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Combined results of theoretical molecular dynamic simulations and in vitro spectroscopic (circular dichroism and fluorescence) studies are presented, providing the atomistic and secondary structure details of the process by which a selected small molecule may destabilize the β-sheet ordered "amyloid" oligomers formed by the model undecapeptide of amyloid β-peptide 25-35 [Aβ(25-35)]. Aβ(25-35) was chosen because it is the shortest fragment capable of forming large β-sheet fibrils and retaining the toxicity of the full length Aβ(1-40/42) peptides. The conformational transition, that leads to the formation of β-sheet fibrils from soluble unordered structures, was found to depend on the environmental conditions, whereas the presence of myricetin destabilizes the self-assembly and antagonizes this conformational shift. In parallel, we analyzed several molecular dynamics trajectories describing the evolution of five monomer fragments, without inhibitor as well as in the presence of myricetin. Other well-known inhibitors (curcumin and (-)-tetracycline), found to be stronger and weaker Aβ(1-42) aggregation inhibitors, respectively, were also studied. The combined in vitro and theoretical studies of the Aβ(25-35) self-assembly and its inhibition contribute to understanding the mechanism of action of well-known inhibitors and the peptide amino acid residues involved in the interaction leading to a rational drug design of more potent new molecules able to antagonize the self-assembly process.

Beside the regulation of fluid distribution, human serum albumin (HSA) carries other activities, such as binding, transport, and detoxification of many molecules. In patients with cirrhosis, HSA exhibits posttranscriptional alterations that likely affect its functions. This study aimed at identifying the structural HSA alterations occurring in cirrhosis and determining their relationship with specific clinical complications and patient survival. One hundred sixty-eight patients with cirrhosis, 35 with stable conditions and 133 hospitalized for acute clinical complications, and 94 healthy controls were enrolled. Posttranscriptional HSA molecular changes were identified and quantified by using a high-performance liquid chromatography/electrospray ionization mass spectrometry technique. Clinical and biochemical parameters were also recorded and hospitalized patients were followed for up to 1 year. Seven HSA isoforms carrying one or more posttranscriptional changes were identified. Altered HSA isoforms were significantly more represented in patients than in healthy controls. Conversely, the native, unchanged HSA isoform was significantly reduced in cirrhosis. Native HSA and most altered isoforms correlated with both Child-Pugh and Model for End-Stage Liver Disease scores. In hospitalized patients, oxidized and N-terminal truncated isoforms were independently associated with ascites, renal impairment, and bacterial infection. Finally, the native HSA and cysteinylated/N-terminal truncated isoforms were predictors of 1-year survival, with greater prognostic accuracy than total serum albumin concentration. Conclusions: Extensive posttranscriptional changes of HSA, involving several molecular sites and increasing in parallel with disease severity, occur in patients with cirrhosis. Altered isoforms are independently associated with specific clinical complications, whereas the residual, native HSA isoform independently predicts patient survival. These findings support the concept of the "effective albumin concentration," which implies that the global HSA function is related not only to its serum concentration, but also to the preservation of its structural integrity. (HEPATOLOGY 2014;60:1851-1860 Abbreviations: 2D, two-dimensional; ACLF, acute on chronic liver failure; ANOVA, analysis of variance; DM, diabetes mellitus; ESI-MS, electrospray ionization mass spectrometry; HCC, hepatocellular carcinoma; HPLC, high-performance liquid chromatography; HSA, native form of human serum albumin; HSA1CYS, cysteinylated human serum albumin; HSA1CYS-DA, cysteinylated and N-terminal truncated (-Asp-Ala) human serum albumin; HSA1CYS1GLYC, cysteinylated and glycosylated human serum albumin; HSA-DA, N-terminal truncated (-Asp-Ala) human serum albumin; HSA1GLYC, glycosylated human serum albumin; HSA-L, C-terminal truncated (-Leu) human serum albumin; HSA1SO 2 H, sulfinilated human serum albumin; LC, liquid chromatography; MELD, Model for End-Stage Liver Disease; Q-TOF, quadruple time of flight; ROC, receiver operating characteristic; SD, st...

The synthesis of multicolor fluorescent oligothiophene N-succinimidyl esters (TSEs) is reported, and their optical properties are discussed with the aid of ab initio calculations. The esters were coupled to proteins and to 3'-amino-modified oligonucleotides in mild conditions and with similar modalities. A comparative study of the bioconjugate of IgG1 anti-CD3 antibody labeled with a blue fluorescent TSE and with fluorescein isothiocyanate (FITC) is reported, showing that the former achieves higher photoluminescence intensity and optical stability than the latter. Fluorescence resonance energy transfer experiments with TSE-labeled oligonucleotides and examples of cellular imaging via TSE-labeled proteins are reported.

We have designed and synthesized a series of 14 hybrid molecules out of the cholinesterase (ChE) inhibitor tacrine and a benzimidazole-based human cannabinoid receptor subtype 2 (hCB2R) agonist and investigated them in vitro and in vivo. The compounds are potent ChE inhibitors, and for the most promising hybrids, the mechanism of human acetylcholinesterase (hAChE) inhibition as well as their ability to interfere with AChE-induced aggregation of β-amyloid (Aβ), and Aβ self-aggregation was assessed. All hybrids were evaluated for affinity and selectivity for hCB1R and hCB2R. To ensure that the hybrids retained their agonist character, the expression of cAMP-regulated genes was quantified, and potency and efficacy were determined. Additionally, the effects of the hybrids on microglia activation and neuroprotection on HT-22 cells were investigated. The most promising in vitro hybrids showed pronounced neuroprotection in an Alzheimer’s mouse model at low dosage (0.1 mg/kg, i.p.), lacking hepatotoxicity even at high dose (3 mg/kg, i.p.).

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Background & Aims. Circulating albumin in cirrhosis can be dysfunctional due to accumulating structural damages, leading to the concept of effective albumin concentration (eAlb) referring to the albumin portion presenting structural and functional integrity. We aimed to estimate eAlb in patients with decompensated cirrhosis and analyze its relationships with albumin function and clinical outcomes as compared to total albumin concentration (tAlb). Approach & Results. We evaluated 319 cirrhotic patients hospitalized for acute decompensation (AD) with and without acute-on-chronic liver failure (ACLF) and 18 age-and sex-comparable outpatients with compensated cirrhosis. tAlb was quantified by standard assay, while eAlb was estimated combining liquid chromatography-electrospray ionization-mass spectrometry and standard methods. Albumin binding and detoxification efficiency were evaluated by electron paramagnetic resonance analysis. Circulating albumin in patients with decompensated cirrhosis displayed multiple structural abnormalities, with reversible oxidation and glycation being the most frequent. As a result, eAlb progressively declined with the worsening of cirrhosis, and was superior to tAlb in stratifying patients between compensated cirrhosis, AD and ACLF, as well as patients with and without complications. Moreover, eALB, but not tAlb, was closely associated with binding capacities in ACLF. Finally, eALB at admission predicted the occurrence of ACLF within 30 days and mortality at 90 days better than tAlb. Conclusion: This large observational study provides the evidence in patients with decompensated cirrhosis that eAlb can be quantified and differentiated from tAlb routinely measured in clinical practice. As compared to tAlb, eAlb is more closely associated with disease severity and albumin dysfunction, and carries a greater prognostic power. These results prompt future research assessing eAlb as a novel biomarker for predicting prognosis and treatment response.