Guiding Transient Peptide Assemblies with Structural Elements Embedded in Abiotic Phosphate Fuels.

Despite great progress in the construction of non-equilibrium systems, most approaches do not consider the structure of the fuel as a critical element to control the processes. Herein, we show that the amino acid side chains (A, F, Nal) in the structure of abiotic phosphates can direct assembly and reactivity during transient structure formation. The fuels bind covalently to substrates and subsequently influence the structures in the assembly process. We focus on the ways in which the phosphate esters guide structure formation and how structures and reactivity cross regulate when constructing assemblies. Through the chemical functionalization of energy-rich aminoacyl phosphate esters, we are able to control the yield of esters and thioesters upon adding dipeptides containing tyrosine or cysteine residues. The structural elements around the phosphate esters guide the lifetime of the structures formed and their supramolecular assemblies. These properties can be further influenced by the peptide sequence of substrates, incorporating anionic, aliphatic and aromatic residues. Furthermore, we illustrate that oligomerization of esters can be initiated from a single aminoacyl phosphate ester incorporating a tyrosine residue (Y). These findings suggest that activated amino acids with varying reactivity and energy contents can pave the way for designing and fabricating structured fuels.

Spontaneous and Selective Peptide Elongation in Water Driven by Aminoacyl Phosphate Esters and Phase Changes.

Nature chose phosphates to activate amino acids, where reactive intermediates and complex machinery drive the construction of polyamides. Outside of biology, the pathways and mechanisms that allow spontaneous and selective peptide elongation in aqueous abiotic systems remain unclear. Herein we work to uncover those pathways by following the systems chemistry of aminoacyl phosphate esters, synthetic counterparts of aminoacyl adenylates. The phosphate esters act as solubility tags, making hydrophobic amino acids and their oligomers soluble in water and enabling selective elongation and different pathways to emerge. Thus, oligomers up to dodecamers were synthesized in one flask and on the minute time scale, where consecutive additions activated autonomous phase changes. Depending on the pathway, the resulting phases initially carry nonpolar peptides and amphiphilic oligomers containing phosphate esters. During elongation and phosphate release, shorter oligomers dominate in solution, while the aggregated phase favors the presence of longer oligomers due to their self-assembly propensity. Furthermore we demonstrated that the solution phases can be isolated and act as a new environment for continuous elongation, by adding various phosphate esters. These findings suggest that the systems chemistry of aminoacyl phosphate esters can activate a selection mechanism for peptide bond formation by merging aqueous synthesis and self-assembly.

Tailorable and Biocompatible Supramolecular-Based Hydrogels Featuring two Dynamic Covalent Chemistries.

Dynamic covalent chemistry (DCC) has proven to be a valuable tool in creating fascinating molecules, structures, and emergent properties in fully synthetic systems. Here we report a system that uses two dynamic covalent bonds in tandem, namely disulfides and hydrazones, for the formation of hydrogels containing biologically relevant ligands. The reversibility of disulfide bonds allows fiber formation upon oxidation of dithiol-peptide building block, while the reaction between NH-NH2 functionalized C-terminus and aldehyde cross-linkers results in a gel. The same bond-forming reaction was exploited for the "decoration" of the supramolecular assemblies by cell-adhesion-promoting sequences (RGD and LDV). Fast triggered gelation, cytocompatibility and ability to "on-demand" chemically customize fibrillar scaffold offer potential for applying these systems as a bioactive platform for cell culture and tissue engineering.

Out-of-Equilibrium Self-Replication Allows Selection for Dynamic Kinetic Stability in a System of Competing Replicators.

Among the key characteristics of living systems are their ability to self-replicate and the fact that they exist in an open system away from equilibrium. Herein, we show how the outcome of the competition between two self-replicators, differing in size and building block composition, is different depending on whether the experiments are conducted in a closed vial or in an open and out-of-equilibrium replication-destruction regime. In the closed system, the slower replicator eventually prevails over the faster competitor. In a replication-destruction regime, implemented through a flow system, the outcome of the competition is reversed and the faster replicator dominates. The interpretation of the experimental observations is supported by a mass-action-kinetics model. These results represent one of the few experimental manifestations of selection among competing self-replicators based on dynamic kinetic stability and pave the way towards Darwinian evolution of abiotic systems.

Two Sides of the Same Coin: Emergence of Foldamers and Self-Replicators from Dynamic Combinatorial Libraries.

The ability of molecules and systems to make copies of themselves and the ability of molecules to fold into stable, well-defined three-dimensional conformations are of considerable importance in the formation and persistence of life. The question of how, during the emergence of life, oligomerization reactions become selective and channel these reactions toward a small number of specific products remains largely unanswered. Herein, we demonstrate a fully synthetic chemical system where structurally complex foldamers and self-replicating assemblies emerge spontaneously and with high selectivity from pools of oligomers as a result of forming noncovalent interactions. Whether foldamers or replicators form depends on remarkably small differences in building block structures and composition and experimental conditions. We also observed the dynamic transformation of a foldamer into a replicator. These results show that the structural requirements/design criteria for building blocks that lead to foldamers are similar to those that lead to replicators. What determines whether folding or replication takes place is not necessarily the type of noncovalent interaction, but only whether they occur intra- or intermolecularly. This work brings together, for the first time, the fields of replicator and foldamer chemistry.

The Endothelial Glycocalyx as a Target of Ischemia and Reperfusion Injury in Kidney Transplantation-Where Have We Gone So Far?

The damage of the endothelial glycocalyx as a consequence of ischemia and/or reperfusion injury (IRI) following kidney transplantation has come at the spotlight of research due to potential associations with delayed graft function, acute rejection as well as long-term allograft dysfunction. The disintegration of the endothelial glycocalyx induced by IRI is the crucial event which exposes the denuded endothelial cells to further inflammatory and oxidative damage. The aim of our review is to present the currently available data regarding complex links between shedding of the glycocalyx components, like syndecan-1, hyaluronan, heparan sulphate, and CD44 with the activation of intricate immune system responses, including toll-like receptors, cytokines and pro-inflammatory transcription factors. Evidence on modes of protection of the endothelial glycocalyx and subsequently maintenance of endothelial permeability as well as novel nephroprotective molecules such as sphingosine-1 phosphate (S1P), are also depicted. Although advances in technology are making the visualization and the analysis of the endothelial glycocalyx possible, currently available evidence is mostly experimental. Ongoing progress in understanding the complex impact of IRI on the endothelial glycocalyx, opens up a new era of research in the field of organ transplantation and clinical studies are of utmost importance for the future.

Self-Sorting in Dynamic Combinatorial Libraries Leads to the Co-Existence of Foldamers and Self-Replicators.

Nature segregates fundamental tasks such as information storage/transmission and catalysis between two different compound classes (e.g. polynucleotides for replication and folded polyamides for catalysis). This division of labor is likely a product of evolution, raising the question of how simpler systems in which replicators and folded macromolecules co-exist may emerge in the transition from chemistry to biology. In synthetic systems, achieving co-existence of replicators and foldamers in a single molecular network remains an unsolved problem. Previous work on dynamic molecular networks has given rise to either self-replicating fibers or well-defined foldamer structures (or completely un-sorted complex systems). We report a system in which two cross-reactive dithiol (nucleobase- and peptide-based) building blocks self-sort into a replicator fiber and foldamer that both emerge spontaneously and co-exist. The self-sorting behavior remains prevalent across different building block ratios as two phases of emergence occur: replicator growth followed by foldamer formation. This is attributed to the autocatalytic formation of the replicator fiber, followed by enrichment of the system in the remaining building block, which is subsequently incorporated into a foldamer.

Spontaneous Emergence of Self-Replicating Molecules Containing Nucleobases and Amino Acids.

The conditions that led to the formation of the first organisms and the ways that life originates from a lifeless chemical soup are poorly understood. The recent hypothesis of "RNA-peptide coevolution" suggests that the current close relationship between amino acids and nucleobases may well have extended to the origin of life. We now show how the interplay between these compound classes can give rise to new self-replicating molecules using a dynamic combinatorial approach. We report two strategies for the fabrication of chimeric amino acid/nucleobase self-replicating macrocycles capable of exponential growth. The first one relies on mixing nucleobase- and peptide-based building blocks, where the ligation of these two gives rise to highly specific chimeric ring structures. The second one starts from peptide nucleic acid (PNA) building blocks in which nucleobases are already linked to amino acids from the start. While previously reported nucleic acid-based self-replicating systems rely on presynthesis of (short) oligonucleotide sequences, self-replication in the present systems start from units containing only a single nucleobase. Self-replication is accompanied by self-assembly, spontaneously giving rise to an ordered one-dimensional arrangement of nucleobase nanostructures.

Complex Molecules That Fold Like Proteins Can Emerge Spontaneously.

Folding can bestow macromolecules with various properties, as evident from nature's proteins. Until now complex folded molecules are the product either of evolution or of an elaborate process of design and synthesis. We now show that molecules that fold in a well-defined architecture of substantial complexity can emerge autonomously and selectively from a simple precursor. Specifically, we have identified a self-synthesizing macrocyclic foldamer with a complex and unprecedented secondary and tertiary structure that constructs itself highly selectively from 15 identical peptide-nucleobase subunits, using a dynamic combinatorial chemistry approach. Folding of the structure drives its synthesis in 95% yield from a mixture of interconverting molecules of different ring sizes in a one-step process. Single-crystal X-ray crystallography and NMR reveal a folding pattern based on an intricate network of noncovalent interactions involving residues spaced apart widely in the linear sequence. These results establish dynamic combinatorial chemistry as a powerful approach to developing synthetic molecules with folding motifs of a complexity that goes well beyond that accessible with current design approaches. The fact that such molecules can form autonomously implies that they may have played a role in the origin of life at earlier stages than previously thought possible.

Co-treatment with a C1B5 peptide of protein kinase Cγ and a low dose of gemcitabine strongly attenuated pancreatic cancer growth in mice through T cell activation.

Although gemcitabine is an effective chemotherapeutic for pancreatic cancer, severe side effects often accompany its use. Since we have discovered that locally administered C1B domain peptides effectively control tumor growth without any side effects, the efficacy of co-treatment with this peptide and a low dose of gemcitabine on the growth of pancreatic cancer was examined. Two- and three-dimensional cell culture studies clarified that a co-treatment with C1B5 peptide and gemcitabine significantly attenuated growth of PAN02 mouse and PANC-1 human pancreatic cancer cells in 2D and 3D cultures. Although treatment with the low dose of gemcitabine alone (76%) or the C1B5 peptide alone (39%) inhibited tumor growth moderately, a co-treatment with C1B5 peptide and a low dose of gemcitabine markedly inhibited the growth of PAN02 autografts in the mouse peritoneal cavity (94% inhibition) without any noticeable adverse effect. The number of peritoneal cavity-infiltrating neutrophils and granzyme B+ lymphocytes was significantly higher in the co-treatment group than in the control group. A significant increase of granzyme B mRNA expression was also detected in human T cells by the co-treatment. Taken together, the current study suggests that C1B5 peptide offers a remarkably effective combination treatment strategy to reduce side effects associated with gemcitabine, without losing its tumoricidal effect.

Antiparallel Dynamic Covalent Chemistries.

The ability to design reaction networks with high, but addressable complexity is a necessary prerequisite to make advanced functional chemical systems. Dynamic combinatorial chemistry has proven to be a useful tool in achieving complexity, however with some limitations in controlling it. Herein we introduce the concept of antiparallel chemistries, in which the same functional group can be channeled into one of two reversible chemistries depending on a controllable parameter. Such systems allow both for achieving complexity, by combinatorial chemistry, and addressing it, by switching from one chemistry to another by controlling an external parameter. In our design the two antiparallel chemistries are thiol-disulfide exchange and thio-Michael addition, sharing the thiol as the common building block. By means of oxidation and reduction the system can be reversibly switched from predominantly thio-Michael chemistry to predominantly disulfide chemistry, as well as to any intermediate state. Both chemistries operate in water, at room temperature, and at mildly basic pH, which makes them a suitable platform for further development of systems chemistry.

Biocatalytic Self-Assembly Cascades.

The properties of supramolecular materials are dictated by both kinetic and thermodynamic aspects, providing opportunities to dynamically regulate morphology and function. Herein, we demonstrate time-dependent regulation of supramolecular self-assembly by connected, kinetically competing enzymatic reactions. Starting from Fmoc-tyrosine phosphate and phenylalanine amide in the presence of an amidase and phosphatase, four distinct self-assembling molecules may be formed which each give rise to distinct morphologies (spheres, fibers, tubes/tapes and sheets). By varying the sequence or ratio in which the enzymes are added to mixtures of precursors, these structures can be (transiently) accessed and interconverted. The approach provides insights into dynamic self-assembly using competing pathways that may aid the design of soft nanostructures with tunable dynamic properties and life times.

Switchable Hydrolase Based on Reversible Formation of Supramolecular Catalytic Site Using a Self-Assembling Peptide.

The reversible regulation of catalytic activity is a feature found in natural enzymes which is not commonly observed in artificial catalytic systems. Here, we fabricate an artificial hydrolase with pH-switchable activity, achieved by introducing a catalytic histidine residue at the terminus of a pH-responsive peptide. The peptide exhibits a conformational transition from random coil to ß-sheet by changing the pH from acidic to alkaline. The ß-sheet self-assembles to form long fibrils with the hydrophobic edge and histidine residues extending in an ordered array as the catalytic microenvironment, which shows significant esterase activity. Catalytic activity can be reversible switched by pH-induced assembly/disassembly of the fibrils into random coils. At higher concentrations, the peptide forms a hydrogel which is also catalytically active and maintains its reversible (de-)activation.

Biocatalytic Pathway Selection in Transient Tripeptide Nanostructures.

Structural adaption in living systems is achieved by competing catalytic pathways that drive assembly and disassembly of molecular components under the influence of chemical fuels. We report on a simple mimic of such a system that displays transient, sequence-dependent formation of supramolecular nanostructures based on biocatalytic formation and hydrolysis of self-assembling tripeptides. The systems are catalyzed by α-chymotrypsin and driven by hydrolysis of dipeptide aspartyl-phenylalanine-methyl ester (the sweetener aspartame, DF-OMe). We observed switch-like pathway selection, with the kinetics and consequent lifetime of transient nanostructures controlled by the peptide sequence. In direct competition, kinetic (rather than thermodynamic) component selection is observed.

GnRH-Gemcitabine conjugates for the treatment of androgen-independent prostate cancer: pharmacokinetic enhancements combined with targeted drug delivery.

Gemcitabine, a drug with established efficacy against a number of solid tumors, has therapeutic limitations due to its rapid metabolic inactivation. The aim of this study was the development of an innovative strategy to produce a metabolically stable analogue of gemcitabine that could also be selectively delivered to prostate cancer (CaP) cells based on cell surface expression of the Gonadotropin Releasing Hormone-Receptor (GnRH-R). The synthesis and evaluation of conjugated molecules, consisting of gemcitabine linked to a GnRH agonist, is presented along with results in androgen-independent prostate cancer models. NMR and ligand binding assays were employed to verify conservation of microenvironments responsible for binding of novel GnRH-gemcitabine conjugates to the GnRH-R. In vitro cytotoxicity, cellular uptake, and metabolite formation of the conjugates were examined in CaP cell lines. Selected conjugates were efficacious in the in vitro assays with one of them, namely, GSG, displaying high antiproliferative activity in CaP cell lines along with significant metabolic and pharmacokinetic advantages in comparison to gemcitabine. Finally, treatment of GnRH-R positive xenografted mice with GSG showed a significant advantage in tumor growth inhibition when compared to gemcitabine.

Differential self-assembly and tunable emission of aromatic peptide bola-amphiphiles containing perylene bisimide in polar solvents including water.

We demonstrate the self-assembly of bola-amphiphile-type conjugates of dipeptides and perylene bisimide (PBI) in water and other polar solvents. Depending on the nature of the peptide used (glycine-tyrosine, GY, or glycine-aspartic acid, GD), the balance between H-bonding and aromatic stacking can be tailored. In aqueous buffer, PBI-[GY]2 forms chiral nanofibers, resulting in the formation of a hydrogel, while for PBI-[GD]2 achiral spherical aggregates are formed, demonstrating that the peptide sequence has a profound effect on the structure formed. In water and a range of other polar solvents, self-assembly of these two PBI-peptides conjugates results in different nanostructures with highly tunable fluorescence performance depending on the peptide sequence employed, e.g., fluorescent emission and quantum yield. Organogels are formed for the PBI-[GD]2 derivative in DMF and DMSO while PBI-[GY]2 gels in DMF. To the best of our knowledge, this is the first successful strategy for using short peptides, specifically, their sequence/structure relationships, to manipulate the PBI nanostructure and consequent optical properties. The combination of controlled self-assembly, varied optical properties, and formation of aqueous and organic gel-phase materials may facilitate the design of devices for various applications related to light harvesting and sensing.

Translation and cross-cultural adaptation of the Kidney Transplant Questionnaire 25 to Greek.

BACKGROUND: Kidney transplantation leads to continuous improvement in the survival rates of kidney transplant recipients (KTRs) and has been established as the treatment of choice for patients with end-stage kidney disease. Health-related quality of life (HRQoL) has become an important outcome measure. It is highly important to develop reliable methods to evaluate HRQoL with disease-specific questionnaires. AIM: To translate the disease-specific instrument Kidney Transplant Questionnaire 25 (KTQ-25) to the Greek language and perform a cross-cultural adaptation. METHODS: The translation and adaptation of the original English version of the KTQ-25 to the Greek language were performed based on the International Quality of Life Assessment. RESULTS: Eighty-four KTRs (59 males; mean age 53.5 ± 10.7 years; mean estimated glomerular filtration rate 47.7 ± 15.1 mL/min/1.73 m2; mean transplant vintage 100.5 ± 83.2 months) completed the Greek version of the KTQ-25 and the 36-item Short-Form Health Survey, and the results were used to evaluate the reliability of the Greek KTQ-25. The Cronbach alpha coefficients for all the KTQ-25 dimensions were satisfactory (physical symptoms = 0.639, fatigue = 0.856, uncertainty/fear = 0.661, appearance = 0.593, emotions = 0.718, total score = 0.708). The statistically significant correlation coefficients among the KTQ-25 dimensions ranged from 0.226 to 0.644. The correlation coefficients of the KTQ-25 dimensions with the SF-36 physical component summary (PCS) ranged from 0.196 to 0.550; the correlation coefficients of the KTQ-25 with the SF-36 mental component summary (MCS) ranged from 0.260 to 0.655; and the correlation coefficients of the KTQ-25 with the total scores with the SF-36 PCS and MCS were 0.455 and 0.613, respectively. CONCLUSION: According to the findings, the Greek version of the KTQ-25 is valid and reliable for administration among kidney transplant patients in Greece.

Disseminated Bacillus Calmette-Guérin (BCG) infection presenting as severe respiratory failure and septic shock.

Introduction: Intravesical Bacillus Calmette-Guérin (BCG) instillation is the most effective adjuvant therapy for superficial urinary bladder carcinoma, prolonging disease-free survival. Although it is usually well tolerated, moderate to severe local or systemic infectious complications, including sepsis involving multiple organs, may occur. Case report: We report the unusual case of a man in his mid '70s who presented with septic shock and severe acute respiratory failure requiring intubation. Lack of response to antibiotics, history of intravesical BCG instillation and consistent imaging findings led to further investigations, with bronchoalveolar lavage (BAL) fluid polymerase chain reaction (PCR) results indicating pneumonitis due to Mycobacterium bovis dissemination. Prompt anti-tuberculosis treatment combined with corticosteroids resulted in significant clinical and radiological improvement, supporting the diagnosis of disseminated BCG infection. Conclusions: Due to its non-specific clinical presentation and the relatively low diagnostic yield of conventional microbiological tests, a high index of suspicion is required for prompt diagnosis and treatment of systemic BCG infection. PCR-based assays for mycobacterial DNA identification may represent a valuable tool facilitating timely diagnosis of this uncommon, yet potentially life-threatening infection.

Longitudinal Assessment of Electrolyte Disorders in a Cohort of Chronic Stable Kidney Transplant Recipients.

BACKGROUND: Kidney transplantation is complicated by various electrolyte disturbances with variable reported prevalence and incidence and of multifactorial pathogenesis. The aim of our study was the retrospective longitudinal assessment of the serum electrolytes in a cohort of stable kidney transplant recipients (KTRs) and the possible associated parameters, including graft function and medications. METHODS: We included 93 stable KTRs under follow-up in our hospital's kidney transplant unit. Serum magnesium, calcium, phosphorus, potassium, sodium, and urine sodium levels were recorded retrospectively during 3 consecutive years. In addition, comorbidities, biochemical parameters, medications, and graft function (estimated glomerular filtration rate (eGFR) using the Chronic Kidney Disease Epidemiology Collaboration equation and 24-hour urinary protein [uTpr]) were recorded. RESULTS: Mean age at baseline was 51 ± 11 years; 64 KTRs were men (68.8%), 17 (18.3%) had diabetes, 79 (85%) had hypertension, and 11 (11.8%) had cardiovascular disease. Mean eGFR and uTpr (mg/24 h) at study initiation were 47.1 ± 13.5 mL/min/1.73 m2 and 369.4 ± 404.2 mg/24 h, respectively. Hypomagnesemia was the most common disturbance observed in 21.7% of KTRs. Patients with hypomagnesemia displayed higher parathyroid hormone levels and more frequently had diabetes. Hypophosphatemia was recorded in 9.7% of KTRs during the first year. Hyperkalemia, hypokalemia, and hypercalcemia were rare (<5%). Mean serum and urine sodium concentration remained stable during the study, whereas urinary sodium levels showed a positive correlation with uTpr (P < .05). CONCLUSIONS: In our cohort of KTRs, there were no significant electrolyte disorders, either in terms of frequency or severity, with hypomagnesemia being the most prevalent disturbance. The identification of potential associated risk factors and clinical data correlations are pivotal for the development of individualized and evidence-based therapeutic approach and decisions.

Discovery of phosphotyrosine-binding oligopeptides with supramolecular target selectivity.

We demonstrate phage-display screening on self-assembled ligands that enables the identification of oligopeptides that selectively bind dynamic supramolecular targets over their unassembled counterparts. The concept is demonstrated through panning of a phage-display oligopeptide library against supramolecular tyrosine-phosphate ligands using 9-fluorenylmethoxycarbonyl-phenylalanine-tyrosine-phosphate (Fmoc-FpY) micellar aggregates as targets. The 14 selected peptides showed no sequence consensus but were enriched in cationic and proline residues. The lead peptide, KVYFSIPWRVPM-NH2 (P7) was found to bind to the Fmoc-FpY ligand exclusively in its self-assembled state with K D = 74 ± 3 µM. Circular dichroism, NMR and molecular dynamics simulations revealed that the peptide interacts with Fmoc-FpY through the KVYF terminus and this binding event disrupts the assembled structure. In absence of the target micellar aggregate, P7 was further found to dynamically alternate between multiple conformations, with a preferred hairpin-like conformation that was shown to contribute to supramolecular ligand binding. Three identified phages presented appreciable binding, and two showed to catalyze the hydrolysis of a model para-nitro phenol phosphate substrate, with P7 demonstrating conformation-dependent activity with a modest k cat/K M = 4 ± 0.3 × 10-4 M-1 s-1.