Old and new strategies in therapy and diagnosis against fungal infections.

Fungal infections represent a serious global health threat. The new emerging pathogens and the spread of different forms of resistance are now hardly challenging the tools available in therapy and diagnostics. With the commonly used diagnoses, fungal identification is often slow and inaccurate, and, on the other hand, some drugs currently used as treatments are significantly affected by the decrease in susceptibility. Herein, the antifungal arsenal is critically summarized. Besides describing the old approaches and their mechanisms, advantages, and limitations, the focus is dedicated to innovative strategies which are designed, identified, and developed to take advantage of the discrepancies between fungal and host cells. Relevant pathways and their role in survival and virulence are discussed as their suitability as sources of antifungal targets. In a similar way, molecules with antifungal activity are reported as potential agents/precursors of the next generation of antimycotics. Particular attention was devoted to biotechnological entities, to their novelty and reliability, to drug repurposing and restoration, and to combinatorial applications yielding significant improvements in efficacy. KEY POINTS: • New antifungal agents and targets are needed to limit fungal morbidity and mortality. • Therapeutics and diagnostics suffer of delays in innovation and lack of targets. • Biologics, drug repurposing and combinations are the future of antifungal treatments.

A biotechnological approach for the production of new protein bioplastics.

The future of biomaterial production will leverage biotechnology based on the domestication of cells as biological factories. Plants, algae, and bacteria can produce low-environmental impact biopolymers. Here, two strategies were developed to produce a biopolymer derived from a bioengineered vacuolar storage protein of the common bean (phaseolin; PHSL). The cys-added PHSL* forms linear-structured biopolymers when expressed in the thylakoids of transplastomic tobacco leaves by exploiting the formation of inter-chain disulfide bridges. The same protein without signal peptide (ΔPHSL*) accumulates in Escherichia coli inclusion bodies as high-molar-mass species polymers that can subsequently be oxidized to form disulfide crosslinking bridges in order to increase the stiffness of the biomaterial, a valid alternative to the use of chemical crosslinkers. The E. coli cells produced 300 times more engineered PHSL, measured as percentage of total soluble proteins, than transplastomic tobacco plants. Moreover, the thiol groups of cysteine allow the site-specific PEGylation of ΔPHSL*, which is a desirable functionality in the design of a protein-based drug carrier. In conclusion, ΔPHSL* expressed in E. coli has the potential to become an innovative biopolymer.

MiR-126 and miR-146a as Melatonin-Responsive Biomarkers for Neonatal Brain Ischemia.

Despite advances in obstetric and neonatal care, challenges remain in early identification of neonates with encephalopathy due to hypoxia-ischemia who are undergoing therapeutic hypothermia. Therefore, there is a deep search for biomarkers that can identify brain injury. The aims of this study were to investigate the serum and brain expressions of two potential biomarkers, miR-126/miR-146a, in a preclinical model of hypoxia-ischemia (HI)-induced brain injury, and to explore their modulation during melatonin treatment. Seven-day-old rats were subjected to permanent ligation of the right carotid artery followed by 2.5 h hypoxia (HI). Melatonin (15 mg/kg) was administered 5 min after HI. Serum and brain samples were collected 1, 6 and 24 h after HI. Results show that HI caused a significant increase in the circulating levels of both miR-126 and miR-146a during the early phase of ischemic brain damage development (i.e. 1 h), with a parallel and opposite pattern in the ischemic cerebral cortex. These effects are not observed 24 h later. Treatment with melatonin restored the HI-induced effects on miR-126/miR-146a expressions, both in the cerebral cortex and in serum. We conclude that miR-126/miR-146a are promising biomarkers of HI injury and demonstrate an associated change in concentration following melatonin treatment.

AFM evaluation of a humanized recombinant antibody affecting C. auris cell wall and stability.

Fungal infections are increasingly impacting on the health of the population and particularly on subjects with a compromised immune system. The resistance phenomenon and the rise of new species carrying sometimes intrinsic and multi-drug resistance to the most commonly used antifungal drugs are greatly concerning healthcare organizations. As a result of this situation, there is growing interest in the development of therapeutic agents against pathogenic fungi. In particular, the Candida genus is responsible for severe life-threatening infections and among its species, C. auris is considered an urgent threat by the Center for Disease Control and Prevention, and is one of the three leading causes of morbidity and mortality worldwide. H5K1 is a humanized monoclonal antibody (hmAb) that selectively binds to ß-1,3-glucans, vital components of the fungal cell wall. It has been previously demonstrated that it is active against Candida species, especially against C. auris, reaching its greatest potential when combined with commercially available antifungal drugs. Here we used atomic force microscopy (AFM) to assess the effects of H5K1, alone and in combination with fluconazole, caspofungin and amphotericin B, on C. auris cells. Through an extensive exploration we found that H5K1 has a significant role in the perturbation and remodeling of the fungal cell wall that is reflected in the loss of whole cell integrity. Moreover, it contributes substantially to the alterations in terms of chemical composition, stiffness and roughness induced specifically by caspofungin and amphotericin B. In addition to this, we demonstrated that AFM is a valuable technique to evaluate drug-microorganism interaction.

Development and in vitro characterization of a humanized scFv against fungal infections.

The resistance and the birth of new intrinsic and multidrug-resistant pathogenic species like C. auris is creating great concern in the antifungal world. Given the limited drug arsenal and the lack of effectiveness of the available compounds, there is an urgent need for innovative approaches. The murine mAb 2G8 was humanized and engineered in silico to develop a single-chain fragment variable (hscFv) antibody against ß-1,3-glucans which was then expressed in E. coli. Among the recombinant proteins developed, a soluble candidate with high stability and affinity was obtained. This selected protein is VL-linker-VH oriented, and it is characterized by the presence of two ubiquitin monomers at the N-terminus and a His tag at the C-terminus. This construct, Ub2-hscFv-His, guaranteed stability, solubility, efficient purification and satisfactory recovery of the recombinant product. HscFv can bind ß-1,3-glucans both as coated antigens and on C. auris and C. albicans cells similarly to its murine parental and showed long stability and retention of binding ability when stored at 4°, -20° and -80° C. Furthermore, it was efficient in enhancing the antifungal activity of drugs caspofungin and amphotericin B against C. auris. The use of biological drugs as antifungals is limited; here we present a promising hscFv which has the potential to be useful in combination with currently available antifungal drugs.

Functionalized 3D scaffolds for engineering the hematopoietic niche.

Hematopoietic stem cells (HSCs) reside in a subzone of the bone marrow (BM) defined as the hematopoietic niche where, via the interplay of differentiation and self-renewal, they can give rise to immune and blood cells. Artificial hematopoietic niches were firstly developed in 2D in vitro cultures but the limited expansion potential and stemness maintenance induced the optimization of these systems to avoid the total loss of the natural tissue complexity. The next steps were adopted by engineering different materials such as hydrogels, fibrous structures with natural or synthetic polymers, ceramics, etc. to produce a 3D substrate better resembling that of BM. Cytokines, soluble factors, adhesion molecules, extracellular matrix (ECM) components, and the secretome of other niche-resident cells play a fundamental role in controlling and regulating HSC commitment. To provide biochemical cues, co-cultures, and feeder-layers, as well as natural or synthetic molecules were utilized. This review gathers key elements employed for the functionalization of a 3D scaffold that demonstrated to promote HSC growth and differentiation ranging from 1) biophysical cues, i.e., material, topography, stiffness, oxygen tension, and fluid shear stress to 2) biochemical hints favored by the presence of ECM elements, feeder cell layers, and redox scavengers. Particular focus is given to the 3D systems to recreate megakaryocyte products, to be applied for blood cell production, whereas HSC clinical application in such 3D constructs was limited so far to BM diseases testing.

Human-rat integrated microRNAs profiling identified a new neonatal cerebral hypoxic-ischemic pathway melatonin-sensitive.

Neonatal encephalopathy (NE) is a pathological condition affecting long-term neurodevelopmental outcomes. Hypothermia is the only therapeutic option, but does not always improve outcomes; hence, researchers continue to hunt for pharmaceutical compounds. Melatonin treatment has benefitted neonates with hypoxic-ischemic (HI) brain injury. However, unlike animal models that enable the study of the brain and the pathophysiologic cascade, only blood is available from human subjects. Therefore, due to the unavailability of neonatal brain tissue, assumptions about the pathophysiology in pathways and cascades are made in human subjects with NE. We analyzed animal and human specimens to improve our understanding of the pathophysiology in human neonates. A neonate with NE who underwent hypothermia and enrolled in a melatonin pharmacokinetic study was compared to HI rats treated/untreated with melatonin. MicroRNA (miRNA) analyses provided profiles of the neonate's plasma, rat plasma, and rat brain cortexes. We compared these profiles through a bioinformatics tool, identifying Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways common to HI brain injury and melatonin treatment. After evaluating the resulting pathways and the literature, to validate the method, the key proteins expressed in HI brain injury were investigated using cerebral cortexes. The upregulated miRNAs in human neonate and rat plasma helped identify two KEGG pathways, glioma and long-term potentiation, common to HI injury and melatonin treatment. A unified neonatal cerebral melatonin-sensitive HI pathway was designed and validated by assessing the expression of protein kinase Cα (PKCα), phospho (p)-Akt, and p-ERK proteins in rat brain cortexes. PKCα increased in HI-injured rats and further increased with melatonin. p-Akt and p-ERK returned phosphorylated to their basal level with melatonin treatment after HI injury. The bioinformatics analyses validated by key protein expression identified pathways common to HI brain injury and melatonin treatment. This approach helped complete pathways in neonates with NE by integrating information from animal models of HI brain injury.

Multitalented Synthetic Antimicrobial Peptides and Their Antibacterial, Antifungal and Antiviral Mechanisms.

Despite the great strides in healthcare during the last century, some challenges still remained unanswered. The development of multi-drug resistant bacteria, the alarming growth of fungal infections, the emerging/re-emerging of viral diseases are yet a worldwide threat. Since the discovery of natural antimicrobial peptides able to broadly hit several pathogens, peptide-based therapeutics have been under the lenses of the researchers. This review aims to focus on synthetic peptides and elucidate their multifaceted mechanisms of action as antiviral, antibacterial and antifungal agents. Antimicrobial peptides generally affect highly preserved structures, e.g., the phospholipid membrane via pore formation or other constitutive targets like peptidoglycans in Gram-negative and Gram-positive bacteria, and glucan in the fungal cell wall. Additionally, some peptides are particularly active on biofilm destabilizing the microbial communities. They can also act intracellularly, e.g., on protein biosynthesis or DNA replication. Their intracellular properties are extended upon viral infection since peptides can influence several steps along the virus life cycle starting from viral receptor-cell interaction to the budding. Besides their mode of action, improvements in manufacturing to increase their half-life and performances are also taken into consideration together with advantages and impairments in the clinical usage. Thus far, the progress of new synthetic peptide-based approaches is making them a promising tool to counteract emerging infections.

A new humanized antibody is effective against pathogenic fungi in vitro.

Invasive fungal infections mainly affect patients undergoing transplantation, surgery, neoplastic disease, immunocompromised subjects and premature infants, and cause over 1.5 million deaths every year. The most common fungi isolated in invasive diseases are Candida spp., Cryptococcus spp., and Aspergillus spp. and even if four classes of antifungals are available (Azoles, Echinocandins, Polyenes and Pyrimidine analogues), the side effects of drugs and fungal acquired and innate resistance represent the major hurdles to be overcome. Monoclonal antibodies are powerful tools currently used as diagnostic and therapeutic agents in different clinical contexts but not yet developed for the treatment of invasive fungal infections. In this paper we report the development of the first humanized monoclonal antibody specific for ß-1,3 glucans, a vital component of several pathogenic fungi. H5K1 has been tested on C. auris, one of the most urgent threats and resulted efficient both alone and in combination with Caspofungin and Amphotericin B showing an enhancement effect. Our results support further preclinical and clinical developments for the use of H5K1 in the treatment of patients in need.

Online information on face masks: analysis of websites in Italian and English returned by different search engines.

OBJECTIVE: Countries have major differences in the acceptance of face mask use for the prevention of COVID-19. This work aims at studying the information online in different countries in terms of information quality and content. DESIGN: Content analysis. METHOD: We analysed 450 webpages returned by searching the string 'are face masks dangerous' in Italy, the UK and the USA using three search engines (Bing, Duckduckgo and Google) in August 2020. The type of website and the stance about masks were assessed by two raters for each language and inter-rater agreement reported as Cohen's kappa. The text of the webpages was collected from the web using WebBootCaT and analysed using a corpus analysis software to identify issues mentioned. RESULTS: Most pages were news outlets, and few (2%-6%) from public health agencies. Webpages with a negative stance on masks were more frequent in Italian (28%) than English (19%). Google returned the highest number of mask-positive pages and Duckduckgo the lowest. Google also returned the lowest number of pages mentioning conspiracy theories and Duckduckgo the highest. Webpages in Italian scored lower than those in English in transparency (reporting authors, their credentials and backing the information with references). When issues about the use of face masks were analysed, mask effectiveness was the most discussed followed by hypercapnia (accumulation of carbon dioxide), contraindication in respiratory disease and hypoxia, with issues related to their contraindications in mental health conditions and disability mentioned by very few pages. CONCLUSIONS: This study suggests that: (1) public health agencies should increase their web presence in providing correct information on face masks; (2) search engines should improve the information quality criteria in their ranking; (3) the public should be more informed on issues related to the use of masks and disabilities, mental health and stigma arising for those people who cannot wear masks.

Online Information of Vaccines: Information Quality, Not Only Privacy, Is an Ethical Responsibility of Search Engines.

The fact that Internet companies may record our personal data and track our online behavior for commercial or political purpose has emphasized aspects related to online privacy. This has also led to the development of search engines that promise no tracking and privacy. Search engines also have a major role in spreading low-quality health information such as that of anti-vaccine websites. This study investigates the relationship between search engines' approach to privacy and the scientific quality of the information they return. We analyzed the first 30 webpages returned searching "vaccines autism" in English, Spanish, Italian, and French. The results show that not only "alternative" search engines (Duckduckgo, Ecosia, Qwant, Swisscows, and Mojeek) but also other commercial engines (Bing, Yahoo) often return more anti-vaccine pages (10-53%) than Google.com (0%). Some localized versions of Google, however, returned more anti-vaccine webpages (up to 10%) than Google.com. Health information returned by search engines has an impact on public health and, specifically, in the acceptance of vaccines. The issue of information quality when seeking information for making health-related decisions also impact the ethical aspect represented by the right to an informed consent. Our study suggests that designing a search engine that is privacy savvy and avoids issues with filter bubbles that can result from user-tracking is necessary but insufficient; instead, mechanisms should be developed to test search engines from the perspective of information quality (particularly for health-related webpages) before they can be deemed trustworthy providers of public health information.