Biological Evaluation and Structural Analysis of Some Aminodiphenylamine Derivatives.

4-Aminodiphenylamino derivatives were investigated for their antioxidant and hydrophobicity character, together with other biological measurements, such as antimicrobial and antibiofilm activity. Among these nine compounds used, we obtained novel derivatives via reaction of the starting material with NBD-chloride. Therefore, we performed a full structural analysis for these compounds, i.e., elemental analysis, IR, UV-Vis, 1H- and 13C-NMR, ESI-MS, X-ray diffraction on single crystal, etc. The hydrophobicity of all the compounds was measured either experimentally using the RP-TLC technique, or via calculation using the fragments method. The other structural characteristics were analyzed, and a correlation between the experimental and computed properties was found. Moreover, the results of the biological evaluation showed that some of the synthesized compounds have antimicrobial and antibiofilm activity.

Anti-Campylobacter Probiotics: Latest Mechanistic Insights.

The Campylobacter genus is the leading cause of human gastroenteritis, with the consumption of contaminated poultry meat as the main route of infection. Probiotic bacteria, such as Lactobacillus, Bacillus, Escherichia coli Nissle, and Bifidobacterium species, have a great immunomodulatory capacity and exhibit antipathogenic effects through various molecular mechanisms. Reducing Campylobacter levels in livestock animals, such as poultry, will have a substantial benefit to humans as it will reduce disease transmissibility through the food chain. Moreover, probiotic-based strategies might attenuate intestinal inflammatory processes, which consequently reduce the severity of Campylobacter disease progression. At a molecular level, probiotics can also negatively impact on the functionality of various Campylobacter virulence and survival factors (e.g., adhesion, invasion), and on the associated colonization proteins involved in epithelial translocation. The current review describes recent in vitro, in vivo, and preclinical findings on probiotic therapies, aiming to reduce Campylobacter counts in poultry and reduce the pathogen's virulence in the avian and human host. Moreover, we focused in particular on probiotics with known anti-Campylobacter activity seeking to understand the biological mechanisms involved in their mode of action.

Structural Bioinformatics Used to Predict the Protein Targets of Remdesivir and Flavones in SARS-CoV-2 Infection.

BACKGROUND: During the current SARS-CoV-2 pandemic, the identification of effective antiviral drugs is crucial. Unfortunately, no specific treatment or vaccine is available to date. OBJECTIVE: Here, we aimed to predict the interactions with SARS-CoV-2 proteins and protein targets from the human body for some flavone molecules (kaempferol, morin, pectolinarin, myricitrin, and herbacetin) in comparison to synthetic compounds (hydroxychloroquine, remdesivir, ribavirin, ritonavir, AMD-070, favipiravir). METHODS: Using MOE software and advanced bioinformatics and cheminformatics portals, we conducted an extensive analysis based on various structural and functional features of compounds, such as their amphiphilic field, flexibility, and steric features. The structural similarity analysis of natural and synthetic compounds was performed using Tanimoto coefficients. The interactions of some compounds with SARS-CoV-2 3CLprotease or RNA-dependent RNA polymerase were described using 2D protein-ligand interaction diagrams based on known crystal structures. The potential targets of considered compounds were identified using the SwissTargetPrediction web tool. RESULTS: Our results showed that remdesivir, pectolinarin, and ritonavir present a strong structural similarity which may be correlated to their similar biological activity. As common molecular targets of compounds in the human body, ritonavir, kaempferol, morin, and herbacetin can activate multidrug resistance-associated proteins, while remdesivir, ribavirin, and pectolinarin appear as ligands for adenosine receptors. CONCLUSION: Our evaluation recommends remdesivir, pectolinarin, and ritonavir as promising anti- SARS-CoV-2 agents.

The in vitro and in vivo anti-virulent effect of organic acid mixtures against Eimeria tenella and Eimeria bovis.

Eimeria tenella and Eimeria bovis are complex parasites responsible for the condition of coccidiosis, that invade the animal gastrointestinal intestinal mucosa causing severe diarrhoea, loss of appetite or abortions, with devastating impacts on the farming industry. The negative impacts of these parasitic infections are enhanced by their role in promoting the colonisation of the gut by common foodborne pathogens. The aim of this study was to test the anti-Eimeria efficacy of maltodextrin, sodium chloride, citric acid, sodium citrate, silica, malic acid, citrus extract, and olive extract individually, in vitro and in combination, in vivo. Firstly, in vitro infection models demonstrated that antimicrobials reduced (p < 0.05), both singly and in combination (AG), the ability of E. tenella and E. bovis to infect MDBK and CLEC-213 epithelial cells, and the virulence reduction was similar to that of the anti-coccidial drug Robenidine. Secondly, using an in vivo broiler infection model, we demonstrated that AG reduced (p = 0.001) E. tenella levels in the caeca and excreted faeces, reduced inflammatory oxidative stress, improved the immune response through reduced ROS, increased Mn-SOD and SCFA levels. Levels of IgA and IgM were significantly increased in caecal tissues of broilers that received 0.5% AG and were associated with improved (p < 0.0001) tissue lesion scores. A prophylactic approach increased the anti-parasitic effect in vivo, and results indicated that administration from day 0, 5 and 10 post-hatch reduced tissue lesion scores (p < 0.0001) and parasite excretion levels (p = 0.002). Conclusively, our in vitro and in vivo results demonstrate that the natural antimicrobial mixture (AG) reduced parasitic infections through mechanisms that reduced pathogen virulence and attenuated host inflammatory events.

Phenotypic and genotypic virulence features of staphylococcal strains isolated from difficult-to-treat skin and soft tissue infections.

Chronic infections represent an important burden on the healthcare system and have a significant impact on the patients' quality of life. While Staphylococcus spp. are commensal bacteria, they can become pathogenic, leading to various types of infections. In this study we aimed to characterize the virulence profiles of staphylococcal strains involved in difficult-to-treat skin and soft tissue infections, from both phenotypic and genotypic points of view. Phenotypic ability of the strains to secrete soluble virulence factors was assessed by a culturing dependent assay and their capacity to develop biofilms on inert substrate was screened by an adapted crystal violet microtiter method. We also tested the presence of several virulence genes by PCR. Most of the studied strains were isolated from purulent secretions of acne lesions and frequently secreted two or three soluble virulence factors. Most frequently secreted soluble virulence factors were caseinase (89%), lipase (71%) and lecithinase (67%). Almost half of the strains produced a well-represented biofilm. The molecular characterization showed the presence of the genes cna, hlg, clfA, and clfB. Staphylococcal strains that produce difficult-to-treat skin and soft tissue infections seem to be characterized by an enhanced ability to produce different soluble virulence factors and to develop biofilms in vitro. Further studies need to be developed in other Staphylococcus spp. infections in order to confirm this hypothesis.

Potential Therapeutic Approaches to Alzheimer's Disease By Bioinformatics, Cheminformatics And Predicted Adme-Tox Tools.

BACKGROUND: Alzheimer's disease (AD) is considered a severe, irreversible and progressive neurodegenerative disorder. Currently, the pharmacological management of AD is based on a few clinically approved acethylcholinesterase (AChE) and N-methyl-D-aspartate (NMDA) receptor ligands, with unclear molecular mechanisms and severe side effects. METHODS: Here, we reviewed the most recent bioinformatics, cheminformatics (SAR, drug design, molecular docking, friendly databases, ADME-Tox) and experimental data on relevant structurebiological activity relationships and molecular mechanisms of some natural and synthetic compounds with possible anti-AD effects (inhibitors of AChE, NMDA receptors, beta-secretase, amyloid beta (Aß), redox metals) or acting on multiple AD targets at once. We considered: (i) in silico supported by experimental studies regarding the pharmacological potential of natural compounds as resveratrol, natural alkaloids, flavonoids isolated from various plants and donepezil, galantamine, rivastagmine and memantine derivatives, (ii) the most important pharmacokinetic descriptors of natural compounds in comparison with donepezil, memantine and galantamine. RESULTS: In silico and experimental methods applied to synthetic compounds led to the identification of new AChE inhibitors, NMDA antagonists, multipotent hybrids targeting different AD processes and metal-organic compounds acting as Aß inhibitors. Natural compounds appear as multipotent agents, acting on several AD pathways: cholinesterases, NMDA receptors, secretases or Aß, but their efficiency in vivo and their correct dosage should be determined. CONCLUSION: Bioinformatics, cheminformatics and ADME-Tox methods can be very helpful in the quest for an effective anti-AD treatment, allowing the identification of novel drugs, enhancing the druggability of molecular targets and providing a deeper understanding of AD pathological mechanisms.

Peripheral immune cell markers in children with recurrent respiratory infections in the absence of primary immunodeficiency.

The immune system of a child has a degree of immaturity that is maintained until 6-7 years of age. Immaturity may be due to age-related functional disorders in the immune response. A healthy child can contract a series of infections which contribute to the maturation of the immune system during the pre-pubertal period. If repeated infections with prolonged or severe complications occur during childhood, the presence of an immunodeficiency should then be considered. Much more frequent than primary immunodeficiency are recurrent infections (frequently involving the upper respiratory tract), which are less severe and occur under the conditions of an immune system with no apparent major defects. A child can present with 4 to 8 episodes of respiratory infections within a year, during the first 5 years of its life. The average duration of infection is 8 days and up to 2 weeks; if the child presents with 3 episodes of acute infections over a period of 6 months, the respiratory infections are then considered recurrent. The aim of this study was to identify the immunological changes or deviations that cause this clinical syndrome in children. For this purpose, 30 children with recurrent respiratory infections and 10 healthy children were included. Immunoglobulin levels were examined and immunophenotyping was performed. We found that the serum immunoglobulin levels were in the normal range in 70% of the children. On the contrary, our data revealed changes in peripheral cell populations, the most important being the decrease in the T-cluster of differentiation (CD)8+ and total B cell percentages and the increase in the number of memory B cells. The data obtained herein indicated that the decrease in the number of total B cells was mainly due to the decrease in the number of naive IgD+ B cells. On the whole, the findings of this study indicate that recurrent respiratory infections may be associated with an altered cellular immune response. In such situations, the investigation of immunological parameters, such as T and B cell subtypes could complete the clinical diagnosis and guide the treatment strategy, thus increasing the quality of life of patients.

Tunable ZnO spheres with high anti-biofilm and antibacterial activity via a simple green hydrothermal route.

A family of distinct ZnO morphologies - hollow, compartmented, core-shell and full solid ZnO spheres, dispersed or interconnected - is obtained by a simple hydrothermal route, in the presence of the starch biopolymer. The zinc-carbonaceous precursors were characterized by infrared spectroscopy, thermal analysis and scanning electron microscopy, while the ZnO spheres, obtained after the thermal processing, were investigated by X-ray diffraction, Raman spectroscopy, scanning electron microscopy, UV-VIS spectroscopy, photoluminescence measurements, antimicrobial, anti-biofilm and flow cytometry tests. The formation mechanism proposed for this versatile synthesis route is based on the gelling ability of amylose, one of the starch template constituents, responsible for the effective embedding of zinc cations into starch prior to its hydrothermal carbonization. The simple variation of the raw materials concentration dictates the type of ZnO spheres. The micro-sized ZnO spheres exhibit high antibacterial and anti-biofilm activity against Gram-positive (Staphylococcus aureus, Bacillus subtilis) and Gram-negative (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) reference and methicillin resistant clinical strains especially for Gram-negative biofilms (P. aeruginosa), demonstrating great potential for new ZnO anti-biofilm formulations.

Antimicrobial and antioxidant activities of alcoholic extracts obtained from vegetative organs of A. retroflexus.

In vitro antimicrobial and antioxidant activities of Amaranthus retroflexus leaves and inflorescence alcoholic (ethanol 70%) extracts of various concentrations ranging from 0.78 to 400 µL/ml were analyzed on different clinical and reference bacterial strains (Staphylococcus aureus, Bacillus subtills, Enterococcus faecalis, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii) and fungal strains (Candida albicans, C. famata, C. utilis, Saccharomyces cerevisiae) using agar disk diffusion method and broth dilution method (MIC determination) for antimicrobial activity and TEAC (Trolox capacity) assay for the evaluation of the antioxidant activity. The adapted diffusion method was used to test the antimicrobial effect of the extracts separately and in combination with a range of standard antibiotics, in order to evidence any synergic effects of A. retroflexus extracts on the antibiotics activity. The extracts showed the highest inhibitory effect against K. pneumoniae and B. subtilis with no activity against S. aureus among the bacterial strains, while in case of the fungal strains the most intensive effect was exhibited against C. famata by both extracts. The A. retroflexus leaves extract improved the ciprofloxacin and ticarcillin-clavulanic acid activity towards the P. aeruginosa clinical strain. The inflorescences extract significantly increased chloramphenicol activity on B. subtilis strain. The antioxidant activity assay showed that the studied extracts exhibited the ability to neutralize the free radicals leading to the conclusion that the tested extracts bear compounds with a broad spectrum of antimicrobial and antioxidant activity that could represent a potential alternative for treating various infectious diseases.

Prevention of microbial biofilms - the contribution of micro and nanostructured materials.

Microbial biofilms are associated with drastically enhanced resistance to most of the antimicrobial agents and with frequent treatment failures, generating the search for novel strategies which can eradicate infections by preventing the persistent colonization of the hospital environment, medical devices or human tissues. Some of the current approaches for fighting biofilms are represented by the development of novel biomaterials with increased resistance to microbial colonization and by the improvement of the current therapeutic solutions with the aid of nano (bio)technology. This special issues includes papers describing the applications of nanotechnology and biomaterials science for the development of improved drug delivery systems and nanostructured surfaces for the prevention and treatment of medical biofilms. Nanomaterials display unique and well-defined physical and chemical properties making them useful for biomedical applications, such as: very high surface area to volume ratio, biocompatibility, biodegradation, safety for human ingestion, capacity to support surface modification and therefore, to be combined with other bioactive molecules or substrata and more importantly being seemingly not attracting antimicrobial resistance. The use of biomaterials is significantly contributing to the reduction of the excessive use of antibiotics, and consequently to the decrease of the emergence rate of resistant microorganisms, as well as of the associated toxic effects. Various biomaterials with intrinsic antimicrobial activity (inorganic nanoparticles, polymers, composites), medical devices for drug delivery, as well as factors influencing their antimicrobial properties are presented. One of the presented papers reviews the recent literature on the use of magnetic nanoparticles (MNP)-based nanomaterials in antimicrobial applications for biomedicine, focusing on the growth inhibition and killing of bacteria and fungi, and, on viral inactivation. The anti-pathogenic activity of the most common types of metallic/metal oxide nanoparticles, as well as the photocontrolled targeted drug-delivery system and the development of traditional Chinese herbs nanoparticles are some of the highlights of another paper of this issue. The applications of synthetic, biodegradable polymers for the improvement of antiinfective therapeutic and prophylactic agents (i.e., antimicrobial and anti-inflammatory agents and vaccines) activity, as well as for the design of biomaterials with increased biocompatibility and resistance to microbial colonization are also discussed, as well as one of the most recent paradigms of the pharmaceutical field and nanobiotechnology, represented by the design of smart multifunctional polymeric nanocarriers for controlled drug delivery. These systems are responding to physico-chemical changes and as a result, they can release the active substances in a controlled and targeted manner. The advantages and limitations of the main routes of polymerization by which these nanovehicles are obtained, as well as the practical appllications in the field of drug nanocarriers are presented. The authors describe the therapeutic applications of dendrimers, which are unimolecular, monodisperse nanocarriers with unique branched tree-like globular structure. The applications of nanotechnology for the stabilization and improved release of anti-pathogenic natural or synthetic compounds, which do not interfere with the microbial growth, but inhibit different features of microbial pathogenicity are also highlighted. We expect this special issue would offer a comprehensive update and give new directions for the design of micro/nano engineered materials to inhibit microbial colonization on the surfaces or to potentiate the efficiency of the current/ novel/alternative antimicrobial agents by improving their bioavailability and pharmacokinetic features.

Water dispersible cross-linked magnetic chitosan beads for increasing the antimicrobial efficiency of aminoglycoside antibiotics.

The aim of this study was to obtain a nano-active system to improve antibiotic activity of certain drugs by controlling their release. Magnetic composite nanomaterials based on magnetite core and cross-linked chitosan shell were synthesized via the co-precipitation method and characterized by Fourier transform infrared spectroscopy (FT-IR), infrared microscopy (IRM), scanning electron microscopy (SEM), dynamic light scattering (DLS), thermogravimetric analysis (TGA) and X-ray diffraction (XRD). The prepared magnetic composite nanomaterials exhibit a significant potentiating effect on the activity of two cationic (kanamycin and neomycin) drugs, reducing the amount of antibiotics necessary for the antimicrobial effect. The increase in the antimicrobial activity was explained by the fact that the obtained nanosystems provide higher surface area to volume ratio, resulting into higher surface charge density thus increasing affinity to microbial cell and also by controlling their release. In addition to the nano-effect, the positive zeta potential of the synthesized magnetite/cross-linked chitosan core/shell magnetic nanoparticles allows for a more favorable interaction with the usually negatively charged cell wall of bacteria. The novelty of the present contribution is just the revealing of this synergistic effect exhibited by the synthesized water dispersible magnetic nanocomposites on the activity of different antibiotics against Gram-positive and Gram-negative bacterial strains. The results obtained in this study recommend these magnetic water dispersible nanocomposite materials for applications in the prevention and treatment of infectious diseases.

Fabrication, characterization and in vitro profile based interaction with eukaryotic and prokaryotic cells of alginate-chitosan-silica biocomposite.

This work is focused on the fabrication of a new drug delivery system based on polyanionic matrix (e.g. sodium alginate), polycationic matrix (e.g. chitosan) and silica network. The FT-IR, SEM, DTA-TG, eukaryotic cell cycle and viability, and in vitro assay of the influence of the biocomposite on the efficacy of antibiotic drugs were investigated. The obtained results demonstrated the biocompatibility and the ability of the fabricated biocomposite to maintain or improve the efficacy of the following antibiotics: piperacillin-tazobactam, cefepime, piperacillin, imipenem, gentamicin, ceftazidime against Pseudomonas aeruginosa ATCC 27853 and cefazolin, cefaclor, cefuroxime, ceftriaxone, cefoxitin, trimethoprim/sulfamethoxazole against Escherichia coli ATCC 25922 reference strains.

Improved antibacterial activity of cephalosporins loaded in magnetic chitosan microspheres.

During the present study, we have evaluated magnetic chitosan as a potential drug delivery device, by specifically determining if chitosan could elute antibiotics in an active form that would be efficacious in inhibiting Staphylococcus aureus and Escherichia coli growth. We have demonstrated that the incorporation of cephalosporins of second, third and fourth generation into magnetic chitosan microspheres can possibly lead to an improved delivery of antibiotics in active forms, probably due to the inherent properties of chitosan.

Hybrid magnetite nanoparticles/Rosmarinus officinalis essential oil nanobiosystem with antibiofilm activity.

Biofilms formed by fungal organisms are associated with drastically enhanced resistance against most antimicrobial agents, contributing to the persistence of the fungi despite antifungal therapy. The purpose of this study is to combine the unique properties of nanoparticles with the antimicrobial activity of the Rosmarinus officinalis essential oil in order to obtain a nanobiosystem that could be pelliculised on the surface of catheter pieces, in order to obtain an improved resistance to microbial colonization and biofilm development by Candida albicans and C. tropicalis clinical strains. The R. officinalis essential oils were extracted in a Neo-Clevenger type apparatus, and its chemical composition was settled by GC-MS analysis. Functionalized magnetite nanoparticles of up to 20 nm size had been synthesized by precipitation method adapted for microwave conditions, with oleic acid as surfactant. The catheter pieces were coated with suspended core/shell nanoparticles (Fe3O4/oleic acid:CHCl3), by applying a magnetic field on nanofluid, while the CHCl3 diluted essential oil was applied by adsorption in a secondary covering treatment. The fungal adherence ability was investigated in six multiwell plates, in which there have been placed catheters pieces with and without hybrid nanoparticles/essential oil nanobiosystem pellicle, by using culture-based methods and confocal laser scanning microscopy (CLSM). The R. officinalis essential oil coated nanoparticles strongly inhibited the adherence ability and biofilm development of the C. albicans and C. tropicalis tested strains to the catheter surface, as shown by viable cell counts and CLSM examination. Due to the important implications of Candida spp. in human pathogenesis, especially in prosthetic devices related infections and the emergence of antifungal tolerance/resistance, using the new core/shell/coated shell based on essential oil of R. officinalis to inhibit the fungal adherence could be of a great interest for the biomedical field, opening new directions for the design of film-coated surfaces with antibiofilm properties.

Mechanisms of ceftazidime and ciprofloxacin transport through porins in multidrug-resistance developed by extended-spectrum beta-lactamase E.coli strains.

Resistance towards antibiotics stands out today as a major issue in the clinical act of treatment of bacterial-generated infections. This process was characterized in proteoliposomes reconstituted from an E.coli strain isolated from invasive infections (blood culture) occurred in patients with a cardio-vascular device admitted for surgery. Fluorescence spectroscopy and patch-clamp technique have been used. Two types of antibiotics have been targeted: ceftazidime and ciprofloxacin. Antibiotics addition in proteoliposomes suspension undergoes a quenching in tryptophan residues from outer membrane porins structure, probably due to the formation of a transient non-fluorescent porin-antibiotic complex. Patch-clamp recordings revealed strong ion current blockages for both antibiotics, reflecting antibiotic-channel interactions but with varying strength of interaction. The present study puts forward the mechanism of multidrug-resistance in extended-spectrum beta-lactamase E.coli strains, as being caused by alterations of the antibiotics transport across the porins of the outer bacterial membrane.

Correlation of anti-Helicobacter pylori cagA IgG antibodies with resistance to first line treatment, bleeding gastroduodenal ulcers and gastric cancer.

UNLABELLED: Helicobacter pylori was recognized in 1994 as a class I carcinogen by the International Agency for Research on Cancer (IARC). The prevalence of H. pylori infection varies from 20 to 50% in industrialized countries to over 80% in developing countries. The cagA strains are more virulent than others, being able to induce morphological changes, vacuolization and degeneration of in vitro cultured cells. AIM: During this study we investigated the possible correlations between the presence of H. pylori cagA (cytotoxin associated gene antigen)-IgG antibodies and the severity of clinical and endoscopical findings. METHODS: Anti-cagA IgG was screened by ELISA in 104 selected patients exhibiting resistance to first line therapy for H. pylori, bleeding gastroduodenal ulcers, non cardia gastric cancer and gastric polyps. RESULTS: A statistically significant association between resistant cases to first line therapy for H. pylori, bleeding gastroduodenal ulcers, non cardia gastric cancer, gastric polyps and cag A Ig G antibodies (p value 0.02 calculated by T-Test) was observed. As Cag A antibodies titer persist up to four months, their level could be an useful marker in detecting previous long-term H pylori infection especially in gastric cancer patients. CONCLUSIONS: CagA positive H. pylori are virulent strains and the cagA IgG antibodies titer is associated with persistence of infection after treatment, upper gastroduodenal ulcers or gastric cancer. The presence of these antibodies, associated with positive biopsy for H. pylori, indicates the need of H. pylori treatment.

[Aflatoxins--health risk factors]. / Aflatoxinele--factori de risc pentru sanatate.

Aflatoxins are secondary metabolites produced by a group of strains, mainly Aspergillus and Penicillium species. These mycotoxins are bifurano-coumarin derivatives group with four major products B1, B2, G1 and G2 according to blue or green fluorescence emitted in ultraviolet light and according to chromatographic separation. After metabolism of aflatoxin B1 and B2 in the mammalian body, result two metabolites M1 and M2 as hydroxylated derivatives of the parent compound. Aflatoxins have high carcinogenic potential, the most powerful carcinogens in different species of animals and humans. International Agency for Research on Cancer has classified aflatoxin B1 in Group I carcinogens. The target organ for aflatoxins is the liver. In chronic poisoning, aflatoxin is a risk to health, for a long term causing cancer (hepatocellular carcinoma), and in acute intoxications aflatoxin is lethal. This work purpose to discuss aflatoxins issue: the synthesis, absorption and elimination of aflatoxins, the toxicity mechanisms, and measures to limit the content of aflatoxins in food