Elaboration on the architecture of pH-sensitive surface charge-adaptive micelles with enhanced penetration and bactericidal activity in biofilms

Background Biofilm formation is one of the main reasons for persistent bacterial infections. Recently, pH-sensitive copolymers have fascinated incredible attention to tackle biofilm-related infections. However, the proper incorporation of pH-sensitive segments in the polymer chains, which could significantly affect the biofilms targeting ability, has not been particularly investigated. Herein, we synthesized three types of pH-sensitive copolymers based on poly (β-amino ester) (PAE), poly (lactic-co-glycolic acid) (PLA) and polyethylene glycol (PEG), PAE-PLA-mPEG (A-L-E), PLA-PAE-mPEG (L-A-E) and PLA-PEG-PAE (L-E-A) to address this issue. Results The three copolymers could self-assemble into micelles (MA-L-E, ML-A-E and ML-E-A) in aqueous medium. Compared with MA-L-E and ML-A-E, placing the PAE at the distal PEG end of PLA-PEG to yield PLA-PEG-PAE (ML-E-A) was characterized with proper triggering pH, fully biofilm penetration, and high cell membrane binding affinity. Further loaded with Triclosan (TCS), ML-E-A/TCS could efficiently kill the bacteria either in planktonic or biofilm mode. We reasoned that PAE segments would be preferentially placed near the surface and distant from the hydrophobic PLA segments. This would increase the magnitude of surface charge-switching capability, as the cationic PAE+ would easily disassociate from the inner core without conquering the additional hydrophobic force arising from covalent linkage with PLA segments, and rapidly rise to the outermost layer of the micellar surface due to the relative hydrophilicity. This was significant in that it could enable the micelles immediately change its surface charge where localized acidity occurred, and efficiently bind themselves to the bacterial surface where they became hydrolyzed by bacterial lipases to stimulate release of encapsulated TCS even a relatively short residence time to prevent rapid wash-out. In vivo therapeutic performance of ML-E-A/TCS was evaluated on a classical biofilm infection model, implant-related biofilm infection. The result suggested that ML-E-A/TCS was effective for the treatment of implant-related biofilm infection, which was proved by the efficient clearance of biofilm-contaminated catheters and the recovery of surrounding infected tissues. Conclusions In summary, elaboration on the architecture of pH-sensitive copolymers was the first step to target biofilm. The ML-E-A structure may represent an interesting future direction in the treatment of biofilm-relevant infections associated with acidity. Graphic abstract

A Tunable Nanoplatform of Nanogold Functionalised with Angiogenin Peptides for Anti-Angiogenic Therapy of Brain Tumours

Angiogenin (ANG), an endogenous protein that plays a key role in cell growth and survival, has been scrutinised here as promising nanomedicine tool for the modulation of pro-/anti-angiogenic processes in brain cancer therapy. Specifically, peptide fragments from the putative cell membrane binding domain (residues 60–68) of the protein were used in this study to obtain peptide-functionalised spherical gold nanoparticles (AuNPs) of about 10 nm and 30 nm in optical and hydrodynamic size, respectively. Different hybrid biointerfaces were fabricated by peptide physical adsorption (Ang60–68) or chemisorption (the cysteine analogous Ang60–68Cys) at the metal nanoparticle surface, and cellular assays were performed in the comparison with ANG-functionalised AuNPs. Cellular treatments were performed both in basal and in copper-supplemented cell culture medium, to scrutinise the synergic effect of the metal, which is another known angiogenic factor. Two brain cell lines were investigated in parallel, namely tumour glioblastoma (A172) and neuron-like differentiated neuroblastoma (d-SH-SY5Y). Results on cell viability/proliferation, cytoskeleton actin, angiogenin translocation and vascular endothelial growth factor (VEGF) release pointed to the promising potentialities of the developed systems as anti-angiogenic tunable nanoplaftforms in cancer cells treatment.

A Tunable Nanoplatform of Nanogold Functionalised With Angiogenin Peptides for Anti-angiogenic Therapy of Brain Tumours

Angiogenin (ANG), an endogenous protein that plays a key role in cell growth and survival, has been scrutinised here as promising nanomedicine tool for the modulation of pro-/ anti-angiogenic processes in brain cancer therapy. Specifically, peptide fragments from the putative cell membrane binding domain (residues 60-68) of the protein were used in this study to obtain peptide-functionalised spherical gold nanoparticles (AuNPs) of about 10 nm and 30 nm in optical and hydrodynamic size, respectively. Different hybrid biointerfaces were fabricated by peptide physical adsorption (Ang60-68) or chemisorption (the cysteine analogous Ang60-68Cys) at the metal nanoparticle surface, and the cellular assays were performed in the comparison with ANG-functionalised AuNPs. Cellular treatments were performed both in basal and in copper-supplemented cell culture medium, to scrutinise the synergic effect of the metal, which is another known angiogenic factor. Two brain cell lines were investigated in parallel, namely tumour glioblastoma (A172) and neuron-like differentiated neuroblastoma (d-SH-SY5Y). Results on cell viability/proliferation, cytoskeleton actin, angiogenin translocation and VEGF release pointed to the promising potentialities of the developed systems as anti-angiogenic tunable nanoplaftforms in cancer cells treatment.

The AC4 Protein of a Cassava Geminivirus Is Required for Virus Infection

Geminiviruses (family Geminiviridae) are among the most devastating plant viruses worldwide, causing severe damage in crops of economic and subsistence importance. These viruses have very compact genomes and many of the encoded proteins are multifunctional. Here, we investigated the role of the East African cassava mosaic Cameroon virus (EACMCV) AC4 on virus infectivity in Nicotiana benthamiana. Results showed that plants inoculated with EACMCV containing a knockout mutation in an AC4 open reading frame displayed symptoms 2 to 3 days later than plants inoculated with wild-type virus, and these plants recovered from infection, whereas plants inoculated with the wild-type virus did not. Curiously, when an additional mutation was made in the knockout mutant, the resulting double mutant virus completely failed to cause any apparent symptoms. Interestingly, the role of AC4 on virus infectivity appeared to be dependent on an encoded N-myristoylation motif that mediates cell membrane binding. We previously showed that EACMCV containing the AC4T38I mutant produced virus progeny characterized by second-site mutations and reversion to wild-type virus. These results were confirmed in this study using additional mutations. Together, these results show involvement of EACMCV AC4 in virus infectivity; they also suggest a role for the combined action of mutation and selection, under prevailing environmental conditions, on begomovirus genetic variation and diversity.

Estrogen inhibition of norepinephrine responsiveness is initiated at the plasma membrane of GnRH-producing GT1-7 cells

The modulatory action of estradiol (E2) on the GnRH network can be exerted indirectly on presynaptic neurons or directly on estrogen receptors (ERs) located within GnRH hypothalamic neurons. Using the GnRH-producing GT1-7 cell line, we have investigated whether E2 is able to modify the response of these cells to norepinephrine (NE) stimulation. A 48-h exposure of GT1-7 cells to 10 nM E2 reduced NE-induced cAMP accumulation. However, 15-min exposure was enough to induce this inhibitory action, provided that a hormone-free period of 48 h after steroid treatment was allowed. Furthermore, this effect was mimicked by E2 coupled to (E-BSA), indicating that it may be exerted through a membrane-mediated mechanism. In addition, competition experiments using E-BSA coupled to fluorescein isothiocyanate (FITC) revealed the presence of cell membrane-binding sites for E2. Binding of E-BSA coupled to FITC was blocked by preincubation of cells with either E2, antiestrogen ICI 182 780, or tamoxifen. Moreover, fluorescence staining of non-permeabilized cells with antibodies against receptors α and β confirmed the presence of both receptor subtypes at the cell membrane. To determine the nature of the ER involved in this response, specific agonists for ERα 4,4′,4′′-(4-propyl-[1H]pyrazole-1,3,5-triyl)tris-phenol (PPT) and ERβ 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) were used. Since PPT, but not DPN, reproduced the effect of E2, it is suggested that estrogen-induced modulatory action on NE responsiveness was mediated by the ERα isoform. Taken together, these results indicate that E2 modulates the adrenergic sensitivity of GT1-7 cells by a mechanism compatible with the activation of membrane-associated ERs.

The growth factor fusion construct containing B-lymphocyte stimulator (BLyS) and the toxin rGel induces apoptosis specifically in BAFF-R–positive CLL cells

The cytokine B lymphocyte stimulator (BLyS) mediates its effect through cell-surface receptors BAFF-R, TACI, and BCMA. BLyS receptors are expressed only on B cells and not present in other normal cells including normal T lymphocytes. Chronic lymphocytic leukemia (CLL) is a B-cell disease and CLL lymphocytes express BLyS receptors. Gelonin, a type 1 ribosome-inactivating toxin, lacks cell membrane binding domain and hence is nontoxic to intact cells. We generated a construct of recombinant gelonin (rGel) fused to BLyS to specifically target quiescent B-CLL lymphocytes. The construct rGel/BLyS specifically binds and internalizes through BAFF-R into CD19+ B-CLL lymphocytes and induces apoptosis at nanomolar concentrations. In contrast, rGel alone was not able to internalize into these leukemic lymphocytes. Mechanistically, the rGel/BLyS construct inhibits protein synthesis with an IC50 of less than 3 nM compared with more than 5000 nM for rGel toxin alone. This rGel/BLyS-mediated decrease in protein synthesis was associated with a decline in short-lived proteins such as MCL-1 and XIAP, the 2 survival proteins in B-CLL. There was a strong relationship between a decrease in these proteins and the cleavage of PARP, a hallmark feature of apoptosis. Taken together, these data suggest that the rGel/BLyS fusion toxin may have potential therapeutic efficacy for B-CLL patients.

Membrane binding of zebrafish actinoporin-like protein: AF domains, a novel superfamily of cell membrane binding domains

Actinoporins are potent eukaryotic pore-forming toxins specific for sphingomyelin-containing membranes. They are structurally similar to members of the fungal fruit-body lectin family that bind cell-surface exposed Thomsen–Friedenreich antigen. In the present study we found a number of sequences in public databases with similarity to actinoporins. They originate from three animal and two plant phyla and can be classified in three families according to phylogenetic analysis. The sequence similarity is confined to a region from the C-terminal half of the actinoporin molecule and comprises the membrane binding site with a highly conserved P-[WYF]-D pattern. A member of this novel actinoporin-like protein family from zebrafish was cloned and expressed in Escherichia coli. It displays membrane-binding behaviour but does not have permeabilizing activity or sphingomyelin specificity, two properties typical of actinoporins. We propose that the three families of actinoporin-like proteins and the fungal fruit-body lectin family comprise a novel superfamily of membrane binding proteins, tentatively called AF domains (abbreviated from actinoporin-like proteins and fungal fruit-body lectins).

Separate Roles of Escherichia coliReplication Proteins in Synthesis and Partitioning of pSC101 Plasmid DNA

ABSTRACT We report here that the Escherichia coli replication proteins DnaA, which is required to initiate replication of both the chromosome and plasmid pSC101, and DnaB, the helicase that unwinds strands during DNA replication, have effects on plasmid partitioning that are distinct from their functions in promoting plasmid DNA replication. Temperature-sensitive dnaB mutants cultured under conditions permissive for DNA replication failed to partition plasmids normally, and when cultured under conditions that prevent replication, they showed loss of the entire multicopy pool of plasmid replicons from half of the bacterial population during a single cell division. As was observed previously for DnaA, overexpression of the wild-type DnaB protein conversely stabilized the inheritance of partition-defective plasmids while not increasing plasmid copy number. The identification of dnaA mutations that selectively affected either replication or partitioning further demonstrated the separate roles of DnaA in these functions. The partition-related actions of DnaA were localized to a domain (the cell membrane binding domain) that is physically separate from the DnaA domain that interacts with other host replication proteins. Our results identify bacterial replication proteins that participate in partitioning of the pSC101 plasmid and provide evidence that these proteins mediate plasmid partitioning independently of their role in DNA synthesis.