Hybrid Verification Research Articles

Histologic subtyping of poorly-differentiated solid lung cancer with molecular testing for epidermal growth factor receptor mutation and ALK gene rearrangement: an analyses of 167 cases

Objective: To study the histological subtyping of poorly differentiated solid lung cancer by using immunohistochemistry and mucin staining along with analysis of epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) gene rearrangement. Methods: Among 827 cases of non-small cell lung cancer at Beijing Hospital from April 2014 to April 2017, 167 cases of solid poorly differentiated lung cancer were identified and histopathologically subtyped by mucin staining (D-PAS) and immunohistochemistry using 10 antibodies (CK7, vimentin, Ki-67, CK5/6, p40, TTF1, Napsin A, CD56, chromogranin A, and synaptophysin). Paraffin embedded tumor samples were subjected to mutation analysis of exons 18, 19, 20 and 21 of the EGFR gene by amplification refractory mutation system (ARMS) method. Immunohistochemistry (Ventana D5F3) for ALK gene rearrangement was performed followed by ALK fluorescence in situ hybridization (FISH) verification. Results: There were 79 females and 88 males in the study cohort. The patient's age ranged from 35 to 77 years (mean 62 years). Cases with solid growth pattern (at least >10%) and without typical histological features of adenocarcinoma, squamous cell carcinoma or neuroendocrine carcinoma were further divided based on immunohistochemistry and mucin stain into 64 cases(38.32%)of adenocarcinoma, 34 cases(20.35%) squamous cell carcinoma, 21 cases(12.57%)large cell neuroendocrine carcinoma, 5 cases(2.99%)combined large cell neuroendocrine carcinoma, 2 cases(1.20%)adenosquamous carcinoma and 41 cases(24.55%)large cell carcinoma. The Ki-67 positive rate ranged from 5% to 65%. Mutations of EGFR were detected in 5 cases (2.99%, 5/167) of adenocarcinoma(19del in 3 cases and L858R in 2 cases). Two cases(1.20%, 2/167) with ALK-rearranged were identified by immunohistochemistry (Ventana D5F3) and confirmed by ALK FISH. Conclusions: Poorly differentiated solid lung cancer without distinct morphological features can be further histologically subtyped by mucin staining and immunohistochemistry. Molecular testing should be performed for accurate molecular target therapy to improve the prognosis.

Assessment of myoblast circular RNA dynamics and its correlation with miRNA during myogenic differentiation.

Myoblast differentiation is a highly complex process that is regulated by proteins as well as by non-coding RNAs. Circular RNAs have been identified as an emerging new class of non-coding RNA in the modulation of skeletal muscle development, whereas their expression profiles and functional regulation in myoblast differentiation remain unknown. In the present study, we performed deep RNA-sequencing of C2C12 myoblasts during cell differentiation and uncovered 37,751 unique circular RNAs derived from 6943 hosting genes. The ensuing qRT-PCR and RNA fluorescence in situ hybridization verification were carried out to confirm the RNA-sequencing results. An unbiased analysis demonstrated dynamic circular RNA expression changes in the process of myoblast differentiation, and the circular RNA abundances were independent from their cognate linear RNAs. Gene ontology analysis showed that many down-regulated circular RNAs were exclusive to cell division and the cell cycle, whereas up-regulated circular RNAs were related to the cell development process. Furthermore, interaction networks of circular RNA-microRNA were constructed. Several microRNAs well-known for myoblast regulation, such as miR-133, miR-24 and miR-23a, were in this network. In summary, this study showed that circular RNA expression dynamics changed during myoblast differentiation. Circular RNAs play a role in regulating the myoblast cell cycle and development by acting as microRNA binding sites to facilitate their regulation of gene expression during myoblast differentiation. These findings open a new avenue for future investigation of this emerging RNA class in skeletal muscle growth and development.

Modelling, formal refinement and partitioning strategies for a small aircraft fuel pump system in Hybrid Event-B

A case study centred on a fuel supply system for a small aircraft is presented in Hybrid Event-B, an extension of conventional Event-B that allows for the modelling and verification of hybrid and cyberphysical systems exhibiting nontrivial continuous behaviour. In contrast to many such case studies, which concentrate predominantly on timing issues, the focus in the present work is on nontrivial physical behaviour, and on the effect that this has on various refinement and partition strategies. More liberal proof obligations are developed to add flexibility to the decomposition process. • Hybrid Event-B modelling of an aircraft fuel system. • Investigation of partitioning strategies. • New verification conditions for decomposition.

Verification of linear hybrid systems with large discrete state spaces using counterexample-guided abstraction refinement

We present a counterexample-guided abstraction refinement ( CEGAR) approach for the verification of safety properties of linear hybrid automata with large discrete state spaces, such as naturally arising when incorporating health state monitoring and degradation levels into the controller design. Such models can – in contrast to purely functional controller models – not be analyzed with hybrid verification engines relying on explicit representations of modes, but require fully symbolic representations for both the continuous and discrete part of the state space. The presented abstraction methods directly work on a symbolic representation of arbitrary non-convex combinations of linear constraints and boolean variables using LinAIGs. Several interpolation methods allow us to compute abstractions consisting of fewer linear constraints, and hence reduce the complexity of the reachable state set computation. In combination with methods that guarantee the preciseness of abstractions, this leads to a significant reduction of the runtimes of the verification process compared with exact verification. • We propose a counterexample guided abstraction refinement approach for linear hybrid systems. • The approach relies on abstraction algorithms replacing the original state set by state sets of simpler shape. • We provide benchmark results showing the relative merits of the approach.

Verification of Power-Management Specification at Early Stages of Power-Constrained Systems Design

Nowadays, power is a dominant factor that constrains highly integrated hardware-systems designs. The implied problems of high power density, causing chip overheating, or limited power source in modern Internet-of-Things devices are most commonly dealt with the use of the dynamic power management. This method enables to use power-reduction techniques, such as clock gating, power gating, or voltage and frequency scaling. Since the adoption of power management is quite difficult in modern complex systems, there are new approaches evolving intended to simplify power-constrained systems design. We have also proposed such an approach, utilizing the system level of design abstraction and increased automation in the design process. In this paper, the proposed hybrid verification approach is described that represents an integral part of the suggested design methodology. It consists of formal and informal techniques, enabling the verification process to begin at the very early specification stage of the system development. Our approach helps a designer to create correct and consistent power-management specification and verifies whether the specified power intent is preserved after design refinement. The continuous automated verification steps can quickly find errors at early design stages and thus reduce the amount of design re-spins, which speeds-up the overall development process.

Microfluidic hydrogel arrays for direct genotyping of clinical samples

A microfluidic hydrogel DNA microarray is developed to overcome the limitations of conventional planar microarrays such as low sensitivity, long overnight hybridization time, lack of a melting verification of proper hybrid, and complicated sample preparation process for genotyping of clinical samples. Unlike our previous prototype hydrogel array which can analyze only single-stranded DNA (ssDNA) targets, the device is the first of its type to allow direct multiplexed single nucleotide polymorphism (SNP) detection of human clinical samples comprising double-stranded DNA (dsDNA). This advance is made possible by incorporating a streptavidin (SA) hydrogel capture/purification element in a double T-junction at the start of the linear hydrogel array structure and fabricating ten different probe DNAs-entrapped hydrogels in microfluidic channels. The purified or unpurified polymerase chain reaction (PCR) products labeled with a fluorophore and a biotin are electrophoresed through the SA hydrogel for binding and purification. After electrophoretic washing, the fluorophore-labeled DNA strand is then thermally released for hybridization capture by its complementary probe gel element. We demonstrate the precise and rapid discrimination of the genotypes of five different clinical targets by melting curve analysis based on temperature-gradient electrophoresis within 3h, which is at least 3-fold decrease in incubation time compared to conventional microarrays. In addition, a 1.7pg (0.024 femtomoles) limit of detection for clinical samples is achieved which is ~100-fold better sensitivity than planar microarrays.

Scene text detection using adaptive color reduction, adjacent character model and hybrid verification strategy

Text detection is a primary task for text recognition and understanding, which can be used in many image analysis techniques. In this paper, we propose an effective scene text detection method including three major steps: connected components (CCs) extraction, character-linking and text/non-text classification. First, for CCs extraction, we design an adaptive color reduction scheme by analyzing image color histogram, which reasonably selects color centers and generates unfixed number of color layers for images in different color complexities. Then, for character-linking, an adjacent character model is built by training an extreme learning machine (ELM), instead of setting various thresholds in previous approaches. Finally, a hybrid text verification strategy is adopted, combining convolutional neural network with ELM for text/non-text classification and performing better than just using one of them. Experimental results on some publicly available datasets illustrate the effectiveness of our method and comparative results with some state-of-the-art algorithms demonstrate our competitiveness.

Scalable and Optimized Hybrid Verification of Embedded Software

The verification of embedded software has become an important subject over the last years. However, neither standalone verification approaches, like simulation-based/formal verification, nor state-of-the-art semiformal verification approaches are able to verify large and complex embedded software with or without hardware dependencies. This work presents a scalable hybrid verification approach for the verification of embedded software using a semiformal algorithm optimized with static parameter assignment (SPA). These algorithms and methodologies like SPA and counterexample guided simulation are used to combine simulation-based and formal verification in a new way. SPA offers a method to interact between dynamic and static verification approaches based on an automated ranking determination of possible function parameters according to the impact on the model size. Furthermore, SPA inserts initialization code for specific function parameters into the source code under test and supports model building and optimization algorithms to reduce the state space. We have successfully applied this optimized hybrid verification methodology to embedded software applications: Motorola's Powerstone Benchmark suite and a complex automotive industrial embedded software. The results show that our approach scales better than standalone software model checkers to reach deep state spaces.

Integrodifferential formulations of the continuous-time random walk for solute transport subject to bimolecular A+B→0 reactions: From micro- to mesoscopic.

We develop continuous-time random walk (CTRW) equations governing the transport of two species that annihilate when in proximity to one another. In comparison with catalytic or spontaneous transformation reactions that have been previously considered in concert with CTRW, both species have spatially variant concentrations that require consideration. We develop two distinct formulations. The first treats transport and reaction microscopically, potentially capturing behavior at sharp fronts, but at the cost of being strongly nonlinear. The second, mesoscopic, formulation relies on a separation-of-scales technique we develop to separate microscopic-scale reaction and upscaled transport. This simplifies the governing equations and allows treatment of more general reaction dynamics, but requires stronger smoothness assumptions of the solution. The mesoscopic formulation is easily tractable using an existing solution from the literature (we also provide an alternative derivation), and the generalized master equation (GME) for particles undergoing A+B→0 reactions is presented. We show that this GME simplifies, under appropriate circumstances, to both the GME for the unreactive CTRW and to the advection-dispersion-reaction equation. An additional major contribution of this work is on the numerical side: to corroborate our development, we develop an indirect particle-tracking-partial-integro-differential-equation (PIDE) hybrid verification technique which could be applicable widely in reactive anomalous transport. Numerical simulations support the mesoscopic analysis.

Development of polymorphic genic-SSR markers by cDNA library sequencing in boxwood, Buxus spp. (Buxaceae).

• Premise of the study: Genic microsatellites or simple sequence repeat (genic-SSR) markers were developed in boxwood (Buxus taxa) for genetic diversity analysis, identification of taxa, and to facilitate breeding.• Methods and Results: cDNA libraries were developed from mRNA extracted from leaves of Buxus sempervirens ‘Vardar Valley’ and sequenced using the Illumina MiSeq system. Approximately 11.9 million base pairs of sequence data were examined and 845 genic-SSRs were identified, including 469 dinucleotide, 360 trinucleotide, seven tetranucleotide, one pentanucleotide, and eight hexanucleotide repeats. Primer pairs were designed for 71 selectively chosen genic-SSRs containing trinucleotide repeat motifs and were used to amplify the corresponding loci in 18 diverse boxwood accessions. Twenty-three primer pairs amplified polymorphic loci, with two to 10 alleles per locus.• Conclusions: These novel polymorphic genic-SSR markers will aid in evaluating genetic diversity of boxwood germplasm and allow verification of hybrids and cultivars for breeding programs.

Efficient resynthesis of oilseed rape (Brassica napus L.) from crosses of winter types B. rapa × B. oleracea via simple ovule culture and early hybrid verification

Resynthesis of Brassica napus is an important tool for the broadening of genetic diversity as well as crop cultivar improvement in oilseed rape. To the best of our knowledge, this is the first study focusing on the crossability, regeneration, and early verification of hybrids between diploid winter turnip rapes (Brassica rapa ssp. oleifera f. biennis), as the female component, and diploid winter curly kales (Brassica oleracea convar. acephalla var. sabellica). Pollination efficiency, mean embryo regeneration per bud and siliqua, as well as their relationships were evaluated in ovule cultures of twenty-four different one-sided crosses between six accessions of B. rapa and four accessions of B. oleracea. Successful germination of the embryos was achieved in 23 combinations. The mean number of embryos per bud reached 0.34. The most productive cross, Svalofs Duro × Kapral, yielded 2.7 embryos per bud in the best replication. A significant and positive correlation (r = 0.26*) was detected between the pollination efficiency (i.e., setting of siliquae) and the number of embryos per siliqua. In the majority of crosses, procedures implemented exhibited better results than in similar published experiments on spring Brassica genotypes, and provides a simplified and less laborious method. The hybrid nature of all regenerants was verified via flow cytometry. The use of bulked samples for analysis was reliable due to highly significant differences in relative DNA content between all hybrid combinations and their respective parental components. Chromosome counting and morphological assessment further corroborated the results of the flow cytometric analysis.

Application of RAPD markers in hybrid verification in coconut

Coconut palms are classified into two major types, viz., 'talls' and 'dwarfs', which mainly differ in their pollination behavior of cross- and self-pollination, respectively. Due to this difference, getting true-to-type progenies of desirable tall and dwarf cultivars has always been a challenge. The conventional practice of selection of seedlings based solely on morphological traits often results in selection of out-crossed seedlings and undesirable off-types. In the present investigation, RAPD markers for the tall/dwarf trait were identified in coconut using a bulked DNA approach. Screening of tall and dwarf palm bulk DNA with 200 primers revealed a RAPD primer OPBA3 which was able to clearly differentiate both the tall and dwarf bulks. For validation, the primer was used to screen individual tall and dwarf coconut palms representing different geographic regions. The primer was also used to screen the parents and validate hybrids of Dwarf x Tall crosses.

Translation from Multisingular Hybrid Petri Nets to Multisingular Hybrid Automata

In this paper, we investigate some important aspects of a new formalism for modelling and verification of hybrid dynamic systems HDS, which is called multisingular hybrid Petri nets MSHPNs. This new hybrid formalism is aimed to bridge the gap between hybrid automata HA and hybrid Petri nets HPNs by equipping the HPN model with the capabilities of HA to control the execution and firing of timed transitions. Practically, MSHPNs can be considered as the counterpart with the same expressive power as multisingular hybrid automata MSHA. In order to analyse MSHPN models, a speed-based partitioning technique has been introduced in which the variable space is partitioned based on the balance of continuous places. In this paper, we formalize the notions of conflicts and conflict resolution and the challenging issue of speed computation. Then, we focus on considering a translation from a bounded MSHPN to a multisingular hybrid automaton that preserves the behavioural semantics of the original MSHPN in terms of weak timed bisimulation. The translation algorithm uses the speed-based partitioning method and obtains a speed-based partitioning hybrid automaton for a given bounded MSHPN. Model checking a timed property for an MSHPN amounts to model checking its equivalent property on the obtained speed-based partitioning hybrid automaton, thus MSHPN models can be analysed using the existing tools. The advantages of the proposed method are twofold: 1 hybrid systems can be described more succinctly and therefore more readably as MSHPNs, and 2 one can use the existing tools like HYTECH to analyse MSHPN models.

Asymmetric Somatic Plant Hybridization: Status and Applications

To create asymmetric somatic hybrids, the genome of the so-called donor protoplast is fragmented prior to protoplast fusion. As a result, only a limited amount of the donor genome is transferred to the fusion product. This technique can circumvent some commonly observed problems related to symmetric fusion and offers a practical breeding tool for asexual hybridization. Genomes are typically fragmented by irradiation, microprotoplast production or application of metabolic inhibitors such as iodoacetamide. Irradiation and microprotoplast production fragment the nuclear genome, whereas iodoacetamide inactivates the cytoplasmic genome. It can therefore be used to introduce cytoplasmic male sterility, an important practical application. For hybrid verification and genome characterization, molecular markers and cytogenetic techniques are applied. This review highlights and discusses progress made during the last decade in spermatophytes asymmetric protoplast fusion.

Kinematic Analysis and Digital Mock-up Based Hybrid Verification about the Industrial Robot with Symmetric Dual Planar Four-Bar Mechanism

In this paper, the context of relative kinematic modeling, and the analysis of symmetric dual four-bar mechanism industrial robot are introduced. For such mechanism, its designation of the representative algorithm, and its simplification, simulation, verification and alternately analysis in Forward Kinematics Problems (FKP) and Inverse Kinematics Problem (IKP) were studied. Via such method, it’s possible to efficiently analyze and solve the both of FKP and IKP of symmetric dual planar four-bar mechanism. Thus this method can be applied for the design, simulation and verification for the robot with similar structure.

A systematic approach to configurable functional verification of HW IP blocks at transaction level

With demand growing by the day, the complexity of electronic devices is constantly increasing. Since simulation is still the most used approach, functional verification has become one of the major bottlenecks in the design and verification flow.In this paper we propose a systematic approach to configurable functional verification of electronic devices. Based on a black box approach, it can be applied to any design where behavior can be expressed by a set of functions. It combines simulation- and assertion-based verification into a hybrid verification. The proposed specification-based coverage metric can be configured ranging from a very rapid to an exhaustive verification. The approach uses Transaction Level (TL) modeling to raise the abstraction level, providing faster verification. The results of the proposed design and verification flow, Intellectual Property (IP) and Test Bench (TB) are reusable.The approach is demonstrated on two case-studies; a video-processing IP block and universal serial bus host controller. The results consider both simulation times and TB generation times.

Cohesive Coverage Management: Simulation Meets Formal Methods

It has been advocated by many experts in design verification that the key to successful verification convergence lies in developing the verification plan with adequate formal rigor. Traditionally, the verification plans for simulation and formal property verification (FPV) are developed in different ways, using different formalisms, and with different coverage goals. In this paper, we propose a framework where the difference between formal properties and simulation test points is diluted by using methods for translating one form of the specification to the other. This allows us to reuse simulation coverage to facilitate formal verification and to reuse proven formal properties to cover simulation test points. We also propose the use of inline assertions in procedural (possibly randomized) test benches, and show that it facilitates the use of hybrid verification techniques between simulation and bounded model checking. We propose the use of promising combinations of formal methods presented in our earlier papers to shape a hierarchical verification flow where simulation and formal methods aim to cover a common design intent specification. The proposed flow is demonstrated using a detailed case study of the ARM AMBA verification benchmark. We believe that the methods presented in this work will stimulate new thought processes and ultimately lead to wider adoption of cohesive coverage management techniques in the design intent validation flow.

Hybrid MPI/OpenMP parallelization of an Euler–Lagrange approach to cavitation modelling

An Euler–Lagrange approach is developed for numerical simulations of cavitating flows. Within this approach the Navier–Stokes equations are solved for the Eulerian liquid/vapor-mixture. Supplementary equations for the bubble size and motion are solved for each of the bubbles/nuclei, composing a discrete vapor phase. Such an approach is computationally demanding when computational grids with several Mio cells and many ten thousand bubbles are considered. The paper reports the development and verification of an efficient hybrid MPI/OpenMP algorithm for coupled Euler–Lagrange simulations. The proposed algorithm facilitates cavitation predictions for challenging industrial applications, such as cavitating marine propellers, in a reasonable amount of wall-clock time.

Convergent Differential Regulation of Parvalbumin in the Brains of Vocal Learners

Spoken language and learned song are complex communication behaviors found in only a few species, including humans and three groups of distantly related birds – songbirds, parrots, and hummingbirds. Despite their large phylogenetic distances, these vocal learners show convergent behaviors and associated brain pathways for vocal communication. However, it is not clear whether this behavioral and anatomical convergence is associated with molecular convergence. Here we used oligo microarrays to screen for genes differentially regulated in brain nuclei necessary for producing learned vocalizations relative to adjacent brain areas that control other behaviors in avian vocal learners versus vocal non-learners. A top candidate gene in our screen was a calcium-binding protein, parvalbumin (PV). In situ hybridization verification revealed that PV was expressed significantly higher throughout the song motor pathway, including brainstem vocal motor neurons relative to the surrounding brain regions of all distantly related avian vocal learners. This differential expression was specific to PV and vocal learners, as it was not found in avian vocal non-learners nor for control genes in learners and non-learners. Similar to the vocal learning birds, higher PV up-regulation was found in the brainstem tongue motor neurons used for speech production in humans relative to a non-human primate, macaques. These results suggest repeated convergent evolution of differential PV up-regulation in the brains of vocal learners separated by more than 65–300 million years from a common ancestor and that the specialized behaviors of learned song and speech may require extra calcium buffering and signaling.

Application of ISSR markers for verification of F1 hybrids in mungbean (Vigna radiata)

Mungbean improvement via hybridization requires the identification of true F(1) hybrids from controlled crosses before further generations of selfing/crossing and selection. We utilized inter-simple sequence repeat (ISSR) markers for identifying putative F(1) hybrids from six cross combinations whose morphological characteristics were very similar to those of their respective female parents and could not be visually discriminated from the self-pollinated progeny. Based on 10 ISSR primers, polymorphisms were found between female and male parents of all six cross combinations. The highest value of genetic differentiation (21.4%) was found between male and female parents of the SUT3 x M5-1 cross. These 10 ISSR primers gave 2.8-25.0% polymorphism between male and female parents, with a mean of 12.1%, and 0-13.0% polymorphism between F(1) hybrid and female parents, with a mean of 4.8%. F(1) hybrids of all six cross combinations could be differentiated from the self-pollinated progeny of their female parents by using only either ISSR 841 or 857 primers, together with the ISSR 835 primer. We conclude that ISSR markers are useful and efficient for identifying mungbean F(1) hybrids in controlled crosses from different genetic background.