Dynamic Superresolution Imaging of Endogenous Proteins on Living Cells at Ultra-High Density

Versatile superresolution imaging methods, able to give dynamic information of endogenous molecules at high density, are still lacking in biological science. Here, superresolved images and diffusion maps of membrane proteins are obtained on living cells. The method consists of recording thousands of single-molecule trajectories that appear sequentially on a cell surface upon continuously labeling molecules of interest. It allows studying any molecules that can be labeled with fluorescent ligands including endogenous membrane proteins on living cells. This approach, named universal PAINT (uPAINT), generalizes the previously developed point-accumulation-for-imaging-in-nanoscale-topography (PAINT) method for dynamic imaging of arbitrary membrane biomolecules. We show here that the unprecedented large statistics obtained by uPAINT on single cells reveal local diffusion properties of specific proteins, either in distinct membrane compartments of adherent cells or in neuronal synapses.     

Adult neurogenic process in the subventricular zone‐olfactory bulb system is regulated by Tau protein under prolonged stress

ObjectivesThe area of the subventricular zone (SVZ) in the adult brain exhibits the highest number of proliferative cells, which, together with the olfactory bulb (OB), maintains constant brain plasticity through the generation, migration and integration of newly born neurons. Despite Tau and its malfunction is increasingly related to deficits of adult hippocampal neurogenesis and brain plasticity under pathological conditions [e.g. in Alzheimer''s disease (AD)], it remains unknown whether Tau plays a role in the neurogenic process of the SVZ and OB system under conditions of chronic stress, a well‐known sculptor of brain and risk factor for AD.Materials and methodsDifferent types of newly born cells in SVZ and OB were analysed in animals that lack Tau gene (Tau‐KO) and their wild‐type littermates (WT) under control or chronic stress conditions.ResultsWe demonstrate that chronic stress reduced the number of proliferating cells and neuroblasts in the SVZ leading to decreased number of newborn neurons in the OB of adult WT, but not Tau‐KO, mice. Interestingly, while stress‐evoked changes were not detected in OB granular cell layer, Tau‐KO exhibited increased number of mature neurons in this layer indicating altered neuronal migration due to Tau loss.ConclusionsOur findings suggest the critical involvement of Tau in the neurogenesis suppression of SVZ and OB neurogenic niche under stressful conditions highlighting the role of Tau protein as an essential regulator of stress‐driven plasticity deficits.     

Characteristics of Acaciamangium shoot apical meristems in natural and in vitro conditions in relation to heteroblasty

Morphological and histocytological characteristics of Acacia mangium shoot apical meristems (SAMs) were assessed in natural and in vitro conditions in relation to heteroblasty. In the natural environment, SAMs with a mature-phyllode morphology were much bigger, contained more cells with larger vacuolated area, or vacuome, and lower nucleoplasmic ratios than those from the juvenile type (Juv). In these latter, nuclei appeared more voluminous, evenly and lightly stained, with clearly distinguishable nucleolei and less abundant chromocenters. In vitro, where reversions from mature to juvenile morphological traits do occur unpredictably, heteroblasty was less obvious in the SAM characteristics examined. In vitro SAMs corresponding to the juvenile and mature types showed similarities with outdoor Juv SAMs, but could be distinguished from these latter by a much larger vacuome that might be induced by the culture conditions. These findings encourage pursuing the investigations at the chromatin and nucleolus level in SAM zones where heteroblasty-related differences have been detected.     

Nitric oxide bioavailability and not production is first altered during the onset of insulin resistance in sucrose-fed rats

Although the role of nitric oxide (NO) in peripheral glucose uptake has been thoroughly described, little is known regarding the alterations in NO metabolism during the early onset of insulin resistance. During this study we investigated the alterations in NO synthesis and bioavailability in a model for dietary modulations of insulin sensitivity. For 6 weeks, rats were fed a standard diet (C), a high-sucrose diet inducing insulin resistance (HS), or high-sucrose diets supplemented with cysteine, which endowed protection against the high-sucrose-induced insulin resistance (Ti). Several markers of NO synthesis and bioavailability were assessed and confronted with markers of insulin sensitivity. After 5 weeks, although urinary cGMP excretion did not differ between the groups, insulin resistance in HS rats was associated with both a significant increase in NO oxidation, as determined by plasma nitrotyrosine concentrations, and in the inducible NO synthase (iNOS)/endothelial NO synthase (iNOS/eNOS) mRNA ratio in skeletal muscle compared with C rats. These alterations were prevented in rats fed the cysteine-rich diets. NO production, as assessed by urinary 15NO3* excretion following a [15N2-(guanido)]-arginine intra-venous bolus, independently and significantly correlated with insulin sensitivity but did not significantly differ between C, HS, and Ti rats; neither did the aortic eNOS protein expression or skeletal muscle insulin-induced eNOS activation. Our results indicate that in this model of dietary modulations of insulin sensitivity (i) NO production accounts for part of total inter-individual variation in insulin sensitivity, but (ii) early diet-related changes in insulin sensitivity are accompanied by changes in NO bioavailability.     

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.     

Long-Lived Antibody and B Cell Memory Responses to the Human Malaria Parasites,Plasmodium falciparum and Plasmodium vivax

Antibodies constitute a critical component of the naturally acquired immunity that develops following frequent exposure to malaria. However, specific antibody titres have been reported to decline rapidly in the absence of reinfection, supporting the widely perceived notion that malaria infections fail to induce durable immunological memory responses. Currently, direct evidence for the presence or absence of immune memory to malaria is limited. In this study, we analysed the longevity of both antibody and B cell memory responses to malaria antigens among individuals who were living in an area of extremely low malaria transmission in northern Thailand, and who were known either to be malaria naïve or to have had a documented clinical attack of P. falciparum and/or P. vivax in the past 6 years. We found that exposure to malaria results in the generation of relatively avid antigen-specific antibodies and the establishment of populations of antigen-specific memory B cells in a significant proportion of malaria-exposed individuals. Both antibody and memory B cell responses to malaria antigens were stably maintained over time in the absence of reinfection. In a number of cases where antigen-specific antibodies were not detected in plasma, stable frequencies of antigen-specific memory B cells were nonetheless observed, suggesting that circulating memory B cells may be maintained independently of long-lived plasma cells. We conclude that infrequent malaria infections are capable of inducing long-lived antibody and memory B cell responses.     

Higher Levels of Neutralizing Antibodies against KSHV in KS Patients Compared to Asymptomatic Individuals from Zambia

Kaposi sarcoma-associated herpesvirus (KSHV) is the etiologic agent for Kaposi Sarcoma (KS), the most common cancer diagnosed in HIV- infected patients. The role of neutralizing antibodies in KS pathogenesis and in KSHV infected individuals is not clearly understood. The goal of this study was to investigate and compare the prevalence and titers of neutralizing antibodies in plasma samples from KS patients and KSHV infected asymptomatic individuals from Zambia, a KS endemic region in sub-Saharan Africa. Plasma samples (N = 267) consisting of KS patients (group 1) and asymptomatic individuals (group 2) were collected from Lusaka, Zambia. A flow cytometry based quantitative neutralization assay utilizing recombinant KSHV expressing GFP was used to detect KSHV neutralizing antibodies. Our results show that the overall prevalence of neutralizing antibodies in KS patients (group 1) was 66.7% which was significantly higher than the prevalence of 6.5% present in KSHV infected asymptomatic individuals (group 2). Total antibody titers as well as neutralizing antibodies titers were found to be significantly higher among KS patients. It is likely that higher neutralizing antibodies prevalence and titers in KS patients result from higher levels of antigenic stimulation over time. This study is first to compare prevalence and titers of neutralizing antibodies in participants with and without disease from a KSHV endemic region.     

Piscine insights into comparisons of anoxia tolerance, ammonia toxicity, stroke and hepatic encephalopathy

Although the number of fish species that have been studied for both hypoxia/anoxia tolerance and ammonia tolerance are few, there appears to be a correlation between the ability to survive these two insults. After establishing this correlation with examples from the literature, and after examining the role Peter Lutz played in catalyzing this convergent interest in two variables, this article explores potential mechanisms underpinning this correlation. We draw especially on the larger body of information for two human diseases with the same effected organ (brain), namely stroke and hepatic encephalopathy. While several dissimilarities exist between the responses of vertebrates to anoxia and hyperammonemia, one consistent observation in both conditions is an overactivation of NMDA receptors or glutamate neurotoxicity. We propose a glutamate excitotoxicity hypothesis to explain the correlation between ammonia and hypoxia resistance in fish. Furthermore, we suggest several experimental paths to test this hypothesis.     

Cu2+TerPy complexes as catalysts of the cleavage of the 5'-cap structure of mRNA

Cu(2+)TerPy is a fairly good catalyst of the cleavage of dinucleoside triphosphates, but its efficiency is not sufficient for use in artificial RNA cleaving enzymes. The present work is aimed at improving the catalysis by Cu(2+)TerPy with additional catalysts. Electrophilic and general acid catalysis have been studied and bifunctional catalysts have been synthesized. The most efficient catalysis was achieved with a Cu(2+)TerPy-dimer.     

ER stress protects from retinal degeneration

The unfolded protein response (UPR) is a specific cellular process that allows the cell to cope with the overload of unfolded/misfolded proteins in the endoplasmic reticulum (ER). ER stress is commonly associated with degenerative pathologies, but its role in disease progression is still a matter for debate. Here, we found that mutations in the ER‐resident chaperone, neither inactivation nor afterpotential A (NinaA), lead to mild ER stress, protecting photoreceptor neurons from various death stimuli in adult Drosophila. In addition, Drosophila S2 cultured cells, when pre‐exposed to mild ER stress, are protected from H₂O₂, cycloheximide‐ or ultraviolet‐induced cell death. We show that a specific ER‐mediated signal promotes antioxidant defences and inhibits caspase‐dependent cell death. We propose that an immediate consequence of the UPR not only limits the accumulation of misfolded proteins but also protects tissues from harmful exogenous stresses.