Position In Sequence Research Articles

Significance of KLK7 expression, polymorphisms, and function in sheep horn growth

BackgroundSheep horns play a critical role in the survival and reproduction of sheep. Research on sheep horns not only aids in comprehending their biological roles but is also vital for developing hornless breeds. Although previous studies have suggested that KLK7 may be associated with keratin growth, there are few studies that have focused on the role of KLK7 in sheep horns. This study aims to elucidate the relationship between KLK7 and sheep horns by analyzing the expression, genetic polymorphisms, and potential functions of KLK7 in sheep horns.ResultsThis study utilized RNA sequencing (RNA-Seq) data to analyze the expression levels of the KLK7 gene across different species, as well as among different breeds, tissue types, and genders in sheep. Potential functional sites of KLK7 were explored using whole-genome sequencing (WGS) data. Allele specific expression sites in the KLK7 gene sequence were identified. Finally, the WGS data were linked with sheep horn length for association analysis, and significantly different single-nucleotide polymorphisms (SNPs) were detected and validated by Kompetitive Allele Specific PCR (KASP) genotyping.ConclusionOur results demonstrate that KLK7 was highly expressed in soft horn and skin tissues, and its expression was significantly higher in small-horned sheep than it was in large-horned sheep, suggesting that KLK7 may have an inhibitory effect on horn growth. By comparing the amino acid sequence of KLK7 with KLK7 sequences in other species, we discovered eight amino acids at specific positions in the KLK7 protein sequence that may have regulatory functions in determining the size of horns in ruminant animals. Thirteen SNPs with F-statistic value (Fst) > 0.15 were identified. By integrating RNA-Seq and WGS data, we discovered two SNPs (g.56695395 T > C and g.56695484A > C) with allele specific expression between the large- and small-horned sheep. The two SNPs were validated and were found to have significantly different (P < 0.05) effects on horn length. Our findings suggest a strong association between KLK7 and sheep horn length, indicating the potential role of KLK7 in inhibiting horn growth and providing novel insights into the functionality of KLK7.

P0588 Efficacy and health-related quality of life associated with second-line vedolizumab after one prior first-line anti-tumour necrosis factor treatment in patients with active ulcerative colitis: A meta-analysis of the GEMINI-1 and VISIBLE-1 trials

Abstract Background Vedolizumab (VDZ) is a monoclonal antibody approved for adults with moderately to severely active ulcerative colitis (UC) and an inadequate response/intolerance to conventional therapy or anti-tumour necrosis factor (TNF) treatment. There is published evidence supporting the use of VDZ as a first-line biologic in patients with UC, but additional efficacy data for second-line VDZ (after first-line anti-TNF treatment) are required to define its optimal position in the treatment sequence. We conducted a meta-analysis of the efficacy and health-related quality of life (HRQoL) associated with second-line VDZ treatment exposure in patients with UC using data from the randomized, placebo (PBO)-controlled phase 3 GEMINI-1/VISIBLE-1 trials. Methods GEMINI-1 (NCT00783718) evaluated intravenous (IV) VDZ as induction/maintenance treatment in patients with active UC.1 VISIBLE-1 (NCT02611830) assessed IV/subcutaneous maintenance VDZ in patients with moderately/severely active UC.2 Patients who received first-line anti-TNF treatment within 5 years before trial enrolment were included in the meta-analysis. The primary outcome was clinical remission (defined as Adapted Mayo Score [AMS] 1 or 2; Figure). Secondary outcomes included corticosteroid-free remission, mucosal healing and HRQoL (measured via the Inflammatory Bowel Disease Questionnaire and EuroQoL-5 Dimension Visual Analogue Scale). Outcomes were assessed at Week 52. Study-specific treatment effects were estimated using generalized linear models. Efficacy of VDZ vs PBO was compared using fixed-effects models. Regression analyses assessed treatment effects, adjusting for disease location/duration and previous corticosteroid use. Results In total, 137 patients from GEMINI-1/VISIBLE-1 were included (VDZ, n=89; PBO, n=48). Patient characteristics were similar across cohorts, but the mean (standard deviation) disease duration ranged from 6.6 (4.4) years (GEMINI-1 VDZ IV cohort) to 9.9 (10.1) years (VISIBLE-1 PBO cohort). Second-line VDZ was associated with a higher likelihood of achieving clinical remission (AMS 1 and 2; Figure) and all secondary outcomes, including improved HRQoL, at Week 52 vs PBO (Table). Regression analysis showed that patients treated with VDZ were 11.35 (95% confidence interval 3.25–39.63) times more likely to achieve clinical remission (defined as AMS 1) vs PBO. Conclusion Second-line VDZ after first-line anti-TNF treatment significantly increased the likelihood of achieving clinical remission, mucosal healing and improved HRQoL at Week 52 vs PBO in patients with UC. The small sample size and heterogeneity observed in some outcomes warrant further research to strengthen these findings.

DsDAP: An efficient method for high-abundance DNA-encoded library construction in mammalian cells.

DNA-encoded libraries are invaluable tools for high-throughput screening and functional genomics studies. However, constructing high-abundance libraries in mammalian cells remains challenging. Here, we present dsDNA-assembly-PCR (dsDAP), a novel Gibson-assembly-PCR strategy for creating DNA-encoded libraries, offering improved flexibility and efficiency over previous methods. We demonstrated this approach by investigating the impact of translation initiation sequences (TIS) on protein expression in HEK293T cells. Both CRISPR-Cas9 and piggyBac systems were employed for genomic integration, allowing comparison of different integration methods. Our results confirmed the importance of specific nucleotides in the TIS region, particularly the preference for adenine at the -3 position in high-expression sequences. We also explored the effects of library dilution on genotype-phenotype correlations. This Gibson-assembly-PCR strategy overcomes limitations of existing methods, such as restriction enzyme dependencies, and provides a versatile tool for constructing high-abundance libraries in mammalian cells. Our approach has broad applications in functional genomics, drug discovery, and the study of gene regulation.

Deep learning–based assessment of missense variants in the COG4 gene presented with bilateral congenital cataract

ObjectiveWe compared the protein structure and pathogenicity of clinically relevant variants of the COG4 gene with AlphaFold2 (AF2), Alpha Missense (AM), and ThermoMPNN for the first time.Methods and analysisThe sequences...

Principles of CRISPR-Cas13 mismatch intolerance enable selective silencing of point-mutated oncogenic RNA with single-base precision.

Single-nucleotide variants (SNVs) are extremely prevalent in human cancers, although most of these remain clinically unactionable. The programmable RNA nuclease CRISPR-Cas13 has been deployed to specifically target oncogenic RNAs. However, silencing oncogenic SNVs with single-base precision remains extremely challenging due to the intrinsic mismatch tolerance of Cas13. Here, we show that introducing synthetic mismatches at precise positions of the spacer sequence enables de novo design of guide RNAs [CRISPR RNAs (crRNAs)] with strong preferential silencing of point-mutated transcripts. We applied these design principles to effectively silence the oncogenic KRAS G12 hotspot, NRAS G12D and BRAF V600E transcripts with minimal off-target silencing of the wild-type transcripts, underscoring the adaptability of this platform to silence various SNVs. Unexpectedly, the SNV-selective crRNAs harboring mismatched nucleotides reduce the promiscuous collateral activity of the RfxCas13d ortholog. These findings demonstrate that the CRISPR-Cas13 system can be reprogrammed to target mutant transcripts with single-base precision, showcasing the tremendous potential of this tool in personalized transcriptome editing.

Applying a cytochrome c oxidase I barcode for Leishmania species typing.

Species delimitation has always been a challenge for taxonomists and for Leishmania studies there is no exception. Herein we attempt to display the usefulness of the mitochondrial gene Cytochrome Oxidase I-coI in classical and barcode-based approaches for Leishmania characterization. A total of 228 samples were analyzed, comprising 28 Leishmania related taxa, mainly from cultures of the Oswaldo Cruz Foundation`s Leishmania Collection. Primers were designed for amplification of coI; sequences were analyzed by distance-based indicators and both the Neighbor Joining and NeighborNet as species grouping techniques. Automatic Barcode Gap Discovery was applied to define species delimitation while for the character-based analysis a software for Barcoding with Logic formulas was employed. Final sequences of 486 bp with 238 parsimonious sites were aligned and edited. Robust groups were formed for most of the genus species, distinctive nucleotide positions in the barcode sequence were observed for 11 of them. A good agreement between the techniques applied and the original characterization was observed. Few species were not distinguished by coI: (i) L. (V.) peruviana, L. (V.) lindenbergi, and L. (V.) utingensis; (ii) L. (L.) venezuelensis and (iii) L. colombiensis and L. equatorensis with identical sequences. Some of these taxa have been, at one time or another, classified as controversial and, for most of them, a higher number of isolates should be studied to properly infer their taxonomic status. CoI represents a mitochondrial target that stands out as a taxonomically important asset with multiple advantages over other genes. This paper corresponds to the first report of coI analysis in Leishmania, a potentially advantageous target for the characterization of this parasite.

Mapping of resistance genes to powdery mildew based on DNA re-sequencing and bulk segregant analysis in Capsicum.

Powdery mildew caused by Leveillula taurica adversely affects the development and growth of pepper plants. However, there have been few reports on the fine mapping and quantitative trait locus (QTLs) gene cloning of resistance genes to powdery mildew in pepper. Herein, an F2 segregating population was constructed using the high resistance material "NuMex Suave Red" and the extremely susceptible material "c89" for bulked segregant analysis and DNA re-sequencing (BSA-seq). Molecular markers were used to achieve fine mapping, followed by expression verification. A major QTL located on chromosome 5 (Chr5, 7.20-11.75Mb) that is associated with resistance to powdery mildew in pepper was mapped using BSA-seq. A narrow interval of 64.86kb encompassing five genes was refined using InDel and KSAP molecular markers developed from the QTL region. Among them, the expression of the ubiquitin-conjugating enzyme E2 gene, Capana05g000392, was significantly upregulated in multiple resistant materials. In addition, there was a single nucleotide polymorphism (SNP) of A/G in the 241st position of the CDS sequence of Capana05g000392, which in turn leads to an amino acid polymorphism of M/V between susceptible parent and resistant parent. Overall, these results indicate that the Capana05g000392 gene may serve as a robust potential factor against powdery mildew in pepper. These findings further elucidate the genetic mechanism of resistance to powdery mildew in pepper and facilitate molecular marker-assisted breeding.

Effects of Suryanamaskara and Sheetali Pranayama on College Students Aggression: A Prospective Interventional Study

Introduction: Students today face a more fast-paced and demanding lifestyle due to heavy workloads and hectic schedules. By using the potential of Suryanamaskara, a dynamic sequence of yoga postures known for its energising and calming effects, along with Sheetali Pranayama, a cooling breathing technique that promotes relaxation and reduces stress, this study contributes to the growing body of research on non pharmacological interventions for emotional regulation in adults. The focus of this research is the effects of Suryanamaskara and Sheetali Pranayama on reducing aggression levels in college students. Aim: To investigate the effects of Suryanamaskara and Sheetali Pranayama in reducing aggression levels in college students. Materials and Methods: This prospective interventional study employed a stratified purposive random sampling design to evaluate the efficacy of Suryanamaskara and Sheetali Pranayama in reducing aggression levels among college students. Voluntary participants were initially screened using the Buss and Perry Aggression Questionnaire (AQ) and those who exhibited high aggression scores were selected for the intervention training programme. A total of 54 participants were randomly assigned to two experimental groups. Group 1 underwent a Suryanamaskara intervention, while Group 2 engaged in Sheetali Pranayama, both incorporating Sukshma Vyayama and a cooling down period within the framework. Intervention sessions lasted 45 minutes, five days a week, for 10 weeks. Aggression levels were assessed preand postintervention and paired sample t-tests and independent sample t-tests were used for statistical analysis. Results: The findings of this study demonstrate a significant reduction in aggression levels among college students following a 10 week yoga intervention programme, with the mean difference in aggression scores between pre- and postintervention being 14.40 for the Suryanamaskara group and 17.29 for the Sheetali Pranayama group, indicating a decrease in aggression levels. Both the Suryanamaskara and Sheetali Pranayama groups exhibited notable improvements in aggression scores as measured by the Buss and Perry AQ. However, a comparative analysis using independent sample t-tests and effect size showed that the pretest t-statistics were -1.59 (p-value=0.11) and for the post-test t-statistics were -1.16 (p-value=0.24), revealing no significant differences between the groups for either pretest or post-test scores (p-value&gt;0.05). Conclusion: This experimental study demonstrated a significant reduction in aggression levels among college students following a 10-week intervention with either Suryanamaskara or Sheetali Pranayama programmes. A comparative analysis revealed equivalent changes in aggression levels between the two groups, indicating that both yogic interventions were equally effective in reducing aggression in college students.

Improving the Potency Prediction for Chemically Modified siRNAs through Insights from Molecular Modeling Focused on Individual Sequence Positions

Improving the Potency Prediction for Chemically Modified siRNAs through Insights from Molecular Modeling Focused on Individual Sequence Positions

Genetic diversity of Tn916 transposon and its association with tetracycline resistance in Staphylococcus aureus isolates

Staphylococcus aureus, a major pathogen responsible for hospital-acquired infections, poses a severe global health threat due to its virulence and increasing antimicrobial resistance. This study confirmed the presence of the tet(M) gene and the Tn916 transposon in tetracycline-resistant S. aureus isolates. We explored genetic polymorphism by analyzing the positions of Tn916 and Shine-Dalgarno sequences in 30 pathogenic S. aureus isolates. Antibiotic resistance profiles against five antibiotics, including tetracycline, were assessed using disc diffusion methods. PCR was utilized to detect the presence of the tet(M) gene and Tn916 transposon, and sequence sizes between these genetic elements were determined using specific primers. The resulting band patterns were converted into a binary matrix, and a phylogenetic tree was constructed using the Past3 software. All S. aureus isolates examined showed tetracycline resistance and contained the tet(M) gene and Tn916 transposon. Analysis of the distances between Tn916 and Shine-Dalgarno sequences revealed diverse band patterns among the strains. Phylogenetic clustering based on PCR band patterns classified the strains into seven distinct clusters, indicating significant genetic diversity and polymorphism. Molecular docking studies supported our findings, suggesting potential applications in developing effective treatments against S. aureus infections.

Ascorbate: a forgotten component in the cytotoxicity of Cu(II) ATCUN peptide complexes

In 1983, Linus Pauling and colleagues reported about enhanced antitumor activity of the Cu(II) complex of the simplest ATCUN (amino terminal Cu(II) and Ni(II)-binding motif) peptide (NH2-Gly-Gly-His-COOH, GGH) in the presence of ascorbate as an additive. In the following 4 decades, structural modifications of this complex were implemented, however, anticancer activity could not be significantly increased. This has led to neglecting the ATCUN motif and its Cu(II) complexes as potential chemotherapeutic agents. Furthermore, the addition of ascorbate with its positive effect on the anticancer activity has fallen into oblivion. In this work, we compared Cu(II) GGH with Cu(II) ATCUN peptides bearing β-Ala instead of Gly at the 2nd position of the peptide sequence regarding their in vitro complex stability and cytotoxicity (MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and annexin V-FITC (fluorescein isothiocyanate) apoptosis assay) towards three cancer cell lines (AGS, HeLa and NCI-N87). Such an exchange of amino acids led to an up to three-fold higher cytotoxic effect in the presence of ascorbate. We thus achieved a significant increase in the otherwise moderate cytotoxicity of Cu(II) ATCUN-like complexes. Lipophilicity assays (n-octanol/water coefficient, log P values) of the studied complexes were used to evaluate differences in the antiproliferative activity.Graphical abstract

Modulation of memory by prism adaptation in healthy subjects

Recent findings suggest that prism adaptation can extend its effects beyond spatial attention, modulating the performance of different cognitive tasks by acting on cerebellar, parietal and temporal-frontal networks. We tested groups of healthy subjects to investigate the effects of rightward vs. leftward prism adaptation vs. neutral lenses exposure in a series of memory tasks, probing either short-term (Digit span, Corsi span) or long-term memory (Supraspan verbal and spatial learning). In the short-term memory tasks, leftward prism adaptation selectively increased verbal span, while rightward prism adaptation increased spatial span. In the long-term memory tasks, leftward prism adaptation selectively increased verbal supraspan, i.e., increased the number of digits in the correct sequence reproduced and reduced the number of repetitions needed to learn the supraspan sequence. On the other hand, rightward prism adaptation selectively increased spatial supraspan, i.e. it increased the number of spatial positions in the correct sequence reproduced and reduced the number of repetitions needed to learn the supraspan sequence. Moreover, rightward, but not leftward, prism adaptation selectively increased supraspan recall after a delay interval, regardless of the stimulus material, i.e., it increased the number of digits or spatial positions recalled after a delay interval. Neutral lenses exposure did not influence any memory task. These findings suggest that prism adaptation can induce both modality/hemispheric-specific and process-specific effects on short-term and long-term explicit memory.

Exploration of the Computational Predicted Internal Tagging Preferred Protein Properties

Protein tags are commonly used in many biological experiments, and adding a tag to an intolerant sequence position can significantly damage the functions of a protein and affect the outcome of an experiment. Recently, an in-house computational prediction method, TagScore, was developed which uses sequence homology to identify non-conservative regions of proteins permissive of tagging. The properties of the proteins that are predicted by TagScore to prefer internal tagging were explored using gene enrichment strategies. Proteins that prefer internal tagging were found to be related to GTPase-associated proteins and sequence features with disordered and polar regions, as predicted by TagScore.

A GCN and Transformer complementary network for skeleton-based action recognition

Graph Convolution Networks (GCNs) have been widely used in skeleton-based action recognition. Although there are significant progress, the inherent limitation still lies in the restricted receptive field of GCN, hindering its ability to extract global dependencies effectively. And the joints that are structurally separated can also have strong correlation. Previous works rarely explore local and global correlations of joints, leading to insufficiently model the complex dynamics of skeleton sequences. To address this issue, we propose a GCN and Transformer complementary network (GTC-Net) that allows parallel communications between GCN and Transformer domains. Specifically, we introduce a graph convolution and self-attention combined module (GAM), which can effectively leverage the complementarity of GCN and self-attention to perceive local and global dependencies of joints for the human body. Furthermore, in order to address the problems of long-term sequence ordering and position detection, we design a position-aware module (PAM), which can explicitly capture the ordering information and unique identity information for body joints of skeleton sequence. Extensive experiments on NTU RGB+D 60 and NTU RGB+D 120 datasets are conducted to evaluate our proposed method. The results demonstrate that our method can achieve competitive results on both datasets.

Semisynthetic Phage Display Library Construction: Design and Synthesis of Diversified Single-Chain Variable Fragments and Generation of Primary Libraries.

Display of antibody fragments on the surface of M13 filamentous bacteriophages is a well-established approach for the identification of antibodies binding to a target of interest. Here, we describe the first of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Libraries. The three-step method involves (1) primary library (PL) construction, (2) filtered library construction, and (3) secondary library construction. The first step, described here, entails design, synthesis, and cloning of four PLs. These PLs are designed with specific properties amenable to therapeutic antibody development using one universal variable heavy (VH) scaffold and four distinct variable light (VL) scaffolds. The scaffolds are diversified in positions that bind both protein and peptide targets identified in antibody-antigen complexes of known structure using the amino acid frequencies found in those positions in known human antibody sequences, avoiding residues that may lead to developability liabilities. The diversified scaffolds are combined with 90 synthetic neutral HCDR3 sequences designed with developable human diversity genes (IGHD) and joining heavy genes (IGHJ) in germline configuration, and assembled as single-chain variable fragments (scFvs) in a VL-linker-VH orientation. The four designed PLs are synthesized using trinucleotide phosphoramidites (TRIMs) and cloned independently into a phagemid vector for M13 pIII display. Quality control of the cloning of the four PLs is also described, which involves sequencing scFvs in each library.

EpiArt: a graphical HLA eplet amino acid repertoire translation reveals the need for an epitope driven revision of allele group nomenclature.

The immune response after transplantation depends on recipient/donor HLA allele mismatches. To enhance our understanding of the relations of HLA alleles in terms of amino-acid polymorphisms and shared epitopes, we assessed pairwise sequence difference between HLA-alleles. We translated amino-acid sequences of confirmed eplets into an atlas of HLA class I and II antigens, followed by visualization of the pairwise allele distances by means of antigen-specific disparity graphs in differential amino-acid space. We obtained an overview of relationships of all alleles of an antigen, corresponding similarity/dissimilarity structures, outliers, alleles with similarity to different antigen groups. Additionally, we calculated prevalence of the amino-acids for each polymorphic sequence position and visualized them in amino-acid motif plots of all alleles belonging to an antigen. Our visualizations show strongly varying intra-group heterogeneity of HLA class I and II alleles, as well as shared inter-group and inter-locus eplets and epitopes, indicating a benefit of epitope-based transplant matching: Single allele recipient/donor mismatches potentially refer to identical eplets, or to a set of multiple mismatched eplets. This data reveals inconsistencies in the HLA group nomenclature and consequently adds a new level of quality to allocation, motivating the definition of tolerable or taboo mismatches.

An interactional linguistics study of Shi ba in Mandarin conversation

From the perspective of interactional linguistics and adopting the methodology of conversation analysis, this study explores some fine-grained interactional uses of shi ba in Mandarin conversation. It first classifies shi ba into unmarked and marked forms generally in terms of their prosodic manifestations with the assistance of Praat, the speech analysis tool. Then it demonstrates that the unmarked shi ba fulfills three interactional functions: requesting confirmation, seeking affiliation and maintaining floor; the marked shi ba registers dissatisfaction and displays affiliation. However, shi ba is basically a tag question, and serves to invite the recipient to proffer certain interactional response. The specific function that shi ba performs in conversation is tied to three decisive factors – its prosodic manifestations, position in specific turns and sequences as well as the relative epistemic status of interlocutors. The study also suggests that there lies a common motivation for the emergence of various interactional functions of shi ba, that is – the demand for an on-line alignment, which reflects the cross-fertilization between language and interaction.

Expression of thermostable MMLV reverse transcriptase in Escherichia coli by directed mutation

The functionality of Moloney murine leukemia virus reverse transcriptase (MMLV RT) will increase with the improvement of its solubility and thermal stability. Introduce directed mutation at specific positions of the MMLV RT sequence and codon optimization is needed to achieve these properties. The two RT coding sequences with (rRT-K) and without directed mutations (rRT-L) were versatility optimized and expressed to analyze the ribonuclease H (RNase H) inactivity and thermostable polymerase activity. For this purpose, the five-point mutations (438–442aa) and three-point mutations (530, 568, and 659 aa) were done at the RT connection domain and RNase H active site, respectively. High expression levels of rRT-L and rRT-K were obtained in E. coli BL21(DE3) and BL21(shuffle) strains, 0.5 mM IPTG concentration at 37 °C, and 8 hours’ post-induction condition. Then, recombinant enzymes were purified and verified by Ni-NTA resin and western blotting. Insilico analysis (IUpred 3.0) showed that the directed mutation in the RNase H domain caused the formation of disorder regions or instability in the RNase H domain of rRT-K compared to rRT-L. The modified RT-PCR and the RT-LAMP reactions proved the RNase H inactivity of rRT-K. In addition, increasing of thermostability of rRT-K compared to rRT-L and commercial RT was evaluated by the RT-PCR and RT-LAMP reactions. The results showed that rRT-K could successfully tolerate 60 ºC in the two methods. This study revealed that the directed mutations and the versatile sequence optimization can promise to produce thermostable commercial enzymes to decrease non-specific one-step RT-PCR and RT-LAMP products.

Optimized design of annular ejector primary nozzle based on orthogonal test method

Abstract Annular ejectors are widely used in aerospace and other applications, and their performance has a significant impact on the overall system. In this paper, the effects of main nozzle area ratio, contraction section angle and diffusion section angle on the performance of annular ejector are investigated by using computational fluid dynamics combined with one-factor experiments, on the basis of which the L9(33) orthogonal table is established to optimise the structure of main nozzle and to screen the main influencing factors. The results show that the flow field inside the annular ejector is extremely complex. The influence of the main nozzle area ratio on the performance of the annular ejector is very great, and the change of the area ratio will lead to the change of the position of the second excitation sequence as well as the Mach number at the outlet of the main nozzle. However, as with the results of the one-way analysis, the effect of the angle of the constriction section on the exit Mach number is negligible.

Design and technological analysis of the multi-position progressive die for the clamp

Abstract The technological process of the clamp part is analyzed, the design of the stamping and bending process for the clamp is introduced, including determination of the operation sequence and operation position parts dimensions. The process is used side blade settles to distance, pilot pin was used in exact positioning, the blanking and bending of the part were carried out continuously. The design of the general structure, layout of the progressive and forming characteristics die for the clamp is introduced in detail, the method for designing and calculating the key parts.