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The appearance of new respiratory virus infections in humans with epidemic or pandemic potential has underscored the urgent need for effective broad-spectrum antivirals (BSAs). Bioactive compounds derived from plants may provide a natural source of new BSA candidates. Here, we investigated the novel phytocomplex formulation SP4™ as a candidate direct-acting BSA against major current human respiratory viruses, including coronaviruses and influenza viruses. SP4™ inhibited the in vitro replication of SARS-CoV-2, hCoV-OC43, hCoV-229E, Influenza A and B viruses, and respiratory syncytial virus in the low-microgram range. Using hCoV-OC43 as a representative respiratory virus, most of the antiviral activity of SP4™ was observed to stem primarily from its dimeric A-type proanthocyanidin (PAC-A) component. Further investigations of the mechanistic mode of action showed SP4™ and its PAC-A-rich fraction to prevent hCoV-OC43 from attaching to target cells and exert virucidal activity. This occurred through their interaction with the spike protein of hCoV-OC43 and SARS-CoV-2, thereby interfering with spike functions and leading to the loss of virion infectivity. Overall, these findings support the further development of SP4™ as a candidate BSA of a natural origin for the prevention of human respiratory virus infections. [ABSTRACT FROM AUTHOR]

Aim: Evaluate the antibacterial efficacy and depth of action into dentinal tubules of different antibiotic mixtures used for dental pulp regeneration (1-9). Methodology: Cylindrical specimens of radicular dentine (n = 72), sterilized with ethylene oxide, have been infected with Enterococcus Faecalis (3 × 107 CFU/mL) for 3 weeks (10). Specimens were randomly assigned to 3 experimental groups (n = 20) plus positive (n = 6) and negative (n = 6) controls. • TRIMIX group (ciprofloxacin, metronidazole and minocycline) • BIMIX group (ciprofloxacin and metronidazole) • TRICLARITRO group (ciprofloxacin, metronidazole and clarithromycin) Each experimental group was divided in two subgroups exposed to antibiotic paste formulations added to macrogol (MG) or ialuronic acid (HA). After 3 weeks o exposition to antibiotic formulations, all specimens were vertically fractured and analyzed with confocal laser scanning microscopy (CLSM) and viability staining (Live/Dead BacLight Viability Stain) to quantitatively evaluate the ratio of dead/live bacteria into dentine tubules (11-12). Volume ratio of red fluorescence (Dead bacteria) was calculated in three-dimensional reconstructions. Differences among groups were analyzed with Kruskall-Wallis and post-hoc Dunn's test (P 0,05). All antibiotic mixtures conveyed by HA showed a better efficacy compared with MG (P 0,05). Conclusions: TRICLARITRO antibiotic mixture showed an effective antibacterial action deep into dentinal tubules, without the risk of tooth crown staining (10).

Streptococcus pyogenes causes a wide spectrum of diseases varying from mild to life threatening, despite antibiotic treatment. Nanoparticle application could facilitate the foreign pathogen fight by increasing the antimicrobial effectiveness and reducing their adverse effects. Here, we designed and produced erythromycin-loaded chitosan nanodroplets (Ery-NDs), both oxygen-free and oxygen-loaded. All ND formulations were characterized for physico-chemical parameters, drug release kinetics, and tested for biocompatibility with human keratinocytes and for their antibacterial properties or interactions with S. pyogenes. All tested NDs possessed spherical shape, small average diameter, and positive Z potential. A prolonged Ery release kinetic from Ery-NDs was demonstrated, as well as a favorable biocompatibility on human keratinocytes. Confocal microscopy images showed ND uptake and internalization by S. pyogenes starting from 3 h of incubation up to 24 h. According to cell counts, NDs displayed long-term antimicrobial efficacy against streptococci significantly counteracting their proliferation up to 24 h, thanks to the known chitosan antimicrobial properties. Intriguingly, Ery-NDs were generally more effective (104–103 log10 CFU/mL), than free-erythromycin (105 log10 CFU/mL), in the direct killing of streptococci, probably due to Ery-NDs adsorption by bacteria and prolonged release kinetics of erythromycin inside S. pyogenes cells. Based on these findings, NDs and proper Ery-NDs appear to be the most promising and skin-friendly approaches for the topical treatment of streptococcal skin infections allowing wound healing during hypoxia. [ABSTRACT FROM AUTHOR]

Introduction: In the vineyard, yeast communities impact the ripening and fermentation of grapes and are influenced by geographical location, climate, and soil characteristics. Despite the great advancement in our knowledge of the vineyard mycobiota, a key step of the process leading to the definition of the vineyard yeast community is still poorly understood: if geography, climate, and soil influence the mycobiota, potentially through selection, where do the yeast originate from, and how can they reach the vineyard? In this perspective, it is currently acknowledged that forests host several yeast species and that insects, particularly social wasps, can vector and maintain the yeasts known to populate the vineyard. Alas, the conveyance, fostered by insects, of yeasts from the forest to the vineyard has not been proven yet. In this study, we aimed to assess the existence of links between a potential natural source of yeasts (woods), the vectors (social wasps), and the composition of the vineyard mycobiota. Methods: For this purpose, the mycobiota of wasps caught in six Italian vineyards were analyzed over 2 years through culturomics approaches. Results: The results clearly indicate that the presence of wooded areas close to vineyards is associated with particular features of the mycobiota vectored by social wasps. Wasps caught in vineyards near wooded areas bear a higher number of yeast cells and higher biodiversity than insects caught in vineyards far from woods. Furthermore, insects caught in vineyards close to woods bear distinctive yeast populations, encompassing species such as Saccharomyces cerevisiae. Discussion: Overall, our work provides fundamental insights into the ecology of the vineyard mycobiota and highlights the need to maintain a vineyard-woodland mosaic landscape, thus preserving the suitable habitat for yeast species relevant to wine-making. [ABSTRACT FROM AUTHOR]

The pharmacological management of influenza virus (IV) infections still poses a series of challenges due to the limited anti-IV drug arsenal. Therefore, the development of new anti-influenza agents effective against antigenically different IVs is therefore an urgent priority. To meet this need, host-targeting antivirals (HTAs) can be evaluated as an alternative or complementary approach to current direct-acting agents (DAAs) for the therapy of IV infections. As a contribution to this antiviral strategy, in this study, we characterized the anti-IV activity of MEDS433, a novel small molecule inhibitor of the human dihydroorotate dehydrogenase (hDHODH), a key cellular enzyme of the de novo pyrimidine biosynthesis pathway. MEDS433 exhibited a potent antiviral activity against IAV and IBV replication, which was reversed by the addition of exogenous uridine and cytidine or the hDHODH product orotate, thus indicating that MEDS433 targets notably hDHODH activity in IV-infected cells. When MEDS433 was used in combination either with dipyridamole (DPY), an inhibitor of the pyrimidine salvage pathway, or with an anti-IV DAA, such as N4-hydroxycytidine (NHC), synergistic anti-IV activities were observed. As a whole, these results indicate MEDS433 as a potential HTA candidate to develop novel anti-IV intervention approaches, either as a single agent or in combination regimens with DAAs. [ABSTRACT FROM AUTHOR]

Background: A few studies suggest that particulate matter (PM) exposure might play a role in bronchiolitis. However, available data are mostly focused on the risk of hospitalization and come from retrospective studies that provided conflicting results. This prospective study investigated the association between PM (PM2.5 and PM10) exposure and the severity of bronchiolitis. Methods: This prospective cohort study was conducted between November 2019 and February 2020 at the pediatric emergency department of the Fondazione IRCCS Ca′ Ospedale Maggiore Policlinico, Milan, Italy. Infants <1 year of age with bronchiolitis were eligible. The bronchiolitis severity score was assessed in each infant and a nasal swab was collected to detect respiratory viruses. The daily PM10 and PM2.5 exposure in the 29 preceding days were considered. Adjusted regression models were employed to evaluate the association between the severity score and PM10 and PM2.5 exposure. Results: A positive association between the PM2.5 levels and the severity score was found at day‐2 (β 0.0214, 95% CI 0.0011–0.0417, p =.0386), day‐5 (β 0.0313, 95% CI 0.0054–0.0572, p =.0179), day‐14 (β 0.0284, 95% CI 0.0078–0.0490, p =.0069), day‐15 (β 0.0496, 95% CI 0.0242–0.0750, p =.0001) and day‐16 (β 0.0327, 95% CI 0.0080–0.0574, p =.0093).Similar figures were observed considering the PM10 exposure and limiting the analyses to infants with respiratory syncytial virus. Conclusion: This study shows for the first time a direct association between PM2.5 and PM10 levels and the severity of bronchiolitis. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19), ranging from asymptomatic conditions to severe/fatal lung injury and multi-organ failure. Growing evidence shows that the nasopharyngeal microbiota composition may predict the severity of respiratory infections and may play a role in the protection from viral entry and the regulation of the immune response to the infection. In the present study, we have characterized the nasopharyngeal bacterial microbiota (BNM) composition and have performed factor analysis in a group of 54 asymptomatic/paucisymptomatic subjects who tested positive for nasopharyngeal swab SARS-CoV-2 RNA and/or showed anti-RBD-IgG positive serology at the enrolment. We investigated whether BNM was associated with SARSCoV-2 RNA positivity and serum anti-RBD-IgG antibody development/maintenance 20-28 weeks after the enrolment. Shannon's entropy a-diversity index [odds ratio (OR) = 5.75, p = 0.0107] and the BNM Factor1 (OR = 2.64, p = 0.0370) were positively associated with serum anti-RBD-IgG antibody maintenance. The present results suggest that BNM composition may influence the immunological memory against SARS-CoV-2 infections. To the best of our knowledge, this is the first study investigating the link between BNM and specific IgG antibody maintenance. Further studies are needed to unveil the mechanisms through which the BNM influences the adaptive immune response against viral infections. [ABSTRACT FROM AUTHOR]

The aim was to evaluate the antibacterial efficacy and penetration depth into dentinal tubules of a solution of chitosan nanodroplets (NDs) loaded with Benzalkonium Chloride (BAK). Seventy-two human single-root teeth with fully formed apex were used. Cylindrical root dentin blocks were longitudinally sectioned and enlarged to a size of a Gates Glidden drill #4. After sterilization, root canals were infected with Enterococcus faecalis ATCC 29212 and further incubated for three weeks. Specimens were assigned to three experimental groups (n = 20), plus positive (n = 6) and negative (n = 6) controls. In the first group, irrigation was achieved with 2 mL of NDs solution loaded with BAK (NDs-BAK), in the second with 2 mL of 5% sodium hypochlorite (NaOCl) and in the last with 2 mL of 2% chlorhexidine (CHX). Specimens were rinsed and vertically fractured. Confocal laser scanning microscopy (CLSM) and viability staining were used to analyze the proportions of dead and live bacteria quantitatively. The volume ratio of red fluorescence (dead) was calculated in 3D reconstructions. Data were analyzed by one-way ANOVA and post hoc Bonferroni tests (p < 0.05). The ratio of red fluorescence over the whole green/red fluorescence resulted in a significant comparison of NDs-BAK with NaOCl (p < 0.01) and NaOCl with CHX (p < 0.01). No differences were found between NDs-BAK and CHX (p > 0.05). The mean depth of efficacy was, respectively: NDs-BAK 325.25 μm, NaOCl 273.36 μm and CHX 246.78 μm with no statistical differences between groups. The NaOCl solution showed the highest antimicrobial efficacy, but nanodroplets with BAK seemed to have the same effect as CHX with a high depth of efficacy. [ABSTRACT FROM AUTHOR]

There is limited knowledge on the origin and development from CD34+ precursors of the ample spectrum of human natural killer (NK) cells, particularly of specialized NK subsets. This study sought to characterize the NK-cell progeny of CD34+DNAM-1brightCXCR4+ and of other precursors circulating in the peripheral blood of patients with chronic viral infections (eg, HIV, hepatitis C virus, cytomegalovirus reactivation). Highly purified precursors were obtained by flow cytometric sorting and cultured in standard NK-cell differentiation media (ie, SCF, FLT3, IL-7, IL-15). Phenotypic and functional analyses on progenies were performed by multiparametric cytofluorimetric assays. Transcriptional signatures of NK-cell progenies were studied by microarray analysis. Inhibition of cytomegalovirus replication was studied by PCR. Unlike conventional CD34+ precursors, Lin−CD34+DNAM-1brightCXCR4+ precursors from patients with chronic infection, rapidly differentiate into cytotoxic, IFN-γ–secreting CD94/NKG2C+KIR+CD57+ NK-cell progenies. An additional novel subset of common lymphocyte precursors was identified among Lin−CD34−CD56−CD16+ cells and characterized by expression of CXCR4 and lack of perforin and CD94. Lin−CD34−CD56−CD16+Perf−CD94−CXCR4+ precursors are also endowed with generation potential toward memory-like NKG2C+NK cells. Maturing NK-cell progenies mediated strong human cytomegalovirus–inhibiting activity. Microarray analysis confirmed a transcriptional signature compatible with NK-cell progenies and with maturing adaptive NK cells. During viral infections, precursors of adaptive NK cells are released and circulate in the peripheral blood. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract Background Human monoclonal antibodies (mAbs) generated as a result of the immune response are likely to be the most effective therapeutic antibodies, particularly in the case of infectious diseases against which the immune response is protective. Human cytomegalovirus (HCMV) is an ubiquitous opportunistic virus that is the most serious pathogenic agent in transplant patients. The available therapeutic armamentarium (e.g. HCMV hyperimmune globulins or antivirals) is associated with severe side effects and the emergence of drug-resistant strains; therefore, neutralizing human mAb may be a decisive alternative in the prevention of primary and re-activated HCMV infections in these patients. Results The purpose of this study was to generate neutralizing mAb against HCMV from the immunological repertoire of immune donors. To this aim, we designed an efficient technology relying on two discrete and sequential steps: first, human B-lymphocytes are stimulated with TLR9-agonists and IL-2; second, after both additives are removed, the cells are infected with EBV. Using this strategy we obtained 29 clones secreting IgG neutralizing the HCMV infectivity; four among these were further characterized. All of the mAbs neutralize the infection in different combinations of HCMV strains and target cells, with a potency ~20 fold higher than that of the HCMV hyperimmune globulins, currently used in transplant recipients. Recombinant human monoclonal IgG1 suitable as a prophylactic or therapeutic tool in clinical applications has been generated. Conclusion The technology described has proven to be more reproducible, efficient and rapid than previously reported techniques, and can be adopted at low overall costs by any cell biology laboratory for the development of fully human mAbs for immunotherapeutic uses.

All humans are colonized by a vast diversity of microbes (bacteria, archaea, protozoa, yeast, and fungi; collectively referred to as the microbiota) and viruses (the virobiota). This latter group includes viruses infecting prokaryotic cells (bacteriophages), viruses infecting eukaryotic-host cells, and virus-derived genetic elements present in host chromosomes. Although these eukaryotic viruses are mostly known to be pathogens, they are also able to establish mutualistic relationships with humans. Little is known about the mutualistic aspects of viral infection. Nevertheless, it is clear that evolution of some animal virus-host interactions has led to benefits in the health of the hosts, as is the case with symbiogenesis and endogenization of retroviruses that has exerted a neuroprotective effect on the human brain, and an important role in the fetal development, thus on the evolution of host species. In this review, we summarize how retroviruses provide amazing examples of cooperative-evolution, i.e., successful exchange between viruses and host, and how, in some cases, the benefits have become essential for the hosts' survival. [ABSTRACT FROM AUTHOR]

Featured Application: Thanks to the use of confocal laser scanning microscopy, it is proved that ciprofloxacin, metronidazole and clarithromycin (3-MIXC) mixed with hyaluronic acid (HA) is a possible alternative to root canal disinfection during regenerative endodontic procedures (REPs) in order to avoid a substantial discoloration of the teeth, mainly due to the chelating effect of the minocycline. Confocal laser scanning microscopy (CLSM) was used to evaluate the antibacterial effect and depth of action of a novel clarithromycin-containing triple antibiotic mixture, which was proposed for root canal disinfection in dental pulp regeneration. A previous study reported that this mixture had no tooth discoloration effects in vitro. After infection with Enterococcus faecalis for 3 weeks, the dentinal tubules in the cylindrical root specimens were exposed to different antibiotic mixtures: ciprofloxacin, metronidazole and minocycline (3-MIX); ciprofloxacin, metronidazole and clarithromycin (3-MIXC) and ciprofloxacin and metronidazole (2-MIX). Each antibiotic formulation was mixed with macrogol (MG) or hyaluronic acid (HA) vehicles. CLSM and viability staining were used to quantitatively analyze the mean depth of the antibacterial effect and the proportions of dead and live bacteria inside the dentinal tubules. The 3-MIX and 3-MIXC demonstrated a similar depth of action. The mean proportion of dead bacteria was similar in the 3-MIX and 3-MIXC groups, and both were statistically higher than that of 2-MIX (p = 0.014). Each antibiotic mixture showed a higher bactericidal efficacy if conveyed with HA, compared to MG (3-MIX, p = 0.019; 3-MIXC, p = 0.013 and 2-MIX, p = 0.0125). The depth of action and the antibacterial efficacy of 3-MIXC seemed comparable with 3-MIX. [ABSTRACT FROM AUTHOR]

Microbial ecosystems, biofilms, resilience, evolution, cross-feeding, cooperation, molecular signaling Generally, cross-feeding, ensuring removal of toxic metabolic products, allows enhanced growth rates, however, these biofilm-living communities can affect also the interaction with the human host (Joshi et al.). Keywords: microbial ecosystems; biofilms; resilience; evolution; cooperation; cross-feeding; molecular signaling EN microbial ecosystems biofilms resilience evolution cooperation cross-feeding molecular signaling 1 3 3 11/26/21 20211124 NES 211124 The idea at the basis of the present Research Topic was to explore how evolution selected microbial behaviors to ensure the generation of more and more complex and sustainable lifestyles that help microbial systems in coping with scarcity and rapidly changing environments. [Extracted from the article]

Background: It has been speculated that the SARS-CoV-2 was already widespread in western countries before February 2020.Methods: We gauged this hypothesis by analysing the nasal swab of infants with either bronchiolitis or a non-infectious disease admitted to the Ospedale Maggiore, Milan (one of the first epicentres of SARS-CoV-2 outbreak in Europe) from November 2019.Results: The SARS-CoV-2 RNA was never detected in 218 infants with bronchiolitis (95 females, median age 4.9 months) and 49 infants (22 females, median age 5.6 months) with a non-infectious disease between November 2019 and February 2020. On the contrary, two infants hospitalised for bronchiolitis between March and April 2020 tested positive for SARS-CoV-2.Conclusions: This study does not support the hypothesis that SARS-CoV-2 was already circulating among infants before the official outbreak of SARS-CoV-2 infection. However, it shows for the first time that SARS-CoV-2 might cause bronchiolitis requiring hospitalisation. [ABSTRACT FROM AUTHOR]

Abstract The identification and validation of new small molecules able to inhibit the replication of human cytomegalovirus (HCMV) remains a priority to develop alternatives to the currently used DNA polymerase inhibitors, which are often burdened by long-term toxicity and emergence of cross-resistance. To contribute to this advancement, here we report on the characterization of the mechanism of action of a bioactive plant-derived alkaloid, berberine (BBR), selected in a previous drug repurposing screen expressly devised to identify early inhibitors of HCMV replication. Low micromolar concentrations of BBR were confirmed to suppress the replication of different HCMV strains, including clinical isolates and strains resistant to approved DNA polymerase inhibitors. Analysis of the HCMV replication cycle in infected cells treated with BBR then revealed that the bioactive compound compromised the progression of virus cycle at a stage prior to viral DNA replication and Early (E) genes expression, but after Immediate-Early (IE) proteins expression. Mechanistic studies in fact highlighted that BBR interferes with the transactivating functions of the viral IE2 protein, thus impairing efficient E gene expression and the progression of HCMV replication cycle. Finally, the mechanism of the antiviral activity of BBR appears to be conserved among different CMVs, since BBR suppressed murine CMV (MCMV) replication and inhibited the transactivation of the prototypic MCMV E1 gene by the IE3 protein, the murine homolog of IE2. Together, these observations warrant for further experimentation to obtain proof of concept that BBR could represent an attractive candidate for alternative anti-HCMV therapeutic strategies. Highlights • We characterize the spectrum of anti-HCMV activity of the bioactive natural compound berberine. • Berberine impairs in vitro replication of clinical and drug-resistant HCMV strains in the low micromolar range. • Berberine prevents viral DNA synthesis and E and L viral genes expression. • The anti-HCMV activity of berberine stems from an interference with the transactivating activity of viral IE2 protein. • Berberine differs from current anti-HCMV drugs, and may represents a promising candidate for new antiviral strategies. [ABSTRACT FROM AUTHOR]

The defense against influenza virus (IV) infections still poses a series of challenges. The current antiviral arsenal against influenza viruses is in fact limited; therefore, the development of new anti-influenza strategies effective against antigenically different viruses is an urgent priority. Bioactive compounds derived from medicinal plants and fruits may provide a natural source of candidates for such broad-spectrum antivirals. In this regard, cranberry (Vaccinium macrocarpon Aiton) extracts on the basis of their recognized anti-adhesive activities against bacteria, may provide potential compounds able to prevent viral attachment to target cells. Nevertheless, only few studies have so far investigated the possible use of cranberry extracts as an antiviral tool. This study focuses on the suitability of a cranberry extract as a direct-acting anti-influenza compound. We show that the novel cranberry extract Oximacro® inhibits influenza A and B viruses (IAV, IBV) replication in vitro because of its high content of A-type proanthocyanidins (PAC-A) dimers and trimers. Mechanistic studies revealed that Oximacro® prevents attachment and entry of IAV and IBV into target cells and exerts a virucidal activity. Oximacro® was observed to interact with the ectodomain of viral hemagglutinin (HA) glycoprotein, thus suggesting the interference with HA functions and a consequent loss of infectivity of IV particles. Fluorescence spectroscopy revealed a reduction in the intrinsic fluorescence of HA protein after incubation with purified dimeric PAC-A (PAC-A2), thus confirming a direct interaction between HA and Oximacro® PAC-A2. In silico docking simulations further supported the in vitro results and indicated that among the different components of the Oximacro® chemical profile, PAC-A2 exhibited the best binding propensity with an affinity below 10 nM. The role of PAC-A2 in the anti-IV activity of Oximacro® was eventually confirmed by the observation that it prevented IAV and IVB replication and caused the loss of infectivity of IV particles, thus indicating PAC-A2 as the major active component of Oximacro®. As a whole, these results suggest Oximacro® as a potential candidate to create novel antiviral agents of natural origin for the prevention of IV infections. [ABSTRACT FROM AUTHOR]

Following primary infection in humans, the human cytomegalovirus (HCMV) persists in a latent state throughout the host's lifetime despite a strong and efficient immune response. If the host experiences some form of immune dysregulation, such as immunosuppression or immunodeficiency, HCMV reactivates, thereby emerging from latency. Thus, in the absence of effective functional immune responses, as occurs in immunocompromised or immunoimmature individuals, both HCMV primary infections and reactivations from latency can cause significant morbidity and mortality. However, even in immunocompetent hosts, HCMV represents a relevant risk factor for the development of several chronic inflammatory diseases and certain forms of neoplasia. HCMV infection may shift between the lytic and latent state, regulated by a delicate and intricate balance between virus-mediated immunomodulation and host immune defenses. Indeed, HCMV is a master in manipulating innate and adaptive host defense pathways, and a large portion of its genome is devoted to encoding immunomodulatory proteins; such proteins may thus represent important virulence determinants. However, the pathogenesis of HCMV-related diseases is strengthened by the activities of bioactive molecules, of both viral and cellular origin, that are secreted from infected cells and collectively named as the secretome. Here, we review the state of knowledge on the composition and functions of HCMV-derived secretomes. In lytic infections of fibroblasts and different types of endothelial cells, the majority of HCMV-induced secreted proteins act in a paracrine fashion to stimulate the generation of an inflammatory microenvironment around infected cells; this may lead to vascular inflammation and angiogenesis that, in turn, foster HCMV replication and its dissemination through host tissues. Conversely, the HCMV secretome derived from latently infected hematopoietic progenitor cells induces an immunosuppressive extracellular environment that interferes with immune recognition and elimination of latently infected cells, thereby promoting viral persistence. Characterization of the composition and biological activities of HCMV secretomes from different types of infected cells will lay the foundation for future advances in our knowledge about the pathogenesis HCMV diseases and may provide targets for the development of novel antiviral intervention strategies. [ABSTRACT FROM AUTHOR]

The human cytomegalovirus (HCMV) US12 gene family includes a group of 10 contiguous genes (US12 to US21) encoding predicted seven-transmembrane-domain (7TMD) proteins that are nonessential for replication within cultured fibroblasts. Nevertheless, inactivation of some US12 family members affects virus replication in other cell types; e.g., deletion of US16 or US18 abrogates virus growth in endothelial and epithelial cells or in human gingival tissue, respectively, suggesting a role for some US12 proteins in HCMV cell tropism. Here, we provide evidence that another member, US20, impacts the ability of a clinical strain of HCMV to replicate in endothelial cells. Through the use of recombinant HCMV encoding tagged versions of the US20 protein, we investigated the expression pattern, localization, and topology of the US20-encoded protein (pUS20). We show that pUS20 is expressed as a partially glycosylated 7TMD protein which accumulates late in infection in endoplasmic reticulum-derived peripheral structures localized outside the cytoplasmic virus assembly compartment (cVAC). US20-deficient mutants generated in the TR clinical strain of HCMV exhibited major growth defects in different types of endothelial cells, whereas they replicated normally in fibroblasts and epithelial cells. While the attachment and entry phases in endothelial cells were not significantly affected by the absence of US20 protein, US20-null viruses failed to replicate viral DNA and express representative E and L mRNAs and proteins. Taken together, these results indicate that US20 sustains the HCMV replication cycle at a stage subsequent to entry but prior to E gene expression and viral DNA synthesis in endothelial cells. [ABSTRACT FROM AUTHOR]

Adaptor protein-3 (AP-3) is a heterotetrameric complex, which regulates vesicular trafficking. Mutations of the β3A subunit cause the Hermansky-Pudlak syndrome type 2 (HPS-2), a rare genetic disease characterized by albinism, platelet defects, and recurrent infections. Likewise, pearl mice, which lack functional AP-3, show several HPS-2 defects. The AP-3 absence results in defective toll-like receptor trafficking and signaling in dendritic cells (DC), but its effect on the efficiency of the in vivo antiviral response is unclear. We evaluated the impact of AP-3 deficiency on the distribution of DC subsets, interferon (IFN) production, and the susceptibility to murine cytomegalovirus (MCMV) infection. Pearl mice showed a distribution and frequency of conventional (cDC) and plasmacytoid DC (pDC) similar to that of wild-type mice both before and after MCMV infection. Moreover, pearl mice controlled MCMV infection even at high virus doses and showed a normal production of IFN-α. Since pDC, but not cDC, from pearl mice showed an impaired IFN-α and tumor necrosis factor-α production in response to prototypic DNA (MCMV and Herpes Simplex virus) or RNA ( Vesicular Stomatitis virus) viruses in vitro, it is likely that MCMV infection can be controlled in vivo independently of an efficient production of IFN-α by pDC, and that the AP-3 complex has a minimal impact on protective antiviral responses. [ABSTRACT FROM AUTHOR]

The article presents information on the investigation of US16 gene of human cytomegalovirus (HCMV) by the ascertaining expression pattern of its product. It is mentioned that US16-deficient mutants were generated in the TR clinical strain of HCMV. It is concluded that a major growth defect was exhibited by the US16-deficient viruses in both retinal pigment epithelial cells and microvascular endothelial cells.

Human interferon (IFN)-inducible IFI16 protein, an innate immune sensor of intracellular DNA, modulates various cell functions, however, its role in regulating virus growth remains unresolved. Here, we adopt two approaches to investigate whether IFI16 exerts pro- and/or anti-viral actions. First, the IFI16 gene was silenced using specific small interfering RNAs (siRNA) in human embryo lung fibroblasts (HELF) and replication of DNA and RNA viruses evaluated. IFI16-knockdown resulted in enhanced replication of Herpesviruses, in particular, Human Cytomegalovirus (HCMV). Consistent with this, HELF transduction with a dominant negative form of IFI16 lacking the PYRIN domain (PYD) enhanced the replication of HCMV. Second, HCMV replication was compared between HELFs overexpressing either the IFI16 gene or the LacZ gene. IFI16 overexpression decreased both virus yield and viral DNA copy number. Early and late, but not immediate-early, mRNAs and proteins were strongly down-regulated, thus IFI16 may exert its antiviral effect by impairing viral DNA synthesis. Constructs with the luciferase reporter gene driven by deleted or site-specific mutated forms of the HCMV DNA polymerase (UL54) promoter demonstrated that the inverted repeat element 1 (IR-1), located between 254 and 243 relative to the transcription start site, is the target of IFI16 suppression. Indeed, electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated that suppression of the UL54 promoter is mediated by IFI16-induced blocking of Sp1-like factors. Consistent with these results, deletion of the putative Sp1 responsive element from the HCMV UL44 promoter also relieved IFI16 suppression. Together, these data implicate IFI16 as a novel restriction factor against HCMV replication and provide new insight into the physiological functions of the IFN-inducible gene IFI16 as a viral restriction factor. [ABSTRACT FROM AUTHOR]

Activation of the IKK/NF-κB signalling pathway is a hallmark of human cytomegalovirus (HCMV) infection. However, its role in regulating major immediate-early promoter (MIEP)-dependent transcription and HCMV replication remains controversial. This study uses a combination of genetic approaches to investigate the effects of cell culture conditions on the importance of virus-induced NF-κB activation during the infection of endothelial cells or fibroblasts. Adenoviral-mediated expression of a dominant-negative mutant of IKK2 kinase (dnIKK2) in human umbilical vein endothelial cells resulted in a strong reduction of IκBα degradation and NF-κB activation following infection with an HCMV clinical isolate. Viral replication was impaired in dnIKK2-expressing cells that were growth-arrested before infection, but not in replicating cells. The inhibitory effect of dnIKK2 was independent from the virus strain and the cell type used, because replication of the laboratory AD169 strain was impaired as well in dnIKK2-expressing quiescent fibroblasts. Moreover, progressive disruption of NF-κB response elements within the MIEP in recombinant HCMV viruses derived from the clinical isolate prevented their replication in quiescent cells but not in actively growing cells. These results demonstrate an essential role of virus-induced IKK/NF-κB activity to trigger both viral IE gene expression and productive replication in quiescent cells. [ABSTRACT FROM AUTHOR]

Background: Milk is considered an important source of bioactive peptides, which can be produced by endogenous or starter bacteria, such as lactic acid bacteria, that are considered effective and safe producers of food-grade bioactive peptides. Among the various types of milk, donkey milk has been gaining more and more attention for its nutraceutical properties. Methods: Lactobacillus rhamnosus 17D10 and Lactococcus lactis subsp. cremoris 40FEL3 were selected for their ability to produce peptides from donkey milk. The endogenous peptides and those obtained after bacterial fermentation were assayed for their antioxidant, antibacterial, and antiviral activities. The peptide mixtures were characterized by means of LC-MS/MS and then analyzed in silico using the Milk Bioactive Peptide DataBase. Results: The peptides produced by the two selected bacteria enhanced the antioxidant activity and reduced E. coli growth. Only the peptides produced by L. rhamnosus 17D10 were able to reduce S. aureus growth. All the peptide mixtures were able to inhibit the replication of HSV-1 by more than 50%. Seventeen peptides were found to have 60% sequence similarity with already known bioactive peptides. Conclusions: A lactic acid bacterium fermentation process is able to enhance the value of donkey milk through bioactivities that are important for human health. [ABSTRACT FROM AUTHOR]

Although coronaviruses (CoVs) have long been predicted to cause zoonotic diseases and pandemics with high probability, the lack of effective anti-pan-CoVs drugs rapidly usable against the emerging SARS-CoV-2 actually prevented a promptly therapeutic intervention for COVID-19. Development of host-targeting antivirals could be an alternative strategy for the control of emerging CoVs infections, as they could be quickly repositioned from one pandemic event to another. To contribute to these pandemic preparedness efforts, here we report on the broad-spectrum CoVs antiviral activity of MEDS433, a new inhibitor of the human dihydroorotate dehydrogenase (hDHODH), a key cellular enzyme of the de novo pyrimidine biosynthesis pathway. MEDS433 inhibited the in vitro replication of hCoV-OC43 and hCoV-229E, as well as of SARS-CoV-2, at low nanomolar range. Notably, the anti-SARS-CoV-2 activity of MEDS433 against SARS-CoV-2 was also observed in kidney organoids generated from human embryonic stem cells. Then, the antiviral activity of MEDS433 was reversed by the addition of exogenous uridine or the product of hDHODH, the orotate, thus confirming hDHODH as the specific target of MEDS433 in hCoVs-infected cells. Taken together, these findings suggest MEDS433 as a potential candidate to develop novel drugs for COVID-19, as well as broad-spectrum antiviral agents exploitable for future CoVs threats. [ABSTRACT FROM AUTHOR]

Emergence of drug resistance and adverse effects often affect the efficacy of nucleoside analogues in the therapy of Herpes simplex type 1 (HSV-1) and type 2 (HSV-2) infections. Host-targeting antivirals could therefore be considered as an alternative or complementary strategy in the management of HSV infections. To contribute to this advancement, here we report on the ability of a new generation inhibitor of a key cellular enzyme of de novo pyrimidine biosynthesis, the dihydroorotate dehydrogenase (DHODH), to inhibit HSV-1 and HSV-2 in vitro replication, with a potency comparable to that of the reference drug acyclovir. Analysis of the HSV replication cycle in MEDS433-treated cells revealed that it prevented the accumulation of viral genomes and reduced late gene expression, thus suggesting an impairment at a stage prior to viral DNA replication consistent with the ability of MEDS433 to inhibit DHODH activity. In fact, the anti-HSV activity of MEDS433 was abrogated by the addition of exogenous uridine or of the product of DHODH, the orotate, thus confirming DHODH as the MEDS433 specific target in HSV-infected cells. A combination of MEDS433 with dipyridamole (DPY), an inhibitor of the pyrimidine salvage pathway, was then observed to be effective in inhibiting HSV replication even in the presence of exogenous uridine, thus mimicking in vivo conditions. Finally, when combined with acyclovir and DPY in checkerboard experiments, MEDS433 exhibited highly synergistic antiviral activity. Taken together, these findings suggest that MEDS433 is a promising candidate as either single agent or in combination regimens with existing direct-acting anti-HSV drugs to develop new strategies for treatment of HSV infections. • We characterize the anti-HSV activity of the new DHODH inhibitor MEDS433 that targets the de novo pyrimidine biosynthesis. • MEDS433 impairs the in vitro replication of HSV-1 and HSV-2 in the nanomolar range. • The anti-HSV activity of MEDS433 is reversed by uracil and orotic acid confirming that it derives from inhibition of DHODH. • The simultaneous treatment with MEDS433 and acyclovir potentiates the anti-HSV activity of the combination. • MEDS433 differs from current anti-HSV drugs, and may represents a promising candidate for new antiviral strategies. [ABSTRACT FROM AUTHOR]

Persistent hypoxia is a main clinical feature of chronic wounds. Intriguingly, oxygen-loaded nanodroplets (OLNDs), filled with oxygen-solving 2H,3H-decafluoropentane and shelled with polysaccharides, have been proposed as a promising tool to counteract hypoxia by releasing a clinically relevant oxygen amount in a time-sustained manner. Here, four different types of chitosan (low or medium weight (LW or MW), glycol-(G-), and methylglycol-(MG-) chitosan) were compared as candidate biopolymers for shell manufacturing. The aim of the work was to design OLND formulations with optimized physico-chemical characteristics, efficacy in oxygen release, and biocompatibility. All OLND formulations displayed spherical morphology, cationic surfaces, ≤500 nm diameters (with LW chitosan-shelled OLNDs being the smallest), high stability, good oxygen encapsulation efficiency, and prolonged oxygen release kinetics. Upon cellular internalization, LW, MW, and G-chitosan-shelled nanodroplets did not significantly affect the viability, health, or metabolic activity of human keratinocytes (HaCaT cell line). On the contrary, MG-chitosan-shelled nanodroplets showed very poor biocompatibility. Combining the physico-chemical and the biological results obtained, LW chitosan emerges as the best candidate biopolymer for future OLND application as a skin device to treat chronic wounds. [ABSTRACT FROM AUTHOR]

The emergence of antibiotic resistance and viruses with high epidemic potential made unexplored marine environments an appealing target source for new metabolites. Marine fungi represent one of the most suitable sources for the discovery of new compounds. Thus, the aim of this work was (i) to isolate and identify fungi associated with the Atlantic sponge Grantia compressa; (ii) to study the fungal metabolites by applying the OSMAC approach (one strain; many compounds); (iii) to test fungal compounds for their antimicrobial activities. Twenty-one fungal strains (17 taxa) were isolated from G. compressa. The OSMAC approach revealed an astonishing metabolic diversity in the marine fungus Eurotium chevalieri MUT 2316, from which 10 compounds were extracted, isolated, and characterized. All metabolites were tested against viruses and bacteria (reference and multidrug-resistant strains). Dihydroauroglaucin completely inhibited the replication of influenza A virus; as for herpes simplex virus 1, total inhibition of replication was observed for both physcion and neoechinulin D. Six out of 10 compounds were active against Gram-positive bacteria with isodihydroauroglaucin being the most promising compound (minimal inhibitory concentration (MIC) 4–64 µg/mL) with bactericidal activity. Overall, G. compressa proved to be an outstanding source of fungal diversity. Marine fungi were capable of producing different metabolites; in particular, the compounds isolated from E. chevalieri showed promising bioactivity against well-known and emerging pathogens. [ABSTRACT FROM AUTHOR]

The article discusses the human cytomegalovirus (HCMV) as a major opportunistic pathogen for immunocompromised individuals. The virus caused mortality and morbidity with congenital defects due to lacking of an effective vaccine. The article also discusses the three independent drug directed against HCMV.

Vancomycin (Vm) currently represents the gold standard against methicillin-resistant Staphylococcus aureus (MRSA) infections. However, it is associated with low oral bioavailability, formulation stability issues, and severe side effects upon systemic administration. These drawbacks could be overcome by Vm topical administration if properly encapsulated in a nanocarrier. Intriguingly, nanobubbles (NBs) are responsive to physical external stimuli such as ultrasound (US), promoting drug delivery. In this work, perfluoropentane (PFP)-cored NBs were loaded with Vm by coupling to the outer dextran sulfate shell. Vm-loaded NBs (VmLNBs) displayed ∼300 nm sizes, anionic surfaces and good drug encapsulation efficiency. In vitro, VmLNBs showed prolonged drug release kinetics, not accompanied by cytotoxicity on human keratinocytes. Interestingly, VmLNBs were generally more effective than Vm alone in MRSA killing, with VmLNB antibacterial activity being more sustained over time as a result of prolonged drug release profile. Besides, VmLNBs were not internalized by staphylococci , opposite to Vm solution. Further US association promoted drug delivery from VmLNBs through an in vitro model of porcine skin. Taken together, these results support the hypothesis that proper Vm encapsulation in US-responsive NBs might be a promising strategy for the topical treatment of MRSA wound infections. [ABSTRACT FROM AUTHOR]

In the absence of efficient preventive vaccines, topical microbicides offer an attractive alternative in the prevention of Herpes simplex type 1 (HSV-1) and type 2 (HSV-2) infections. Because of their recognized anti-adhesive activity against bacterial pathogens, cranberry ( Vaccinium macrocarpon Ait.) extracts may represent a natural source of new antiviral microbicides. However, few studies have addressed the applications of cranberry extract as a direct-acting antiviral agent. Here, we report on the ability of the novel cranberry extract Oximacro ® and its purified A-type proanthocyanidins (PACs-A), to inhibit HSV-1 and HSV-2 replication in vitro . Analysis of the mode of action revealed that Oximacro ® prevents adsorption of HSV-1 and HSV-2 to target cells. Further mechanistic studies confirmed that Oximacro ® and its PACs-A target the viral envelope glycoproteins gD and gB, thus resulting in the loss of infectivity of HSV particles. Moreover, Oximacro ® completely retained its anti-HSV activity even at acidic pHs (3.0 and 4.0) and in the presence of 10% human serum proteins; conditions that mimic the physiological properties of the vagina - a potential therapeutic location for Oximacro ® . Taken together, these findings indicate Oximacro ® as an attractive candidate for the development of novel microbicides of natural origin for the prevention of HSV infections. [ABSTRACT FROM AUTHOR]

We recently reported that some clinically approved antifungal drugs are potent inhibitors of human cytomegalovirus (HCMV). Here, we report the broad-spectrum activity against HCMV of isavuconazole (ICZ), a new extended-spectrum triazolic antifungal drug. ICZ inhibited the replication of clinical isolates of HCMV as well as strains resistant to the currently available DNA polymerase inhibitors. The antiviral activity of ICZ against HCMV could be linked to the inhibition of human cytochrome P450 51 (hCYP51), an enzyme whose activity we previously demonstrated to be required for productive HCMV infection. Moreover, time-of-addition studies indicated that ICZ might have additional inhibitory effects during the first phase of HCMV replication. Importantly, ICZ showed synergistic antiviral activity in vitro when administered in combination with different approved anti-HCMV drugs at clinically relevant doses. Together, these results pave the way to possible future clinical studies aimed at evaluating the repurposing potential of ICZ in the treatment of HCMV-associated diseases. • Isavuconazole (ICZ) is a clinically approved antifungal that inhibits human cytomegalovirus (HCMV) replication. • ICZ is a broad-spectrum HCMV inhibitor, including clinical isolates and drug-resistant strains within its clinical range. • ICZ inhibits in vitro the activity of human CYP51 enzyme, required for productive HCMV infection. • ICZ acts synergistically with approved anti-HCMV drugs, indicating a repurposing potential in controlling HCMV. [ABSTRACT FROM AUTHOR]

Elimination of virus-infected cells by cytotoxic lymphocytes is triggered by activating receptors, among which NKG2D and DNAM-1/CD226 play an important role. Their ligands, that is, MHC class I-related chain (MIC) A/B and UL16-binding proteins (ULBP)1-6 (NKG2D ligand), Nectin-2/CD112, and poliovirus receptor (PVR)/CD155 (DNAM-1 ligand), are often induced on virus-infected cells, although some viruses, including human CMV (HCMV), can block their expression. In this study, we report that infection of different cell types with laboratory or low-passage HCMV strains upregulated MICA, ULBP3, and PVR, with NKG2D and DNAM-1 playing a role in NK cell-mediated lysis of infected cells. Inhibition of viral DNA replication with phosphonoformic acid did not prevent ligand upregulation, thus indicating that early phases of HCMV infection are involved in ligand increase. Indeed, the major immediate early (IE) proteins IE1 and IE2 stimulated the expression of MICA and PVR, but not ULBP3. IE2 directly activated promoter via its MICA binding to an IE2-responsive element that we identified within the promoter and that is conserved among different alleles of MICA. Both IE proteins were instead required for PVR upregulation via a mechanism independent of IE DNA binding activity. Finally, inhibiting IE protein expression during HCMV infection confirmed their involvement in ligand increase. We also investigated the contribution of the DNA damage response, a pathway activated by HCMV and implicated in ligand regulation. However, silencing of ataxia telangiectasia mutated, ataxia telangiectasia and Rad3--related protein, and DNA-dependent protein kinase did not influence ligand expression. Overall, these data reveal that MICA and PVR are directly regulated by HCMV IE proteins, and this may be crucial for the onset of an early host antiviral response. [ABSTRACT FROM AUTHOR]

The repressor element-1 silencing transcription factor (REST) has emerged as a key controller of neuronal differentiation and has been shown to play a critical role in the expression of the neuronal phenotype; however, much has still to be learned about its role at specific developmental stages and about the functional targets affected. Among these targets, calcium signaling mechanisms are critically dependent on the developmental stage and their full expression is a hallmark of the mature, functional neuron. We have analyzed the role played by REST in GN11 cells, an immortalized cell line derived from gonadotropin hormone releasing hormone (GnRH) neurons at an early developmental stage, electrically non-excitable and with a strong migratory activity. We show for the first time that functional voltage-dependent calcium channels are expressed in wild type GN11 cells; down-regulation of REST by a silencing approach shifts these cells towards a more differentiated phenotype, increasing the functional expression of P/Q-type channels and reducing their migratory potential. [ABSTRACT FROM AUTHOR]