Nuclear shield: a multi-enzyme task-force for nucleus protection

Background

In eukaryotic cells the nuclear envelope isolates and protects DNA from molecules that could damage its structure or interfere with its processing. Moreover, selected protection enzymes and vitamins act as efficient guardians against toxic compounds both in the nucleoplasm and in the cytosol. The observation that a cytosolic detoxifying and antioxidant enzyme i.e. glutathione transferase is accumulated in the perinuclear region of the rat hepatocytes suggests that other unrecognized modalities of nuclear protection may exist. Here we show evidence for the existence of a safeguard enzyme machinery formed by an hyper-crowding of cationic enzymes and proteins encompassing the nuclear membrane and promoted by electrostatic interactions.

Methodology/Principal Findings

Electron spectroscopic imaging, zeta potential measurements, isoelectrofocusing, comet assay and mass spectrometry have been used to characterize this surprising structure that is present in the cells of all rat tissues examined (liver, kidney, heart, lung and brain), and that behaves as a “nuclear shield”. In hepatocytes, this hyper-crowding structure is about 300 nm thick, it is mainly formed by cationic enzymes and the local concentration of key protection enzymes, such as glutathione transferase, catalase and glutathione peroxidase is up to seven times higher than in the cytosol. The catalytic activity of these enzymes, when packed in the shield, is not modified and their relative concentrations vary remarkably in different tissues. Removal of this protective shield renders chromosomes more sensitive to damage by oxidative stress. Specific nuclear proteins anchored to the outer nuclear envelope are likely involved in the shield formation and stabilization.

Conclusions/Significance

The characterization of this previously unrecognized nuclear shield in different tissues opens a new interesting scenario for physiological and protection processes in eukaryotic cells. Selection and accumulation of protection enzymes near sensitive targets represents a new safeguard modality which deeply differs from the adaptive response which is based on expression of specific enzymes.     

Gd-EOB-DTPA-Enhanced MR Guidance in Thermal Ablation of Liver Malignancies

Objective

To evaluate the potency of Gd-EOB-DTPA to support hepatic catheter placement in laser ablation procedures by quantifying time-dependent delineation effects for instrumentation and target tumor within liver parenchyma. Monitoring potential influence on online MR thermometry during the ablation procedure is a secondary aim.

Materials and Methods

30 cases of MR-guided laser ablation were performed after i.v. bolus injection of gadoxetic acid (0.025 mmol/Kg Gd-EOB-DTPA; Bayer Healthcare, Berlin, Germany). T₁-weighted GRE sequences were used for applicator guidance (FLASH 3D) in the catheter placement phase and for therapy monitoring (FLASH 2D) in the therapy phase. SNR and consecutive CNR values were measured for elements of interest plotted over time both for catheter placement and therapy phase and compared with a non-contrast control group of 19 earlier cases. Statistical analysis was realized using the paired Wilcoxon test.

Results

Sustainable signal elevation of liver parenchyma in the contrast-enhanced group was sufficient to silhouette both target tumor and applicator against the liver. Differences in time dependent CNR alteration were highly significant between contrast-enhanced and non-contrast interventions for parenchyma and target on the one hand (p = 0.020) and parenchyma and instrument on the other hand (p = 0.002). Effects lasted for the whole procedure (monitoring up to 60 min) and were specific for the contrast-enhanced group. Contrasting maxima were seen after median 30 (applicator) and 38 (tumor) minutes, in the potential core time of a multineedle procedure. Contrast influence on T1 thermometry for real-time monitoring of thermal impact was not significant (p = 0.068–0.715).

Conclusion

Results strongly support anticipated promotive effects of Gd-EOB-DTPA for MR-guided percutaneous liver interventions by proving and quantifying the delineating effects for therapy-relevant elements in the procedure. Time benefit, cost effectiveness and oncologic outcome of the described beneficiary effects will have to be part of further investigations.     

Glial precursor cell transplantation therapy for neurotrauma and multiple sclerosis

Traumatic injury to the brain or spinal cord and multiple sclerosis (MS) share a common pathophysiology with regard to axonal demyelination. Despite advances in central nervous system (CNS) repair in experimental animal models, adequate functional recovery has yet to be achieved in patients in response to any of the current strategies. Functional recovery is dependent, in large part, upon remyelination of spared or regenerating axons. The mammalian CNS maintains an endogenous reservoir of glial precursor cells (GPCs), capable of generating new oligodendrocytes and astrocytes. These GPCs are upregulated following traumatic or demyelinating lesions, followed by their differentiation into oligodendrocytes. However, this innate response does not adequately promote remyelination. As a result, researchers have been focusing their efforts on harvesting, culturing, characterizing, and transplanting GPCs into injured regions of the adult mammalian CNS in a variety of animal models of CNS trauma or demyelinating disease. The technical and logistic considerations for transplanting GPCs are extensive and crucial for optimizing and maintaining cell survival before and after transplantation, promoting myelination, and tracking the fate of transplanted cells. This is especially true in trials of GPC transplantation in combination with other strategies such as neutralization of inhibitors to axonal regeneration or remyelination. Overall, such studies improve our understanding and approach to developing clinically relevant therapies for axonal remyelination following traumatic brain injury (TBI) or spinal cord injury (SCI) and demyelinating diseases such as MS.     

Individually Adapted Imagery Improves Brain-Computer Interface Performance in End-Users with Disability

Brain-computer interfaces (BCIs) translate oscillatory electroencephalogram (EEG) patterns into action. Different mental activities modulate spontaneous EEG rhythms in various ways. Non-stationarity and inherent variability of EEG signals, however, make reliable recognition of modulated EEG patterns challenging. Able-bodied individuals who use a BCI for the first time achieve - on average - binary classification performance of about 75%. Performance in users with central nervous system (CNS) tissue damage is typically lower. User training generally enhances reliability of EEG pattern generation and thus also robustness of pattern recognition. In this study, we investigated the impact of mental tasks on binary classification performance in BCI users with central nervous system (CNS) tissue damage such as persons with stroke or spinal cord injury (SCI). Motor imagery (MI), that is the kinesthetic imagination of movement (e.g. squeezing a rubber ball with the right hand), is the "gold standard" and mainly used to modulate EEG patterns. Based on our recent results in able-bodied users, we hypothesized that pair-wise combination of "brain-teaser" (e.g. mental subtraction and mental word association) and "dynamic imagery" (e.g. hand and feet MI) tasks significantly increases classification performance of induced EEG patterns in the selected end-user group. Within-day (How stable is the classification within a day?) and between-day (How well does a model trained on day one perform on unseen data of day two?) analysis of variability of mental task pair classification in nine individuals confirmed the hypothesis. We found that the use of the classical MI task pair hand vs. feed leads to significantly lower classification accuracy - in average up to 15% less - in most users with stroke or SCI. User-specific selection of task pairs was again essential to enhance performance. We expect that the gained evidence will significantly contribute to make imagery-based BCI technology become accessible to a larger population of users including individuals with special needs due to CNS damage.     

The Ultrastructure of the Compound Eye of Two Species of Marine Ostracodes (Crustacea: Ostracoda: Cypridinidae)

Ultrastructurally, the compound eyes of the luminescent marine ostracodes Vargula graminkola and V. tsujii are similar. These ostracodes have two lateral compound eyes, with relatively few ommatidia (13 and 20 respectively). They exhibit apposition type compound eyes as seen in many other arthropods. Each ommatidium includes: a flat, ectodermal cuticular covering, corneagen cells, two long cone cells that give rise to a large conspicuous crystalline cone, retinular cells, pigment cells, a microvillar rhabdom and proximal axonal neurons. The axons merge to form an optic nerve that extends into the brain through a short, muscular stalk that is surrounded externally by a cuticle. The number of retinular cells is typically six per ommatidium in V. graminicola and eight per ommatidium in V. tsujii. Screening pigment cells surround each ommatidium forming a layer that is about 5–15 pigment granules thick. In addition to pigment cells, the cytoplasm of the retinular cells includes numerous screening pigment granules. In light/dark adaptation, there are no obvious morphological differences in the orientation of the rhabdom or in the organization of the screening pigments. Both Vargula species studied are nocturnally active and bioluminescent suggesting that these eyes are capable receptors of the bright conspecific luminescence.     

miR-125b Acts as a Tumor Suppressor in Breast Tumorigenesis via Its Novel Direct Targets ENPEP,CK2-α, CCNJ,and MEGF9

MicroRNAs (miRNAs) play important roles in diverse biological processes and are emerging as key regulators of tumorigenesis and tumor progression. To explore the dysregulation of miRNAs in breast cancer, a genome-wide expression profiling of 939 miRNAs was performed in 50 breast cancer patients. A total of 35 miRNAs were aberrantly expressed between breast cancer tissue and adjacent normal breast tissue and several novel miRNAs were identified as potential oncogenes or tumor suppressor miRNAs in breast tumorigenesis. miR-125b exhibited the largest decrease in expression. Enforced miR-125b expression in mammary cells decreased cell proliferation by inducing G2/M cell cycle arrest and reduced anchorage-independent cell growth of cells of mammary origin. miR-125b was found to perform its tumor suppressor function via the direct targeting of the 3’-UTRs of ENPEP, CK2-α, CCNJ, and MEGF9 mRNAs. Silencing these miR-125b targets mimicked the biological effects of miR-125b overexpression, confirming that they are modulated by miR-125b. Analysis of ENPEP, CK2-α, CCNJ, and MEGF9 protein expression in breast cancer patients revealed that they were overexpressed in 56%, 40–56%, 20%, and 32% of the tumors, respectively. The expression of ENPEP and CK2-α was inversely correlated with miR-125b expression in breast tumors, indicating the relevance of these potential oncogenic proteins in breast cancer patients. Our results support a prognostic role for CK2-α, whose expression may help clinicians predict breast tumor aggressiveness. In particular, our results show that restoration of miR-125b expression or knockdown of ENPEP, CK2-α, CCNJ, or MEGF9 may provide novel approaches for the treatment of breast cancer.     

Transposon-assisted cloning and traceless mutagenesis of adenoviruses: Development of a novel vector based on species D

Until recently, adenovirus (Ad)-mediated gene therapy was almost exclusively based on human Ad type 5 (Ad5). Preexisting immunity and the limited, coxsackievirus and adenovirus receptor-dependent tropism of Ad5 stimulated attempts to exploit the natural diversity in tropism of the other 50 known human Ad serotypes. Aiming in particular at immunotherapy and vaccination, we have screened representative serotypes from different Ad species for their ability to infect dendritic cells. Ad19a, an Ad from species D, was selected for development as a new vector for vaccination and cancer gene therapy. To clone and manipulate its genome, we have developed a novel methodology, coined "exposon mutagenesis," that allows the rapid and precise introduction of virtually any genetic alteration (deletions, point mutations, or insertions) into recombinant Ad bacterial artificial chromosomes. The versatility of the system was exemplified by deleting the E3 region of Ad19a, by specifically knocking out expression of a species-specific E3 gene, E3/49K, and by reinserting E3/49K into an E3 null Ad19a mutant. The technology requires only limited sequence information and is applicable to other Ad species. Therefore, it should be extremely valuable for the analysis of gene functions from any Ad species. In addition, a basic, replication-defective E1- and E3-deleted Ad19a vector expressing GFP (Ad19aGFP) was generated. This new vector based on species D Ads exhibits a very promising tropism for lymphoid and muscle cells and shows great potential as an alternative vector for transduction of cell types that are resistant to or only poorly transduced by conventional Ad5-based vectors.     

Retention of a bean phaseolin/maize gamma-Zein fusion in the endoplasmic reticulum depends on disulfide bond formation

Most seed storage proteins of the prolamin class accumulate in the endoplasmic reticulum (ER) as large insoluble polymers termed protein bodies (PBs), through mechanisms that are still poorly understood. We previously showed that a fusion between the Phaseolus vulgaris vacuolar storage protein phaseolin and the N-terminal half of the Zea mays prolamin gamma-zein forms ER-located PBs. Zeolin has 6 Cys residues and, like gamma-zein with 15 residues, is insoluble unless reduced. The contribution of disulfide bonds to zeolin destiny was determined by studying in vivo the effects of 2-mercaptoethanol (2-ME) and by zeolin mutagenesis. We show that in tobacco (Nicotiana tabacum) protoplasts, 2-ME enhances interactions of newly synthesized proteins with the ER chaperone BiP and inhibits the secretory traffic of soluble proteins with or without disulfide bonds. In spite of this general inhibition, 2-ME enhances the solubility of zeolin and relieves its retention in the ER, resulting in increased zeolin traffic. Consistently, mutated zeolin unable to form disulfide bonds is soluble and efficiently enters the secretory traffic without 2-ME treatment. We conclude that disulfide bonds that lead to insolubilization are a determinant for PB-mediated protein accumulation in the ER.     

Farnesylated Nuclear Proteins Kugelkern and Lamin Dm0 Affect Nuclear Morphology by Directly Interacting with the Nuclear Membrane

Nuclear shape changes are observed during a variety of developmental processes, pathological conditions, and ageing. The mechanisms underlying nuclear shape changes in the above-mentioned situations have mostly remained unclear. To address the molecular mechanism behind nuclear shape changes, we analyzed how the farnesylated nuclear envelope proteins Kugelkern and lamin Dm0 affect the structure of the nuclear membrane. We found that Kugelkern and lamin Dm0 affect nuclear shape without requiring filament formation or the presence of a classical nuclear lamina. We also could show that the two proteins do not depend on a group of selected inner nuclear membrane proteins for their localization to the nuclear envelope. Surprisingly, we found that farnesylated Kugelkern and lamin Dm0 protein constructs change the morphology of protein-free liposomes. Based on these findings, we propose that farnesylated proteins of the nuclear membrane induce nuclear shape changes by being asymmetrically inserted into the phospholipid bilayer via their farnesylated C-terminal part.