Computational approaches to understand interactions between zinc finger proteins and DNA

This dissertation seeks to improve C2H2 zinc finger protein (ZFP) engineering through computational and experimental studies. Engineered ZFPs that are able to bind any desired DNA sequence in a genome have great potential for basic biological research and biomedical applications. Current methods to engineer ZFPs are either unreliable, due to high failure rates, or inaccessible to some researchers, due to high demands of labor, time and expertise. We created a web-accessible database that organizes zinc finger modules (ZFMs) and ZFPs from our lab, our collaborators\u27 labs and the literature. This database helps researchers by providing information about the existence of ZFMs or ZFPs and their performance in DNA binding. We then described rules that govern the interaction of ZFPs and DNA by analyzing the finger sequences in the ZFM pools created by Oligomerized Pool ENgineering (OPEN), a recently developed, reliable, rapid and publicly available ZFP engineering method. These rules will be valuable in the improvement of the OPEN method and the understanding of the interaction between engineered ZFPs and DN

Predictive and prognostic value of aurora kinase A combined with tumor-infiltrating lymphocytes in medullary thyroid carcinoma

BackgroundAurora kinase A (AURKA) and tumor-infiltrating lymphocytes (TILs) are both known to play an essential role in tumorigenesis. However, the expression and prognostic value of the AURKA and TILs in medullary thyroid carcinoma (MTC) have not yet been investigated.Patients and methodsSurgical specimens and clinical data of 137 patients diagnosed with MTC were collected. AURKA expression and TILs infiltration were quantified by immunohistochemistry and hematoxylin-eosin staining. Subsequently, the prognostic value of AURKA expression and TIL infiltration in MTC was evaluated.ResultsAURKA was highly expressed in patients with multifocal tumor, cervical lymph node metastasis, and an advanced TNM stage, indicating a high probability of recurrence. AURKA further exhibited a positive correlation with TILs (R = 0.44, P < 0.001). High expression of AURKA combined with a low numbers of TILs (AURKAhigh/TILslow) was identified as an independent prognostic factor for biochemical recurrence (odds ratio: 4.57, 95% confidence interval: 1.54–14.66, P < 0.01) and recurrence-free survival (hazard ratio: 3.64, 95% confidence interval: 1.52–8.71, P < 0.001). The combination of AURKA and TILs apparently improves the prognostic value for biochemical recurrence (area under the curve: 0.751) and structural recurrence (area under the curve: 0.836) of MTC. Notably, AURKAhigh/TILslow demonstrated a high value for prediction of distant or unresectable locoregional recurrence, with an overall accuracy of 86.9%.ConclusionAURKAhigh is associated with the MTC malignancy. The combination of AURKAhigh/TILslow was identified as novel independent prognostic marker in MTC, predicting incurable disease recurrence with high accuracy

Application of the Kaiser score by MRI in patients with breast lesions by ultrasound and mammography

PURPOSEThis study aimed to verify whether the use of the Kaiser score can improve the diagnostic performance in breast magnetic resonance imaging (MRI) for suspicious lesions and avoid further invasive diagnostic approaches.METHODSThis retrospective study enrolled 97 patients who underwent breast MRI before undergoing breast biopsy or surgery. Evaluations were conducted on all MRI images individually by 2 radiologists using the Kaiser score. Neither radiologist had the knowledge of the final histopathological diagnosis. The ability of the Kaiser score in diagnosis was established via a receiver performing characteristic (ROC) analysis, which was measured by the area under the ROC curve (AUC). Youden index was used to define the optimal cutoff value. Kaiser score categories were dichotomized into positive (cutoff score > 4) and negative scores (cutoff score ≤ 4). Cohen’s kappa coefficient was used to analyze the inter-rater agreement.RESULTSHistopathology revealed 56 malignant and 41 benign lesions. The AUC for all lesions evaluated by the Kaiser score was 0.992 (95% CI: 0.981-1.0) and 0.958 (95% CI: 0.920-0.996) for 2 radiologists, respectively. Inter-rater agreement of the dichotomized Kaiser score was excellent (κ=0.894, P < .001). A total of 20 lesions (33.8%) previously categorized as BI-RADS 4 were reduced to BI-RADS 2/3 (19 benign lesions and 1 malignant lesion).CONCLUSIONThe Kaiser score is a valuable auxiliary diagnostic tool for improving the diagnostic ability of radiologists, whose experiences in breast MRI are diverse. In some cases, the application of the Kaiser score could possibly avoid unnecessary breast biopsies

Partial preservation of the normal thyroid gland based on tumor diameter may be possible in small medullary thyroid carcinoma: a two-center 15-year retrospective study

BackgroundAt present, there are some controversies in the formulation of surgical protocol for small medullary thyroid carcinoma(s-MTC). We wanted to explore the feasibility of normal thyroid gland retention in small medullary thyroid carcinoma based on different tumor diameters and its prognostic impact on the tumor.MethodsThe data of patients with stage T1 MTC treated at Tianjin Cancer Hospital and Sichuan Cancer Hospital from 2006 to 2021 were analyzed. The tumor diameters of 0.5 cm and 1.0 cm were used as dividing points. The outcomes were tumor recurrence, metastasis, or patient death. Survival was estimated by the Kapan–Meier curve.ResultsA total of 121 T1 s-MTC patients were included, including 55 with total thyroidectomy (TT) and 66 with subthyroidectomy (Sub-TT). There were eleven cases of tumor recurrence and metastasis, and four patients died. When the tumor diameter was 1.0 cm as the cut-off point, tumor diameter (p = 0.010), TT (p = 0.028), unilateral and bilateral type (p = 0.009), and TNM staging (p = 0.007) had significant effects on progression-free survival (PFS). The tumor diameter, unilateral and bilateral type, and TT were risk factors for the prognosis of T1 MTC (p &lt; 0.05).ConclusionThe tumor diameter of 1.0 cm can be used as a cut-off point for stage T1 MTC. Alt-hough there was no significant difference in overall survival (OS) between T1a and T1b in patients, tumor diameter significantly influenced PFS. TT is not necessary for patients with sporadic MTC with T1a

Predicting success of oligomerized pool engineering (OPEN) for zinc finger target site sequences

<p>Abstract</p> <p>Background</p> <p>Precise and efficient methods for gene targeting are critical for detailed functional analysis of genomes and regulatory networks and for potentially improving the efficacy and safety of gene therapies. Oligomerized Pool ENgineering (OPEN) is a recently developed method for engineering C2H2 zinc finger proteins (ZFPs) designed to bind specific DNA sequences with high affinity and specificity <it>in vivo</it>. Because generation of ZFPs using OPEN requires considerable effort, a computational method for identifying the sites in any given gene that are most likely to be successfully targeted by this method is desirable.</p> <p>Results</p> <p>Analysis of the base composition of experimentally validated ZFP target sites identified important constraints on the DNA sequence space that can be effectively targeted using OPEN. Using alternate encodings to represent ZFP target sites, we implemented Naïve Bayes and Support Vector Machine classifiers capable of distinguishing "active" targets, i.e., ZFP binding sites that can be targeted with a high rate of success, from those that are "inactive" or poor targets for ZFPs generated using current OPEN technologies. When evaluated using leave-one-out cross-validation on a dataset of 135 experimentally validated ZFP target sites, the best Naïve Bayes classifier, designated ZiFOpT, achieved overall accuracy of 87% and specificity⁺of 90%, with an ROC AUC of 0.89. When challenged with a completely independent test set of 140 newly validated ZFP target sites, ZiFOpT performance was comparable in terms of overall accuracy (88%) and specificity⁺(92%), but with reduced ROC AUC (0.77). Users can rank potentially active ZFP target sites using a confidence score derived from the posterior probability returned by ZiFOpT.</p> <p>Conclusion</p> <p>ZiFOpT, a machine learning classifier trained to identify DNA sequences amenable for targeting by OPEN-generated zinc finger arrays, can guide users to target sites that are most likely to function successfully <it>in vivo</it>, substantially reducing the experimental effort required. ZiFOpT is freely available and incorporated in the Zinc Finger Targeter web server (<url>http://bindr.gdcb.iastate.edu/ZiFiT</url>).</p

A Roadmap for Functional Structural Variants in the Soybean Genome

Gene structural variation (SV) has recently emerged as a key genetic mechanism underlying several important phenotypic traits in crop species. We screened a panel of 41 soybean (Glycine max) accessions serving as parents in a soybean nested association mapping population for deletions and duplications in more than 53,000 gene models. Array hybridization and whole genome resequencing methods were used as complementary technologies to identify SV in 1528 genes, or approximately 2.8%, of the soybean gene models. Although SV occurs throughout the genome, SV enrichment was noted in families of biotic defense response genes. Among accessions, SV was nearly eightfold less frequent for gene models that have retained paralogs since the last whole genome duplication event, compared with genes that have not retained paralogs. Increases in gene copy number, similar to that described at the Rhg1 resistance locus, account for approximately one-fourth of the genic SV events. This assessment of soybean SV occurrence presents a target list of genes potentially responsible for rapidly evolving and/or adaptive traits

A Roadmap for Functional Structural Variants in the Soybean Genome

Gene structural variation (SV) has recently emerged as a key genetic mechanism underlying several important phenotypic traits in crop species. We screened a panel of 41 soybean (Glycine max) accessions serving as parents in a soybean nested association mapping population for deletions and duplications in more than 53,000 gene models. Array hybridization and whole genome resequencing methods were used as complementary technologies to identify SV in 1528 genes, or approximately 2.8%, of the soybean gene models. Although SV occurs throughout the genome, SV enrichment was noted in families of biotic defense response genes. Among accessions, SV was nearly eightfold less frequent for gene models that have retained paralogs since the last whole genome duplication event, compared with genes that have not retained paralogs. Increases in gene copy number, similar to that described at the Rhg1 resistance locus, account for approximately one-fourth of the genic SV events. This assessment of soybean SV occurrence presents a target list of genes potentially responsible for rapidly evolving and/or adaptive traits

Rapid “Open-Source” Engineering of Customized Zinc-Finger Nucleases for Highly Efficient Gene Modification

Summary—Custom-made zinc-finger nucleases (ZFNs) can induce targeted genome modifications with high efficiency in cell types including Drosophila, C. elegans, plants, and humans. A bottleneck in the application of ZFN technology has been the generation of highly specific engineered zincfinger arrays. Here we describe OPEN (Oligomerized Pool ENgineering), a rapid, publicly available strategy for constructing multi-finger arrays, which we show is more effective than the previously published modular assembly method. We used OPEN to construct 37 highly active ZFN pairs which induced targeted alterations with high efficiencies (1 to 50%) at 11 different target sites located within three endogenous human genes (VEGF-A, HoxB13, CFTR), an endogenous plant gene (tobacco SuRA), and a chromosomally-integrated EGFP reporter gene. In summary, OPEN provides an “opensource” method for rapidly engineering highly active zinc-finger arrays, thereby enabling broader practice, development, and application of ZFN technology for biological research and gene therapy

Computational approaches to understand interactions between zinc finger proteins and DNA

This dissertation seeks to improve C2H2 zinc finger protein (ZFP) engineering through computational and experimental studies. Engineered ZFPs that are able to bind any desired DNA sequence in a genome have great potential for basic biological research and biomedical applications. Current methods to engineer ZFPs are either unreliable, due to high failure rates, or inaccessible to some researchers, due to high demands of labor, time and expertise. We created a web-accessible database that organizes zinc finger modules (ZFMs) and ZFPs from our lab, our collaborators' labs and the literature. This database helps researchers by providing information about the existence of ZFMs or ZFPs and their performance in DNA binding. We then described rules that govern the interaction of ZFPs and DNA by analyzing the finger sequences in the ZFM pools created by Oligomerized Pool ENgineering (OPEN), a recently developed, reliable, rapid and publicly available ZFP engineering method. These rules will be valuable in the improvement of the OPEN method and the understanding of the interaction between engineered ZFPs and DNA</p

Joint Dedusting and Enhancement of Top-Coal Caving Face via Single-Channel Retinex-Based Method with Frequency Domain Prior Information

Affected by the uneven concentration of coal dust and low illumination, most of the images captured in the top-coal caving face have low definition, high haze and serious noise. In order to improve the visual effect of underground images captured in the top-coal caving face, a novel single-channel Retinex dedusting algorithm with frequency domain prior information is proposed to solve the problem that Retinex defogging algorithm cannot effectively defog and denoise, simultaneously, while preserving image details. Our work is inspired by the simple and intuitive observation that the low frequency component of dust-free image will be amplified in the symmetrical spectrum after adding dusts. A single-channel multiscale Retinex algorithm with color restoration (MSRCR) in YIQ space is proposed to restore the foggy approximate component in wavelet domain. After that the multiscale convolution enhancement and fast non-local means (FNLM) filter are used to minimize noise of detail components while retaining sufficient details. Finally, a dust-free image is reconstructed to the spatial domain and the color is restored by white balance. By comparing with the state-of-the-art image dedusting and defogging algorithms, the experimental results have shown that the proposed algorithm has higher contrast and visibility in both subjective and objective analysis while retaining sufficient details