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Osmolytes are organic solutes that change the protein folding landscape shifting the equilibrium towards the folded state. Herein, we use osmolytes to probe the structuring and aggregation of the intrinsically disordered mutant Huntingtin (mHtt) vis-a-vis the pathogenicity of mHtt on transcription factor function and cell survival. Using an inducible PC12 cell model of Huntington’s disease (HD), we show that stabilizing polyol osmolytes drive the aggregation of Htt103Q Exon1 -EGFP from a diffuse ensemble into inclusion bodies (IBs), whereas the destabilizing osmolyte urea does not. This effect of stabilizing osmolytes is innate, generic, countered by urea, and unaffected by HSP70 and HSC70 knockdown. A qualitatively similar result of osmolyte-induced mHtt IB formation is observed in a conditionally immortalized striatal neuron model of HD, and IB formation correlates with improved survival under stress. Increased expression of diffuse mHtt sequesters the CREB transcription factor to repress CREB-reporter gene activity. This repression is mitigated either by stabilizing osmolytes, which deplete diffuse mHtt or by urea, which negates protein–protein interaction. Our results show that stabilizing polyol osmolytes promote mHtt aggregation, alleviate CREB dysfunction, and promote survival under stress to support the hypothesis that lower molecular weight entities of disease protein are relevant pathogenic species in neurodegeneration.

A great deal of attention has been focused on nanoparticles for cancer therapy, with the promise of tumor-selective delivery. However, despite intense work in the field over many years, the biggest obstacle to this vision remains extremely low delivery efficiency of nanoparticles into tumors. Due to the cost, time, and impact on the animals for in vivo studies, the nanoparticle field predominantly uses cellular uptake assays as a proxy to predict in vivo outcomes. Extensive research has focused on decreasing macrophage uptake in vitro as a proxy to delay nanoparticle accumulation in the mononuclear phagocytic system (MPS), mainly the liver and spleen, and thereby increase tumor accumulation. We have recently reported novel synthetic methods employing small molecule crosslinkers for the controlled assembly of small nanoparticles into larger aggregates and found that these nanoaggregates had remarkably high surface coverage and low cell uptake, even in macrophages. We further found that this extremely low cellular uptake could be recapitulated on solid gold nanoparticles by densely coating their surface with small molecules. Here we report our studies on the biodistribution and clearance of these materials in comparison to more conventional PEGylated gold nanoparticles. It was expected that the remarkably low macrophage uptake in vitro would translate to extended blood circulation time in vivo, but instead we found no correlation between either surface coverage or in vitro macrophage cell uptake and in vivo blood circulation. Gold nanoaggregates accumulate more rapidly and to a higher level in the liver compared to control gold nanoparticles. The lack of correlation between in vitro macrophage uptake and in vivo blood circulation suggests that the field must find other in vitro assays to use as a primary proxy for in vivo outcomes or use direct in vivo experimentation as a primary assay.

Induction of the heat shock response (HSR) and increased expression of the heat shock proteins (HSPs) provide mechanisms to ensure proper protein folding, trafficking, and disposition. The importance of HSPs is underscored by the understanding that protein mis-folding and aggregation contribute centrally to the pathogenesis of neurodegenerative diseases. We used a cell-based hsp70-luciferease reporter gene assay system to identify agents that modulate the HSR and show here that clinically relevant concentrations of the FDA-approved ALS drug riluzole significantly increased the heat shock induction of hsp70-luciferse reporter gene. Immuno-Western and -cytochemical analysis of HSF1 show that riluzole increased the amount of cytosolic HSF1 to afford a greater activation of HSF1 upon heat shock. The increased HSF1 contributed centrally to the cytoprotective activity of riluzole as hsf1 gene knockout negated the synergistic activity of riluzole and conditioning heat shock to confer cell survival under oxidative stress. Evidence of a post-transcriptional mechanism for the increase in HSF1 include: quantitation of mRNA(hsf1) by RT-PCR showed no effect of either heat shock or riluzole treatment; riluzole also increased the expression of HSF1 from a CMV-promoter; analysis of the turnover of HSF1 by pulse chase and immunoprecipitation show that riluzole slowed the decay of [(35)S]labeled-HSF1. The effect of riluzole on HSF1 was qualitatively different from that of MG132 and chloroquine, inhibitors of the proteasome and lysosome, respectively, and appeared to involve the chaperone-mediated autophagy pathway as RNAi-mediated knockdown of CMA negated its effect. We show that riluzole increased the amount of HSF1 to amplify the HSR for cytoprotection. Our study provides novel insight into the mechanism that regulates HSF1 turnover, and identifies the degradation of HSF1 as a target for therapeutics intervention.

Weibel Palade bodies (WPBs) are vesicles found in endothelial cells which carry the multimeric protein von Willebrand factor (VWF). As cellular confluency has been shown to influence the number of WPBs in endothelial cells, we propose to test two methods of attaining endothelial cell confluence to inform on the relevancy of cellular culture methods when analyzing endothelial WPBs. We test these cellular culture methods in two endothelial cell types, human umbilical vein endothelial cells (HUVECs) and endothelial colony forming cells (ECFCs). One method maintains a constant incubation time of 96 hrs. while varying the seeding density. The second method maintains a constant seeding density of 30,000 cells/cm 2 while varying incubation time. In comparing these two methods, we evaluate the nuclei count, total WPB count, and WPB/nuclei count for each. Our results show that there is a trend of increasing nuclei count, total WPB count, and WPB/nuclei count as incubation time and seeding density increases. However, there is no difference in WPB/nuclei quantification whether confluency is reached via a constant seeding density or a constant incubation time. In addition, we show that confluency plays a major role in WPB/nuclei generation as we demonstrate higher WPB/nuclei counts in confluent cultures compared to sub-confluent cultures.

BackgroundIn-office vocal fold injections (VFI) are an effective treatment for glottic insufficiency. The primary objective of this study was to assess if patients reported decisional regret after VFI. Secondary objectives included determining if variables were associated with lower decisional regret.MethodsCase–control study of patients who underwent in-office VFIs for glottic insufficiency from August 2017 to December 2019 at a tertiary laryngology clinic. Participants completed the validated Decision Regret Scale (DRS). Demographic data, clinician’s perceptual analysis with GRBAS (Grade, Roughness, Breathiness, Asthenia, Strain), and patient’s self-reported Voice Handicap Index-10 (VHI-10) were analyzed. Nonparametric tests as well as univariate and multiple logistics regression were performed.ResultsOf patients eligible, 75% (136/182) completed the DRS (mean age 65.4 years (SD 13.9), 58.1% male). Eighty-three (61.0%) reported no decisional regret, thirty-three (24.3%) reported mild decisional regret, and twenty (14.7%) reported moderate to strong decisional regret. Improvement in most recent VHI-10 (Kendall correlation coefficient tau = 0.156, p = 0.029), Grade of voice (tau = 0.236, p value = 0.002) and Breathiness of voice (tau = 0.150, p = 0.044) were associated with lower DRS. Multivariate logistics regression results showed that the change in Grade of voice (OR 9.9, p < 0.01), Roughness (OR 0.2, p < 0.01) and Breathiness (OR 0.2, p < 0.03) were significantly associated with DRS.ConclusionThe majority of patients had no or mild decisional regret after in-office VFI for glottic insufficiency. Both patients who reported less vocal handicap after VFI and clinician-noted improvements in perceptual evaluation of voice after VFI were associated with significantly lower decisional regret.

[This corrects the article DOI: 10.1371/journal.pone.0298133.].

Alterations in von Willebrand factor (VWF) have an important role in human health and disease. Deficiency of VWF is associated with symptoms of bleeding and excesses of VWF are associated with thrombotic outcomes. Understanding the mechanisms that drive VWF regulation can lead to a better understanding of modulation of VWF levels in humans. We identified clusterin ( CLU ) as a potential candidate regulator of VWF based on a single cell RNA sequencing (scRNA-seq) analysis in control endothelial cells (ECs) and von Willebrand disease (VWD) endothelial colony-forming-cells (ECFCs). We found that patients with deficiencies of VWF (von Willebrand disease, VWD) had decreased CLU expression and ECs with low VWF expression also had low CLU expression. Based on these findings, we sought to evaluate the role of CLU in the regulation of VWF, specifically as it relates to VWD. As CLU is primarily thought to be a golgi protein involved in protein chaperoning, we hypothesized that knockdown of CLU would lead to decreases in VWF and alterations in Weibel-Palade bodies (WPBs). We used both siRNA- and CRISPR-Cas9-based approaches to modulate CLU in human umbilical vein endothelial cells (HUVECs) and evaluated VWF protein levels, VWF mRNA copy number, and WPB quantity and size. We demonstrated that siRNA-based knockdown of CLU resulted in decreases in VWF content in cellular lysates and supernatants, but no significant change in WPB quantity or size. A CRISPR-Cas9-based knockdown of CLU demonstrated similar findings of decreases in intracellular VWF content but no significant change in WPB quantity or size. Our data suggests that CLU knockdown is associated with decreases in cellular VWF content but does not affect VWF mRNA levels or WPB quantity or size.

Amyloid beta (Aβ) deposits in the retina of the Alzheimer’s disease (AD) eye may provide a useful diagnostic biomarker for AD. This study focused on the relationship of Aβ with macroglia and microglia, as these glial cells are hypothesized to play important roles in homeostasis and clearance of Aβ in the AD retina. Significantly higher Aβ load was found in AD compared to controls, and specifically in the mid-peripheral region. AD retina showed significantly less immunoreactivity against glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) compared to control eyes. Immunoreactivity against ionized calcium binding adapter molecule-1 (IBA-1), a microglial marker, demonstrated a higher level of microgliosis in AD compared to control retina. Within AD retina, more IBA-1 immunoreactivity was present in the mid-peripheral retina, which contained more Aβ than the central AD retina. GFAP co-localized rarely with Aβ, while IBA-1 co-localized with Aβ in more layers of control than AD donor retina. These results suggest that dysfunction of the Müller and microglial cells may be key features of the AD retina.

IntroductionThe immunocompromised host (ICH) is at increased risk for a range of opportunistic and healthcare-associated infections (OI/HAIs). With increased use of novel therapeutics and prolonged survival, the malignancy and transplant population is a particularly vulnerable and expanding subgroup. In the absence of a coordinated Australian infection surveillance programme, estimates of the prevalence of OI/HAIs for the high-risk ICH population are yet to be established. Approaches to infection prevention and control (IPC) are also non-standardised across healthcare facilities (HCFs). This study aims to provide data on key pathogen prevalence and comparative IPC and infection monitoring practice amongst the Australian cancer/transplant population to inform future consensus ICH-specific policy.Methods and analysisThe first multi-site adapted point prevalence survey (PPS) for OI/HAIs in the high-risk ICH population will be conducted across several Australian public HCFs providing inpatient (IP) care for adult transplant (solid organ/haematopoietic stem-cell)±malignancy (haematological/oncological) patients. Surveillance methodology using the European Centre for Disease Prevention and Control (ECDC) PPS protocol modified for the ICH will be applied. ICH-adapted ECDC and Centres for Disease Control and Prevention (CDC)/National Healthcare Safety Network (NHSN) surveillance case definitions will be used for key HAIs and diagnostic criteria for select OIs. Potentially eligible cancer and transplant patients will be identified for sampling by active antimicrobial use. Infection data, patient-level risks and correlates for HCF impact will be collected from medical records. To contextualise infectious rates, IPC and surveillance strategy will be explored through qualitative interviews with IPC personnel at each sampling site. The prevalence of infection will be approximated from the proportion with infection in the sample screened, and descriptive data analysis will be used to support the expected outcomes of this study, which includes providing a unique insight into infectious disease trends alongside current IPC and surveillance processes within this highly specialised population.Ethics and disseminationEthical approval has been obtained from the Peter MacCallum Cancer Centre (PMCC) Human Research Ethics Committee (HREC/112164/PMCC) via the National Mutual Acceptance Scheme. Research findings will be disseminated through peer-review publication and conference presentation and contribute to future work on consensus IPC and surveillance guidelines.

Platelets from patients with myeloproliferative neoplasms (MPNs) exhibit a hyperreactive phenotype. Here, we found elevated P-selectin exposure and platelet-leukocyte aggregates indicating activation of platelets from essential thrombocythemia (ET) patients. Single-cell RNA-seq analysis of primary samples revealed significant enrichment of transcripts related to platelet activation, mTOR, and oxidative phosphorylation in ET patient platelets. These observations were validated via proteomic profiling. Platelet metabolomics revealed distinct metabolic phenotypes consisting of elevated ATP generation accompanied by increases in the levels of multiple intermediates of the tricarboxylic acid cycle, but lower α-ketoglutarate (α-KG) in MPN patients. Inhibition of PI3K/AKT/mTOR signaling significantly reduced metabolic responses and hyperreactivity in MPN patient platelets, while α-KG supplementation markedly reduced oxygen consumption and ATP generation. Ex vivo incubation of platelets from both MPN patients and Jak2 V617F-knockin mice with α-KG supplementation significantly reduced platelet activation responses. Oral α-KG supplementation of Jak2 V617F mice decreased splenomegaly and reduced hematocrit, monocyte, and platelet counts. Finally, α-KG treatment significantly decreased proinflammatory cytokine secretion from MPN CD14+ monocytes. Our results reveal a previously unrecognized metabolic disorder in conjunction with aberrant PI3K/AKT/mTOR signaling that contributes to platelet hyperreactivity in MPN patients.