Critical Roles for Rictor/Sin1 Complexes in Interferon-dependent Gene Transcription and Generation of Antiproliferative Responses

We provide evidence that type I IFN-induced STAT activation is diminished in cells with targeted disruption of the Rictor gene, whose protein product is a key element of mTOR complex 2. Our studies show that transient or stable knockdown of Rictor or Sin1 results in defects in activation of elements of the STAT pathway and reduced STAT-DNA binding complexes. This leads to decreased expression of several IFN-inducible genes that mediate important biological functions. Our studies also demonstrate that Rictor and Sin1 play essential roles in the generation of the suppressive effects of IFNα on malignant erythroid precursors from patients with myeloproliferative neoplasms. Altogether, these findings provide evidence for critical functions for Rictor/Sin1 complexes in type I IFN signaling and the generation of type I IFN antineoplastic responses.     

A Polysaccharide Virulence Factor from Aspergillus fumigatus Elicits Anti-inflammatory Effects through Induction of Interleukin-1 Receptor Antagonist

The galactosaminogalactan (GAG) is a cell wall component of Aspergillus fumigatus that has potent anti-inflammatory effects in mice. However, the mechanisms responsible for the anti-inflammatory property of GAG remain to be elucidated. In the present study we used in vitro PBMC stimulation assays to demonstrate, that GAG inhibits proinflammatory T-helper (Th)1 and Th17 cytokine production in human PBMCs by inducing Interleukin-1 receptor antagonist (IL-1Ra), a potent anti-inflammatory cytokine that blocks IL-1 signalling. GAG cannot suppress human T-helper cytokine production in the presence of neutralizing antibodies against IL-1Ra. In a mouse model of invasive aspergillosis, GAG induces IL-1Ra in vivo, and the increased susceptibility to invasive aspergillosis in the presence of GAG in wild type mice is not observed in mice deficient for IL-1Ra. Additionally, we demonstrate that the capacity of GAG to induce IL-1Ra could also be used for treatment of inflammatory diseases, as GAG was able to reduce severity of an experimental model of allergic aspergillosis, and in a murine DSS-induced colitis model. In the setting of invasive aspergillosis, GAG has a significant immunomodulatory function by inducing IL-1Ra and notably IL-1Ra knockout mice are completely protected to invasive pulmonary aspergillosis. This opens new treatment strategies that target IL-1Ra in the setting of acute invasive fungal infection. However, the observation that GAG can also protect mice from allergy and colitis makes GAG or a derivative structure of GAG a potential treatment compound for IL-1 driven inflammatory diseases.     

Development and validation of a new Ppo-A1 marker useful for marker-assisted selection in tetraploid wheats

Polyphenol oxidase (PPO) activity is a major cause of undesirable brown color of semolina. In tetraploid wheat, the Ppo-A1 gene is significantly involved in the phenotypic expression of PPO activity. The main goal of this study was to develop and validate a more efficient marker for Ppo-A1 to facilitate marker-assisted selection for low PPO activity in tetraploid wheat breeding programs. A large tetraploid wheat collection, including durum cultivars, domesticated and wild accessions, was used to evaluate the PPO activity. The heritability values indicated that the phenotypic expression of PPO activity was mainly due to genotypic effect. PPO18, and a new marker named MG18, were used to study the Ppo-A1 allelic variation in a tetraploid wheat collection. PPO18 analysis detected four alleles (Ppo-A1b, Ppo-A1e, Ppo-A1f and Ppo-A1g). The high frequency of Ppo-A1g (no PCR product) detected in the tetraploid wheat collection, led to the development of a new genome-specific Ppo-A1 marker (MG18). MG18 analysis identified the same alleles as PPO18 which were associated with low or high PPO activity. The new MG18 marker was more efficient than PPO18 in detecting the four different alleles of Ppo-A1 in the tetraploid wheat collection. Indeed, the accessions assigned to the Ppo-A1g group, according to PPO18, when tested with MG18, were better classified in the four alleles of the Ppo-A1 gene. The MG18 analysis proved that the PPO18 marker overestimated the number of accessions with Ppo-A1g. Therefore, MG18 can be applied to large-scale marker-assisted selection for PPO activity in durum breeding programs.     

Nailfold videocapillaroscopy micro-haemorrhage and giant capillary counting as an accurate approach for a steady state definition of disease activity in systemic sclerosis

Introduction

Nailfold videocapillaroscopy (NVC) in systemic sclerosis (SSc) is a procedure commonly used for patient classification and subsetting, but not to define disease activity (DA). This study aimed to evaluate whether the number of micro-haemorrhages (MHE), micro-thrombosis (MT), giant capillaries (GC), and normal/dilated capillaries (Cs) in NVC could predict DA in SSc.

Methods

Eight-finger NVC was performed in 107 patients with SSc, and the total number of MHE/MT, GC, and the mean number of Cs were counted and defined as number of micro-haemorrhages (NEMO), GC and Cs scores, respectively. The European Scleroderma Study Group (ESSG) index constituted the gold standard for DA assessment, and scores ≥3.5 and =3 were considered indicative of high and moderate activity, respectively.

Results

NEMO and GC scores were positively correlated with ESSG index (R = 0.65, P <0.0001, and R = 0.47, P <0.0001, respectively), whilst Cs score showed a negative correlation with that DA index (R = −0.30, P <0.001). The area under the curve (AUC) of receiver operating characteristic plots, obtained by NEMO score sensitivity and specificity values in classifying patients with ESSG index ≥3.5, was significantly higher than the corresponding AUC derived from either GC or Cs scores (P <0.03 and P <0.0006, respectively). A modified score, defined by the presence of a given number of MHE/MT and GC, had a good performance in classifying active patients (ESSG index ≥3, sensitivity 95.1%, specificity 84.8%, accuracy 88.7%).

Conclusions

MHE/MT and GC appear to be good indicators of DA in SSc, and enhances the role of NVC as an easy technique to identify active patients.     

Rapid methods to extract DNA and RNA from Cryptococcus neoformans

Extraction of nucleic acids from the pathogenic yeast Cryptococcus neoformans is normally hampered by a thick and resistant capsule, accounting for at least 70% of the whole cellular volume. This paper presents procedures based on mechanical cell breakage to extract DNA and RNA from C. neoformans and other capsulated species. The proposed system for DNA extraction involves capsule relaxation by means of a short urea treatment and bead beating. These two steps allow a consistent extraction even from strains resistant to other procedures. Yield and quality of DNA obtained with the proposed method were higher than those obtained with two earlier described methods. This protocol can be extended to every yeast species and particularly to those difficult to handle for the presence of a capsule. RNA purification is accomplished using an original lysing matrix and the FastPrep System (Bio101) after a preliminary bead beating treatment. Yields range around 1 mg RNA from 15 ml overnight culture (10(9) cells), RNA appears undegraded, making it suitable for molecular manipulations.     

Rapamycin Rescues TDP-43 Mislocalization and the Associated Low Molecular Mass Neurofilament Instability

TDP-43 is a nuclear protein involved in exon skipping and alternative splicing. Recently, TDP-43 has been identified as the pathological signature protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis. In addition, TDP-43-positive inclusions are present in Parkinson disease, dementia with Lewy bodies, and 30% of Alzheimer disease cases. Pathological TDP-43 is redistributed from the nucleus to the cytoplasm, where it accumulates. An ∼25-kDa C-terminal fragment of TDP-43 accumulates in affected brain regions, suggesting that it may be involved in the disease pathogenesis. Here, we show that overexpression of the 25-kDa C-terminal fragment is sufficient to cause the mislocalization and cytoplasmic accumulation of endogenous full-length TDP-43 in two different cell lines, thus recapitulating a key biochemical characteristic of TDP-43 proteinopathies. We also found that TDP-43 mislocalization is associated with a reduction in the low molecular mass neurofilament mRNA levels. Notably, we show that the autophagic system plays a role in TDP-43 metabolism. Specifically, we found that autophagy inhibition increases the accumulation of the C-terminal fragments of TDP-43, whereas inhibition of mTOR, a key protein kinase involved in autophagy regulation, reduces the 25-kDa C-terminal fragment accumulation and restores TDP-43 localization. Our results suggest that autophagy induction may be a valid therapeutic target for TDP-43 proteinopathies.TDP-43 (transactive response DNA-binding protein 43) is a conserved and ubiquitously expressed nuclear protein with a theoretical molecular mass of ∼44 kDa. It is encoded by the TARDBP gene on chromosome 1, which is made of six exons that can be alternatively spliced to yield 11 different isoforms, with the mRNA encoding TDP-43 being the major species (1). Functionally, TDP-43 appears to be involved in exon skipping and alternative splicing (2, 3), and it has also been shown to link different types of nuclear bodies (4). Structural studies have confirmed the presence of two RNA recognition motifs (RRM1 and RRM2) and a glycine-rich C-terminal tail, which is thought to mediate protein-protein interaction (5).Recently, TDP-43 has been shown to be the major pathological protein in a wide range of disorders referred to as TDP-43 proteinopathies (6–8). These include frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U),² motor neuron disease, and amyotrophic lateral sclerosis (ALS). These last two disorders have been directly linked to mutations in TDP-43 (9, 10). In addition, TDP-43-positive inclusions are present in Parkinson disease, dementia with Lewy bodies, and 30% of Alzheimer disease cases (11–14). Sporadic and familial forms of FTLD-U and ALS are characterized by cytoplasmic accumulation of insoluble, hyperphosphorylated, ubiquitinated, and proteolytically cleaved C-terminal fragments in affected brain and spinal cord regions. The cytoplasmic accumulation of TDP-43 is associated with a depletion of nuclear TDP-43 (8, 15–21). These data suggest that some of these TDP-43 proteinopathies may share common mechanisms of pathogenesis.FTLD-U is caused by loss-of-function mutations in the progranulin gene, which lead, by an unknown mechanism, to the accumulation of cytoplasmic TDP-43 inclusions (22, 23). Notably, the TDP-43 inclusions in the ALS and FTLD-U brains are enriched with TDP-43 C-terminal fragments (8, 19). It has been suggested that the C-terminal fragments can be obtained by caspase-dependent cleavage of the full-length protein (24). However, it remains to be established if these fragments play a role in the disease pathogenesis.TDP-43 proteinopathies are characterized by the accumulation of abnormally modified TDP-43, suggesting that dysfunction in the intracellular quality control systems (ubiquitin-proteasome system and the autophagy-lysosome system) may be involved in the disease pathogenesis. The autophagic system is a conserved intracellular system designed for the degradation of long-lived proteins and organelles in lysosomes (25, 26). Three types of autophagy have been described: macroautophagy, microautophagy, and chaperon-mediated autophagy. Whereas macroautophagy and microautophagy involve the “in bulk” degradation of regions of the cytosol (27, 28), chaperon-mediated autophagy is a more selective pathway, and only proteins with a lysosomal targeting sequence are degraded (29). Cumulative evidence has suggested that an age-dependent decrease in the autophagy-lysosome system may account for the accumulation of abnormal proteins during aging (30, 31).Macroautophagy is induced when an isolation membrane is formed surrounding cytosolic components, forming an autophagic vacuole, which will eventually fuse with lysosomes for protein/organelle degradation. Induction of the isolation membrane is negatively regulated by mTOR (mammalian target of rapamycin) (32). It has been shown that increasing autophagy activation by mTOR inhibitors has beneficial effects in neurodegeneration (33–35).     

1α,25-Dihydroxyvitamin D3 inhibits CD40L-induced pro-inflammatory and immunomodulatory activity in Human Monocytes

CD40 ligand (CD40L) stimulation induces proinflammatory and immunomodulatory activity in monocytes. Here, we report on the effects of the steroid hormone 1α,25-dihydroxyvitamin D3 (1,25D3) on human blood monocytes that have been stimulated with the CD40L ligand. Co-treatment of CD40L-stimulated monocytes with 1,25D3 resulted in reduced production and secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-1β, as well as in reduced expression of the surface co-stimulatory molecules CD80 and CD86. In addition, costimulation of CD4+ T lymphocytes by monocytes co-treated with CD40L and 1,25D3 resulted in reduced cell proliferation and diminished interferon (IFN)-γ but enhanced IL-10 production by CD4+ T cells. Finally, 1,25D3 interfered with the ability of CD40L to rescue monocytes from apoptosis induced by serum withdrawal. These findings suggest that 1,25D3 may regulate the interaction of monocytes with T cells or other cell types that express CD40L, thus influencing the outcome of the immune or inflammatory response.