Hsp90 governs dispersion and drug resistance of fungal biofilms

Fungal biofilms are a major cause of human mortality and are recalcitrant to most treatments due to intrinsic drug resistance. These complex communities of multiple cell types form on indwelling medical devices and their eradication often requires surgical removal of infected devices. Here we implicate the molecular chaperone Hsp90 as a key regulator of biofilm dispersion and drug resistance. We previously established that in the leading human fungal pathogen, Candida albicans, Hsp90 enables the emergence and maintenance of drug resistance in planktonic conditions by stabilizing the protein phosphatase calcineurin and MAPK Mkc1. Hsp90 also regulates temperature-dependent C. albicans morphogenesis through repression of cAMP-PKA signalling. Here we demonstrate that genetic depletion of Hsp90 reduced C. albicans biofilm growth and maturation in vitro and impaired dispersal of biofilm cells. Further, compromising Hsp90 function in vitro abrogated resistance of C. albicans biofilms to the most widely deployed class of antifungal drugs, the azoles. Depletion of Hsp90 led to reduction of calcineurin and Mkc1 in planktonic but not biofilm conditions, suggesting that Hsp90 regulates drug resistance through different mechanisms in these distinct cellular states. Reduction of Hsp90 levels led to a marked decrease in matrix glucan levels, providing a compelling mechanism through which Hsp90 might regulate biofilm azole resistance. Impairment of Hsp90 function genetically or pharmacologically transformed fluconazole from ineffectual to highly effective in eradicating biofilms in a rat venous catheter infection model. Finally, inhibition of Hsp90 reduced resistance of biofilms of the most lethal mould, Aspergillus fumigatus, to the newest class of antifungals to reach the clinic, the echinocandins. Thus, we establish a novel mechanism regulating biofilm drug resistance and dispersion and that targeting Hsp90 provides a much-needed strategy for improving clinical outcome in the treatment of biofilm infections

Incremental Prognostic Value of Echocardiographic Strain and Its Association with Mortality in Cancer Patients

Background Left ventricular global longitudinal systolic strain (GLS) has been shown to be superior to ejection fraction in detecting subclinical dysfunction in patients with cancer and predicting mortality in patients with cardiovascular disease. Cancer-related fatigue is common in the later stages of neoplastic malignancies and may be indicative of nonovert heart failure. The aim of this study was to determine whether reduced strain by echocardiography was associated with all-cause mortality in a cancer cohort. Methods In this retrospective study, 120 patients with cancer undergoing or scheduled to undergo chemotherapy and with normal ejection fractions (>50%) underwent assessments of GLS. GLS was derived by averaging all speckle-tracking strain segments of the left ventricle. Results Over an average follow-up period of 21.6 ± 13.9 months, 57 of 120 patients died. Univariate predictors of all-cause mortality (P < .10) were Eastern Cooperative Oncology Group performance status, male sex, hematologic malignancy, β-blocker use, and GLS. Multivariate analysis of all significant univariate variables showed that Eastern Cooperative Oncology Group performance status (hazard ratio, 2.12; 95% confidence interval, 1.54–2.92; P < .001), male sex (hazard ratio, 1.93; 95% confidence interval, 1.14–3.27; P = .014), and GLS (hazard ratio, 0.89; 95% confidence interval, 0.81–0.97; P = .012) were significantly and independently associated with mortality. Stepwise analysis of the multivariate associations showed an increase in the global χ2 value after adding GLS (P = .011) to significant clinical variables. Conclusions Eastern Cooperative Oncology Group performance status, male sex, and GLS were significantly associated with all-cause mortality in patients with cancer with normal ejection fractions receiving chemotherapy. Adding GLS to significant clinical variables provided incremental prognostic information

Presence of extracellular DNA in candida albicans biofilm matrix and its role in biofilm structure and antifungal susceptibility

Biofilms are structurally complex microconsortia of surface adhering cells embedded within an extracellular matrix (ECM) composed of substances produced and secreted by cells or derived from cell lysis. One of the recently discovered bacterial biofilms ECM components is the extracellular DNA (eDNA). Although the investigation on eDNA in fungal biofilms is scarce, preliminary studies suggest that eDNA may play a role in biofilms formed by the opportunistic fungal pathogen Candida albicans. Thus, the present study aimed at determining the eDNA content of C. albicans SC5314 biofilm ECM and the effect of DNase I treatment on biofilm formation and biofilm cells susceptibility to antifungals, as indicators of the role of eDNA in C. albicans biofilms. Results from our experiments showed that the ECM of C. albicans biofilms formed under conditions of flow for 48 h contained 3045.4 ± 227.3 ng eDNA/mg of protein. Additionally, using a microtiter plate model, we observed that different DNase treatments (0.02 - 2 mg/ml) did not affect further biofilm development by C. albicans adherent cells. However, DNase (> 0.03 mg/ml) promoted a general biomass reduction on C. albicans preformed biofilms. Finally, DNase (0.13 mg/ml) did not change C. albicans biofilm cells susceptibility to fluconazole, but increased their susceptibility to amphotericin B and caspofungin, as indicated by the lower SMIC compared to biofilms grown without DNase. This work presents evidence for the role of eDNA in C. albicans biofilm integrity and antifungal resistance consistent with eDNA being a key element of the ECM

Physiologic Expression of the Candida albicans Pescadillo Homolog Is Required for Virulence in a Murine Model of Hematogenously Disseminated Candidiasis

Morphogenetic conversions contribute to the pathogenesis of Candida albicans invasive infections. Many studies to date have convincingly demonstrated a link between filamentation and virulence; however, relatively little is known regarding the role of the filament-to-yeast transition during the pathogenesis of invasive candidiasis. We previously identified the C. albicans pescadillo homolog (PES1) as essential during yeast growth and growth of lateral yeast on hyphae but not during hyphal growth. Furthermore, we demonstrated that PES1 is required for virulence in vivo in a Galleria mellonella larva model of candidiasis. Here, we have used a regulatable tetO-PES1/pes1 strain to assess the contribution of C. albicansPES1 to pathogenesis in the commonly used and clinically relevant murine model of hematogenously disseminated candidiasis. Our results indicate that a physiologically controlled level of PES1 expression is required for full virulence in this animal model, with virulence defects observed both when PES1 is overexpressed and and when it is depleted. The pathogenetic defect of cells depleted of PES1 is not due to a general growth defect, as demonstrated by the fact that PES1-depleted cells still kill Caenorhabditis elegans as efficiently as the wild type due to hyphal outgrowth through worm tissues. Our results suggest a critical role of lateral yeast growth in the ability of C. albicans to normally proliferate within tissues, as well as a pivotal role for Pes1 in the normal developmental cycle of C. albicans within the mammalian host during infection

Sensing and analysis of angular vibrations of tremor

Tremor is defined as the unintentional involuntary muscle movements involving rhythmic oscillations of any part of body like legs, hands, arms and limbs. This thesis deals with the most common tremor that occurs in hands. Tremor is not life threatening but greatly affects quality of life. The main purpose of this research is to identify a feedback control technique for the suppression of Essential Tremor. Tremor can be defined as the nonlinear and non stationary phenomenon which is the most common movement disorder affecting more than 4% of elderly people. In this research, a tremor is simulated using a stepper motor and is being sensed using a gyroscope. The Arduino UNO board is used to obtain the tremor data that is being sensed by a gyroscope. Designing and implementing the controller based on the obtained tremor characteristics is the central part of the research. The simulation results are analyzed in the National Instruments LABVIEW software and another stepper motor is used to control the tremor. The long term results are expected to lead toward a suppressed tremor without disturbing the voluntary motion of the hand

A novel flow cytometric protocol for assessment of yeast cell adhesion

Microbial adhesion is a field of recognized relevance and, as such, an impressive array of tools has been developed to understand its molecular mechanisms and ultimately for its quantification. Some of the major limitations found within these methodologies concern the incubation time, the small number of cells analyzed, and the operator's subjectivity. To overcome these aspects, we have developed a quantitative method to measure yeast cells' adhesion through flow cytometry. In this methodology, a suspension of yeast cells is mixed with green fluorescent polystyrene microspheres (uncoated or coated with host proteins). Within 2 h, an adhesion profile is obtained based on two parameters: percentage and cells-microsphere population's distribution pattern. This flow cytometry protocol represents a useful tool to quantify yeast adhesion to different substrata in a large scale, providing manifold data in a speedy and informative manner

Adapting Single-View View Synthesis with Multiplane Images for 3D Video Chat

Activities like one-on-one video chatting and video conferencing with multiple participants are more prevalent than ever today as we continue to tackle the pandemic. Bringing a 3D feel to video chat has always been a hot topic in Vision and Graphics communities. In this thesis, we have employed novel view synthesis in attempting to turn one-on-one video chatting into 3D. We have tuned the learning pipeline of Tucker and Snavely\u27s single-view view synthesis paper — by retraining it on MannequinChallenge dataset — to better predict a layered representation of the scene viewed by either video chat participant at any given time. This intermediate representation of the local light field — called a Multiplane Image (MPI) — may then be used to rerender the scene at an arbitrary viewpoint which, in our case, would match with the head pose of the watcher in the opposite, concurrent video frame. We discuss that our pipeline, when implemented in real-time, would allow both video chat participants to unravel occluded scene content and peer into each other\u27s dynamic video scenes to a certain extent. It would enable full parallax up to the baselines of small head rotations and/or translations. It would be similar to a VR headset\u27s ability to determine the position and orientation of the wearer\u27s head in 3D space and render any scene in alignment with this estimated head pose. We have attempted to improve the performance of the retrained model by extending MannequinChallenge with the much larger RealEstate10K dataset. We present a quantitative and qualitative comparison of the model variants and describe our impactful dataset curation process, among other aspects

Game and player: C. albicans biofilm lifestyle and extracellular DNA

DNA is as a structural component of bacterial biofilms extracellular matrix (ECM). Although evidences have shown that DNA may play a role in C. albicans biofilms, further studies are required to understand the contribution of extracellular DNA (eDNA) in C. albicans biofilm lifestyle. Herein we aimed to determine the eDNA content of C. albicans SC5314 biofilm ECM and the effect of DNase I and exogenous DNA treatments on biofilm formation and biofilm cells susceptibility to antifungals. First, for eDNA estimation in C. albicans biofilm ECM, biofilms were formed under flow conditions for 48 h. ECM was isolated and its DNA and protein contents were determined. Second, DNase (0.02 - 2 mg/ml) and exogenous DNA (10 - 2560 ng/ml) were added at different stages of biofilm development (microtiter plate model under static conditions). The effect of 24 h treatments was evaluated in terms of biofilm biomass by crystal violet assay (A550). Third, for antifungal testing, biofilms (in 96-well plates) were challenged with amphotericin B (0.06 - 16 mg/l), caspofungin (0.008 to 2 mg/l), and fluconazole (4 - 1024 mg/l) alone or in combination with DNase (0.125 mg/ml) or exogenous DNA (320 ng/ml). Sessile minimum inhibitory concentrations (SMIC) were determined at 80 % inhibition compared to drug-free controls using the XTT reduction assay. RPMI medium was used in all the assays. On one hand, C. albicans biofilms ECM contained 3045.4 ± 227.3 ng eDNA/mg of protein. On the other hand, DNase or exogenous DNA treatments did not affect further biofilm development by C. albicans adherent cells. In contrast, DNase (> 0.03 mg/ml) promoted a general biomass reduction on C. albicans preformed biofilms, as indicated by the reduction of A550 compared with the control. Furthermore addition of exogenous DNA (> 160 ng/ml) to preformed biofilms led to an increase in biofilm biomass, similarly assessed by the higher A550 readings compared with control biofilms. Finally, DNase I (0.125 mg/ml) did not change C. albicans biofilm cells susceptibility to fluconazole, but increased their susceptibility to amphotericin B and caspofungin, as indicated by the lower SMIC compared to biofilms grown without DNase. In contrast, exogenous DNA (320 ng/ml) did not affect C. albicans biofilm cells susceptibility against these antifungals