Comprehensive Molecular Approach Research Articles

Comparison of Clinicopathogenomic Features and Treatment Outcomes of EGFR and HER2 Exon 20 Insertion Mutations in Non-Small Cell Lung Cancer: Single-Institution Experience.

Exon 20 insertion mutations (E20ins) in epidermal growth factor receptor (EGFR) or human epidermal growth factor receptor 2 (HER2) in non-small cell lung cancer (NSCLC) patients has become more important with emergence of novel agents targeting E20ins. Advanced/Metastatic NSCLC patients with E20ins were included. EGFR E20ins was identified by two methods, next-generation sequencing (NGS) or real-time polymerase chain reaction (PCR), while HER2 E20ins was done by NGS only. Between December 2013 and July 2021, E20ins were identified in 107 patients at Asan Medical Center; 67 EGFR E20ins and 40 HER2 E20ins. Out of 32 patients with EGFR E20ins who had tested both PCR and NGS, 17 were identified only through NGS and the other 15 through both tests, giving a discordance rate of 53.1%. There was no clinically significant difference in clinicopathologic features between EGFR and HER2 E20ins; both were observed more frequently in adenocarcinoma, female and never-smokers. Brain metastases were evident at diagnosis in 31.8% of EGFR E20ins and 27.5% of HER2 E20ins, respectively. Platinum-based doublets demonstrated objective response rates (ORR) of 13.3% with a median progression-free survival (PFS) of 4.2 months for EGFR E20ins and 35.3% with 4.7 months for HER2 E20ins, respectively. In contrast, novel EGFR E20ins-targeted agents exhibited an ORR of 46.2% with a median PFS of 5.4 months, while HER2-targeted agents showed an ORR of 50% with that of 7.0 months. Identification of EGFR and HER2 E20ins is more important as their targeted therapies improved outcomes. Upfront NGS test as a comprehensive molecular approach is strongly warranted.

The critical role of the phytosterols in modulating tumor microenvironment via multiple signaling: A comprehensive molecular approach.

Cancer is the leading cause of mortality and morbidity worldwide, and its cases are rapidly increasing every year. Several factors contribute to the development of tumorigenesis. including radiation, dietary lifestyle, smoking, environmental, and genetic factors. The cell cycle is regulated by a variety of molecular signaling proteins. However, when the proteins involved in the cell cycle regulation are altered, cellular growth and proliferation are significantly affected. Natural products provide an important source of new drug development for a variety of ailments. including cancer. Phytosterols (PSs) are an important class of natural compounds reported for numerous pharmacological activities, including cancer. Various PSs, such as ergosterol, stigmasterol, sitosterol, withaferin A, etc., have been reported for their anti-cancer activities against a variety of cancer by modulating the tumor microenvironment via molecular signaling pathways discussed within the article. These signaling pathways are associated with the production of pro-inflammatory mediators, growth factors, chemokines, and pro-apoptotic and anti-apoptotic genes. These mediators and their upstream signaling are very active within the variety of tumors and by modulating these signalings, thus PS exhibits promising anti-cancer activities. However, further high-quality studies are needed to firmly establish the clinical efficacy as well the safety of the phytosterols.

Robust methylation-based classification of brain tumours using nanopore sequencing.

DNA methylation-based classification of cancer provides a comprehensive molecular approach to diagnose tumours. In fact, DNA methylation profiling of human brain tumours already profoundly impacts clinical neuro-oncology. However, current implementation using hybridisation microarrays is time consuming and costly. We recently reported on shallow nanopore whole-genome sequencing for rapid and cost-effective generation of genome-wide 5-methylcytosine profiles as input to supervised classification. Here, we demonstrate that this approach allows us to discriminate a wide spectrum of primary brain tumours. Using public reference data of 82 distinct tumour entities, we performed nanopore genome sequencing on 382 tissue samples covering 46 brain tumour (sub)types. Using bootstrap sampling in a cohort of 55 cases, we found that a minimum set of 1000 random CpG features is sufficient for high-confidence classification by ad hoc random forests. We implemented score recalibration as a confidence measure for interpretation in a clinical context and empirically determined a platform-specific threshold in a randomly sampled discovery cohort (N = 185). Applying this cut-off to an independent validation series (n = 184) yielded 148 classifiable cases (sensitivity 80.4%) and demonstrated 100% specificity. Cross-lab validation demonstrated robustness with concordant results across four laboratories in 10/11 (90.9%) cases. In a prospective benchmarking (N = 15), the median time to results was 21.1h. In conclusion, nanopore sequencing allows robust and rapid methylation-based classification across the full spectrum of brain tumours. Platform-specific confidence scores facilitate clinical implementation for which prospective evaluation is warranted and ongoing.

Characterization and Genetic Diversity of Bacillus cereus Strains Isolated from Baby Wipes.

Bacillus cereus, a ubiquitous environmental microorganism known to cause foodborne illness, was isolated from samples taken from imported baby wipes from two different countries. These strains were characterized using a comprehensive molecular approach involving endpoint PCR, whole genome sequencing (WGS), comparative genomics, and biochemical analyses. A multiplex endpoint PCR assay was used to identify the enterotoxins: hemolysin BL, nonhemolytic enterotoxin, cytotoxin K, and enterotoxin FM toxin genes. Phylogenetically, the strains clustered into two major groups according to sequence type (ST) and singleton. We used the Center for Food Safety and Applied Nutrition (CFSAN) GalaxyTrakr BTyper computational tool to characterize the strains further. As an additional means of characterization, we investigated the possible role of carbohydrate transport systems and their role in nutrient uptake by performing a BLAST analysis of the 40 B. cereus genomes recovered from baby wipes. This study outlines a multifaceted workflow that uses the analysis of enterotoxigenic potential, bioinformatics, genomic diversity, genotype, phenotype, and carbohydrate utilization as a comprehensive strategy to characterize these B. cereus strains isolated from baby wipes and further our understanding of the phylogenetic relatedness of strains associated with baby wipe production facilities that could potentially pose an infection risk to a vulnerable infant population.

A comprehensive molecular approach in fish allergy: Usefulness in daily clinical practice

Background and aimsDiagnosis of fish may represent an important challenge for the allergists. This study aimed to evaluate the diagnostic sensitivity of an in vitro multiplex assay using a comprehensive panel of fish allergens and the cross-reactivity patterns between different molecular components. Methods56 subjects with fish allergy were enrolled. All patients underwent specific IgE measurement using the Allergy Explorer-Alex 2™ multiplex assay (Macroarray Diagnostics, Vienna, Austria) ResultsThe single ß-parvalbumins Clu h 1, Cyp c 1, Gad m 1, Sal s 1, Sco s 1, Thu a 1 and Xyp g 1 scored positive in 75.0%, 67.8%, 62.5%, 80.3%,80.3%, 78.8% and 73,2% patients, respectively. 14.3% scored positive for the α-parvalbumin (Raj c-parvalbumin), and 16.1% for the aldolase + enolase (Gad m 2 + 3) components. 92.8% reacted to at least one ß-parvalbumin and 96.4% to at least one of the allergens tested. Overall sensitivity was higher than that obtained using commercial extracts of cod, salmon and tuna for skin prick test (75.8%) and IgE detection (92.3%). ConclusionsThe Alex 2 showed high diagnostic sensitivity and it might be used as an additional assay to investigate the cross-reactivity patterns between different molecular components, looking for potentially safe fish species.

Analysis of the mitochondrial genome of the Indian darter, Anhinga melanogaster, suggests a species status taxonomic rank.

Anhinga melanogaster is a carnivorous water bird native to many Asian countries. A. melanogaster is part of the Old World clade of darters. There is currently significant debate about the organization of the Old World clade due to morphological and genetic ambiguities. It is essential to establish the taxonomic status of A. melanogaster because it was recently listed by the International Union for Conservation of Nature (IUCN) as a near threatened species. The present study utilized a comprehensive molecular approach of the complete mitogenome of A. melanogaster to resolve its taxonomic status within the genus Anhinga. The mitogenome of A. melanogaster comprised of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and a control region. A partially duplicated cytochrome b gene and control region were also present. Duplicated mitogenomic segments and phylogenetic analyses suggest that A. melanogaster, A. novaehollandiae, A. rufa and A. anhinga should be considered distinct species within the Old World clade of darters. The present study provides new insights into the mitogenome features of A. melanogaster and its evolutionary relationship within the genus, Anhinga.

A comprehensive molecular approach to the detection of drug-type versus fiber-type hemp varieties

A comprehensive molecular approach to the detection of drug-type versus fiber-type hemp varieties

A comprehensive molecular approach to the detection of drug-type versus fiber-type hemp varieties

The availability of molecular markers able to distinguish drug-type from fiber-type Cannabis sativa cultivars would allow fast and cheap analysis of any plant specimen, including seeds and leaves. Several approaches to this issue have been described, mainly using polymorphisms in the genes coding for tetrahydrocannabinol acid synthase or cannabidiolic acid synthase. Some studies reported sequencing of these genes from small groups of hemp varieties belonging to both chemotypes, showing the occurrence of specific DNA signatures. However, the effectiveness of the corresponding primers to discriminate among chemotypes has been validated on a limited number of cultivars, or not tested at all. Here we report a thorough in silico analysis of available gene sequences for both synthases, showing the existence of hypervariable regions at 3’ and 5’ ends. This notwithstanding, some possible signatures were identified, and 12 putatively specific primer pairs were designed and tested on 16 fiber-type and 11 drug-type varieties. In most cases inconsistent results were obtained, further strengthening the high genetic variability of these genes in hemp germplasm, yet some highly informative polymorphisms were identified. Potentiality and perspectives of this approach are discussed.

A Comprehensive Multi-Omic Approach Reveals a Relatively Simple Venom in a Diet Generalist, the Northern Short-Tailed Shrew, Blarina brevicauda.

Animals that use venom to feed on a wide diversity of prey may evolve a complex mixture of toxins to target a variety of physiological processes and prey-defense mechanisms. Blarina brevicauda, the northern short-tailed shrew, is one of few venomous mammals, and is also known to eat evolutionarily divergent prey. Despite their complex diet, earlier proteomic and transcriptomic studies of this shrew’s venom have only identified two venom proteins. Here, we investigated with comprehensive molecular approaches whether B. brevicauda venom is more complex than previously understood. We generated de novo assemblies of a B. brevicauda genome and submaxillary-gland transcriptome, as well as sequenced the salivary proteome. Our findings show that B. brevicauda’s venom composition is simple relative to their broad diet and is likely limited to seven proteins from six gene families. Additionally, we explored expression levels and rate of evolution of these venom genes and the origins of key duplications that led to toxin neofunctionalization. We also found three proteins that may be involved in endogenous self-defense. The possible synergism of the toxins suggests that vertebrate prey may be the main target of the venom. Further functional assays for all venom proteins on both vertebrate and invertebrate prey would provide further insight into the ecological relevance of venom in this species.

63P - Clinical impact of a comprehensive molecular approach in advanced gynecologic tumors

63P - Clinical impact of a comprehensive molecular approach in advanced gynecologic tumors

The all age asthma cohort (ALLIANCE) - from early beginnings to chronic disease: a longitudinal cohort study

BackgroundAsthma and wheezing disorders in childhood and adulthood are clinically heterogeneous regarding disease presentation, natural course, and response to treatment. Deciphering common disease mechanisms in distinct subgroups requires harmonized molecular (endo-) phenotyping of both children and adult patients with asthma in a prospective, longitudinal setting.MethodsThe ALL Age Asthma Cohort (ALLIANCE) of the German Center for Lung Research (DZL) is a prospective, multi-center, observational cohort study with seven recruiting sites across Germany. Data are derived from four sources: (a) patient history from medical records, (b) standardized questionnaires and structured interviews, (c) telephone interviews, and (d) objective measurements. Objective measurements include amongst others lung function and quantitative assessment of airway inflammation and exhaled breath, peripheral blood, skin, nasal, pharyngeal, and nasopharyngeal swabs, nasal secretions, primary nasal epithelial cells, and induced sputum. In cases, objective measurements and biomaterial collection are performed regularly, while control subjects are only examined once at baseline.DiscussionThe standardized and detailed collection of epidemiological and physiological data, and the molecular deep phenotyping of a comprehensive range of biomaterials in a considerable number of study participants across all ages are the outstanding characteristics of this multi-center cohort. Despite extensive biomaterial sampling, and a recruitment strategy that also includes pre-school children as young as 6 months, attrition is low. In children 83.9%, and in adults 90.5% attended the 12-month follow-up. The earliest time-point to include cases, however, is disease manifestation. Therefore, unraveling mechanisms that drive disease onset is limited, as this question can only be answered in a population-based birth cohort. Nonetheless, ALLIANCE offers a unique, integrative and inter-disciplinary framework with a comprehensive molecular approach in a prospective and identical fashion across ages in order to identify biomarkers and predictors for distinct childhood wheeze and asthma trajectories as well as their further course during adulthood. Ultimately, this approach aims to translate its most significant findings into clinical practice, and to improve asthma transition from adolescence to adulthood.Trial registrationNCT02496468 for pediatric arm, NCT02419274 for adult arm.

Amyloid fibril polymorphism: a challenge for molecular imaging and therapy.

The accumulation of misfolded proteins (MPs), both unique and common, for different diseases is central for many chronic degenerative diseases. In certain patients, MP accumulation is systemic (e.g. TTR amyloid), and in others, this is localized to a specific cell type (e.g. Alzheimer's disease). In neurodegenerative diseases, NDs, it is noticeable that the accumulation of MP progressively spreads throughout the nervous system. Our main hypothesis of this article is that MPs are not only markers but also active carriers of pathogenicity. Here, we discuss studies from comprehensive molecular approaches aimed at understanding MP conformational variations (polymorphism) and their bearing on spreading of MPs, MP toxicity, as well as MP targeting in imaging and therapy. Neurodegenerative disease (ND) represents a major and growing societal challenge, with millions of people worldwide suffering from Alzheimer's or Parkinson's diseases alone. For all NDs, current treatment is palliative without addressing the primary cause and is not curative. Over recent years, particularly the shape-shifting properties of misfolded proteins and their spreading pathways have been intensively researched. The difficulty in addressing ND has prompted most major pharma companies to severely downsize their nervous system disorder research. Increased academic research is pivotal for filling this void and to translate basic research into tools for medical professionals. Recent discoveries of targeting drug design against MPs and improved model systems to study structure, pathology spreading and toxicity strongly encourage future studies along these lines to provide an opportunity for selective imaging, prognostic diagnosis and therapy.

Identification of Nicotiana benthamiana microRNAs and their targets using high throughput sequencing and degradome analysis.

BackgroundNicotiana benthamiana is a widely used model plant species for research on plant-pathogen interactions as well as other areas of plant science. It can be easily transformed or agroinfiltrated, therefore it is commonly used in studies requiring protein localization, interaction, or plant-based systems for protein expression and purification. To discover and characterize the miRNAs and their cleaved target mRNAs in N. benthamiana, we sequenced small RNA transcriptomes and degradomes of two N. benthamiana accessions and validated them by Northern blots.ResultsWe used a comprehensive molecular approach to detect and to experimentally validate N. benthamiana miRNAs and their target mRNAs from various tissues. We identified 40 conserved miRNA families and 18 novel microRNA candidates and validated their target mRNAs with a genomic scale approach. The accumulation of thirteen novel miRNAs was confirmed by Northern blot analysis. The conserved and novel miRNA targets were found to be involved in various biological processes including transcription, RNA binding, DNA modification, signal transduction, stress response and metabolic process. Among the novel miRNA targets we found the mRNA of REPRESSOR OF SILENCING (ROS1). Regulation of ROS1 by a miRNA provides a new regulatory layer to reinforce transcriptional gene silencing by a post-transcriptional repression of ROS1 activity.ConclusionsThe identified conserved and novel miRNAs along with their target mRNAs also provides a tissue specific atlas of known and new miRNA expression and their cleaved target mRNAs of N. benthamiana. Thus this study will serve as a valuable resource to the plant research community that will be beneficial well into the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2209-6) contains supplementary material, which is available to authorized users.

Molecular phylogenetics and phylogeographic structure of Sorex bedfordiae based on mitochondrial and nuclear DNA sequences

The southeastern margin of the Tibetan Plateau is characterized by complex topography and a discontinuous landscape, creating a sky island situation. However, the way topography shapes genetic structures and demographic histories of endemic species has not been well studied. We examined the phylogeographic pattern and demographic histories of Sorex bedfordiae, a dispersal-limited small mammal, using three nuclear genes [1977bp] and two mitochondrial genes [1794bp] with comprehensive molecular approaches. We recovered five well-supported clades whose distributions are along mountain ridges and roughly subdivided by large rivers. Demographic expansions in the middle Pleistocene were strongly supported by both nuclear and mitochondrial genes. Our results support the hypothesis that sky island topography and river systems strongly affect the genetic structure of non-aquatic terrestrial species. We further clarify that S. bedfordiae and S. cylindricauda are valid sibling species, whereas S. excelsus is most likely a geographic subspecies of S. bedfordiae.

Muscular dystrophy with marked Dysferlin deficiency is consistently caused by primary dysferlin gene mutations.

Dysferlin is a 237-kDa transmembrane protein involved in calcium-mediated sarcolemma resealing. Dysferlin gene mutations cause limb-girdle muscular dystrophy (LGMD) 2B, Miyoshi myopathy (MM) and distal myopathy of the anterior tibialis. Considering that a secondary Dysferlin reduction has also been described in other myopathies, our original goal was to identify cases with a Dysferlin deficiency without dysferlin gene mutations. The dysferlin gene is huge, composed of 55 exons that span 233 140 bp of genomic DNA. We performed a thorough mutation analysis in 65 LGMD/MM patients with ≤20% Dysferlin. The screening was exhaustive, as we sequenced both genomic DNA and cDNA. When required, we used other methods, including real-time PCR, long PCR and array CGH. In all patients, we were able to recognize the primary involvement of the dysferlin gene. We identified 38 novel mutation types. Some of these, such as a dysferlin gene duplication, could have been missed by conventional screening strategies. Nonsense-mediated mRNA decay was evident in six cases, in three of which both alleles were only detectable in the genomic DNA but not in the mRNA. Among a wide spectrum of novel gene defects, we found the first example of a ‘nonstop' mutation causing a dysferlinopathy. This study presents the first direct and conclusive evidence that an amount of Dysferlin ≤20% is pathogenic and always caused by primary dysferlin gene mutations. This demonstrates the high specificity of a marked reduction of Dysferlin on western blot and the value of a comprehensive molecular approach for LGMD2B/MM diagnosis.

Pirellula and OM43 are among the dominant lineages identified in an Oregon coast diatom bloom

Although bacterioplankton and phytoplankton are generally perceived as closely linked in marine systems, specific interactions between discrete bacterioplankton and phytoplankton populations are largely unknown. However, measurements of bacterioplankton distributions during phytoplankton blooms may indicate specific microbial lineages that are responding to phytoplankton populations, and potentially controlling them by producing allelopathic compounds. Here we use a comprehensive molecular approach to identify, characterize and quantify bacterioplankton community responses to an Oregon coast diatom bloom. Total DAPI counts increased by nearly sevenfold in bloom samples, reaching 5.7 x 10(9) cells l(-1), and lineage-specific cell counts using fluorescence in situ hybridization (FISH) indicated that Bacteria accounted for approximately 89% of observed increases. Several dominant members of the bacterial community present outside the bloom (SAR11 and SAR86) did not contribute significantly to observed increases in bloom samples. Clone library and FISH data indicated that uncultured planctomycetes most closely related to Pirellula, and members of the OM43 clade of beta proteobacteria, reached 0.5 x 10(8) and 1.2 x 10(8) cells l(-1), respectively, and were among the dominant lineages in bloom samples.

A short history of the initial application of anti-5-BrdU to the detection and measurement of S phase.

Because this article is a bit of history rather than ascientific paper, we will depart from the formality of ajournalarticle.Thisarticlehastwopurposes.Thefirstistogive credit to two deceased individuals, Albert Castro,M.D., and Howard Gratzner, Ph.D. (1). Castro was the keyperson who was able to produce a polyclonal antibodythat was specific for bromodeoxyuridine (BrdU). For 10years, Gratzner took the lead in converting an idea into afunctional assay that affected DNA analysis and led to thedevelopment of many other assays with similar method-ologies. The second is to inform young scientists that (a)inventions or creations have utility in fields other than thepurpose for which they were created; (b) it is possible todo useful work in an institution that certainly would nothave been classified as one of the best; and (c) the pres-ence of talented people who are willing to collaborate isinvaluable.Scientifically, Gratzner is best known for the develop-ment of the first monoclonal antibody (mAb) to BrdU andiododeoxyuridine. This work was the culmination of pre-vious efforts with Robert Leif to develop polyclonal anti-bodies, which detected BrdU with variable specificity.Collaborations with scientists at the Lawrence LivermoreLaboratory encouraged the development of a widely usedflow cytometric test for the simultaneous measurementsof DNA and BrdU. The use of mAbs to halogenated pyri-midines was a significant scientific advance and currentlyis an integral tool for the analysis by flow and digitalmicroscopy of the cell cycle.On a personal level, Gratzner’s enthusiasm for scienceand his intelligence, curiosity, loyal interactions with fel-low collaborators, and friendship are sorely missed by allwhoknewhim.Hewasgenuinelyawarmandnicepersonwho was liked by almost everyone with whom he came incontact (1).Castro was a jovial man who interacted well with hiscollaborators. He was generous and very knowledgeable.He ran an immunodiagnostics laboratory at the universi-ties of Oregon and Miami and was an expert in the pro-duction of antibodies in animals. His warm smile, gener-ous giving of his knowledge, common sense, enthusiasmfor scientific research, and ability to get the job done wereessential to this project.Because this is a story of events that happened approx-imately 30 years ago and the existing records are limited,the accuracy of this article is limited by our memory andthose of our colleagues who have kindly helped with thisproject. Of course, this is our version of the events.Cell division and DNA replication are fundamental tobiology and specifically to the biology of cancer. Thisarticledescribeshowasimpleprocedureforthedetectionand quantitation of DNA synthesis was developed. Theprecise determination of when S phase occurs in the cellcycle is of use to maximize the selective killing of tumorcells with cycle-specific chemotherapeutic drugs. Ofgreater significance, studies of the control of the cellcycle, including its detour into apoptosis, can provideextremely useful insights into the creation of new thera-peutic regimens.The original process to detect S-phase cells included theuse of tritiated thymidine with autoradiography to mea-sure the labeling index (percentage of S-phase cells)and/or the fraction of labeled mitotic cells. Before and atthe infancy of flow cytometry, it was believed that thequantitative DNA analysis of normal and neoplastic cellsmight provide an objective marker for the diagnosis ofneoplasia. These measurements were performed with amicroscope on Feulgen-stained cells. In a normal popula-tion, it was established that there were two predominantpeaks in the DNA distribution, with a ratio of 1:2 in DNAcontent, and that cells that were synthesizing DNA (Sphase) were scattered in between, with a relative DNAcontent between 1 and 2. The detection of tritiated thy-

Expression of axolotl RNA-binding protein during development of the Mexican axolotl.

Amphibians occupy a central position in phylogeny between aquatic and terrestrial vertebrates and are widely used as model systems for studying vertebrate development. We have undertaken a comprehensive molecular approach to understand the early events related to embryonic development in the Mexican axolotl, Ambystoma mexicanum, which is an exquisite animal model for such explorations. Axolotl RBP is a RNA-binding protein which was isolated from the embryonic Mexican axolotl by subtraction hybridization and was found to show highest similarity with human, mouse, and Xenopus cold-inducible RNA-binding protein (CIRP). The reverse transcriptase polymerase chain reaction (RT-PCR) analysis suggests that it is expressed in most of the axolotl tissues except liver; the expression level appears to be highest in adult brain. We have also determined the temporal and spatial pattern of its expression at various stages of development. RT-PCR and in situ hybridization analyses indicate that expression of the AxRBP gene starts at stage 10-12 (gastrula), reaches a maxima around stage 15-20 (early tailbud), and then gradually declines through stage 40 (hatching). In situ hybridization suggests that the expression is at a maximum in neural plate and neural fold at stage 15 (neurula) of embryonic development.