Compression stiffening of fibrous networks with stiff inclusions.

Tissues commonly consist of cells embedded within a fibrous biopolymer network. Whereas cell-free reconstituted biopolymer networks typically soften under applied uniaxial compression, various tissues, including liver, brain, and fat, have been observed to instead stiffen when compressed. The mechanism for this compression-stiffening effect is not yet clear. Here, we demonstrate that when a material composed of stiff inclusions embedded in a fibrous network is compressed, heterogeneous rearrangement of the inclusions can induce tension within the interstitial network, leading to a macroscopic crossover from an initial bending-dominated softening regime to a stretching-dominated stiffening regime, which occurs before and independently of jamming of the inclusions. Using a coarse-grained particle-network model, we first establish a phase diagram for compression-driven, stretching-dominated stress propagation and jamming in uniaxially compressed two- and three-dimensional systems. Then, we demonstrate that a more detailed computational model of stiff inclusions in a subisostatic semiflexible fiber network exhibits quantitative agreement with the predictions of our coarse-grained model as well as qualitative agreement with experiments.

Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies.

Horseradish peroxidase (HRP), an enzyme omnipresent in biotechnology, is still produced from hairy root cultures, although this procedure is time-consuming and only gives low yields. In addition, the plant-derived enzyme preparation consists of a variable mixture of isoenzymes with high batch-to-batch variation preventing its use in therapeutic applications. In this study, we present a novel and scalable recombinant HRP production process in Escherichia coli that yields a highly pure, active and homogeneous single isoenzyme. We successfully developed a multi-step inclusion body process giving a final yield of 960 mg active HRP/L culture medium with a purity of ≥99% determined by size-exclusion high-performance liquid chromatography (SEC-HPLC). The Reinheitszahl, as well as the activity with 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 3,3',5,5'-tetramethylbenzidine (TMB) as reducing substrates, are comparable to commercially available plant HRP. Thus, our preparation of recombinant, unglycosylated HRP from E. coli is a viable alternative to the enzyme from plant and highly interesting for therapeutic applications.

Hippocampal α-synuclein pathology correlates with memory impairment in multiple system atrophy.

Recent post-mortem studies reported 22-37% of patients with multiple system atrophy can develop cognitive impairment. With the aim of identifying associations between cognitive impairment including memory impairment and α-synuclein pathology, 148 consecutive patients with pathologically proven multiple system atrophy were reviewed. Among them, 118 (79.7%) were reported to have had normal cognition in life, whereas the remaining 30 (20.3%) developed cognitive impairment. Twelve of them had pure frontal-subcortical dysfunction, defined as the presence of executive dysfunction, impaired processing speed, personality change, disinhibition or stereotypy; six had pure memory impairment; and 12 had both types of impairment. Semi-quantitative analysis of neuronal cytoplasmic inclusions in the hippocampus and parahippocampus revealed a disease duration-related increase in neuronal cytoplasmic inclusions in the dentate gyrus and cornu ammonis regions 1 and 2 of patients with normal cognition. In contrast, such a correlation with disease duration was not found in patients with cognitive impairment. Compared to the patients with normal cognition, patients with memory impairment (pure memory impairment: n = 6; memory impairment + frontal-subcortical dysfunction: n = 12) had more neuronal cytoplasmic inclusions in the dentate gyrus, cornu ammonis regions 1-4 and entorhinal cortex. In the multiple system atrophy mixed pathological subgroup, which equally affects the striatonigral and olivopontocerebellar systems, patients with the same combination of memory impairment developed more neuronal inclusions in the dentate gyrus, cornu ammonis regions 1, 2 and 4, and the subiculum compared to patients with normal cognition. Using patients with normal cognition (n = 18), frontal-subcortical dysfunction (n = 12) and memory impairment + frontal-subcortical dysfunction (n = 18), we further investigated whether neuronal or glial cytoplasmic inclusions in the prefrontal, temporal and cingulate cortices or the underlying white matter might affect cognitive impairment in patients with multiple system atrophy. We also examined topographic correlates of frontal-subcortical dysfunction with other clinical symptoms. Although no differences in neuronal or glial cytoplasmic inclusions were identified between the groups in the regions examined, frontal release signs were found more commonly when patients developed frontal-subcortical dysfunction, indicating the involvement of the frontal-subcortical circuit in the pathogenesis of frontal-subcortical dysfunction. Here, investigating cognitive impairment in the largest number of pathologically proven multiple system atrophy cases described to date, we provide evidence that neuronal cytoplasmic inclusion burden in the hippocampus and parahippocampus is associated with the occurrence of memory impairment in multiple system atrophy. Further investigation is necessary to identify the underlying pathological basis of frontal-subcortical dysfunction in multiple system atrophy.

Prevalence of inclusion body disease and associated comorbidity in captive collections of boid and pythonid snakes in Belgium.

Inclusion body disease (IBD) is caused by reptarenaviruses and constitutes one of the most notorious viral diseases in snakes. Although central nervous system disease and various other clinical signs have been attributed to IBD in boid and pythonid snakes, studies that unambiguously reveal the clinical course of natural IBD and reptarenavirus infection are scarce. In the present study, the prevalence of IBD and reptarenaviruses in captive snake collections and the correlation of IBD and reptarenavirus infection with the clinical status of the sampled snakes were investigated. In three IBD positive collections, long-term follow-up during a three- to seven-year period was performed. A total of 292 snakes (178 boas and 114 pythons) from 40 collections in Belgium were sampled. In each snake, blood and buffy coat smears were evaluated for the presence of IBD inclusion bodies (IB) and whole blood was tested for reptarenavirus RNA by RT-PCR. Of all tested snakes, 16.5% (48/292) were positive for IBD of which all were boa constrictors (34.0%; 48/141) and 17.1% (50/292) were reptarenavirus RT-PCR positive. The presence of IB could not be demonstrated in any of the tested pythons, while 5.3% (6/114) were reptarenavirus positive. In contrast to pythons, the presence of IB in peripheral blood cells in boa constrictors is strongly correlated with reptarenavirus detection by RT-PCR (P<0.0001). Although boa constrictors often show persistent subclinical infection, long-term follow-up indicated that a considerable number (22.2%; 6/27) of IBD/reptarenavirus positive boas eventually develop IBD associated comorbidities.

Efficient nanowire-assisted electroporation and cellular inclusion release of microalgal cells achieved by a low voltage.

A mild and low-energy cell disruption method with high efficiency has growing application potential in both the extraction of high-value microalgal products and the inactivation of microalgal cells. Conventional technologies available have disadvantages including high energy consumption, the use of chemicals and so on. Here, this study developed an efficient microalgal cell disruption method using the copper oxide nanowire (CuONW)-modified three-dimensional (3D) copper foam electrodes with a low applied voltage. Electrodes with nanowires synthesized at 400 °C, the optimal preparation temperature, achieved efficient microalgal cell electroporation. Microalgal cells were completely inactivated and disrupted at the voltage of 2 V with the hydraulic retention time (HRT) of 10 s. Scanning electron microscopy (SEM) images showed obvious electroporation damage on the cell surface upon electroporation-treatment (2 V, 30 s). The amount of released cellular inclusion increased significantly with prolonged HRT and the energy consumption of this technology was only 0.014 kWh/kg via the treatment of 2 V and 10 s. This study provided a novel, energy-efficient and chemical-free technique for both microalgal products extraction and cell inactivation.

Positive in vitro wound healing effects of functional inclusion bodies of a lipoxygenase from the Mexican axolotl.

BACKGROUND: AmbLOXe is a lipoxygenase, which is up-regulated during limb-redevelopment in the Mexican axolotl, Ambystoma mexicanum, an animal with remarkable regeneration capacity. Previous studies have shown that mammalian cells transformed with the gene of this epidermal lipoxygenase display faster migration and wound closure rate during in vitro wound healing experiments. RESULTS: In this study, the gene of AmbLOXe was codon-optimized for expression in Escherichia coli and was produced in the insoluble fraction as protein aggregates. These inclusion bodies or nanopills were shown to be reservoirs containing functional protein during in vitro wound healing assays. For this purpose, functional inclusion bodies were used to coat cell culture surfaces prior cell seeding or were added directly to the medium after cells reached confluence. In both scenarios, AmbLOXe inclusion bodies led to faster migration rate and wound closure, in comparison to controls containing either no AmbLOXe or GFP inclusion bodies. CONCLUSIONS: Our results demonstrate that AmbLOXe inclusion bodies are functional and may serve as stable reservoirs of this enzyme. Nevertheless, further studies with soluble enzyme are also necessary in order to start elucidating the exact molecular substrates of AmbLOXe and the biochemical pathways involved in the wound healing effect.

The Relevance of Iron in the Pathogenesis of Multiple System Atrophy: A Viewpoint.

Iron is essential for cellular development and maintenance of multiple physiological processes in the central nervous system. The disturbance of its homeostasis leads to abnormal iron deposition in the brain and causes neurotoxicity via generation of free radicals and oxidative stress. Iron toxicity has been established in the pathogenesis of Parkinson's disease; however, its contribution to multiple system atrophy (MSA) remains elusive. MSA is characterized by cytoplasmic inclusions of misfolded α-synuclein (α-SYN) in oligodendrocytes referred to as glial cytoplasmic inclusions (GCIs). Remarkably, the oligodendrocytes possess high amounts of iron, which together with GCI pathology make a contribution toward MSA pathogenesis likely. Consistent with this observation, the GCI density is associated with neurodegeneration in central autonomic networks as well as olivopontocerebellar and striatonigral pathways. Iron converts native α-SYN into a ß-sheet conformation and promotes its aggregation either directly or via increasing levels of oxidative stress. Interestingly, α-SYN possesses ferrireductase activity and α-SYN expression underlies iron mediated translational control via RNA stem loop structures. Despite a correlation between progressive putaminal atrophy and iron accumulation as well as clinical decline, it remains unclear whether pathologic iron accumulation in MSA is a secondary event in the cascade of neuronal degeneration rather than a primary cause. This review summarizes the current knowledge of iron in MSA and gives evidence for perturbed iron homeostasis as a potential pathogenic factor in MSA-associated neurodegeneration.

Chlamydia trachomatis regulates growth and development in response to host cell fatty acid availability in the absence of lipid droplets.

Chlamydia trachomatis (Ct) is a Gram-negative obligate intracellular pathogen of humans that causes significant morbidity from sexually transmitted and ocular diseases globally. Ct acquires host fatty acids (FA) to meet the metabolic and growth requirements of the organism. Lipid droplets (LDs) are storehouses of FAs in host cells and have been proposed to be a source of FAs for the parasitophorous vacuole, termed inclusion, in which Ct replicates. Previously, cells devoid of LDs were shown to produce reduced infectious progeny at 24 hr postinfection (hpi). Here, although we also found reduced progeny at 24 hpi, there were significantly more progeny at 48 hpi in the absence of LDs compared to the control wild-type (WT) cells. These findings were confirmed using transmission electron microscopy where cells without LDs were shown to have significantly more metabolically active reticulate bodies at 24 hpi and significantly more infectious but metabolically inert elementary bodies at 48 hpi than WT cells. Furthermore, by measuring basal oxygen consumption rates (OCR) using extracellular flux analysis, Ct infected cells without LDs had higher OCRs at 24 hpi than cells with LDs, confirming ongoing metabolic activity in the absence of LDs. Although the FA oleic acid is a major source of phospholipids for Ct and stimulates LD synthesis, treatment with oleic acid, but not other FAs, enhanced growth and led to an increase in basal OCR in both LD depleted and WT cells, indicating that FA transport to the inclusion is not affected by the loss of LDs. Our results show that Ct regulates inclusion metabolic activity and growth in response to host FA availability in the absence of LDs.

Spinal motor neuron protein supersaturation patterns are associated with inclusion body formation in ALS.

Amyotrophic lateral sclerosis (ALS) is a heterogeneous degenerative motor neuron disease linked to numerous genetic mutations in apparently unrelated proteins. These proteins, including SOD1, TDP-43, and FUS, are highly aggregation-prone and form a variety of intracellular inclusion bodies that are characteristic of different neuropathological subtypes of the disease. Contained within these inclusions are a variety of proteins that do not share obvious characteristics other than coaggregation. However, recent evidence from other neurodegenerative disorders suggests that disease-affected biochemical pathways can be characterized by the presence of proteins that are supersaturated, with cellular concentrations significantly greater than their solubilities. Here, we show that the proteins that form inclusions of mutant SOD1, TDP-43, and FUS are not merely a subset of the native interaction partners of these three proteins, which are themselves supersaturated. To explain the presence of coaggregating proteins in inclusions in the brain and spinal cord, we observe that they have an average supersaturation even greater than the average supersaturation of the native interaction partners in motor neurons, but not when scores are generated from an average of other human tissues. These results suggest that inclusion bodies in various forms of ALS result from a set of proteins that are metastable in motor neurons, and thus prone to aggregation upon a disease-related progressive collapse of protein homeostasis in this specific setting.

FUS inclusions disrupt RNA localization by sequestering kinesin-1 and inhibiting microtubule detyrosination.

Cytoplasmic inclusions of the RNA-binding protein fused in sarcoma (FUS) represent one type of membraneless ribonucleoprotein compartment. Formation of FUS inclusions is promoted by amyotrophic lateral sclerosis (ALS)-linked mutations, but the cellular functions affected upon inclusion formation are poorly defined. In this study, we find that FUS inclusions lead to the mislocalization of specific RNAs from fibroblast cell protrusions and neuronal axons. This is mediated by recruitment of kinesin-1 mRNA and protein within FUS inclusions, leading to a loss of detyrosinated glutamate (Glu)-microtubules (MTs; Glu-MTs) and an inability to support the localization of RNAs at protrusions. Importantly, dissolution of FUS inclusions using engineered Hsp104 disaggregases, or overexpression of kinesin-1, reverses these effects. We further provide evidence that kinesin-1 affects MT detyrosination not through changes in MT stability, but rather through targeting the tubulin carboxypeptidase enzyme onto specific MTs. Interestingly, other pathological inclusions lead to similar outcomes, but through apparently distinct mechanisms. These results reveal a novel kinesin-dependent mechanism controlling the MT cytoskeleton and identify loss of Glu-MTs and RNA mislocalization as common outcomes of ALS pathogenic mutations.

Functional inclusion bodies produced in the yeast Pichia pastoris.

BACKGROUND: Bacterial inclusion bodies (IBs) are non-toxic protein aggregates commonly produced in recombinant bacteria. They are formed by a mixture of highly stable amyloid-like fibrils and releasable protein species with a significant extent of secondary structure, and are often functional. As nano structured materials, they are gaining biomedical interest because of the combination of submicron size, mechanical stability and biological activity, together with their ability to interact with mammalian cell membranes for subsequent cell penetration in absence of toxicity. Since essentially any protein species can be obtained as IBs, these entities, as well as related protein clusters (e.g., aggresomes), are being explored in biocatalysis and in biomedicine as mechanically stable sources of functional protein. One of the major bottlenecks for uses of IBs in biological interfaces is their potential contamination with endotoxins from producing bacteria. RESULTS: To overcome this hurdle, we have explored here the controlled production of functional IBs in the yeast Pichia pastoris (Komagataella spp.), an endotoxin-free host system for recombinant protein production, and determined the main physicochemical and biological traits of these materials. Quantitative and qualitative approaches clearly indicate the formation of IBs inside yeast, similar in morphology, size and biological activity to those produced in E. coli, that once purified, interact with mammalian cell membranes and penetrate cultured mammalian cells in absence of toxicity. CONCLUSIONS: Structurally and functionally similar from those produced in E. coli, the controlled production of IBs in P. pastoris demonstrates that yeasts can be used as convenient platforms for the biological fabrication of self-organizing protein materials in absence of potential endotoxin contamination and with additional advantages regarding, among others, post-translational modifications often required for protein functionality.

Ultrastructural and cytogenetic analyses of mature human oocyte dysmorphisms with respect to clinical outcomes.

PURPOSE: The study aimed to describe the ultrastructure of two human mature oocyte intracytoplasmic dysmorphisms, the bull-eye inclusion and the granular vacuole, with evaluation of clinical outcomes after intracytoplasmic sperm injection (ICSI) treatment. METHODS: We retrospectively evaluated 4099 consecutive ICSI cycles during the period 2003-2013. Three groups were compared: controls, those with a bulls-eye inclusion, and those with granular vacuoles. Oocyte dysmorphisms were evaluated by transmission electron microscopy and in situ fluorescence hybridization (FISH). Detailed data on demographic and stimulation characteristics, as well as on embryological, clinical, and newborn outcomes, are fully presented. RESULTS: The bull-eye inclusion is a prominent smooth round structure containing trapped vesicles, being surrounded by lipid droplets. The presence of this dysmorphism in the oocyte cohort had no clinical impact except when transferred embryos were exclusively derived from dysmorphism oocytes. The granular vacuole is delimited by a discontinuous double membrane and contains lipid droplets and vesicles. As FISH analysis revealed the presence of chromosomes, they probably represent pyknotic nuclei. The presence of this dysmorphism in the oocyte cohort had no clinical impact except when at least one transferred embryo was derived from dimorphic oocytes. CONCLUSIONS: Poor clinical outcomes were observed with transfer of embryos derived from dysmorphism oocytes, although without causing gestation or newborn problems. The bull-eye inclusion and granular vacuoles may thus be new prognostic factors for clinical outcomes.

Stepwise acquirement of hallmark neuropathology in FUS-ALS iPSC models depends on mutation type and neuronal aging.

Autosomal-dominant mutations within the gene FUS (fused in sarcoma) are responsible for 5% of familial cases of amyotrophic lateral sclerosis (ALS). The FUS protein is physiologically mainly located in the nucleus, while cytoplasmic FUS aggregates are pathological hallmarks of FUS-ALS. Data from non-neuronal cell models and/or models using heterologous expression of FUS mutants suggest cytoplasmic FUS translocation as a pivotal initial event which leads to neurodegeneration depending on a second hit. Here we present the first human model of FUS-ALS using patient-derived neurons carrying endogenous FUS mutations leading to a benign (R521C) or a more severe clinical phenotype (frameshift mutation R495QfsX527). We thereby showed that the severity of the underlying FUS mutation determines the amount of cytoplasmic FUS accumulation and cellular vulnerability to exogenous stress. Cytoplasmic FUS inclusions formed spontaneously depending on both, severity of FUS mutation and neuronal aging. These aggregates showed typical characteristics of FUS-ALS including methylated FUS. Finally, neurodegeneration was not specific to layer V cortical neurons perfectly in line with the current model of disease spreading in ALS. Our study highlights the value and usefulness of patient-derived cell models in FUS-ALS.

Models of lipid droplets growth and fission in adipocyte cells.

Lipid droplets (LD) are spherical cellular inclusion devoted to lipids storage. It is well known that excessive accumulation of lipids leads to several human worldwide diseases like obesity, type 2 diabetes, hepatic steatosis and atherosclerosis. LDs' size range from fraction to one hundred of micrometers in adipocytes and is related to the lipid content, but their growth is still a puzzling question. It has been suggested that LDs can grow in size due to the fusion process by which a larger LD is obtained by the merging of two smaller LDs, but these events seems to be rare and difficult to be observed. Many other processes are thought to be involved in the number and growth of LDs, like the de novo formation and the growth through additional neutral lipid deposition in pre-existing droplets. Moreover the number and size of LDs are influenced by the catabolism and the absorption or interaction with other organelles. The comprehension of these processes could help in the confinement of the pathologies related to lipid accumulation. In this study the LDs' size distribution, number and the total volume of immature (n=12), mature (n=12, 10-days differentiated) and lipolytic (n=12) 3T3-L1 adipocytes were considered. More than 11,000 LDs were measured in the 36 cells after Oil Red O staining. In a previous work Monte Carlo simulations were used to mimic the fusion process alone between LDs. We found that, considering the fusion as the only process acting on the LDs, the size distribution in mature adipocytes can be obtained with numerical simulation starting from the size distribution in immature cells provided a very high rate of fusion events. In this paper Monte Carlo simulations were developed to mimic the interaction between LDs taking into account many other processes in addition to fusion (de novo formation and the growth through additional neutral lipid deposition in pre-existing droplets) in order to reproduce the LDs growth and we also simulated the catabolism (fission and the decrease through neutral lipid exit from pre-existing droplets) to reproduce their size reduction observed in lipolytic conditions. The results suggest that each single process, considered alone, can not be considered the only responsible for the size variation observed, but more than one of them, playing together, can quite well reproduce the experimental data.

Inactivation of adenosine A2A receptors reverses working memory deficits at early stages of Huntington's disease models.

Cognitive impairments in Huntington's disease (HD) are attributed to a dysfunction of the cortico-striatal pathway and significantly affect the quality of life of the patients, but this has not been a therapeutic focus in HD to date. We postulated that adenosine A(2A) receptors (A(2A)R), located at pre- and post-synaptic elements of the cortico-striatal pathways, modulate striatal neurotransmission and synaptic plasticity and cognitive behaviors. To critically evaluate the ability of A(2A)R inactivation to prevent cognitive deficits in early HD, we cross-bred A(2A)R knockout (KO) mice with two R6/2 transgenic lines of HD (CAG120 and CAG240) to generate two double transgenic R6/2-CAG120-A(2A)R KO and R6/2-CAG240-A(2A)R KO mice and their corresponding wild-type (WT) littermates. Genetic inactivation of A(2A)R prevented working memory deficits induced by R6/2-CAG120 at post-natal week 6 and by R6/2-CAG240 at post-natal month 2 and post-natal month 3, without modifying motor deficits. Similarly the A2(A)R antagonist KW6002 selectively reverted working memory deficits in R6/2-CAG240 mice at post-natal month 3. The search for possible mechanisms indicated that the genetic inactivation of A(2A)R did not affect ubiquitin-positive neuronal inclusions, astrogliosis or Thr-75 phosphorylation of DARPP-32 in the striatum. Importantly, A(2A)R blockade preferentially controlled long-term depression at cortico-striatal synapses in R6/2-CAG240 at post-natal week 6. The reported reversal of working memory deficits in R6/2 mice by the genetic and pharmacological inactivation of A(2A)R provides a proof-of-principle for A(2A)R as novel targets to reverse cognitive deficits in HD, likely by controlling LTD deregulation.

Dynamic droplets: the role of cytoplasmic inclusions in stress, function, and disease.

Neurodegenerative diseases and other proteinopathies constitute a class of several dozen illnesses etiologically linked to pathological protein misfolding and aggregation. Because of this strong association with disease pathology, cell death, and aging, accumulation of proteins in aggregates or aggregation-associated structures (inclusions) has come to be regarded by many as a deleterious process, to be avoided if possible. Recent work has led us to see inclusion structures and disordered aggregate-like protein mixtures (which we call dynamic droplets) in a new light: not necessarily as a result of a pathological breakdown of cellular order, but as an elaborate cellular architecture regulating function and stress response. In this review, we discuss what is currently known about the role of inclusion structures in cellular homeostasis, stress response, toxicity, and disease. We will focus on possible mechanisms of aggregate toxicity, in contrast to the homeostatic function of several inclusion structures.

Automated generation of tissue-specific three-dimensional finite element meshes containing ellipsoidal cellular inclusions.

Finite element analysis provides a means of describing cellular mechanics in tissue, which can be useful in understanding and predicting physiological and pathological changes. Many prior studies have been limited to simulations of models containing single cells, which may not accurately describe the influence of mechanical interactions between cells. It is desirable to generate models that more accurately reflect the cellular organisation in tissue in order to evaluate the mechanical function of cells. However, as the model geometry becomes more complicated, manual model generation can become laborious. This can be prohibitive if a large number of distinct cell-scale models are required, for example, in multiscale modelling or probabilistic analysis. Therefore, a method was developed to automatically generate tissue-specific cellular models of arbitrary complexity, with minimal user intervention. This was achieved through a set of scripts, which are capable of generating both sample-specific models, with explicitly defined geometry, and tissue-specific models, with geometry derived implicitly from normal statistical distributions. Models are meshed with tetrahedral (TET) elements of variable size to sufficiently discretise model geometries at different spatial scales while reducing model complexity. The ability of TET meshes to appropriately simulate the biphasic mechanical response of a single-cell model is established against that of a corresponding hexahedral mesh for an illustrative use case. To further demonstrate the flexibility of this tool, an explicit model was developed from three-dimensional confocal laser scanning image data, and a set of models were generated from a statistical cellular distribution of the articular femoral cartilage. The tools presented herein are free and openly accessible to the community at large.

[Implication of PML nuclear bodies in intrinsic and innate immunity]. / Implication des corps nucléaires PML dans l'immunité intrinsèque et innée.

PML/TRIM19 is the organizer of PML nuclear bodies (NB), large multiprotein structures associated to the nuclear matrix, which recruit a great number of proteins and which are implicated in various cellular processes including antiviral defense. The conjugation of PML to SUMO is required for the formation and function of PML NB. Alternative splicing from a single PML gene generates several PML isoforms (PMLI to PMLVIIb), each harboring a specific carboxy-terminal region. This variability allows each isoform to recruit different partners and thus confers them specific functions. PML gene is directly induced by interferon and certain PML isoforms are implicated in its antiviral properties, as they display intrinsic antiviral activities against RNA or DNA viruses. One isoform, PMLIV, is also implicated in innate immunity by enhancing IFN-ß production during a viral infection. Here we review recent findings on PML/TRIM19 implication in interferon response and antiviral defense, at the interface between intrinsic and innate immunity.

Preconditioning stimulus of proteasome inhibitor enhances aggresome formation and autophagy in differentiated SH-SY5Y cells.

The abnormal accumulation of protein aggregates is a dominant pathological feature common in neurodegenerative diseases. Autophagy contributes to the processing of aggregated proteins resistant to proteasomal degradation. Autophagic degradation is multi-step process, and especially aggresome formation is a specific and active cellular process for appropriate autophagy-mediated protein homeostasis mechanism. Here, we showed that preconditioning of cells with a non-toxic low dose of MG132 induced autophagy, using an in vitro experimental model that closely represents the characteristics of the autophagy pathway under proteasome inhibition. Clear and large aggresome-like protein accumulation was observed in the perinuclear region of differentiated SH-SY5Y cells with preconditioning stimulus. This results in up-regulation of autophagosome formation and turnover and degradation of intracellular ubiquitinated and p62-bound protein aggregates. Pretreatment with low dose of MG132 attenuated proteinopathy-related cytotoxicity. Together, our experimental model could provide a proper in vitro system for studying the autophagy-related pathophysiology of neurodegeneration, especially therapeutic targeting of intracellular aggresome-like aggregates formation.

Electron tomography and cryo-SEM characterization reveals novel ultrastructural features of host-parasite interaction during Chlamydia abortus infection.

Chlamydia (C.) abortus is a widely spread pathogen among ruminants that can be transmitted to women during pregnancy leading to severe systemic infection with consecutive abortion. As a member of the Chlamydiaceae, C. abortus shares the characteristic feature of an obligate intracellular biphasic developmental cycle with two morphological forms including elementary bodies (EBs) and reticulate bodies (RBs). In contrast to other chlamydial species, C. abortus ultrastructure has not been investigated yet. To do so, samples were fixed by high-pressure freezing and processed by different electron microscopic methods. Freeze-substituted samples were analysed by transmission electron microscopy, scanning transmission electron microscopical tomography and immuno-electron microscopy, and freeze-fractured samples were analysed by cryo-scanning electron microscopy. Here, we present three ultrastructural features of C. abortus that have not been reported up to now. Firstly, the morphological evidence that C. abortus is equipped with the type three secretion system. Secondly, the accumulation and even coating of whole inclusion bodies by membrane complexes consisting of multiple closely adjacent membranes which seems to be a C. abortus specific feature. Thirdly, the formation of small vesicles in the periplasmic space of RBs in the second half of the developmental cycle. Concerning the time point of their formation and the fact that they harbour chlamydial components, these vesicles might be morphological correlates of an intermediate step during the process of redifferentiation of RBs into EBs. As this feature has also been shown for C. trachomatis and C. pneumoniae, it might be a common characteristic of the family of Chlamydiaceae.