,输卵管绝育术占29.1%,输精管绝育术占3.5%,放置皮下填埋剂术占0.1%。全省人工流产例数呈下降趋势,突变点出现在2018年;人工流产活产比相对稳定,年均人工流产活产比为76.5%。从区域层面看,全省与珠三角地区长效可逆避孕方法例数和人工流产例数的变化趋势呈现出平行关系,粤东、粤西和粤北地区长效可逆避孕方法例数变化趋势与人工流产例数变化趋势的关联各异。结论广东省放置宫内节育器术在长效避孕方法中占主导地位,输卵管绝育术次之,长效避孕责任主要由女性群体承担。区域间长效可逆避孕方法与人工流产变化趋势呈现出不同的关系,有必要深入了解导致这些地区差异的因素,促进不同区域避孕服务的有效落实,避免发生意外妊娠。此外,建议地区积极探讨将避孕服务与妇女全生命周期服务整合,提高妇女生殖健康水平。