Water Extracts from Industrial Hemp Waste Inhibit the Adhesion and Development of Candida Biofilm and Showed Antioxidant Activity on HT-29 Colon Cancer Cells.

The evolution of regulatory perspectives regarding the health and nutritional properties of industrial hemp-based products (Cannabis sativa L.) has pushed research to focus on the development of new methods for both the extraction and formulation of the bioactive compounds present in hemp extracts. While the psychoactive and medicinal properties of hemp-derived cannabinoid extracts are well known, much less has been investigated on the functional and antimicrobial properties of hemp extracts. Within the hemp value chain, various agricultural wastes and by-products are generated. These materials can be valorised through eco-innovations, ultimately promoting sustainable economic development. In this study, we explored the use of waste from industrial light cannabis production for the extraction of bioactive compounds without the addition of chemicals. The five extracts obtained were tested for their antimicrobial activity on both planktonic and sessile cells of pathogenic strains of the Candida albicans, Candida parapsilosis, and Candida tropicalis species and for their antioxidant activity on HT-29 colon cancer cells under oxidative stress. Our results demonstrated that these extracts display interesting properties both as antioxidants and in hindering the development of fungal biofilm, paving the way for further investigations into the sustainable valorisation of hemp waste for different biomedical applications.

Metabolomic and Proteomic Changes in Candida albicans Biofilm in Response to Zosteric Acid Treatment.

Zosteric acid (ZA) is a secondary metabolite of the seagrass Zostera marina, with antibiofilm activity against fungi. Information concerning its mechanisms of action is lacking and this limits the development of more potent derivatives based on the same target and activity structure. The aim of this work was to investigate the ZA mode of action by analyzing the metabolic status of Candida albicans biofilm and its protein expression profile upon ZA treatment. Fourier-Transform Infrared Spectroscopy confirmed that ZA modified the metabolomic response of treated cells, showing changes in the spectral regions, mainly related to the protein compartment. Nano Liquid Chromatography-High-Resolution Mass Spectrometry highlighted that 10 proteins were differentially expressed in the C. albicans proteome upon ZA treatment. Proteins involved in the biogenesis, structure and integrity of cell walls as well as adhesion and stable attachment of hyphae were found downregulated, whereas some proteins involved in the stress response were found overexpressed. Additionally, ZA was involved in the modulation of non-DNA-based epigenetic regulatory mechanisms triggered by reactive oxygen species. These results partially clarified the ZA mechanism of action against fungi and provided insight into the major C. albicans pathways responsible for biofilm formation.

One year in review 2021: fibromyalgia.

Fibromyalgia (FM) is a syndrome of unknown aetiology characterised by chronic pain, fatigue, and disturbed sleep. This review presents and summarises the 2020 literature on FM by retrieving all articles indexed in PubMed between 1 January 2020 and 31 December 2020. The attention of the scientific community towards FM is constantly growing, and this year's review is focused on the diagnostic, pathogenetic and therapeutic aspects of this syndrome. In particular, the treatment options for FM, both pharmacological and non-pharmacological, have been extensively studied.

How does temperature trigger biofilm adhesion and growth in Candida albicans and two non-Candida albicans Candida species?

BACKGROUND: Biofilm formation on biotic and abiotic surfaces is finely regulated by genetic factors but also by oxygen concentration, pH, temperature and other environmental factors, already extensively explored for bacterial biofilms. Much less is known about fungal biofilm, that is considered a virulence factor for Candida pathogenic species among the few fungal species able to grow and survive at high temperatures such as 37°C as well as those induced by fever. The resistance to high temperatures coupled with the ability to form biofilm are threatening factors of these fungal species that could severely impact at an epidemiological level. OBJECTIVES: In this framework, we decided to study the thermal tolerance of biofilms formed by three medical relevant species such as Candida albicans and two non-Candida albicans Candida species. METHODS: Thirty nosocomial strains were investigated for their ability to adhere and grow in proximity and over body temperature (from 31 to 43°C), mimicking different environmental conditions or severe febrile-like reactions. RESULTS: Candida sessile cells reacted to different temperatures showing a strain-specific response. It was observed that the attachment and growth respond differently to the temperature and that mechanism of adhesion has different outputs at high temperature than the growth. CONCLUSIONS: This strain-dependent response is probably instrumental to guarantee the best success to cells for the infection, attachment and growth to occur. These observations reinforce the concept of temperature as a major trigger in the evolution of these species especially in this period of increasing environmental temperatures and excessive domestic heating.

Candida palmioleophila isolation in Italy from two cases of systemic infection, after a CHROMagar and Vitek system mis-identification as C. albicans.

A correct, fast, reliable identification method is pivotal in nosocomial environments to guide treatment strategies, whereas misidentification might lead to treatment failure. For routine identifications the Vitek system and CHROMagar are widely used but not always reliable, especially now with an increasing number of new emerging fungal pathogens that need careful identification. Here we describe two cases of candidemia, due to Candida palmioleophila previously misidentified as Candida albicans by using the Vitek2 system and CHROMagar. The first case is a 54-year-old man with an infected ulcer in the lower right limb, treated with a targeted therapy using a central venous catheter (CVC). After two months he developed a CVC-related candidemia MDR identified as C. albicans. The second case is a 2-month-old male baby that was admitted to the neonatal unit with acute respiratory failure due to a severe community-acquired bilateral pneumonia; blood cultures were all positive for C. albicans MDR. The isolated strains where re-identified with Maldi-Tof and DNA sequencing as C. palmioleophila. From the identification point of view, CHROMagar can be clearly misleading, especially because CHROMagar types currently available are not designed to discriminate new emerging species, suggesting that systems other than MALDI-TOF and marker sequencing may be inadequate even for routine identification and could contribute to producing misleading identifications and therapeutically wrong practices, leading to failures and patient death.

α-synuclein and synapsin III cooperatively regulate synaptic function in dopamine neurons.

The main neuropathological features of Parkinson's disease are dopaminergic nigrostriatal neuron degeneration, and intraneuronal and intraneuritic proteinaceous inclusions named Lewy bodies and Lewy neurites, respectively, which mainly contain α-synuclein (α-syn, also known as SNCA). The neuronal phosphoprotein synapsin III (also known as SYN3), is a pivotal regulator of dopamine neuron synaptic function. Here, we show that α-syn interacts with and modulates synapsin III. The absence of α-syn causes a selective increase and redistribution of synapsin III, and changes the organization of synaptic vesicle pools in dopamine neurons. In α-syn-null mice, the alterations of synapsin III induce an increased locomotor response to the stimulation of synapsin-dependent dopamine overflow, despite this, these mice show decreased basal and depolarization-dependent striatal dopamine release. Of note, synapsin III seems to be involved in α-syn aggregation, which also coaxes its increase and redistribution. Furthermore, synapsin III accumulates in the caudate and putamen of individuals with Parkinson's disease. These findings support a reciprocal modulatory interaction of α-syn and synapsin III in the regulation of dopamine neuron synaptic function.

Toll Like Receptor 4 Affects the Cerebral Biochemical Changes Induced by MPTP Treatment.

The etiology and pathogenesis of Parkinson's disease (PD) are still unclear. However, multiple lines of evidence suggest a critical role of the toll like receptor 4 (TLR4) in inflammatory response and neuronal death. Neuroinflammation may be associated with the misfolding and aggregation of proteins accompanied by a change in their secondary structure. Recent findings also suggest that biochemical perturbations in cerebral lipid content could contribute to the pathogenesis of central nervous system (CNS) disorders, including PD. Thus, it is of great importance to determine the biochemical changes that occur in PD. In this respect, Fourier Transform Infrared (FTIR) spectroscopy represents a useful tool to detect molecular alterations in biological systems in response to stress stimuli. By relying upon FTIR approach, this study was designed to elucidate the potential role of TLR4 in biochemical changes induced by methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin in a mouse model of PD. The analysis of the FTIR spectra was performed in different brain regions of both wild type (WT) and toll like receptor 4-deficient (TLR4-/-) mice. It revealed that each brain region exhibited a characteristic molecular fingerprint at baseline, with no significant differences between genotypes. Conversely, WT and TLR4-/- mice showed differential biochemical response to MPTP toxicity, principally related to lipid and protein composition. These differences appeared to be characteristic for each brain area. Furthermore, the present study showed that WT mice resulted more vulnerable than TLR4-/- animals to striatal dopamine (DA) depletion following MPTP treatment. These results support the hypothesis of a possible involvement of TLR4 in biochemical changes occurring in neurodegeneration.

Invasive listeriosis in a patient with several episodes of antibiotic associated colitis presumably due to Clostridium difficile.

A 62-year-old man developed a blood stream infection and meningitis due to Listeria monocytogenes, 20 days after an episode of pseudo-membranous colitis. The patient, hospitalized for the first time for transurethral prostatectomy, was readmitted 20 days later with watery diarrhea. Pseudo-membranous colitis was diagnosed and treated successfully, without testing for Clostridium difficile infection (CDI). After 15 more days, the patient developed again diarrhea, fever and confusion. Hospitalized again, blood and cerebrospinal fluid cultures resulted positive for L. monocytogenes. The patient was treated successfully and a diagnosis of recurrent CDI was confirmed following culture and nucleic acid amplification assays both positive for C. difficile. This is the first report of an invasive listeriosis after CDI underlines the importance of taking greater awareness in complicated blood stream infections that may arise after CDI.

Ionic Conductivity as a Tool To Study Biocidal Activity of Sulfobetaine Micelles against Saccharomyces cerevisiae Model Cells.

Zwitterionic sulfobetaine surfactants are used in pharmaceutical or biomedical applications for the solubilization and delivery of hydrophobic molecules in aqueous medium or in biological environments. In a screening on the biocidal activity of synthetic surfactants on microbial cells, remarkable results have emerged with sulfobetaine amphiphiles. The interaction between eight zwitterionic sulfobetaine amphiphiles and Saccharomyces cerevisiae model cells was therefore analyzed. S. cerevisiae yeast cells were chosen, as they are a widely used unicellular eukaryotic model organism in cell biology. Conductivity measurements were used to investigate the interaction between surfactant solution and cells. Viable counts measurements were performed, and the mortality data correlated with the conductivity profiles very well, in terms of the inflection points (IPs) observed in the curves and in terms of supramolecular properties of the aggregates. A Fourier transform infrared (FTIR)-based bioassay was then performed to determine the metabolomic stress-response of the cells subjected to the action of zwitterionic surfactants. The surfactants showed nodal concentration (IPs) with all the techniques in their activities, corresponding to the critical micellar concentrations of the amphiphiles. This is due to the pseudocationic behavior of sulfobetaine micelles, because of their charge distribution and charge densities. This behavior permits the interaction of the micellar aggregates with the cells, and the structure of the surfactant monomers has impact on the mortality and the metabolomic response data observed. On the other hand, the concentrations that are necessary to provoke a biocidal activity do not promote these amphiphiles as potential antimicrobial agents. In fact, they are much higher than the ones of cationic surfactants.

First Case of Trichoderma longibrachiatum CIED (Cardiac Implantable Electronic Device)-Associated Endocarditis in a Non-immunocompromised Host: Biofilm Removal and Diagnostic Problems in the Light of the Current Literature.

BACKGROUND: Trichoderma species are saprophytic filamentous fungi producing localized and invasive infections that are cause of morbidity and mortality, especially in immunocompromised patients, causing up to 53% mortality. Non-immunocompromised patients, undergoing continuous ambulatory peritoneal dialysis, are other targets of this fungus. Current molecular diagnostic tools, based on the barcode marker ITS, fail to discriminate these fungi at the species level, further increasing the difficulty associated with these infections and their generally poor prognosis. CASE REPORT: We report on the first case of endocarditis infection caused by Trichoderma longibrachiatum in a 30-year-old man. This patient underwent the implantation of an implantable cardioverter defibrillator in 2006, replaced in 2012. Two years later, the patient developed fever, treated successfully with amoxicillin followed by ciprofloxacin, but an echocardiogram showed large vegetation onto the ventricular lead. After CIED extraction, the patient had high-grade fever. The culturing of the catheter tip was positive only in samples deriving from sonication according to the 2014 ESCMID guidelines, whereas the simple washing failed to remove the biofilm cells from the plastic surface. Subsequent molecular (ITS sequencing) and microbiological (macromorphology) analyses showed that the vegetation was due to T. longibrachiatum. CONCLUSIONS: This report showed that T. longibrachiatum is an effective threat and that sonication is necessary for the culturing of vegetations from plastic surfaces. Limitations of the current barcode marker ITS, and the long procedures required by a multistep approach, call for the development of rapid monophasic tests.

Phenotypic and molecular diversity of Meyerozyma guilliermondii strains isolated from food and other environmental niches, hints for an incipient speciation.

Meyerozyma guilliermondii is a yeast species widely isolated from several natural environments and from fruit; in medical microbiology it is known as the teleomorph of the opportunistic pathogen Candida guilliermondii, which causes about 2% of the human blood infections. This yeast is also promising in a variety of biotechnological applications as vitamins production and post-harvest control. The question if isolates from different sources are physiologically and genetically similar, or if the various environments induced significant differences, is crucial for the understanding of this species structure and to select strains appropriate for each application. This question was addressed using LSU and ITS sequencing for taxonomic assignment, i-SSR (GACA4) for the molecular characterization and FTIR for the metabolomic fingerprint. All data showed that fruit and environmental isolates cluster separately with a general good agreement between metabolomics and molecular analysis. An additional RAPD analysis was able to discriminate strains according to the isolation position within the pineapple fruit. Although all strains are members of the M. guilliermondii species according to the current standards, the distribution of large variability detected suggests that some specialization occurred in the niches inhabited by this yeast and that food related strains can be differentiated from the medical isolates.

Candida milleri species reveals intraspecific genetic and metabolic polymorphisms.

Candida milleri, together with Candida humilis, is the most representative yeast species found in type I sourdough ecosystems. In this work, comparison of the ITS region and the D1/D2 domain of 26S rDNA gene partial sequences, karyotyping, mtDNA-RFLP analysis, Intron Splice Site dispersion (ISS-PCR) and (GTG)5 microsatellite analyses, assimilation test of different carbohydrates, and metabolome assessment by FT-IR analysis, were investigated in seventeen strains isolated from four different companies as well as in type strains CBS6897(T) and CBS5658(T). Most isolates were ascribed to C. milleri, even if a strong relatedness was confirmed with C. humilis as well, particularly for three strains. Genetic characterization showed a high degree of intraspecific polymorphism since 12 different genotypes were discriminated. The number of chromosomes varied from 9 to 13 and their size ranged from less than 0.3 to over 2 Mbp. Phenotypic traits let to recognize 9 different profiles of carbon sources assimilation. FT-IR spectra from yeast cells cultivated in different media and collected at different growth phases revealed a diversity of behaviour among strains in accordance with the results of PCR-based fingerprinting. A clear evidence of the polymorphic status of C. milleri species is provided thus representing an important feature for the development of technological applications in bakery industries.

Assessment of safety and efficiency of nitrogen organic fertilizers from animal-based protein hydrolysates--a laboratory multidisciplinary approach.

BACKGROUND: Protein hydrolysates or hydrolysed proteins (HPs) are high-N organic fertilizers allowing the recovery of by-products (leather meal and fluid hydrolysed proteins) otherwise disposed of as polluting wastes, thus enhancing matter and energy conservation in agricultural systems while decreasing potential pollution. Chemical and biological characteristics of HPs of animal origin were analysed in this work to assess their safety, environmental sustainability and agricultural efficacy as fertilizers. Different HPs obtained by thermal, chemical and enzymatic hydrolytic processes were characterized by Fourier transform infrared spectroscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis, and their safety and efficacy were assessed through bioassays, ecotoxicological tests and soil biochemistry analyses. RESULTS: HPs can be discriminated according to their origin and hydrolysis system by proteomic and metabolomic methods. Three experimental systems, soil microbiota, yeast and plants, were employed to detect possible negative effects exerted by HPs. The results showed that these compounds do not significantly interfere with metabolomic activity or the reproductive system. CONCLUSION: The absence of toxic and genotoxic effects of the hydrolysates prepared by the three hydrolytic processes suggests that they do not negatively affect eukaryotic cells and soil ecosystems and that they can be used in conventional and organic farming as an important nitrogen source derived from otherwise highly polluting by-products.

Yamadazyma terventina sp. nov., a yeast species of the Yamadazyma clade from Italian olive oils.

During an investigation of olive oil microbiota, three yeast strains were found to be divergent from currently classified yeast species according to the sequences of the D1/D2 domain of the gene encoding the rRNA large subunit (LSU) and the internal transcribed spacer region including the gene for 5.8S rRNA. Phylogenetic analysis revealed that these strains, designated CBS 12509, CBS 12510(T) and CBS 12511, represent a novel anascosporogenous species described herein as Yamadazyma terventina sp. nov; the type strain is DAPES 1924(T) (= CBS 12510(T) = NCAIM Y.02028(T)). This novel species was placed in the Yamadazyma clade, with Yamadazyma scolyti, Candida conglobata and Candida aaseri as closest relatives. Y. terventina differs from the above-mentioned species in the ability to strongly assimilate dl-lactate and weakly assimilate ethanol.

MinION Sequencing of Yeast Mock Communities To Assess the Effect of Databases and ITS-LSU Markers on the Reliability of Metabarcoding Analysis.

Microbial communities play key roles both for humans and the environment. They are involved in ecosystem functions, maintaining their stability, and provide important services, such as carbon cycle and nitrogen cycle. Acting both as symbionts and as pathogens, description of the structure and composition of these communities is important. Metabarcoding uses ribosomal DNA (rDNA) (eukaryotic) or rRNA gene (prokaryotic) sequences for identification of species present in a site and measuring their abundance. This procedure requires several technical steps that could be source of bias producing a distorted view of the real community composition. In this work, we took advantage of an innovative "long-read" next-generation sequencing (NGS) technology (MinION) amplifying the DNA spanning from the internal transcribed spacer (ITS) to large subunit (LSU) that can be read simultaneously in this platform, providing more information than "short-read" systems. The experimental system consisted of six fungal mock communities composed of species present at various relative amounts to mimic natural situations characterized by predominant and low-frequency species. The influence of the sequencing platform (MinION and Illumina MiSeq) and the effect of different reference databases and marker sequences on metagenomic identification of species were evaluated. The results showed that the ITS-based database provided more accurate species identification than LSU. Furthermore, a procedure based on a preliminary identification with standard reference databases followed by the production of custom databases, including only the best outputs of the first step, is proposed. This additional step improved the estimate of species proportion of the mock communities and reduced the number of ghost species not really present in the simulated communities. IMPORTANCE Metagenomic analyses are fundamental in many research areas; therefore, improvement of methods and protocols for the description of microbial communities becomes more and more necessary. Long-read sequencing could be used for reducing biases due to the multicopy nature of rDNA sequences and short-read limitations. However, these novel technologies need to be assessed and standardized with controlled experiments, such as mock communities. The interest behind this work was to evaluate how long reads performed identification and quantification of species mixed in precise proportions and how the choice of database affects such analyses. Development of a pipeline that mitigates the effect of the barcoding sequences and the impact of the reference database on metagenomic analyses can help microbiome studies go one step further.

Metabolic Syndrome and Its Components Have a Different Presentation and Impact as Cardiovascular Risk Factors in Psoriatic and Rheumatoid Arthritis.

Patients with chronic inflammatory arthritis have a higher cardiovascular (CV) risk than the general population. Traditional CV risk factors are clearly implicated, while the impact of metabolic syndrome (MetS) is less defined. The aim of this study was to compare MetS prevalence and impact on the CV risk in psoriatic arthritis (PsA) versus rheumatoid arthritis (RA). A retrospective analysis of real-world data of PsA and RA patients referred to a rheumatology clinic was conducted. The following data were extracted and compared: demographic data; clinical data; presence of traditional CV risk factors and MetS. Univariate and multivariate models were used to compare the impact of MetS and its components in patients with PsA versus RA. Overall, 170 patients were included (PsA: 78; RA; 92). The two groups differed significantly in mean age, disease duration, and presence of MetS, while other variables were comparable. Univariate and multivariate analysis identified distinct predictors of MetS in PsA (hypertension) and RA (dyslipidemia). The history of CV events was similar in the two groups. Predictors of CV events were MetS and most of its components in PsA, while dyslipidemia was the strongest predictor in RA. These associations were stronger in PsA than in RA. In conclusion, the impact of MetS and its components is different in PsA and RA. The association of these risk factors with CV events is stronger in PsA than in RA. This suggests the implication of different mechanisms, which may require distinct strategies for the prevention of CV events in PsA and RA.

Renewing Lost Genetic Variability with a Classical Yeast Genetics Approach.

Due to their long domestication time course, many industrial Saccharomyces cerevisiae strains are adopted in numerous processes mostly for historical reasons instead of scientific and technological needs. As such, there is still significant room for improvement for industrial yeast strains relying on yeast biodiversity. This paper strives to regenerate biodiversity with the innovative application of classic genetic methods to already available yeast strains. Extensive sporulation was indeed applied to three different yeast strains, specifically selected for their different origins as well as backgrounds, with the aim of clarifying how new variability was generated. A novel and easy method to obtain mono-spore colonies was specifically developed, and, to reveal the extent of the generated variability, no selection after sporulation was introduced. The obtained progenies were then tested for their growth in defined mediums with high stressor levels. A considerable and strain-specific increase in both phenotypic and metabolomic variability was assessed, and a few mono-spore colonies were found to be of great interest for their future exploitation in selected industrial processes.

Evolution of Rheumatoid-Arthritis-Associated Interstitial Lung Disease in Patients Treated with JAK Inhibitors: A Retrospective Exploratory Study.

BACKGROUND: The aim of this multicenter retrospective study was to investigate the effectiveness and safety of the available JAK-inhibitors (JAKi) in patients with rheumatoid arthritis (RA) and interstitial lung disease (ILD). METHODS: We retrospectively analyzed patients with classified RA and RA-ILD undergoing JAKi in 6 Italian tertiary centers from April 2018 to June 2022. We included patients with at least 6 months of active therapy and one high-resolution chest tomography (HRCT) carried out within 3 months of the start of JAKi treatment. The HRCT was then compared to the most recent one carried out within 3 months before the last available follow-up appointment. We also kept track of the pulmonary function tests. RESULTS: We included 43 patients with RA-ILD and 23 males (53.48%) with a median age (interquartile range, IQR) of 68.87 (61.46-75.78) treated with JAKi. The median follow-up was 19.1 months (11.03-34.43). The forced vital capacity remained stable in 22/28 (78.57%) patients, improved in 3/28 (10.71%) and worsened in 3/28 (10.71%). The diffusing capacity of lung for carbon monoxide showed a similar trend, remaining stable in 18/25 (72%) patients, improving in 2/25 (8%) and worsening in 5/25 (20%). The HRCT remained stable in 37/43 (86.05) cases, worsened in 4/43 (9.30%) and improved in the last 2 (4.65%). DISCUSSION: This study suggests that JAKi therapy might be a safe therapeutic option for patients with RA-ILD in a short-term follow-up.

Candida coquimbonensis sp. nov., a link between Australian and Nearctic/Neotropical Phaffomyces.

A novel species of ascomycetous yeast, Candida coquimbonensis sp. nov., from the necrotic tissue of cacti in Chile and Australia is described. C. coquimbonensis sp. nov. is closely related and phenotypically similar to Phaffomyces opuntiae. There is no overlap in the geographical distribution between C. coquimbonensis and any species in the Phaffomyces clade. However, this is the first member of the clade to be collected in both native (Chile) and non-native (Australia) cactus habitats. The type strain of C. coquimbonensis sp. nov. is TSU 00-206.4B(T) ( = CBS 12348(T) = USCFST 12-103(T)).

Kazachstania ichnusensis sp. nov., a diploid homothallic ascomycetous yeast from Sardinian lentisk rhizosphere.

During an investigation of yeast biota in the rhizosphere of lentisk in Sardinian semi-arid areas, a strain was isolated that could not be assigned to any known species. The sequence of the D1/D2 domain of the large subunit rDNA gene revealed that the strain belonged to the genus Kazachstania and was phylogenetically related to a clade including Kazachstania aerobia, Kazachstania servazzii, Kazachstania solicola and Kazachstania unispora. The novel isolate differed from members of this clade in its ability to assimilate D-glucono-1,5-lactone and its very weak fermentation of glucose and sucrose; its assimilation profile was unique within the genus Kazachstania. Monosporal colonies were able to sporulate, indicating that the species is homothallic. It is proposed that the isolate represents a novel species, Kazachstania ichnusensis sp. nov., with LCF 1675(T) (=CBS 11859(T)) as type strain.