High-throughput sequencing for B cell receptor (BCR) repertoire provides useful insights for the adaptive immune system. With the continuous development of the BCR-seq technology, many efforts have ...been made to develop methods for analyzing the ever-increasing BCR repertoire data. In this review, we comprehensively outline different BCR repertoire library preparation protocols and summarize three major steps of BCR-seq data analysis,
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., V(D)J sequence annotation, clonal phylogenetic inference, and BCR repertoire profiling and mining. Different from other reviews in this field, we emphasize background intuition and the statistical principle of each method to help biologists better understand it. Finally, we discuss data mining problems for BCR-seq data and with a highlight on recently emerging multiple-sample analysis.
Cytidine deaminases as DNA mutators play important roles in immunity and genome stability. Here, we present a high-throughput protocol for deamination of long single-stranded (ss) DNA or oligo pools ...containing complex sequences. We describe steps for the preparation of both enzyme (activation-induced deaminase, AID) and ssDNA substrates, the deamination reaction, uracil-friendly amplification, and data analysis. This assay can be used to determine the intrinsic mutation profile of a single antibody gene or a pool of selected regions on genomic DNA.
For complete details on the use and execution of this protocol, please refer to Wang et al. (2023).1
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•Stepwise high-throughput assay for AID/APOBEC cytidine deaminase activity•Reveal antibody variable exon mutation profiles in vitro•Intrinsic mutation profiling of up to 600K nt DNA in one reaction•Simplified optimization and evaluation of the antibody sequence
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Cytidine deaminases as DNA mutators play important roles in immunity and genome stability. Here, we present a high-throughput protocol for deamination of long single-stranded (ss) DNA or oligo pools containing complex sequences. We describe steps for the preparation of both enzyme (activation-induced deaminase, AID) and ssDNA substrates, the deamination reaction, uracil-friendly amplification, and data analysis. This assay can be used to determine the intrinsic mutation profile of a single antibody gene or a pool of selected regions on genomic DNA.
Breakthrough infection of SARS-CoV-2 is a serious challenge, as increased infections were documented in fully-vaccinated individuals. Recipients with poor antibody response are highly vulnerable to ...reinfection, whereas those with strong antibody responses achieve sterilizing immunity. Thus far, biomarkers associated with levels of vaccine-elicited antibody response are still lacking. Here, we studied the antibody response of age- and gender-controlled healthy cohort, who received inactivated SARS-CoV-2 vaccines and profiled the B cell receptor repertoires in longitudinally consecutive samples. Upon vaccination, all vaccinated individuals displayed a convergent antibody response with shared common antibody clones and public neutralizing antibodies. Strikingly, poor vaccine-responders are distinguishable from strong vaccine-responders by a biased V-usage before vaccination and IgG to IgM mRNA ratio. These findings reveal molecular signatures associated with the different levels of vaccine-induced antibody response, which could be further developed into biomarkers for the design of vaccination strategies.
Base editors (BEs) introduce base substitutions without double-strand DNA cleavage. Besides precise substitutions, BEs generate low-frequency 'stochastic' byproducts through unclear mechanisms. Here, ...we performed in-depth outcome profiling and genetic dissection, revealing that C-to-G BEs (CGBEs) generate substantial amounts of intermediate double-strand breaks (DSBs), which are at the centre of several byproducts. Imperfect DSB end-joining leads to small deletions via end-resection, templated insertions or aberrant transversions during end fill-in. Chromosomal translocations were detected between the editing target and off-targets of Cas9/deaminase origin. Genetic screenings of DNA repair factors disclosed a central role of abasic site processing in DSB formation. Shielding of abasic sites by the suicide enzyme HMCES reduced CGBE-initiated DSBs, providing an effective way to minimize DSB-triggered events without affecting substitutions. This work demonstrates that CGBEs can initiate deleterious intermediate DSBs and therefore require careful consideration for therapeutic applications, and that HMCES-aided CGBEs hold promise as safer tools.
Somatic hypermutation (SHM), initiated by activation-induced cytidine deaminase (AID), generates mutations in the antibody-coding sequence to allow affinity maturation. Why these mutations ...intrinsically focus on the three nonconsecutive complementarity-determining regions (CDRs) remains enigmatic. Here, we found that predisposition mutagenesis depends on the single-strand (ss) DNA substrate flexibility determined by the mesoscale sequence surrounding AID deaminase motifs. Mesoscale DNA sequences containing flexible pyrimidine-pyrimidine bases bind effectively to the positively charged surface patches of AID, resulting in preferential deamination activities. The CDR hypermutability is mimicable in in vitro deaminase assays and is evolutionarily conserved among species using SHM as a major diversification strategy. We demonstrated that mesoscale sequence alterations tune the in vivo mutability and promote mutations in an otherwise cold region in mice. Our results show a non-coding role of antibody-coding sequence in directing hypermutation, paving the way for the synthetic design of humanized animal models for optimal antibody discovery and explaining the AID mutagenesis pattern in lymphoma.
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•AID activity on single-strand DNA per se shapes the CDR-preferential SHM profile•Non-coding function of DNA at the mesoscale level directs AID deamination in vivo•Flexibility of single-strand DNA substrate contributes to AID substrate preference•DNA alteration can make a cold FR hot
Somatic hypermutation in antibody-coding sequences preferentially occurs within specific regions; this study uncovers that the intrinsic single-stranded DNA flexibility within the regions confers the specificity.
Abstract
The main purpose of this paper is to provide readers with a guide to mathematical modeling. The idea of establishing a model is roughly as follows: extract the probability factors of the ...research object, analyze the research object itself, observe the changes of the research object, and observe the effects of environmental changes on the research object. In this article, taking the 2021 American Mathematical Competition in Modeling (MCM) as an example, using the proposed modeling guidelines, a mathematical model was established to describe the decomposition of woody fiber by fungi, and the independent variable was set to hyphal extension rate, temperature conditions, climate conditions, etc. Set the dependent variable to decomposition rate, and fit a series of logarithmic expression and exponential expression according to the data provided by references
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. Establish a community evolution model to realize the dynamic evolution of the interaction between different types of fungi, fungi with a higher competitive ranking will have a higher hyphal extension rate than the original without the interaction. As a result, fungi with a higher competitive ranking is in a dominant position in the colony, and fungi with a lower competitive ranking is in a subordinate or inferior position in the colony. In order to use the relative advantages and disadvantages of various fungi in different environments to predict the exact composition of fungi in the colony, considering the influence of initial climatic conditions on the evolution of the community, and bring about relative changes in the competitive ranking of different types of fungi. The model example in this article reflects the correctness of the modeling guidelines.
Abstract
Background
The mechanics of double key loop (DKL) are not well defined, and this finite element study was designed to explore its force system.
Methods
A simplified 3-dimensional finite ...element model of single and double key loops with an archwire between the lateral incisor and second premolar was established in Ansys Workbench 17.0. Activation in Type-1 (retraction at the distal end), Type-2 (retraction at the distal key) and Type-3 (Type-2 plus ligation between keys) was simulated. The vertical force, load/deflection ratio and moment/force ratio of stainless-steel and titanium-molybdenum alloy (TMA) loops were calculated and compared.
Results
The double key loop generated approximately 40% of the force of a single key loop. Type-2 loading of DKL showed a higher L/D ratio than Type-1 loading with a similar M/F ratio. Type-3 loading of DKL showed the highest M/F ratio with a similar L/D ratio as single key loop. The M/F ratio in Type-3 loading increased with the decreasing of retraction force. The DKL of TMA produced approximately 40% of the force and moment compared with those of SS in all loading types. When activated at equal distances below 1 mm, the M/F ratios of SS and TMA DKL with equal preactivation angles were almost the same.
Conclusion
The M/F ratio on anterior teeth increases with the preactivation angle and deactivation of DKL. The M/F ratio at a certain distance of activation mainly depends on the preactivation angle instead of the wire material. TMA is recommended as a substitute for SS in DKL for a lower magnitude of force.
ZmSTK2_USP, located on the long arm of chromosome 4, belongs to the serine/threonine kinase gene in maize. The sequence analysis of 2100 bp upstream from the start codon ATG has shown that it ...contains cis-element motifs and two types of anther/pollen-specific promoter elements (GTGA and AGAAA), suggesting that it is the pollen-specific promoter. To investigate the function of ZmSTK2_USP promoter, the GUS gene fusion system was employed. In proZmSTK2_USP-GUS genetically modified plants, GUS activity was detected in mature pollen grains and pollen tubes but not found in other floral and vegetative tissues. These results show that proZmSTK2_USP is the pollen-specific promoter and drives pollen-specific activity during the middle stage of pollen development until pollen maturation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The proteomic analysis of serine/threonine kinase (STK) genes was conducted using various bioinformatics tools. Eight members of STK gene family were obtained from NCBI having more than 80% ...similarity with STK1 and STK2. The results of analysis revealed that their domains and conserved motifs has similar positions on the sequence and possibly perform similar functions. The results of expansion mode of STK showed that it belongs to tandem repeat mode. The phylogenetic tree revealed that STKs in maize existed before the species differentiation. The STKs in maize belong to paralog relationship, which shows that they might have common ancestral genes, representing that USP domain in STKs was already present before species differentiation. Thus the present study would be helpful in better understanding of genetic evolutionary relationship of homological sequences of STK1 and STK2 with its other members, and the similarities as well as differences between the structure and function of each member of the STK gene family.
Breast cancer is the most common cancer in women. Obtaining the tumor part of breast ultrasound image is of great significance for medical assistance. Breast ultrasound images have the ...characteristics of variable tumor morphology, more shadows, and blurred borders. Therefore, image preprocessing is usually required before segmentation. However, the traditional image preprocessing method is difficult to effectively distinguish the tumor area and tissue shadow in the ultrasound image, which affects the segmentation result of the tumor part. Therefore, this paper proposes an ultrasound image preprocessing method based on texture features. First, extract the different texture feature images of the breast ultrasound image, and then concatenate the original image and two different texture feature images together to form a new 3-channel RGB image. In the experiment, combining different preprocessing methods and different segmentation methods, the segmentation results of breast ultrasound images are evaluated. Compared with traditional preprocessing methods, the preprocessing methods proposed in this paper have improved in all segmentation evaluation indexes. The experimental results show that the Intersection-over-Union (IoU) and Dice-Similarity-Coefficient (DSC) increased to 0.6022 and 0.7554 respectively.
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