No abstract

Many evolutionarily conserved microRNAs (miRNAs) in plants regulate transcription factors with key functions in development. Hence, mutations in the core components of the miRNA biogenesis machinery cause strong growth defects. An essential aspect of miRNA biogenesis is the precise excision of the small RNA from its precursor. In plants, miRNA precursors are largely variable in size and shape and can be processed by different modes. Here, we optimized an approach to detect processing intermediates during miRNA biogenesis. We characterized a miRNA whose processing is triggered by a terminal branched loop. Plant miRNA processing can be initiated by internal bubbles, small terminal loops or branched loops followed by dsRNA segments of 15–17 bp. Interestingly, precision and efficiency vary with the processing modes. Despite the various potential structural determinants present in a single a miRNA precursor, DCL1 is mostly guided by a predominant structural region in each precursor in wild-type plants. However, our studies in fiery1, hyl1 and se mutants revealed the existence of cleavage signatures consistent with the recognition of alternative processing determinants. The results provide a general view of the mechanisms underlying the specificity of miRNA biogenesis in plants.

The electroactivity of electrogenerated polypyrrole films was degraded by applying consecutive anodic potential pulses (overoxidation). After each degradation step, the electroactivity of the material was recorded by voltammetric and chronoamperometric measurements. The charge consumed during each degradation step was calculated by the difference between the degradation and the control chronoamperograms. The diffusion coefficients of the counterions within the material were obtained from the control chronoamperograms by applying the electrochemically stimulated conformational-relaxation model. As the materials became increasingly degraded, slower oxidation processes were observed, and decreasing diffusion coefficients were obtained. These observations point to growing cross linking between neighboring polymeric chains during the degradation process, thus reducing conjugation lengths and increasing the rigidity of the conformational electro-chemo-stimulated movements on the chains. An electroactivity degradation of 50% induces a 15% increase in the weight of the material, pointing to the presence of rigid and oxidized islands entrapped by cross-linking points that prevent any ionic interchanges.

No abstract

Reliable diffusion coefficients, D, for the diffusion of perchlorate anions into polypyrrole films during polymeric oxidation were obtained from chronoamperometric results. Two different models were used to calculate D: the Cottrell equation and the electrochemically stimulated conformational relaxation (ESCR) model. As expected, the initial Cottrell hypothesis was far from swelling/shrinking polymeric electrodes and the obtained D range was from 10(-10) to 10(-6) cm(2) s(-1). The ESCR model, based on the internal diffusion that takes place from regions where the steady state of oxidation has already been reached to regions where the oxidation is only just beginning, provided values of D ranging from 0.4 x 10(-9) to 2.2 x 10(-9) cm(2) s(-1), which is close to the values expected for a gel. When a constant amplitude is kept for the potential step, D increases with increasing initial anodic potentials, i.e., from increasingly swollen films. When it is stepped to the same oxidation potential, D decreases when starting from more cathodic potentials, i.e., from a more compact structure. These changes in D can be attributed to (i) swelling processes during oxidation, giving a gel-like structure; (ii) compacting processes at increasing cathodic potentials; (iii) the increasing thickness of the film during oxidation; and (iv) a decrease in film viscosity during the swelling process.

SUMMARY Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a severe neuropsychiatric disorder mediated by autoantibodies against the GluN1 subunit of the NMDAR. Patients’ antibodies cause crosslinking and internalization of NMDAR, but the synaptic events leading to depletion of NMDAR are poorly understood. Using super-resolution microscopy, we studied the effects of the autoantibodies on the nanoscale distribution of NMDAR in cultured neurons. Our findings show that, under control conditions, NMDARs form nanosized objects and patients’ antibodies increase the clustering of synaptic and extrasynaptic receptors inside the nano-objects. This clustering is subunit specific and predominantly affects GluN2B-NMDARs. Following internalization, the remaining surface NMDARs return to control clustering levels but are preferentially retained at the synapse. Monte Carlo simulations using a model in which antibodies induce NMDAR cross-linking and disruption of interactions with other proteins recapitulated these results. Finally, activation of EphB2 receptor partially antagonized the antibody-mediated disorganization of the nanoscale surface distribution of NMDARs.

We synthesize poly(N-isopropylacrylamide) (PNIPAM) gels with different sizes in the micrometer scale by a slight variation of a recent emulsion polymerization method (ref 1). The procedure is different than that typically used for obtaining macroscopic PNIPAM hydrogels. The resultant minigel suspension is polydisperse thus allowing the swelling kinetics for different gel sizes to be studied; we do so at temperatures below the volume-transition temperature by wetting with water previously dried particles. The resultant swelling is followed by optical video microscopy. We find that the characteristic swelling time scales with the inverse of the particle dimension squared, in agreement with theoretical predictions (ref 2). The proportionality constant is the network diffusion coefficient D, which for the minigels under consideration appears to be in between that of PNIPAM macrogels and the self-diffusion coefficient of water.

Since their discovery in the mid-eighties, the main papillomavirus oncoproteins E6 and E7 have been recalcitrant to high-resolution structure analysis. However, in the last decade a wealth of three-dimensional information has been gained on both proteins whether free or complexed to host target proteins. Here, we first summarize the diverse activities of these small multifunctional oncoproteins. Next, we review the available structural data and the new insights they provide about the evolution of E6 and E7, their multiple interactions and their functional variability across human papillomavirus (HPV) species.