MALS: an efficient strategy for multiple site-directed mutagenesis employing a combination of DNA amplification, ligation and suppression PCR

<p>Abstract</p> <p>Background</p> <p>Multiple approaches for the site-directed mutagenesis (SDM) have been developed. However, only several of them are designed for simultaneous introduction of multiple nucleotide alterations, and these are time consuming. In addition, many of the existing multiple SDM methods have technical limitations associated with type and number of mutations that can be introduced, or are technically demanding and require special chemical reagents.</p> <p>Results</p> <p>In this study we developed a quick and efficient strategy for introduction of multiple complex mutations in a target DNA without intermediate subcloning by using a combination of connecting SDM and suppression PCR. The procedure consists of sequential rounds, with each individual round including PCR amplification of target DNA with two non-overlapping pairs of oligonucleotides. The desired mutation is incorporated at the 5' end of one or both internal oligonucleotides. DNA fragments obtained during amplification are mixed and ligated. The resulting DNA mixture is amplified with external oligonucleotides that act as suppression adapters. Suppression PCR limits amplification to DNA molecules representing full length target DNA, while amplification of other types of molecules formed during ligation is suppressed. To create additional mutations, an aliquot of the ligation mixture is then used directly for the next round of mutagenesis employing internal oligonucleotides specific for another region of target DNA.</p> <p>Conclusion</p> <p>A wide variety of complex multiple mutations can be generated in a short period of time. The procedure is rapid, highly efficient and does not require special chemical reagents. Thus, MALS represents a powerful alternative to the existing methods for multiple SDM.</p

A designed amphiphilic peptide containing the silk fibroin motif as a potential carrier of hydrophobic drugs

The amphiphilic peptide is becoming attractive as a potential drug carrier to improve the dissolvability of hydrophobic drugs in an aqueous system; thus, facilitating drug uptake by target cells. Here, we report a novel designed amphiphilic peptide, Ac-RADAGAGARADAGAGA-NH2, which was able to stabilize pyrene, a hydrophobic model drug we chose to study in aqueous solution. This designed peptide formed a colloidal suspension by encapsulating pyrene inside the peptide–pyrene complex. Egg phosphatidylcholine (EPC) vesicles were used to mimic cell bilayer membranes. We found that pyrene was released from the peptide coating into the EPC vesicles by mixing the colloidal suspension with EPC vesicles, which was followed by steady fluorescence spectra as a function of time. A calibration curve for the amount of pyrene released into the EPC vesicles at a given time was used to determine the final concentration of pyrene released into the lipid vesicles from the peptide–pyrene complex. The release rate of the peptide–pyrene complex was calculated to quantify the transfer of pyrene into EPC vesicles.China. Ministry of Education (Sichuan University, National "985 Project"

A modified eCK model with stronger security for tripartite authenticated key exchange

Since Bellare and Rogaway presented the first formal security model for authenticated key exchange (AKE) protocols in 1993, many formal security models have been proposed. The extended Canetti-Krawczyk (eCK) model proposed by LaMacchia et al. is currently regarded as the strongest security model for two-party AKE protocols. In this paper, we first generalize the eCK model for tripartite AKE protocols, called teCK model, and enhance the security of the new model by adding a new reveal query. In the teCK model, the adversary has stronger powers, and can learn more secret information. Then we present a new tripartite AKE protocol based on the NAXOS protocol, called T-NAXOS protocol, and analyze its security in the teCK model under the random oracle assumption

Efficient privacy preserving predicate encryption with fine-grained searchable capability for cloud storage

With the fast development in Cloud storage technologies and ever increasing use of Cloud data centres, data privacy and confidentiality has become a must. Indeed, Cloud data centres store each time more sensitive data such as personal data, organizational and enterprise data, transactional data, etc. However, achieving confidentiality with flexible searchable capability is a challenging issue. In this article, we show how to construct an efficient predicate encryption with fine-grained searchable capability. Predicate Encryption (PEPE) can achieve more sophisticated and flexible functionality compared with traditional public key encryption. We propose an efficient predicate encryption scheme by utilizing the dual system encryption technique, which can also be proved to be IND-AH-CPA (indistinguishable under chosen plain-text attack for attribute-hiding) secure without random oracle. We also carefully analyse the relationship between predicate encryption and searchable encryption. To that end, we introduce a new notion of Public-Key Encryption with Fine-grained Keyword Search (PEFKSPEFKS). Our results show that an IND-AH-CPA secure PE scheme can be used to construct an IND-PEFKS-CPA (indistinguishable under chosen plain-text attack for public-key encryption with fine-grained keyword search) secure PEFKSPEFKS scheme. A new transformation of PE-to-PEFKS is also proposed and used to construct an efficient PEFKSPEFKS scheme based on the transformation from the proposed PEPE scheme. Finally, we design a new framework for supporting privacy preserving predicate encryption with fine-grained searchable capability for Cloud storage. Compared to most prominent frameworks, our framework satisfies more features altogether and can serve as a basis for developing such frameworks for Cloud data centres.Peer ReviewedPostprint (author's final draft

Local bone metabolism balance regulation via double-adhesive hydrogel for fixing orthopedic implants

© 2021 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)The effective osteointegration of orthopedic implants is a key factor for the success of orthopedic surgery. However, local metabolic imbalance around implants under osteoporosis condition could jeopardize the fixation effect. Inspired by the bone structure and the composition around implants under osteoporosis condition, alendronate (A) was grafted onto methacryloyl hyaluronic acid (H) by activating the carboxyl group of methacryloyl hyaluronic acid to be bonded to inorganic calcium phosphate on trabecular bone, which is then integrated with aminated bioactive glass (AB) modified by oxidized dextran (O) for further adhesion to organic collagen on the trabecular bone. The hybrid hydrogel could be solidified on cancellous bone in situ under UV irradiation and exhibits dual adhesion to organic collagen and inorganic apatite, promoting osteointegration of orthopedic implants, resulting in firm stabilization of the implants in cancellous bone areas. In vitro, the hydrogel was evidenced to promote osteogenic differentiation of embryonic mouse osteoblast precursor cells (MC3T3-E1) as well as inhibit the receptor activator of nuclear factor-κ B ligand (RANKL)-induced osteoclast differentiation of macrophages, leading to the upregulation of osteogenic-related gene and protein expression. In a rat osteoporosis model, the bone-implant contact (BIC) of the hybrid hydrogel group increased by 2.77, which is directly linked to improved mechanical stability of the orthopedic implants. Overall, this organic-inorganic, dual-adhesive hydrogel could be a promising candidate for enhancing the stability of orthopedic implants under osteoporotic conditions.This work was supported by the National Key R&D Program of China (2020YFA0908200), National Natural Science Foundation of China (82120108017), Six talent peaks project in Jiangsu Province (WSW-018). This work was financed by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project “Institute for Research and Innovation in Health Sciences” UID/BIM/04293/2019.info:eu-repo/semantics/publishedVersio