A SARS-CoV-2 full genome sequence of the B.1.1 lineage sheds light on viral evolution in Sicily in late 2020.

To investigate the influence of geographic constrains to mobility on SARS-CoV-2 circulation before the advent of vaccination, we recently characterized the occurrence in Sicily of viral lineages in the second pandemic wave (September to December 2020). Our data revealed wide prevalence of the then widespread through Europe B.1.177 variant, although some viral samples could not be classified with the limited Sanger sequencing tools used. A particularly interesting sample could not be fitted to a major variant then circulating in Europe and has been subjected here to full genome sequencing in an attempt to clarify its origin, lineage and relations with the seven full genome sequences deposited for that period in Sicily, hoping to provide clues on viral evolution. The obtained genome is unique (not present in databases). It hosts 20 single-base substitutions relative to the original Wuhan-Hu-1 sequence, 8 of them synonymous and the other 12 encoding 11 amino acid substitutions, all of them already reported one by one. They include four highly prevalent substitutions, NSP12:P323L, S:D614G, and N:R203K/G204R; the much less prevalent S:G181V, ORF3a:G49V and N:R209I changes; and the very rare mutations NSP3:L761I, NSP6:S106F, NSP8:S41F and NSP14:Y447H. GISAID labeled this genome as B.1.1 lineage, a lineage that appeared early on in the pandemic. Phylogenetic analysis also confirmed this lineage diagnosis. Comparison with the seven genome sequences deposited in late 2020 from Sicily revealed branching leading to B.1.177 in one branch and to Alpha in the other branch, and suggested a local origin for the S:G118V mutation.

Pilot Investigation of SARS-CoV-2 Variants in the Island of Sicily Prior to and in the Second Wave of the COVID-19 Pandemic.

After 2 years of the COVID-19 pandemic, we continue to face vital challenges stemming from SARS-CoV-2 variation, causing changes in disease transmission and severity, viral adaptation to animal hosts, and antibody/vaccine evasion. Since the monitoring, characterization, and cataloging of viral variants are important and the existing information on this was scant for Sicily, this pilot study explored viral variants circulation on this island before and in the growth phase of the second wave of COVID-19 (September and October 2020), and in the downslope of that wave (early December 2020) through sequence analysis of 54 SARS-CoV-2-positive samples. The samples were nasopharyngeal swabs collected from Sicilian residents by a state-run one-health surveillance laboratory in Palermo. Variant characterization was based on RT-PCR amplification and sequencing of four regions of the viral genome. The B.1.177 variant was the most prevalent one, strongly predominating before the second wave and also as the wave downsized, although its relative prevalence decreased as other viral variants, particularly B.1.160, contributed to virus circulation. The occurrence of the B.1.160 variant may have been driven by the spread of that variant in continental Europe and by the relaxation of travel restrictions in the summer of 2020. No novel variants were identified. As sequencing of the entire viral genome in Sicily for the period covered here was restricted to seven deposited viral genome sequences, our results shed some light on SARS-CoV-2 variant circulation during that wave in this insular region of Italy which combines its partial insular isolation with being a major entry point for the African immigration.

Icariin in Combination with Amoxycillin-Clavulanate and Ampicillin, but Not Vancomycin, Increases Antibiotic Sensitivity and Growth Inhibition against Methicillin-Resistant Staphylococcus aureus.

The widespread irrational use of antibiotics in recent years has resulted in an increase in the detection of multi-resistant bacterial strains, particularly methicillin-resistant Staphylococcus aureus (MRSA). The use of natural derivatives such as flavonoids is postulated as one of the most promising avenues to solve this emerging public health problem. The objective of the present study is to characterize the antimicrobial activity of icariin, a flavonoid compound isolated from a variety of plants of the Epimedium genus, against human and animal clinical MRSA isolates. Our study found that icariin alone did not have any antimicrobial effect on S. aureus or MRSA clinical isolates. However, icariin enhanced the effect of amoxycillin-clavulanate or ampicillin, whereas no effect was seen when used in combination with vancomycin. Specifically, co-incubation of S. aureus with amoxycillin-clavulanate plus icariin resulted in an increased proportion of dead cells, suggesting that this flavonoid potentially increases antimicrobial activity when used in combination with the beta-lactam antibiotic amoxycillin-clavulanate. Furthermore, we demonstrate that co-incubation of S. aureus with AmoxyClav plus icariin resulted in increased membrane disruption and growth inhibition. This study demonstrates the potential utility of icariin in permitting lower antibiotic therapeutic doses in alignment with strategies to reduce the spread of antibiotic resistance. Further research is required to determine the optimum concentration of icariin and to define clinically relevant combinations of flavonoid and antibiotic.

Salivary biomarkers and their efficacies as diagnostic tools for Oral Squamous Cell Carcinoma: Systematic review and meta-analysis.

BACKGROUND: More than 90% of malignant tumors of the head and neck are oral squamous cell carcinomas. Early detection of oral squamous cell carcinoma using salivary biomarkers could prevent malignant transformations and enhance patient survival. METHODS: A systematic search in MEDLINE and the Central Register of Controlled Trials and meta-analysis were undertaken to identify the screening potential of six salivary biomarkers for early oral squamous cell carcinoma detection: interleukins IL-8 and IL1-ß, DUSP-1 and S100P messenger RNAs, and miR125a and miR200a microRNAs. RESULTS: The sensitivities of IL-8 (0.41; 95%CI 0.19-0.99), IL1-ß (0.26; 95%CI 0.19-0.99), DUSP-1 (0.61; 95%CI 0.01-0.98), and S100P (0.67; 95%CI 0.32-0.99) were calculated. Specificities of the biomarkers analyzed were found to be IL-8 (0.69; 95%CI 0.66-0.99), IL1-ß (0.47; 95%CI 0.46-0.90), DUSP-1 (0.75; 95%CI 0.33-1), and S100P (0.73; 95%CI 0.18-0.99). CONCLUSIONS: Early detection of oral squamous cell carcinoma was best achieved by screening for salivary messenger RNA DUSP-1 and S100P. Further investigation is required into miRNAs as novel biomarkers.

Herbal Extracts with Antifungal Activity against Candida albicans: A Systematic Review.

In the era of antimicrobial resistance, fungal pathogens are not an exception. Several strategies, including antimicrobial stewardship programs and high throughput screening of new drugs, are being implemented. Several recent studies have demonstrated the effectiveness of plant compounds with antifungal activity. In this systematic review, we examine the use of natural compounds as a possible avenue to fight fungal infections produced by Candida albicans, the most common human fungal pathogen. Electronic literature searches were conducted through PubMed/MEDLINE, Cochrane, and Science Direct limited to the 5 years. A total of 131 articles were included, with 186 plants extracts evaluated. Although the majority of the natural extracts exhibited antifungal activities against C. albicans (both in vivo and in vitro), the strongest antifungal activity was obtained from Lawsonia inermis, Pelargonium graveolens, Camellia sinensis, Mentha piperita, and Citrus latifolia. The main components with proven antifungal activities were phenolic compounds such as gallic acid, thymol, and flavonoids (especially catechin), polyphenols such as tannins, terpenoids and saponins. The incorporation of nanotechnology greatly enhances the antifungal properties of these natural compounds. Further research is needed to fully characterize the composition of all herbal extracts with antifungal activity as well as the mechanisms of action of the active compounds.

Dental black plaque: metagenomic characterization and comparative analysis with white-plaque.

Extrinsic black dental staining is an external dental discoloration of bacterial origin, considered a special form of dental plaque. Currently, there is no definitive therapeutic option for eliminating black stain. This study employed 16S rRNA metagenomics to analyze black stain and white-plaque samples from 27 adult volunteers. Study objectives were to: describe the microbial diversity of adult black stain samples; characterize their taxonomic profile; compare the microbiomes of black stain versus white-plaque from adult volunteers and propose a functional map of the black stain microbiome using PICRUSt2. The black stain microbiome was poorer in species diversity as compared to white-plaque. The five most abundant genera in black stain were Capnocytophaga, Leptotrichia, Fusobacterium, Corynebacterium and Streptococcus. Functional analysis of microbial species revealed conserved and consistent clustering of functional pathways within and between black stain and white-plaque microbiomes. We describe enrichment of heme biosynthetic pathways in black stain. Our results suggest that the dysbiosis in black stain resembles "orally healthy" communities. The increased abundance of heme biosynthetic pathways suggests that heme-dependent iron sequestration and subsequent metabolism are key for black stain formation. Further research should decipher the regulation of heme biosynthetic genes and characterize the temporal sequence leading to colonization and dysbiosis.

Raising awareness about microbial antibiotic resistance in undergraduate dental students: a research-based strategy for teaching non-laboratory elements of a microbiology curriculum.

BACKGROUND: Resistance to antimicrobial agents has become a problem in modern society. Antibiotic resistant bacteria undermine the prevention and treatment of infections. Undergraduate dental students in Europe are required to receive information in aspects of microbiology relevant for dental practice, including oral microbial pathogens and resistance mechanisms against antimicrobial compounds. The objective of this study was to implement a research-based strategy to aid the understanding of the increase in antimicrobial resistance in undergraduate dental student training. The primary outcome of this project is the efficacious delivery of the learning objectives. METHODS: Ten volunteer undergraduate student "ambassadors" were recruited to manage the project with assistance from lead academics. Student ambassadors were a source of peer learning for their colleagues. The project consisted of three phases: Pre-project preparation (in which the ambassadors received special instruction and training); Practical experience (in which the ambassadors worked with volunteer student colleagues to carry out the project); Public presentation of results (in which ambassadors presented study results at a scientific conference of their choosing). RESULTS: A total of 1164 students volunteered for the project, corresponding to an average participation rate of 76.4% students per year of the course. Following final debriefing, student participants and ambassadors were strongly positive in their evaluation of the achievement of 8 key student learning objectives. The results demonstrate that most volunteers improved their knowledge related to antimicrobial resistance mechanisms in microbiology. Additional benefits of participation in this project included an improvement in dental knowledge and ethics in biomedical research for the student volunteers, whilst the student ambassadors reported improved knowledge about critical thinking and study design, as well as a deeper understanding about microbiological analysis methods. CONCLUSIONS: To the best of our knowledge, this the first instance of the application of project-based methodologies to the teaching of a traditionally non-laboratory component of a subject taught in the dentistry curriculum. Results from both students and ambassadors highlighted the increase in dental knowledge and an increased awareness of antimicrobial resistance as the key outcomes of this project.

Risk factors for the presence of dental black plaque.

In order to evaluate risk factors related to the presence of extrinsic dental black stain, a total of 94 orally healthy volunteers (47 individuals with dental black stain and 47 individuals without dental black stain) were recruited from ten different dental clinics in Valencia and Castellón (Spain). Data regarding their oral hygiene, dietary habits, and oral health status were gathered by questionnaire. Samples of dental plaque, saliva and drinking water were collected for chemical analysis. Three factors were found to be statistically significantly associated with dental black stain, (i) consuming water with high iron content, (ii) consuming water with high pH, and (iii) having a high salivary pH. Other factors such as smoking, taking iron supplements or consuming caffeinated drinks were not found to be risk factors for the presence of black stain. A multivariate logistic regression analysis showed that drinking tap or osmosis-purified water and lower levels of salivary iron increase the risk of having dental black stain. Overall, several risk factors for the presence of dental black stain have been identified. The main modifiable risk factor identified in this study was the consumption of tap or osmosis drinking water.

Modulation of salivary cytokines in response to alcohol, tobacco and caffeine consumption: a pilot study.

It has been shown that long-term stimulant consumption alters the biological and microbiological status of the oral cavity. We present a pilot study describing stimulant-specific oral immunomodulation in the oral cavity. Changes in salivary cytokine levels in response to long-term alcohol, tobacco and caffeine were identified. Volunteers were recruited from amongst the patients visiting University Dental Clinic of CEU Cardenal Herrera University (Alfara del Patriarca, Spain). Participants were grouped according to their self-reported levels of consumption of either caffeine, alcohol or tobacco (control group volunteers were non-consumers of all three). Informed consent was provided and stimulated saliva samples were obtained and assayed for interleukin-1α IL-1α), Tumor Necrosis Factor α (TNF-α) and Interferon γ IFN-γ). Long-term, high-level consumers of alcohol or tobacco exhibited elevated salivary concentrations of the three inflammatory cytokines with respect to control values. Specifically, IL-1α was found to be elevated in alcohol users whilst IFN-γ concentration higher in tobacco users versus controls. Long-term caffeine consumers displayed elevated levels of IFN-γ and TNF-α, whereas IL-1α levels were reduced with respect to control volunteers. This pilot study demonstrates that salivary cytokines can be modulated in response to quantity and duration of alcohol, caffeine or tobacco consumption.

Identification of vacuole defects in fungi.

Fungal vacuoles are involved in a diverse range of cellular functions, participating in cellular homeostasis, degradation of intracellular components, and storage of ions and molecules. In recent years there has been a significant increase in the number of studies linking these organelles with the regulation of growth and control of cellular morphology, particularly in those fungal species able to undergo yeast-hypha morphogenetic transitions. This has contributed to the refinement of previously published protocols and the development of new techniques, particularly in the area of live-cell imaging of membrane trafficking events and vacuolar dynamics. The current review outlines recent advances in the imaging of fungal vacuoles and assays for characterization of trafficking pathways, and other physiological activities of this important cell organelle.

Pseudohypha budding patterns of Candida albicans.

Pseudohyphal growth of Candida albicans has been recognized as a morphological growth form that exhibits characteristics that are distinct from those of the budding yeast phase and true hyphal form of this pathogenic fungus. In Saccharomyces cerevisiae, pseudohypha growth involves synchronous unipolar cell divisions that are a modification of the bipolar budding pattern of diploid cells. While pseudohyphae of C. albicans also exhibit unipolar cell divisions, live cell imaging demonstrated departures from the normal unipolar pattern. Buds occasionally followed a bipolar or axial budding event in which buds could be formed from the proximal or distal ends of a parent pseudohypha. This extends the known morphological repertoire of cell division patterns in C. albicans pseudohyphal cells.

Calcium homeostasis is required for contact-dependent helical and sinusoidal tip growth in Candida albicans hyphae.

Hyphae of the dimorphic fungus, Candida albicans, exhibit directional tip responses when grown in contact with surfaces. On hard surfaces or in liquid media, the trajectory of hyphal growth is typically linear, with tip re-orientation events limited to encounters with topographical features (thigmotropism). In contrast, when grown on semisolid surfaces, the tips of C. albicans hyphae grow in an oscillatory manner to form regular two-dimensional sinusoidal curves and three-dimensional helices. We show that, like thigmotropism, initiation of directional tip oscillation in C. albicans hyphae is severely attenuated when Ca2+ homeostasis is perturbed. Chelation of extracellular Ca2+ or deletion of the Ca2+ transporters that modulate cytosolic [Ca2+] (Mid1, Cch1 or Pmr1) did not affect hyphal length but curve formation was severely reduced in mid1Delta and cch1Delta and abolished in pmr1Delta. Sinusoidal hypha morphology was altered in the mid1Delta, chs3Delta and heterozygous pmr1Delta/PMR1 strains. Treatments that affect cell wall integrity, changes in surface mannosylation or the provision of additional carbon sources had significant but less pronounced effects on oscillatory growth. The induction of two- and three-dimensional sinusoidal growth in wild-type C. albicans hyphae is therefore the consequence of mechanisms that involve Ca2+ influx and signalling rather than gross changes in the cell wall architecture.

Candida albicans ABG1 gene is involved in endocytosis.

The human fungal pathogen Candida albicans undergoes reversible morphogenetic transitions between yeast, hyphal and pseudohyphal forms. The fungal vacuole actively participates in differentiation processes and plays a key role supporting hyphal growth. The ABG1 gene of C. albicans encodes an essential protein located in the vacuolar membranes of both yeast and hyphae. Using fluorescence microscopy of a green fluorescent protein-tagged version of Abg1p, a fraction of the protein was detected in hyphal tips, not associated with vacuolar membranes. Live cell imaging of emerging germ tubes showed that Abg1p migrated to the polarized growth site and colocalized with endocytic vesicles. Phenotypic analysis of a methionine-regulated conditional mutant confirmed that Abg1p is involved in endocytosis.

Vacuole inheritance regulates cell size and branching frequency of Candida albicans hyphae.

Hyphal growth of Candida albicans is characterized by asymmetric cell divisions in which the subapical mother cell inherits most of the vacuolar space and becomes cell cycle arrested in G1, while the apical daughter cell acquires most of the cell cytoplasm and progresses through G1 into the next mitotic cell cycle. Consequently, branch formation in hyphal compartments is delayed until sufficient cytoplasm is synthesized to execute the G1 'START' function. To test the hypothesis that this mode of vacuole inheritance determines cell cycle progression and therefore the branching of hyphae, eight tetracycline-regulated conditional mutants were constructed that were affected at different stages of the vacuole inheritance pathway. Under repressing conditions, vac7, vac8 and fab1 mutants generated mycelial compartments with more symmetrically distributed vacuoles and increased branching frequencies. Repression of VAC1, VAM2 and VAM3 resulted in sparsely branched hyphae, with large vacuoles and enlarged hyphal compartments. Therefore, during hyphal growth of C. albicans the cell cycle, growth and branch formation can be uncoupled, resulting in the investment of cytoplasm to support hyphal extension at the expense of hyphal branching.

Vacuolar dynamics during the morphogenetic transition in Candida albicans.

Morphogenetic transitions between the yeast, pseudohyphal and true hyphal forms of the fungal pathogen Candida albicans are coordinated with the cell cycle. In true hyphae, large vacuoles are formed in subapical compartments that influence compartment size and hence cell cycle progression and branching frequency. Here, live-cell imaging techniques were applied to study the dynamic process of vacuolar segregation in the three different morphological forms of C. albicans. We show the presence of a filamentous vacuole segregation structure in pseudohyphae and true hyphae that differs from that of a stream of vacuole vesicles typical of vacuole inheritance in yeast cells. In growing hyphae, retrograde transport of the vacuoles led to apparent capture and adhesion of vacuoles to supapical septa, and septation events sometimes led to the bisection of large vacuoles that occupied sites of cytokinesis. Inhibitor studies also demonstrated that vacuole motility in C. albicans was actin dependent and microtubule independent.

Vacuoles and fungal biology.

Fungal vacuoles have long been recognised as versatile organelles, involved in many aspects of protein turnover, cellular homeostasis, membrane trafficking, signalling and nutrition. Recent research has also revealed an expanding repertoire of physiological functions for fungal vacuoles that are vital for fungal growth, differentiation, symbiosis and pathogenesis. Vacuole-mediated long-distance nutrient transporting systems have been shown to facilitate mycelial foraging and long-distance communication in saprophytes and mycorrhizal fungi. Some hyphae of plant and human fungal pathogens can grow under severely nutrient-limited conditions by expanding the vacuolar space rather than synthesising new cytoplasm and organelles. Autophagy has been recognised as a crucial process in plant pathogens for the initiation of appressorium formation. These studies demonstrate the importance of fungal vacuoles as organelles that are essential for many of the attributes that define the activities and roles of fungi in their natural environments.

Rapid PCR-based test for identifying Candida albicans by using primers derived from the pH-regulated KER1 gene.

A PCR-based method in combination with a simple, reliable and inexpensive DNA extraction procedure for rapid detection of Candida albicans clinical isolates is described here. The extraction protocol is based on a combination of chemical (NaOH and detergents) and physical (boiling) treatments, thus avoiding many of the problems inherent in the currently available DNA extraction protocols (basically the use of expensive and/or toxic chemical reagents), and may be useful for daily clinical routine. The PCR-based system described here uses a single pair of primers (SC1F and SC1R) deduced from the C. albicans-specific KER1 gene sequence. These primers amplify a 670-bp fragment of the KER1 gene. All the clinical C. albicans isolates generated the expected 670-bp amplicon. Other non-albicans Candida species, including the azole-resistant C. krusei and C. glabrata, and the very closely related C. dubliniensis, failed to amplify any DNA fragment. The PCR results reported here suggest that amplification with SC1F and SC1R primers is species specific and, consequently, may be useful for specifically identifying C. albicans strains.

ABG1, a novel and essential Candida albicans gene encoding a vacuolar protein involved in cytokinesis and hyphal branching.

Immunoscreening of a Candida albicans expression library resulted in the isolation of a novel gene encoding a 32.9-kDa polypeptide (288 amino acids), with 27.7% homology to the product of Saccharomyces cerevisiae YGR106c, a putative vacuolar protein. Heterozygous mutants in this gene displayed an altered budding growth pattern, characterized by the formation of chains of buds, decreasingly in size towards the apex, without separation of the daughter buds. Consequently, this gene was designated ABG1. A conditional mutant for ABG1 with the remaining allele under the control of the MET3 promoter did not grow in the presence of methionine and cysteine, demonstrating that ABG1 was essential for viability. Western analysis revealed the presence of a major 32.9-kDa band, mainly in a particulate fraction (P40) enriched in vacuoles, and tagging with green fluorescent protein confirmed that Abg1p localized to the vacuole. Vacuole inheritance has been linked to the regulation of branching frequency in C. albicans. Under repressing conditions, the conditional mutant had an increased frequency of branching under hyphal inducing conditions and an altered sensitivity to substances that interfered with cell wall assembly. Repression of ABG1 in the conditional mutant strain caused disturbance of normal size and number of vacuoles both in yeast and mycelial cells and also in the asymmetric vacuole inheritance associated with the characteristic pattern of germ tubes and branching in C. albicans. These observations indicate that ABG1 plays a key role in vacuole biogenesis, cytokinesis, and hyphal branching.