Wild-type Fragment Research Articles

Numb Suppresses Notch-Dependent Activation of Enhancer of split during Lateral Inhibition in the Drosophila Embryonic Nervous System.

The role of Drosophila numb in regulating Notch signaling and neurogenesis has been extensively studied, with a particular focus on its effects on the peripheral nervous system (PNS). Previous studies based on a single loss-of-function allele of numb, numb1, showed an antineurogenic effect on the peripheral nervous system (PNS), which revealed that the wild-type numb suppresses Notch signaling. In the current study, we examined whether this phenotype is consistently observed in loss-of-function mutations of numb. Two more numb alleles, numbEY03840 and numbEY03852, were shown to have an antineurogenic phenotype in the PNS. We also found that introducing a wild-type numb genomic fragment into numb1 homozygotes rescued their antineurogenic phenotype. These results demonstrated that loss-of-function mutations of numb universally induce this phenotype. Many components of Notch signaling are encoded by maternal effect genes, but no maternal effect of numb was observed in this study. The antineurogenic phenotype of numb was found to be dependent on the Enhancer of split (E(spl)), a downstream gene of Notch signaling. We found that the combination of E(spl) homozygous and numb1 homozygous suppressed the neurogenic phenotype of the embryonic central nervous system (CNS) associated with the E(spl) mutation. In the E(spl) allele, genes encoding basic helix-loop-helix proteins, such as m5, m6, m7, and m8, remain. Thus, in the E(spl) allele, derepression of Notch activity by numb mutation can rescue the neurogenic phenotype by increasing the expression of the remaining genes in the E(spl) complex. We also uncovered a role for numb in regulating neuronal projections. Our results further support an important role for numb in the suppression of Notch signaling during embryonic nervous system development.

An ultrasensitive DNA-enhanced amplification method for detecting cfDNA drug-resistant mutations in non-small cell lung cancer with selective FEN-assisted degradation of dominant somatic fragments.

Blood cell-free DNA (cfDNA) can be a new reliable tool for detecting epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) patients. However, the currently reported cfDNA assays have a limited role in detecting drug-resistant mutations due to their deficiencies in sensitivity, stability, or mutation detection rate. We developed an Archaeoglobus fulgidus-derived flap endonuclease (Afu FEN)-based DNA-enhanced amplification system of mutated cfDNA by designing a pair of hairpin probes to anneal with wild-type cfDNA to form two 5'-flaps, allowing for the specific cleavage of wild-type cfDNA by Afu FEN. When the dominant wild-type somatic cfDNA fragments were cleaved by structure-recognition-specific Afu FEN, the proportion of mutated cfDNA in the reaction system was greatly enriched. As the amount of mutated cfDNA in the system was further increased by PCR amplification, the mutation status could be easily detected through first-generation sequencing. In a mixture of synthetic wild-type and T790M EGFR DNA fragments, our new assay still could detect T790M mutation at the fg level with remarkably high sensitivity. We also tested its performance in detecting low variant allele frequency (VAF) mutations in clinical samples from NSCLC patients. The plasma cfDNA samples with low VAF (0.1 and 0.5 %) could be easily detected by DNA-enhanced amplification. This system with enhanced amplification of mutated cfDNA is an effective tool used for the early screening and individualized targeted therapy of NSCLC by providing arapid, sensitive, and economical way for the detection of drug-resistant mutations in tumors.

Development of a Fast and Quantitative FLT3-TKD Mutation Prototype Using Lab in a Cartridge™ Technology

Introduction Acute myeloid leukemia (AML) is a heterogenous hematologic malignancy characterized by the rapid proliferation of abnormal myeloid cells in the bone marrow and blood. Fast and reliable diagnostic tests are imperative for the advancement of targeted therapies in the treatment of AML. FLT3 (FMS-like tyrosine kinase3) is one of the most frequent genetic alterations in AML and is present in approximately one-third of newly diagnosed patients. Considering the targeted therapies, such as midostaurin and gilteritinib available for FLT3 mutated AML, accurate and timely diagnosis of FLT3 mutations is vital for guiding treatment decisions and improving patient outcomes. The current diagnostic methods to detect FLT3-TKD mutations require a series of complex steps including blood sample handling, plate-based PCR, enzyme digestion and capillary electrophoresis or sequencing which can be time-consuming and labor-intensive. We have developed and evaluated the performance of a FLT3-TKD prototype using Cepheid's automated cartridge technology that demonstrates significantly less hands-on time and faster turnaround. Objectives We have developed a novel, fast and simplified qPCR-based FLT3-TKD prototype using Cepheid's GeneXpert® automated platform for detecting 14 mutations, including 7 mutations present in codon D835 and another 7 mutations in I836. The FLT3-TKD prototype is an easy-to-use sample in/result out assay with minimal hands-on time and time to results in less than 3 hours, and without the use of restriction enzymes or capillary electrophoresis. This assay is designed for quantitative detection of FLT3-TKD mutated DNA (D835 & I836) using ABL1 as an endogenous control in peripheral blood (PB) of AML patients. This assay utilizes the special chemistry, special design primers, and an optimized wild-type (WT) blocker to specifically detect SNPs and indels. With the combination of WT blocker and unique design of special design primers we were able to selectively amplify the rare mutations in the presence of abundant closely related WT fragments. Results Using this prototype, we were able to detect and quantify all 14 mutations (7 of D835 and 7 of I836) with an estimated LoD of AR 0.02 (Ratio of TKD mutation copies/ABL copies in PB background) in PB samples spiked-in with positive templates. ABL1 serves as a reference gene to normalize the WT DNA present in the background. Plasmid DNA with individual mutations was generated and spiked into the PB to assess the dynamic range of the test. We observed good alignment of dCt-AR (deltaCt-Allelic ratio) regression curve among all 14 mutations within the dynamic range tested (0.02-0.33). With pre-defined thresholds, our assay specificity has been above 95% among 20 screened EDTA donor blood samples. To further assess the sensitivity of this prototype, we also evaluated a CRISPR-CAS engineered cell line for the D835 G>A mutation. Using this cell line, we were successful in detecting the mutation down to AR of 0.008 in a PB background. For quantification analysis we prepared a linear panel of the most prevalent mutation D835(G>T) in the PB background and were able to quantify all mutations within the range of AR 0.008-to 0.83. Conclusions Here we describe the development and evaluation of an automated cartridge-based FLT3-TKD prototype that can detect 14 FLT3-TKD mutations. The cartridge prototype is preloaded with sample processing reagents and qPCR reagents with a turn-around time of less than 3 hours including offboard peripheral blood processing. When tested with contrived samples generated with either plasmid DNA or engineered cell lines spiked into blood, all 14 tested FLT3-TKD mutations were detected within a dynamic range of 0.02 to 0.33 AR with a specificity of > 95% in healthy donor whole blood background. With optimized primer and WT blocker design this prototype showed high sensitivity & specificity. We tested a broader dynamic range for the most prevalent mutation, D835 G>T, and were able to detect from AR of 0.008 to 0.83 AR. Additionally, since the prototype uses the GeneXpert® platform and current universal lysate sample prep procedure, the sample can potentially be used across all Cepheid Hematology Oncology products. This prototype significantly reduces the hands-on time with future potential to provide clinicians with fast and accurate detection of FLT3 mutation status that can aid prognostic evaluation and treatment in AML patients.

A modified high-resolution melting-based assay (HRM) to identify the SARS-CoV-2 N501Y variant

High-resolution melting (HRM) analysis is a PCR–based method that can be used as a screening assay to identify SARS-CoV-2 variants. However, conventional HRM assays hardly detect slight melting temperature differences at the A–T to T–A transversion. As the N501Y substitution results from A–T to T–A transversion in A23063, few or no studies have shown that a conventional HRM assay can identify N501Y variants. This study successfully developed an HRM assay for identifying the N501Y mutation. Two HRM assays were used in the N501 site because the discrimination results were affected by the virus copy numbers. One is a conventional HRM assay (detectable at 103–106 copies/mL) and the other is a modified HRM assay by adding the wild-type fragment (detectable at 105–1010 copies/mL). Using viral RNAs from cultured variants (Alpha, Beta, and Gamma), a modified HRM assay correctly identified three N501Y variants because of high-copy-number RNAs in those viral samples. The sensitivity and specificity of the N501Y assay were 93.3% and 100%, respectively, based on 209 clinical samples (105 for N501; 104 for N501Y). These results suggest that our HRM-based assay is a powerful tool for rapidly identifying various SARS-CoV-2 variants.

Rapid Identification of SARS-CoV-2 Omicron BA.5 Spike Mutation F486V in Clinical Specimens Using a High-Resolution Melting-Based Assay.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant BA.5 emerged as of February 2022 and replaced the earlier Omicron subvariants BA.1 and BA.2. COVID-19 genomic surveillance should be continued as new variants seem to subsequently appear, including post-BA.5 subvariants. A rapid assay is needed to differentiate between the currently dominant BA.5 variant and other variants. This study successfully developed a high-resolution melting (HRM)-based assay for BA.4/5-characteristic spike mutation F486V detection and demonstrated that our assay could discriminate between BA.1, BA.2, and BA.5 subvariants in clinical specimens. The mutational spectra at two regions (G446/L452 and F486) for the variant-selective HRM analysis was the focus of our assay. The mutational spectra used as the basis to identify each Omicron subvariant were as follows: BA.1 (G446S/L452/F486), BA.2 (G446/L452/F486), and BA.4/5 (G446/L452R/F486V). Upon mutation-coding RNA fragment analysis, the wild-type fragments melting curves were distinct from those of the mutant fragments. Based on the analysis of 120 clinical samples (40 each of subvariants BA.1, BA.2, and BA.5), this method's sensitivity and specificity were determined to be more than 95% and 100%, respectively. These results clearly demonstrate that this HRM-based assay is a simple screening method for monitoring Omicron subvariant evolution.

Diagnosis of polycystic kidney disease in dogs

A test system has been developed for the diagnosis of polycystic kidney disease in dogs, including primers, probes, and a stub oligonucleotide for another allele. A solution of a control plasmid with a wild-type gene fragment, with a fragment of the mutant allele of the gene, or a mixture of both, imitating a heterozygote, was used as a template. The system is based on the TaqMan method. This test system allows for early diagnosis of animals with mutant alleles in order to monitor the state of health during ontogenesis, as well as exclude these dogs from breeding before their puberty.

A phase 1 trial of D2C7-it in combination with an Fc-engineered anti-CD40 monoclonal antibody (2141-V11) administered intratumorally via convection-enhanced delivery for adult patients with recurrent malignant glioma (MG).

e14015 Background: D2C7 immunotoxin (D2C7-IT) is a dual-specific recombinant immunotoxin comprising an EGFR wild-type and mutant-specific (EGFRvIII) monoclonal antibody (Ab) fragment and a genetically engineered form of the Pseudomonas exotoxin. When injected directly into the tumor by convection enhanced delivery (CED), immunotoxins induce both direct tumor killing and secondary immune responses by activation of CD4+ and CD8+ T-cells. Tumor-associated macrophages (TAMs) are the most prominent glioma-infiltrating immune cells and constitute up to 40% of the tumor mass. Upon binding of D2C7-IT to EGFR and cellular internalization, the Pseudomonas exotoxin moiety of the D2C7-IT kills residual GBM cells, upregulates proinflammatory CD40, and induces pattern recognition receptor pathway transcriptome expression. This potentially creates a proinflammatory glioma microenvironment where TAM activation may be further stimulated by sequential CED of 2141-V11, an Fc engineered anti-human CD40 agonist antibody developed at Rockefeller University. We are conducting a first in human trial of the combination of D2C7-IT + 2141-V11 administered via CED in recurrent MG patients. Methods: Eligibility includes adult patients with recurrence of a solitary supratentorial WHO grade 3 or 4 MG; ≥ 4 weeks after chemotherapy, bevacizumab or study drug; adequate organ function; and KPS ≥ 70%. Cohorts of 3 patients are treated with increasing levels of 2141-V11 to determine the maximum tolerated dose (MTD) of the compound administered intratumorally in conjunction with D2C7-IT. Dose escalation and de-escalation are managed using a modified Bayesian optimal interval (BOIN) design to identify the MTD. Intratumoral administration of D2C7-IT via CED (4612 ng/mL over 72 hours) is followed by a 7-hour infusion of 2141-V11, both infused at 0.5 mL/hr. 2141-V11 is dose-escalated to determine the MTD when combined with D2C7-IT. Four dose levels (DL) are planned: #1: 0.70 mg; #2: 2.0 mg; #3: 7.0 mg; #4: 21.0 mg. Results: As of February 7, 2022, three patients were treated at DL1 and DL2, and two patients at DL3. No DLTs have been observed, and all eight patients remain alive and in observation on study after 7.0, 6.5, 6.0, 4.4, 2.8, 2.4, 0.9 and 0.5 months. Early signs of tumor response have been observed, with one patient at DL1 and 2 patients at DL2 without radiographic evidence of active tumor. Grade 2 or higher AEs due to D2C7-IT and/or 2141-V11 include: headache (grade 3, n = 1; grade 2, n = 2); paresthesia (grade 3, n = 1; grade 2, n = 1); dysphasia (grade 3, n = 1); pyramidal tract disorder (grade 3, n = 1; grade 2, n = 1); and depressed level of consciousness (grade 2, n = 1). Enrollment is ongoing. Conclusions: Intratumoral administration of D2C7-IT + 2141-V11 via CED is safe, and encouraging efficacy results have been observed. Clinical trial information: NCT04547777.

Next-Generation Sequencing-Based Analysis of Urine Cell-Free mtDNA Reveals Aberrant Fragmentation and Mutation Profile in Cancer Patients.

Many studies have demonstrated the high efficacy of cell-free nuclear DNA in cancer diagnostics. Compared to nuclear DNA, mitochondrial DNA (mtDNA) exhibits distinct characteristics, including multiple copies per cell and higher mutation frequency. However, the potential applicability of cell-free mtDNA (cf-mtDNA) in plasma and urine remains poorly investigated. Here, we comprehensively analyzed the fragmentomic and mutational characteristics of cf-mtDNA in urine and plasma samples from controls and cancer patients using next-generation sequencing. Compared to plasma cf-mtDNA, urine cf-mtDNA exhibited increased copy numbers and wider spread in fragment size distributions. Based on 2 independent animal models, urine cf-mtDNA originated predominantly from local shedding and transrenal excretion. Further analysis indicated an enhanced fragmentation of urine cf-mtDNA in renal cell carcinoma (RCC) and colorectal cancer (CRC) patients. Using the mtDNA sequence of peripheral blood mononuclear cells for reference, the mutant fragments were shorter than wild-type fragments in urine cf-mtDNA. Size selection of short urine cf-mtDNA fragments (<150 bp) significantly enhanced the somatic mutation detection. Our data revealed remarkably different base proportions of fragment ends between urine and plasma cf-mtDNA that also were associated with fragment size. Moreover, both RCC and CRC patients exhibited significantly higher T-end and lower A-end proportions in urine cf-mtDNA than controls. By integrating the fragmentomic and mutational features of urine cf-mtDNA, our nomogram model exhibited a robust efficacy for cancer diagnosis. Our proof-of-concept findings revealed aberrant fragmentation and mutation profiles of urine cf-mtDNA in cancer patients that have diagnostic potential.

Scaling-up a fragment-based protein-protein interaction method using a human reference interaction set.

Protein–protein interactions (PPIs) are essential in understanding numerous aspects of protein function. Here, we significantly scaled and modified analyses of the recently developed all‐vs‐all sequencing (AVA‐Seq) approach using a gold‐standard human protein interaction set (hsPRS‐v2) containing 98 proteins. Binary interaction analyses recovered 20 of 47 (43%) binary PPIs from this positive reference set (PRS), comparing favorably with other methods. However, the increase of 20× in the interaction search space for AVA‐Seq analysis in this manuscript resulted in numerous changes to the method required for future use in genome‐wide interaction studies. We show that standard sequencing analysis methods must be modified to consider the possible recovery of thousands of positives among millions of tested interactions in a single sequencing run. The PRS data were used to optimize data scaling, auto‐activator removal, rank interaction features (such as orientation and unique fragment pairs), and statistical cutoffs. Using these modifications to the method, AVA‐Seq recovered >500 known and novel PPIs, including interactions between wild‐type fragments of tumor protein p53 and minichromosome maintenance complex proteins 2 and 5 (MCM2 and MCM5) that could be of interest in human disease.

SuperSelective primer pairs for sensitive detection of rare somatic mutations

SuperSelective primers, by virtue of their unique design, enable the selective exponential amplification of rare DNA fragments containing somatic mutations in the presence of abundant closely related wild-type DNA fragments. However, when a SuperSelective primer is used in conjunction with a conventional reverse primer, linear amplification of the abundant wild-type fragments occurs, and this may lead to a late arising signal that can be confused with the late arising signal from the rare mutant fragments. We have discovered that the use of a pair of SuperSelective primers, one specific for the target mutation in a plus strand, and the other specific for the same mutation in the complementary minus strand, but both possessing 3′-terminal nucleotides that are complementary to the mutation, significantly suppresses the linear amplification of the related wild-type sequence, and prevents the generation of false mutant sequences due to mis-incorporation by the DNA polymerase. As a consequence, the absence of mutant fragments in a sample does not give rise to a false-positive signal, and the presence of mutant fragments in a sample is clearly distinguishable as a true-positive signal. The use of SuperSelective primer pairs should enhance the sensitivity of multiplex PCR assays that identify and quantitate somatic mutations in liquid biopsies obtained from patients with cancer, thereby enabling the choice of a targeted therapy, the determination of its effectiveness over time, and the substitution of a more appropriate therapy as new mutations arise.

Complementation of the tomato HWS gene with its Arabidopsis counterpart demonstrates conservation of the gene function between both species.

The HAWAIIAN SKIRT (HWS) gene was originally described in Arabidopsis for the characteristic fusion of sepals in the mutant. A tomato line mutated in the putative ortholog gene was isolated in a previous study. The tomato hws-1 mutant showed facultative parthenocarpy and produced fruits with elevated Brix, revealing the gene as a hopeful resource for crop improvement. To confirm the orthology relationship between the Arabidopsis and tomato HWS genes, the hws-1 mutant was complemented with either the tomato wild-type genomic fragment or the Arabidopsis sequence of the gene. In both complementation experiments, defective phenotypes of hws-1 are rescued, albeit to different extents. Recovery of these phenotypes, which include parthenocarpic fruit production, increased Brix, loss of leaflet serration, alteration of bud and petal shape, firmly establishes SlHWS as an ortholog of the originally described HWS in Arabidopsis. This work indicates that the function of HWS is likely to be conserved in a wide range of plant species.

Lesion Genotype Modifies High-Fat Diet Effects on Endometriosis Development in Mice.

Endometriosis is a chronic, estrogen-dependent gynecologic disorder that affects reproductive-aged women and to a lesser extent, post-menopausal women on hormone therapy. The condition is associated with systemic and local immune dysfunctions. While its underlying mechanisms remain poorly understood, endometriosis has a genetic component and propensity for the disease is subject to environmental, nutritional, and lifestyle influences. Previously, we showed that high-fat diet (HFD) increased ectopic lesion numbers, concurrent with systemic and peritoneal changes in inflammatory and oxidative stress status, in immunocompetent recipient mice ip administered with endometrial fragments null for Krüppel-like factor 9 gene. Herein, we determined whether HFD modifies lesion parameters, when recipient peritoneal environment is challenged with ectopic wild-type (WT) endometrial fragments, the latter simulating retrograde menstruation common in women during the menstrual period. WT endometrium-recipient mice fed HFD (45% kcal from fat) showed reduced lesion incidence, numbers, and volumes, in the absence of changes in systemic ovarian steroid hormone and insulin levels, relative to those fed the control diet (CD, 17% kcal from fat). Lesions from HFD- and CD-fed recipients demonstrated comparable gene expression for steroid hormone receptors (Esr and Pgr) and cytokines (Il-6, Il-8, and CxCL4) and similar levels of DNA oxidative biomarkers. HFD moderately altered serum (3-nitrotyrosine and methionine/homocysteine) and peritoneal (reduced glutathione/oxidized glutathione) pro-oxidative status but had no effect on peritoneal inflammatory (tumor necrosis factor α and tumor necrosis factor receptor 1) mediators. Results indicate that lesion genotype modifies dietary effects on disease establishment and/or progression and if translated, could be important for provision of nutritional guidelines to women with predisposition to, or affected by endometriosis.

Comparison of Circulating Tumor DNA Recovery from Plasma and Serum

Background: Circulating tumor DNA (ctDNA) has emerged as a promising biomarker for noninvasive disease detection and tumor genotyping in patients with a wide range of malignancies. Circulating tumor DNA often comprises less than 1% of the total cell-free DNA (cfDNA) pool; maximizing recovery of tumor DNA is therefore critical for disease detection and characterization. Levels of ctDNA are dependent upon features of each tumor type, including disease burden (Bettegowda et al., Sci Transl Med 2014; Newman et al., Nat Med 2014). However, ctDNA levels are also potentially sensitive to other analytical considerations, such as the biological source material. In this study, we sought to directly compare plasma and serum samples for ctDNA detection.Methods: We applied Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) to 33 pretreatment serum and 75 pretreatment plasma samples from patients with diffuse large B cell lymphoma (DLBCL), with matched tumor in 40 cases. In addition, we sequenced matched serum and plasma samples from a single phlebotomy in 3 cases. Germline DNA was also sequenced. Plasma and serum samples were assessed for differences in cfDNA concentration, number of mutations identified, and mean tumor allele fraction (AF). We further compared the size distribution between mutated and wild-type DNA fragments and between plasma and serum samples. Finally, we compared the recovery rate of tumor-biopsy confirmed mutations in plasma and serum samples.Results: Serum samples had a higher median concentration of cfDNA per mL compared to matched plasma (serum: 481 ng/mL, range 270-499; plasma: 17.7 ng/mL, range 8.4-30). This discrepancy was also seen across the full cohort (serum: 718 ng/mL, IQR 458-1248; plasma: 33.3 ng/mL, IQR 33.3-73.1; p < 0.0001). Despite higher cfDNA concentration, fewer mutations were identified in serum than in plasma in both paired blood samples (serum: 22 mutations per sample, range 0-299; plasma: 186 mutations per sample, range 60-459) and in the full cohort (serum: 50 mutations per sample, IQR 17.8-162; plasma: 184 mutations per sample, IQR 72-331; p < 0.0001). Serum samples also had lower tumor AF than plasma samples in the paired (serum: 0.85 %AF, range 0.71-1.49; plasma 2.8 %AF, range 1.9-8.22) and full cohorts (serum: 0.40 %AF, IQR 0.04-0.98; plasma: 2.91% AF, IQR 0.88-6.05; p = 0.003; Figure 1A). However, when quantitated as a concentration of tumor molecules per mL of sample, serum and plasma had similar levels of ctDNA (serum: 476 haploid genome equivalents (hGE) per mL, IQR 95-1085; plasma: 292 hGE per mL, IQR 82-990; p = 0.91; Figure 1B). This suggests the higher concentration of total cfDNA found in serum consists mostly of non-tumor derived molecules.In serum, non-mutated molecules were significantly longer compared to molecules harboring tumor mutations (non-mutated: 319 bp, IQR 291-359; mutated: 269 bp, IQR 243-304; p < 0.0001; Figure 1C). This pattern was not seen in plasma (Figure 1D). Interestingly, serum samples also had a longer mean cfDNA fragment length compared to plasma samples (serum: 319 bp, IQR 291-358; plasma: 235 bp, IQR 219-251; p < 0.0001), consistent with the finding that serum contains a higher proportion of non-tumor-derived cfDNA. Finally, when considering mutations identified in tumor biopsies, the percentage of tumor mutations recovered in matched plasma was significantly higher than that in matched serum (serum: 0.4% tumor mutations, IQR 0-40%; plasma: 62% tumor mutations, IQR 28-85%; p = 0.0005; Figure 1E).Conclusions: Plasma and serum contain equivalent absolute quantities of ctDNA. However, plasma contains a higher fraction of ctDNA than serum, facilitating mutation detection and noninvasive genotyping. When possible, blood samples used for cancer detection should be obtained and stored as plasma to optimize ctDNA recovery.Figure 1. Comparison of serum and plasma samples.(A) Mean tumor AF is higher in plasma than in serum (p = 0.003) (B) Total quantity of tumor-derived DNA is equivalent between plasma and serum samples (C) In serum, non-tumor-derived cfDNA has a longer average fragment length than ctDNA (p < 0.0001) (D) In plasma, ctDNA and non-tumor-derived cfDNA show overlapping fragment distributions (E) A greater number of mutations seen in tumor biopsies are recovered in paired plasma samples as compared to paired serum samples (p = 0.0005) [Display omitted] DisclosuresNeelapu:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Poseida Therapeutics, Inc: Research Funding; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karus: Research Funding; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis Inc.: Research Funding. Nastoupil:TG Therapeutics: Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Honoraria; Janssen: Honoraria, Research Funding; Karus Therapeutics: Research Funding; Celgene: Honoraria, Research Funding. Diehn:Novartis: Consultancy; Roche: Consultancy; Quanticel Pharmaceuticals: Consultancy; Varian Medical Systems: Research Funding. Alizadeh:Roche: Consultancy; Genentech: Consultancy; Celgene: Consultancy; CiberMed: Consultancy; Gilead: Consultancy.

Application of Nanotechnology for Sensitive Detection of Low-Abundance Single-Nucleotide Variations in Genomic DNA: A Review.

Single-nucleotide polymorphisms (SNPs) are the simplest and most common type of DNA variations in the human genome. This class of attractive genetic markers, along with point mutations, have been associated with the risk of developing a wide range of diseases, including cancer, cardiovascular diseases, autoimmune diseases, and neurodegenerative diseases. Several existing methods to detect SNPs and mutations in body fluids have faced limitations. Therefore, there is a need to focus on developing noninvasive future polymerase chain reaction (PCR)–free tools to detect low-abundant SNPs in such specimens. The detection of small concentrations of SNPs in the presence of a large background of wild-type genes is the biggest hurdle. Hence, the screening and detection of SNPs need efficient and straightforward strategies. Suitable amplification methods are being explored to avoid high-throughput settings and laborious efforts. Therefore, currently, DNA sensing methods are being explored for the ultrasensitive detection of SNPs based on the concept of nanotechnology. Owing to their small size and improved surface area, nanomaterials hold the extensive capacity to be used as biosensors in the genotyping and highly sensitive recognition of single-base mismatch in the presence of incomparable wild-type DNA fragments. Different nanomaterials have been combined with imaging and sensing techniques and amplification methods to facilitate the less time-consuming and easy detection of SNPs in different diseases. This review aims to highlight some of the most recent findings on the aspects of nanotechnology-based SNP sensing methods used for the specific and ultrasensitive detection of low-concentration SNPs and rare mutations.

The \u03c354 system directly regulates bacterial natural product genes

Bacterial-derived polyketide and non-ribosomal peptide natural products are crucial sources of therapeutics and yet little is known about the conditions that favor activation of natural product genes or the regulatory machinery controlling their transcription. Recent findings suggest that the σ54 system, which includes σ54-loaded RNA polymerase and transcriptional activators called enhancer binding proteins (EBPs), might be a common regulator of natural product genes. Here, we explored this idea by analyzing a selected group of putative σ54 promoters identified in Myxococcus xanthus natural product gene clusters. We show that mutations in putative σ54-RNA polymerase binding regions and in putative Nla28 EBP binding sites dramatically reduce in vivo promoter activities in growing and developing cells. We also show in vivo promoter activities are reduced in a nla28 mutant, that Nla28 binds to wild-type fragments of these promoters in vitro, and that in vitro binding is lost when the Nla28 binding sites are mutated. Together, our results indicate that M. xanthus uses σ54 promoters for transcription of at least some of its natural product genes. Interestingly, the vast majority of experimentally confirmed and putative σ54 promoters in M. xanthus natural product loci are located within genes and not in intergenic sequences.

Improved EGFR mutation detection sensitivity after enrichment by Cas9/sgRNA digestion and PCR amplification.

The detection of circulating tumor DNA is important in cancer research and clinical practice. In the present study, we aimed to improve the sensitivity of downstream mutation detection of next-generation sequencing using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system to selectively target wild-type fragments but with low or no cleavage activity to mutant fragments, followed by amplification using polymerase chain reaction. We selected different mutant sites of epidermal growth factor receptor gene (EGFR)-exon19 deletions in patients with lung cancer and constructed mixed templates of mutant and wild-type DNA comprising ratios of 10% to 0.01% to test the effectiveness of the enrichment method. The results showed that after CRISPR/Cas9 enrichment, a low concentration of mutant DNA fragments (0.01%) could be detected by Sanger sequencing, which represented a 1000-fold increase compared with the untreated samples. We further verified the feasibility of the introduced method and obtained similar results in clinical samples from patients with non-small cell lung cancer, indicating that this method has the potential to detect low copy number mutations at the early stage.

Effect of substitutions of key residues on the stability and the insecticidal activity of Vip3Af from Bacillus thuringiensis

Modern agriculture demands for more sustainable agrochemicals to reduce the environmental and health impact. The whole process of the discovery and development of new active substances or control agents is sorely slow and expensive. Vegetative insecticidal proteins (Vip3) from Bacillus thuringiensis are specific toxins against caterpillars with a potential capacity to broaden the range of target pests. Site-directed mutagenesis is one of the most approaches used to test hypotheses on the role of different amino acids on the structure and function of proteins. To gain a better understanding of the role of key amino acid residues of Vip3A proteins, we have generated 12 mutants of the Vip3Af1 protein by site-directed mutagenesis, distributed along the five structural domains of the protein. Ten of these mutants were successfully expressed and tested for stability and toxicity against three insect pests (Spodoptera frugiperda, Spodoptera littoralis and Grapholita molesta). The results showed that, to render a wild type fragment pattern upon trypsin treatment, position 483 required an acidic residue, and position 552 an aromatic residue. Regarding toxicity, the change of Met34 to Lys34 significantly increased the toxicity of the protein for one of the three insect species tested (S. littoralis), whereas the other residue substitutions did not improve, or even decreased, insect toxicity, confirming their key role in the structure/function of the protein.

The Role of the Cysteine Fragments of the Nickel Binding Loop in the Activity of the Ni(II)-Containing SOD Enzyme.

Detailed equilibrium, spectroscopic, and SOD activity studies are reported on nickel(II) complexes formed with the N-terminally free HHDLPCGVY-NH2 (NiSODHH) and HCDLPHGVY-NH2 (NiSODHC) peptides mimicking the nickel binding loop in NiSOD. In these model peptides, cysteine was incorporated in different positions in order to gain better insight into the role of the cysteine residues in NiSOD. The results are compared with those obtained with the wild-type fragment of NiSOD. The complex formation equilibria of nickel(II) with the two peptides exhibit different features. In the case of NiSODHH, the ligand field of the (NH2,NIm,NIm,S-) donor set is not strong enough to cause spin pairing and an octahedral paramagnetic complex is formed under physiological conditions. In contrast, NiSODHC forms a square-planar diamagnetic complex with (NH2,N-,S-,NIm) donors which exhibits remarkable SOD activity. Our results unambiguously prove that the presence of cysteine in the secondary position of the peptide chain is crucial to establish the square-planar geometry in the reduced form of NiSOD, while the distant cysteine affects the redox properties of the Ni(II)/Ni(III) couple. Compared to the model systems, the Ni(II) complex with the wild-type fragment of NiSOD exhibits superior SOD activity. This confirms that both cysteinyl residues are essential in the efficient degradation of superoxide ion. The enzyme mimetic complexes are also capable of assisting the decomposition of superoxide ion; however, they show considerably smaller catalytic activity due to the absence of one of the cysteine residues.

Polymorphism study of BMP-15 (FecXR) and GDF-9 gene related with twinning in cattle

Natural mutations in prolificacy genes in some species especially sheep has been shown transforming growth factor beta (TGF-β) super family ligands such as growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and their type I receptor (bone morphogenetic protein receptor, BMPR1B) crucial for ovulation and as well as for increasing litter size. Mutations in any of these genes increased prolificacy in carrier. The present investigation was carried out for identification of fecundity genes in cattle, with comparison of DNA fragment between dams bearing twin and singleton and to see inheritance pattern of these fragments in their twin progenies. The animals belonged to six breeds viz. pure (Sahiwal and Hariana) and three crossbreeds (Karan Swiss, Karan Fires, Holstein Friesian) besides other cross breeds of unknown lineage selected from field. The study involved analysis of genome related to twinning using molecular markers (PCR) on BMP-15 (FecXR) gene and A competitive technique called tetra-primer amplification refractory mutation system-PCR was adapted to type G1 locus mutation GDF-9 fecundity gene. PCR analysis of the FecXR allele of the BMP-15 gene detected the presence of wild-type allele in the dams and their progenies. An allele specific primer used for detection of GDF-9 gene mutation, identified only wild-type and control fragment which revealed the absence of the mutant fragment for this gene locus. The study revealed monomorphic pattern of the two fecundity genes BMP-15 and GDF-9 genes, which has no effect on twinning in cattle.

96 Evaluation of CRISPR/Cas9 alternative delivery in parthenogenetic porcine embryos

Porcine genetic editing is considered promising in biomedical research, particularly for xenotransplantation. However, invitro porcine embryo production is less efficient than in other animal models. Thus, we aimed to perform a rapid optimization essay by producing parthenogenetic porcine embryos to evaluate transgenesis delivery and CRISPR/Cas9 editing efficiency. First, PCX-enhanced green fluorescent protein (EGFP) plasmid was microinjected (30ng μL−1) into a diploid parthenogenic zygote with (lipo+) or without lipofectamine (lipo−), since it has been shown that this transfection reagent improves transgene delivery and increases its expression in bovine embryos (Vichera et al. 2011 Reprod. Domest. Anim. 46, 214-220; https://doi.org/10.1111/j.1439-0531.2010.01642.x). Briefly, invitro-matured oocytes were electrically activated, followed by incubation in synthetic oviductal fluid medium containing 6-dimethylaminopyridine. Embryos were invitro cultured until Day 7, and EGFP-positive (EGFP+) embryos were corroborated at Day 5. Data were analysed using Fisher's exact test. Cleavage rates were no different among groups (EGFP/lipo−: 40%, n=28; EGFP/lipo+: 45%, n=41; control: 41, 3%, n=45; P&amp;gt;0.05). Also, no significant difference in the percentage of EGFP+ embryos was observed between EGFP/lipo+ (31%, n=13) and EGFP/Lipo− (18%, n=5) groups. Although blastocyst rate showed no statistical difference among groups, a lower blastocyst percentage tendency was observed in the EGFP/Lipo+ group compared with the control group (EGFP/Lipo+: 5%, n=2; control: 20%, n=9; P=0.051), suggesting that the presence of lipofectamine and EGFP plasmid may affect embryo development. Next, two guides (single guide (sg) RNA) were designed for the internal regions of GGTA1, CMAH, and VWF target genes, involved in hyperacute rejection and coagulation in xenotransplantation. A liposome-DNA mixture was used: DNA for sgRNA and Cas9, with and without lipofectamine (10× dilution; CRISPR/lipo+, CRISPR/lipo−), diluted to half concentration with 10% polyvinylpyrrolidone, resulting in a final concentration of 20ng ul−1 for all sgRNA and 40ng ul−1 for Cas9. A total of 2 pL of the mixtures was microinjected into the diploid parthenogenetic zygotes which were invitro cultured until Day 7. Genetic editing was corroborated by analysing the presence of a double cut directed by the two sgRNA designed for the target genes, resulting in an amplicon with lower molecular weight compared to the wild-type PCR fragment. No differences in cleavage or blastocyst rates were observed among groups (blastocyst rates: CRISPR/Lipo−: 12%, n=13; CRISPR/Lipo+: 10%, n=7; control: 15%, n=18; P&amp;gt;0.05). Finally, one embryo showed a single deletion for GGTA1 and another showed a double deletion in GGTA1 and VWF genes, out of seven embryos from the CRISPR/Lipo+ group. No gene deletion was confirmed in any of the embryos from the CRISPR/Lipo− group. These results are preliminary data (more experiments are currently being done) suggesting that microinjection of CRISPR/Cas9 with lipofectamine could be used as an alternative delivery system, since it seems to have no impact on porcine embryo development.