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Antibiofilm Activity of the Brown Alga Halidrys siliquosa against Clinically Relevant Human Pathogens.

The marine brown alga Halidrys siliquosa is known to produce compounds with antifouling activity against several marine bacteria. The aim of this study was to evaluate the antimicrobial and antibiofilm activity of organic extracts obtained from the marine brown alga H. siliquosa against a focused panel of clinically relevant human pathogens commonly associated with biofilm-related infections. The partially fractionated methanolic extract obtained from H. siliquosa collected along the shores of Co. Donegal; Ireland; displayed antimicrobial activity against bacteria of the genus Staphylococcus; Streptococcus; Enterococcus; Pseudomonas; Stenotrophomonas; and Chromobacterium with MIC and MBC values ranging from 0.0391 to 5 mg/mL. Biofilms of S. aureus MRSA were found to be susceptible to the algal methanolic extract with MBEC values ranging from 1.25 mg/mL to 5 mg/mL respectively. Confocal laser scanning microscopy using LIVE/DEAD staining confirmed the antimicrobial nature of the antibiofilm activity observed using the MBEC assay. A bioassay-guided fractionation method was developed yielding 10 active fractions from which to perform purification and structural elucidation of clinically-relevant antibiofilm compounds

A Replicative In Vitro Assay for Drug Discovery against Leishmania donovani.

The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis, a disease potentially fatal if not treated. Current available treatments have major limitations, and new and safer drugs are urgently needed. In recent years, advances in high-throughput screening technologies have enabled the screening of millions of compounds to identify new antileishmanial agents. However, most of the compounds identified in vitro did not translate their activities when tested in in vivo models, highlighting the need to develop more predictive in vitro assays. In the present work, we describe the development of a robust replicative, high-content, in vitro intracellular L. donovani assay. Horse serum was included in the assay media to replace standard fetal bovine serum, to completely eliminate the extracellular parasites derived from the infection process. A novel phenotypic in vitro infection model has been developed, complemented with the identification of the proliferation of intracellular amastigotes measured by EdU incorporation. In vitro and in vivo results for miltefosine, amphotericin B, and the selected compound 1 have been included to validate the assay

Unravelling the rate of action of hits in the Leishmania donovani box using standard drugs amphotericin B and miltefosine

In recent years, the neglected diseases drug discovery community has elected phenotypic screening as the key approach for the identification of novel hit compounds. However, when this approach is applied, important questions related to the mode of action for these compounds remain unanswered. One of such questions is related to the rate of action, a useful piece of information when facing the challenge of prioritising the most promising hit compounds. In the present work, compounds of the "Leishmania donovani box" were evaluated using a rate of action assay adapted from a replicative intracellular high content assay recently developed. The potency of each compound was determined every 24 hours up to 96 hours, and standard drugs amphotericin B and miltefosine were used as references to group these compounds according to their rate of action. Independently of this biological assessment, compounds were also clustered according to their minimal chemical scaffold. Comparison of the results showed a complete correlation between the chemical scaffold and the biological group for the vast majority of compounds, demonstrating how the assay was able to bring information on the rate of action for each chemical series, a property directly linked to the mode of action. Overall, the assay here described permitted us to evaluate the rate of action of the "Leishmania donovani box" using two of the currently available drugs as references and, also, to propose a number of fast-acting chemical scaffolds present in the box as starting points for future drug discovery projects to the wider scientific community. The results here presented validate the use of this assay for the determination of the rate of action early in the discovery process, to assist in the prioritisation of hit compounds

Snapshot Profiling of the Antileishmanial Potency of Lead Compounds and Drug Candidates against Intracellular <i>Leishmania donovani</i> Amastigotes, with a Focus on Human-Derived Host Cells

This study characterised in vitro potencies of anti-leishmanial agents against intracellular Leishmania donovani amastigotes in primary human macrophages, obtained with or without CD14-positive monocyte enrichment, phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 cells and mouse peritoneal exudate macrophages (PEMs). Host cell dependent potency was confirmed for pentavalent and trivalent antimony. Fexinidazole was inactive against intracellular amastigotes across the host cell panel. Fexinidazole sulfone, (R)-PA-824, (S)-PA-824 and VL-2098 displayed similar potency in all host cells tested

Design, synthesis and activity evaluation of antioxidant peptides

Reactive Oxygen Species (ROS) produced during normal aerobic metabolism, if not promptly removed by the detoxification mechanisms of the cells, can easily react with cell components, especially lipids, producing secondary cytotoxic molecules called reactive carbonyl species (RCSs). RCSs exhibit significant chemical reactivity and can cause protein modification and dysfunction. One of the most important ReSs is 4-hydroxinonenal (HNE), an unsaturated aldehyde that has been strongly linked to Alzheimer'S disease (AD). A new series of dipeptide histidyl hydrazide analogues of carnosine was prepared, with the aim of producing molecules with enhanced HNE scavenging activity. These compounds were demonstrated to scavenge HNE and to protect SH-SY5Y cells from HNE-induced tOxlcity and were superior in action to carnosine. The synthesis of an analogue of the best compound of the series containing caffeic acid, resulted in the generation of a new molecule exhibiting complete retention of HNE-scavenging activity and also possessing free Radical Scavenging Activity (RSA). Mitochondria are the major sites of high levels of oxidative stress and the targeting of a reactive carbonyl scavenger directly to these organelles would result in extinguishing the primary source of RCS, thus arresting any consequent cellular damage. In the present work, the possibility of specifying the cellular localization of histidyl hydrazide was investigated using previously described mitochondria penetrating peptides (MPPs) and new examples of such sequences I modified to be more resistant to protease degradation. The ligation of histidyl hydrazide to a previously reported MPP was successful in targeting the former to the mitochondria of HeLa cells. Finally. a particular chemical ligation approach was investigated for the development of a system of linking a common peptide vector and a variety of cargo molecules of choice through facile in situ coupling, without requiring the de novo synthesis of a new chemical conjugate in each instance.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

3D printing of gelatin/chitosan biodegradable hybrid hydrogel: Critical issues due to the crosslinking reaction, degradation phenomena and process parameters

Hydrogel materials are being investigated for application as scaffolds in tissue engineering owing to their many advantages, such as high water content, softness and flexibility similar to many soft tissues, tuneable physical, chemical, and biological properties, excellent biocompatibility and biodegradability, and extensive framework for cell proliferation and survival. During the past decade, because of the great versatility offered in terms of processing approach, material selection, and customization, 3D printing has become a leading technology used to fabricate hydrogel scaffolds. Furthermore, high reproducibility and unparalleled control over structural and compositional characteristics make additive manufacturing the preferred technology for the fabrication of biodegradable hydrogel scaffolds. However, the production time could become critical in relation to any crosslinking reactions and degradation that may occur in the hydrogel and make the printing process unstable. In this study an analysis of the critical issues due to the crosslinking reaction and degradation phenomena have been executed following a statistical approach. In particular, three different experimental campaign demonstrate how the printing process became instable due to the mentioned phenomena. Finally, a procedure was developed to print gelatine-based biocompatible hydrogels with chitosan and functionalized polyethylene glycol as a cross linker (G-PEG-CH). In order to reach the printing temperature, the hydrogel mixture was initially stored in the refrigerator at 4°C for 12 h, followed by a 10 min incubation in warm water at 40°C. Based on this procedure, a filament strand of 950 μm with a standard deviation in the range of 20% was obtained by imposing a printing pressure of 1 bar and a printing speed of 100 mm/s at a temperature of 28 °C

Drinking Water Contamination Due to Internal Corrosion of Copper Distribution Pipes

The present paper deals with water quality modification in metal distribution pipes, caused by the development of electrochemical corrosion processes. More particularly it is aimed at investigating metal release during stagnation of water in copper pipes. At this aim several experiments are developed, varying water quality characteristics and stagnation length, to ascertain the dependence of copper release on both water composition and contact lime between water and copper. Copper is chosen as plumbing material because of its wide use in drinking water distribution systems all around the world, and because of its recognized toxicity at high dosages. The effect of different concentrations of bicarbonates and sulphates on metal release is studied, assuming stagnation periods ranging between 15 and 90 minutes. A special corrosion cell is used as experimental apparatus, and the so called corrosion potential stagnation flow (CPSF) theory is followed for copper concentration monitoring, which means that copper concentration is monitored not only by direct measuring it, but also by measuring the corrosion potential of the metal surface during stagnation episodes. Experimental data allow to verify the formation of a protective layer on copper surface which lowers the corrosion rate, reducing the metal release. This layer is more likely to be formed at higher bicarbonate and sulphate concentrations. The effect of longer stagnation times results to be not dissimilar from that observed varying water quality characteristics. The corrosion potential always exhibits a typical and reproducible trend: it decreases at the beginning of each stagnation period, and then slightly increases once more. The minimum reached by the corrosion potential is function of the water quality, being less pronounced for higher alkalinity values and higher sulphate concentrations, and more pronounced in the apposite case. This trend is well explained by the development of the corrosion process and by the formation of the over mentioned protective layer. The experimental data allow to validate the CPSF theory, showing the existence of a direct correlation between the potential at the end of each stagnation period, and the corresponding total copper concentration in the stagnated water