Early Proliferation Does Not Prevent the Loss of Oligodendrocyte Progenitor Cells during the Chronic Phase of Secondary Degeneration in a CNS White Matter Tract

Partial injury to the central nervous system (CNS) is exacerbated by additional loss of neurons and glia via toxic events known as secondary degeneration. Using partial transection of the rat optic nerve (ON) as a model, we have previously shown that myelin decompaction persists during secondary degeneration. Failure to repair myelin abnormalities during secondary degeneration may be attributed to insufficient OPC proliferation and/or differentiation to compensate for loss of oligodendrocyte lineage cells (oligodendroglia). Following partial ON transection, we found that sub-populations of oligodendroglia and other olig2+ glia were differentially influenced by injury. A high proportion of NG2+/olig2–, NG2+/olig2+ and CC1−/olig2+ cells proliferated (Ki67+) at 3 days, prior to the onset of death (TUNEL+) at 7 days, suggesting injury-related cues triggered proliferation rather than early loss of oligodendroglia. Despite this, a high proportion (20%) of the NG2+/olig2+ OPCs were TUNEL+ at 3 months, and numbers remained chronically lower, indicating that proliferation of these cells was insufficient to maintain population numbers. There was significant death of NG2+/olig2– and NG2−/olig2+ cells at 7 days, however population densities remained stable, suggesting proliferation was sufficient to sustain cell numbers. Relatively few TUNEL+/CC1+ cells were detected at 7 days, and no change in density indicated that mature CC1+ oligodendrocytes were resistant to secondary degeneration in vivo. Mature CC1+/olig2– oligodendrocyte density increased at 3 days, reflecting early oligogenesis, while the appearance of shortened myelin internodes at 3 months suggested remyelination. Taken together, chronic OPC decreases may contribute to the persistent myelin abnormalities and functional loss seen in ON during secondary degeneration.     

Synergistic Antitumor Effect between Gefitinib and Fractionated Irradiation in Anaplastic Oligodendrogliomas Cannot Be Predicted by the Egfr Signaling Activity

In high-grade gliomas, the identification of patients that could benefit from EGFR inhibitors remains a challenge, hindering the use of these agents. Using xenografts models, we evaluated the antitumor effect of the combined treatment “gefitinib + radiotherapy” and aimed to identify the profile of responsive tumors. Expression of phosphorylated proteins involved in the EGFR-dependent signaling pathways was analyzed in 10 glioma models. We focused on three models of anaplastic oligodendrogliomas (TCG2, TCG3 and TCG4) harboring high levels of phospho-EGFR, phospho-AKT and phospho-MEK1. They were treated with gefitinib (GEF 75 mg/kg/day x 5 days/week, for 2 weeks) and/or fractionated radiotherapy (RT: 5x2Gy/week for 2 weeks). Our results showed that GEF and/or RT induced significant tumor growth delays. However, only the TCG3 xenografts were highly responsive to the combination GEF+RT, with ∼50% of tumor cure. Phosphoproteins analysis five days after treatment onset demonstrated in TCG3 xenografts, but not in TCG2 model, that the EGFR-dependent pathways were inhibited after GEF treatment. Moreover, TCG3-bearing mice receiving GEF monotherapy exhibited a transient beneficial therapeutic response, rapidly followed by tumor regrowth, along with a major vascular remodeling. Taken together, our data evoked an “EGFR-addictive” behavior for TCG3 tumors. This study confirms that combination of gefitinib with fractionated irradiation could be a potent therapeutic strategy for anaplastic oligodendrogliomas harboring EGFR abnormalities but this treatment seems mainly beneficial for “EGFR-addictive” tumors. Unfortunately, neither the usual molecular markers (EGFR amplification, PTEN loss) nor the basal overexpression of phosphoproteins were useful to distinguish this responsive tumor. Evaluating the impact of TKIs on the EGFR-dependent pathways during the treatment might be more relevant, and requires further validation.     

Suppression of Hepcidin Expression and Iron Overload Mediate Salmonella Susceptibility in Ankyrin 1 ENU-Induced Mutant

Salmonella, a ubiquitous Gram-negative intracellular bacterium, is a food borne pathogen that infects a broad range of hosts. Infection with Salmonella Typhimurium in mice is a broadly recognized experimental model resembling typhoid fever in humans. Using a N-ethyl-N-nitrosurea (ENU) mutagenesis recessive screen, we report the identification of Ity16 (Immunity to Typhimurium locus 16), a locus responsible for increased susceptibility to infection. The position of Ity16 was refined on chromosome 8 and a nonsense mutation was identified in the ankyrin 1 (Ank1) gene. ANK1 plays an important role in the formation and stabilization of the red cell cytoskeleton. The Ank1^Ity16/Ity16 mutation causes severe hemolytic anemia in uninfected mice resulting in splenomegaly, hyperbilirubinemia, jaundice, extramedullary erythropoiesis and iron overload in liver and kidneys. Ank1^Ity16/Ity16 mutant mice demonstrated low levels of hepcidin (Hamp) expression and significant increases in the expression of the growth differentiation factor 15 (Gdf15), erythropoietin (Epo) and heme oxygenase 1 (Hmox1) exacerbating extramedullary erythropoiesis, tissue iron deposition and splenomegaly. As the infection progresses in Ank1^Ity16/Ity16, the anemia worsens and bacterial load were high in liver and kidneys compared to wild type mice. Heterozygous Ank1^+/Ity16 mice were also more susceptible to Salmonella infection although to a lesser extent than Ank1^Ity16/Ity16 and they did not inherently present anemia and splenomegaly. During infection, iron accumulated in the kidneys of Ank1^+/Ity16 mice where bacterial loads were high compared to littermate controls. The critical role of HAMP in the host response to Salmonella infection was validated by showing increased susceptibility to infection in Hamp-deficient mice and significant survival benefits in Ank1 ^+/Ity16 heterozygous mice treated with HAMP peptide. This study illustrates that the regulation of Hamp and iron balance are crucial in the host response to Salmonella infection in Ank1 mutants.     

Massive Sorghum Collection Genotyped with SSR Markers to Enhance Use of Global Genetic Resources

Large ex situ collections require approaches for sampling manageable amounts of germplasm for in-depth characterization and use. We present here a large diversity survey in sorghum with 3367 accessions and 41 reference nuclear SSR markers. Of 19 alleles on average per locus, the largest numbers of alleles were concentrated in central and eastern Africa. Cultivated sorghum appeared structured according to geographic regions and race within region. A total of 13 groups of variable size were distinguished. The peripheral groups in western Africa, southern Africa and eastern Asia were the most homogeneous and clearly differentiated. Except for Kafir, there was little correspondence between races and marker-based groups. Bicolor, Caudatum, Durra and Guinea types were each dispersed in three groups or more. Races should therefore better be referred to as morphotypes. Wild and weedy accessions were very diverse and scattered among cultivated samples, reinforcing the idea that large gene-flow exists between the different compartments. Our study provides an entry to global sorghum germplasm collections. Our reference marker kit can serve to aggregate additional studies and enhance international collaboration. We propose a core reference set in order to facilitate integrated phenotyping experiments towards refined functional understanding of sorghum diversity.     

The Calpain/Calpastatin System Has Opposing Roles in Growth and Metastatic Dissemination of Melanoma

Conventional calpains are ubiquitous cysteine proteases whose activity is promoted by calcium signaling and specifically limited by calpastatin. Calpain expression has been shown to be increased in human malignant cells, but the contribution of the calpain/calpastatin system in tumorigenesis remains unclear. It may play an important role in tumor cells themselves (cell growth, migration, and a contrario cell death) and/or in tumor niche (tissue infiltration by immune cells, neo-angiogenesis). In this study, we have used a mouse model of melanoma as a tool to gain further understanding of the role of calpains in tumor progression. To determine the respective importance of each target, we overexpressed calpastatin in tumor and/or host in isolation. Our data demonstrate that calpain inhibition in both tumor and host blunts tumor growth, while paradoxically increasing metastatic dissemination to regional lymph nodes. Specifically, calpain inhibition in melanoma cells limits tumor growth in vitro and in vivo but increases dissemination by amplifying cell resistance to apoptosis and accelerating migration process. Meanwhile, calpain inhibition restricted to host cells blunts tumor infiltration by immune cells and angiogenesis required for antitumor immunity, allowing tumor cells to escape tumor niche and disseminate. The development of highly specific calpain inhibitors with potential medical applications in cancer should take into account the opposing roles of the calpain/calpastatin system in initial tumor growth and subsequent metastatic dissemination.     

Preoperative Diffusion-Weighted Imaging of Single Brain Metastases Correlates with Patient Survival Times

Background

MRI-based diffusion-weighted imaging (DWI) visualizes the local differences in water diffusion in vivo. The prognostic value of DWI signal intensities on the source images and apparent diffusion coefficient (ADC) maps respectively has not yet been studied in brain metastases (BM).

Methods

We included into this retrospective analysis all patients operated for single BM at our institution between 2002 and 2010, in whom presurgical DWI and BM tissue samples were available. We recorded relevant clinical data, assessed DWI signal intensity and apparent diffusion coefficient (ADC) values and performed histopathological analysis of BM tissues. Statistical analyses including uni- and multivariate survival analyses were performed.

Results

65 patients (34 female, 31 male) with a median overall survival time (OS) of 15 months (range 0–99 months) were available for this study. 19 (29.2%) patients presented with hyper-, 3 (4.6%) with iso-, and 43 (66.2%) with hypointense DWI. ADCmean values could be determined in 32 (49.2%) patients, ranged from 456.4 to 1691.8*10⁻⁶ mm²/s (median 969.5) and showed a highly significant correlation with DWI signal intensity. DWI hyperintensity correlated significantly with high amount of interstitial reticulin deposition. In univariate analysis, patients with hyperintense DWI (5 months) and low ADCmean values (7 months) had significantly worse OS than patients with iso/hypointense DWI (16 months) and high ADCmean values (30 months), respectively. In multivariate survival analysis, high ADCmean values retained independent statistical significance.

Conclusions

Preoperative DWI findings strongly and independently correlate with OS in patients operated for single BM and are related to interstitial fibrosis. Inclusion of DWI parameters into established risk stratification scores for BM patients should be considered.     

3D Microstructural Architecture of Muscle Attachments in Extant and Fossil Vertebrates Revealed by Synchrotron Microtomography

Background

Firm attachments binding muscles to skeleton are crucial mechanical components of the vertebrate body. These attachments (entheses) are complex three-dimensional structures, containing distinctive arrangements of cells and fibre systems embedded in the bone, which can be modified during ontogeny. Until recently it has only been possible to obtain 2D surface and thin section images of entheses, leaving their 3D histology largely unstudied except by extrapolation from 2D data. Entheses are frequently preserved in fossil bones, but sectioning is inappropriate for rare or unique fossil material.

Methodology/Principal Findings

Here we present the first non-destructive 3D investigation, by propagation phase contrast synchrotron microtomography (PPC-SRµCT), of enthesis histology in extant and fossil vertebrates. We are able to identify entheses in the humerus of the salamander Desmognathus from the organization of bone-cell lacunae and extrinsic fibres. Statistical analysis of the lacunae differentiates types of attachments, and the orientation of the fibres, reflect the approximate alignment of the muscle. Similar histological structures, including ontogenetically related pattern changes, are perfectly preserved in two 380 million year old fossil vertebrates, the placoderm Compagopiscis croucheri and the sarcopterygian fish Eusthenopteron foordi.

Conclusions/Significance

We are able to determine the position of entheses in fossil vertebrates, the approximate orientation of the attached muscles, and aspects of their ontogenetic histories, from PPC-SRµCT data. Sub-micron microtomography thus provides a powerful tool for studying the structure, development, evolution and palaeobiology of muscle attachments.     

Widespread Occurrence of Chemical Residues in Beehive Matrices from Apiaries Located in Different Landscapes of Western France

Background

The honey bee, Apis mellifera, is frequently used as a sentinel to monitor environmental pollution. In parallel, general weakening and unprecedented colony losses have been reported in Europe and the USA, and many factors are suspected to play a central role in these problems, including infection by pathogens, nutritional stress and pesticide poisoning. Honey bee, honey and pollen samples collected from eighteen apiaries of western France from four different landscape contexts during four different periods in 2008 and in 2009 were analyzed to evaluate the presence of pesticides and veterinary drug residues.

Methodology/Findings

A multi-residue analysis of 80 compounds was performed using a modified QuEChERS method, followed by GC-ToF and LC−MS/MS. The analysis revealed that 95.7%, 72.3% and 58.6% of the honey, honey bee and pollen samples, respectively, were contaminated by at least one compound. The frequency of detection was higher in the honey samples (n = 28) than in the pollen (n = 23) or honey bee (n = 20) samples, but the highest concentrations were found in pollen. Although most compounds were rarely found, some of the contaminants reached high concentrations that might lead to adverse effects on bee health. The three most frequent residues were the widely used fungicide carbendazim and two acaricides, amitraz and coumaphos, that are used by beekeepers to control Varroa destructor. Apiaries in rural-cultivated landscapes were more contaminated than those in other landscape contexts, but the differences were not significant. The contamination of the different matrices was shown to be higher in early spring than in all other periods.

Conclusions/Significance

Honey bees, honeys and pollens are appropriate sentinels for monitoring pesticide and veterinary drug environmental pollution. This study revealed the widespread occurrence of multiple residues in beehive matrices and suggests a potential issue with the effects of these residues alone or in combination on honey bee health.