Structural progression of amyloid-ß Arctic mutant aggregation in cells revealed by multiparametric imaging.

The 42-amino-acid ß-amyloid (Aß42) is a critical causative agent in the pathology of Alzheimer's disease. The hereditary Arctic mutation of Aß42 (E22G) leads to increased intracellular accumulation of ß-amyloid in early-onset Alzheimer's disease. However, it remains largely unknown how the Arctic mutant variant leads to aggressive protein aggregation and increased intracellular toxicity. Here, we constructed stable cell lines expressing fluorescent-tagged wildtype (WT) and E22G Aß42 to study the aggregation kinetics of the Arctic Aß42 mutant peptide and its heterogeneous structural forms. Arctic-mutant peptides assemble and form fibrils at a much faster rate than WT peptides. We identified five categories of intracellular aggregate-oligomers, single fibrils, fibril bundles, clusters, and aggresomes-that underline the heterogeneity of these Aß42 aggregates and represent the progression of Aß42 aggregation within the cell. Fluorescence-lifetime imaging (FLIM) and 3D structural illumination microscopy (SIM) showed that all aggregate species displayed highly compact structures with strong affinity between individual fibrils. We also found that aggregates formed by Arctic mutant Aß42 were more resistant to intracellular degradation than their WT counterparts. Our findings uncover the structural basis of the progression of Arctic mutant Aß42 aggregation in the cell.

Anisotropic nonlinear refractive index measurement of a photorefractive crystal via spatial self-phase modulation.

We show that the refractive index modification photoinduced in a biased nonlinear photorefractive crystal can be accurately measured and controlled by means of a background incoherent illumination and an external electric field. The proposed easy-to-implement method is based on the far-field measurement of the diffraction patterns of a laser beam propagating through a self-defocusing medium undergoing spatial self-phase modulation. For various experimental conditions, both saturation intensity and maximum refractive index modification have been measured. We also clearly evidence and characterise the anisotropic nonlinear response of the crystal in the stationary regime.

Nanoscopic insights into seeding mechanisms and toxicity of α-synuclein species in neurons.

New strategies for visualizing self-assembly processes at the nanoscale give deep insights into the molecular origins of disease. An example is the self-assembly of misfolded proteins into amyloid fibrils, which is related to a range of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. Here, we probe the links between the mechanism of α-synuclein (AS) aggregation and its associated toxicity by using optical nanoscopy directly in a neuronal cell culture model of Parkinson's disease. Using superresolution microscopy, we show that protein fibrils are taken up by neuronal cells and act as prion-like seeds for elongation reactions that both consume endogenous AS and suppress its de novo aggregation. When AS is internalized in its monomeric form, however, it nucleates and triggers the aggregation of endogenous AS, leading to apoptosis, although there are no detectable cross-reactions between externally added and endogenous protein species. Monomer-induced apoptosis can be reduced by pretreatment with seed fibrils, suggesting that partial consumption of the externally added or excess soluble AS can be significantly neuroprotective.

Extracellular monomeric tau protein is sufficient to initiate the spread of tau protein pathology.

Understanding the formation and propagation of aggregates of the Alzheimer disease-associated Tau protein in vivo is vital for the development of therapeutics for this devastating disorder. Using our recently developed live-cell aggregation sensor in neuron-like cells, we demonstrate that different variants of exogenous monomeric Tau, namely full-length Tau (hTau40) and the Tau-derived construct K18 comprising the repeat domain, initially accumulate in endosomal compartments, where they form fibrillar seeds that subsequently induce the aggregation of endogenous Tau. Using superresolution imaging, we confirm that fibrils consisting of endogenous and exogenous Tau are released from cells and demonstrate their potential to spread Tau pathology. Our data indicate a greater pathological risk and potential toxicity than hitherto suspected for extracellular soluble Tau.

Possible loss of the chloroplast genome in the parasitic flowering plant Rafflesia lagascae (Rafflesiaceae).

Rafflesia is a genus of holoparasitic plants endemic to Southeast Asia that has lost the ability to undertake photosynthesis. With short-read sequencing technology, we assembled a draft sequence of the mitochondrial genome of Rafflesia lagascae Blanco, a species endemic to the Philippine island of Luzon, with ∼350× sequencing depth coverage. Using multiple approaches, however, we were only able to identify small fragments of plastid sequences at low coverage depth (<2×) and could not recover any substantial portion of a chloroplast genome. The gene fragments we identified included photosynthesis and energy production genes (atp, ndh, pet, psa, psb, rbcL), ribosomal RNA genes (rrn16, rrn23), ribosomal protein genes (rps7, rps11, rps16), transfer RNA genes, as well as matK, accD, ycf2, and multiple nongenic regions from the inverted repeats. None of the identified plastid gene sequences had intact reading frames. Phylogenetic analysis suggests that ∼33% of these remnant plastid genes may have been horizontally transferred from the host plant genus Tetrastigma with the rest having ambiguous phylogenetic positions (<50% bootstrap support), except for psaB that was strongly allied with the plastid homolog in Nicotiana. Our inability to identify substantial plastid genome sequences from R. lagascae using multiple approaches--despite success in identifying and developing a draft assembly of the much larger mitochondrial genome--suggests that the parasitic plant genus Rafflesia may be the first plant group for which there is no recognizable plastid genome, or if present is found in cryptic form at very low levels.

Early and prolonged continuous hypertonic saline infusion in patients with acute liver failure.

PURPOSE: To study patient characteristics, physiological changes, and outcomes associated with prolonged continuous hypertonic saline (HTS) infusion in acute liver failure (ALF). MATERIALS AND METHODS: Retrospective observational cohort study of adult patients with ALF. We collected clinical, biochemical, and physiological data six hourly for the first week, daily until day 30 or hospital discharge, and weekly, when documented, until day 180. RESULTS: Of 127 patients, 85 received continuous HTS. Compared with non-HTS patients they were more likely to receive continuous renal replacement therapy (CRRT) (p < 0.001) and mechanical ventilation (p < 0.001). Median HTS duration was 150 (Interquartile range (IQR): 84-168) hours, delivering a median 2244 (IQR: 979-4610) mmol sodium load. Median peak sodium concentration was 149 mmol/L vs 138 mmol/L in non-HTS patients (p < 0.001). The median rate of sodium increase with infusion was 0.1 mmol/L/h and median rate of decrease during weaning was 0.1 mmol/L every 6 h. Median lowest pH value was 7.29 vs. 7.35 in non-HTS patients. Survival of HTS patients was 72.9% overall and 72.2% without transplantation. CONCLUSIONS: In ALF patients, the prolonged administration of HTS infusion was not associated with severe hypernatremia or rapid shifts in serum sodium upon commencement, delivery, or weaning.

Retrospective, real-life study of venetoclax plus azacitidine or low-dose cytarabine in French patients with acute myeloid leukemia ineligible for intensive chemotherapy.

BACKGROUND: Recently, the combination of venetoclax plus a hypomethylating agent (HMA; azacitidine ordecitabine) or low-dose cytarabine (LDAC) showed promise in Phase III trials in previously untreated AML. In France at the time of this study, venetoclax was not yet approved for AML and there were therefore no formal usage recommendations. Here we report the first study in a French cohort that assessed venetoclax in combination with existing treatments for AML under real-life conditions. METHOD: This retrospective, real-life study collected data on venetoclax use and management in a French cohort with acute myeloid leukemia (AML) ineligible for intensive chemotherapy. RESULT: Of 118 patients, 81 were in second line/beyond (71.6% also hypomethylating agent [HMA]; 23.5% lowdose cytarabine [LDAC]) and 37 in first line. For venetoclax initiation, 57.3% underwent ramp up and 74.6% were hospitalized. Median venetoclax duration was 2.5 months (range 0.03-16.2). With all treatment lines and regimens, most common grade 3/4 adverse events were hematologic (overall 96.4% of patients) and infections (57.1%). Dosage adjustments for drug interactions and safety varied between centers. In second-line/beyond, median progression-free survival was 4.0 months (95% confidence interval [CI] 2.7-12.8) with venetoclax-HMA and 3.4 months (1.3-8.9) with venetoclax-LDAC; overall response rate was 51.9% and 41.2%, respectively. Thus, we showed that venetoclax-based treatment yields promising findings in patients with AML, but to address treatment complexity, practice harmonization is needed.

Outcome after allogeneic stem cell transplantation with haploidentical versus HLA-matched donors in patients with higher-risk MDS.

Allogeneic hematopoietic stem cell transplantation remains the best curative option for higher-risk myelodysplastic syndrome. The presence of monosomal karyotype and/or complex karyotype abnormalities predicts inferior survival after allo-SCT in MDS patients. Haploidentical allo-SCT has been increasingly used in acute leukemia (AL) and has similar results as using HLA-matched donors, but data on higher-risk MDS is sparse. We compared outcomes in 266 patients with higher-risk MDS after HLA-matched sibling donor (MSD, n = 79), HLA-matched unrelated donor (MUD, n = 139) and HLA haploidentical donor (HID, n = 48) from 2010 to 2019. Median donor age differed between the three groups (p < 0.001). The overall survival was significantly different between the three groups with a better OS observed in the MUD group (p = 0.014). This observation could be explained by a higher progression-free survival with MUD (p = 0.014). The cumulative incidence of grade 2-4 acute GvHD was significantly higher in the HID group (p = 0.051). However, in multivariable analysis, patients transplanted using an HID had comparable mortality to patients transplanted using a MUD (subdistribution hazard ratio [sHR]: 0.58 [0.32-1.07]; p = 0.080) and a MSD ([sHR]: 0.56 [0.28-1.11]; p = 0.094). MUD do not remain a significant positive predictor of survival, suggesting that beyond the donor-recipient HLA matching, the donor age might impact recipient outcome.

Prospective evaluation of clinical and biological markers to predict the outcome of invasive pulmonary aspergillosis in hematological patients.

Early evaluation of treatment efficacy in invasive aspergillosis (IA), a leading cause of morbidity and mortality in hematological patients, remains a challenge. We conducted a prospective study to evaluate the performance of different markers in predicting the outcome of patients with IA. Both clinical and biological criteria were assessed 7, 14, 21, and 45 days after inclusion in the study, and mortality was assessed at day 60. The association between baseline data and their evolution and the day 45 response to treatment was analyzed. A total of 57 patients (4 with proven, 44 with probable, and 9 with possible aspergillosis according to the revised EORTC/MSG [European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group] definitions) were included. At day 45, 30 patients (53%) were determined to be responders, 25 (44%) were nonresponders, and 2 were not able to be evaluated. Twenty patients died within the 60 days of follow-up. We found that a poor day 45 outcome was associated with patients who had high baseline serum galactomannan (GM) antigen levels and those receiving steroids at the time of IA. A consistently negative serum GM index was associated with a good outcome, and the day 14 clinical evaluation was predictive of the day 45 outcome. No association was found between Aspergillus antibodies or DNA detection and patients' outcome. We conclude that the GM index value at diagnosis of IA, GM index kinetics, and clinical evaluation at day 14 are good markers for predicting the outcome of patients with IA and should be taken into account for adapting antifungal treatment.

Characterisation of serpin polymers in vitro and in vivo.

Neuroserpin is a member of the serine protease inhibitor or serpin superfamily of proteins. It is secreted by neurones and plays an important role in the regulation of tissue plasminogen activator at the synapse. Point mutations in the neuroserpin gene cause the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. This is one of a group of disorders caused by mutations in the serpins that are collectively known as the serpinopathies. Others include α(1)-antitrypsin deficiency and deficiency of C1 inhibitor, antithrombin and α(1)-antichymotrypsin. The serpinopathies are characterised by delays in protein folding and the retention of ordered polymers of the mutant serpin within the cell of synthesis. The clinical phenotype results from either a toxic gain of function from the inclusions or a loss of function, as there is insufficient protease inhibitor to regulate important proteolytic cascades. We describe here the methods required to characterise the polymerisation of neuroserpin and draw parallels with the polymerisation of α(1)-antitrypsin. It is important to recognise that the conditions in which experiments are performed will have a major effect on the findings. For example, incubation of monomeric serpins with guanidine or urea will produce polymers that are not found in vivo. The characterisation of the pathological polymers requires heating of the folded protein or alternatively the assessment of ordered polymers from cell and animal models of disease or from the tissues of humans who carry the mutation.

Prevalence, severity, duration and resolution of cholestasis after acute liver failure.

OBJECTIVE: Persistent cholestasis may follow acute liver failure (ALF), but its course remains unknown. We aimed to describe the prevalence, onset, severity, duration and resolution of post-ALF cholestasis. DESIGN: Cohort of 127 adult patients with ALF at a liver transplantation centre identified using electronic databases. We obtained laboratory data every 6 hours for the first week, daily until day 30 and weekly, when documented, until day 180. RESULTS: Median age was 40.7 (IQR 31.0-52.4) years, median peak alanine aminotransferase level was 5494 (2521-8819) U/L and 87 (68.5%) cases had paracetamol toxicity. Overall, 12.6% underwent transplantation (3.4% for paracetamol vs 32.5% for non-paracetamol; p<0.001). Ninety-day mortality was 20.7% for paracetamol versus 30.0% for non-paracetamol patients. All non-transplanted survivors reached a bilirubin level>50 µmol/L, which peaked 3.5 (1.0-10.1) days after admission at 169.0 (80.0-302.0) µmol/L. At hospital discharge, 18.8% of patients had normal bilirubin levels and, at a median follow-up time from admission to last measurement of 16 (10-30) days, 46.9% had normal levels. Similarly, there was an increase in alkaline phosphatase (ALP) (207.0 (148.0-292.5) U/L) and gamma-glutamyl transferase (GGT) (336.0 (209.5-554.5) U/L) peaking at 4.5 days, with normalised values in 40.3% and 8.3% at hospital discharge. CONCLUSION: Post-ALF cholestasis is ubiquitous. Bilirubin, ALP and GGT peak at 3 to 5 days and, return to baseline in the minority of patients at median follow-up of 16 days. These data inform clinical expectations of the natural course of this condition.

Pressure support ventilation in intensive care patients receiving prolonged invasive ventilation.

Background: To our knowledge, the use and management of pressure support ventilation (PSV) in patients receiving prolonged (≥ 7 days) invasive mechanical ventilation has not previously been described. Objective: To collect and analyse data on the use and management of PSV in critically ill patients receiving prolonged ventilation. Design, setting and participants: We performed a multicentre retrospective observational study in Australia, with a focus on PSV in patients ventilated for ≥ 7 days. Main outcome measures: We obtained detailed data on ventilator management twice daily (8am and 8pm moments) for the first 7 days of ventilation. Results: Among 143 consecutive patients, 90/142 (63.4%) had received PSV by Day 7, and PSV accounted for 40.5% (784/1935) of ventilation moments. The most common pressure support level was 10 cmH2O (352/780) observations [45.1%]) with little variation over time, and 37 of 114 patients (32.4%) had no change in pressure support. Mean tidal volume during PSV was 8.3 (7.0-9.5) mL/kg predicted bodyweight (PBW) compared with 7.5 (7.0-8.3) mL/kg PBW during mandatory ventilation (P < 0.001). For 74.6% (247/331) of moments, despite a tidal volume of more than 8 mL/kg PBW, the pressure support level was not changed. Among 122 patients exposed to PSV, 97 (79.5%) received likely over-assistance according to rapid shallow breathing index criteria. Of 784 PSV moments, 411 (52.4%) were also likely over-assisted according to rapid shallow breathing index criteria, and 269/346 (77.7%) having no subsequent adjustment of pressure support. Conclusions: In patients receiving prolonged ventilation, almost two-thirds received PSV, which accounted for 40.5% of mechanical ventilation time. Half of the PSV-treated patients were exposed to high tidal volume and two-thirds to likely over-assistance. These observations provide evidence that can be used to inform interventional studies of PSV management.

Controlled excitation of scar modes in passive and active multimode chaotic fiber.

A multimode optical fiber with a D-shaped cross section is an experimental paradigm of a wave system with chaotic ray dynamics. We show that seldom but usable modes, called scar modes, localized along some particular direction of the geometric trajectories, can be selectively excited. We report numerical simulations that demonstrate the importance of the so-called self-focal point in the scar mode selection process. We use a localized illumination in a passive fiber, or a localized gain in a ytterbium-doped fiber, located in the vicinity of this special point to control scar mode selection.

Intrinsically aggregation-prone proteins form amyloid-like aggregates and contribute to tissue aging in Caenorhabditis elegans.

Reduced protein homeostasis leading to increased protein instability is a common molecular feature of aging, but it remains unclear whether this is a cause or consequence of the aging process. In neurodegenerative diseases and other amyloidoses, specific proteins self-assemble into amyloid fibrils and accumulate as pathological aggregates in different tissues. More recently, widespread protein aggregation has been described during normal aging. Until now, an extensive characterization of the nature of age-dependent protein aggregation has been lacking. Here, we show that age-dependent aggregates are rapidly formed by newly synthesized proteins and have an amyloid-like structure resembling that of protein aggregates observed in disease. We then demonstrate that age-dependent protein aggregation accelerates the functional decline of different tissues in C. elegans. Together, these findings imply that amyloid-like aggregates contribute to the aging process and therefore could be important targets for strategies designed to maintain physiological functions in the late stages of life.

Superfluid motion and drag-force cancellation in a fluid of light.

Quantum fluids of light merge many-body physics and nonlinear optics, revealing quantum hydrodynamic features of light when it propagates in nonlinear media. One of the most outstanding evidence of light behaving as an interacting fluid is its ability to carry itself as a superfluid. Here, we report a direct experimental detection of the transition to superfluidity in the flow of a fluid of light past an obstacle in a bulk nonlinear crystal. In this cavityless all-optical system, we extract a direct optical analog of the drag force exerted by the fluid of light and measure the associated displacement of the obstacle. Both quantities drop to zero in the superfluid regime characterized by a suppression of long-range radiation from the obstacle. The experimental capability to shape both the flow and the potential landscape paves the way for simulation of quantum transport in complex systems.

C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction.

Alpha-synuclein is known to bind to small unilamellar vesicles (SUVs) via its N terminus, which forms an amphipathic alpha-helix upon membrane interaction. Here we show that calcium binds to the C terminus of alpha-synuclein, therewith increasing its lipid-binding capacity. Using CEST-NMR, we reveal that alpha-synuclein interacts with isolated synaptic vesicles with two regions, the N terminus, already known from studies on SUVs, and additionally via its C terminus, which is regulated by the binding of calcium. Indeed, dSTORM on synaptosomes shows that calcium mediates the localization of alpha-synuclein at the pre-synaptic terminal, and an imbalance in calcium or alpha-synuclein can cause synaptic vesicle clustering, as seen ex vivo and in vitro. This study provides a new view on the binding of alpha-synuclein to synaptic vesicles, which might also affect our understanding of synucleinopathies.

Detection of pyrethroid resistance gene in head lice in schoolchildren from Bobigny, France.

The head louse, Pediculus humanus capitis (De Geer), is an hematophagous ectoparasite that affects mainly children. Resistance to insecticides belonging to pyrethroids and other pediculicides, such as malathion, is responsible for frequently reported treatment failures. Recent studies showed that a M815I-T929I-L932F kdr-like mutation in the voltage-gated sodium channel alpha-subunit gene was associated with permethrin resistance in head lice from several countries worldwide. We searched for the presence ofpyrethroid resistance gene in head lice populations obtained in schoolchildren in an urban area of France. All the 15 primary schools of Bobigny, a city located 3 km north of Paris, were selected to participate. Of 3,493 children enrolled, 3,345 (95.8%) children were screened for head lice by using fine-toothed antilouse combs. Live head lice were detected in 112 (3.3%) of children screened. A subsample of 90 lice was processed for DNA study. The amplification of a 332-bp portion of the kdr-like gene spanning the codon 929 was performed, and polymerase chain reaction products were submitted to the restriction enzyme SspI. Twenty of these lice (22.2%) were homozygous susceptible, 33 (36.7%) were homozygous resistant, and 37 (41.1%) were heterozygotes. Globally, the frequency of the T929I mutation was 0.57. The prevalence of pediculosis in schoolchildren of Bobigny seemed relatively low in comparison with findings of other European studies. The presence of the T929I mutation associated with permethrin resistance probably reflected the frequent local use of this insecticide. Further studies are now required to evaluate the prevalence of the kdr-like mutant allele in head lice in French schools.

Advanced imaging of tau pathology in Alzheimer Disease: New perspectives from super resolution microscopy and label-free nanoscopy.

Alzheimer's disease (AD) is the main cause of dementia in the elderly population. Over 30 million people worldwide are living with dementia and AD prevalence is projected to increase dramatically in the next two decades. In terms of neuropathology, AD is characterized by two major cerebral hallmarks: extracellular ß-amyloid (Aß) plaques and intracellular Tau inclusions, which start accumulating in the brain 15-20 years before the onset of symptoms. Within this context, the scientific community worldwide is undertaking a wide research effort to detect AD pathology at its earliest, before symptoms appear. Neuroimaging of Aß by positron emission tomography (PET) is clinically available and is a promising modality for early detection of Aß pathology and AD diagnosis. Substantive efforts are ongoing to develop advanced imaging techniques for early detection of Tau pathology. Here, we will briefly describe the key features of Tau pathology and its heterogeneity across various neurodegenerative diseases bearing cerebral Tau inclusions (i.e., tauopathies). We will outline the current status of research on Tau-specific PET tracers and their clinical development. Finally, we will discuss the potential application of novel super-resolution and label-free techniques for investigating Tau pathology at the experimental level and their potential application for AD diagnosis. Microsc. Res. Tech. 79:677-683, 2016. © 2016 Wiley Periodicals, Inc.

ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function.

The mechanisms by which mutations in FUS and other RNA binding proteins cause ALS and FTD remain controversial. We propose a model in which low-complexity (LC) domains of FUS drive its physiologically reversible assembly into membrane-free, liquid droplet and hydrogel-like structures. ALS/FTD mutations in LC or non-LC domains induce further phase transition into poorly soluble fibrillar hydrogels distinct from conventional amyloids. These assemblies are necessary and sufficient for neurotoxicity in a C. elegans model of FUS-dependent neurodegeneration. They trap other ribonucleoprotein (RNP) granule components and disrupt RNP granule function. One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation. Nuclear FUS granules may be similarly affected. Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins.

BRCA1 haploinsufficiency leads to altered expression of genes involved in cellular proliferation and development.

The assessment of BRCA1 and BRCA2 coding sequences to identify pathogenic mutations associated with inherited breast/ovarian cancer syndrome has provided a method to identify high-risk individuals, allowing them to seek preventative treatments and strategies. However, the current test is expensive, and cannot differentiate between pathogenic variants and those that may be benign. Focusing only on one of the two BRCA partners, we have developed a biological assay for haploinsufficiency of BRCA1. Using a series of EBV-transformed cell lines, we explored gene expression patterns in cells that were BRCA1 wildtype compared to those that carried (heterozygous) BRCA1 pathogenic mutations. We identified a subset of 43 genes whose combined expression pattern is a sensitive predictor of BRCA1 status. The gene set was disproportionately made up of genes involved in cellular differentiation, lending credence to the hypothesis that single copy loss of BRCA1 function may impact differentiation, rendering cells more susceptible to undergoing malignant processes.