Rab12 Localizes to Shiga Toxin‐Induced Plasma Membrane Invaginations and Controls Toxin Transport

Several exogenous and endogenous cargo proteins are internalized independently of clathrin, including the bacterial Shiga toxin. The mechanisms underlying early steps of clathrin‐independent uptake remain largely unknown. In this study, we have designed a protocol to obtain gradient fractions containing Shiga toxin internalization intermediates. Using stable isotope labeling with amino acids in cell culture (SILAC) and quantitative mass spectrometry, Rab12 was found in association with these very early uptake carriers. The localization of the GTPase on Shiga toxin‐induced plasma membrane invaginations was shown by fluorescence microscopy in cells transfected with GFP‐Rab12. Furthermore, using a quantitative biochemical assay, it was found that the amount of receptor‐binding B‐subunit of Shiga toxin reaching the trans‐Golgi/TGN membranes was decreased in Rab12‐depleted cells, and that cells were partially protected against intoxication by Shiga‐like toxin 1 under these conditions. These findings demonstrate the functional importance of Rab12 for retrograde toxin trafficking. Among several other intracellular transport pathways, only the steady‐state localizations of TGN46 and cation‐independent mannose‐6‐phosphate receptor were affected. These data thus strongly suggest that Rab12 functions in the retrograde transport route.      

Role of HLA DRB1*15 and HLA DRB1*16alleles in the genetic susceptibility to develop systemic lupus erythematosus (SLE) after Chikungunya and Zika viruses infection in México

Systemic lupus erythematosus (SLE) is a clinically and genetically heterogeneous disease particularly prevalent in Mexico. Althoughits etiology is unknown, genetic factors strongly influence its presenceas well as triggering factors, such as viral infections, including Cytomegalovirus and Epstein-Barr virus. Here,the study presents the appearance of de novoSLE (patients who did not present SLE before de virus infection, corroborated by serological analysis and negative for antinuclear antibodies) cases in Mexicans who live near the southern border of Mexico, who presented clinical symptoms of arthritic, hematological, mucocutaneous and renal SLE, after Zika and/ or Chikungunya virus infection. Low resolution class Ⅱ HLA typing was performed, which found a significantly increased frequency of HLA DRB1*02 (15 and 16)when compared to a group of 99 healthy individuals (P =0.001, OR=4.5, IC95% 1.8~11.0). All the patients were diagnosed with SLE 1 to 3 years after being confirmed with the Zika, and/or Chikungunya infection. At the point of acute viral infection, none of the patients presented clinical signs or symptoms of autoimmunity or were negative for antinuclear antibodies. In genetically susceptible individuals, Zika and Chikungunya viral infection can trigger SLE.     

Spectral sensitivity of mollies: comparing surface- and cave-dwelling Atlantic mollies, Poecilia mexicana

The visual pigments of cones and rods in three species of mollies, Poecilia mexicana , Poecilia latipinna and their asexual hybrid Poecilia formosa , were examined using microspectrophotometry. In P. mexicana , populations from extreme photic habitats were used: one population originated from a clear water habitat, one from a milky water habitat and another from a completely dark cave. Ultraviolet-sensitive cones were found in all species. Differences in the λ_max values of the visual pigments were small between species and among the three P. mexicana populations, but dark-reared cave fishes showed appreciably higher variance. The hybrid species P. formosa showed a highly variable long wavelength cone absorbance, ranging from 528·9 to 598·5 nm, suggesting multiple opsin expression or chromophore mixing.     

Pleomorphic Ensembles: Formation of Large Clusters Composed of Weakly Interacting Multivalent Molecules

Molecular interactions of importance to cell biology are subject to sol-gel transitions: large clusters of weakly interacting multivalent molecules (gel phase) are produced at a critical concentration of monomers. Examples include cell-cell and cell-matrix adhesions, nucleoprotein bodies, and cell signaling platforms. We use the term pleomorphic ensembles (PEs) to describe these clusters, because they have dynamic compositions and sizes and have rapid turnover of their molecular constituents; this plasticity can be highly responsive to cellular signals. The classical polymer physical chemistry theory developed by Flory and Stockmayer provides a brilliant framework for treating multivalent interactions for simple idealized systems. But the complexity and variability of PEs challenges existing modeling approaches. Here we describe and validate a computational algorithm that extends the Flory-Stockmayer formalism to overcome the limitations of analytic theories. We divide the problem by deterministically calculating the fraction of bound sites for each type of binding site, followed by the stochastic assignment of the bonds to a finite number of molecules. The method allows for high valency within many different kinds of interacting molecules and site types, permits simulation of steady-state distributions, as well as assembly kinetics, and can treat cooperative binding within one of the interacting molecules. We then apply our method to the analysis of interactions in the nephrin-Nck-N-Wasp signaling system, demonstrating how multivalent layered scaffolds produce PEs at low monomer concentrations despite weak binding interactions. We show how the experimental data for this system are most consistent with synergistic cooperative interactions between Nck and N-Wasp.     

Single-Voxel Recording of Voltage Transients in Dendritic Spines

We report sensitive recording of membrane potential in single dendritic spines in cortical neurons within a brain slice using two-photon excitation and a new, fluorinated, intracellularly loaded organic dye, di-2-AN(F)EPPTEA. With a two-photon excitation wavelength of 1060 nm, we achieve voltage sensitivity of >16% change in fluorescence per 100 mV. By targeting single spines in single-voxel recordings, we attain excellent single/noise quality, with back-propagating action potentials (bAPs) visible in single sweeps while recording at 10 kHz. This recording rate allows us to reliably assess fast bAP dynamics on single sweeps including bAP rise times of 0.5 ms. The amplitude and propagation delays of the bAPs are similar among different spines located within the same dendritic region, and this is true despite large differences in spine size. The interregion differences in bAP waveforms in spines vary in relation to their distance from the soma and the caliber of their parent dendrites.     

Microspectrophotometry of the photoreceptors of palaeognathous birds — the emu and the tinamou

Summary With the aid of a microspectrophotometer the visual pigments and oil globules in the retina of the emu (Dromiceius novae-hollandiae), the brushland tinamou (Nothoprocta c. cinerascens) and the Chilean tinamou (Nothoprocta perdicaria sanborni) were characterized. All three of these palaeognathous birds contain in their rods a typical rhodopsin with _max near 500 nm. Each of these birds has cones containing iodopsin-like visual pigments with _max in the 560–570 nm spectral region. No unequivocal evidence was obtained for the presence of cone pigments other than this iodopsin-like pigment, although one cell thought to be a cone, and containing a visual pigment with _max near 498 nm, was observed in the retina of the brushland tinamou. The oil globule systems of the three palaeognathous species are identical to each other and are much simpler than is typical for neognathous birds in that only two different types of globule are present, one with _T50 at 508 nm and another with _T50 at 568 nm. Comparison of the data with observations made on neognathous species indicates (1) that palaeognathous birds probably have poorer color discrimination capabilities than neognathous birds and (2) that the tinamou is more closely related to the ratites than to the galliform species.This study was supported, in part, by NIH Grant No. EY01839 (A.J. Sillman), NIH Grant No. EY00323 (W.N. McFarland) and NSF Grant No. 78-07657 (E.R. Loew). The authors thank E. Clinite, R. Dunford, C. Murphy, R. Riis and D. Weathers for their valuable assistance. Thanks also go to R.E. Burger for his gift of the emus.     

A Systematic Critical Appraisal of Non-Pharmacological Management of Rheumatoid Arthritis with Appraisal of Guidelines for Research and Evaluation II

Clinical practice guidelines (CPGs) have been developed to summarize evidence about the management of rheumatoid arthritis (RA) and facilitate the uptake of evidence-based knowledge by consumers, health professionals, health administrators and policy makers. The objectives of this review was to assess the quality of CPGS on non-pharmacological management of RA with a standardized and validated instrument - the Appraisal of Guidelines for Research and Evaluation (AGREE II) tool and summarize the key recommendations from these CPGs. Scientific literature databases from 2001 to 2013 were systematically searched and a total of 13 CPGs for RA was identified. Only a minority of AGREE II domains were effectively addressed by the CPGS. Scope and purpose was effectively addressed in 10 out of 13 CPGs, stakeholder involvement in 11 CPGs, rigor of development in 6 CPGs, clarity/presentation in 9 CPGs, editorial independence in 1 CPGs, and applicability in none of the CPGs. The overall quality of the included CPGs according to the 7-point AGREE II scoring system was 4.8±1.04. Patient education/self-management, aerobic, dynamic and stretching exercises were the commonly recommended for the non-pharmacological management of RA by the high-quality CPGs. The general clinical management recommendations tended to be similar among high-quality CPGs. Non-pharmacological management interventions were superficially addressed in more than half of the selected CPGs. CPGs creators should use the AGREE II criteria when developing guidelines. Innovative and effective methods of CPGs implementation to users are needed to ultimately enhance the quality of life of arthritic individuals. In addition, it was difficult to establish between strongly recommended, recommended and weakly recommended, as there is no consensus between the strength of the recommendations between the appraised CPGs.     

Assessing combined methylation-sensitive high resolution melting and pyrosequencing for the analysis of heterogeneous DNA methylation

Heterogeneous DNA methylation leads to difficulties in accurate detection and quantification of methylation. Methylation-sensitive high resolution melting (MS-HRM) is unique among regularly used methods for DNA methylation analysis in that heterogeneous methylation can be readily identified, although not quantified, by inspection of the melting curves. Bisulfite pyrosequencing has been used to estimate the level of heterogeneous methylation by quantifying methylation levels present at individual CpG dinucleotides. Sequentially combining the two methodologies using MS-HRM to screen the amplification products prior to bisulfite pyrosequencing would be advantageous. This would not only replace the quality control step using agarose gel analysis prior to the pyrosequencing step but would also provide important qualitative information in its own right. We chose to analyze DAPK1 as it is an important tumor suppressor gene frequently heterogeneously methylated in a number of malignancies, including chronic lymphocytic leukemia (CLL). A region of the DAPK1 promoter was analyzed in ten CLL samples by MS-HRM. By using a biotinylated primer, bisulfite pyrosequencing could be used to directly analyze the samples. MS-HRM revealed the presence of various extents of heterogeneous DAPK1 methylation in all CLL samples. Further analysis of the biotinylated MS-HRM products by bisulfite pyrosequencing provided quantitative information for each CpG dinucleotide analyzed, and confirmed the presence of heterogeneous DNA methylation. Whereas each method could be used individually, MS-HRM and bisulfite pyrosequencing provided complementary information for the assessment of heterogeneous methylation.Key words: MS-HRM, pyrosequencing, digital PCR, heterogeneous DNA methylation, DAPK1, chronic lymphocytic leukemia     

Internalization and down-regulation of the ALK receptor in neuroblastoma cell lines upon monoclonal antibodies treatment

Recently, activating mutations of the full length ALK receptor, with two hot spots at positions F1174 and R1275, have been characterized in sporadic cases of neuroblastoma. Here, we report similar basal patterns of ALK phosphorylation between the neuroblastoma IMR-32 cell line, which expresses only the wild-type receptor (ALK(WT)), and the SH-SY5Y cell line, which exhibits a heterozygous ALK F1174L mutation and expresses both ALK(WT) and ALK(F1174L) receptors. We demonstrate that this lack of detectable increased phosphorylation in SH-SY5Y cells is a result of intracellular retention and proteasomal degradation of the mutated receptor. As a consequence, in SH-SY5Y cells, plasma membrane appears strongly enriched for ALK(WT) whereas both ALK(WT) and ALK(F1174L) were present in intracellular compartments. We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALK(WT). We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation. In contrast, antagonist mAb induced ALK internalization and recycling to the plasma membrane. Importantly, we correlate this differential trafficking of ALK in response to mAb with the recruitment of the ubiquitin ligase Cbl and ALK ubiquitylation only after agonist stimulation. This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization.     

In situ optical mapping of voltage and calcium in the heart

Electroanatomic mapping the interrelation of intracardiac electrical activation with anatomic locations has become an important tool for clinical assessment of complex arrhythmias. Optical mapping of cardiac electrophysiology combines high spatiotemporal resolution of anatomy and physiological function with fast and simultaneous data acquisition. If applied to the clinical setting, this could improve both diagnostic potential and therapeutic efficacy of clinical arrhythmia interventions. The aim of this study was to explore this utility in vivo using a rat model. To this aim, we present a single-camera imaging and multiple light-emitting-diode illumination system that reduces economic and technical implementation hurdles to cardiac optical mapping. Combined with a red-shifted calcium dye and a new near-infrared voltage-sensitive dye, both suitable for use in blood-perfused tissue, we demonstrate the feasibility of in vivo multi-parametric imaging of the mammalian heart. Our approach combines recording of electrophysiologically-relevant parameters with observation of structural substrates and is adaptable, in principle, to trans-catheter percutaneous approaches.