Development and Validation of a Clinical Trial Patient Stratification Assay That Interrogates 27 Mutation Sites in MAPK Pathway Genes

Somatic mutations identified on genes related to the cancer-developing signaling pathways have drawn attention in the field of personalized medicine in recent years. Treatments developed to target a specific signaling pathway may not be effective when tumor activating mutations occur downstream of the target and bypass the targeted mechanism. For instance, mutations detected in KRAS/BRAF/NRAS genes can lead to EGFR-independent intracellular signaling pathway activation. Most patients with these mutations do not respond well to anti-EGFR treatment. In an effort to detect various mutations in FFPE tissue samples among multiple solid tumor types for patient stratification many mutation assays were evaluated. Since there were more than 30 specific mutations among three targeted RAS/RAF oncogenes that could activate MAPK pathway genes, a custom designed Single Nucleotide Primer Extension (SNPE) multiplexing mutation assay was developed and analytically validated as a clinical trial assay. Throughout the process of developing and validating the assay we overcame many technical challenges which include: the designing of PCR primers for FFPE tumor tissue samples versus normal blood samples, designing of probes for detecting consecutive nucleotide double mutations, the kinetics and thermodynamics aspects of probes competition among themselves and against target PCR templates, as well as validating an assay when positive control tumor tissue or cell lines with specific mutations are not available. We used Next Generation sequencing to resolve discordant calls between the SNPE mutation assay and Sanger sequencing. We also applied a triplicate rule to reduce potential false positives and false negatives, and proposed special considerations including pre-define a cut-off percentage for detecting very low mutant copies in the wild-type DNA background.     

Assessment of the repellent effect of Lippia alba essential oil and major monoterpenes on the cattle tick Rhipicephalus microplus

The control of Rhipicephalus microplus (Ixodida: Ixodidae) is achieved using synthetic acaricides. However, resistant tick populations are widespread around the world. Plant essential oils can act as repellents, keeping ticks away from hosts and decreasing the selection pressure on synthetic acaricides. The aim of this study was to evaluate the in vitro repellent effect of Lippia alba essential oil on R. microplus larvae. Leaves from two L. alba genotypes maintained under the same agronomic and environmental conditions were collected. Essential oil was extracted by hydrodistillation and analysed by gas chromatography–mass spectrometry (GC‐MS). The major monoterpenes detected in the chemical analysis were commercially acquired and tested. For the repellency test, a glass rod was vertically fixed to measure active climbing of approximately 30 R. microplus larvae aged 14–21 days in response to essential oils and monoterpenes. Repellency was evaluated at 1 h, 3 h and 5 h after treatment. Variation in repellent action was detected between the genotypes. The major monoterpenes identified in the essential oils (limonene and carvone) showed low repellent effects in comparison with intact essential oils. Thus, the present results showed that L. alba essential oil contains bioactive compounds with great repellent activity against ticks that varies according to the plant genotype.     

Synergism of thymol,carvacrol and eugenol in larvae of the cattle tick,Rhipicephalus microplus,and brown dog tick,Rhipicephalus sanguineus

The effects of combinations of the monoterpenes thymol and carvacrol and the phenylpropanoid eugenol in larvae of Rhipicephalus microplus (Canestrini, 1888) (Acari: Ixodidae) and Rhipicephalus sanguineus sensu lato (s.l.) (Acari: Ixodidae) were assessed by the larval packet test. The CompuSyn program was used to make qualitative assessments of the effects (synergistic, additive and antagonistic) of the associations. The effects of all combinations tested against R. microplus larvae were synergistic, with combination indices (CIs) <0.70. When tested against R. sanguineus, eight of the mixtures showed a synergistic effect (CI < 0.70); only the carvacrol + thymol mixture at LC₅₀ presented a moderate synergistic effect, with CIs between 0.70–0.90. This study is the first to determine the effects of the interactions of these substances in the control of these two tick species. The combinations of carvacrol + thymol, carvacrol + eugenol and thymol + eugenol have synergistic effects in R. microplus and R. sanguineus s.l. larvae.     

安徽大别山木本植物幼树小枝薄壁组织组成特征初探

木本植物次生木质部轴向薄壁组织和射线薄壁组织在物质存储和转运等功能中起着重要的作用, 剖析次生木质部薄壁组织组成有助于深入探究其功能, 而小枝木质部薄壁组织组成特征还缺乏研究。该研究以天马国家级自然保护区内的18种木本植物幼树为研究对象, 测算了各物种小枝木质部薄壁组织的组成含量并检测了其谱系信号, 结合有关树干薄壁组织含量数据集, 初步探讨木本植物小枝木质部薄壁组织含量特征。研究结果表明: (1) 18种木本植物幼树小枝的薄壁组织总含量为9.96%-18.56%, 平均为14.80%; 其中射线薄壁组织含量为7.74%-15.45%, 高于轴向薄壁组织的含量(1.13%-7.49%); (2)小枝中薄壁组织总含量呈低于树干的趋势, 其中小枝射线薄壁组织含量低于树干, 而轴向薄壁组织含量高于树干, 这可能是由器官差异和生活史阶段差异造成的; (3)轴向薄壁组织的含量具有显著的谱系信号, 即物种亲缘关系越近其含量越相近。该研究初步验证了木本植物次生木质部薄壁组织的谱系信号, 同时暗示了器官和生活史阶段差异对薄壁组织含量具有重要影响。     

功能矫形前伸青春期大鼠下颌后翼外肌MyoD、myogenin 的表达

目的:探讨"应力-生长(改建)"在细胞水平上的体现,为功能矫形治疗和矫治效果的保持提供新思路和实验依据。方法:本实验选用20只4周龄,雄性SD大鼠随机分为8组。其中实验组大鼠经戊巴比妥麻醉后佩戴上颌斜面导板,对照组未佩用。依据时间不同又分为四组:1d,7d,14d,21d。采用RT-PCR技术分析各组大鼠翼外肌组织中肌分化相关基因MyoD、myogenin mRNA的表达变化。结果:未施加功能矫形力的大鼠翼外肌组织MyoD表达伴随其生长发育呈现递减趋势,实验组在第7 d出现表达上调。同时,力学刺激后实验组动物myogenin的表达与对照组相比较在14 d组出现明显上调。结论:功能矫形力作用于翼外肌组织可以诱导MyoD和myogenin的表达上调进而诱导成肌细胞的分化。     

Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones

Neutrophils play an important role in innate immunity by defending the host organism against invading microorganisms. Antimicrobial activity of neutrophils is mediated by release of antimicrobial peptides, phagocytosis as well as formation of neutrophil extracellular traps (NET). These structures are composed of DNA, histones and granular proteins such as neutrophil elastase and myeloperoxidase. This study focused on the influence of NET on the host cell functions, particularly on human alveolar epithelial cells as the major cells responsible for gas exchange in the lung. Upon direct interaction with epithelial and endothelial cells, NET induced cytotoxic effects in a dose-dependent manner, and digestion of DNA in NET did not change NET-mediated cytotoxicity. Pre-incubation of NET with antibodies against histones, with polysialic acid or with myeloperoxidase inhibitor but not with elastase inhibitor reduced NET-mediated cytotoxicity, suggesting that histones and myeloperoxidase are responsible for NET-mediated cytotoxicity. Although activated protein C (APC) did decrease the histone-induced cytotoxicity in a purified system, it did not change NET-induced cytotoxicity, indicating that histone-dependent cytotoxicity of NET is protected against APC degradation. Moreover, in LPS-induced acute lung injury mouse model, NET formation was documented in the lung tissue as well as in the bronchoalveolar lavage fluid. These data reveal the important role of protein components in NET, particularly histones, which may lead to host cell cytotoxicity and may be involved in lung tissue destruction.     

Seed germination of Pilosocereus arrabidae (Cactaceae) from a semiarid region of south‐east Brazil

We evaluated the effect of temperature regimes (six constant and four alternating temperatures), light qualities (five red : far red ratios) and water potentials (ΨW; seven NaCl and polyethylene glycol 6000 [PEG] solutions) on the percentage and germination rate, as well as the post‐seminal development morphology, that allow Pilosocereus arrabidae seeds to germinate in a hot semiarid climate on the south‐eastern Brazilian coast. The results showed that seeds germinated similarly between constant and alternating temperatures, with an optimal germination at 25/20°C and 20°C. Pilosocereus arrabidae seeds were photoblastic positive and the final germination percentage was inhibited at low red : far red ratios. Maximum germination was obtained in distilled water (0 MPa) and decreases of Ψ_W in the solutions reduced the germination, which was lower in NaCl than in iso‐osmotic PEG solutions. Germination inhibition appears to be osmotic because the recovery response was high when non‐germinated seeds from both iso‐osmotic solutions were transferred to water. Seeds of P. arrabidae are small and germination is phaneroepigeal. Despite the slow growth typically seen in seedlings and adults of Cactaceae, germination in this species depends on the ability of the seeds to appropriately sense and react to environmental cues that correlate with times and places under low‐risk growth conditions.     

甲功五项化学发光免疫分析的基质效应对临床测值的影响

目的:探索评价基质效应在化学发光免疫分析中对甲状腺功能五项指标的影响。方法:选取甲状腺功能五项高值血清,用10种基质牛血清、马血清、山羊血清、水解明胶、BSA、PBS、生理盐水、正常人血清、甲减人血清、甲亢人血清分别对T3、T4、FT3、FT4、TSH的高值血清进行倍比稀释,观察基质效应,另将10种基质用考马斯亮兰法检测蛋白含量,分析蛋白含量与基质效应的关系。结果:T3项目牛血清、水解明胶、BSA有明显基质效应;T4和FT3项目牛血清、水解明胶、BSA、PBS、生理盐水有明显基质效应;FT4项目牛血清、马血清、水解明胶、BSA、PBS、生理盐水有明显基质效应;TSH项目没有发现基质效应,正常人血清、甲减人血清和甲亢人血清对甲状腺功能五项无基质效应。检测结果显示蛋白含量多少与基质效应无关。结论:人血清基质是用于稀释样本,基质效应最小的液体,针对个体差异性进行的选择,稀释T3、T4、FT3、FT4高值血清选择甲减人血清,稀释TSH高值血清选择甲亢人血清,可以得到较为满意的结果。     

Phylogenetic relationships of nonbiting midges in the subfamily Tanypodinae (Diptera: Chironomidae) inferred from morphology

The nonbiting midge subfamily Tanypodinae represents one of the most diverse lineages of Chironomidae. Despite the wide distribution and high diversity of tanypodine chironomids, the evolutionary history of the subfamily remains poorly understood. Here, we present the first phylogenetic analysis of the subfamily Tanypodinae based on morphological data. Cladistic analyses were conducted using 86 morphological characters from 115 species belonging to 54 tanypodine genera, including the eight currently recognised tribes: Anatopyniini, Clinotanypodini, Coelopyniini, Macropelopiini, Natarsiini, Pentaneurini, Procladiini and Tanypodini. We use characters from fourth‐instar larvae, pupae and adults of both sexes. We examine the effects of implied weighting by reanalysing the data with varying values of concavity constant (k). Our analysis supports the monophyly of Tanypodinae with Podonominae as its sister group. All previously proposed tribes are recovered as monophyletic assemblages under a wide range of weighting factors. Under these conditions, the genus Fittkauimyia is the sister group of the remaining Macropelopiini and is erected as a new monobasic tribe, Fittkauimyiini trib.n . The tribe Pentaneurini is recovered as monophyletic with some internal relationships resolved. The genus Paramerina, recovered as sister of Reomyia + Zavrelimyia, is formally synonymised with Zavrelimyia syn.n. , based on morphological similarity in all three life stages and treated as a subgenus of the latter. Finally, the recently suggested synonymies of Gressittius and Guassutanypus with Alotanypus and the establishment of the subgenera Conchapelopia (Helopelopia), Macropelopia (Bethbilbeckia), Monopelopia (Cantopelopia), Thienemannimyia (Hayesomyia) and Zavrelimyia (Reomyia and Schineriella) are investigated. Our results support all proposed changes, except for the subgenus‐level status of Helopelopia and Cantopelopia. We suggest re‐establishment of Helopelopia as a genus, but refrain from promoting genus‐level status of Cantopelopia at present because the apparent sister‐relationship between Monopelopia + Nilotanypus likely is due to wing vein reduction caused by miniaturisation. This published work has been registered in ZooBank, http://zoobank.org/urn:lsid:zoobank.org:pub:DF012C17‐AFB3‐4904‐83DC‐30DD94D0B376 .     

Genetic relationships among populations of Aedes aegypti from Uruguay and northeastern Argentina inferred from ISSR‐PCR data

Aedes aegypti (L.) (Diptera: Culicidae), the main vector of yellow fever and dengue viruses, was eradicated from Argentina between 1955 and 1963, but reinvaded the country in 1986. In Uruguay, the species was reintroduced in 1997. In this study we used highly polymorphic inter‐simple sequence repeats (ISSR) markers to analyse the genetic structure of Ae. aegypti populations from Uruguay and northeastern Argentina to identify possible colonization patterns of the vector. Overall genetic differentiation among populations was high (F_ST = 0.106) and showed no correlation with geographic distance, which is consistent with the short time since the reintroduction of the species in the area. Differentiation between pairs of Argentine populations (F_ST 0.072 to 0.221) was on average higher than between Uruguayan populations (F_ST?0.044 to 0.116). Bayesian estimation of population structure defined four genetic clusters and most populations were admixtures of two of them: Mercedes and Treinta y Tres (Uruguay) were mixtures of clusters 1 and 3; Salto (Uruguay) and Paraná (Argentina) of clusters 1 and 4; Fray Bentos (Uruguay) of clusters 2 and 3, and Gualeguaychú (Argentina) of clusters 2 and 3. Posadas and Buenos Aires in Argentina were fairly genetically homogeneous. Our results suggest that Ae. aegypti recolonized Uruguay from bordering cities in Argentina via bridges over the Uruguay River and also from Brazil.