High level expression of human basic fibroblast growth factor in Escherichia coli: Evaluating the effect of the GC content and rare codons within the first 13 codons

High-level expression of recombinant human basic fibroblast growth factor in Escherichia coli presents research opportunities such as analysis of hbFGF expression after translation initiation region (TIR) mutagenesis. In our study, hbfgf-cDNA was expressed in three stains of E. coli comprising OrigamiB (DE3), BL21 (DE3) and modified strain carrying copies for rare codon tRNAs (BL21 (DE3)-codonplus- RP). During the course of these experiments, we investigated the role of rare codon replacement and ofGC content reduction in N-terminal, just downstream of the ATG start codon. As standardized procedure, two forward primers were designed for modification of N-terminal of hbfgf-cDNA. Nterminally modified genes were PCR amplified and cloned into the expression vector, pET-22b.Meanwhile, wild-type gene remarkably expressed in all the strains especially in codon plus strain, rare codon substituted hbFGF gene construct surprisingly displayed undetectable levels of protein production; modified gene construct with reduction in GC content of the first 13 codons contributes to 2.5 folds increased expression level. In addition, recombinant hbFGF were purified and the biological activity of the recombinant growth factor was demonstrated by its ability to stimulate proliferation of NIH/3T3 cells. Purified rhbFGF exhibited proliferative activity comparable to commercial rhbFGF

PEGylated systems in pharmaceutics

This review addresses the use of poly(ethylene glycol) (PEG) and PEG conjugation for the design of novel dosage forms and the modification of biomolecules. The peculiarities of PEGylated nanoparticles, liposomes, proteins, enzymes, and small drug and polyelectrolyte molecules and their influence on systemic drug delivery, including overcoming of various biological barriers and adhesion to mucosal tissues (mucoadhesion), are considered

Evaluation of the effect of crocetin on antitumor activity of doxorubicin encapsulated in PLGA nanoparticles

Objective(s): The current study reports investigation of codelivery by PLGA nanoparticles (NPs) loaded with crocetin (Cro), a natural carotenoid dicarboxylicHYPERLINK “http://en.wikipedia.org/wiki/Carboxylic_acid” acid that is found in the crocus flower, and Doxorubicin (DOX). Materials and Methods: Double emulsion/solvent evaporation method was used for preparation of PLGA nanoparticles containing Dox and Cro. Characterizations of prepared NPs were investigated by atomic force microscopy (AFM) and dynamic light scattering analysis. In vitro Cytotoxicity of DOX and Cro loaded PLGA NPs (PLGA-DOX-Cro) on MCF-7 cell line was evaluated using MTT test. Flow cytometry experiments were implemented to distinguish cells undergoing apoptosis from those undergoing necrosis. Furthermore the expression of caspase 3 was examined by western blot analysis. Results: The prepared formulations had size of 150- 300 nm. Furthermore, PLGA-DOX-Cro nanoparticles inhibited MCF-7 tumor cells growth more efficiently than either DOX or Cro alone at the same concentrations, as quantified by MTT assay and flow cytometry. Studies on cellular uptake of DOX-Cro-NPs demonstrated that NPs were effectively taken up by MCF-7 tumor cells. Conclusion: This study suggested that DOX-Cro-NPs may have promising applications in breast cancer therapy

Molecular analysis and the toxin, MSCRAMM, and biofilm genes of methicillin-resistant Staphylococcus aureus strains isolated from pemphigus wounds: A study based on SCCmec and dru typing

Introduction: Pemphigus is a chronic autoimmune blistering disease. Pemphigus blisters can damage the natural skin barrier and increase the risk of life-threatening conditions. Colonization of pemphigus wounds with methicillin-resistant Staphylococcus aureus (MRSA) prolongs wound healing and increases mortality rate. Assessing MRSA prevalence, types, and toxin and adhesion genes can facilitate the detection of MRSA strains which cause infections, selection of appropriate treatments, and healing of pemphigus wounds. This study aimed to determine the SCCmec, the direct repeat unit (dru) types (dts), and the toxin, MSCRAMM, and biofilm genes of MRSA strains isolated from pemphigus wounds. Methods: In this cross-sectional study, 118 S. aureus isolates were gathered from 118 patients with pemphigus. MRSA detection was performed using the mecA gene. Using the polymerase chain reaction method, all MRSA isolates were assessed for the presence of the sea, seb, sec, tst, eta, pvl, hla, hlb, MSCRAMM, and ica genes. Typing and subtyping were performed through respectively SCCmec typing and dru typing methods. The Bionumerics software was used for analyzing the data and drawing the minimum spanning tree. Findings: From 118 S. aureus isolates, 51 were MRSA. SCCmec typing revealed the prevalence of SCCmec II with a prevalence of 64.7 (33 out of 51 isolates) and SCCmec III with a prevalence of 35.3 (18 out of 51 isolates). Dru typing indicated seven dts, namely dts 10a, 10g, 10m, 13i, 8h, 8i, and 9ca in two main clusters. The dt9ca was a new dru type and was registered in the dru-typing database (www.dru-typing.org). The prevalence rates of the hla, sea, and sec genes in MRSA isolates were respectively 54.9, 27.4, and 1.9, while the hlb, seb, eta, and pvl genes were not detected at all. Only one MRSA with SCCmec III and dt10a carried the tst encoding gene. MSCRAMM gene analysis revealed the high prevalence of the eno (31.3) and the fib (21.5) genes. The prevalence rates of the icaA and icaD biofilm formation genes were 3.9 and 5.8, respectively. There were no significant differences between the two detected SCCmec types and between the two detected dts clusters respecting the prevalence of the encoding genes of virulence factors and MSCRAMMs. Conclusion: The toxin genes hla and sea are prevalent among MRSA strains with SCCmec II and III isolated from pemphigus wounds. The most prevalent dts are dt10a and dt10g among MRSA with SCCmec III and dt8h and dt8i among MRSA with SCCmec II. © 2020 Elsevier B.V

Emerging era of “somes”: polymersomes as versatile drug delivery carrier for cancer diagnostics and therapy

Over the past two decades, polymersomes have been widely investigated for the delivery of diagnostic and therapeutic agents in cancer therapy. Polymersomes are stable polymeric vesicles, which are prepared using amphiphilic block polymers of different molecular weights. The use of high molecular weight amphiphilic copolymers allows for possible manipulation of membrane characteristics, which in turn enhances the efficiency of drug delivery. Polymersomes are more stable in comparison with liposomes and show less toxicity in vivo. Furthermore, their ability to encapsulate both hydrophilic and hydrophobic drugs, significant biocompatibility, robustness, high colloidal stability, and simple methods for ligands conjugation make polymersomes a promising candidate for therapeutic drug delivery in cancer therapy. This review is focused on current development in the application of polymersomes for cancer therapy and diagnosis. Graphical abstract