Gold Labeling Research Articles

How do sustainability standards consider biodiversity?

Sustainability certification schemes and standards are meant to prevent a range of unacceptable socioeconomic and environmental consequences, such as threats to biodiversity. While there is wide support for conserving biodiversity, operationalizing this support in the form of guiding principles, criteria/indicators, and legislation is complicated. This study investigates how and to what extent 26 sustainability standards (eleven for forest management, nine for agriculture and six biofuel‐related) consider biodiversity, by assessing how they seek to prevent actions that can threaten biodiversity as well as how they support actions aimed at biodiversity conservation. For this purpose, a benchmark standard was developed, meant to represent a case with very high ambitions concerning biodiversity conservation. Of the assessed standards, the biofuel‐related standards demonstrated the highest level of compliance with the benchmark. On average, they complied with 72% of the benchmark's component criteria, compared to 61% for the agricultural standards and 60% for the forestry standards. Fairtrade, Sustainable Agriculture Network/Rainforest Alliance (SAN/RA), Roundtable on Sustainable Palm Oil (RSPO), and Roundtable on Responsible Soy (RTRS) were particularly stringent, while Green Gold Label S5 (GGLS5), PEOLG, Global Partnership for Good Agricultural Practices (GLOBALGAP), European Union Organic (EU Organic), National Organic Program (NOP), Green Gold Label S2 (GGLS2), and International Sustainability & Carbon Certification (ISCC) were particularly unstringent. All eleven forestry standards, six of the nine agricultural standards, and all six biofuel‐related standards addressed ecosystem conversion, ranging from requiring that high conservation value areas be identified and preserved to requiring full protection. Finally, key barriers to, and challenges for, certification schemes are discussed and recommendations are made for further development of sustainability standards. WIREs Energy Environ 2015, 4:26–50. doi: 10.1002/wene.118This article is categorized under: Bioenergy > Economics and Policy Bioenergy > Climate and Environment

Rebuttal from Peter M. Lalley, Paul M. Pilowsky, Hubert V. Forster and Edward J. Zuperku

Our opponents contend that nanolitre microinjections into preBotC may not target critical preBotC neurons, and claim that drug diffusion from microdialysis probes located 0.5–1.1 mm from preBotC are superior (Montandon et al. 2011). Microinjection studies rely on coordinates and functional properties of the preBotC (mixture of ventral respiratory column (VRC) I-and E-neurons) in addition to rapid onset tachypnoeic responses (Mustapic et al. 2010) to d,l-homocysteic acid (DLH) microinjections (Schwarzacher et al. 1995; Monnier et al. 2003; Krolo et al. 2005). With reverse microdialysis, the drug is continuously released and targets a large volume due to diffusion in a complex interstitial space, which is not localized to a specific target (Hocht et al. 2013), e.g. only neurokinin-1 receptor-containing VRC neurons. To address this limitation ‘correlation maps’ based on probe distances from points in medullary 3-D space and corresponding response latencies were used to suggest the preBotC as the exclusive target. However, this method does not provide unequivocal results. Several points of evidence from a number of studies reinforce the validity and accuracy of microinjections into targeted regions of the pons and medulla, and their local effectiveness. Extensive bilateral naloxone microinjections in the preBotC region were ineffective (n = 15, Mustapic et al. 2010), but naloxone microinjections (∼540 nl each) into the parabrachial region produced rapid (30–40 s) and complete reversal of systemic opioid (remifentanil)-induced bradypnoea (Prkic et al. 2012). Contrary to the opinion put forth by Montandon et al. (2011), it is unlikely that this reversal was due to antagonism of endogenously released opioids since the amount of reversal matched the amount (72 ± 4%) of remifentanil-induced bradypnoea in both decerebrate (n = 9) and isoflurane-anaesthetized (n = 12) preparations. Furthermore, the parabrachial region is distinctly different from the rostral ventromedial medullary pain pathways (Phillips et al. 2012). In contrast, Montandon et al. (2011) dialysed naloxone (300 μm) for 45 min prior to i.v. fentanyl injections, allowing antagonist concentrations (>50 nm) to reach μ-opioid receptors at great distances. The suggestion (Montandon et al. 2011) that endomorphin-1 was injected into BotC in the Lonergan et al. (2003) study, thereby causing tachypnoea, is incorrect. PreBotC was identified by field potential mapping and albumin–colloidal gold labelling at opioid injection sites. Moreover, in decerebrate dogs, all DAMGO (n = 16) preBotC microinjections produced tachypnoea. Considering the limitations of reverse dialysis to target a specific site compared to the precision of microinjections, we conclude that preBotC μ-opioid receptors do not mediate the bradypnoea induced by systemic μ-opioid administration.

RETENTIVENESS OF VARIOUS LUTING AGENTS USED WITH IMPLANT SUPPORTED- PROSTHESIS: AN INVITRO STUDY

Abstract ABSTRACT:Purpose: Desired retrievability of cemented implant-supported fixed prosthesis makes the retentive strength of cementing agents an important consideration. The aim of the study was to evaluate the retentiveness of purposely-designed implant cement and to compare its retentiveness with dental cements that are commonly used with implant systems.Materials and method: Ten implant analogs were embedded in auto-polymerizing acrylic resin blocks and titanium abutments were attached to them. 50 standardized copings were waxed directly on the abutment and casted. The cements used were: 1. resin-bonded zinc oxide eugenol cement (Kalzinol, DPI), 2. purposely-designed implant cement (Premier implant cement), 3. zinc phosphate cement (DeTrey Zinc, Dentsply), 4. zinc polycarboxylate cement (Poly-F, Dentsply) and 5. glass ionomer cement (GC Gold Label, GC corporation, Japan). After cementation, each sample was subjected to a pull-out test using universal testing machine and loads required to remove the crowns were recorded. The mean values and standard deviations of cement failure loads were analyzed using ANOVA and Bonferroni test.Results: The mean values (±SD) of loads at failure (n = 10) for various cements were as follows (N): resin-bonded zinc oxide eugenol cement 394.62 (±9.76), Premier implant cement 333.86 (±18.91), zinc phosphate cement 629.30 (±20.65), zinc polycarboxylate cement 810.08 (±11.52) and glass ionomer cement 750.17 (±13.78).Conclusions: The results do not suggest that one cement type is better than another, but they do provide a ranking order of the cements in their ability to retain the prosthesis and to facilitate its easy retrievability.

Medical decision support using machine learning for early detection of late-onset neonatal sepsis

The objective was to develop non-invasive predictive models for late-onset neonatal sepsis from off-the-shelf medical data and electronic medical records (EMR). The data used in this study are from 299 infants admitted to the neonatal intensive care unit in the Monroe Carell Jr. Children's Hospital at Vanderbilt and evaluated for late-onset sepsis. Gold standard diagnostic labels (sepsis negative, culture positive sepsis, culture negative/clinical sepsis) were assigned based on all the laboratory, clinical and microbiology data available in EMR. Only data that were available up to 12 h after phlebotomy for blood culture testing were used to build predictive models using machine learning (ML) algorithms. We compared sensitivity, specificity, positive predictive value and negative predictive value of sepsis treatment of physicians with the predictions of models generated by ML algorithms. The treatment sensitivity of all the nine ML algorithms and specificity of eight out of the nine ML algorithms tested exceeded that of the physician when culture-negative sepsis was included. When culture-negative sepsis was excluded both sensitivity and specificity exceeded that of the physician for all the ML algorithms. The top three predictive variables were the hematocrit or packed cell volume, chorioamnionitis and respiratory rate. Predictive models developed from off-the-shelf and EMR data using ML algorithms exceeded the treatment sensitivity and treatment specificity of clinicians. A prospective study is warranted to assess the clinical utility of the ML algorithms in improving the accuracy of antibiotic use in the management of neonatal sepsis.

Detection of KIR6 family protein in rat heart and liver mitochondria by immunoelectron microscopy

The electron microscopic study of thin sections of rat liver and heart using commercial specific antibodies against KIR.6.2 and secondary antibodies conjugated with colloidal gold was performed. It was found that the gold-labeled protein is localized in mitochondria of cardiomyocytes and hepatocytes but not in rough and smooth endoplasmic reticulum of hepatic cells and myofibrils of myocardium. In rat heart and liver mitochondria, the gold label was mainly located in mitochondrial cristae and was not found in mitochondrial matrix and intermembrane space. The data indicate that in heart and liver mitochondria there exists a protein similar in structure to the channel-forming subunit of a cytoplasmic potassium channel, KIR6.2. This is also supported by the presence of common modulators of cytoplasmic and mitochondrial ATP-dependent potassium channels. A possible role of the protein as a subunit of the mitochondrial ATP-dependent potassium channel is discussed.

Localization of aristolochic acid in mouse kidney tissues by immunohistochemistry using an anti-AA-I and AA-II monoclonal antibody.

Aristolochic acids (AAs) are found in herbal medicines of Aristolochiaceae plants, including Aristolochia and Asarum species. AAs are associated with a rapidly progressive interstitial nephritis, which is called aristolochic acid nephropathy (AAN). However, the in-situ localization of AAs in the target organ, the kidney, has not been investigated yet. In the present study, the accumulation of aristolochic acid I (AA-I) in mouse kidney was revealed by immunoperoxidase light microscopy as well as colloidal gold immunoelectron microscopy (IEM) based on an anti-AA-I and AA-II monoclonal antibody (mAb). Male BALB/c mice were treated with 1.25 or 2.50 mg kg(-1) of AA-I per day for 5 days. Paraffin sections and ultra-thin sections of kidney tissue were respectively prepared. Under light microscopy, the apical surface of proximal tubules was strongly stained for AA-I, whereas no obvious immunostaining was found in the distal tubules and glomerulus, which remained relatively intact. Under electron microscopy, epithelial cells of the proximal tubules, distal tubules and collecting tubules were broken to various degrees. Gold labeling in the proximal and distal tubules was stronger than that in the collecting tubules. In renal tubules, immunogold signals of AA-I tended to accumulate in the mitochondria and peroxisomes, though the signals could be observed all over the cell. Gold signals were also found in the erythrocytes of glomeruli. The MAb against AA-I and AA-II provides a clue for the identification of proteins or factors which might interact with AA-I and thus induce targeted damage of kidney.

Implementation of scanning probe microscopy for the solution of molecular diagnostics tasks

We present new approaches to improve the efficiency of DNA by scanning probe microscopy using a highly specific hybridization on affine surfaces and nanostructures of gold as a labels. Scanning probe microscopy allows to register of individual acts of hybridization by the detection of gold labels on the surface affinity followed by automatic calculation of the total.

Microvesicles : What's Plasma Made of?

Microvesicles (MVs) are cell-derived membrane fragments which are present in plasma and other body fluids. In plasma, MVs participate in physiological processes of hemostasis and inflammation. MVs contain cell-specific molecules and are present at elevated levels in various diseases, which has raised the hypothesis of their potential application as disease biomarkers. The characterization of MVs is however hampered by their small size, estimated from 50 nm to 1 μm, and by limitations of the methods currently used for their analysis.Our aim is to provide a comprehensive description of MVs from plasma of healthy individuals and answer basic questions concerning MVs: 1) What do MVs from plasma look like?; 2) What is their size distribution?; 3) How many of them expose phosphatidylserine (PS)?; 4) How many of them derive from erythrocytes?, from platelets?; or 5) What is their concentration?By combining cryo-Electron Microscopy (EM) and receptor-specific gold labeling, the morphology, size and phenotype of MVs from normal plasma were characterized. MVs present three morphologies, consisting of spherical vesicles, of 40 nm −1 μm in diameter, tubular vesicles, of 1-5 μm in length, and large membrane fragments, 1-8 μm wide. The sub-population of pro-coagulant MVs that expose PS, identified by labeling with Annexin-A5-conjugated gold nanoparticles, was found to form a minority of MVs, about 25%, in contrast with the current theory of MV formation. MVs derived from the main blood cell populations, erythrocytes, platelets and leukocytes, were identified by immuno-gold labeling. Finally, concentrations of MVs were determined by a novel quantitative approach based on MV sedimentation on EM grids.This study (in revision) provides a detailed description of MVs from normal plasma, novel insights on mechanisms of MV formation, and will serve as a reference for further studies of MVs in pathological situations.

A hybrid paper and microfluidic chip with electrowetting valves and colorimetric detection

Sequential fluid delivery with minimized external equipment is vital towards a point-of-care diagnostic device. In this work, we have further developed the On-chip Electrowetting Valves concept for the sequential delivery of the reagents to the reaction site in a miniaturized capillary-driven microfluidic chip. Specifically, a disposable polymeric microfluidic device was developed containing capillary force driven microchannels. The device was fabricated using laser ablation and inkjet printing and required no external pumping equipment. The assay was conducted on the microchip containing microfluidic channels with embedded electrowetting valves and a porous membrane patterned with capture molecules and colloidal gold labels. To conduct the assay, the microchip was connected with a low voltage supply which was capable of sequentially opening the valves, delivering the sample and the rinsing reagent to generate visual results. Using T7 bacteriophage as a model, we have demonstrated the development of the device, operation of the valves and execution of the automated assay.

Positively charged nanogold label allows the observation of fine cell filopodia and flagella in solution by atmospheric scanning electron microscopy

Optical microscopy is generally the first choice to observe microbes and cells. However, its resolution is not always sufficient to reveal specific target structures, such as flagella and pili, which are only nanometers wide. ASEM is an attractive higher resolution alternative, as the sample is observed in aqueous solution at atmospheric pressure. Sample pretreatment for ASEM only comprises simple tasks including fixation, gold labeling, and reagent exchange, taking less than 1 h in total. The lengthy sample pretreatments often required for more classical electron microscopies, such as embedding and dehydration, are unnecessary, and native morphology is preserved. In this study, positively charged nanogold particles were used to label the surfaces of bacteria and cultured animal cells, exploiting their net negative surface charge. After gold enhancement to increase the size of the nanogold particles, ASEM imaging of the bacteria in aqueous solution revealed pili and delicate spiral flagella. This natural shape contrasts starkly with images of dried flagella recorded by standard SEM. Positively charged nanogold labeled the plasma membrane of cultured COS7 cells, and after enhancement allowed filopodia as thin as 100 nm in diameter to be clearly visualized. Based on these studies, ASEM combined with positively charged nanogold labeling promises to become an important tool for the study of cell morphology and dynamics in the near future.

Actin cytoskeleton reorganization correlates with polarization of secretory vesicle and cell morphology in the degranulation of mast cell subtypes in human colon tissues

Mast cells play a central role in the intestinal immune response. To investigate the relationship between degranulation, cell polarization and the reorganization of actin cytoskeleton of mast cells, we used fluorescence or gold labeling methods to identify different mast cell subtypes in human colon. The reorganization of filamentous actin was visualized and then the polarization of secretory vesicles, as well as cell surfaces, was analyzed by fluorescence microscopy and electron microscopy. Our results first showed a diversity of filamentous actin assembly or disassembly within the contacting cell membrane of different mast cell subtypes. The polarization and degranulation of secretory vesicles was not only accompanied with the assembly and disassembly of filamentous actin at the cell periphery, but also with changes of cell surface polarization. Our study provides an insight into the local membranous structures and suggested correlations of cytoskeleton arrangement with the polarization of secretory vesicles and cell surface configuration during mast cell degranulation.

A comparative study of modified confirmatory techniques and additional immuno-based methods for non-conclusive autolytic Bovine spongiform encephalopathy cases

BackgroundIn the framework of the Bovine Spongiform Encephalopathy (BSE) surveillance programme, samples with non-conclusive results using the OIE confirmatory techniques have been repeatedly found. It is therefore necessary to question the adequacy of the previously established consequences of this non-conclusive result: the danger of failing to detect potentially infected cattle or erroneous information that may affect the decision of culling or not of an entire bovine cohort. Moreover, there is a very real risk that the underreporting of cases may possibly lead to distortion of the BSE epidemiological information for a given country.In this study, samples from bovine nervous tissue presenting non-conclusive results by conventional OIE techniques (Western blot and immunohistochemistry) were analyzed. Their common characteristic was a very advanced degree of autolysis. All techniques recommended by the OIE for BSE diagnosis were applied on all these samples in order to provide a comparative study. Specifically, immunohistochemistry, Western blotting, SAF detection by electron microscopy and mouse bioassay were compared. Besides, other non confirmatory techniques, confocal scanning microscopy and colloidal gold labelling of fibrils, were applied on these samples for confirming and improving the results.ResultsImmunocytochemistry showed immunostaining in agreement with the positive results finally provided by the other confirmatory techniques. These results corroborated the suitability of this technique which was previously developed to examine autolysed (liquified) brain samples. Transmission after inoculation of a transgenic murine model TgbovXV was successful in all inocula but not in all mice, perhaps due to the very scarce PrPsc concentration present in samples.Electron microscopy, currently fallen into disuse, was demonstrated to be, not only capable to provide a final diagnosis despite the autolytic state of samples, but also to be a sensitive diagnostic alternative for resolving cases with low concentrations of PrPsc.ConclusionsDemonstration of transmission of the disease even with low concentrations of PrPsc should reinforce that vigilance is required in interpreting results so that subtle changes do not go unnoticed. To maintain a continued supervision of the techniques which are applied in the routine diagnosis would prove essential for the ultimate eradication of the disease.

Mechanical performance of encapsulated restorative glass-ionomer cements for use with Atraumatic Restorative Treatment (ART)

The Atraumatic Restorative Treatment (ART) approach was suggested to be a suitable method to treat enamel and dentine carious lesions in patients with disabilities. The use of a restorative glass-ionomer with optimal mechanical properties is, therefore, very important.Objective:To test the null-hypotheses that no difference in diametral tensile, compressive and flexural strengths exists between: (1) The EQUIA system and (2) The Chemfil Rock (encapsulated glass-ionomers; test materials) and the Fuji 9 Gold Label and the Ketac Molar Easymix (hand-mixed conventional glass-ionomers; control materials); (3) The EQUIA system and Chemfil Rock. Material and Methods:Specimens for testing flexural (n=240) and diametral tensile (n=80) strengths were prepared according to standardized specifications; the compressive strength (n=80) was measured using a tooth-model of a class II ART restoration. ANOVA and Tukey B tests were used to test for significant differences between dependent and independent variables. Results:The EQUIA system and Chemfil Rock had significantly higher mean scores for all the three strength variables than the Fuji 9 Gold Label and Ketac Molar Easymix (α=0.05). The EQUIA system had significant higher mean scores for diametral tensile and flexural strengths than the Chemfil Rock (α=0.05). Conclusion:The two encapsulated high-viscosity glass-ionomers had significantly higher test values for diametral tensile, flexural and compressive strengths than the commonly used hand-mixed high-viscosity glass-ionomers.

Competitive immunochromatographic assay for the detection of the organophosphorus pesticide EPN

An immunochromatographic assay (ICA) based on competitive antigen-coated format and colloidal gold label was developed for the detection of the organophosphorus insecticide O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN). The ICA test strip consisted of a membrane with a detection zone, a sample pad and an absorbent pad. The membrane was separately coated with EPN hapten-OVA conjugate (test line) and anti-mouse IgG (control line). As the competition is between the migrating analyte and the immobilised analyte hapten for the binding sites of the migrating antibody–colloidal gold (Ab–CG) conjugate, this study suggests that the relative migration speed between the two migrating substances is a critically important factor for the sensitive detection by competitive ICA. Based on this criterion, a nitrocellulose (NC) membrane was chosen as the most suitable for EPN ICA. The detection limit of the ICA for EPN standard and EPN spiked into agricultural samples were 10−2 and 5×10−2 µg mL−1, respectively.

A Rapid, Multiplexed, High-Throughput Flow-Through Membrane Immunoassay: A Convenient Alternative to ELISA.

This paper describes a rapid, high-throughput flow-through membrane immunoassay (FMIA) platform. A nitrocellulose membrane was spotted in an array format with multiple capture and control reagents for each sample detection area, and assay steps were carried out by sequential aspiration of sample and reagents through each detection area using a 96-well vacuum manifold. The FMIA provides an alternate assay format with several advantages over ELISA. The high surface area of the membrane permits high label concentration using gold labels, and the small pores and vacuum control provide rapid diffusion to reduce total assay time to ~30 min. All reagents used in the FMIA are compatible with dry storage without refrigeration. The results appear as colored spots on the membrane that can be quantified using a flatbed scanner. We demonstrate the platform for detection of IgM specific to lipopolysaccharides (LPS) derived from Salmonella Typhi. The FMIA format provides analytical results comparable to ELISA in less time, provides integrated assay controls, and allows compensation for specimen-to-specimen variability in background, which is a particular challenge for IgM assays.

Novel gold labeling reagent to localize protein in macromolecular assemblies

Electron microscopy (EM) is a leading technique to elucidate structure of macromolecular assemblies. Obtaining high‐resolution information by EM is often hindered by sample compositional and conformational heterogeneity. It is challenging and error prone to build an informative model into a low‐resolution 3‐dimensional reconstruction. Thus, there is demand to expand upon current approaches used for accurate protein localization to aid model building. We report use of a labeling reagent consisting of tris‐nitrilotriacetic acid (tris‐NTA) conjugated with a monofunctionalized gold nanoparticle that site‐specifically labels protein termini fused to a His‐tag. Multivalent binding of tris‐NTA to a His‐tag via complexed Ni(II) ion results into subnanomolar affinity and a defined 1:1 stoichiometry. Precise localization of a protein is further ensured by the reagents conformationally restricted linker and monofunctionalized small Au nanoparticle. We have employed the label to localize His‐tagged proteins in a T. thermophiles ATPase, RuvB, and in the E. coli 50S ribosomal subunit. Our mono‐tris‐NTA‐Au conjugate specifically binds to the target protein in these assemblies, exhibiting stoichiometric labeling. Our new Au labeling reagent improves upon current labeling approaches and should have extensive applicability in sites‐pecific labeling of His‐tag protein termini in macromolecular assemblies.

Evaluation of push‐out bond strength of four luting agents and SEM observation of the dentine/fibreglass bond interface

To assess the dentine/fibreglass-bonded interface by scanning electron microscopy and the push-out bond strength of four luting cements. Forty root-filled human canines, with similar root lengths, were restored with fibreglass posts and randomly divided into four groups according to the cements employed (CG Gold Label I, RelyX ARC, BisCem and RelyX U100). After standardized post-space preparation, the root dentine was pre-treated for dual-polymerizing resin cements and untreated for the other cements. The mixed luting cement paste was inserted into root canals with a spiral filler and applied to the post-surface that was seated into the root canal. After 7 days, the teeth were sectioned perpendicular to their long axis into 1-mm-thick sections and submitted to a push-out test (1 mm min(-1)). Statistical analyses were carried out by two-way anova and Tukey's tests (P < 0.05). The bond strength was significantly affected by the type of cement (P < 0.001) and by the longitudinal region of the root canal (P < 0.031). All cements exhibited gaps at the dentine/cement interface. The self-adhesive materials and the glass ionomer cements had the highest push-out bond strength values when compared with the dual-curing resin cement.

High resolution helium ion scanning microscopy of the rat kidney.

Helium ion scanning microscopy is a novel imaging technology with the potential to provide sub-nanometer resolution images of uncoated biological tissues. So far, however, it has been used mainly in materials science applications. Here, we took advantage of helium ion microscopy to explore the epithelium of the rat kidney with unsurpassed image quality and detail. In addition, we evaluated different tissue preparation methods for their ability to preserve tissue architecture. We found that high contrast, high resolution imaging of the renal tubule surface is possible with a relatively simple processing procedure that consists of transcardial perfusion with aldehyde fixatives, vibratome tissue sectioning, tissue dehydration with graded methanol solutions and careful critical point drying. Coupled with the helium ion system, fine details such as membrane texture and membranous nanoprojections on the glomerular podocytes were visualized, and pores within the filtration slit diaphragm could be seen in much greater detail than in previous scanning EM studies. In the collecting duct, the extensive and striking apical microplicae of the intercalated cells were imaged without the shrunken or distorted appearance that is typical with conventional sample processing and scanning electron microscopy. Membrane depressions visible on principal cells suggest possible endo- or exocytotic events, and central cilia on these cells were imaged with remarkable preservation and clarity. We also demonstrate the use of colloidal gold probes for highlighting specific cell-surface proteins and find that 15 nm gold labels are practical and easily distinguishable, indicating that external labels of various sizes can be used to detect multiple targets in the same tissue. We conclude that this technology represents a technical breakthrough in imaging the topographical ultrastructure of animal tissues. Its use in future studies should allow the study of fine cellular details and provide significant advances in our understanding of cell surface structures and membrane organization.

Regulation of Postsynaptic Retrograde Signaling by Presynaptic Exosome Release

Retrograde signals from postsynaptic targets are critical during development and plasticity of synaptic connections. These signals serve to adjust the activity of presynaptic cells according to postsynaptic cell outputs and to maintain synaptic function within a dynamic range. Despite their importance, the mechanisms that trigger the release of retrograde signals and the role of presynaptic cells in this signaling event are unknown. Here we show that a retrograde signal mediated by Synaptotagmin 4 (Syt4) is transmitted to the postsynaptic cell through anterograde delivery of Syt4 via exosomes. Thus, by transferring an essential component of retrograde signaling through exosomes, presynaptic cells enable retrograde signaling.

Structure and mechanism of the PilF DNA transformation ATPase from Thermus thermophilus

Many Gram-negative bacteria contain specific systems for uptake of foreign DNA, which play a critical role in the acquisition of antibiotic resistance. The TtPilF (PilF ATPase from Thermus thermophilus) is required for high transformation efficiency, but its mechanism of action is unknown. In the present study, we show that TtPilF is able to bind to both DNA and RNA. The structure of TtPilF was determined by cryoelectron microscopy in the presence and absence of the ATP analogue p[NH]ppA (adenosine 5′-[β,γ-imido]triphosphate), at 10 and 12 Å (1 Å=0.1 nm) resolutions respectively. It consists of two distinct N- and C-terminal regions, separated by a short stem-like structure. Binding of p[NH]ppA induces structural changes in the C-terminal domains, which are transmitted via the stem to the N-terminal domains. Molecular models were generated for the apoenzyme and p[NH]ppA-bound states in the C-terminal regions by docking of a model based on a crystal structure from a closely related enzyme. Analysis of DNA binding by electron microscopy, using gold labelling, localized the binding site to the N-terminal domains. The results suggest a model in which DNA uptake by TtPilF is powered by ATP hydrolysis, causing conformational changes in the C-terminal domains, which are transmitted via the stem to take up DNA into the cell.