BackgroundCandida 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.ResultsBiofilm 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.ConclusionsOur 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.

Survivin is an inhibitor of apoptosis protein, overexpressed in most human malignancies and implicated in mitosis regulation and preservation of cell viability. In order to investigate the prevalence and clinical significance of survivin in early-stage non-small cell lung carcinoma (NSCLC), survivin mRNA levels and protein expression were evaluated, using quantitative real-time RT-PCR and immunohistochemistry, respectively, in a series of 83 patients with stage I (IA and IB) surgically resected NSCLC. Detectable survivin mRNA levels could be demonstrated in all non-neoplastic lung tissue samples and in the tumours analysed. Survivin mRNA levels were elevated in 80 carcinomas (96%) compared to normal lung (p = 0.008). Among all tumours, survivin transcripts were present at a higher level in squamous cell carcinomas (p = 0.0022). Cytoplasmic and nuclear immunoreactivity was found in 70% and 80% of tumours, respectively and both were present in 54%. Cytoplasmic immunoreactivity correlated with tumour stage (p = 0.019). Survivin expression levels did not correlate with patient survival. In one specimen, cytoplasmic and focal nuclear immunostaining was observed in dysplastic bronchial squamous metaplasia. These results document that survivin overexpression is almost always present in early-stage NSCLC, suggesting that this protein may play a role in lung tumourigenesis. This ubiquitous expression makes survivin an appealing new target for novel therapies in lung cancer. In addition, this study also documents that survivin overexpression could be exploited for diagnostic purposes and that quantitative real-time RT-PCR can be a useful tool for evaluating survivin activation in NSCLC.

Aims: A considerable proportion of patients affected by coronavirus respiratory disease (COVID-19) develop cardiac injury. The viral impact in cardiomyocytes deserves, however, further investigations, especially in asymptomatic patients. Methods: We investigated for SARS-CoV-2 presence and activity in heart tissues of six consecutive COVID-19 patients deceased from respiratory failure showing no signs of cardiac involvement and with no history of heart disease. Cardiac autopsy samples were collected within 2 h after death, and then analysed by digital PCR, Western blot, immunohistochemistry, immunofluorescence, RNAScope, and transmission electron microscopy assays. Results: The presence of SARS-CoV-2 into cardiomyocytes was invariably detected in all assays. A variable pattern of cardiomyocyte injury was observed, spanning from absence of cell death and subcellular alterations hallmarks, to intracellular oedema and sarcomere ruptures. In addition, we found active viral transcription in cardiomyocytes, by detecting both sense and antisense SARS-CoV-2 spike RNA. Conclusions: In this autopsy analysis of patients with no clinical signs of cardiac involvement, the presence of SARS-CoV-2 in cardiomyocytes has been detected, determining variable patterns of intracellular damage. These findings suggest the need for cardiologic surveillance in surviving COVID-19 patients not displaying a cardiac phenotype.

Burkholderia multivorans is a significant health threat to persons with cystic fibrosis (CF). Infections are difficult to treat as this pathogen is inherently resistant to multiple antibiotics. Susceptibility testing of isolates obtained from CF respiratory cultures revealed that single agents selected from different antibiotic classes were unable to inhibit growth. However, all isolates were found to be susceptible to ceftazidime when combined with the novel non-β-lactam β-lactamase inhibitor, avibactam (all minimum inhibitor concentrations (MICs) were ≤8 mg/L of ceftazidime and 4 mg/L of avibactam). Furthermore, a major β-lactam resistance determinant expressed in B. multivorans, the class A carbapenemase, PenA was readily inhibited by avibactam with a high k2/K of (2 ± 1) × 106 μM−1 s−1 and a slow koff of (2 ± 1) × 10−3 s−1. Mass spectrometry revealed that avibactam formed a stable complex with PenA for up to 24 h and that avibactam recyclized off of PenA, re-forming the active compound. Crystallographic analysis of PenA–avibactam revealed several interactions that stabilized the acyl–enzyme complex. The deacylation water molecule possessed decreased nucleophilicity, preventing decarbamylation. In addition, the hydrogen-bonding interactions with Lys-73 were suggestive of a protonated state. Thus, Lys-73 was unlikely to abstract a proton from Ser-130 to initiate recyclization. Using Galleria mellonella larvae as a model for infection, ceftazidime–avibactam was shown to significantly (p < 0.001) improve survival of larvae infected with B. multivorans. To further support the translational impact, the ceftazidime–avibactam combination was evaluated using susceptibility testing against other strains of Burkholderia spp. that commonly infect individuals with CF, and 90% of the isolates were susceptible to the combination. In summary, ceftazidime–avibactam may serve as a preferred therapy for people that have CF and develop Burkholderia spp. infections and should be considered for clinical trials.

Skin malignant melanoma (MM) is an aggressive cancer with an increasing incidence with limited therapies in advanced stages. Tumor-associated macrophages (TAMs) are the major immune constituent of the MM microenvironment and contribute toward its prognosis. TAMs' characterization and localization in human cancer is important to understand cancer progression and to identify molecular personalized therapies. M2 TAMs in stage I-II MMs are associated with worse prognostic parameters. A comprehensive M1-macrophage and M2-macrophage intratumoral localization and quantification in all stages of skin MMs is documented here with its clinical significance. To highlight immune pathways and possible early indicators of MM progression, we evaluated the number of M1 and M2 TAMs and intratumoral distribution in a large series of skin MMs. CD68 double immunostaining with MRP8-14 or inducible nitric oxide synthase (M1 macrophages) and with CD163 or CD204 (M2 macrophages) was performed in 94 stage I-IV skin MMs with a long duration of follow-up. The accumulation and distribution of M1 and M2 TAMs in intratumoral nests, stroma, and at the invasive front was correlated with clinicopathological variables. Since the early stage of MMs, M1 intratumoral macrophages were fewer than the M2 population; their recruitment was rapidly and progressively overwhelmed by an increase in M2 TAMs during MM progression. Independent of their intratumoral distribution, the accumulation of both M1 and M2 TAMs is associated with poor prognostic indicators and patients' survival. M1-recruited macrophages shift to the M2 phenotype early in MM development, possibly induced by high inducible nitric oxide synthase intratumoral increase peculiarly occurring since the initial MM stages. M2-recruited TAMs overwhelm M1 accumulation in all stages of MM progression, thus favoring neoplastic growth and dissemination. Independent of their intratumoral distribution, the prevalent accumulation of M2 TAMs in MM is statistically confirmed to be a poor indicator of patients' outcome and a potential target of immune therapies.