Rapid Probing of Biological Surfaces with a Sparse-Matrix Peptide Library

Finding unique peptides to target specific biological surfaces is crucial to basic research and technology development, though methods based on biological arrays or large libraries limit the speed and ease with which these necessary compounds can be found. We reasoned that because biological surfaces, such as cell surfaces, mineralized tissues, and various extracellular matrices have unique molecular compositions, they present unique physicochemical signatures to the surrounding medium which could be probed by peptides with appropriately corresponding physicochemical properties. To test this hypothesis, a naïve pilot library of 36 peptides, varying in their hydrophobicity and charge, was arranged in a two-dimensional matrix and screened against various biological surfaces. While the number of peptides in the matrix library was very small, we obtained “hits” against all biological surfaces probed. Sequence refinement of the “hits” led to peptides with markedly higher specificity and binding activity against screened biological surfaces. Genetic studies revealed that peptide binding to bacteria was mediated, at least in some cases, by specific cell-surface molecules, while examination of human tooth sections showed that this method can be used to derive peptides with highly specific binding to human tissue

Role of the unstructured N-terminal domain of the human Apurinic/Apyrimidinic Endonuclease 1 (hAPE1) in the modulation of its interaction with nucleic acids and Nucleophosmin (NPM1)

The hAPE1 (human apurinic/apyrimidinic endonuclease 1) is an essential enzyme, being the main abasic endonuclease in higher eukaryotes.However, there is strong evidence to showthat hAPE1 can directly bind specific gene promoters, thus modulating their transcriptional activity, even in the absence of specific DNA damage. Recent findings, moreover, suggest a role for hAPE1 in RNA processing, which is modulated by the interaction with NPM1 (nucleophosmin). Independent domains account for many activities of hAPE1; however, whereas the endonuclease and the redox-active portions of the protein arewell characterized, a better understanding of the role of the unstructured N-terminal region is needed. In the present study, we characterized the requirements for the interaction of hAPE1 with NPM1 and undamaged nucleic acids.We show that DNA/RNA secondary structure has an impact on hAPE1 binding in the absence of damage. Biochemical studies, using the isolated N-terminal region of the protein, reveal that the hAPE1 N-terminal domain represents an evolutionary gain of function, since its composition affects the protein\u2019s stability and ability to interact with both nucleic acids and NPM1. Although required, however, this region is not sufficient itself to stably interact with DNA or NPM1

Role of the unstructured N-terminal domain of the hAPE1 (human apurinic/apyrimidinic endonuclease 1) in the modulation of its interaction with nucleic acids and NPM1 (nucleophosmin).

The hAPE1 (human apurinic/apyrimidinic endonuclease 1) is an essential enzyme, being the main abasic endonuclease in higher eukaryotes. However, there is strong evidence to show that hAPE1 can directly bind specific gene promoters, thus modulating their transcriptional activity, even in the absence of specific DNA damage. Recent findings, moreover, suggest a role for hAPE1 in RNA processing, which is modulated by the interaction with NPM1 (nucleophosmin). Independent domains account for many activities of hAPE1; however, whereas the endonuclease and the redox-active portions of the protein arewell characterized, a better understanding of the role of the unstructured N-terminal region is needed. In the present study, we characterized the requirements for the interaction of hAPE1 with NPM1 and undamaged nucleic acids. We show that DNA/RNA secondary structure has an impact on hAPE1 binding in the absence of damage. Biochemical studies, using the isolated N-terminal region of the protein, reveal that the hAPE1 N-terminal domain represents an evolutionary gain of function, since its composition affects the protein's stability and ability to interact with both nucleic acids and NPM1. Although required, however, this region is not sufficient itself to stably interact with DNA or NPM1. © The Authors Journal compilation © 2013 Biochemical Society

Role of mutual interactions in the chemical and thermal stability of nucleophosmin NPM1 domains

Nucleophosmin (NPM1) is a key factor involved in fundamental biological processes. Mutations involving the NPM1 gene are the most frequent molecular alterations in acute myeloid leukemia. Here we report a biophysical characterization of NPM1 and of its domains in order to gain insights into the role that inter-domain interactions plays in the protein stabilization. Thermal denaturation analyses show that the N-terminal domain is endowed with an exceptional thermal stability, as it does not unfold in the investigated temperature range (20-105\ub0C). This finding is corroborated by chemical denaturation experiments showing that this domain is not significantly affected by the addition of 8M urea. These results are consistent with the chaperone function of NPM1. In line with literature data, the other folded domain of the NPM1, a 3-helix bundle domain located at the C-terminus, shows a lower stability. Interestingly, the chemical and thermal stability of this latter domain, which embeds natural mutations related to acute myeloid leukemia, is influenced by the presence of other regions of the protein. Small but significant stabilizations of the C-terminal 3-helix bundle are provided by the adjacent unfolded fragment as well as by the rest of the protein

Enhanced Drug Delivery into Cell Cytosol via Glycoprotein H-Derived Peptide Conjugated Nanoemulsions

The key role of nanocarriers in improving the pharmacological properties of commonly used drugs is recognized worldwide. It is also known that in the development of new effective nanocarriers the use of targeting moieties integrated on their surface is essential. Herein, we propose a nanocarrier based on an oil in water nanoemulsion coated with a membranotropic peptide derived from the glycoprotein H of Herpes simplex virus I, known as gH625, in order to reduce endolysosomal accumulation and to enhance cytosolic localization. In addition, we show an enhanced anti-inflammatory activity of curcumin, a bioactive compound isolated from the Curcuma longa plant, when loaded into our engineered nanocarriers. This effect is a consequence of a higher uptake combined with a high curcumin preservation exerted by the active nanocapsules compared to control ones. When loaded into our nanocapsules, indeed, curcumin molecules are directly internalized into the cytosol rather than into lysosomes. Further, in order to extend the in vitro experimental setting with a more complex model and to explore the possibility to use our nanocarriers for further biological applications, we tested their performance in a 3D sprouting angiogenesis model. Finally, we show promising preliminary in vivo results by assessing the anti-inflammatory properties of the proposed nanocarrier

Regional HIV Surveillance in Italy: a starting point for the future national system

BACKGROUND: the Italian National HIV Surveillance, instituted by the Ministerial Decree of March 31st, 2008, is based on 21 regional surveillance systems and adopts an essential data collection form with a definite data flow. The unification of HIV and AIDS surveillance systems and the implementation of an identical data collection form are priorities of the Italian National HIV/AIDS Action Plan 2017 (PNAIDS). OBJECTIVES: to describe the 21 regional HIV surveillance systems and to verify the feasibility of their unification. METHODS: in March 2017, a questionnaire containing 13 questions was sent to all the regional representatives of the 21 surveillance systems. The main questions were about timeliness, data flow, and quality of the system. The quality was measured through a subjective evaluation expressed by the regional referent through scores from 1 (minimum) to 10 (maximum) regarding four indicators (regional coverage, timeliness, correctness, and completeness of the data). RESULTS: more than half of the regional systems use a computerized data collection method. Some of these regions have not completely adapted to the data collection form contained in the Decree and other regions declare a undernotification of the system. The majority of the regions record a slight notification delay by the reporting centres. Some regions report gaps in the completeness of the data received by the reporting centres. CONCLUSIONS: the main strengths of the HIV surveillance system are computerization of the systems and slightly reporting delay. Regarding the quality of the regional systems and its data, the study reports a good self-evaluation. This study also showed useful indications to improve the national HIV surveillance system, such as the unification of HIV surveillance with the AIDS surveillance and the implementation of a unique national system, as suggested by guidelines of the PNAIDS 2017