Synaptically Released Matrix Metalloproteinase Activity in Control of Structural Plasticity and the Cell Surface Distribution of GluA1-AMPA Receptors

Synapses are particularly prone to dynamic alterations and thus play a major role in neuronal plasticity. Dynamic excitatory synapses are located at the membranous neuronal protrusions called dendritic spines. The ability to change synaptic connections involves both alterations at the morphological level and changes in postsynaptic receptor composition. We report that endogenous matrix metalloproteinase (MMP) activity promotes the structural and functional plasticity of local synapses by its effect on glutamate receptor mobility and content. We used live imaging of cultured hippocampal neurons and quantitative morphological analysis to show that chemical long-term potentiation (cLTP) induces the permanent enlargement of a subset of small dendritic spines in an MMP-dependent manner. We also used a superresolution microscopy approach and found that spine expansion induced by cLTP was accompanied by MMP-dependent immobilization and synaptic accumulation as well as the clustering of GluA1-containing AMPA receptors. Altogether, our results reveal novel molecular and cellular mechanisms of synaptic plasticity.     

MR Angiography Follow-Up 10 Years after Cryptogenic Nonperimesencephalic Subarachnoid Hemorrhage

ObjectivesLong-term magnetic resonance angiography (MRA) follow-up studies regarding cryptogenic nonperimesencephalic subarachnoid hemorrhage (nSAH) are scarce. This single-centre study identified all patients with angiographically verified cryptogenic nSAH from 1998 to 2007: The two main objectives were to prospectively assess the incidence of de novo aneurysm with 3.0-MRI years after cryptogenic nSAH in patients without evidence for further hemorrhage, and retrospectively assess patient demographics and outcome.MethodsFrom prospectively maintained report databases all patients with angiographically verified cryptogenic nSAH were identified. 21 of 29 patients received high-resolution 3T-MRI including time-of-flight and contrast-enhanced angiography, 10.2 ± 2.8 years after cryptogenic nSAH. MRA follow-up imaging was compared with initial digital subtraction angiography (DSA) and CT/MRA. Post-hemorrhage images were related to current MRI with reference to persistent lesions resulting from delayed cerebral ischemia (DCI) and post-hemorrhagic siderosis. Patient-based objectives were retrospectively abstracted from clinical databases.Results29 patients were identified with cryptogenic nSAH, 17 (59%) were male. Mean age at time of hemorrhage was 52.9 ± 14.4 years (range 4 – 74 years). 21 persons were available for long-term follow-up. In these, there were 213.5 person years of MRI-follow-up. No de novo aneurysm was detected. Mean modified Rankin Scale (mRS) during discharge was 1.28. Post-hemorrhage radiographic vasospasm was found in three patients (10.3%); DCI-related lesions occurred in one patient (3.4%). Five patients (17.2%) needed temporary external ventricular drainage; long-term CSF shunt dependency was necessary only in one patient (3.4%). Initial DSA retrospectively showed a 2 x 2 mm aneurysm of the right distal ICA in one patient, which remained stable. Post-hemorrhage siderosis was detected 8.1 years after the initial bleeding in one patient (4.8%).ConclusionPatients with cryptogenic nSAH have favourable outcomes and do not exhibit higher risks for de novo aneurysms. Therefore the need for long-term follow up after cryptogenic nSAH is questionable.     

Pili-Induced Clustering of N. gonorrhoeae Bacteria

Type IV pili (Tfp) are prokaryotic retractable appendages known to mediate surface attachment, motility, and subsequent clustering of cells. Tfp are the main means of motility for Neisseria gonorrhoeae, the causative agent of gonorrhea. Tfp are also involved in formation of the microcolonies, which play a crucial role in the progression of the disease. While motility of individual cells is relatively well understood, little is known about the dynamics of N. gonorrhoeae aggregation. We investigate how individual N. gonorrhoeae cells, initially uniformly dispersed on flat plastic or glass surfaces, agglomerate into spherical microcolonies within hours. We quantify the clustering process by measuring the area fraction covered by the cells, number of cell aggregates, and their average size as a function of time. We observe that the microcolonies are also able to move but their mobility rapidly vanishes as the size of the colony increases. After a certain critical size they become immobile. We propose a simple theoretical model which assumes a pili-pili interaction of cells as the main clustering mechanism. Numerical simulations of the model quantitatively reproduce the experimental data on clustering and thus suggest that the agglomeration process can be entirely explained by the Tfp-mediated interactions. In agreement with this hypothesis mutants lacking pili are not able to form colonies. Moreover, cells with deficient quorum sensing mechanism show similar aggregation as the wild-type bacteria. Therefore, our results demonstrate that pili provide an essential mechanism for colony formation, while additional chemical cues, for example quorum sensing, might be of secondary importance.     

Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells

Specific types of human papillomaviruses (HPVs) cause cervical cancer. Cervical cancers exhibit aberrant cellular microRNA (miRNA) expression patterns. By genome-wide analyses, we investigate whether the intracellular and exosomal miRNA compositions of HPV-positive cancer cells are dependent on endogenous E6/E7 oncogene expression. Deep sequencing studies combined with qRT-PCR analyses show that E6/E7 silencing significantly affects ten of the 52 most abundant intracellular miRNAs in HPV18-positive HeLa cells, downregulating miR-17-5p, miR-186-5p, miR-378a-3p, miR-378f, miR-629-5p and miR-7-5p, and upregulating miR-143-3p, miR-23a-3p, miR-23b-3p and miR-27b-3p. The effects of E6/E7 silencing on miRNA levels are mainly not dependent on p53 and similarly observed in HPV16-positive SiHa cells. The E6/E7-regulated miRNAs are enriched for species involved in the control of cell proliferation, senescence and apoptosis, suggesting that they contribute to the growth of HPV-positive cancer cells. Consistently, we show that sustained E6/E7 expression is required to maintain the intracellular levels of members of the miR-17~92 cluster, which reduce expression of the anti-proliferative p21 gene in HPV-positive cancer cells. In exosomes secreted by HeLa cells, a distinct seven-miRNA-signature was identified among the most abundant miRNAs, with significant downregulation of let-7d-5p, miR-20a-5p, miR-378a-3p, miR-423-3p, miR-7-5p, miR-92a-3p and upregulation of miR-21-5p, upon E6/E7 silencing. Several of the E6/E7-dependent exosomal miRNAs have also been linked to the control of cell proliferation and apoptosis. This study represents the first global analysis of intracellular and exosomal miRNAs and shows that viral oncogene expression affects the abundance of multiple miRNAs likely contributing to the E6/E7-dependent growth of HPV-positive cancer cells.     

Binding Affinity,Specificity and Comparative Biodistribution of the Parental Murine Monoclonal Antibody MX35 (Anti-NaPi2b) and Its Humanized Version Rebmab200

The aim of this preclinical study was to evaluate the characteristics of the monoclonal antibody Rebmab200, which is a humanized version of the ovarian-specific murine antibody MX35. This investigation contributes to the foundation for future clinical α-radioimmunotherapy of minimal residual ovarian cancer with ²¹¹At-Rebmab200. Here, the biodistribution of ²¹¹At-Rebmab200 was evaluated, as was the utility of ^99mTc-Rebmab200 for bioimaging. Rebmab200 was directly compared with its murine counterpart MX35 in terms of its in-vitro capacity for binding the immobilized NaPi2B epitope and live cells; we also assessed its biodistribution in nude mice carrying subcutaneous OVCAR-3 tumors. Tumor antigen and cell binding were similar between Rebmab200 and murine MX35, as was biodistribution, including normal tissue uptake and in-vivo tumor binding. We also demonstrated that ^99mTc-Rebmab200 can be used for single-photon emission computed tomography of subcutaneous ovarian carcinomas in tumor-bearing mice. Taken together, our data support the further development of Rebmab200 for radioimmunotherapy and diagnostics.     

Surficial gains and subsoil losses of soil carbon and nitrogen during secondary forest development

Reforestation of formerly cultivated land is widely understood to accumulate above‐ and belowground detrital organic matter pools, including soil organic matter. However, during 40 years of study of reforestation in the subtropical southeastern USA, repeated observations of above‐ and belowground carbon documented that significant gains in soil organic matter (SOM) in surface soils (0–7.5 cm) were offset by significant SOM losses in subsoils (35–60 cm). Here, we extended the observation period in this long‐term experiment by an additional decade, and used soil fractionation and stable isotopes and radioisotopes to explore changes in soil organic carbon and soil nitrogen that accompanied nearly 50 years of loblolly pine secondary forest development. We observed that accumulations of mineral soil C and N from 0 to 7.5 cm were almost entirely due to accumulations of light‐fraction SOM. Meanwhile, losses of soil C and N from mineral soils at 35 to 60 cm were from SOM associated with silt and clay‐sized particles. Isotopic signatures showed relatively large accumulations of forest‐derived carbon in surface soils, and little to no accumulation of forest‐derived carbon in subsoils. We argue that the land use change from old field to secondary forest drove biogeochemical and hydrological changes throughout the soil profile that enhanced microbial activity and SOM decomposition in subsoils. However, when the pine stands aged and began to transition to mixed pines and hardwoods, demands on soil organic matter for nutrients to support aboveground growth eased due to pine mortality, and subsoil organic matter levels stabilized. This study emphasizes the importance of long‐term experiments and deep measurements when characterizing soil C and N responses to land use change and the remarkable paucity of such long‐term soil data deeper than 30 cm.     

QTL and quantitative genetic analysis of beak morphology reveals patterns of standing genetic variation in an Estrildid finch

The intra- and interspecific diversity of avian beak morphologies is one of the most compelling examples for the power of natural selection acting on a morphological trait. The development and diversification of the beak have also become a textbook example for evolutionary developmental biology, and variation in expression levels of several genes is known to causally affect beak shape. However, until now, no genomic polymorphisms have been identified, which are related to beak morphology in birds. QTL mapping does reveal the location of causal polymorphisms, albeit with poor spatial resolution. Here, we estimate heritability and genetic correlations for beak length, depth and width and perform a QTL linkage analysis for these traits based on 1404 informative single-nucleotide polymorphisms genotyped in a four-generation pedigree of 992 captive zebra finches (Taeniopygia guttata). Beak size, relative to body size, was sexually dimorphic (larger in males). Heritability estimates ranged from 0.47 for beak length to 0.74 for beak width. QTL mapping revealed four to five regions of significant or suggestive genome-wide linkage for each of the three beak dimensions (nine different regions in total). Eight out of 11 genes known to influence beak morphology are located in these nine peak regions. Five QTL do not cover known candidates demonstrating that yet unknown genes or regulatory elements may influence beak morphology in the zebra finch.     

Establishment of introduced antagonistic bacteria in the rhizosphere of transgenic potatoes and their effect on the bacterial community

In a field release experiment, rifampicin resistant mutants of two antagonistic plant-associated bacteria were used for seed tuber inoculation of transgenic T4 lysozyme expressing potatoes, transgenic control potatoes and non-transgenic parental potatoes. The T4 lysozyme tolerant Pseudomonas putida QC14-3-8 was originally isolated from the tuber surface (geocaulosphere) of T4 lysozyme producing plants and showed in vitro antibacterial activity to the bacterial pathogen Erwinia carotovora ssp. atroseptica. The T4 lysozyme sensitive Serratia grimesii L16-3-3 was originally isolated from the rhizosphere of parental potatoes and showed in vitro antagonism toward the plant pathogenic fungus Verticillium dahliae. The establishment of the inoculated bacteria in the rhizosphere and geocaulosphere of the different plant lines was monitored over one growing season to assess the effect of T4 lysozyme produced by transgenic potato plants on the survival of both inoculants. Both introduced isolates were able to colonize the rhizo- and geocaulosphere of transgenic plants and non-transgenic parental plants, and established in the rhizosphere at levels of ca. log(10) 5 colony forming units g(-1) fresh weight of root. During flowering of plants, significantly more colony counts of the T4 lysozyme tolerant P. putida were recovered from transgenic T4 lysozyme plants than from the transgenic control and the parental line. At this time, the highest level of T4 lysozyme (% of total soluble protein) was detected. Effects of the inoculants on the indigenous microbial community were monitored by analysis of PCR-amplified fragments of the 16S rRNA genes of the whole bacterial community after separation by denaturing gradient gel electrophoresis (DGGE). At any sampling time, the DGGE pattern of rhizosphere and geocaulosphere communities did not show differences between the inoculated and non-inoculated potatoes. Neither of the introduced strains became a dominant member of the bacterial community. This work was the first approach to assess the establishment of plant growth promoting rhizobacteria and potential biocontrol agents on transgenic plants.     

Concomitant Detection of HER2 Protein and Gene Alterations by Immunohistochemistry (IHC) and Silver Enhanced In Situ Hybridization (SISH) Identifies HER2 Positive Breast Cancer with and without Gene Amplification

Introduction

HER2 status assessment became a mandatory test assay in breast cancer, giving prognostic and predictive information including eligibility for adjuvant anti-HER2 therapy. Precise and reliable assessment of HER2 status is therefore of utmost importance. In this study we analyzed breast cancer samples by a novel technology for concomitant detection of the HER2 protein and gene copy number.

Methods

Tissue microarrays containing 589 invasive breast cancer samples were analyzed with a double immunohistochemistry (IHC) and silver labeled in situ hybridization (SISH) assay simultaneously detecting HER2 protein and gene copy number in the same tumor cells. This bright-field assay was analyzed using scores according to the modified ASCO guidelines and the results were correlated with patient prognosis.

Results

Overall concordance rate between protein expression and the presence of gene amplification was 98%. Fifty-seven of 60 tumors (95%) with IHC score 3+, 6 of 10 tumors with IHC score 2+ (60%) and only 3 of 519 tumors (0.6%) with IHC score 0/1+ were amplified by SISH. Patients with gene amplification despite IHC score 0/1+ had a tendency for worse overall survival (p = 0.088, reaching nearly statistical significance) compared to IHC score 0/1+ without amplification. In contrast, there was no difference in overall survival in IHC score 3+/2+ tumors with and without gene amplification.

Conclusions

The novel double IHC and SISH assay for HER2 is efficient in the identification of breast cancer with discordant HER2 protein and HER2 gene status, especially for the prognostically relevant groups of HER2 protein negative tumors with HER2 amplification and HER2 protein positive tumors without HER2 amplification. Breast cancer without HER2 amplification among IHC score 2+/3+ tumors (10% in our cohort) suggests that other mechanisms than gene amplification contribute to protein overexpression in these cells.