Whole-genome analysis suggesting probiotic potential and safety properties of Pediococcus pentosaceus DSPZPP1, a promising LAB strain isolated from traditional fermented sausages of the Basilicata region (Southern Italy).

Introduction: Many lactic acid bacteria (LAB) strains are currently gaining attention in the food industry and various biological applications because of their harmless and functional properties. Given the growing consumer demand for safe food, further research into potential probiotic bacteria is beneficial. Therefore, we aimed to characterize Pediococcus pentosaceus DSPZPP1, a LAB strain isolated from traditional fermented sausages from the Basilicata region of Southern Italy. Methods: In this study, we analyzed the whole genome of the P. pentosaceus DSPZPP1 strain and performed in silico characterization to evaluate its applicability for probiotics and use in the food industry. Results and Discussion: The whole-genome assembly and functional annotations revealed many interesting characteristics of the DSPZPP1 strain. Sequencing raw reads were assembled into a draft genome of size 1,891,398 bp, with a G + C content of 37.3%. Functional annotation identified 1930 protein-encoding genes and 58 RNAs including tRNA, tmRNA, and 16S, 23S, and 5S rRNAs. The analysis shows the presence of genes that encode water-soluble B-group vitamins such as biotin, folate, coenzyme A, and riboflavin. Furthermore, the analysis revealed that the DSPZPP1 strain can synthesize class II bacteriocin, penocin A, adding importance to the food industry for bio-enriched food. The DSPZPP1 genome does not show the presence of plasmids, and no genes associated with antimicrobial resistance and virulence were found. In addition, two intact bacteriophages were identified. Importantly, the lowest probability value in pathogenicity analysis indicates that this strain is non-pathogenic to humans. 16 s rRNA-based phylogenetic analysis and comparative analysis based on ANI and Tetra reveal that the DSPZPP1 strain shares the closest evolutionary relationship with P. pentosaceus DSM 20336 and other Pediococcus strains. Analysis of carbohydrate active enzymes (CAZymes) identified glycosyl transferases (GT) as a main class of enzymes followed by glycoside hydrolases (GH). Our study shows several interesting characteristics of the isolated DSPZPP1 strain from fermented Italian sausages, suggesting its potential use as a promising probiotic candidate and making it more appropriate for selection as a future additive in biopreservation.

Potential PDE4B inhibitors as promising candidates against SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an RNA virus belonging to the coronavirus family responsible for coronavirus disease 2019 (COVID-19). It primarily affects the pulmonary system, which is the target of chronic obstructive pulmonary disease (COPD), for which many new compounds have been developed. In this study, phosphodiesterase 4 (PDE4) inhibitors are being investigated. The inhibition of PDE4 enzyme produces anti-inflammatory and bronchodilator effects in the lung by inducing an increase in cAMP concentrations. Piclamilast and rolipram are known selective inhibitors of PDE4, which are unfortunately endowed with common side effects, such as nausea and emesis. The selective inhibition of the phosphodiesterase 4B (PDE4B) subtype may represent an intriguing technique for combating this highly contagious disease with fewer side effects. In this article, molecular docking studies for the selective inhibition of the PDE4B enzyme have been carried out on 21 in-house compounds. The compounds were docked into the pocket of the PDE4B catalytic site, and in most cases, they were almost completely superimposed onto piclamilast. Then, in order to enlarge our study, drug-likeness prediction studies were performed on the compounds under study.

Antimicrobial and antioxidant properties and quantitative screening of phytochemicals of Fraxinus excelsior L. and Eschscholtzia californica Cham. mother tinctures.

The antioxidant and antimicrobial activities of Fraxinus excelsior L. and Eschscholtzia californica Cham. mother tinctures against a range of foodborne bacteria were investigated to determine the major components and to analyse the action spectrum and antimicrobial effectiveness of the extracts. Results demonstrated a significant antioxidant activity of Fraxinus excelsior L. and a lower activity of Eschscholtzia californica Cham. and a good chemical phenolic composition with the highest content of flavonoids. The Fraxinus excelsior L. and Eschscholtzia californica Cham. mother tinctures demonstrated a middle-high antimicrobial activity against, respectively, 66.67% and 43.33% of all tested bacteria. The inhibitory activity showed a moderate effect on the growth of the sensitive strains in presence of extracts minimum inhibitory concentration. The synergistic actions of bioactive compounds detected in the extracts might be on the basis of antioxidant and biological activities observed and should be used in pharmaceutical, food preservation, alternative medicine and natural therapies fields.

Are Nutraceuticals Effective in COVID-19 and Post-COVID Prevention and Treatment?

The beginning of the end or the end of the beginning? After two years mastered by coronavirus disease 19 (COVID-19) pandemic, we are now witnessing a turnaround. The reduction of severe cases and deaths from COVID-19 led to increasing importance of a new disease called post-COVID syndrome. The term post-COVID is used to indicate permanency of symptoms in patients who have recovered from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Immune, antiviral, antimicrobial therapies, as well as ozone therapy have been used to treat COVID-19 disease. Vaccines have then become available and administered worldwide to prevent the insurgence of the disease. However, the pandemic is not over yet at all given the emergence of new omicron variants. New therapeutic strategies are urgently needed. In this view, great interest was found in nutraceutical products, including vitamins (C, D, and E), minerals (zinc), melatonin, probiotics, flavonoids (quercetin), and curcumin. This review summarizes the role of nutraceuticals in the prevention and/or treatment of COVID-19 disease and post-COVID syndrome.

Thidiazuron: New Trends and Future Perspectives to Fight Xylella fastidiosa in Olive Trees.

These days, most of our attention has been focused on the COVID-19 pandemic, and we have often neglected what is happening in the environment. For instance, the bacterium Xylella fastidiosa re-emerged as a plant pathogen of global importance in 2013 when it was first associated with an olive tree disease epidemic in Italy, called Olive Quick Decline Syndrome (OQDS), specifically caused by X. fastidiosa subspecies pauca ST53, which affects the Salento olive trees (Apulia, South-East Italy). This bacterium, transmitted by the insect Philaenus spumarius, is negatively reshaping the Salento landscape and has had a very high impact in the production of olives, leading to an increase of olive oil prices, thus new studies to curb this bacterium are urgently needed. Thidiazuron (TDZ), a diphenylurea (N-phenyl-1,2,3-thiadiazol-5-yl urea), has gained considerable attention in recent decades due to its efficient role in plant cell and tissue culture, being the most suitable growth regulator for rapid and effective plant production in vitro. Its biological activity against bacteria, fungi and biofilms has also been described, and the use of this low-cost compound to fight OQDS may be an intriguing idea.

Metagenomic monitoring of soil bacterial community after the construction of a crude oil flowline.

This study aimed to assess the metagenomic changes of soil bacterial community after constructing a crude oil flowline in Basilicata region, Italy. Soils identified a total of 56 taxa at the phylum level and 485 at the family level, with a different taxa distribution, especially in samples collected on 2014. Since microbiological diversity occurred in the soils collected after 2013 (the reference year), we performed a differential abundance analysis using DESeq2 by GAIA pipeline. In the forest area, 14 phyla and 126 families were differentially abundant (- 6.06 < logFC > 7.88) in 2014 compared to 2013. Nine families were differentially abundant in 2015, with logFC between - 3.16 and 4.66, while 20 families were significantly more abundant and 16 less abundant in 2016, with logFC between - 6.48 and 6.45. In the cultivated area, 33 phyla and 260 families showed differential abundance in 2014. In the next year (2015), 14 phyla were significantly more abundant and 19 less abundant, while 29 families were substantially more abundant and 139 less abundant, with fold changes ranging between - 5.67 and 4.01. In 2016, 33 phyla showed a significantly different abundance, as 14 were more abundant and 19 decreased, and 81 families showed a significantly increased amount with logFC between - 5.31 and 5.38. These results hypothesise that the analysed site is an altered soil where the development of particular bacterial groups attends to bioremediation processes, naturally occurring to restore optimal conditions.

Bis-Thiourea Quaternary Ammonium Salts as Potential Agents against Bacterial Strains from Food and Environmental Matrices.

In recent years, the phenomenon of antibiotic resistance in hospitals, communities and the environment has increasingly grown, so antibiotic resistance has become an urgent problem that requires a decisive and global intervention. Incorrect/unnecessary use of antibiotics contributes to increase the ability of microorganisms to develop resistance faster and faster. Research efforts must, therefore, be made to ensure a future in which antibiotic drugs will still be useful in combating infectious diseases. The search for new antibacterial compounds is fundamental. In this study, the antimicrobial activity of the compounds was evaluated against selected bacterial strains from food and environmental matrices by using the Agar Well Diffusion Assay. A total of thirty-six Gram-positive and Gram-negative bacteria were employed to determine the action spectrum and the antimicrobial effectiveness of a small series of thiourea derivatives. Results showed that the highest activities were found for compounds 1 and 4. The important role of the alkyl chain length and/or guanidine moiety in the width of action spectrum was evidenced. Further studies will allow evaluating the efficacy of the inhibiting action and the molecular mechanisms underlying this activity in order to identify compounds capable of counteracting the phenomenon of antibiotic resistance and to identify possible future applications of these newly synthesized compounds that have shown a high bactericidal action potential.

A Phenylacetamide Resveratrol Derivative Exerts Inhibitory Effects on Breast Cancer Cell Growth.

Resveratrol (RSV) is a natural compound that displays several pharmacological properties, including anti-cancer actions. However, its clinical application is limited because of its low solubility and bioavailability. Here, the antiproliferative and anti-inflammatory activity of a series of phenylacetamide RSV derivatives has been evaluated in several cancer cell lines. These derivatives contain a monosubstituted aromatic ring that could mimic the RSV phenolic nucleus and a longer flexible chain that could confer a better stability and bioavailability than RSV. Using MTT assay, we demonstrated that most derivatives exerted antiproliferative effects in almost all of the cancer cell lines tested. Among them, derivative 2, that showed greater bioavailability than RSV, was the most active, particularly against estrogen receptor positive (ER+) MCF7 and estrogen receptor negative (ER-) MDA-MB231 breast cancer cell lines. Moreover, we demonstrated that these derivatives, particularly derivative 2, were able to inhibit NO and ROS synthesis and PGE2 secretion in lipopolysaccharide (LPS)-activated U937 human monocytic cells (derived from a histiocytoma). In order to define the molecular mechanisms underlying the antiproliferative effects of derivative 2, we found that it determined cell cycle arrest at the G1 phase, modified the expression of cell cycle regulatory proteins, and ultimately triggered apoptotic cell death in both breast cancer cell lines. Taken together, these results highlight the studied RSV derivatives, particularly derivative 2, as promising tools for the development of new and more bioavailable derivatives useful in the treatment of breast cancer.

New Insights into the Exploitation of Vitis vinifera L. cv. Aglianico Leaf Extracts for Nutraceutical Purposes.

The leaves of Vitis vinifera L. have been used for a long time in traditional medicine for the treatment of many ailments. Grape polyphenols, indeed, have been demonstrated to be able to defend against oxidative stress, responsible for various disorders such as cancer, diabetes and neurodegenerative diseases. The effects of different extraction techniques, Soxhlet (SOX), Accelerated Solvent (ASE 40, ASE 50) and Ultrasound Assisted Extraction (UAE) were studied in this work to evaluate their impact on the chemical profile and bioactive potential of Vitis vinifera L. (cv. Aglianico) leaf extracts. The phytochemical profile was investigated by HPLC-DAD and 9 phenolic compounds were identified and quantified in the extract. Moreover, the antioxidant, anticholinesterase and antityrosinase activities were evaluated. In detail, the total polyphenol content and antioxidant activity (2,2-diphenyl-1-picrylhydrazyl, Oxygen Radical Absorbance Capacities and ß-Carotene Bleaching assays) were evaluated and compared to assess the Relative Antioxidant Capacity Index (RACI). To test the inhibitory activity of extracts towards cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition assays were performed. SOX and ASE 50 have shown the highest value of RACI, 0.76 and 0.65, respectively. Regarding enzymatic inhibitory activity, ASE 50 (IC50 = 107.16 ± 8.12 µg/mL) and SOX (IC50 = 171.34 ± 12.12 µg/mL) extracts exhibited the highest AChE and BChE inhibitory activity, respectively, while UAE (IC50 = 293.2 ± 25.6 µg/mL, followed by SOX (IC50 = 302.5 ± 38.3 µg/mL) showed the highest tyrosinase inhibition value. Our results demonstrated for the first time that Aglianico leaves are important sources of phenols that could be used to prevent oxidative stress and be potentially helpful in diseases treatable with tyrosinase and cholinesterase inhibitors, like myasthenia gravis or Alzheimer's.

Carbazole Derivatives as Kinase-Targeting Inhibitors for Cancer Treatment.

Protein Kinases (PKs) are a heterogeneous family of enzymes that modulate several biological pathways, including cell division, cytoskeletal rearrangement, differentiation and apoptosis. In particular, due to their crucial role during human tumorigenesis and cancer progression, PKs are ideal targets for the design and development of effective and low toxic chemotherapeutics and represent the second group of drug targets after G-protein-coupled receptors. Nowadays, several compounds have been claimed to be PKs inhibitors, and some of them, such as imatinib, erlotinib and gefitinib, have already been approved for clinical use, whereas more than 30 others are in various phases of clinical trials. Among them, some natural or synthetic carbazole-based molecules represent promising PKs inhibitors due to their capability to interfere with PK activity by different mechanisms of action including the ability to act as DNA intercalating agents, interfere with the activity of enzymes involved in DNA duplication, such as topoisomerases and telomerases, and inhibit other proteins such as cyclindependent kinases or antagonize estrogen receptors. Thus, carbazoles can be considered a promising this class of compounds to be adopted in targeted therapy of different types of cancer.

Inhibition of Human Topoisomerase†II by N,N,N-Trimethylethanammonium Iodide Alkylcarbazole Derivatives.

Chemotherapy is used for the treatment of all stages of breast cancer, including the metastatic stage of the disease. Treatment regimens are generally tailored for each patient's particular situation. However, chemotherapeutic agents are the leading cause of serious drug-related adverse effects; moreover, drug resistance often occurs. In this study, we designed and synthesized a new series of N-alkylcarbazoles derived from ellipticine, an alkaloid with a carbazole skeleton initially used in the treatment of metastatic breast cancer and later dismissed because of poor aqueous solubility and severe side effects. After evaluating the binding modes of our class of newly synthesized compounds with human topoisomerase II (hTopo II), we performed hTopo II decatenation assays, identifying compound 4 f (2-(4-((3-chloro-9H-carbazol-9-yl)pentyl)piperazin-1-yl)-N,N,N-trimethylethanammonium iodide) as a good inhibitor. Moreover, 4 f and 4 g (2-(4-((3-chloro-9H-carbazol-9-yl)hexyl)piperazin-1-yl)-N,N,N-trimethylethanammonium iodide) showed a good anti-proliferative activity toward breast cancer cells, causing apoptosis by activation of the caspase pathway. Interestingly, the activity of these two compounds on triple-negative MDA-MB-231 cells, which tend to be highly metastatic and aggressive, is strictly connected to the observed inhibition of hTopo II.

Chloro-1,4-dimethyl-9H-carbazole Derivatives Displaying Anti-HIV Activity.

BACKGROUND: Despite the progress achieved by anti-retroviral drug research in the last decades, the discovery of novel compounds endowed with selective antiviral activity and reduced side effects is still a necessity. At present, the most urgent requirement includes the improvement of HIV (Human Immunodeficiency Virus) prevention and sexual transmission and the development of new drugs to treat the chronic lifelong infection. METHODS: Six chloro-1,4-dimethyl-9H-carbazoles (2a,b-4a,b) have been prepared following opportunely modified known chemical procedures and tested in luciferase and Escherichia coli ß-galactosidase expressing CD4⁺, CXCR4⁺, CCR5⁺ TZM-bl cells. RESULTS AND CONCLUSION: a preliminary biological investigation on the synthesized small series of chloro-1,4-dimethyl-9H-carbazoles has been carried out. Among all tested compounds, a nitro-derivative (3b) showed the most interesting profile representing a suitable lead for the development of novel anti-HIV drugs.

In Vitro Effects of Some Botanicals with Anti-Inflammatory and Antitoxic Activity.

Several extrinsic factors, like drugs and chemicals, can foster autoimmunity. Tetracyclines, in particular oxytetracycline (OTC), appear to correlate with the emergence of immune-mediated diseases. Accumulation of OTC, the elective drug for gastrointestinal and respiratory infectious disease treatment in broiler chickens, was reported in chicken edible tissues and could represent a potential risk for pets and humans that could assume this antibiotic as residue in meat or in meat-derived byproducts. We investigated the in vitro anti-inflammatory properties of a pool of thirteen botanicals as a part of a nutraceutical diet, with proven immunomodulatory activity. In addition, we evaluated the effect of such botanicals in contrasting the in vitro proinflammatory toxicity of OTC. Our results showed a significant reduction in interferon- (INF-) γ production by human and canine lymphocytes in presence of botanicals ((⁎) p < 0.05). Increased INF-γ production, dependent on 24-hour OTC-incubation of T lymphocytes, was significantly reduced by the coincubation with Haematococcus pluvialis, with Glycine max, and with the mix of all botanicals ((⁎) p < 0.05). In conclusion, the use of these botanicals was shown to be able to contrast OTC-toxicity and could represent a new approach for the development of functional foods useful to enhance the standard pharmacological treatment in infections as well as in preventing or reducing the emergence of inflammatory diseases.

Assessment of the genetic polymorphism and physiological characterization of indigenous Oenococcus oeni strains isolated from Aglianico del Vulture red wine.

The aim of this study was a reliable intra-species discrimination and strain biodiversity in Oenococcus oeni populations of two different Aglianico wineries by molecular, biochemical, and physiological characterization. Pulsed field gel electrophoresis (PFGE) analysis revealed a high polymorphism related to the origin (winery) of strains, while differential display PCR (DD-PCR) allowed a further discrimination of strains from the same winery. Moreover, the heterogeneity of these natural populations was investigated by capillary electrophoresis and enzymatic assays. A variability related to a different surface charge distribution was observed among strains, linked to their origin. Malolactic activity study evidenced strain-specific differences in malic acid degradation, and then, only the presence of L(-)-malic acid in the medium induced the mle gene. This study provided evidences on the importance of intra-species biodiversity of malolactic bacterial populations in wine ecosystems, as each wine possess peculiar winemaking conditions and physical-chemical properties which make specific the bacterial survival and growth. This study highlighted a great biodiversity among O. oeni strains that can be also winery specific. Such biodiversity within a certain winery and winemaking area is important for selecting malolactic starters, and strain-specific trait identification is especially important to match individual strains to specific industrial process.

Genotypic and technological diversity of Brevibacterium linens strains for use as adjunct starter cultures in 'Pecorino di Filiano' cheese ripened in two different environments.

Twenty-two Brevibacterium linens strains isolated from 'Pecorino di Filiano' cheese ripened in two different environments (natural cave and storeroom) were characterized and differentiated for features of technological interest and by genotypic methods, in order to select strains with specific features to be used as surface starter cultures. Results showed significant differences among strains on the basis of physiological and technological features, indicating heterogeneity within the species. A middle-low level of proteolytic activity was observed in 27.3 % of strains, while a small group (9.1 %) showed a high ability. Lipolytic activity was observed at three different temperatures and the highest value was detected at 20 °C with 13.6 % of strains, while an increase in temperature produced a slightly lower lipolysis in all strains. The evaluation of diacetyl production revealed that only 22.8 % of strains showed this ability, and most of them were isolated from product ripened in the natural cave. All strains exhibited only leu-aminopeptidase activity, with values more elevated in strains coming from the natural cave product. The combined analysis of genotypic results with the data obtained by the features of technological interest study established that the random amplified polymorphic DNA (RAPD) clusters obtained were composed not only of different genotypes but of different profiles based on technological properties too. This study demonstrated the importance of the ripening environment that affects the typical features of the artisanal product, leading to the selection of a specific surface microflora. Characterized strains could be associated within surface starters to standardize the production process of cheese, but preserving its typical organoleptic and sensory characteristics and improving the quality of the final product.

Efficient recovery of whole cell proteins in Oenococcus oeni--a comparison of different extraction protocols for high-throughput malolactic starter applications.

In this study, we compared different total protein extraction protocols to achieve highly efficient isolation and purification of total proteins for the specific protein profiling of Oenococcus oeni. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns obtained for the different extraction protocols revealed not only a qualitative similar protein pattern but also quantitative variations with different intensity bands depending on the extraction method used. The selected extraction method added with sonication proved to work extremely well and efficiently and was able to obtain a high-resolution 2-D electrophoresis (2-DE) map. Prominent spots were successfully identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry and corresponded to 76 different proteins involved in the main metabolic pathways. The approach allowed to achieve a protein profiling specific for O. oeni from Aglianico wine with numerous characterized protein products corresponding to many different O. oeni genes and associated with main cellular pathways. Further investigations of the 2-DE protein expression profile will provide useful and interesting information on the molecular mechanisms at the protein level responsible for growth and survival of O. oeni in wine.

Fluorescent differential display analysis of Lactobacillus sakei strains under stress conditions.

Lactobacillus (Lb.) sakei is widely used as starter in the production process of Italian fermented sausages and its growth and survival are affected by various factors such as temperature, pH and salt concentration. We studied the behaviour of Lb. sakei strains under various growth conditions relative to acid, osmotic and heat stress treatments by a novel fluorescent differential display (FDD) technique. This study obtained the development and the optimization of a technique that allows the identification of genome expression changes, associated with differential microbial behaviour under different stress conditions with a better stress response definition and a better discrimination of starter cultures. DNA sequence information from the FDD products provided an important tool to assess and observe the response to a variety of environmental stimuli and the adaptation to bacterial stress. Our work provided an innovative FDD method, with a high level of reproducibility and quality for studying and probing the knowledge of the relation between differential genome expression and different stresses tolerance.

Fingerprinting analysis of Oenococcus oeni strains under stress conditions.

Oenococcus oeni strains from traditional Italian red wines of the Basilicata region were investigated on the basis of their physiological and molecular response to different temperatures and ethanol concentrations. All strains were highly resistant to different ethanol concentrations and it has been observed that 7% ethanol was able to stimulate the growth of strains in wine, and 12-13% of ethanol allowed their proliferation. Moreover, strain tolerances to 18 and 42 degrees C were observed. Fingerprinting analysis with fluorescent differential display-PCR and investigation of changes in gene expression during the tolerance process were carried out. The expression gene pattern reflects mechanisms involved in tolerance to environmental conditions. This study establishes and validates a method that enables, with a high reproducibility, different gene expression identification under stress conditions in lactic acid bacteria.

Development and optimization of a fluorescent differential display PCR system for analyzing the stress response in Lactobacillus sakei strains.

Lactobacillus sakei is widely used as starter in the production process of Italian fermented sausages and its growth and survival are affected by various factors. We studied the differential expression of genome in response to different stresses by the fluorescent differential display (FDD) technique. This study resulted in the development and optimization of an innovative technique, with a high level of reproducibility and quality, which allows the identification of gene expression changes associated with different microbial behaviours under different growth conditions.