Preparation and target protease identification of a cyanobacterial serine protease inhibitor, arthropin

Objective·To prepare a high-purity cyanobacterial serine protease inhibitor, screen its target proteases, and detect its inhibitory activity.Methods·A novel serine protease inhibitor from Arthrospira platensis was identified in the Expanded Human Oral Microbiome Database (eHOMD) by amino acid sequence alignment and named as arthropin. The fusion expression vector pSUMO3-arthropin was constructed and transferred into Escherichia coli (E. coli) BL21(DE3) system for fusion protein expression. The recombinant arthropin was purified by a four-step chromatographic purification approach of nickel affinity chromatography, enzymatic digestion, reverse nickel affinity chromatography, and anion exchange chromatography. In addition, the recombinant arthropin was co-incubated with 14 serine proteases such as activated factor Ⅸ (FⅨa), FⅩa, FⅪa, activated protein C (APC) and kallikrein 1 (KLK1), respectively, and then analyzed by SDS-PAGE. The inhibitory rate of arthropin on KLK1 was assayed with kinetic methods. The crystallization conditions of the recombinant arthropin were screened preliminarily, and the suitable crystals were picked for X-ray diffraction to collect the data. Finally, a sub-stable structure model of arthropin was predicted with AlphaFlod Colab.Results·SDS-PAGE showed that the fused arthropin was successfully expressed in the E. coli BL21(DE3) system, and following purification, the high-purity recombinant arthropin, the relative molecular mass of which was similar to the theoretical value (45 800), was obtained. The co-incubation analysis of recombinant arthropin with 14 serine proteases revealed that arthropin was able to form stable covalent complexes with 9 proteases, including FⅩa, APC, FⅨa, FⅪa, trypsin, cathepsin G, KLK1, KLK7 and thrombin. Arthropin inhibited KLK1 with a second-order association rate constant of 1.7×103 L/(mol·s). Moreover, the recombinant arthropin crystalised under the condition of 25% PEG MME 550, 0.1 mol/L MES (pH 6.5) and 0.01 mol/L ZnCl2 , and the crystals preliminarily diffracted to a resolution of 10 Å (1 Å=1×10-10 m). The analysis of the structure predicted by AlphaFlod Colab revealed that arthropin had the classical structural features of the inhibitory serpin.Conclusion·Arthropin, a serpin from Arthrospira platensis, was successfully obtained with high purity and a broad-spectrum of serine protease inhibition, but at a low inhibitory rate

Multi-index fuzzy comprehensive evaluation model with information entropy of alfalfa salt tolerance based on LiDAR data and hyperspectral image data

Rapid, non-destructive and automated salt tolerance evaluation is particularly important for screening salt-tolerant germplasm of alfalfa. Traditional evaluation of salt tolerance is mostly based on phenotypic traits obtained by some broken ways, which is time-consuming and difficult to meet the needs of large-scale breeding screening. Therefore, this paper proposed a non-contact and non-destructive multi-index fuzzy comprehensive evaluation model for evaluating the salt tolerance of alfalfa from Light Detection and Ranging data (LiDAR) and HyperSpectral Image data (HSI). Firstly, the structural traits related to growth status were extracted from the LiDAR data of alfalfa, and the spectral traits representing the physical and chemical characteristics were extracted from HSI data. In this paper, these phenotypic traits obtained automatically by computation were called Computing Phenotypic Traits (CPT). Subsequently, the multi-index fuzzy evaluation system of alfalfa salt tolerance was constructed by CPT, and according to the fuzzy mathematics theory, a multi-index Fuzzy Comprehensive Evaluation model with information Entropy of alfalfa salt tolerance (FCE-E) was proposed, which comprehensively evaluated the salt tolerance of alfalfa from the aspects of growth structure, physiology and biochemistry. Finally, comparative experiments showed that: (1) The multi-index FCE-E model based on the CPT was proposed in this paper, which could find more salt-sensitive information than the evaluation method based on the measured Typical Phenotypic Traits (TPT) such as fresh weight, dry weight, water content and chlorophyll. The two evaluation results had 66.67% consistent results, indicating that the multi-index FCE-E model integrates more information about alfalfa and more comprehensive evaluation. (2) On the basis of the CPT, the results of the multi-index FCE-E method were basically consistent with those of Principal Component Analysis (PCA), indicating that the multi-index FCE-E model could accurately evaluate the salt tolerance of alfalfa. Three highly salt-tolerant alfalfa varieties and two highly salt-susceptible alfalfa varieties were screened by the multi-index FCE-E method. The multi-index FCE-E method provides a new method for non-contact non-destructive evaluation of salt tolerance of alfalfa

Role of OCT4 in cisplatin treatment of testicular embryonal carcinoma

Purpose: To determine the role of embryonal transcription factor OCT4 in cisplatin treatment of testicular embryonal carcinoma.Methods: In vitro assays were employed to assess the effect of cisplatin treatment on testicular embryonal carcinoma cell lines under OCT4 silencing. Following treatment with 500 ng/μL cisplatin, MTT assay was used to examine cell proliferation of 2012-EP and 833K-E cells with or without OCT silencing, while wound healing assay was used to examine cell migration ability. Transwell assay and crystal violet staining were employed to measure cell invasive capacity, whereas the distribution pattern of cell cycle was assessed by flow cytometry. The expression levels of several critical components in tumorigenicity related pathways with or without OCT silencing were determined by Western-blot analysis.Results: Cisplatin enhanced OCT4-silenced cell viability at all concentration (p < 0.01) when compared to control cells. Upon treatment with 500 ng/μL cisplatin, OCT4-silenced cells showed 2- to 3-fold enhancement in cell proliferation (p < 0.001), 2-fold increase in cell migration capacity (p < 0.001), and about 1.5-fold enhancement in invasive capacity (p < 0.001) when compared to control cells. In addition, OCT4 silencing upregulated the expression level of the proteins involved in cell proliferation, cell mobility, cancer metastasis and cell cycle control.Conclusion: The results suggest that OCT4 may serve as a therapeutic target for testicular embryonal carcinoma treatment in combination with cisplatin by modulating OCT4 expression level. This physiological evidence indicates that OCT4 downregulation contributes to cisplatin resistance in chemotherapy and subsequent disease relapse.Keywords: OCT4, Cisplatin resistance, Testicular embryonal carcinoma, Chemotherap

A fingerprint based crypto-biometric system for secure communication

To ensure the secure transmission of data, cryptography is treated as the most effective solution. Cryptographic key is an important entity in this procedure. In general, randomly generated cryptographic key (of 256 bits) is difficult to remember. However, such a key needs to be stored in a protected place or transported through a shared communication line which, in fact, poses another threat to security. As an alternative, researchers advocate the generation of cryptographic key using the biometric traits of both sender and receiver during the sessions of communication, thus avoiding key storing and at the same time without compromising the strength in security. Nevertheless, the biometric-based cryptographic key generation possesses few concerns such as privacy of biometrics, sharing of biometric data between both communicating users (i.e., sender and receiver), and generating revocable key from irrevocable biometric. This work addresses the above-mentioned concerns. In this work, a framework for secure communication between two users using fingerprint based crypto-biometric system has been proposed. For this, Diffie-Hellman (DH) algorithm is used to generate public keys from private keys of both sender and receiver which are shared and further used to produce a symmetric cryptographic key at both ends. In this approach, revocable key for symmetric cryptography is generated from irrevocable fingerprint. The biometric data is neither stored nor shared which ensures the security of biometric data, and perfect forward secrecy is achieved using session keys. This work also ensures the long-term security of messages communicated between two users. Based on the experimental evaluation over four datasets of FVC2002 and NIST special database, the proposed framework is privacy-preserving and could be utilized onto real access control systems.Comment: 29 single column pages, 8 figure

Expression of DNMT1 and DNMT3a Are Regulated by GLI1 in Human Pancreatic Cancer

BACKGROUND AND AIMS: GLI1, as an indispensable transcriptional factor of Hedgehog signaling pathway, plays an important role in the development of pancreatic cancer (PC). DNA methyltransferases (DNMTs) mediate the methylation of quantity of tumor-related genes. Our study aimed to explore the relationship between GLI1 and DNMTs. METHODS: Expressions of GLI1 and DNMTs were detected in tumor and adjacent normal tissues of PC patients by immunohistochemistry (IHC). PANC-1 cells were treated by cyclopamine and GLI1-siRNA, while BxPC-3 cells were transfected with overexpression-GLI1 lentiviral vector. Then GLI1 and DNMTs expression were analyzed by qRT-PCR and western blot (WB). Then we took chromatin immunoprecipitation (ChIP) to demonstrate GLI1 bind to DNMT1. Finally, nested MSP was taken to valuate the methylation levels of APC and hMLH1, when GLI1 expression altered. RESULTS: IHC result suggested the expressions of GLI1, DNMT1 and DNMT3a in PC tissues were all higher than those in adjacent normal tissues (p<0.05). After GLI1 expression repressed by cyclopamine in mRNA and protein level (down-regulation 88.1±2.2%, 86.4±2.2%, respectively), DNMT1 and DNMT3a mRNA and protein level decreased by 91.6%±2.2% and 83.8±4.8%, 87.4±2.7% and 84.4±1.3%, respectively. When further knocked down the expression of GLI1 by siRNA (mRNA decreased by 88.6±2.1%, protein decreased by 63.5±4.5%), DNMT1 and DNMT3a mRNA decreased by 80.9±2.3% and 78.6±3.8% and protein decreased by 64.8±2.8% and 67.5±5.6%, respectively. Over-expression of GLI1 by GLI1 gene transfection (mRNA increased by 655.5±85.9%, and protein increased by 272.3±14.4%.), DNMT1 and DNMT3a mRNA and protein increased by 293.0±14.8% and 578.3±58.5%, 143.5±17.4% and 214.0±18.9%, respectively. ChIP assays showed GLI1 protein bound to DNMT1 but not to DNMT3a. Results of nested MSP demonstrated GLI1 expression affected the DNA methylation level of APC but not hMLH1 in PC. CONCLUSION: DNMT1 and DNMT3a are regulated by GLI1 in PC, and DNMT1 is its direct target gene

Structural insights into molecular mechanism for N6-adenosine methylation by MT-A70 family methyltransferase METTL4

METTL4 belongs to a subclade of MT-A70 family members of methyltransferase (MTase) proteins shown to mediate N6-adenosine methylation for both RNA and DNA in diverse eukaryotes. Here, we report that Arabidopsis METTL4 functions as U2 snRNA MTase for N6−2’-O-dimethyladenosine (m6Am) in vivo that regulates flowering time, and specifically catalyzes N6-methylation of 2’-O-methyladenosine (Am) within a single-stranded RNA in vitro. The apo structures of full-length Arabidopsis METTL4 bound to S-adenosyl-L-methionine (SAM) and the complex structure with an Am-containing RNA substrate, combined with mutagenesis and in vitro enzymatic assays, uncover a preformed L-shaped, positively-charged cavity surrounded by four loops for substrate binding and a catalytic center composed of conserved residues for specific Am nucleotide recognition and N6-methylation activity. Structural comparison of METTL4 with the mRNA m6A enzyme METTL3/METTL14 heterodimer and modeling analysis suggest a catalytic mechanism for N6-adenosine methylation by METTL4, which may be shared among MT-A70 family members

Identification and treatment of intestinal malrotation with midgut volvulus in childhood: a multicenter retrospective study

BackgroundIntestinal malrotation is a rare condition, and its delayed diagnosis can lead to fatal consequences. This study aimed to investigate the identification and treatment of malrotation in children.MethodsClinical data, imaging, operative findings, and early postoperative outcomes of 75 children with malrotation were retrospectively analyzed.ResultsThe mean age was 6.18 ± 4.93 days and 51.26 ± 70.13 months in the neonatal group (56 patients) and non-neonatal group (19 patients), respectively. Sixty-seven patients were under the age of 1 year at the time of diagnosis. The occurrence of bilious vomiting and jaundice was significantly higher in the neonatal group (89.29%) than that in the non-neonatal group (37.5%), p &lt; 0.05 and p &lt; 0.01, respectively. The incidence of abnormal ultrasound (US) findings was 97.30% and 100%, respectively, and the sensitivities of the upper gastrointestinal series were 84.21% and 87.5%, respectively. Sixty-six (88%) patients had midgut volvulus, including in utero volvulus (two patients) and irreversible intestinal ischemia (four patients). Most neonates (89.29%) underwent open Ladd's procedure with a shorter operative time (p &lt; 0.01). Reoperation was performed for postoperative complications (four patients) or missed comorbidities (two patients).ConclusionsNon-bilious vomiting was the initial symptom in &gt;10% of neonates and nearly 40% of non-neonates. This highlights the importance for emergency physicians and surgeons to be cautious about ruling out malrotation in patients with non-bilious vomiting. Utilizing US can obviate the need for contrast examinations owing to its higher diagnostic accuracy and rapid diagnosis and can be recommended as a first-line imaging technique. Additionally, open surgery is still an option for neonatal patients

Identification of RegIV as a Novel GLI1 Target Gene in Human Pancreatic Cancer

GLI1 is the key transcriptional factor in the Hedgehog signaling pathway in pancreatic cancer. RegIV is associated with regeneration, and cell growth, survival, adhesion and resistance to apoptosis. We aimed to study RegIV expression in pancreatic cancer and its relationship to GLI1.GLI1 and RegIV expression were evaluated in tumor tissue and adjacent normal tissues of pancreatic cancer patients and 5 pancreatic cancer cell lines by qRT-PCR, Western blot, and immunohistochemistry (IHC), and the correlation between them. The GLI1-shRNA lentiviral vector was constructed and transfected into PANC-1, and lentiviral vector containing the GLI1 expression sequence was constructed and transfected into BxPC-3. GLI1 and RegIV expression were evaluated by qRT-PCR and Western blot. Finally we demonstrated RegIV to be the target of GLI1 by chromatin immunoprecipitation (CHIP) and electrophoretic mobility shift assays (EMSA).The results of IHC and qRT-PCR showed that RegIV and GLI1 expression was higher in pancreatic cancer tissues versus adjacent normal tissues (p<0.001). RegIV expression correlated with GLI1 expression in these tissues (R = 0.795, p<0.0001). These results were verified for protein (R = 0.939, p = 0.018) and mRNA expression (R = 0.959, p = 0.011) in 5 pancreatic cancer cell lines. RegIV mRNA and protein expression was decreased (94.7±0.3%, 84.1±0.5%; respectively) when GLI1 was knocked down (82.1±3.2%, 76.7±2.2%; respectively) by the RNAi technique. GLI1 overexpression in mRNA and protein level (924.5±5.3%, 362.1±3.5%; respectively) induced RegIV overexpression (729.1±4.3%, 339.0±3.7%; respectively). Moreover, CHIP and EMSA assays showed GLI1 protein bound to RegIV promotor regions (GATCATCCA) in pancreatic cancer cells.GLI1 promotes RegIV transcription by binding to the RegIV gene promoter in pancreatic cancer