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The thorough understanding of the physiological and pathological processes mediated by extracellular vesicles (EVs) is challenged by purification methods which are cumbersome, not reproducible, or insufficient to yield homogeneous material. Chromatography based on both ion-exchange and immune-capture can represent an effective method to improve EV purification and successive analysis.
Cell culture supernatant was used as a model sample for assessing the capacity of anion-exchange chromatography to separate distinct EV fractions and to isolate nanobodies by direct panning on whole EVs to recover binders specific for the native conformation of EV-surface epitopes and suitable to develop EV immune-capture reagents.
Anion-exchange chromatography of cell culture supernatant separated distinct protein-containing fractions and all of them were positive for CD9, a biomarker associated to some EVs. This suggested the existence of several EV fractions but did not help in separating EVs from other contaminants. We further isolated several nanobodies instrumental for implementing immune-affinity protocols. These were able to immobilize EVs from both cell culture supernatant and biological samples, to be used in ELISA, flow-cytometry, and immune-purification.
Here we report the first successful isolation of anti-EV nanobodies for the use in immunoaffinity-based EV capture by panning a phage library directly on partially purified EVs. This achievement paves the way for the application of direct EV panning for the discovery of novel antibody-vesicle surface biomarker pairs and represents the preliminary requirement for the development of selective immune-capture that, in combination with anion-exchange chromatography, can simplify the systematic stratification of EV sub-populations and their individual characterization.
Extracellular vesicles (EVs) have been identified in many biological fluids such as blood, urine, cerebrospinal fluid, milk, ascites. The present scientific interest for EVs stems from the discovery that they play a crucial role in paracrine and long-distance cell–cell communication in physiological and pathological conditions as different as coagulation, inflammation, regenerative and differentiation processes, immune system modulation, tumor growth and metastasis [1–7]. EVs specifically deliver their cargos thanks to surface displayed proteins that have affinity for target-cell receptors . Their stable lipid bilayer forms a relatively large internal volume in which regulatory messengers (nucleic acids, lipids, proteins, and metabolites) are protected during transport and finally released by internalization or direct fusion with target cell membranes [9, 10]. Since EVs are easily accessible in biological fluids, they are evaluated as diagnostic and prognostic biomarkers in liquid biopsy assays [11, 12]. However, this perspective will be realized only after having established reliable and reproducible purification methods for eliminating contaminants and, possibly, discriminating among EV sub-classes carrying distinct molecular information . Comparative surveys of EV purification methodologies indicate that EVs can be obtained highly pure by a combination of density gradient ultracentrifugation and size-exclusion chromatography or ultrafiltration, but fractionations of EV subclasses is mostly dependent on affinity techniques [14–20].
Although size exclusion chromatography is a standard step during EV purification, other chromatographic techniques have not been generally assessed. The major reason is the large EV diameter that could lead to rapid clogging of resin-based columns. Nevertheless, ion-exchange chromatography can be used to recover EVs, as recently demonstrated with different anion exchange setups [21–23]. Particularly interesting are monolith columns since their structural stability and the possibility to obtain pores of variable diameter make them compatible with EV immobilization and recovery . By using such material, there is the chance to exploit the difference in EV charges and to elute separately distinct EV classes. Sub-population specificity could be improved by coupling orthogonal chromatographic separation methods such as ion-exchange and immunoaffinity. The limiting factor for the development of immune-based capture is the identification of reliable and possibly inexpensive binders specific for EV epitopes, optimally ones that are able to discriminate among vesicle sub-groups. Conventional antibodies—generally poly- and monoclonal antibodies of the IgG class—have been successfully used for immune-affinity purification of EV sub-groups . Nevertheless, some of their characteristics are unwanted, such as the quality differences among lots, their long isolation procedure and elevated production costs, their genetic instability, and the low homogeneity after labelling/functionalization [25, 26]. Single-domain antibodies (VHHs, nanobodies) have reduced mass (14 kDa in comparison to the 150 kDa of an IgG), are structurally stable, simple to engineer and label at specific residues, inexpensive to produce in bacteria and their clonality remains constant over the time . These characteristics make them appreciated and effective reagents in different applications such as oncology, infectious, inflammatory, and neurodegenerative diseases . A further advantage is that large pre-immune libraries are available and can be directly panned against both soluble antigens and whole cells [29–33]. We speculated that also EVs could be used as material for direct panning and that this approach would represent an advantage because it is rapid and would enable the isolation of binders for the native conformation of the antigen exposed on the EV surface. This is critical because it has been reported that the antigen present on EVs can be slightly modified with respect to that expressed in the original cell . In this paper we demonstrate: (i) the possibility to separate EV-containing fractions according to their retention in a monolith anion-exchange column; (ii) the feasibility of direct panning on EV-enriched fraction to isolate nanobodies that recognize membrane epitopes on such vesicles. Now that the technical conditions have been established, the method will enable to isolate further binders for different antigens and this antibody pool will allow for more detailed characterization of EV fractions. In combination with anionic-exchange chromatography, immune-capture will contribute to the stratification of EV populations.
Liquid chromatography performed using a monolith anion-exchange column and applied to medium from cancer cell culture supernatant resulted in the separation of three distinct elution peaks (Fig. 1a). Apparently, all the three fractions contained EVs because they were positive for CD9 when analyzed by flow cytometry (Fig. 1). An EV enrichment step obtained submitting the cell culture supernatant to ultrafiltration and successive precipitation by means of the Total Exosome Isolation kit  induced a significant modification of the chromatographic elution profile (Fig. 1b). Four peaks were detected, with peaks 1 and 3 sharing the same retention volume of the homologous peaks eluted from the untreated cell culture supernatant. Peaks 2 and 4 have no direct correspondence in the elution profile from the untreated cell culture supernatant (the retention time of peak 2 differed in the two samples, peak 4 was detected only in the second sample) but were also positive for CD9 (Additional file 1: Figure S1). Strikingly, the precipitation step significantly reduced the protein content of peak 1 (from 60 to 3 mAU), while the protein content of peak 3 remained constant (15 mAU). The thorough characterization of the molecular content of the different fractions will be the object of a further project, but these preliminary results already enabled to establish that EV surface charge is sufficient to separate distinct sub-classes. Their further characterization relies on the availability of another orthogonal purification method, such as immunopurification exploiting antibodies for specific surface antigens.
An effective method for isolating antibodies able to recognize an antigen discriminating between two very close cell types is the blind in vitro selection known as differential panning. A pre-immune library is first incubated with the control population and all the antibodies bound to the membranes are discharged (depletion step). The unbound fraction is then challenged with the target cells and the antibodies that remain in solution are discharged, whereas the bound antibodies are recovered after elution (enrichment step) . The approach has the further advantage of selecting binders that recognize the native conformation of the antigen because the selection is performed directly on whole cells and not on recombinant protein. We argued that it could be possible to adapt the protocol used for cells to EVs. We used the EV-enriched fraction corresponding to peak 1 obtained from chromatography of HEK293 supernatant for depletion and the material from peak 1 obtained from chromatography of SKBR3 supernatant for enrichment. In both cases, EV-enriched samples were bound to magnetic beads to simplify the washing steps. After two rounds of panning, 92 clones were analysed by phage ELISA on EVs recovered from both SKBR3 and HEK293 cells (Fig. 2a). This screening method uses directly culture media enriched in secreted phages and, despite the fact that the phage quantity is not normalized among the samples, is sufficient to discriminate between negative (14) and positive clones (78) which recognized EVs from both cell lines, usually with a preference for one of them. We did not identify binders with exclusive specificity for SKBR3 and selected 10 clones with high signal among those that apparently bound better to SKBR3-derived EVs. The sequencing results indicated the presence of five unique sequences. These underwent further validation by flow-cytometry in combination with commercial antibodies against CD9 (Fig. 2b). The data indicated two distinct binding behaviours for the nanobody clones. Whereas the two antibodies seemed to bind independently when the commercial anti-CD9 was tested with B1, F7, and D5, in the presence of the clones H1 and H6 the binding of the anti-CD9 appeared to be inhibited. Since CD9 is one of the canonical EV biomarkers, showing that we selected anti-CD9 nanobodies would indicate that direct panning on EVs is not only feasible and yields functional binders, but that at least part of them are directed against EVs, despite the low purity of the material used for the panning . A consideration is necessary here to explain the lack of SKBR3-selective binders. We imputed it to the elevated loss of EVs from the beads during the washing steps that we observed. Under these conditions the library depletion was probably insufficient for eliminating clones able to recognize shared epitopes.
To prove that H1 and H6 were anti-CD9 antibodies, these clones were first sub-cloned in an expression vector that allows the production of fusion proteins composed of nanobody, eGFP, and C-term 6xHis tag. The preliminary small-scale expression test showed that the constructs were produced in soluble form in E. coli (data not shown) and was used to set the optimal growth conditions. Purification of VHH-GFP constructs (42 kDa) from soluble bacterial fractions by metal-affinity chromatography resulted in non-homogenous preparations (Additional file 2: Figure S2) which needed an anion-exchange chromatographic step to separate contaminants from the fluorescent immunoreagents (Additional file 2: Figure S2). Final yields were in the range of 10 mg of homogeneous VHH-GFP per litre of medium and these purified constructs were used in competition experiments at flow-cytometry. Binding comparison to EVs derived from different cell lines confirmed that some clones (H1 and B1) had clearly differing binding preferences, an indication that they recognize different antigens on the EVs (Additional file 3: Figure S3A–C).
Latex beads coated with EV-enriched fractions recovered from HEK293, SKBR3, and Jurkat cells were tested for their CD9 positivity with a commercial monoclonal antibody (Additional file 3: Figure S3D), then were incubated either with anti-CD9–PE antibodies or with both anti-CD9–PE and VHH-GFP antibodies (Fig. 3). As seen by the shift in FL2 fluorescence intensity, the addition of H1 nanobodies significantly decreased the binding of anti-CD9 to beads coated with EVs. This was confirmed with EVs derived from all the three cell types and the H6 clone. In terms of labelled beads (Fig. 3), the presence of H1-GFP inhibited the CD9 binding from 36.86 ± 9.95 to 11.46 ± 1.82% (HEK293-EVs) and from 45.78 ± 8.42 to 12.18 ± 3.89% (SKBR3-EVs), while H6-VHH competition resulted in a decrease of anti-CD9 bound beads from 49.55 ± 4.21 to 13.40 ± 1.73% (HEK293-EVs) and from 52.86 ± 5.14 to 7.59 ± 0.86% (SKBR3-EVs). Controls such as an irrelevant nanobody (nbVHH) and the B1 clone which was assessed as a CD9-neg but EV-pos in the preliminary screening (Fig. 2b) did not affect significantly the CD9 binding to the vesicle-coated beads (Fig. 3). The capacity of the monovalent VHHs to compete successfully with the bivalent commercial monoclonal IgG was observed at conditions of almost 200 times molarity excess for the nanobodies in solution.
The results of the competition experiments seemed to indicate that H1 and H6 are anti-CD9 nanobodies. This feature enables their use as specific binders for EVs for improving the stability of their binding (to beads, to ELISA plate, to biosensor surfaces, etc.) and their selective capture when contaminated EV fractions are available. As a proof-of-principle, H1/H6 nanobodies were used for immune-purification of EVs from both cell culture supernatant and human serum. The nanobodies were coated onto latex beads and used to bind the EVs present in the samples. EVs were then released by lowering the buffer pH. Beads were incubated with 20 µg of EV-fraction proteins recovered from supernatant of HEK293, SKBR3, and Jurkat cells. The yields (9–11 µg) were constant for all the samples. In the case of human plasma, immune-purification resulted in 42 µg of exosomal protein per 250 µL of undiluted plasma. A qualitative evaluation of the recovered EVs was performed by TEM (Fig. 4). This analysis identified vesicles with diameter between 50 and 200 nm in all the observed samples.
Next, we evaluated if the nanobody-mediated capture could increase the amount of EVs stably bound to the latex beads used for flow-cytometry. Both H1 and H6 coated beads were used in combinations with samples of cell culture supernatant collected from HEK293 and SKBR3 cells and the immune-captured EVs were used to bind anti-CD9–PE antibodies. The amount of captured EVs was finally assessed indirectly by measuring the PE-dependent fluorescence intensity (Fig. 5a). A clear increase was measured for both tested nanobodies, with constant higher fluorescence values obtained when using material from SKBR3 cell cultures. Both nanobodies bound to latex beads were successful in capturing EVs also from human plasma of healthy donors (Fig. 5b). Despite the elevated background signal and the low concentration of EVs in these samples , the fluorescence value increased from 5.37% (control) to 14.05% (H1-coated beads) and from 4.93% (control) to 14.89% (H6-coated beads). An irrelevant nanobody (nb-VHH) used as a control was not able to capture vesicles from human plasma. Purified H1 and H6 nanobodies were also able to bind strongly to the fraction 1 separated by IEX chromatography and linked to latex beads (Additional file 4: Figure S4), in agreement with the data previously obtained with monoclonal anti-CD9 that confirmed the presence of EVs in this sample.
Next we assessed the possibility to improve the reproducibility of EV ELISA by using H1- and H6-mediated nanobody immune-capture instead of direct EV coating on microplate surface to reduce the vesicle removal during the washing steps. In the experimental setting, EVs from cell culture supernatant were first immobilized in the microplate wells – directly or by means of anti-CD9 nanobodies-and later used to screen phage displayed nanobodies according to their affinities for such EVs (Fig. 5c). Bound phages were visualized using anti-M13 HRP antibodies. The false negative (EVs from HEK293 cells) and the high standard deviation (EVs from SKBR3) values obtained by direct EV coating indicated highly unstable conditions and/or consequent low data reproducibility. H1/H6-mediated EV immune-capture eliminated the variability and enabled to collect reproducible data. Probably, EVs directly coated on the microplate plastic are apparently prone to be washed away and adopting this methodology resulted in misleading results. In detail, the absorbance (A450) values describing the E10 clone binding capacity to HEK293-derived EVs passed from 0.18 ± 0.05 (direct coating) to 2.60 ± 0.35 and 2.49 ± 0.53 for H1- and H6-mediated EV capture, respectively. In the case of the SKBR3 EVs, direct coating yielded an A450 value of 1.16 ± 1.46, whereas nanobody-mediated EV coating resulted in A450 values of 2.49 ± 0.53 and 1.73 ± 0.31.
The involvement of EVs in both physiological and pathological processes  makes them promising diagnostic and prognostic biomarkers [12, 22]. In the last years research aimed at standardizing the EV purification and characterization methods as well as stratifying EV sub-populations for improving the reliability of the diagnostic information. This development led to an increasing interest for affinity-, and specifically for immune-affinity-based purification methods. However, the elevated development and production costs of reagents, such as monoclonal antibodies suitable for specific EV biomarkers, slowed down this approach implementation. In this work we clearly demonstrate the feasibility of isolating nanobodies by panning directly against EV-enriched fractions. This methodology is fast and inexpensive in comparison to conventional hybridoma protocols (Additional file 5: Table S1). Furthermore, it presents two additional major advantages: (i) the selected binders recognize EV-epitopes in their native conformation; (ii) potentially, it enables to isolate binders against new biomarkers. The first benefit was directly exploited in the present work. We had noticed that the major drawback of direct panning on EVs was due to the poor binding of the vesicles to solid surfaces, namely latex beads during the panning and plastic microplates during ELISA. This condition impaired the application of a successful differential panning to isolate nanobodies which can discriminate between subgroups. When we used the two selected nanobodies which apparently compete with CD9 (Fig. 3) for the immunocapture of the vesicles to the solid surfaces, EV binding was strongly stabilized and experimental variability was drastically reduced (Fig. 5). This is a critical accomplishment since it allows for the standardization and reproducibility of the selection procedure and will enable the implementation of differential panning protocols to fish for nanobodies that can discriminate between exosome sub-populations, as we already successfully performed with whole cells . It is probable that an accurate EV profiling will require the analysis of a large amount of both qualitative and quantitative biomarkers [37, 38], namely of several corresponding antibodies. As already demonstrated in this work with the constructs VHH-GFP, the straight-forward bacterial production of immune-fluorescent fusions can provide inexpensive application-friendly reagents  for multi-dimensional vesicle characterization. These immune-reagents can be directly used with complex samples such as human plasma without any pre-fractionation (Fig. 5b).
In this work we also showed that anion-exchange chromatography can be used for preliminary separation of EV fractions. The presence of several fractions demonstrates the heterogeneity of vesicular content and existence of several sub-populations even in a simplified system such as conditioned cell culture supernatant. Large preparative anion-exchange purification could provide the material necessary for: (i) the characterization of the molecular content of the different fractions, both in terms of EV sub-types and "contaminants" such as soluble proteins; (ii) performing differential panning and isolation of binders which selectively recognize the EV fraction present in only one elution peak or distinguish the same peak in material obtained from different samples, discriminating between clinical relevant and non-relevant markers. Advantages of monolithic supports for ion chromatography include better mass transfer properties, the ability to be manufactured with a wide range of pore sizes, the relative ease of scaling up and scaling down, and the low back pressure even at very high flow [39, 40]. Monoliths can be functionalized to perform different separation modes [41, 42], including immune-affinity, and therefore should be further considered for miniaturized multi-components for EV-based diagnostics .
In this work we report the first case of successful isolation of anti-EV nanobodies by direct panning of a phage library on partially purified EVs. This achievement enables stable immunoaffinity-based EV capture and consequently simplifies the future discovery of novel antibody-vesicle surface biomarker pairs that will be instrumental for the systematic stratification of EV sub-populations and their individual characterization.
Human embryonic kidney (HEK-293), breast adenocarcinoma (SKBR3), and T cell lymphoma (Jurkat) cells were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA). HEK-293 and SKBR3 cells were grown in Dulbecco's modified minimal essential medium (DMEM) supplemented with 10% v/v heat inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco, Thermo-Fisher Scientific) at 37 °C under a 5% CO2/95% air atmosphere at constant humidity. Jurkat cells were grown in RPMI medium supplemented with 10% v/v heat inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco, Thermo-Fisher Scientific) at 37 °C under a 5% CO2/95% air atmosphere at constant humidity.
SKBR3 and HEK293 cells were grown in DMEM medium supplemented with FBS (not EV-free) until they reached 70–80% confluence. Jurkat cells were grown in RPMI medium supplemented with FBS (not EV-free) for 3 days. The culture medium was then removed and the cells rinsed with PBS before the addition of FBS-free DMEM medium in the case of HEK-293 and SKBR3, or FBS-free RPMI in the case of Jurkat cells. After 24 h, the conditioned cell culture supernatant was transferred into 50 mL polypropylene tubes and centrifuged 30 min at 300×g at 4 °C to pellet floating cells and filtered through 0.45 µM syringe filter to remove cell debris and aggregates. The supernatant was collected into a sterile glass bottle and used for chromatographic separation using an ÄKTA pure 25 system (GE Healthcare) in combination with a 1 mL CIM QA-1-monolith column with pores 6 µm in diameter (BIAseparations) equilibrated in 20 mM Tris–HCl, pH 8.03. Two millilitre of exhausted media were used, corresponding to a culture of roughly 1 million cells, diluted 1:10 in 20 mM Tris–HCl buffer, pH 8. After a 10 mL washing step with the equilibration buffer, a 20 mL linear salt gradient (0–100%) was obtained by adding 20 mM Tris–HCl, pH 8.03, 2 M NaCl at a flow rate of 5 mL/min. The eluted material was monitored detecting the absorbance at 280 nm. As an alternative, an EV-enriched fraction was purified using the Total Exosome Isolation kit (Life Technologies, Thermo-Fisher Scientific), according to the manufacturer's instruction and ultrafiltration using an ultra-centrifugal cartridge with cut-off of 100 kDa (Amicon, Merck).
Panning was performed following a modified version of previously described methods . Nanobody-displaying phages (3 × 1011)—diluted in 1 mL of PBS containing 2% skimmed milk—were first depleted by incubating them twice 30 min in the presence of 25 µL of milk-blocked naked beads and each time the bound fraction was discharged. The unbound phage fraction was transferred to the HEK293 EV-coated beads and incubated for 1 h. EV-coated beads were prepared using epoxy magnetic beads (25 µL; Life Technologies, Thermo-Fisher Scientific). Epoxy magnetic beads were washed in PBS and finally resuspended in 25 µL of the same buffer before adding 2 µg of the EV-enriched fraction recovered by chromatographic separation of the material present in HEK293 cell culture supernatant. After overnight incubation, the still reactive sites on bead surfaces were blocked 30 min at room temperature with 1 mL of PBS, 2% milk powder. Pre-selected phages were incubated with EV-coated beads for 1 h at room temperature and the unbound fraction was recovered. The depletion procedure as repeated and finally the unbound phage fraction was incubated with beads coated with EV-enriched fraction recovered by chromatographic separation of SKBR3 culture media (see above). After 20 washing cycles in PBS, bound phages were eluted by adding 1 mL of 200 mM glycine, pH 2.2 containing 1 mg/mL BSA and neutralized in 150 µL of 1 M Tris–HCl, pH 9.1. The phages were used to infect TG1 cells that were spread over 2xTY Petri dishes. The resulting colonies were recovered the successive day and the corresponding rescued phages used for a second panning cycle.
The phages produced by the colonies recovered after the second panning cycle were screened by ELISA for their capacity to bind EVs. An irrelevant clone was used as a negative control. Microtiter plates (96-well, MaxiSorp, Nunc™) were coated by incubating overnight at 4 °C with 2 μg/well (100 µL/well in PBS) of total protein from EV-enriched fractions. Plates were blocked (1 h at 37 °C in PBS buffer plus 5% (w/v) skimmed milk) and phages pre-incubated 1 h at 37 °C in PBS buffer plus 1% (w/v) skimmed milk. One hundred microliter per well of blocked phages were added to the plates and incubated 1 h at room temperature. After washing (4 times × 5 min in PBS), 100 μL of mouse anti-M13 antibodies conjugated with HRP (dilution 1:5000, GE Healthcare) were added to each well and incubated 1 h at 37 °C. Reporter signal was obtained by adding 100 µL of TMB solution (Sigma Aldrich) after another washing step (4 times × 5 min in PBS). The colour reaction was stopped by 50 μL of 2 NaH2SO4. Absorbance at 405 nm was recorded by using a HTS7000 Bioassay reader (Perkin Elmer).
Nanobody sequences were subcloned into a modified pET14b vector using NcoI and NotI enzymes to obtain fusion constructs in which nanobodies are linked at their C-terminal with both eGFP and 6xHis tag . These vectors were transformed into E. coli BL21 (DE3) hosting the plasmid for the expression of sulfhydryl oxidase and DsbC [44, 45]. Antibodies were produced as previously described with some modifications . Briefly, 2 mL of over-night pre-culture were used to inoculate 500 mL of LB broth in the presence of the 100 µg/mL ampicillin and 25 µg/mL chloramphenicol. Bacteria were grown at 37 °C until OD600nm reached 0.4. Sulfhydryl oxidase and DsbC expression was induced by adding 0.5% (g/mL culture) of arabinose and the temperature was lowered to 30 °C. After 30 min, 0.1 mM of IPTG was added to induce antibody expression, the bacteria were grown overnight at 21 °C, harvested, and frozen. Bacterial pellet was resuspended in 20 mL of 100 mM Tris–HCl, pH8, 500 mM NaCl, 2.5 mM MgCl2. Lysozyme (0.5 mg/mL) and DNAse (3U) were added and the lysate was kept 30 min at room temperature. Samples were sonicated and finally centrifuged at 18,000×g for 20 min at 4 °C. Supernatant was filtered (0.45 µM) and loaded on a 5 mL HiTrap Talon Crude column (GE Healthcare) connected to a chromatographic ÄKTA pure 25 system (GE Healthcare). The column was equilibrated with 50 mM phosphate buffer, pH 7.4, containing 500 mM NaCl and 5 mM imidazole, while bound proteins were eluted using 50 mM phosphate buffer, pH 7.4, containing 500 mM NaCl and 150 mM imidazole. Fractions containing VHH-GFP constructs were pooled and buffer was exchanged into 30 mM Tris–HCl buffer, pH 8.3, by using a 5 mL HiTrap Desalting column (GE Healthcare) in combination with a chromatographic ÄKTA pure 25 system (GE Healthcare). Further purification of the VHH-GFP construct was achieved using a Mini Q 4.6/50 ion exchange column (GE Healthcare) mounted on a chromatographic ÄKTA purifier ten system (GE Healthcare). Samples were loaded on a column pre-equilibrated with loading buffer (30 mM Tris buffer, pH 8.3) and eluted using 30 mM Tris buffer, pH 8.3, 1 M NaCl. Elution was achieved by using a linear 0–100% gradient (10 column volumes). Antibody concentration was determined by Bradford colorimetric assay  while the presence of contaminants was assessed by SDS-PAGE (14%, denaturing conditions).
Flow-cytometry was performed by using either phages or purified nanobodies. In the first case, the protocol started with the coating of aldehyde/sulphate latex beads 4% w/v, 4 µm (Thermo Fisher Scientific) with EVs. Specifically, 25 µL of latex beads were coated overnight at 4 °C with 2 µg of total protein from EV-enriched fraction in PBS. Beads were washed 3 times with PBS and blocked first for 30 min at room temperature with 200 mM glycine and then 30 min in PBS plus 5% (w/v) skimmed milk. Phages were pre-incubated 1 h at 37 °C in PBS buffer plus 1% (w/v) skimmed milk and 100 µL of the blocked phages were incubated with 25 µL beads for 45 min at room temperature. Beads were washed 3 times in PBS before adding 100 μL of mouse anti-M13 antibodies (dilution 1:100, GE Healthcare). After 30 min at room temperature, beads were washed 4 times in PBS and 100 μL of anti-mouse PE-labelled antibodies (dilution 1:100, BD Biosciences) were added. After 30 min at room temperature and another washing step as above, 100 μL of anti-CD9 APC-labelled antibodies (BD-Biosciences, dilution 1:100, final concertation 16.7 nM) were added. Beads were incubated 20 min at room temperature before washing and flow-cytometry analysis by using a FACSCanto II instrument (BD Biosciences) collecting around 1000 events/s. A blue solid state 200 mW laser at 488 nm and a 100 mW red laser at 640 nm were used for excitation. The emission was detected with 561 nm (FL2, PE) and 660 nm (FL3, APC) filter. The positive beads were gated on the FL2-PE, FL3-APC plot. APC and PE specific fluorescence was assessed as the signal increase with respect to negative control (autofluorescence of beads without coated EVs).
When purified VHH-GFP constructs were used for detection of EVs, 25 µL of aldehyde/sulfate latex blocked beads containing EVs were incubated with 100 µg of nanobodies (2.5 μM) in 1% BSA solution in PBS for 1 h at room temperature. Beads were washed 3 times in PBS before adding 100 μL of mouse anti-CD9 PE-labelled antibodies (dilution 1:100, GE Healthcare) and were then incubated for 45 min at room temperature. After a washing step as described before, beads were analysed using FACS Calibur (BD Biosciences). A blue solid state 200 mW laser at 488 nm was used for excitation. The emission was detected with 525 nm (FL1, GFP) and 561 nm (FL2, PE) filter. The positive beads were gated on the FL1-GFP, SSC plot and FL2-PE, SSC plot. GFP and/or PE specific fluorescence was assessed as the signal increase with respect to negative control (autofluorescence of beads without coated EVs).
EVs were captured using purified VHH-GFP constructs in both ELISA and flow-cytometry format assays. For ELISA, microtiter plates (96-well, MaxiSorp, Nunc™) were coated by incubating 10 μg/well of VHH-GFP (100 µL/well in PBS) overnight at 4 °C. Plates were blocked (1 h at 37 °C in PBS buffer plus 5% (w/v) skimmed milk) after which 0.1 µg of total protein from EV fraction in PBS with 1% milk (w/v) was added and incubated 1 h at 21 °C. After washing (4 × 5 min, PBS), 100 µL/well of blocked phages were added to the plates and incubated 1 h at room temperature. After washing (4 × 5 min in PBS), 100 μL of mouse anti-M13 antibodies conjugated with HRP (dilution 1:5000, GE Healthcare) were added to each well and incubated 1 h at 37 °C. Signal was developed after another washing step by the addition of 100 µL of TMB solution (Sigma Aldrich) and 1 h incubation at 37 °C. The colour reaction was stopped by 50 μL of 2 NaH2SO4. Absorbance was monitored at 405 nm in a HTS7000 Bioassay reader (Perkin Elemer).
For flow-cytometry, 25 µL of latex beads were coated overnight at 4 °C with 50 µg of VHH-GFP. Beads were washed three times with PBS and blocked first for 30 min at room temperature with 200 mM glycine and then 30 min in PBS plus 5% (w/v) skimmed milk. Beads were incubated with 10 µg of total protein from EV enriched fraction in PBS with 1% milk (w/v)/10 µL of VHH-coated beads or with 1:2 diluted plasma pool from healthy volunteers for 1 h at room temperature. Beads were washed 3 times in PBS before adding 100 μL of mouse anti-CD9 PE-labelled antibodies (dilution 1:100, GE Healthcare) and incubated for 45 min at room temperature. After washing steps as described before, beads were analysed using FACS Calibur (BD Biosciences). The analysis rate was around 1000 events s-1. A blue solid state 200 mW laser at 488 nm was used for excitation, the emission was detected at 561 nm (FL2, PE). The positive beads were gated on the FL2-PE, SSC plot. Specific fluorescence was assessed as the signal increase with respect to negative control (autofluorescence of beads without coated EVs).
Twenty microliter of latex beads were coated overnight at 4 °C with 100 µg of H1/H6-GFP. Beads were washed three times with PBS and blocked first for 30 min at room temperature with 200 mM glycine and then 30 min in PBS plus 5% (w/v) skimmed milk. Beads were incubated 1 h at room temperature with: (i) 20 µg of total protein from cell supernatant EV enriched fraction in PBS with 1% milk (w/v); (ii) plasma pool from healthy volunteers diluted 1:2 in the same buffer. Beads were washed 5 times in PBS before elution in 100 µL of 200 mM glycine, pH 2.2 and immediate neutralization in 15 µL of 1 M Tris–HCl, pH 9.1. Eluted vesicles were stabilized by the addition of 25 mM trehalose and their morphology was evaluated by TEM after sample negative staining. Four microliter of vesicle suspension were adsorbed on carbon/formvar coated 400 mesh nickel grids (Electron Microscopy Sciences, Fort Washington, USA). After 10 min, the sample excess was removed with filter paper and immediately replaced by 4 μL of staining agent (uranyl acetate diluted 1:3 in distilled water), which was allowed to settle 10 min. Grids were washed 5 times in distilled water, the water excess was removed with filter paper, and then observed in a Philips CM 10 (FEI, Eindhoven, The Netherlands) TEM, operated at 80 kV.
Statistical analysis was performed using GraphPad Prism v7 for Windows (San Diego). A significance level of p ≤ 0.05 was used for analysis of variance, implemented using the Kruskal–Wallis test followed by the Tukey's post hoc test (p ≤ 0.05). Correlation between different parameters was performed at significance level of p ≤ 0.05.
MP and AdM conceived the experiments and wrote the manuscript, MP performed the panning and biochemical experiments, EM prepared the clones, BT performed the FACS experiments, AdM designed the project. All authors read and approved the final manuscript.
This work was supported by the Slovenia-Serbia bilateral ARRS program (Project "Isolation of recombinant nanobodies specific for exosome subclasses", BI-RS/16-17-014), by the Slovenia-Belgium ARRS-FWO program (Project "Identification of recombinant nanobodies for exosome immune-capture in breast cancer diagnosis", ARRS/N4-0046-5100-1/2015-59) and by the Grant No. 172049 from Ministry of Education, Science and Technological Development of the Republic of Serbia. The authors wish to thank An Hendrix for her valuable comments and Rita Musetti for her assistance (TEM images).
12934_2017_856_MOESM1_ESM.tif Additional file 1: Figure S1. Chromatographic separation of kit-purified EV-enriched fraction from culture media. Kit-precipitated EVs present in SKBR3 cell culture supernatant were separated using a large-pore anion-exchange monolith column. All the four separated fractions were analyzed by flow-cytometry and resulted positive for the EV marker CD9.
12934_2017_856_MOESM2_ESM.tif Additional file 2: Figure S2. Purification strategy of VHH-GFP constructs. H1-GFP and H6-GFP constructs were expressed in E. coli and purified from the soluble fraction using immobilized metal-affinity chromatography. The corresponding chromatograms are reported together with the SDS-gels of total bacterial lysate (lane 1) and elution fractions 10–12 (lanes 2, 3, 4). After desalting, the samples underwent IEX purification and the eluted fractions were separated by SDS-PAGE (H6, lanes 1–10; H1, lanes 11–19; MW molecular weight markers).
12934_2017_856_MOESM3_ESM.tif Additional file 3: Figure S3. Antibody differential binding to cell-derived EVs. EVs derived from HEK-293, SKBR3, and Jurkat cells were used to evaluate the binding preferences of the nanobodies H1 and B1 compared with the binding of the irrelevant clone nbVHH (A–C). The binding capacity of a commercial anti-CD9 antibody was tested with the same cell lines (D). Bars indicate median percentage of positively stained EV coated beads with anti-CD9–PE antibodies to H1-GFP, B1-GFP, and non-binding VHH coated beads with respect to autofluorescence of unstained EV coated beads. The error bars indicate standard deviations for triplicate measurements.
12934_2017_856_MOESM4_ESM.tif Additional file 4: Figure S4. Anti-exosome nanobodies bind EV-fractions separated by chromatography. Flow cytometry experiments show that both H1 and H6 strongly bind to exosomes present in the fraction 1 separated by IEX chromatography.
12934_2017_856_MOESM5_ESM.doc Additional file 5: Table S1. Comparison of indicative times and costs necessary to produce monoclonal antibodies.
Development of a separation method for exosomes by means of large pores monolithic supports. [http://www.biaseparations.com/support/posters/product/775-development-of-a-separation-method-for-exosomes-by-means-of-large-pore-monolithic-columns].


text:
Homepage Home > Patient praise for "superb service" during Autism Awareness Month
Patient praise for "superb service" during Autism Awareness Month
A woman with Asperger syndrome has praised the "superb service" she received from autism experts at Norfolk and Suffolk NHS Foundation Trust, saying she's "not sure she'd be here today" without their help and support.
Sue Penkosky-Cornwell, who lives in Stowmarket, has shared her experience during Autism Awareness Month, which aims to raise awareness of the condition which affects more than 700,000 people across the UK.
The 47-year-old was finally diagnosed with Asperger syndrome following referral to the Trust's Suffolk Autism Diagnostic Service, after suspecting she was suffering from the illness for nearly a decade.
Since then, she has been given the help and support she need to cope with Asperger syndrome, which is a form of autism causing difficulties with communication and interaction.
"To have the diagnosis confirmed has made a significant difference to my attitudes towards my abilities and my self-perceived failures," said Sue.
"It gave my partner, who was kept involved during the whole diagnostic process, a chance to learn a lot more about me, which means I have a whole new and understanding support system both at home and in my wider family. It made me learn a lot more about myself, and gave me the much-needed confirmation that I needed to seek adaptations at work. It also opened the way to some fantastic post-diagnostic services that have made me evaluate my life, given me the ability to arrest control over difficult phases of depression and given me an unprecedented new level of self-confidence.
"The Autism Diagnostic Service was absolutely wonderful. The lead clinical psychologist, Dr Colm Magee, was friendly and warm and made the two-and-a-half hour appointment very easy and comfortable for me.
"He treated me as someone who mattered in the system, and left me feeling that I hadn't been going slowly insane all these years."
The Suffolk Autism Diagnostic Service caters for patients aged 18 and over, and offers discussion and counselling for people who may be on the autistic spectrum but do not have a diagnosis.
Launched 18 months ago, it has already had more than 300 referrals, while colleagues in the service's youth team, which has been running a year, have clocked up nearly 200 referrals.
The multi-disciplinary team of experienced clinicians complete a comprehensive assessment – sometimes during several different appointments – so that they can make sure patients and their families receive an accurate diagnosis. They then provide advice, support and – where appropriate – counselling to help patients manage their condition and cope with any challenges they have been facing, as well as signposting them to other agencies.
"I've had two follow-up appointments for the post-diagnosis process, have attended classes for newly-diagnosed Asperger adults and plan to go along to social groups the service has arranged. The staff seem to go out of their way to help people on the autism spectrum," added Sue.
"More recently during a meltdown, I was able to call the centre and receive direct support from senior staff, which was an enormous help in calming me down, as well as help with coping strategies and a way forward. Without them, I'm not sure I'd be here today.
"There are clearly areas of the NHS that are silently excellent. The Autism Diagnostic Service in Suffolk is one of them and I hope that they're recognised for their superb service."
Dr Colm Magee, Clinical Lead and Clinical Psychologist with the service, said: "We have been overwhelmed by the excellent feedback we have received since the service launched 18 months ago. The number of referrals we have received so far show there was a real need for this dedicated service, and we are delighted to be making such a difference to people's lives.
"Previously, patients had to travel to London or Cambridge for a specialist diagnosis. We have tried to make our service as convenient as possible by offering clinics across the county so that people can get the help they need closer to home.
"We have also designed the service carefully to make it autism friendly. For example, we don't offer to shake hands as people with the condition don't often like being touched. We'll also take plenty of breaks to make it that little bit easier for people.
"So far, we have diagnosed people of all ages, ranging from 18 to 73. Although the diagnosis won't change what has happened in their lives, it can help them to make sense of their past, which is really important. It is also vital in helping them to learn the coping mechanisms which will allow them to tackle any difficulties they are facing and enjoy a good quality of life."
Anyone who thinks they could benefit from a referral should speak to their GP.
For more information on Autism visit:
www.autism.org.uk
Patient-praise-for-superb-service-during-Autism-Awareness-Month

redpajama_set_name: RedPajamaCommonCrawl


text:
using System;
using System.Collections.Generic;
using System.Linq;
using System.Text;
using System.Threading.Tasks;
using System.Xml;

namespace StudyToolReader
{

    /// <summary>
    /// Represents a set of questions and answers
    /// </summary>
    public class Questions
    {

        /// <summary>
        /// Gets/Sets the name of the section.  Used for organizing sets of StudyToolReader.Questions.
        /// </summary>
        public string section { get; set; }

        /// <summary>
        /// Gets/Sets the list of questions.
        /// </summary>
        public List<Question> questions { get; set; }

        /// <summary>
        /// Uniqueidentifier of the set of Questions.
        /// </summary>
        public Guid Id { get; set; }

        /// <summary>
        /// Uniqueidentifier of the Class this set of Questions belongs to.
        /// </summary>
        public Guid ClassId { get; set; }

        /// <summary>
        /// Initializes the set of questions to their default values.
        /// </summary>
        public Questions()
        {
            this.section = "";
            this.questions = new List<Question>();
            this.Id = Guid.NewGuid();
        }

        /// <summary>
        /// Initializes the set of questions with the data from the System.Xml.XmlNode.
        /// </summary>
        /// <param name="node">XmlNode Containing the data.</param>
        public Questions(XmlNode node)
        {
            try { this.section = node.Attributes["section"].InnerText; }
            catch { this.section = ""; }

            this.questions = new List<Question>();
            foreach(XmlNode q in node.ChildNodes)
            {
                try { this.questions.Add(new Question(q)); }
                catch { }
            }
            this.Id = Guid.NewGuid();
        }

        /// <summary>
        /// Initializes the set of questions with the given set of questions and section.
        /// </summary>
        /// <param name="questions">List of questions.</param>
        /// <param name="section">Name of the section.</param>
        public Questions(List<Question> questions, string section)
        {
            this.questions = questions;
            this.section = section;
            this.Id = Guid.NewGuid();
        }

        /// <summary>
        /// Creates an System.Xml.XmlNode from the Questions
        /// </summary>
        /// <param name="xdoc">XmlDocument needed to create the node for.</param>
        /// <returns>Returns the created node.</returns>
        public XmlNode ToXmlNode(XmlDocument xdoc)
        {
            XmlNode node = xdoc.CreateElement("questions", null);
            XmlAttribute s = xdoc.CreateAttribute("section", null);
            s.InnerText = this.section;

            node.Attributes.Append(s);

            foreach (Question q in this.questions)
            {
                try { node.AppendChild(q.ToXmlNode(xdoc)); }
                catch { }
            }

            return node;
        }
    }
}

redpajama_set_name: RedPajamaGithub


Spacious 1.5 acre lot features 3 homes and is good for both owner occupy or long term investor. It can also be repurposed into a mix of owner occupy and guest house or management housing for your orchard. The main house is a well kept 4 bed/2.5 bath house with attached garage. The second is a nice little 4 bed/2 bath mfg home and the third is a 2 bed/1 bath mfg home. Also included are a 616 sq ft block outbuilding, a massive 3900 sq ft workshop and an ample 32 bed H2A approved bunk facility.


Our Longest 0% Balance Transfer.
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Made a plain garlic and mozzarella pizza the other day. Used a Presto Pizzazz Rotating Open-Air Oven for 10 minutes (probably should have gone 12 to add more crispiness) -- result was excellent.
Am trying to figure out how to use the shells to make calzones, but they will crack if I try to bend them. May end up just using two shells as a sandwich and let the Presto unit do its work.
We had a pizza I made using this crust tonight. It was very good and more like the pizzas we are used to in SE MA. We are now in SE FL and can't get what we consider a good pizza. This crust is thin and crisp, just like we love. It was done in no time and also gone in no time!
Best Pizza shells!! My family loves them!
I live in Florida but I was born in Baltimore. I was on a visit and was introduce to the Sun of Italy Italian Spice. It smells so good. I found out it is sold online. That is when I saw the pizza shells. How convenient they are! I put them in the freezer and thaw them when I need them. Thin and Crispy! My family loves it when I say it's "Pizza Night" I recently had guest over and I made pizza. It was a hit!
As promised, the crust crisps nicely and has lovely brown bubbles on the bottom. Is nice for doing "gourmet" pizzas.
Click the button below to add the 3 Pk. Thin & Crispy Pizza Shells to your wish list.


2018 was a record year at Wicklow Wolf with rapid growth that saw us begin construction on our new brewery, launch a new website, brew a shed load of great and interesting beers, establish our own in-house distribution company – Alpha Beer and Cider Distribution, open new export markets and now we are delighted to announce that we have welcomed Master Brewer John Allen to the Wicklow Wolf team.
John will take the role of Production Manager and will oversee the development of our new 17,000 sq ft, 35hL BrauKon brewery in Newtownmountkennedy, Co. Wicklow.
"We at Wicklow Wolf are absolutely thrilled to have John join our team as Production Manager. John has a very impressive pedigree in brewing in both Australia and the UK. We are on a journey to grow our business and our brand in line with the key principles that we hold highly, the first and foremost being the quality and consistency of the beer that we produce. John is a big addition which will help us to move to the next stage in our adventure. We look forward to brewing more great beer and of course having a bit of craic with John leading the way!" says Managing Director, Quincey Fennelly.
John, a Roscommon native, returns to Ireland after spending the last 12 years in some of the world's leading breweries. He began his brewing career after graduating from Maynooth with a degree in Biotechnology, his passion for brewing blossomed which led him to completing a Masters of Brewing at the Heriot-Watt University in Edinburgh.
John has spent the last 6 years at BrewDog in Scotland where he rose to the role of Brewing Manager. John's extensive brewing background extends to time in Diageo's R&D sector just outside of London, Fuller's in London, and Little Creatures in Australia.
Wicklow Wolf and John will be at Craft Beer Rising (https://www.craftbeerrising.co.uk) in London this February pouring some new Wicklow Wolf limited edition beers. Stop by and have a beer with John and the team. We will also be hosting a number of Tap Takeover's around Ireland in the coming months.
Born and Bred in County Wicklow, Ireland, Wicklow Wolf was founded in 2014 by Quincey Fennelly and Simon Lynch. Four years on, Wicklow Wolf has grown from strength to strength. Our multi-award winning beers include our Alpha Pack (core range), Endangered Species (Limited Editions), Crossbreed Series (Collaborations) and The Locavore Project (Fresh Hop Seasonal) – visit www.wicklowwolf.com for more about our range.
At Wicklow Wolf we are very proud of having our very own 10 acre hop farm located in the Wicklow mountains. The hop farm are overseen by the expert eye of co-owner Simon Lynch. Simon has spent over 20 years working in the horticulture business and his passion, expertise and drive is what helps to make our hop farm flourish. The Wicklow Wolf Hop Farm is the first commercial hop farm in ireland in over 20 years. Here we grow over 12 varieties including Chinook, Cascade, Perle, bramling cross, Fuggle. Challenger, Apollo.
Wicklow Wolf is working on exciting plans for our new, purpose built brewery, opening in 2019. We have begun construction on the new home of Wicklow Wolf. Located in Newtownmountkennedy, Co. Wicklow, this new brewery will be state of the art. Built on a 2 acre site the new 17,000 sq ft brewery will be home to our new 35hl brewhouse, tanks, canning line and bottling line.


Pack a lunch and bring your baseball gear! Let's have fun and work on a number of baseball fundamentals, (hitting, pitching, stealing bases, and taking ground balls in the cage). We also enjoy taking the game outside with a run to the beach, where fly balls are thrown and diving is encouraged! Team whiffle ball & tug-o-war on a grassy field makes for a great time too!
Book Online & use promo code "early summer" for $50.00 off before June 1!
This camp is specifically designed for ages 6-12. Pack a lunch and bring your baseball gear! Let's have fun and work on a number of baseball fundamentals, (hitting, pitching, stealing bases, and taking ground balls in the cage). We also enjoy taking the game outside with a run to the beach, where fly balls are thrown and diving is encouraged! Team whiffle ball & tug-o-war on a grassy field makes for a great time too! Aftercare is available for $15 per hour.
Please bring water, lunch, baseball gear and be ready for a ton of fun!
This camp is specifically designed for ages 12-17, with the focus being on strength training, conditioning, defensive reps, and swing analysis/analytics on the HitTrax.
The day starts with a work out at beautiful Crissy Beach West where the sand is used to develop proper movement patterns, speed, and agility. Exercises are continued at the batting cage where we focus on core and explosive strength.
Fieldwork and throwing marks the beginning of baseball practice, where we wrap up by batting on the HitTrax where the swing is recorded and sent home for review.
Please bring water, lunch, baseball gear, and a willingness to work hard!


text:
module BlockIo

  class Key

    def self.generate
      # returns a new key
      Bitcoin::Key.generate(Bitcoin::Key::TYPES[:compressed]) # compressed
    end

    def self.from_private_key_hex(priv_key_hex)
      # returns Bitcoin::Key (compressed)
      # quirky behavior from bitcoinrb 0.7.0: use IntegerOctetString.encode on private key (integer) first
      Bitcoin::Key.new(:priv_key => ECDSA::Format::IntegerOctetString.encode(priv_key_hex.to_i(16), 32).bth, :key_type => Bitcoin::Key::TYPES[:compressed])
    end

    def self.from_passphrase(passphrase)
      # ATTENTION: use BlockIo::Key.new to generate new private keys. Using passphrases is not recommended due to lack of / low entropy.
      # create a private/public key pair from a given passphrase
      # use a long, random passphrase. your security depends on the passphrase's entropy.

      raise Exception.new('Must provide passphrase at least 8 characters long.') if passphrase.nil? or passphrase.length < 8

      hashed_key = Helper.sha256([passphrase].pack('H*')) # must pass bytes to sha256

      # modding is for backward compatibility with legacy bitcoinjs
      BlockIo::Key.from_private_key_hex((hashed_key.to_i(16) % ECDSA::Group::Secp256k1.order).to_s(16))
    end

    def self.from_wif(wif)
      # returns a new key extracted from the Wallet Import Format provided

      Bitcoin::Key.from_wif(wif)

    end

  end

end


He enjoys spending most of his spare time with his family and he relishes all things computers. He enjoys bicycling and is always looking for events to participate in. Press here to learn more about Dr. Medina.
Kimberlee Harjula Peterson was born and raised in St George. She attended Georges Valley High School and then attended the University of Southern Maine obtaining an Associate of Science in Business Administration. After moving home, she began working in a local dental office. She loved the field of dentistry and decided to further her education by returning to school earning her degree in Dental Hygiene. She started with Dr. Medina after first getting her license and has recently returned.
Lanita Medina, Dr. Medina's wife, worked as an RN at the Infant Heart Transplant Center and Pediatrics at Loma Linda University Medical Center in California before moving to Maine. Her favorite job is being a mom to Kandice, Benjamin, Krista and Kami, and enjoyed the years she was able to homeschool them.
Pictures have always been an important part of her life, and as she made scrapbook albums for each of her kids, she realized how much meaning the photos and their stories were in the children's lives, helping to affirm them, pass on family values and provide great entertainment. Other people started asking how this was done and she became a Creative Memories Consultant and Personal Photo Organizer. She has been actively teaching others how to organize, preserve and enjoy their photos for 22 years. Through the years she has taught Photo organizing classes, held workshops and large Scrapbooking retreats. For more information on an upcoming retreat visit www.croptoberfest.com; You may want to see ideas on what you can do with your photos at www.mycmsite.com/lanitamedina.
I truly enjoy my job here as a dental hygienist; each day brings something new. I love working with children from their first tooth all the way into adult hood. I will help your child learn the skills necessary for good oral hygiene and work with your family to help keep those healthy habits going home.
Originally I am from Massachusetts but moved to Maine to pursue my education and dental hygiene license and decided to stay. I love living in Maine, even through the harsh winters and mud season! My husband Marty and I have settled into the Augusta area with our daughter Alice and our fur baby (cat), Wally. When I'm not in the dental office, I can be found outside walking with my family, at the beach or doing something crafty around the house.
I look forward to meeting with you and being part of your good experience here with us! Press here to learn about-about Laura.


The Information Commissioner has announced that The Home Office is to be monitored for three months over concerns that it takes too long to respond to Freedom of Information requests from the public.
The information commissioner said that he would be looking at responses to FOI requests received between 1 July and 30 September 2013. From my own experiences with The Home Office on What Do They Know, I am well aware of the problem, and have infact raised about 30 complaints to the ICO in relation to various Freedom of Information requests that I myself have filed to The Home Office.
It is, however, NOT just The Home Office that is to be monitored. The ICO's announcement also states that the period will also monitor South Tyneside Council and Sussex Police.


text:
<menu xmlns:android="http://schemas.android.com/apk/res/android"
    xmlns:app="http://schemas.android.com/apk/res-auto"
    xmlns:tools="http://schemas.android.com/tools"
    tools:context="com.gorbin.androidsocialnetworksextended.asne.MainActivity" >
    <item android:id="@+id/action_delete_friend"
        android:title="@string/delete_friend"
        app:showAsAction="always" />
</menu>


New study finds marijuana may reverse cognitive aging in mice.
It can calm you down, give you the munchies, make you giggle, and, according to a new study, marijuana can also reverse brain aging and improve cognitive functioning.
Researchers at the University of Bonn and Hebrew University published a study earlier this year in Nature Medicine that found that giving mice a regular, low dose of tetrahydrocannabinol (THC), one of the main active ingredients found in marijuana, can reverse age-related cognitive declines in the brain.
In the study, the team gave young, middle-aged and elderly mice a steady, small dose of THC. A control group received no THC. After a month, they tested the mice's ability to perform certain cognitive tasks, like navigating mazes.
The young mice in the control groups performed better at these cognitive tasks than the middle aged and elderly mice in the same group. They also performed better than the young mice who received the doses of THC. But the middle aged and elderly mice that received THC performed as well as the young mice in the control group.
Additional studies showed that administering THC boosted the number of connections between brain cells in the hippocampus, and altered the gene-expression pattern in the hippocampus. The hippocampus is involved in forming memories. "This is something we absolutely did not expect: the old animals [that received] THC looked most similar to the young untreated control mice," said Zimmer.
THC is one of many cannabinoids, a plant compound found in the Cannabis sativa plant. These compounds mimic our brain's endogenous cannabinoid system, a biochemical pathway in the human (and mouse) brain that can activate receptors in the brain that modulate neural activity. The system develops gradually during childhood, increases activity in adolescence, then steadily declines with age.


text:
Selected Poems David Harsent
A selection of David Harsent's work from across his career
paperback ebook
In an illustrious career, David Harsent has published eight collections of poetry, from A Violent County in 1969, to Legion, winner of the Forward Prize in 2005. This selection, made by the author himself, draws upon the full arc of his career and offers an outstanding concentration of, and introduction to, the full range and powers of this distinguished poet.
Harsent has an unusual claim to notice: you begin every poem with the certain prospect of pleasure and surprise.
Martin Amis, Observer
Few poets today can command such tension or such fluency of line.
Times Literary Supplement
DavidHarsent
David Harsent has published eleven collections of poetry. Night (2011) was a Poetry Book Society Choice, was shortlisted for the Costa, Forward and T. S. Eliot poetry prizes and won the Griffin International Poetry Prize. The most recent, Fire Songs, was published in 2014 and won the T. S. Eliot Prize.
More books by David Harsent
A sequence of intricately patterned verses that capture the lyrical, dream-like intensity of a mind at night.
Vivid, fragment-like poems from the major award-winning poet – now in paperback.
New series of vivid, distilled, fragment-like poems from the major award-winning poet – unlike any of his previous collections.
A reissue of award-winning poet David Harsent's acclaimed 1998 book, which describes a circular journey in a sequence of 25 poems.
Bird's Idea of Flight
Fire Songs is the new collection from award-winning poet David Harsent.
Fire Songs
Night is David Harsent's follow-up to his Forward Prize-winning collection Legion.
Legion by David Harsent is a distinguished successor to his previous collection, Marriage.
Marriage consists of two sequences of poems. The first is loosely based on the relationship between Pierre Bonnard and his muse…
The Early Poems and Juvenilia of Philip Larkin gives an unprecedented insight into the formation of one of the most remarkable…
Early Poems and Juvenilia
The first book for 16 years from a giant of the poetry stage.
The Noise of a Fly
Douglas Dunn
A selection of the best contemporary poetry published in the UK over 2021.
The Forward Book of Poetry 2022
Various Poets


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Jilamatomba chat rooms are awesome not only due to the reason that its packed up with open minded social people but also because those people are given a moderated platform to share their views, meet new strangers, participate in interesting discussions, make friends and find their dream partner for relationship.
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text:
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    <meta http-equiv="Content-Type" content="text/html;charset=utf-8" />
    <meta name="content-language" content="en-AU" />
    <title>{% block title %}Small DMS Test{% endblock %}</title>
    <link rel="stylesheet" type="text/css" href="/media/css/screen.css" media="screen" />
    {% block extra_head %}{% endblock %}

Django-DMS :: Small DMS app test
{% block content %}{% endblock content %}

{% block footer %}{% endblock %}

{% block final_javascript %}{% endblock %}


text:
Home » Last Call: The Age of Ageism
more... Gigging Advice • Pro Advice • How-Tos • April 2015 • Last Call
Last Call: The Age of Ageism
John Bohlinger
Despite their average age of 68, the Eagles are still filling arenas around the world.
By the time Arnold Schwarzenegger left office as governor of California, the state's coffers were empty. During his tenure, California resorted to issuing IOUs to cover their bills for the first time since the Great Depression. The Governator's successor was a spry, 71-year-old Jerry Brown who turned the Golden State around nearly immediately after taking office.
Now 76, Governor Moonbeam has proven to be one of the most effective public servants in recent American history, and yet the Democrats will not even consider him as a potential candidate for the presidency. Why? Because they say he is too old. Granted, the average life span in the U.S. hovers around 78 (that's behind 34 other countries, interestingly). But even if JB is pushing up daisies in four years, it only took him a few years to bring California from poverty to prosperity. Brown could possibly do the same for the U.S. but he will not be given the chance because of ageism, the last publicly accepted form of discrimination.
Sure, small-minded folks discriminate against others for all sorts of silly reasons: race, gender, sexual orientation, religious beliefs, affinity for wearing Speedos, use of solid-state amplifiers, etc. However, the general public usually recognizes discrimination as a bad thing. But that's not the case when it comes to age. Even the music industry—which in theory is driven by creativity and open-mindedness—continues to narrow what is considered a viable age for artists. Today, if you're a musician who hasn't released something by age 27, most record labels believe you've exceeded your expiration date.
Now before all of you aspiring, undiscovered, over-28 musicians get depressed, stop. First of all, music is its own reward. Second, labels control only a tiny piece of the music-money pie. Third, older guitarists rule rock 'n' roll—the musical category that really matters to most of us reading PG.
Today, if you're a musician who hasn't released something by age 27, most record labels believe you've exceeded your expiration date.
Although no labels want to roll the dice on signing anything but children, the world clearly is willing to pay big to see middle-aged and above musicians perform. In spite of increasingly poor album sales in all genres, touring took in $20 billion in 2014, making it perhaps the most lucrative year ever. Surprisingly, geezers were one of the main draws.
Let's look at the Billboard list of 15 top 2014 tours: The Rolling Stones came in at No. 3, with an average age of 71. The Eagles—each member is well past his mid-60s—is right behind at No. 4. In the No. 6 slot is 72-year-old Paul McCartney, and 65-year-old Billy Joel is No. 11. Springsteen, also 65 but looking lots younger than Joel, hit No. 13. Coming in at No. 15 was Elton John at an impish 67. One Direction, Justin Timberlake, Katy Perry, Bruno Mars, Beyoncé, Lady Gaga, Michael Bublé, and Luke Bryan round out the list. Which means that if you were on a top tour in 2014, you were a senior-citizen rocker, a pop act, or a thirtysomething country star who sounds like a rock act.
Okay, so apparently tours run on either glitter or prune juice, but what does all this mean? Blinded by their discriminatory view of acts over 27, dying record labels struggle to keep their doors open. Old rockers make solid dough on the road while putting out albums recognized by critics and ticket-buying fans as being superior. So what if ageism limits what's being signed? Rock was born out of rebellion and creativity, so why should it care about the discriminatory views of corporations? Good music gets heard by those who care enough to find it. Corporations and political parties enforce age discrimination, but people remain strangely loyal to musicians, no matter how saggy and wrinkly they get.
The trick is to connect with your audience. In an Off Camera with Sam Jones interview, Dave Grohl offers this advice to aspiring musicians on how to accomplish this: "Go play live. I don't understand the industry ... where music is headed, but I just know that when you walk into a club and you see a band that blows you away, you are going to follow that band ... buy their CD, find them online, see them the next time they come to play. Fuck product placement, labels, A&R people, and all that bullshit—it doesn't matter. If you back that up with the idea that playing your great songs in your great band is enough reward for you, then you're set. But you've got to be badass."
YouTube It
Watch Grohl's entire interview with Sam Jones:
John Bohlinger is a Nashville-based multi-instrumentalist best known for leading the band on NBC's Nashville Star, serving as music director for the CMT Awards and specials on PBS and GAC, and of course for his appearances in PG Rig Rundown, Review Demo, Axes & Artifacts, and What Bohlinger Plays videos.
Recent Articles by John Bohlinger
Last Call: Live, Without a Net
First Look: Aclam Dr. Robert
Rig Rundown: Lukas Nelson & Promise of the Real
Rig Rundown: Arielle
Last Call: Space Is Music, Too
Last Call: The One Rule of Showbiz and Why It's Good for You
Last Call: The Ballad of Mandolin Mike
Last Call: The Ups and Downs of Touring
Last Call: The Appeal of Dweedly-Dweedly
Last Call: The Journey of Frodo Douchebaggins


The Hayesbrook Year 8's and 9's played a tennis fixture last night (8th June) away to Bennett Memorial, with some encouraging results.
Luke Macro, Ben Barnett and Kieran Skinner all participated in the Year 10 fixture, so were playing a year up and were unfortunate to lose their singles and doubles matches.
We had positive results with the Year 8 fixture, however, as James Curry and George Denning both won their singles matches 6-2. Oli Smith, who was also playing a year up, won his match emphatically, with the final score 6-0.
George and David Crouch played a close doubles match, but came out losing 6-3 in a tight encounter. However James Curry and Oli Smith won their doubles match 6-2 to ensure Hayesbrook won the tie 8-4.
The boys have their last fixture of the season at home to Knole Academy next Wednesday.


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We encourage you to review our methodology for more information on how Smart Saver works.


text:
Bombing Probe Shines Spotlight On Amnesty Law
Legislation let suspect in New York City blast remain in United States illegally for 5 years
By Ron Scherer Staff writer of The Christian Science Monitor
MOHAMMED SALAMEH, a suspect in the World Trade Center explosion, entered the United States on a six-month tourist visa in 1988. Five years later, he was still here. The reason: Like many others, he had found a way to abuse the immigration system.
Over the past five years, Mr. Salameh has applied for immigration amnesty under a variety of programs he was never eligible for. Once he had applied, however, he was guaranteed work permits and amnesty until the Immigration and Naturalization Service (INS) could rule on his applications.
A similar technicality apparently allowed Sheikh Omar Abdul Rahman, a radical Muslim cleric who worshipped at the same mosque as Salameh, to remain in the US even after the government had determined that his application for asylum had been fraudulently obtained.
In Salameh's case, it took the INS nearly five years to decide he was not eligible for any of the programs he had applied for.
Salameh's initial foray into the US immigration system came only five months after he moved to the US. He applied for amnesty under a 1986 law that set up the Special Agricultural Worker (SAW) program. That program permitted illegal immigrants who worked in agriculture between 1984 and 1986 to apply for amnesty. Even though Salameh was not in the US until after 1986, he applied under SAW and was allowed to remain in the US until the case was resolved.
Salameh was not alone. Immigration lawyers report that people who entered the US as late as 1992 applied for amnesty under the 1986 law, formally known as the Immigration Reform and Control Act. There are still thousands of cases pending.
"Congress did not foresee the level of fraud would be unbelievably high," says Edwin Rubin, a Newark, N.J., lawyer and former president of the American Immigration Lawyers Association.
Sen. Alan Simpson (R) of Wyoming, a leading congressional expert on immigration, said in an interview that it is a "terribly abused program."
In New Jersey, for example, criminal rings sold falsified documents which stated the individual had worked on a farm. Senator Simpson says people claiming to be agricultural workers could not answer a simple question, such as, Where do watermelons grow? "They thought watermelons grow on trees," Simpson recounts.
While the government initially expected to get 350,000 amnesty applications, 1.3 million poured in. A report by the Commission on Agricultural Workers issued in early March found that, to date, over 1 million applications had been approved. But INS lawyers believe that at least 700,000 of those were fraudulent.
While the INS was trying to cope with the applications, a judge ruled that the INS rules on another program - which legalized workers who had been in the country before 1982 - were too restrictive. The INS was forced to reopen the application period. With the court-ordered extensions, Mr. Rubin says, "this program has created dozens of millionaires," as lawyers and shady operators found ways to sell false documentation or just collect filing fees.
Just before the INS denied Salameh amnesty status under SAW - three years after he applied - he applied for the other amnesty program. It took the INS another 15 months to deny his second application.
It's unclear how Salameh even obtained a tourist visa. He was only 19 years old, unmarried, and reportedly making $50 a month in Jordan. There is apparently no way to find out how he received his visa, since the records on visas are destroyed after one year. A US consular official says, "We don't automatically refuse entry because of a low income. Possibly he presented what looked like adequate income.... Possibly someone took a chance on him and blew it."
Normally, the details of an individual's immigration file are considered confidential. But the Newark (N.J.) Star Ledger reported the details of Salameh's case on March 10. The next day, the INS confirmed the newspaper's account.
The Star Ledger quoted anonymous INS officials as saying they were convinced Salameh's application was "a work of fiction but there was nothing we could do about it." That is because, under the SAW amnesty program, no one could be deported on the basis of his application unless criminal fraud was involved. And, according to INS lawyers, the Justice Department, which has the responsibility for prosecuting criminal fraud, decided to go after criminal rings, not individuals.
The only way the INS could have deported Salameh was to wait for him to break the law or to catch him during a random raid. It never did find him, and now he is charged in connection with the World Trade Center bombing, which killed five people and injured more than 1,000.
Duke Austin, an INS spokesman, defends his agency by noting that it annually inspects 21 million people entering the US on temporary visas. Half a million of them overstay their visas, he says. "Once they enter with a valid visa, no one tracks them as they move about the country. The US is an open society," he says.
Simpson says the problems are compounded by an efficient underground information network. "People know which programs to gimmick," he says.
How will Chile's President-elect Bachelet tackle immigration reform?
Immigration reform: What the last 'path to citizenship' did for immigrants
How immigrants may qualify to stay under Obama 'amnesty'


As winter looms, fall can be an ideal time to go plant shopping, even in our climate. The key is to choose healthy plants that look like they've been growing well all summer. It's a gamble to try to r.
Hamilton aquarium shop owner Cliff Frankis said the plant had been sold in stores "years ago". Tough biosecurity laws now prohibited its sale and distribution. salvinia and another invasive weed, w.
Plant all bulbs of a kind at the same depth when grouped together, so they will bloom at the same time and attain the same height. For planting clumps of hyacinth bulbs in beds and borders, dig a hole large enough to hold all the bulbs in one group or drift.
I am not sure whether it's gardeners anxious to extend the growing season – or those who market to gardeners – but paperwhite and amaryllis bulb kits are stacked for sale like fruitcakes. crocus an.
Inform the public to never dump aquarium contents into a natural water body; legislation to ban the sale of hyacinth as an aquarium or pond plant may reduce its.
Water hyacinth, any aquatic plant of the genus Eichhornia of the pickerelweed family (Pontederiaceae), consisting of about five species, native primarily to tropical America. Some species float in shallow water; others are rooted in muddy stream banks and lakeshores.
Dec 22, 2017. It's too late to purchase tulip and hyacinth bulbs from area nurseries. Here in Louisiana, we generally do not plant spring flowering bulbs as.
Prepared Hyacinths are specially treated to enable them to flower at Christmas. Our Prepared Hyacinths are not despatched until early September to ensure they have their full treatment and are at their best for Christmas flowering.
Hyacinth Bean 'Ruby Moon' is a fragrant vine that's perfect for covering trellises and fence lines, 'Ruby Moon' is a one of a kind beauty! Buy farm-fresh plants online!
wood hyacinth, spider lilies and more. A limited selection of special trees and shrubs will also be available for sale. Highlighting the conference will be featured speakers Keith Kridler and Greg Gra.
Plant the largest bulbs at their proper depth, cover them with soil and then plant the next largest at a shallower level (e.g. plant lilies at 25 cm and tulips at 15 cm). Small species and cultivars s.
CLEAR LAKE — Central Gardens of North Iowa's 14th annual perennial plant sale and Gardening. Lawn and Entrance Plaza. The sale features hundreds of winter-hardy perennials, including iris, bleeding.
Now's the time to plant if you want to see a beautiful show of flowers next. comprising sapphire shades of anemone, allium, iris, crocus, hyacinth, chionodoxa and muscari. 73 flower bulbs. 10 Alliu.
Fall-Planted flower bulbs are the surest signs that spring has arrived, with some bulbs even blooming when there's still snow on the ground. If you want to herald spring with a burst of color, you need to plan ahead because these bulbs must be planted in fall.
The name "hyacinth" itself can be traced back to remote antiquity, a relict of a. which possessed a blue flower, the plant later developing bud variations of white.
Purple Hyacinth Bean is often grown as a flowering plant, although most of the plant is edible. With shades of purple in the leaves, stems, flowers and showy seed pods, it is undeniably eye-catching. Purple hyacinth bean is a very vigorous grower and the vines are a popular choice for a quick cover.
Learn how to plant, grow, and care for hyacinth bulbs with our planting guide. Be sure to visit our shop and browse our hyacinth bulb collection!
In Florida, the Florida Noxious Weed List identifies plants that require a permit to possess and that are restricted for sale or commercial use. plant rule covering other species such as water hyac.
Buy Hyacinth bulbs: Delivery by Waitrose Garden in association with Crocus.
Plant hyacinth bulbs in fall in a sunny location. Showy and fragrant flowers emerge in mid-spring and come in an assortment of colors. Colors often seen are purple hyacinth flowers, pink hyacinth flowers, yellow hyacinth flowers, white hyacinth flowers, peach hyacinth flowers and even true blue hyacinth.
Water-hyacinth is a floating plant that has clusters of leaves with spongy stalks arising from a base of dark purple feathery roots. The leaf clusters are often linked.
The recent ban on the sale and distribution of a popular water garden plant in Arkansas is an example of trying to strike an appropriate balance. The plant in question is water hyacinth (Eichhornia crassipes), a floating perennial herb that has become a serious.
Other invaders, such as the water hyacinth, were brought into the country purely to decorate fishponds. Now this floating water plant reproduces wildly. inevitably reopen with new exotic animals fo.
WARNING: Water hyacinth is a highly invasive species in areas with mild winters.The plants are banned in several states. Once they enter waterways, the plants grow and reproduce to form dense mats that choke out native species.
The gel is 100 percent organic and made from renewable energy sources – water hyacinth, sawdust. modify enzymes that have the potential to break down cellulose in plants or biomass," says Oye. "Fos.
Floating pond plants will spread across the waters' surface providing shade and coverage for the water gardens inhabitants. Their roots absorb nutrients from the water that might otherwise cause algae and provide hiding places for baby fish. Water Hyacinth and Water lettuce grow best in.
A real breakthrough in breeding, this rare and unusual Hyacinth makes a dramatic specimen plant for your spring beds, borders and containers; producing highly fragrant black-purple blooms. Plant Hyacinth 'Midnight Mystic'® in groups for a truly breathtaking display.
Gardeners looking forward to tulip, hyacinth and daffodil blooms next spring need to set. Dormant bulbs are lifted from the field in the summer, packaged for sale in the autumn and are available fo.
The best quality Hyacinth bulbs for sale!. Plant all flower bulbs in well-drained garden soil to prevent rot. Buy Hyacinth bulbs online in our online webshop.
In addition to developers on the hyacinth waiting list, other builders who recently reserved sewer service through a pre-sale of future capacity also were allowed to buy service right away instead of.
The grocery store had large racks of potted hyacinth bulbs that will bloom inside this winter. A bulb vendor had unusual amaryllis bulbs for sale and I left with two. I will pot them up soon and en.
This popular floating pond plant has beautiful lavender-blue flowers, with fleshy, dark green leaves. Water Hyacinths spread across the water garden providing coverage and.
Muscari (Muscari ameniacum), sometimes called by the old-fashioned name "grape hyacinth," blooms indoors if you first. You can find muscari bulbs for sale in fall, which is perfect timing if you wa.
I plant Clover in. Moss Phlox, Grape Hyacinth, Clover, Chives, and Dandelions. Baby birds are hatching about the same time that the butterfly larvae are coming out. Both are hungry. Some magazines.
I attended a presentation by Gabriela Chavarria, a world authority on bees, at the Plant Select annual meeting at the Denver. Mid-spring bloomers like grape hyacinth, fritillaria and Indian hyacint.


Jurassic Apartments - Jurassic Apartments Holiday Home sleeps 6 people, and is 1 mile from Weymouth.
The Haybarn sleeps 12 people, and is 34 miles from Weymouth.
Amberley View sleeps 4 people, and is 19 miles from Weymouth.
Broad Lodge sleeps 6 people, and is 19 miles from Weymouth.
Downwood Vineyard sleeps 16 people, and is 26 miles from Weymouth.
New Inn Farmhouse sleeps 12 people, and is 32 miles from Weymouth.
Leam House sleeps 8 people, and is 7 miles from Weymouth.
Armswell House sleeps 18 people, and is 19 miles from Weymouth.
Bedborough Farm Cottages - The Old Dairy sleeps 5 people, and is 34 miles from Weymouth.
Jurassic Apartments - Martleaves House sleeps 12 people, and is 1 mile from Weymouth.
Jurassic Apartments - Jurassic Apartments Bungalow sleeps 4 people, and is 1 mile from Weymouth.
Bedborough Farm Cottages - The Parlour sleeps 5 people, and is 34 miles from Weymouth.