Pushing the Boundaries of Structural Heterogeneity with the Lipid A of Marine Bacteria Cellulophaga.

Marine bacteria, which are often described as chemical gold, are considered an exceptional source of new therapeutics. Considerable research interest has been given to lipopolysaccharides (LPSs), the main components of the Gram-negative outer membrane. LPS and its lipid A portion from marine bacteria are known to exhibit a tricky chemistry that has been often associated with intriguing properties such as behaving as immune adjuvants or anti-sepsis molecules. In this scenario, we report the structural determination of the lipid A from three marine bacteria within the Cellulophaga genus, which showed to produce an extremely heterogenous blend of tetra- to hexa-acylated lipid A species, mostly carrying one phosphate and one D-mannose on the glucosamine disaccharide backbone. The ability of the three LPSs in activating TLR4 signaling revealed a weaker immunopotential by C. baltica NNO 15840T and C. tyrosinoxydans EM41T , while C. algicola ACAM 630T behaved as a more potent TLR4 activator.

Campylobacter jejuni bacteremia in Italian pediatric patients with acute lymphoblastic leukemia: Report of two cases.

Infections caused by Campylobacter jejuni are rarely associated with extraintestinal complications. C. jejuni bacteremia is difficult to detect in patients with hematological malignancies undergoing chemotherapy where the choice of appropriate antibiotic treatment is extremely important. We report two cases of C. jejuni bacteremia in Italian pediatric patients affected by acute lymphoblastic leukemia (ALL). Agreeing with the most recent epidemiological data, both clinical isolates showed a typical phenotypic antimicrobial resistance patterns with combined resistance to ciprofloxacin and tetracycline. To our knowledge, this is the first report of C. jejuni isolation from the blood of ALL pediatric patients in Italy, and it provides important epidemiological information on this rare infection.

Epstein-Barr Virus Seroprevalence and Primary Infection at the University Hospital Luigi Vanvitelli of Naples from 2007 to 2017.

Epstein-Barr virus (EBV) is a common herpesvirus that may cause asymptomatic infection or various diseases, such as mononucleosis, lymphoproliferative disorders and several cancers. Our objective was to estimate the prevalence of EBV among patients hospitalized in "Luigi Vanvitelli" University Hospital in the last 10 years. Our results showed that EBV seroprevalence in our geographical area was 65%. Seroprevalence increased gradually with age with no significant difference between females (49.42%) and males (50.58%). The seropositivity for primary infection was higher in patients about 5 years old, while seropositivity for past infection was predominant in patients of about 35 years old. These results underline that children in our country are still exposed to EBV. The development and the deeper use of an EBV vaccine in the early years of life could represent the solution for this infection.

Non-dermatophytic onychomycosis diagnostic criteria: an unresolved question.

Non-dermatophytic moulds (NDMs) have been increasingly recognised as causative agents of onychomycosis. The diagnosis of onychomycosis is most often obtained by microscopic observation of nail specimens where fungal elements can be detected and cultured by standard mycological techniques. Direct microscopic examination does not always result positive in NDM onychomycosis; therefore to perform a correct diagnosis, a proper mycological culture is often required. The purpose of our study was to evaluate the role of direct microscopic examination in the NDM onychomycosis diagnosis. The results show that only 57.2% of the specimens from onychomycosis patients could be properly diagnosed showing positivity to both direct microscopic examination and NDMs culture isolation in two or more subsequent inoculations, while 42.8% of analysed specimens with a negative direct microscopic examination, showed NDMs growth after three or more subsequent inoculations. The large proportion of false negatives (more than 42%) could be related to the duration of the infection and/or to the experience and skills of the personnel dedicated to specimen collection. We point out the need for thoroughly evaluating all specimens showing cultural growth in at least three subsequent medium inoculations, whatever the result of the microscopic examination, in order to reduce false-negative rates. This strategy would allow for more accurate diagnosis of this mycosis.

Marine Antimicrobial Peptides: Nature Provides Templates for the Design of Novel Compounds against Pathogenic Bacteria.

The discovery of antibiotics for the treatment of bacterial infections brought the idea that bacteria would no longer endanger human health. However, bacterial diseases still represent a worldwide treat. The ability of microorganisms to develop resistance, together with the indiscriminate use of antibiotics, is mainly responsible for this situation; thus, resistance has compelled the scientific community to search for novel therapeutics. In this scenario, antimicrobial peptides (AMPs) provide a promising strategy against a wide array of pathogenic microorganisms, being able to act directly as antimicrobial agents but also being important regulators of the innate immune system. This review is an attempt to explore marine AMPs as a rich source of molecules with antimicrobial activity. In fact, the sea is poorly explored in terms of AMPs, but it represents a resource with plentiful antibacterial agents performing their role in a harsh environment. For the application of AMPs in the medical field limitations correlated to their peptide nature, their inactivation by environmental pH, presence of salts, proteases, or other components have to be solved. Thus, these peptides may act as templates for the design of more potent and less toxic compounds.

Exploitation of viral properties for intracellular delivery.

Nanotechnology is an expanding area of study with potentially pivotal applications in a discipline as medicine where new biomedical active molecules or strategies are continuously developing. One of the principal drawbacks for the application of new therapies is the difficulty to cross membranes that represent the main physiological barrier in our body and in all living cells. Membranes are selectively permeable and allow the selective internalization of substances; generally, they form a highly impermeable barrier to most polar and charged molecules, and represent an obstacle for drug delivery, limiting absorption to specific routes and mechanisms. Viruses provide attracting suggestions for the development of targeted drug carriers as they have evolved naturally to deliver their genomes to host cells with high fidelity. A detailed understanding of virus structure and their mechanisms of entry into mammalian cells will facilitate the development and analysis of virus-based materials for medical applications.

Antimicrobial efficacy of Punica granatum Lythraceae peel extract against pathogens belonging to the ESKAPE group.

The improper use and abuse of antibiotics have led to an increase in multidrug-resistant (MDR) bacteria resulting in a failure of standard antibiotic therapies. To date, this phenomenon represents a leading public health threat of the 21st century which requires alternative strategies to fight infections such as the identification of new molecules active against MDR strains. In the last 20 years, natural extracts with biological activities attracted scientific interest. Following the One Health Approach, natural by-products represent a sustainable and promising alternative solution. Consistently, the aim of the present study was to evaluate the antimicrobial activity of hydro-alcoholic pomegranate peel extract (PPE) against MDR microorganisms belonging to Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. "ESKAPE" group pathogens. Through semiquantitative and quantitative methods, the PPE showed effective antimicrobial activity against Gram-positive and Gram-negative MDR bacteria. The kinetics of bactericidal action of PPE highlighted that microbial death was achieved in a time- and dose-dependent manner. High concentrations of PPE exhibited antioxidant activity, providing a protective effect on cellular systems and red blood cell membranes. Finally, we report, for the first time, a significant intracellular antibacterial property of PPE as highlighted by its bactericidal action against the staphylococcal reference strain and its bacteriostatic effect against clinical resistant strain in the HeLa cell line. In conclusion, due to its characterized content of polyphenolic compounds and antioxidant activity strength, the PPE could be considered as a therapeutic agent alone or in conjunction with standard antibiotics against challenging infections caused by ESKAPE pathogens.

Peptide-lipid interactions: experiments and applications.

The interactions between peptides and lipids are of fundamental importance in the functioning of numerous membrane-mediated cellular processes including antimicrobial peptide action, hormone-receptor interactions, drug bioavailability across the blood-brain barrier and viral fusion processes. Moreover, a major goal of modern biotechnology is obtaining new potent pharmaceutical agents whose biological action is dependent on the binding of peptides to lipid-bilayers. Several issues need to be addressed such as secondary structure, orientation, oligomerization and localization inside the membrane. At the same time, the structural effects which the peptides cause on the lipid bilayer are important for the interactions and need to be elucidated. The structural characterization of membrane active peptides in membranes is a harsh experimental challenge. It is in fact accepted that no single experimental technique can give a complete structural picture of the interaction, but rather a combination of different techniques is necessary.

Corrigendum: Intracellular Delivery: Exploiting Viral Membrane Topic Peptide.

The authors declare that after the publication of the article, it was noticed that two citations were inadvertently omitted. The references have now been included as [74b] and [74c]: [74] (b) Vitiello, M.; Galdiero, M.; Galdiero, M. Inhibition of Viral-Induced Membrane Fusion by Peptides. Protein Pep. Lett., 2009, 16(7), 786-793. (c) Galdiero, S.; Falanga, A.; Vitiello, M.; D'Isanto, M.; Cantisani, M.; Kampanaraki, A.; Benedetti, E.; Browne, H.; Galdiero, M. Peptides containing membrane-interacting motifs inhibit herpes simplex virus type 1 infectivity. Peptides, 2008, 29(9), 1461- 1471. The authors would like to include this reference in the online version of the article to ensure completeness.

Structure and orientation of the gH625-644 membrane interacting region of herpes simplex virus type 1 in a membrane mimetic system.

Glycoprotein H (gH) of the herpes simplex virus type 1 is involved in the complex mechanism of membrane fusion of the viral envelope with host cells. The virus requires four glycoproteins (gB, gD, gH, gL) to execute fusion and the role played by gH remains mysterious. Mutational studies have revealed several regions of gH ectodomain required for fusion and identified the segment from amino acid 625 to 644 as the most fusogenic region. Here, we studied the behavior in a membrane-mimicking DPC micellar environment of a peptide encompassing this region (gH625-644) and determined its NMR solution structure and its orientation within the micelles.

Molecular Epidemiology of Genital Infections in Campania Region: A Retrospective Study.

This study provides updated information on the prevalence and co-infections caused by genital microorganisms and pathogens: Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, Ureaplasma urealyticum, Trichomonas vaginalis, and Gardnerella vaginalis, by retrospectively analyzing a cohort of patients living in the Naples metropolitan area, Campania region, Southern Italy. To investigate the genital infections prevalence in clinical specimens (vaginal/endocervical swabs and urines) collected from infertile asymptomatic women and men from November 2018 to December 2020, we used a multiplex real-time PCR assay. Of the 717 specimens collected, 302 (42.1%) resulted positive for at least one of the targets named above. Statistically significant differences in genital prevalence of selected microorganisms were detected in both women (62.91%) and men (37.08%). G. vaginalis and U. parvum represented the most common findings with an 80.2% and 16.9% prevalence in vaginal/endocervical swabs and first-voided urines, respectively. Prevalence of multiple infections was 18.18% and 8.19% in women and men, respectively. The most frequent association detected was the co-infection of G. vaginalis and U. parvum with 60% prevalence. Our epidemiological analysis suggests different infection patterns between genders, highlighting the need to implement a preventative screening strategy of genital infections to reduce the complications on reproductive organs.

Alcohol-Based Hand Sanitizers: Does Gelling Agent Really Matter?

Hand hygiene, social distancing, and face covering are considered the first protection against Coronavirus spreading. The high demand during the COVID-19 emergency has driven a frenetic production and marketing of hand sanitizer gels. Nevertheless, the effect of the gelling agent and its amount on the effectiveness of alcohol-based hand sanitizers (ABHSs) needs to be clarified. We presented a systematic study on the effect of the characteristics and concentration of the most employed excipients on the properties and antimicrobial activity of ABHSs. Three different gelling agents, carbopol, hydroxypropylmethylcellulose (HPMC), and hydroxyethylcellulose (HEC), at four different concentrations were used to prepare ABHSs. Viscosity, spreadability, delivery from commercial dispensers, evaporation rate, rubbing time, and hand distribution of the ABHSs were then explored. Biocidal activity of selected ABHSs was evaluated in vitro on ATCC and clinical strains. The studied ABHS can be considered bioactive and comfortable. Nevertheless, the cellulose polymers and ethanol interactions led to a slight but significant reduction in the biocidal activity compared with carbopol-based formulations. Our results underline the importance of the gelling agent properties and support the choice of carbopol as one of the best thickener agents in ABHS formulations.

The presence of a single N-terminal histidine residue enhances the fusogenic properties of a Membranotropic peptide derived from herpes simplex virus type 1 glycoprotein H.

Herpes simplex virus type 1 (HSV-1)-induced membrane fusion remains one of the most elusive mechanisms to be deciphered in viral entry. The structure resolution of glycoprotein gB has revealed the presence of fusogenic domains in this protein and pointed out the key role of gB in the entry mechanism of HSV-1. A second putative fusogenic glycoprotein is represented by the heterodimer comprising the membrane-anchored glycoprotein H (gH) and the small secreted glycoprotein L, which remains on the viral envelope in virtue of its non-covalent interaction with gH. Different domains scattered on the ectodomain of HSV-1 gH have been demonstrated to display membranotropic characteristics. The segment from amino acid 626 to 644 represents the most fusogenic region identified by studies with synthetic peptides and model membranes. Herein we have identified the minimal fusogenic sequence present on gH. An enlongation at the N terminus of a single histidine (His) has proved to profoundly increase the fusogenic activity of the original sequence. Nuclear magnetic resonance (NMR) studies have shown that the addition of the N-terminal His contributes to the formation and stabilization of an alpha-helical domain with high fusion propensity.

Role of membranotropic sequences from herpes simplex virus type I glycoproteins B and H in the fusion process.

The entry of enveloped viruses involves attachment followed by close apposition of the viral and plasma membranes. Then, either on the cell surface or in an endocytotic vesicle, the two membranes fuse by an energetically unfavourable process requiring the destabilisation of membrane microenvironment in order to release the viral nucleocapsid into the cytoplasm. The core fusion machinery, conserved throughout the herpesvirus family, involves glycoprotein B (gB) and the non-covalently associated complex of glycoproteins H and L (gH/gL). Both gB and gH possess several hydrophobic domains necessary for efficient induction of fusion, and synthetic peptides corresponding to these regions are able to associate to membranes and induce fusion of artificial liposomes. Here, we describe the first application of surface plasmon resonance (SPR) to the study of the interaction of viral membranotropic peptides with model membranes in order to enhance our molecular understanding of the mechanism of membrane fusion. SPR spectroscopy data are supported by tryptophan fluorescence, circular dichroism and electron spin resonance spectroscopy (ESR). We selected peptides from gB and gH and also analysed the behaviour of HIV gp41 fusion peptide and the cationic antimicrobial peptide melittin. The combined results of SPR and ESR showed a marked difference between the mode of action of the HSV peptides and the HIV fusion peptide compared to melittin, suggesting that viral-derived membrane interacting peptides all act via a similar mechanism, which is substantially different from that of the non-cell selective lytic peptide melittin.

P2 porin and loop L7 from Haemophilus influenzae modulate expression of IL-6 and adhesion molecules in astrocytes.

During neuropathological conditions such as infections and degenerative diseases, astrocytes can be activated by infiltrating immune cells. Activated astrocytes can produce chemokines, cytokines and adhesion molecules. In this study, the production of IL-6 and adhesion molecules intercellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1) and E-selectin by human astroglioma cells stimulated with Gram-negative surface components was investigated. Haemophilus influenzae type b porin P2 and its selected active peptide, loop L7, were found to induce MEK1-MEK2/ mitogen-activated protein kinase phosphorylation in U87-MG cells as demonstrated by ELISA, and up-regulate cellular adhesion molecule and interleukin-6 (IL-6) production as shown by RT-PCR and ELISA. Using two potent and selective inhibitors of MEK activation by Raf-1 (PD-098059) and p38 (SB-203580), it was also demonstrated that both ERK1/2 and p38 pathways play key roles in the production of IL-6 as well as in ICAM-1, VCAM-1 and E-selectin expression by Hib porin.

Silver nanoparticles as potential antiviral agents.

Virus infections pose significant global health challenges, especially in view of the fact that the emergence of resistant viral strains and the adverse side effects associated with prolonged use continue to slow down the application of effective antiviral therapies. This makes imperative the need for the development of safe and potent alternatives to conventional antiviral drugs. In the present scenario, nanoscale materials have emerged as novel antiviral agents for the possibilities offered by their unique chemical and physical properties. Silver nanoparticles have mainly been studied for their antimicrobial potential against bacteria, but have also proven to be active against several types of viruses including human imunodeficiency virus, hepatitis B virus, herpes simplex virus, respiratory syncytial virus, and monkey pox virus. The use of metal nanoparticles provides an interesting opportunity for novel antiviral therapies. Since metals may attack a broad range of targets in the virus there is a lower possibility to develop resistance as compared to conventional antivirals. The present review focuses on the development of methods for the production of silver nanoparticles and on their use as antiviral therapeutics against pathogenic viruses.

The Unusual Lipid A Structure and Immunoinhibitory Activity of LPS from Marine Bacteria Echinicola pacifica KMM 6172T and Echinicola vietnamensis KMM 6221T.

Gram-negative bacteria experiencing marine habitats are constantly exposed to stressful conditions dictating their survival and proliferation. In response to these selective pressures, marine microorganisms adapt their membrane system to ensure protection and dynamicity in order to face the highly mutable sea environments. As an integral part of the Gram-negative outer membrane, structural modifications are commonly observed in the lipopolysaccharide (LPS) molecule; these mainly involve its glycolipid portion, i.e., the lipid A, mostly with regard to fatty acid content, to counterbalance the alterations caused by chemical and physical agents. As a consequence, unusual structural chemical features are frequently encountered in the lipid A of marine bacteria. By a combination of data attained from chemical, MALDI-TOF mass spectrometry (MS), and MS/MS analyses, here, we describe the structural characterization of the lipid A isolated from two marine bacteria of the Echinicola genus, i.e., E. pacifica KMM 6172T and E. vietnamensis KMM 6221T. This study showed for both strains a complex blend of mono-phosphorylated tri- and tetra-acylated lipid A species carrying an additional sugar moiety, a d-galacturonic acid, on the glucosamine backbone. The unusual chemical structures are reflected in a molecule that only scantly activates the immune response upon its binding to the LPS innate immunity receptor, the TLR4-MD-2 complex. Strikingly, both LPS potently inhibited the toxic effects of proinflammatory Salmonella LPS on human TLR4/MD-2.

Microbiological Evaluation and Sperm DNA Fragmentation in Semen Samples of Patients Undergoing Fertility Investigation.

Fifteen percent of male infertility is associated with urogenital infections; several pathogens are able to alter the testicular and accessory glands' microenvironment, resulting in the impairment of biofunctional sperm parameters. The purpose of this study was to assess the influence of urogenital infections on the quality of 53 human semen samples through standard analysis, microbiological evaluation, and molecular characterization of sperm DNA damage. The results showed a significant correlation between infected status and semen volume, sperm concentration, and motility. Moreover, a high risk of fragmented sperm DNA was demonstrated in the altered semen samples. Urogenital infections are often asymptomatic and thus an in-depth evaluation of the seminal sample can allow for both the diagnosis and therapy of infections while providing more indicators for male infertility management.

Proteomic analysis of human U937 cell line activation mediated by Haemophilus influenzae type b P2 porin and its surface-exposed loop 7.

The virulence of Haemophilus influenzae type b (Hib) has been attributed to a variety of potential factors associated with its cell surface, including lipopolysaccharides (LPS) and major outer membrane proteins (OMPs). P2 porin, one of the best-characterized porins in terms of its functional characteristics, is the most abundant OMP in Hib and has also been shown to possess proinflammatory activity. To characterize the role played by bacterial surface components in disease onset and development, the proteomic profiling of human U937 cell line activated by H. influenzae type b P2 porin and its most active surface-exposed loop (L7) was performed by means of two-dimensional electrophoresis and mass spectrometry. The study provided a list of candidate proteins with potential relevance in the host immune and inflammatory response. Most of the differentially expressed proteins are involved in metabolic processes, remodelling of cytoskeleton, stress response and signal transduction pathways. The results constitute the basis for dissecting signal transduction cascades activated by P2 stimulation and gain insights into the molecular events involved in the modulation of pathogen-host cell interactions.

Novel synthetic, salt-resistant analogs of human beta-defensins 1 and 3 endowed with enhanced antimicrobial activity.

Human beta-defensins (hBDs) are antimicrobial peptides of human innate immunity. The antibacterial activities of hBDs 1, 2, and 4 but not the activity of hBD3 are impaired by high salt levels. We have designed and synthesized seven novel hBD analogs, constituted by different domains of hBD1 (which is constitutively expressed in humans) and of hBD3 (which is induced by microorganisms and inflammatory factors in humans), that would maintain and potentially increase the wild-type antimicrobial activities and be salt resistant. We have compared the antibacterial, antiviral, and chemotactic activities of the analogs with those of hBD1 and hBD3. We show that the hBD1 internal region and the hBD3 C-terminal region are critical for antibacterial activity also at high salt concentrations, whereas deletion of the N-terminal region of hBD3 results in an increase in antibacterial activity. All analogs inhibited herpes simplex virus; antiviral activity was enhanced by the hBD1 internal region and the hBD3 C-terminal region. Wild-type and analog peptides were chemotactic for granulocytes and monocytes, irrespective of the salt concentrations. These new peptides may have therapeutic potential.