Fluorescence of KCl Aqueous Solution: A Possible Spectroscopic Signature of Nucleation.

Ion pairing in water solutions alters both the water hydrogen-bond network and ion solvation, modifying the dynamics and properties of electrolyte water solutions. Here, we report an anomalous intrinsic fluorescence of KCl aqueous solution at room temperature and show that its intensity increases with the salt concentration. From the ab initio density functional theory (DFT) and time-dependent DFT modeling, we propose that the fluorescence emission could originate from the stiffening of the hydrogen bond network in the hydration shell of solvated ion-pairs that suppresses the fast nonradiative decay and allows the slower radiative channel to become a possible decay pathway.

Anomalous Intrinsic Fluorescence of HCl and NaOH Aqueous Solutions.

The unique properties of liquid water mainly arise from its hydrogen bond network. The geometry and dynamics of this network play a key role in shaping the characteristics of soft matter, from simple solutions to biosystems. Here we report an anomalous intrinsic fluorescence of HCl and NaOH aqueous solutions at room temperature that shows important differences in the excitation and emission bands between the two solutes.

A FTIR microspectroscopy study of the structural and biochemical perturbations induced by natively folded and aggregated transthyretin in HL-1 cardiomyocytes.

Protein misfolding and aggregation are associated with a number of human degenerative diseases. In spite of the enormous research efforts to develop effective strategies aimed at interfering with the pathogenic cascades induced by misfolded/aggregated peptides/proteins, the necessary detailed understanding of the molecular bases of amyloid formation and toxicity is still lacking. To this aim, approaches able to provide a global insight in amyloid-mediated physiological alterations are of importance.

Chemical space guided discovery of antimicrobial bridged bicyclic peptides against and its biofilms.

Herein we report the discovery of antimicrobial bridged bicyclic peptides (AMBPs) active against , a highly problematic Gram negative bacterium in the hospital environment. Two of these AMBPs show strong biofilm inhibition and dispersal activity and enhance the activity of polymyxin, currently a last resort antibiotic against which resistance is emerging. To discover our AMBPs we used the concept of chemical space, which is well known in the area of small molecule drug discovery, to define a small number of test compounds for synthesis and experimental evaluation.

Biochemical and Electrophysiological Modification of Amyloid Transthyretin on Cardiomyocytes.

Transthyretin (TTR) amyloidoses are familial or sporadic degenerative conditions that often feature heavy cardiac involvement. Presently, no effective pharmacological therapy for TTR amyloidoses is available, mostly due to a substantial lack of knowledge about both the molecular mechanisms of TTR aggregation in tissue and the ensuing functional and viability modifications that occur in aggregate-exposed cells. TTR amyloidoses are of particular interest regarding the relation between functional and viability impairment in aggregate-exposed excitable cells such as peripheral neurons and cardiomyocytes.

In situ characterization of protein aggregates in human tissues affected by light chain amyloidosis: a FTIR microspectroscopy study.

Light chain (AL) amyloidosis, caused by deposition of amyloidogenic immunoglobulin light chains (LCs), is the most common systemic form in industrialized countries. Still open questions, and premises for developing targeted therapies, concern the mechanisms of amyloid formation in vivo and the bases of organ targeting and dysfunction. Investigating amyloid material in its natural environment is crucial to obtain new insights on the molecular features of fibrillar deposits at individual level.

The polyphenol Oleuropein aglycone hinders the growth of toxic transthyretin amyloid assemblies.

Transthyretin (TTR) is involved in a subset of familial or sporadic amyloid diseases including senile systemic amyloidosis (SSA), familial amyloid polyneuropathy and cardiomyopathy (FAP/FAC) for which no effective therapy has been found yet. These conditions are characterized by extracellular deposits primarily found in the heart parenchyma and in peripheral nerves whose main component are amyloid fibrils, presently considered the main culprits of cell sufferance. The latter are polymeric assemblies grown from misfolded TTR, either wt or carrying one out of many identified mutations.

Biochemical nature of Russell Bodies.

Professional secretory cells produce and release abundant proteins. Particularly in case of mutations and/or insufficient chaperoning, these can aggregate and become toxic within or amongst cells. Immunoglobulins (Ig) are no exception.

Wild type beta-2 microglobulin and DE loop mutants display a common fibrillar architecture.

Beta-2 microglobulin (β2m) is the protein responsible for a pathologic condition known as dialysis related amyloidosis. In recent years an important role has been assigned to the peptide loop linking strands D and E (DE loop) in determining β2m stability and amyloid propensity. Several mutants of the DE loop have been studied, showing a good correlation between DE loop geometrical strain, protein stability and aggregation propensity.

Paclitaxel is incorporated by mesenchymal stromal cells and released in exosomes that inhibit in vitro tumor growth: a new approach for drug delivery.

Mesenchymal stromal cells (MSCs) have been proposed for delivering anticancer agents because of their ability to home in on tumor microenvironment. We found that MSCs can acquire strong anti-tumor activity after priming with Paclitaxel (PTX) through their capacity to uptake and then release the drug. Because MSCs secrete a high amount of membrane microvesicles (MVs), we here investigated the role of MVs in the releasing mechanism of PTX.

A Fourier transform infrared spectroscopy study of cell membrane domain modifications induced by docosahexaenoic acid.

Background: Detergent resistant membranes (DRMs) are a useful model system for the in vitro characterization of cell membrane domains. Indeed, DRMs provide a simple model to study the mechanisms underlying several key cell processes based on the interplay between specific cell membrane domains on one hand, and specific proteins and/or lipids on the other. Considering therefore their biological relevance, the development of methods capable to provide information on the composition and structure of membrane domains and to detect their modifications is highly desirable.

Dendron synthesis and carbohydrate immobilization on a biomaterial surface by a double-click reaction.

The synthesis of new dendrons and their immobilization on collagen patches via thiol-ene photoclick reaction, followed by chemoselective alkoxyamino-carbonyl conjugation to carbohydrates is presented. XPS, FTIR, and ELLA assays confirmed the effectiveness of the collagen multivalent neoglycosylation.

Fourier transform infrared spectroscopy as a method to study lipid accumulation in oleaginous yeasts.

Background: Oleaginous microorganisms, such as different yeast and algal species, can represent a sustainable alternative to plant oil for the production of biodiesel. They can accumulate fatty acids (FA) up to 70% of their dry weight with a predominance of (mono)unsaturated species, similarly to what plants do, but differently from animals. In addition, their growth is not in competition either with food, feed crops, or with agricultural land.

Reciprocal influence of protein domains in the cold-adapted acyl aminoacyl peptidase from Sporosarcina psychrophila.

Acyl aminoacyl peptidases are two-domain proteins composed by a C-terminal catalytic α/β-hydrolase domain and by an N-terminal β-propeller domain connected through a structural element that is at the N-terminus in sequence but participates in the 3D structure of the C-domain. We investigated about the structural and functional interplay between the two domains and the bridge structure (in this case a single helix named α1-helix) in the cold-adapted enzyme from Sporosarcina psychrophila (SpAAP) using both protein variants in which entire domains were deleted and proteins carrying substitutions in the α1-helix. We found that in this enzyme the inter-domain connection dramatically affects the stability of both the whole enzyme and the β-propeller.

Why and how protein aggregation has to be studied in vivo.

The understanding of protein aggregation is a central issue in different fields of protein science, from the heterologous protein production in biotechnology to amyloid aggregation in several neurodegenerative and systemic diseases. To this goal, it became more and more evident the crucial relevance of studying protein aggregation in the complex cellular environment, since it allows to take into account the cellular components affecting protein aggregation, such as chaperones, proteases, and molecular crowding. Here, we discuss the use of several biochemical and biophysical approaches that can be employed to monitor protein aggregation within intact cells, focusing in particular on bacteria that are widely employed as microbial cell factories.

Effects of methanol on a methanol-tolerant bacterial lipase.

Methanol is often employed in biocatalysis with the purpose of increasing substrates solubility or as the acyl acceptor in transesterification reactions, but inhibitory effects are observed in several cases. We have studied the influence of methanol on the catalytic activity and on the conformation of the lipase from Burkholderia glumae, which is reported to be highly methanol tolerant if compared with other lipases. We detected highest activity in the presence of 50-70 % methanol.

Synthesis and characterization of designed BMHP1-derived self-assembling peptides for tissue engineering applications.

The importance of self-assembling peptides (SAPs) in regenerative medicine is becoming increasingly recognized. The propensity of SAPs to form nanostructured fibers is governed by multiple forces including hydrogen bonds, hydrophobic interactions and π-π aromatic interactions among side chains of the amino acids. Single residue modifications in SAP sequences can significantly affect these forces.

Biophysical characterization of Met-G-CSF: effects of different site-specific mono-pegylations on protein stability and aggregation.

The limited stability of proteins in vitro and in vivo reduces their conversion into effective biopharmaceuticals. To overcome this problem several strategies can be exploited, as the conjugation of the protein of interest with polyethylene glycol, in most cases, improves its stability and pharmacokinetics. In this work, we report a biophysical characterization of the non-pegylated and of two different site-specific mono-pegylated forms of recombinant human methionyl-granulocyte colony stimulating factor (Met-G-CSF), a protein used in chemotherapy and bone marrow transplantation.

Fourier transform infrared spectroscopy of intrinsically disordered proteins: measurement procedures and data analyses.

A peculiar property of intrinsically disordered proteins (IDPs), or of intrinsically disordered domains, is the absence of a well-defined three dimensional structure under native conditions. Moreover, IDPs usually acquire a specific structure in the presence of different interactors. In this framework, Fourier transform infrared (FTIR) spectroscopy is a powerful tool to assess the disordered character of a protein and to study its induced folding.

Fourier transform infrared microspectroscopy of complex biological systems: from intact cells to whole organisms.

Fourier transform infrared (FTIR) microspectroscopy is a powerful tool for the study of complex biological systems. Indeed, it is employed to characterize intact cells, tissues, and whole model organisms such as nematodes, since it allows to obtain a chemical fingerprint of the sample under investigation, giving information on the molecular composition and structures. The successful application of this technique for the in situ study of biological processes requires specific sample preparations, in order to obtain reliable and reproducible results.

Ethidium bromide as a marker of mtDNA replication in living cells.

Mitochondrial DNA (mtDNA) in tumor cells was found to play an important role in maintaining the malignant phenotype. Using laser scanning confocal fluorescence microscopy (LSCFM) in a recent work, we reported a variable fluorescence intensity of ethidium bromide (EB) in mitochondria nucleoids of living carcinoma cells. Since when EB is bound to nucleic acids its fluorescence is intensified; a higher EB fluorescence intensity could reflect a higher DNA accessibility to EB, suggesting a higher mtDNA replication activity.

Structure, stability, and aggregation of β-2 microglobulin mutants: insights from a Fourier transform infrared study in solution and in the crystalline state.

β-2 microglobulin (β2m) is an amyloidogenic protein involved in dialysis-related amyloidosis. We report here the study of the structural properties of the protein in solution and in the form of single crystals by Fourier transform infrared (FTIR) spectroscopy and microspectroscopy. The investigation has been extended to four β2m mutants previously characterized by x-ray crystallography: Asp(53)Pro, Asp(59)Pro, Trp(60)Gly, and Trp(60)Val.

Concepts and tools to exploit the potential of bacterial inclusion bodies in protein science and biotechnology.

Cells have evolved complex and overlapping mechanisms to protect their proteins from aggregation. However, several reasons can cause the failure of such defences, among them mutations, stress conditions and high rates of protein synthesis, all common consequences of heterologous protein production. As a result, in the bacterial cytoplasm several recombinant proteins aggregate as insoluble inclusion bodies.

Compaction properties of an intrinsically disordered protein: Sic1 and its kinase-inhibitor domain.

IDPs in their unbound state can transiently acquire secondary and tertiary structure. Describing such intrinsic structure is important to understand the transition between free and bound state, leading to supramolecular complexes with physiological interactors. IDP structure is highly dynamic and, therefore, difficult to study by conventional techniques.