Biofilms formed by Candida albicans bloodstream isolates display phenotypic and transcriptional heterogeneity that are associated with resistance and pathogenicity

Background: Candida albicans infections have become increasingly recognised as being biofilm related. Recent studies have shown that there is a relationship between biofilm formation and poor clinical outcomes in patients infected with biofilm proficient strains. Here we have investigated a panel of clinical isolates in an attempt to evaluate their phenotypic and transcriptional properties in an attempt to differentiate and define levels of biofilm formation.<p></p> Results: Biofilm formation was shown to be heterogeneous; with isolates being defined as either high or low biofilm formers (LBF and HBF) based on different biomass quantification. These categories could also be differentiated using a cell surface hydrophobicity assay with 24 h biofilms. HBF isolates were more resistance to amphotericin B (AMB) treatment than LBF, but not voriconazole (VRZ). In a Galleria mellonella model of infection HBF mortality was significantly increased in comparison to LBF. Histological analysis of the HBF showed hyphal elements intertwined indicative of the biofilm phenotype. Transcriptional analysis of 23 genes implicated in biofilm formation showed no significant differential expression profiles between LBF and HBF, except for Cdr1 at 4 and 24 h. Cluster analysis showed similar patterns of expression for different functional classes of genes, though correlation analysis of the 4 h biofilms with overall biomass at 24 h showed that 7 genes were correlated with high levels of biofilm, including Als3, Eap1, Cph1, Sap5, Plb1, Cdr1 and Zap1.<p></p> Conclusions: Our findings show that biofilm formation is variable amongst C. albicans isolates, and categorising isolates depending on this can be used to predict how pathogenic the isolate will behave clinically. We have shown that looking at individual genes in less informative than looking at multiple genes when trying to categorise isolates at LBF or HBF. These findings are important when developing biofilm-specific diagnostics as these could be used to predict how best to treat patients infected with C. albicans. Further studies are required to evaluate this clinically.<p></p&gt

Evidence of SARS-CoV-2 in nasal brushings and olfactory mucosa biopsies of COVID-19 patients

The aim of the present study is to detect the presence of SARS-CoV-2 of patients affected by COVID-19 in olfactory mucosa (OM), sampled with nasal brushing (NB) and biopsy, and to assess whether a non-invasive procedure, such as NB, might be used as a large-scale procedure for demonstrating SARS-CoV-2 presence in olfactory neuroepithelium. Nasal brushings obtained from all the COVID-19 patients resulted positive to SARS-CoV-2 immunocytochemistry while controls were negative. Double immunofluorescence showed that SARS-CoV-2 positive cells included supporting cells as well as olfactory neurons and basal cells. OM biopsies showed an uneven distribution of SARS-CoV-2 positivity along the olfactory neuroepithelium, while OM from controls were negative. SARS-CoV-2 was distinctively found in sustentacular cells, olfactory neurons, and basal cells, supporting what was observed in NB. Ultrastructural analysis of OM biopsies showed SARS-CoV-2 viral particles in the cytoplasm of sustentacular cells. This study shows the presence of SARS-CoV-2 at the level of the olfactory neuroepithelium in patients affected by COVID-19. For the first time, we used NB as a rapid non-invasive tool for assessing a potential neuroinvasion by SARS-CoV-2 infection

Unexpected distribution of CA19.9 and other type 1 chain Lewis antigens in normal and cancer tissues of colon and pancreas: Importance of the detection method and role of glycosyltransferase regulation

Background: CA19.9 antigen has been assumed as an abundant product of cancer cells, due to the reactivity found by immunohistochemical staining of cancer tissues with anti-CA19.9 antibody. Methods: Expression and biosynthesis of type 1 chain Lewis antigens in the colon and the pancreas were studied by immunodetection in tissue sections and lysates, quantification of glycosyltransferase transcripts, bisulfite sequencing, and chromatin immunoprecipitation assays. Results: CA19.9 was poorly detectable in normal colon mucosa and almost undetectable in colon cancer, while it was easily detected in the pancreatic ducts, togetherwith Lewis b antigen, under both normal and cancer conditions. B3GALT5 transcripts were down-regulated in colon cancer, while they remained expressed in pancreatic cancer. Even ST3GAL3 transcript appeared well expressed in the pancreas but poorly in the colon, irrespective of normal or cancer conditions. CpG islands flanking B3GALT5 native promoter presented an extremely low degree of methylation in pancreatic cancer with respect to colon cancer. In a DNA region about 1 kb away fromthe B3GALT5 retroviral promoter, a stretch of CG dinucleotides presented a methylation pattern potentially associated with transcription. Such a DNA region and the transcription factor binding site provided overlapping results by chromatin immunoprecipitation assays, corroborating the hypothesis. Conclusions: CA19.9 appears as a physiological product whose synthesis strongly depends on the tissue specific and epigenetically-regulated expression of B3GALT5 and ST3GAL3. General significance: CA19.9 and other Lewis antigens acquire tumor marker properties in the pancreas due to mechanisms giving rise to reabsorption into vessels and elevation in circulating levels.Background CA19.9 antigen has been assumed as an abundant product of cancer cells, due to the reactivity found by immunohistochemical staining of cancer tissues with anti-CA19.9 antibody. Methods Expression and biosynthesis of type 1 chain Lewis antigens in the colon and the pancreas were studied by immunodetection in tissue sections and lysates, quantification of glycosyltransferase transcripts, bisulfite sequencing, and chromatin immunoprecipitation assays. Results CA19.9 was poorly detectable in normal colon mucosa and almost undetectable in colon cancer, while it was easily detected in the pancreatic ducts, together with Lewis b antigen, under both normal and cancer conditions. B3GALT5 transcripts were down-regulated in colon cancer, while they remained expressed in pancreatic cancer. Even ST3GAL3 transcript appeared well expressed in the pancreas but poorly in the colon, irrespective of normal or cancer conditions. CpG islands flanking B3GALT5 native promoter presented an extremely low degree of methylation in pancreatic cancer with respect to colon cancer. In a DNA region about 1\ua0kb away from the B3GALT5 retroviral promoter, a stretch of CG dinucleotides presented a methylation pattern potentially associated with transcription. Such a DNA region and the transcription factor binding site provided overlapping results by chromatin immunoprecipitation assays, corroborating the hypothesis. Conclusions CA19.9 appears as a physiological product whose synthesis strongly depends on the tissue specific and epigenetically-regulated expression of B3GALT5 and ST3GAL3. General significance CA19.9 and other Lewis antigens acquire tumor marker properties in the pancreas due to mechanisms giving rise to reabsorption into vessels and elevation in circulating levels

Myriocin treatment of CF lung infection and inflammation: complex analyses for enigmatic lipids

Our aim was to use quantitative and qualitative analyses to gain further insight into the role of ceramide in cystic fibrosis (CF). Sphingolipid ceramide is a known inflammatory mediator, and its accumulation in inflamed lung has been reported in different types of emphysema, chronic obstructive pulmonary disease and CF. CF is caused by a mutation of the chloride channel and associated with hyperinflammation of the respiratory airways and high susceptibility to ongoing infections. We have previously demonstrated that de novo ceramide synthesis is enhanced in lung inflammation and sustains Pseudomonas aeruginosa pulmonary infection in a CF murine model. We used liquid chromatography and matrix-assisted laser desorption/ionization (MALDI) imaging coupled with mass spectrometry, confocal laser scan microscopy and histology analyses to reveal otherwise undecipherable information. We demonstrated that (i) upregulated ceramide synthesis in the alveoli is strictly related to alveolar infection and inflammation, (ii) alveolar ceramide (C16) can be specifically targeted by nanocarrier delivery of the ceramide synthesis inhibitor myriocin (Myr) and (iii) Myr is able to downmodulate pro-inflammatory lyso-PC, favouring an increase in anti-inflammatory PCs. We concluded that Myr modulates alveolar lipids milieu, reducing hyperinflammation and favouring anti-microbial effective response in CF mouse model

Telomerase in differentiated thyroid cancer: promoter mutations, expression and localization

Telomerase-reverse-transcriptase (TERT) promoter mutations have been recently described in tumors. In the present large series, TERT mutations were found in 12% of papillary thyroid cancers (PTCs) and in 14% of follicular thyroid cancers (FTCs), and were found to significantly correlate with older age at diagnosis and poorer outcome. Interestingly, the prognostic value of TERT mutations resulted to be significantly stronger than that of BRAF(V600E). Moreover, the outcome was not different among tumors with isolated TERT mutation and those with coexistent mutations (TERT/BRAF in PTCs or TERT/RAS in FTCs). TERT rs2853669 polymorphism was found in 44.4% of tumors. At WB, TERT was significantly more expressed in tumors than in normal samples, being the highest levels of expression recorded in TERT mutated cases. At IHC, in tumors and in metastatic lymph-nodes TERT staining was significantly higher in the cytoplasm than in the nucleus, whereas in normal tissue the degree of staining did not differ in the two cellular compartments. In conclusion, TERT mutations were shown to strongly correlate with a poorer outcome in differentiated thyroid tumors, and neither BRAF nor RAS mutation were found to confer an additional effect in the disease persistence. TERT protein was found to be more expressed in neoplastic than in normal tissues, and to display a different cellular localization, suggesting that it could contribute to thyroid cancer progression by mechanisms taking place in the cytoplasm

Jagged Ligands Enhance the Pro-Angiogenic Activity of Multiple Myeloma Cells

Multiple myeloma (MM) is an incurable plasma cell malignancy arising primarily within the bone marrow (BM). During MM progression, different modifications occur in the tumor cells and BM microenvironment, including the angiogenic shift characterized by the increased capability of endothelial cells to organize a network, migrate and express angiogenic factors, including vascular endothelial growth factor (VEGF). Here, we studied the functional outcome of the dysregulation of Notch ligands, Jagged1 and Jagged2, occurring during disease progression, on the angiogenic potential of MM cells and BM stromal cells (BMSCs). Jagged1–2 expression was modulated by RNA interference or soluble peptide administration, and the effects on the MM cell lines’ ability to induce human pulmonary artery cells (HPAECs) angiogenesis or to indirectly increase the BMSC angiogenic potential was analyzed in vitro; in vivo validation was performed on a zebrafish model and MM patients’ BM biopsies. Overall, our results indicate that the MM-derived Jagged ligands (1) increase the tumor cell angiogenic potential by directly triggering Notch activation in the HPAECs or stimulating the release of angiogenic factors, i.e., VEGF; and (2) stimulate the BMSCs to promote angiogenesis through VEGF secretion. The observed pro-angiogenic effect of Notch activation in the BM during MM progression provides further evidence of the potential of a therapy targeting the Jagged ligands