Development and optimization of topical antimicrobial agents based on human antimicrobial peptides

New antimicrobial compounds are urgently needed to fight infective diseases caused by antibiotic-resistant human pathogens. AntiMicrobial Peptides (AMPs) are attractive candidates as they can both directly kill microbes and modulate immune response. Surprisingly, many human proteins whose functions are not necessarily related to host defense can behave as sources of AMPs. Some examples are lactoferrin, lysozyme or thrombin. Since these AMPs are hidden in large proteins, they can be defined “cryptic”. In order to identify by a rational approach further human proteins carrying new cryptic AMPs, a scoring function allowing a quantitative prediction of antibacterial activity was developed and validated analyzing the sequence of already known AMP releasing proteins. Using this method to screen about 4'000 human extracellular proteins, a wide list of potential new AMPs was obtained. The main aim of the present PhD thesis is to select and characterize the most pharmacologically interesting potential AMPs in order to find candidates suited for the development of novel human antimicrobial and anti-inflammatory agents. Firstly, I developed a novel cost-effective fusion system, particularly well-suited for the production of toxic peptides in E. coli. To avoid AMPs toxicity toward bacterial host, I rationally designed a carrier protein starting from a Rana pipiens ribonuclease, named onconase (ONC), that is expressed at high level (200 mg/L of culture) as inclusion bodies in E. coli. In order to optimize this method, a well-known AMP derived from human thrombin, named GKY20 was used as model peptide. Moreover, I demonstrated that this strategy also allows to prepare peptides with a N-terminal cysteine residues that could find many applications in basic and applied research like for example preparation of labeled peptides, protein ligation for the semi-synthesis of modified proteins, immobilization on supports etc. Using this procedure I prepared and characterized several new human AMPs from 20 to almost 50 amino acids with yields of at least 10 mg/L of culture. Therefore, our method is complementary to chemical synthesis being particularly well-suited to prepare peptide longer than 20-25 residues. All these peptides show a broad antimicrobial activity on GRAM-negative and GRAM-positive bacteria, antibiofilm and immunomodulatory activity. Very interestingly, they proved to be both non-toxic on eukaryotic cells, providing AMPs of human origin particularly well suited to develop anti-infective drugs

Descriptive analysis of postmarket surveillance data for hip implants

Purpose: Recent safety issues involving medical devices have highlighted the need for better postmarket surveillance (PMS) evaluation. This article aims to describe and to assess the quality of the PMS data for a medical device and, finally, to provide recommendations to improve the data gathering process. Methods: A descriptive analysis of medical device reports (MDRs) on the use of MRA, a specific type of hip implant replacement submitted to the Food and Drug Administration Manufacturer and User Facility Device Experience database from 1 January 2008 to 31 December 2017. The number of reports was described as the number of MDRs per unique MDR number and stratified by different variables. The quality was assessed by the level of completeness of the collected PMS data. Results: The total number of reports related to MRA was 2377, and the number of MDRs per year ranged between 84 in 2009 and 452 in 2017. Most of the reports were reported by manufacturer Depuy Johnson & Johnson and were reported by a physician. In 44.9% of the reports, the device problem was reported as “Unknown.” When the device problem was known, in the majority of cases, it was related to an implant fracture. The quality of the collected data was assessed as low due to missing information. Conclusion: The underlying data should meet high quality standards to generate more evidence and to ensure a timely signal generation. This case study shows that the completeness and quality of the MDRs can be improved. The authors propose the development of tools to ensure a more dynamic complaint data collection to contribute to this enhancement

Evaluating the Safety Profile of Non-Active Implantable Medical Devices Compared with Medicines

Recent safety issues involving non-active implantable medical devices (NAIMDs) have highlighted the need for better pre-market and post-market evaluation. Some stakeholders have argued that certain features of medicine safety evaluation should also be applied to medical devices. Our objectives were to compare the current processes and methodologies for the assessment of NAIMD safety profiles with those for medicines, identify potential gaps, and make recommendations for the adoption of new methodologies for the ongoing benefit–risk monitoring of these devices throughout their entire life cycle. A literature review served to examine the current tools for the safety evaluation of NAIMDs and those for medicines. We searched MEDLINE using these two categories. We supplemented this search with Google searches using the same key terms used in the MEDLINE search. Using a comparative approach, we summarized the new product design, development cycle (preclinical and clinical phases), and post-market phases for NAIMDs and drugs. We also evaluated and compared the respective processes to integrate and assess safety data during the life cycle of the products, including signal detection, signal management, and subsequent potential regulatory actions. The search identified a gap in NAIMD safety signal generation: no global program exists that collects and analyzes adverse events and product quality issues. Data sources in real-world settings, such as electronic health records, need to be effectively identified and explored as additional sources of safety information, particularly in some areas such as the EU and USA where there are plans to implement the unique device identifier (UDI). The UDI and other initiatives will enable more robust follow-up and assessment of long-term patient outcomes. The safety evaluation system for NAIMDs differs in many ways from those for drugs, but both systems face analogous challenges with respect to monitoring real-world usage. Certain features of the drug safety evaluation process could, if adopted and adapted for NAIMDs, lead to better and more systematic evaluations of the latter

Host defense peptides identified in human apolipoprotein B as natural food bio-preservatives: Evaluation of their biosafety and digestibility

The employment of chemical agents in the food industry is raising several concerns by consumers and is leading to an increasing interest in natural food preservatives. Among alternatives, host defense peptides (HDPs) have attracted great interest for their ability to preserve food samples from contamination without altering their quality, taste, and organoleptic properties. Recently, we evaluated the applicability of ApoB-derived peptides as novel food bio-preservatives and demonstrated their ability to prevent chicken meat sample contamination when immobilized on chitosan films. To perform a further step towards the applicability of these peptides in the food field, here we evaluated peptides biosafety and digestibility. To do this, we used a multidisciplinary approach including the evaluation of the peptides' toxicity and antimicrobial activity, the analysis of resistance phenotype development, an in silico prediction of the peptides' susceptibility to proteases and the evaluation of the peptides' stability in simulated gastric and intestinal fluids. ApoB-derived peptides were found to be nontoxic when tested on human gastric carcinoma cells SNU-1 and on human colon-rectal adenocarcinoma cells HT-29, and not to induce resistance phenotype in Salmonella strains. Bioinformatic analyses showed that the peptides are susceptible to several proteases, as also confirmed by experiments in simulated gastric and intestinal fluids. Altogether these findings open interesting perspectives to the future applicability of ApoB-derived peptides as novel food biopreservatives

Novel Antimicrobial Strategies to Prevent Biofilm Infections in Catheters after Radical Cystectomy: A Pilot Study

Catheter-associated infections in bladder cancer patients, following radical cystectomy or ureterocutaneostomy, are very frequent, and the development of antibiotic resistance poses great challenges for treating biofilm-based infections. Here, we characterized bacterial communities from catheters of patients who had undergone radical cystectomy for muscle-invasive bladder cancer. We evaluated the efficacy of conventional antibiotics, alone or combined with the human ApoB-derived antimicrobial peptide r(P)ApoBLAla, to treat ureteral catheter-colonizing bacterial communities on clinically isolated bacteria. Microbial communities adhering to indwelling catheters were collected during the patients' regular catheter change schedules (28 days) and extracted within 48 h. Living bacteria were characterized using selective media and biochemical assays. Biofilm growth and novel antimicrobial strategies were analyzed using confocal laser scanning microscopy. Statistical analyses confirmed the relevance of the biofilm reduction induced by conventional antibiotics (fosfomycin, ceftriaxone, ciprofloxacin, gentamicin, and tetracycline) and a well-characterized human antimicrobial peptide r(P)ApoBLAla (1:20 ratio, respectively). Catheters showed polymicrobial communities, with Enterobactericiae and Proteus isolates predominating. In all samples, we recorded a meaningful reduction in biofilms, in both biomass and thickness, upon treatment with the antimicrobial peptide r(P)ApoBLAla in combination with low concentrations of conventional antibiotics. The results suggest that combinations of conventional antibiotics and human antimicrobial peptides might synergistically counteract biofilm growth on ureteral catheters, suggesting novel avenues for preventing catheter-associated infections in patients who have undergone radical cystectomy and ureterocutaneostomy

The new paradigm of Network Medicine to analyse breast cancer phenotypes

Breast cancer (BC) is a heterogeneous and complex disease as witnessed by the existence of different subtypes and clinical characteristics that poses significant challenges in disease management. The complexity of this tumor may rely on the highly interconnected nature of the various biological processes as stated by the new paradigm of Network Medicine. We explored The Cancer Genome Atlas (TCGA)-BRCA data set, by applying the network-based algorithm named SWItch Miner, and mapping the findings on the human interactome to capture the molecular interconnections associated with the disease modules. To characterize BC phenotypes, we constructed protein–protein interaction modules based on “hub genes”, called switch genes, both common and specific to the four tumor subtypes. Transcriptomic profiles of patients were stratified according to both clinical (immunohistochemistry) and genetic (PAM50) classifications. 266 and 372 switch genes were identified from immunohistochemistry and PAM50 classifications, respectively. Moreover, the identified switch genes were functionally characterized to select an interconnected pathway of disease genes. By intersecting the common switch genes of the two classifications, we selected a unique signature of 28 disease genes that were BC subtype-independent and classification subtype-independent. Data were validated both in vitro (10 BC cell lines) and ex vivo (66 BC tissues) experiments. Results showed that four of these hub proteins (AURKA, CDC45, ESPL1, and RAD54L) were over-expressed in all tumor subtypes. Moreover, the inhibition of one of the identified switch genes (AURKA) similarly affected all BC subtypes. In conclusion, using a network-based approach, we identified a common BC disease module which might reflect its pathological signature, suggesting a new vision to face with the disease heterogeneity

The activation of the cannabinoid receptor type 2 reduces neutrophilic protease-mediated vulnerability in atherosclerotic plaques

Aims The activation of cannabinoid receptor type 2 (CB2)-mediated pathways might represent a promising anti-atherosclerotic treatment. Here, we investigated the expression of the endocannabinoid system in human carotid plaques and the impact of CB2 pharmacological activation on markers of plaque vulnerability in vivo and in vitro. Methods and results The study was conducted using all available residual human carotid tissues (upstream and downstream the blood flow) from our cohort of patients symptomatic (n = 13) or asymptomatic (n = 27) for ischaemic stroke. Intraplaque levels of 2-arachidonoylglycerol, anandamide N-arachidonoylethanolamine, N-palmitoylethanolamine, N-oleoylethanolamine, and their degrading enzymes (fatty acid amide hydrolase and monoacylglycerol lipase) were not different in human plaque portions. In the majority of human samples, CB1 (both mRNA and protein levels) was undetectable. In downstream symptomatic plaques, CB2 protein expression was reduced when compared with asymptomatic patients. In these portions, CB2 levels were inversely correlated (r = −0.4008, P = 0.0170) with matrix metalloprotease (MMP)-9 content and positively (r = 0.3997, P = 0.0174) with collagen. In mouse plaques, CB2 co-localized with neutrophils and MMP-9. Treatment with the selective CB2 agonist JWH-133 was associated with the reduction in MMP-9 content in aortic root and carotid plaques. In vitro, pre-incubation with JWH-133 reduced tumour necrosis factor (TNF)-α-mediated release of MMP-9. This effect was associated with the reduction in TNF-α-induced ERK1/2 phosphorylation in human neutrophils. Conclusion Cannabinoid receptor type 2 receptor is down-regulated in unstable human carotid plaques. Since CB2 activation prevents neutrophil release of MMP-9 in vivo and in vitro, this treatment strategy might selectively reduce carotid vulnerability in human

The Effect of Supply Chain Disruptions on Business Post COVID-19

There are many reasons for experiencing supply chain disruptions. The reasons could be miscommunication between the factory and the warehouse, miscommunication between the warehouse and the stores, or miscommunication between the stores and the customers. We investigated these possible disruptions throughout this paper with the help of a questionnaire. We further investigated the effect of various problems that may occur with the company stock which resulted in supply chain disruptions. There have been many papers written about the effect of the disruptions regarding these problems. With the goal of finding out the tactical approach from the company affects the value of the stock, we investigated this further. Additionally, this paper examined the nature of the tactical standings of the company and the effects on supply chain disruptions and the position of the company stock. Based on the responses to the questionnaire, we found how the tactical elements affect the supply chain disruptions. We also showed the effect of the supply chain disruptions on the company stock

TCGA-TCIA Impact on Radiogenomics Cancer Research: A Systematic Review

In the last decade, the development of radiogenomics research has produced a significant amount of papers describing relations between imaging features and several molecular ‘omic signatures arising from next-generation sequencing technology and their potential role in the integrated diagnostic field. The most vulnerable point of many of these studies lies in the poor number of involved patients. In this scenario, a leading role is played by The Cancer Genome Atlas (TCGA) and The Cancer Imaging Archive (TCIA), which make available, respectively, molecular ‘omic data and linked imaging data. In this review, we systematically collected and analyzed radiogenomic studies based on TCGA-TCIA data. We organized literature per tumor type and molecular ‘omic data in order to discuss salient imaging genomic associations and limitations of each study. Finally, we outlined the potential clinical impact of radiogenomics to improve the accuracy of diagnosis and the prediction of patient outcomes in oncology