Double Restriction Research Articles

Moorean and Tahitian Partula tree snail survival after a mass extinction: New genomic insights using museum specimens

Natural history museum collections provide a biodiversity window into the past and are of particular importance to the study of extinction-impacted clades such as the Pacific Island tree snail family Partulidae. Deliberate introduction of the predatory rosy wolf snail Euglandina rosea in the late 20th century led to the extinction/extirpation of 55/61 Society Island Partulidae species. In this study, we phylogenomically investigated the inter-relationships of the three surviving Society Island valley Partula species: P. taeniata (Moorea), P. clara and P. hyalina (Tahiti). All three formed a distinct clade in earlier mitochondrial phylogenies. Using Next Generation Sequencing (NGS) double digested Restriction Associated DNA sequencing (ddRADseq), we found that 46-year-old lyophilized museum specimens produced similar numbers of reads, sequencing depth, and loci as 10-year old ethanol-preserved collections. Phylogenomic trees indicated that Tahitian P. clara and P. hyalina are the result of a single founding lineage from Moorea, contrasting previous mitochondrial results and clarifying the enigmatic taxonomic status of P. c. incrassa. Our study highlights the utility and viability of NGS techniques for museum specimens and their increased resolution of evolutionary patterns. Sampling will be expanded to include the remaining Society Island partulid taxa to further explore the evolutionary history of this radiation.

Expression, purification and kinetic characterization of recombinant benzoate dioxygenase from Rhodococcus ruber UKMP-5M.

In this study, benzoate dioxygenase from Rhodococcus ruber UKMP-5M was catalyzed by oxidating the benzene ring to catechol and other derivatives. The benzoate dioxygenase (benA gene) from Rhodococcus ruber UKMP-5M was then expressed, purified, characterized, The benA gene was amplified (642 bp), and the product was cloned into a pGEM-T vector. The recombinant plasmid pGEMT-benA was digested by double restriction enzymes BamHI and HindIII to construct plasmid pET28b-benA and was then ligated into Escherichia coli BL21 (DE3). The recombinant E. coli was induced with 0.5 mM isopropyl β-D-thiogalactoside (IPTG) at 22˚C to produce benzoate dioxygenase. The enzyme was then purified by ion exchange chromatography after 8 purification folds. The resulting product was 25 kDa, determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Benzoate dioxygenase activity was found to be 6.54 U/mL and the optimal pH and temperature were 8.5 and 25°C, respectively. Maximum velocity (Vmax) and Michaelis constant (Km) were 7.36 U/mL and 5.58 µM, respectively. The end metabolite from the benzoate dioxygenase reaction was cyclohexane dione, which was determined by gas chromatography mass spectrometry (GC-MS).

Construction of epithelial membrane protein 1 eukaryotic expression vector and its influence on migration and invasion of human oral tongue squamous carcinoma cells

This study aimed to construct a eukaryotic expression vector pEGFP-N1-EMP1 of epithelial mem-brane protein 1 (EMP1) and investigate its influence on migration and invasion of human oral tongue squamous carcinoma cells. The human EMP1 gene was amplified by reverse transcription polymerase chain reaction and then ligated into the pEGFP-N1 vector by double restriction endonuclease digestion to construct pEGFP-N1-EMP1 recombinant plasmid. After sequencing identification, pEGFP-N1-EMP1 recombinant plasmid and pEGFP-N1 plasmid were transfected into human oral tongue squamous carcinoma Tb3.1 cell line. The expression of green fluorescent protein in cells was observed after transfection using an inverted fluorescence microscope. The overexpression of EMP1 mRNA was identified at 24, 48, and 72h after transfection by real-time fluorescence quantitative polymerase chain reaction. The effect of EMP1 overexpression on migration and invasion of Tb3.1 cells was detected by Transwell assay. The full-length EMP1 gene sequence was successfully obtained. Sequence analysis showed that the EMP1 gene was inserted into the pEGFP-N1 vector correctly. Green fluorescence was observed in the transfected cells under fluorescence microscopy. The results of real-time fluorescence quantitative polymerase chain reaction indicated that the expression of EMP1 at 24h after pEGFP-N1-EMP1 transfection was significantly higher than the other groups. Transwell assays indicated that overexpression of the EMP1 gene could significantly inhibit the migration and invasion ability of Tb3.1 cells. The eukaryotic expression vector of EMP1 was successfully constructed, and EMP1 overexpression was confirmed to inhibit the migration and inva-sion of oral tongue squamous carcinoma cells in vitro. This study laid a foundation for further investigation on the influence of the EMP1 gene on the metastasis of oral tongue squamous carcinoma and its molecular mechanism. .

Insights into the Electronic Structure of Molecules from Generalized Valence Bond Theory

In this article we describe the unique insights into the electronic structure of molecules provided by generalized valence bond (GVB) theory. We consider selected prototypical hydrocarbons as well as a number of hypervalent molecules and a set of first- and second-row valence isoelectronic species. The GVB wave function is obtained by variationally optimizing the orbitals and spin coupling in the valence bond wave function. The GVB wave function is a generalization of the Hartree-Fock (HF) wave function, lifting the double occupancy restriction on a subset of the HF orbitals as well as the associated orthogonality and spin coupling constraints. The GVB wave function includes a major fraction (if not all) of the nondynamical correlation energy of a molecule. Because of this, GVB theory properly describes bond formation and can answer one of the most compelling questions in chemistry: How are atoms changed by molecular formation? We show that GVB theory provides a unified description of the nature of the bonding in all of the above molecular species as well as contributing new insights into the well-known, but poorly understood, first-row anomaly.

Prokaryotic Expression of the Oligopeptide Transporter hPepT2(560-663)

Peptide transporter 2 is a high affinity and low capacity transporter, that has recently been found in the mammalian kidney, brain, lung, and mammary glands, which is known to transport dipeptides and tripeptides and to reabsorb small peptides generated by luminal peptidases in the kidney. In addition, peptide transporter 2 can transport peptide-like drugs and play important roles in the transshipment and positioning of drugs. In this paper, an E.coli BL21(DE3) containing pET30a(+)-hPepT2(560-663) recombinant plasmid was obtained by using PCR, double restriction enzyme digestion, ligation, and transformation. Then, the effect of changing both the induction time and induction dose using Isopropyl β-D-1-Thiogalactopyranoside (IPTG) on the bacterial expression of hPepT2(560-663) was detected by SDS-PAGE electrophoresis, the optimum expression conditions (at 37°) were identified as: 3 h and a working concentration of 0.5 mM Isopropyl β-D-1-Thiogalactopyranoside. Finally, we purified the target protein hPepT2(560-663) via Sephadex G-75 gel chromatography. These results will facilitate in future studies on the nature and function of the protein.

Double Fuzzy Implications-Based Restriction Inference Algorithm

The main condition of the differently implicational inferencealgorithm is reconsidered from a contrary direction, which motivatesa new fuzzy inference strategy, called the double fuzzyimplications-based restriction inference algorithm. New restrictioninference principle is proposed, which improves the principle of thefull implication restriction inference algorithm. Furthermore,focusing on the new algorithm, we analyze the basic property of itssolution, and then obtain its optimal solutions aiming at theproblems of fuzzy modus ponens (FMP) as well as fuzzy modus tollens(FMT). Lastly, comparing with the full implication restrictioninference algorithm, the new algorithm can make the inferencecloser, and generate more, better specific inference algorithms.

A set of 204 SNP and INDEL markers for Bigleaf maple (Acer macrophyllum Pursch)

Novel single nucleotide polymorphisms (SNPs) and insertion/deletions (INDELs) were identified for the Bigleaf maple (Acer macrophyllum Pursch) using a combined next generation sequencing approach on the Ion Torrent PGM system with genotyping on the MassARRAY® iPLEX™ platform. Five hundred and ninety-eight putative loci were identified through sequencing of DNA fragments following a double restriction enzyme digest method. Two hundred and four polymorphic loci (199 SNPs and five INDELs) were successfully amplified across 65 individuals from seven populations across the native range of the species. Twenty-nine loci showed evidence of deviation from Hardy–Weinberg Equilibrium, and 85 were significantly linked. Expected heterozygosity ranged from 0.015 to 0.472 and F ST from 0.011 to 0.359. These genetic resources will prove useful for future studies into the population genetics and phylogeography of this important and iconic timber species.

Some combinatorial applications of the q-Riordan matrix

Recently, the authors developed a q-analogue for Riordan matrices by means of Eulerian generating functions of the form g(z)=∑n≥0gnzn/n!q where n!q is the q-factorial. We apply this concept to give q-analogues of some familiar objects from the set partitions with double restrictions on blocks, namely the (r,s)-Bessel numbers of both types. By setting r=0 and letting s→∞, these numbers may be reduced to the q-Stirling numbers of both kinds. Several algebraic formulas for the q-analogues are also derived using combinatorial methods together with the concept of q-Riordan matrices. In particular, q-analogues of the classical Bessel numbers of both kinds and their combinatorial interpretations are obtained.

Management accunting its constraints

The organizational transformation that happened in the world economy recent decade was accompanied by the concepts of less-profitable production and flexible system. The concepts implicitly provide a production and working capacity that is consistent with demand and current capacity of acceptance. The origin of limited management was higher according to efficiency and imbalance between resources and demand for the resources. There are double restrictions for the private institutions and in all Irish economic fields. Methodology: The international institutions face with the global limitations about less-profitable productive models. The non-commercial institutes are facing new problems that are related to the lack of employees and other resources while there were a recession and low efficiency about resources. Goldratt in her book providing theories about constrains provides new fields in economic perspective (Goldratt and Cooks, 1993). These managing ideas of best salesman are describing as an exciting story that describes the manager’s attempts to prevent the loss caused by performance and the bankruptcy of the company. Results: In the story, Goldratt and Descooks attacked to the price finished accounting as an “enemy to productivity” rather than using developed holistic techniques as on-time production and statistic control of the process. In this article, the relative usefulness of the theory of constraints related to accounting business managing methods is explained to use the resources and decision-making. Conclusion: The article, also examines the spread and evaluation of constrains and reactions that cause accounting and managing culture. Survival of the price finished management that is explaining against the continuous attacks is consistent with the probabilities by Goldratt relating to regulations ability to make changes and obligatory developments as training programs.

Utilizing Ligation Independent Cloning in an Undergraduate Biochemistry Laboratory Course

As a continuation of the transformation of our laboratory course to a more research focused experience we tested whether the Ligation Independent Cloning (LIC) technique would be suitable for students with little to no prior experience in molecular biology methodologies. To produce an expression system for for E. coli alkaline phosphatase (PhoA) we cloned its gene into the pETHSUL vector kindly donated by Dr. Patrick Loll. The construct expresses a fusion protein where a His‐tagged SUMO protein is fused to PhoA. The sumo moiety can be proteolytically released from PhoA following affinity column purification. We compare the ease of building the pETHSUL‐PhoA construct and enzyme activity data for the purified enzyme with an expression system developed previously in our class using a classical double restriction enzyme sub‐cloning into a pET29a vector that incorporates a His‐tag at the C‐terminus of PhoA.

Research on Bearing Capacity of Concrete Filled Double Skin (CHS Inner and SHS Outer) under Eccentric Compression

The ultimate load calculation formula for the solid multi barrel tube-confined concrete columns (CHS inner and SHS outer) was derived based on the Unified Strength Theory (UST) in this paper. The influence of intermediate principal stress and the double restriction effect and the decrease of longitudinal stress were considered in the formula. Based on the axial compression load and consider the eccentricity ratio and slenderness ratio, by introducing the reduction factor of concrete strength and the equivalent restriction reduction factor, based on unified strength theory solutions of thick tube, combined with concrete filled skin tube unified theory, deduce the calculation formula of eccentric compression bearing capacity of concrete filled steel tubular columns and analysis the influence complication.

Ambiguity resolution with double troposphere parameter restriction for long range reference stations in NRTK System

The correct ambiguity resolution between reference stations is the key to calculate the high precision Network Real-Time Kinematic (NRTK) differential information. For long range reference stations (≧50 km), the double difference troposphere model residuals should be considered as the parameters being solved, but this will aggravate the ill conditioning of ambiguity resolution (AR) model between reference stations; as a result, the ambiguity fixing becomes more difficult for the case of long range station ambiguity resolution. In the paper, a new method with double troposphere parameters restriction is put forward for ambiguity resolution of long range reference stations. The proposed method applies GPT2 model, which is called the state of the art empirical troposphere model, to form a high precision troposphere a priori estimation to provide high accuracy double difference troposphere delay estimation. Based on the principles of TIKHONOV regularisation, a regularisation criterion for the double difference restriction model is then built. The difference between the troposphere estimation and the truth value is used as a restriction parameter to improve the estimation of the unknown parameters and optimisation of the ambiguity search range. Trials verify the significant reduction in the ill conditioning of the parametric resolution functions when the double troposphere restriction model is applied. The success rate of ambiguity resolution within 60 s is above 98% for baselines over 80 km, which is an immense improvement from conventional methods.

Methionine and cystine double deprivation stress suppresses glioma proliferation via inducing ROS/autophagy

Cancer cells are highly dependent on methionine and cystine (Met–Cys) for survival and proliferation. However, the molecular mechanism is not fully clear. The present study is to investigate the effects of Met–Cys deprivation on glioma cells proliferation. The results showed that Met–Cys double deprivation had synergistic action on elevating ROS level, decreased GSH level and inhibition of glioma cell proliferation. Moreover, both of them deprivation triggered autophagy of glioma cells both in vitro and in vivo. Importantly, Met–Cys double restriction diet inhibited growth of glioma. These results provided a new regulation mechanism of Met–Cys metabolism on affecting glioma cell proliferation, suggesting that targeting Met–Cys metabolism may be a potential strategy for glioma therapy.

Genotyping the GALNT14 gene by joint analysis of two linked single nucleotide polymorphisms using liver tissues for clinical and geographical comparisons.

A GALNT14 single nucleotide polymorphism, rs9679162, has recently been found to be capable of predicting chemotherapy responses in patients with far-advanced hepatocellular carcinoma (HCC). In the present study, a novel assay was designed and genotyping was performed on 244 surgically removed liver tissues. This assay employed two polymerase chain reaction (PCR)-generated restriction enzyme sites to simultaneously determine the genotypes of two adjacent single nucleotide polymorphisms (SNPs), rs9679162 and rs6752303, on the GALNT14 gene. Genotypes determined by this assay reached 100% concordance with those detected by the direct sequencing method. Clinical analysis showed that the TT genotype of rs9679162 was lower in percentage among patients with virus-originated HCC compared with those with non-viral HCC (22.57 vs. 47.06%, respectively; P=0.023). The proportion of the TT genotype in the 244 HCC patients (24.18%) did not deviate significantly from those of two public-domain (HapMap) Chinese cohorts from Denver, Colorado, USA (28.44%) and Beijing, China (30.15%) (P>0.05). The proportion of the TT genotype was significantly higher in Japanese and African populations (42.11–54.55%; P<0.0001) but significantly lower in an Italian cohort (7.84%; P=0.0004). In conclusion, the novel PCR-generated double restriction enzyme sites method could correctly determine the genotypes of two target SNPs in GALNT14 in liver tissues. The TT genotype was associated with the non-viral etiology of HCC. A marked variation in ethnicity was found for the distribution of this genotype.

A Method of Multi-View Images 3D Restructuring of Discrete Line Segments

In this paper, the average reprojection geometrical errors on all images of a spatial straight line is taken as the restructuring and optimization target function to ensure the optimal result to be acquired; a spatial straight line is expressed by use of the Plücker coordinates for the proposal of the analytical method of correcting the double linear restriction on the Plücker coordinates of noise-included spatial straight lines; during the optimization process, to ensure parameters that meet double linear restriction, the iteration renewal process is expressed by at least 4 parameters, to increase the precision of restructuring results. Experiments results derived from simulation data and real images have all demonstrated the high efficiency and precision of the algorithm proposed in this paper.

Lubricating Grease Shear Flow and Boundary Layers in a Concentric Cylinder Configuration

Grease is extensively used to lubricate various machine elements such as rolling bearings, seals, and gears. Understanding the flow dynamics of grease is relevant for the prediction of grease distribution for optimum lubrication and for the migration of wear and contaminant particles. In this study, grease flow is visualized using microparticle image velocimetry (μPIV). The experimental setup includes a concentric cylinder configuration with a rotating shaft to simulate the grease flow in a double restriction seal geometry with two different grease pocket sizes. It is shown that the grease is partially yielded in the large grease pocket geometry and fully yielded in the small grease pocket. For the small grease pocket, it is shown that three distinct grease flow layers are present: a high shear rate region close to the stationary wall, a bulk flow layer, and a high shear rate boundary region near the rotating shaft. The grease shear thinning behavior and its wall slip effects have been identified. The μPIV experimental results have been compared with a numerical model for both the large and small gap size. It is shown that the flow is close to one-dimensional in the center of the small pocket. A one-dimensional analytical model based on the Herschel-Bulkley rheology model has been developed, showing good agreement with the measured velocity profiles in the small grease pocket. Furthermore, wall slip effects and shear banding are observed, where the latter imply that using the assumption of uniform shear in conventional concentric cylinder rheometers may result in erroneous rheological results.

Effect of combo mutation of core promoter on expression of proliferation cell nuclear antigen, nuclear factor-κB and p21

Objective The A1762T/G1764A/T1753A/T1768A combo mutant core promoter was constructed through the gene synthesis,and infected into L-02 cells.The expression of proliferation cell nuclear antigen (Ki-67),nuclear factor-κB (NF-κB) and p21 was investigated.Methods In order to obtain recombinant plasmid pLenO-RTP-CM-HBX,the pLenO-RTP carrier and pUC57-CM-HBX were connected directly after double restriction enzyme digestion.The recombinant plasmid was transfected into 293T cells for virus packaging through calcium turn method,and then infected into L-02.The expression of HBXFlag,Ki-67,NF-κB and p21 was detected by Western blotting.Results The pLenO-RTP-CM-HBX core promoter lentivirus carrier could infected into L-02 cells with high efficiency (＞ 95％).The HBX-Flag could be transcribed,translated,synthetized and secreted in L-02 cells after transfection.The red fluorescence intensity did not change obviously with the extension of incubation time.The Ki-67,NF-κB,p21 expression gray in mutant group were 2.103 ±0.346,2.201 ±0.486,0.771 ±0.338; the expressions gray in light group were 0.221 ± 0.016,0.232 ± 0.117,2.032 ± 0.215 ; and the expressions gray of Ki-67,NF-κB,p21 in blank control group were 0.323 ±0.017,0.334 ±0.207,2.141 ±0.305.The expression of Ki-67 and NF-κB in mutant group was increased,and that of p21 was reduced compared to light group and the blank control group (P ＜ 0.05).There was no significant difference between the light group and the blank control group.Conclusion The combo mutation of core promoter can influence the expression of Ki-67,NF-κB and p21. Key words: Hepatitis B virus; Core promoter; Combo mutation

Polycarbonate foams with tailor-made cellular structures by controlling the dissolution temperature in a two-step supercritical carbon dioxide foaming process

Closed-cell polycarbonate foams were prepared using a two-step foaming process, which consisted of the initial dissolution of supercritical CO2 (scCO2) into PC foaming precursors and their later expansion by heating using a double contact restriction method. The effects of the parameters of both CO2 dissolution and heating stages on the cellular structure characteristics as well as on the physical aging of PC in the obtained foams were investigated. A higher amount of CO2 was dissolved in PC with increasing the dissolution temperature from 80 to 100°C, with similar CO2 desorption trends and diffusion coefficients being found for both conditions. PC foams displayed an isotropic-like microcellular structure at a dissolution temperature of 80°C. It was shown that it is possible to reduce their density while keeping their microcellular structure with increasing the heating time. On contrary, when dissolving CO2 at 100°C and later expanding, PC foams presented a cellular morphology with bigger cells and with an increasingly higher cell elongation in the vertical growth direction with increasing the heating time. Comparatively, PC foams obtained by dissolving CO2 at 100°C presented a more marked physical aging after CO2 dissolution and foaming, although this effect could be reduced and ultimately suppressed with increasing the heating time.

Effects of KAP-1 in promoting the epithelial-mesenchymal transition of pancreatic cancer cell line Capan-2

Objective To investigate the effects of KAP-1 in promoting the epithelial-mesenchymal transition (EMT) of pancreatic cancer cell line Capan-2.Methods The lentiviral vector of LV-plenti-GFP-KAP-1 was constructed.Capan-2 cells were divided into the experimental group (cells transfected by lentiviral vector of LVplenti-GFP-KAP-1),negative control group (cells transfected by empty vector) and blank control group 1 (cells cultured in 1640 medium plus 10％ fetal calf serum).Capan-2 cells in the experimental group were subdivided into the miR-100-5p inhibitor transfection group (cells transfected with miR-100-5p inhibitor),empty vector control group (cells transfected with microRNAs inhibitor),blank control group 2 (cells cultured in 1640 medium plus 10％ fetal calf serum).The lentivirus was identified by double endonuclease restriction and sequencing,and the virus titer was detected.The morphological changes of the cells were observed after transfecting lentiviral vector of LV-plenti-GFP-KAP-1 to the Capan-2 cells.The expressions of KAP-1,genes of EMT proteins and mRNA of miR-100-5p were detected by real-time quantitative polymerase chain reaction.The protein expressions of KAP-1,EMT proteins in all the groups were detected by Western blot.The measurement data were presented by mean ± standard deviation,and were analyzed using the analysis of variance.Results The lentiviral vector of LV-plentiGFP-KAP-1 was successfully constructed,and the virus titer was 2 × 108 TU/ml.Compared with the control group,the mesenchymal transition of the Capan-2 cells was detected in the experimental group after transfecting the Capan-2 cells with lentiviral vector of LV-plenti-GFP-KAP-1 for 48 hours.The relative mRNA expressions of KAP-1,N-cadherin,vimentin,E-cadherin,miR-100-5p were 1.77 ± 0.83,2.62 ± 0.71,2.50 ± 0.21,7.20 ± 1.17 and 1.81 ±0.40 in the experimental group,5.03 ±0.29,5.07 ±1.53,3.83 ±0.57,7.83 ±0.78,7.01 ± 0.96 in the negative control group,5.13 ± 1.14,5.81 ± 1.49,4.92 ± 0.90,3.07 ± 0.36,6.87 ± 0.35 in the blank control group 1,with significant difference among the 3 groups (F =5.99,7.62,7.88,6.62,4.64,P ＜0.05).The relative mRNA expression of KAP-1 in the miR-100-5p inhibitor transfection group,empty vector control group,blank control group 2 were 1.56 ± 0.42,4.89 ± 0.61,5.20 ± 0.38,with significant difference among the 3 groups (F =5.14,P ＜ 0.05).The relative mRNA expressions of vimentin in the miR-100-5p inhibitor transfection group,empty vector control group,blank control group 2 were 3.10 ± 1.37,3.44 ± 0.94,3.08 ±1.16,with no significant difference among the 3 groups (F =0.49,P ＞ 0.05).The results of western blot showed that the relative protein expressions of KAP-1,N-cadherin,vimentin,E-cadherin were 2.77 ± 1.99,1.31 ±0.38,4.25 ± 0.63,0.62 ± 0.06 in the experimental group,0.83 ± 0.46,0.41 ± 0.37,1.03 ± 0.33,1.17 ± 0.45 in the negative control group,0.71 ± 0.26,0.08 ± 0.04,1.37 ± 0.92,3.04 ± 0.65 in the blank control group 1,with significant difference among the 3 groups (F =5.54,4.68,3.19,8.18,P ＜ 0.05).The relative protein expression of KAP-1 in the miR-100-5p inhibitor transfection group,empty vector control group,blank control group 2 were 2.27 ±0.71,0.56 ±0.43,0.61 ±0.39,with significant difference among the 3 groups (F =4.81,P ＜0.05).The relative protein expressions of vimentin in the miR-100-5p inhibitor transfection group,empty vector control group,blank control group 2 were 3.19 ± 0.55,3.93 ± 0.06,3.61 ± 0.73,with no significant difference among the 3 groups (F =0.04,P ＞ 0.05).Conclusion KAP-1 promotes the EMT of Capan-2 cells by specifically down-regulating the miR-100-5p expression. Key words: Pancreatic neoplasms; KAP-1 ; Epithelial-mesenchymal transition ; microRNAs

O contexto religioso-político da contraposição entre pirronismo e academia na "Apologia de Raymond Sebond"

Montaigne faz um ataque pirrônico ao conceito acadêmico de verossimilhança ou probabilidade na Apologia de Raymond Sebond. O ataque é paradoxal porque Montaigne parece seguir o verossímil na própria Apologia e em diversos outros ensaios. Para resolver este problema exegético proponho uma dupla restrição do escopo do ataque à verossimilhança. Por um lado, mostro que o ataque visa mais a leitura epistêmica da verossimilhança proposta por Filo de Larissa do que ao conceito original de ordem exclusivamente prática de Carnéades. Por outro, situo-o em um contexto político-religioso bem específico. O ataque pirrônico à verossimilhança é a estratégia oferecida por Montaigne à rainha católica de Navarra e irmã do rei da França, Marguerite de Valois, para eventual uso nas polêmicas religiosas em sua corte majoritariamente protestante de Nérac. Esta contextualização soluciona também outros problemas exegéticos da Apologia, como o da defesa paradoxal de Sebond, a inconsistência aparente entre as respostas de Montaigne às duas objeções feitas ao livro de Sebond, e o problema do fideísmo.