Ultrasonication-enhanced gelation properties of a versatile amphiphilic formamidine-based gelator exhibiting both organogelation and hydrogelation abilities.

We describe the preparation of a novel amphiphilic gelator built from a formamidine core, which is able to form a variety of physical organogels and hydrogels at concentrations ranging from 15 to 150 mg mL-1. Interestingly, ultrasound treatment of isotropic solutions (i.e., gel-precursor) resulted in a remarkable enhancement of the gelation kinetics as well as the gelation scope and characteristic gel properties (e.g., critical gelation concentration, gel-to-sol transition temperature, viscoelastic moduli) in comparison to the heating-cooling protocol typically used to obtain supramolecular gels. Thermoreversibility, thixotropy, injectability and multistimuli responsiveness are some of the most relevant functionalities of these gels. Electron microscopy imaging revealed the formation of entangled networks made of fibers of nanometer diameters and micrometer lengths, with different morphological features depending on the solvent. Insights into the driving forces for molecular aggregations were obtained from FTIR, NMR, PXRD and computational studies. The results suggest a major stabilization of the fibers through additive N-HO hydrogen bonds, in combination with hydrophobic interactions, over π-π stacking interactions.

Transfection of Antisense Oligonucleotides Mediated by Cationic Vesicles Based on Non-Ionic Surfactant and Polycations Bearing Quaternary Ammonium Moieties.

Three different ionene polymers with varying quaternary ammonium moieties were used as a proof of concept for the formulation of antisense oligonucleotides, which are capable of inhibiting Renilla luciferase messenger ribonucleic acid (mRNA). Cationic vesicles, consisting of cationic polymer, antisense oligonucleotide (Luc) and non-ionic surfactant polysorbate 80, were investigated regarding their ζ potential, cytotoxicity and transfection efficiency. Deoxyribonucleic acid- (DNA) forming complexes in the presence of cationic vesicles were also investigated in terms of small-angle X-ray scattering (SAXS). The studied cationic vesicles showed very little, if any, toxicity against HeLa cells. Transfection abilities proved to vary strongly depending on the present quaternary ammonium moiety.

Aromatic ionene topology and counterion-tuned gelation of acidic aqueous solutions.

Unusual gelation of acidic solutions was achieved using polycations bearing quaternary ammonium moieties. These ionene polymers are based on a disubstituted phenylene dibenzamide core, which allows the construction of different topomers (i.e. ortho-1, meta-2 and para-3). The topology of the polymers was found to play a key role on their aggregation behaviour both in pure water and in a variety of aqueous acidic solutions leading to the formation of stable acidic gels. Specifically, ortho-1 showed superior gelation ability than the analogues meta-2 and para-3 in numerous solutions of different pH and ionic strengths. Lower critical gelation concentrations, higher gel-to-sol transition temperatures and faster gelation were usually observed for ortho-1 regardless the solvent system. Detailed computational molecular dynamic simulations revealed a major role of the counterion (Cl-) and specific polymerpolymer interactions. In particular, hydrogen bonds, N-Hπ interactions and intramolecular π-π stacking networks are distinctive in ortho-1. In addition, counterions located at internal hydration regions also affect to such polymerpolymer interactions, acting as binders and, therefore, providing additional stability.

Antimicrobial and Hemolytic Studies of a Series of Polycations Bearing Quaternary Ammonium Moieties: Structural and Topological Effects.

A series of polycations bearing quaternary ammonium moieties have shown antimicrobial activity against the Gram-negative bacterium Escherichia coli. Different polymer topologies governed by a disubstituted aromatic core as well as different diamine-based linkers were found to influence the antimicrobial properties. Moreover, the hemolytic activity against human red blood cells was measured and demonstrated good biocompatibility and selectivity of these polycations for bacteria over mammalian cells.

Supramolecular phase-selective gelation by peptides bearing side-chain azobenzenes: effect of ultrasound and potential for dye removal and oil spill remediation.

Phase selective gelation (PSG) of organic phases from their non-miscible mixtures with water was achieved using tetrapeptides bearing a side-chain azobenzene moiety. The presence of the chromophore allowed PSG at the same concentration as the minimum gelation concentration (MGC) necessary to obtain the gels in pure organic phases. Remarkably, the presence of the water phase during PSG did not impact the thermal, mechanical, and morphological properties of the corresponding organogels. In the case of miscible oil/water mixtures, the entire mixture was gelled, resulting in the formation of quasi-hydrogels. Importantly, PSG could be triggered at room temperature by ultrasound treatment of the mixture or by adding ultrasound-aided concentrated solution of the peptide in an oil-phase to a mixture of the same oil and water. Moreover, the PSG was not affected by the presence of salts or impurities existing in water from natural sources. The process could be scaled-up, and the oil phases (e.g., aromatic solvents, gasoline, diesel fuel) recovered almost quantitatively after a simple distillation process, which also allowed the recovery and reuse of the gelator. Finally, these peptidic gelators could be used to quantitatively remove toxic dyes from aqueous solutions.

Non-invasive and continuous monitoring of the sol-gel phase transition of supramolecular gels using a fast (open-ended coaxial) microwave sensor.

An open coaxial re-entrant microwave sensor has been used for the non-invasive and continuous monitoring of the sol-gel transition of physical gels characterized by different gelation mechanisms, solvents, compositions, and stabilities. Comparison of measurements by differential scanning calorimetry allowed the identification of the phase transition by a change in the dielectric properties of the material over time.

Amide-triazole isosteric substitution for tuning self-assembly and incorporating new functions into soft supramolecular materials.

The proof-of-concept for the modular synthesis of new functional soft gel materials based on amide-triazole isosteric replacement has been demonstrated. A coassembly approach of isosteric amino acid-based hydrogelators was fruitfully applied for fine-tuning the release of entrapped drugs.

Dissolvable metallohydrogels for controlled release: evidence of a kinetic supramolecular gel phase intermediate.

Two metallohydrogels based on an amino acid-based ligand and Zn(II) salts were synthesized. These hydrogels show an uncommon, reversible, time-dependent transformation from the opaque to transparent state. These hydrogels also exhibit gradual dissolution in water (pH ≤ 7) over time. A water-soluble stimulant, caffeine, could be in situ loaded into the hydrogels and slowly released during dissolution.

Amino acid-based multiresponsive low-molecular weight metallohydrogels with load-bearing and rapid self-healing abilities.

A multiresponsive metallohydrogel based on an amino acid-derived low molecular weight (LMW) ligand and a Zn(II) salt was prepared. This hydrogel showed remarkable shape-persistent, self-standing, load-bearing and self-healing properties, which is uncommon in LMW hydrogels.

Multistimuli-responsive supramolecular organogels formed by low-molecular-weight peptides bearing side-chain azobenzene moieties.

This work demonstrates that the incorporation of azobenzene residues into the side chain of low-molecular-weight peptides can modulate their self-assembly process in organic solvents leading to the formation of stimuli responsive physical organogels. The major driving forces for the gelation process are hydrogen bonding and π-π interactions, which can be triggered either by thermal or ultrasound external stimuli, affording materials having virtually the same properties. In addition, a predictive model for gelation of polar protic solvent was developed by using Kamlet-Taft solvent parameters and experimental data. The obtained viscoelastic materials exhibited interconnected multistimuli responsive behaviors including thermal-, photo-, chemo- and mechanical responses. All of them displayed thermoreversability with gel-to-sol transition temperatures established between 33-80 °C and gelation times from minutes to several hours. Structure-property relationship studies of a designed peptide library have demonstrated that the presence and position of the azobenzene residue can be operated as a versatile regulator to reduce the critical gelation concentration and enhance both the thermal stability and mechanical strength of the gels, as demonstrated by comparative dynamic rheology. The presence of N-Boc protecting group in the peptides showed also a remarkable effect on the formation and properties of the gels. Despite numerous examples of peptide-based gelators known in the literature, this is the first time in which low-molecular-weight peptides bearing side chain azobenzene units are used for the synthesis of "intelligent" supramolecular organogels. Compared with other approaches, this strategy is advantageous in terms of structural flexibility since it is compatible with a free, unprotected amino terminus and allows placement of the chromophore at any position of the peptide sequence.

Programming of marginal zone B-cell fate by basic Kruppel-like factor (BKLF/KLF3).

Splenic marginal zone (MZ) B cells are a lineage distinct from follicular and peritoneal B1 B cells. They are located next to the marginal sinus where blood is released. Here they pick up antigens and shuttle the load onto follicular dendritic cells inside the follicle. On activation, MZ B cells rapidly differentiate into plasmablasts secreting antibodies, thereby mediating humoral immune responses against blood-borne type 2 T-independent antigens. As Krüppel-like factors are implicated in cell differentiation/function in various tissues, we studied the function of basic Krüppel-like factor (BKLF/KLF3) in B cells. Whereas B-cell development in the bone marrow of KLF3-transgenic mice was unaffected, MZ B-cell numbers in spleen were increased considerably. As revealed in chimeric mice, this occurred cell autonomously, increasing both MZ and peritoneal B1 B-cell subsets. Comparing KLF3-transgenic and nontransgenic follicular B cells by RNA-microarray revealed that KLF3 regulates a subset of genes that was similarly up-regulated/down-regulated on normal MZ B-cell differentiation. Indeed, KLF3 expression overcame the lack of MZ B cells caused by different genetic alterations, such as CD19-deficiency or blockade of B-cell activating factor-receptor signaling, indicating that KLF3 may complement alternative nuclear factor-κB signaling. Thus, KLF3 is a driving force toward MZ B-cell maturation.

Non-conservative homologous recombination in human B lymphocytes is promoted by activation-induced cytidine deaminase and transcription.

During secondary immunoglobulin (Ig) diversification in vertebrates, the sequence of the variable region of Ig genes may be altered by templated or non-templated mechanisms. In both cases, cytidine deamination by activation-induced cytidine deaminase (AID) in the transcribed Ig loci leads to DNA lesions, which are repaired by conservative homologous recombination (HR) during Ig gene conversion, or by non-templated mutagenesis during somatic hypermutation. The molecular basis for the differential use of these two pathways in different species is unclear. While experimental ablation of HR in avian cells performing Ig gene conversion may promote a switch to somatic hypermutation, the activity of HR processes in intrinsically hypermutating mammalian cells has not been measured to date. Employing a functional HR assay in human germinal centre like B cell lines, we detect elevated HR activity that can be enhanced by transcription and AID. Products of such recombination events mostly arise through non-conservative HR pathways, while the activity of conservative HR is low to absent. Our results identify non-conservative HR as a novel DNA transaction pathway promoted by AID and suggest that somatic hypermutation in germinal centre B cells may be based on a physiological suppression of conservative HR.

Involvement of Rad18 in somatic hypermutation.

Somatic hypermutation of Ig genes is initiated by transcription-coupled cytidine deamination in Ig loci. Error-prone processing of the resultant DNA lesions is thought to cause extensive mutagenesis, but it is presently an enigma how and why error-prone rather than error-free repair pathways are recruited. During DNA replication, recruitment of error-prone translesion polymerases may be mediated by Rad6/Rad18-mediated ubiquitination of proliferating cell nuclear antigen, a major switchboard controlling the fidelity of DNA lesion bypass in eukaryotes. By inactivation of Rad18 in the DT40 B cell line, we show that the Rad6 pathway is involved in somatic hypermutation in these cells. Our findings imply that targeted recruitment of mutagenic polymerases by the Rad6 pathway contributes to the complex process of somatic hypermutation and provide a framework for more detailed mechanistic studies of the mutagenesis phase of secondary Ig diversification.

Structural phylogenetic analysis of activation-induced deaminase function.

In mammals, activation-induced deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) of Ig genes. SHM and CSR activities require separate regions within AID. A chromosome region maintenance 1 (CRM1)-dependent nuclear export signal (NES) at the AID C terminus is necessary for CSR, and has been suggested to associate with CSR-specific cofactors. CSR appeared late in AID evolution, during the emergence of land vertebrates from bony fish, which only display SHM. Here, we show that AID from African clawed frog (Xenopus laevis), but not pufferfish (Takifugu rubripes), can induce CSR in AID-deficient mouse B cells, although both are catalytically active in bacteria and mammalian cell systems, albeit at decreased level. Like mammalian AID, Takifugu AID is actively exported from the cell nucleus by CRM1, and the Takifugu NES can substitute for the equivalent region in human AID, indicating that all the CSR-essential NES motif functions evolutionarily predated CSR activity. We also show that fusion of the Takifugu AID catalytic domain to the entire human noncatalytic domain restores activity in mammalian cells, suggesting that AID features mapping within the noncatalytic domain, but outside the NES, influence its function.

Episomal vectors to monitor and induce somatic hypermutation in human Burkitt-Lymphoma cell lines.

Somatic hypermutation (SHM) occurs at a specific B-cell differentiation stage, during the germinal centre reaction, and provides a means to diversify and shape the antibody repertoire of the adaptive immune system. Burkitt-Lymphoma (BL) is a germinal centre derived B-cell malignancy. Presumably deregulation of the somatic hypermutation- and/or class switch recombination process causes a translocation between the myc-locus and one of the Ig-loci, which is characteristic for BL. We show here that we developed an episomal-based vector system to monitor and induce AID-dependent somatic hypermutation in human cell lines. Exemplarily, we utilized this system to investigate the hypermutation status of various BL cell lines. Analysis of the influence of Ig-specific cis-regulatory elements for the mutability of a green fluorescent protein (GFP) reporter transgene revealed, that the presence of Ig-enhancers is required for an efficient targeting of a constitutively transcribed GFP transgene. In contrast, a transcriptional inducible GFP transgene was hypermutated by overexpression of activation-induced cytidine deaminase (AID), even in the absence of Ig-specific sequences. This observation verifies that overexpression of AID in non hypermutating BL cell lines as well as the expression of endogenous AID in the hypermutating BL cell line Raji can overcome the target restriction of AID.

Stringent doxycycline-dependent control of gene activities using an episomal one-vector system.

Conditional expression systems are of pivotal importance for the dissection of complex biological phenomena. Here, we describe a novel EBV-derived episomally replicating plasmid (pRTS-1) that carries all the elements for conditional expression of a gene of interest via Tet regulation. The vector is characterized by (i) low background activity, (ii) high inducibility in the presence of doxycycline (Dox) and (iii) graded response to increasing concentrations of the inducer. The chicken beta actin promoter and an element of the murine immunoglobin heavy chain intron enhancer drive constitutive expression of a bicistronic expression cassette that encodes the highly Dox-sensitive reverse tetracycline controlled transactivator rtTA2(S)-M2 and a Tet repressor-KRAB fusion protein (tTS(KRAB)) (silencer) placed downstream of an internal ribosomal entry site. The gene of interest is expressed from the bidirectional promoter P(tet)bi-1 that allows simultaneous expression of two genes, of which one may be used as surrogate marker for the expression of the gene of interest. Tight down regulation is achieved through binding of the silencer tTS(KRAB) to P(tet)bi-1 in the absence of Dox. Addition of Dox releases repression and via binding of rtTA2(S)-M2 activates P(tet)bi-1.

Proviral integration of an Abelson-murine leukemia virus deregulates BKLF-expression in the hypermutating pre-B cell line 18-81.

The transcription factor BKLF (basic Krüppel-like factor, KLF3) is a member of the Krüppel-like factors (KLF) family. KLF members harbor a characteristic C-terminal zinc-finger DNA-binding domain and bind preferentially to CACCC-motifs. BKLF is highly expressed in haematopoietic and erythoid cells and works either as repressor or activator of transcription in various genes. BKLF-deficient mice display myeloproliferative disorders and abnormalities in haematopoiesis. Other members of the KLF-family such as GKLF and BCL11A have been implicated in tumorigenesis, however, for BKLF such association has not yet been demonstrated. We report here that a single Abelson-murine leukemia virus (A-MuLV) provirus is present in the genome of the hypermutating murine pre-B cell line 18-81. The provirus has integrated into the locus of the transcription factor BKLF. In contrast to other A-MuLV transformed pre-B cell lines, BKLF is highly transcribed in cell line 18-81. BKLF transcripts originate from the retroviral long terminal repeats (LTRs) and BKLF protein can be detected by gel shift retardation assay. We hypothesize on a potential role of BKLF deregulation in tumorigenesis and/or in the induction of somatic hypermutation in cell line 18-81.

Activation-induced cytidine deaminase fails to induce a mutator phenotype in the human pre-B cell line Nalm-6.

Activation-induced cytidine deaminase (AID) plays a key role in the induction of somatic hypermutation and class switching at the immunoglobulin loci of B lymphocytes. AID overexpression can induce a mutator phenotype in lymphoid and nonlymphoid cell lines, suggesting that AID by itself is sufficient to trigger hypermutation and class switching. AID expression in vivo is considered to be restricted to germinal center B lymphocytes, yet AID expression is also seen in many B cell lymphomas, hinting at a potential role for the development of these malignancies. We used a GFP-based reversion assay to efficiently evaluate the activation of mutator phenotypes. As expected, AID overexpression in the human Burkitt lymphoma cell line BL70 caused hypermutation. Surprisingly, AID overexpression in the human pre-B cell line Nalm-6 failed to induce a detectable mutator phenotype, indicating that Nalm-6 cells are probably lacking an essential factor(s) to confer AID-induced mutagenesis. This finding supports the concept that AID overexpression by itself must not automatically lead to the onset of a mutator phenotype. In addition, treating Nalm-6 transfectants with thymidine, a potential mutagenic drug, caused profound mutation rates on the GFP transgene. Thus, the GFP-based mutation assay might prove a powerful tool to study protein- and chemical-induced mutator phenotypes in cell lines.

Dual reporter system to dissect cis- and trans-effects influencing the mutation rate in a hypermutating cell line.

Activation induced cytidine deaminase (AID) plays a key role in the induction of somatic hypermutation and class switching in the immunoglobulin genes of B-lymphocytes. AID expression by itself is sufficient to induce a GC-basepair biased mutator phenotype in lymphoid and non-lymphoid cell lines. Nevertheless a network of cis-regulatory elements and additional trans-factor proteins seems to govern the molecular mechanism of somatic hypermutation. To address the nature of mutation rate changes observed in the hypermutating pre-B cell line 18-81, we extended our previously described green fluorescent protein (GFP) reversion-system. Introducing an additional mutation reporter transgene enables us to discriminate between cis- and trans-factor caused alterations in the mutator phenotype. We show here that in cell line 18-81 the mutation rate declines upon prolonged periods of cell culture. The gradual loss of the mutator phenotype in cell line 18-81 is due to the downregulation of endogenous AID expression and can be reconstituted by overexpression of human AID protein. A correlation between AID mRNA levels and mutation rates is evident and even small changes in AID expression levels cause a significant effect on the mutability of the reporter transgenes.