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The formation of vesicles is essential for many biological processes, in particular for the trafficking of membrane proteins within cells. The Endosomal Sorting Complex Required for Transport (ESCRT) directs membrane budding away from the cytosol. Unlike other vesicle formation pathways, the ESCRT-mediated budding occurs without a protein coat. Here, we propose a minimal model of ESCRT-induced vesicle budding. Our model is based on recent experimental observations from direct fluorescence microscopy imaging that show ESCRT proteins colocalized only in the neck region of membrane buds. The model, cast in the framework of membrane elasticity theory, reproduces the experimentally observed vesicle morphologies with physically meaningful parameters. In this parameter range, the minimum energy configurations of the membrane are coatless buds with ESCRTs localized in the bud neck, consistent with experiment. The minimum energy configurations agree with those seen in the fluorescence images, with respect to both bud shapes and ESCRT protein localization. On the basis of our model, we identify distinct mechanistic pathways for the ESCRT-mediated budding process. The bud size is determined by membrane material parameters, explaining the narrow yet different bud size distributions in vitro and in vivo. Our membrane elasticity model thus sheds light on the energetics and possible mechanisms of ESCRT-induced membrane budding.
Author Summary
Lipid membranes enclose the cytosol of biological cells and compartmentalize their interior. Vesicles are used to transport membrane proteins between cellular compartments. The ESCRT protein machinery induces the creation of such vesicles away from the cytosol. The resulting vesicles are uncoated by protein. Upon vesicle scission and release into the endosome, the ESCRT proteins are recycled into the cytosol. We develop a membrane-elasticity model that captures this budding process. The model reproduces the vesicle morphologies observed in fluorescence microscopy images, and identifies the energetic driving force of vesiculation. We also characterize possible mechanisms of ESCRT-induced membrane budding. The size of the resulting vesicles is determined by membrane material parameters, explaining the narrow yet different bud size distributions in vitro and in vivo. Our membrane elasticity model thus provides insight into the energetics and mechanisms of uncoated vesicle formation.
Citation: Różycki B, Boura E, Hurley JH, Hummer G (2012) Membrane-Elasticity Model of Coatless Vesicle Budding Induced by ESCRT Complexes. PLoS Comput Biol 8(10): e1002736. https://doi.org/10.1371/journal.pcbi.1002736
Editor: Vijay S. Pande, Stanford University, United States of America
Received: March 20, 2012; Accepted: August 23, 2012; Published: October 18, 2012
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This research was supported by a Marie Curie International Outgoing Fellowship within the 7th European Community Framework Programme (B.R.), the Intramural AIDS Targeted Antiviral Program (J.H.H.), and the Intramural Research Program of the NIH, NIDDK (B.R., E.B., J.H.H., G.H.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Lipid membranes enclose the cytosol of biological cells and compartmentalize their interior. The structure and contents of cellular membranes are actively controlled to sustain the vital functions of the cell. Transport vesicles are used to traffic membrane-bound proteins between cellular compartments. The best-characterized pathways of vesicle formation include those facilitated by BAR domain proteins [1] and by the coat protein clathrin with its adaptors [2]. Coat proteins impose their curved shape onto the membrane, thereby promoting vesicle curvature in an intuitively straightforward and computationally well-characterized process [3]–[5]. In the degradative transport of membrane proteins from endosomes to lysosomes (Fig. 1), small patches of the endosomal membrane bud into the interior (lumen) of the endosome and detach, forming intralumenal vesicles (ILVs) [6], [7]. This pathway is catalyzed by the cytosolic Endosomal Sorting Complex Required for Transport (ESCRT) [8]–[10]. The ESCRT proteins are not internalized in ILVs, but rather recycled continuously in the cytosol [11], [12]. To avoid the consumption of ESCRT proteins within the ILVs, these vesicles contain no protein coat to template their shape. Rather, these vesicles are initially formed as buds whose necks are stabilized by an assembly of ESCRT-I and -II [13]. As the assembly matures with the incorporation of ESCRT-III, the bud neck is cleaved from the cytosolic side, leaving ESCRTs in the cytosol and the detached spherical ILVs in the lumen of the endosome. Because this process involves no protein coat, the shape and energy of the mature buds must be governed primarily by membrane mechanical properties.
The ESCRT proteins assemble on the membrane side opposite the vesicle bud (C) and do not enter the ILV lumen for possible recycling (D). Energy input from ATP hydrolysis accelerates disassembly of the ESCRT machinery (D), but is not required in steps (A–C).
Until recently, the membrane remodeling functions of ESCRT were attributed only to ESCRT-III proteins, which assemble on liposomes in vitro [14], induce the ILV formation in giant unilamellar vesicles (GUVs) [12] and cause the plasma membrane to bud and tubulate when overexpressed in cells [15]. Based on these experimental observations, membrane deformations induced by ESCRT-III have been modeled [12] and studied theoretically [16]. An unresolved problem of these models was their inability to explain how the ESCRT-III proteins can be recycled from self-induced buds or tubes.
Recent in vitro experiments showed, however, that ESCRT-I and -II together are responsible for vesicle budding [13]. In these experiments, vesicle budding could be induced at physiological concentrations (15 nM) of ESCRT-I and -II. Fluorescence microscopy showed ESCRT-I and -II to be colocalized in the neck region of the buds (Fig. 1C), where they were shown to recruit ESCRT-III. The latter proteins then induced membrane scission [12]. Importantly, the ESCRT proteins were not found in the bud lumen. In this way, the ESCRT machinery that facilitates membrane budding and scission is not consumed in the process of ILV formation (Fig. 1D). Moreover, with ESCRT binding restricted to the neck region, the principal part of vesicle buds in vitro is thus bare lipid membrane.
Fluorescence microscopy [13] also showed that membrane-bound ESCRT proteins form microdomains on vesicle membranes (Fig. 1B and 1C). The lipid composition of these domains likely differs from that in the ESCRT-free membrane portions because ESCRTs bind specifically to certain charged lipids (PI3P) and could employ raft-favoring lipids and cholesterol to facilitate membrane budding [17], [18].
These two experimental observations of (i) the formation of ESCRT microdomains on the membrane and (ii) the formation of coatless buds (with ESCRT proteins localized in the bud neck) jointly form the basis for a phenomenological model of ESCRT-induced budding. Observation (i) provides us with the starting point for the budding process and motivates a mechanism that can provide the large energy necessary for membrane deformation. Observation (ii) determines the end point of the budding pathway. In our model, we assume that the ESCRT-I-II supercomplexes have an enhanced affinity for binding to saddle-shaped membrane regions, and that a line tension acts on the outer boundary of an ESCRT-sequestered membrane domain. We cast our model in the framework of membrane elasticity theory. A bending elastic model of lipid bilayers was previously used to study the energetics of a possible mechanism of ESCRT-III induced fission of nascent vesicles [19]. Here we employ a similar approach to study the ESCRT-I-II induced formation of vesicle buds.
The analytical solutions of our model allow us to map out different membrane morphologies over a range of possible physical parameters. We identify a regime of membrane bending parameters and line tensions for which the minimum energy configurations are coatless membrane buds with ESCRTs localized in the bud neck. These minimum energy configurations closely resemble the fluorescence images observed in experiment [13]. Within our model, we also identify energetically and kinetically feasible budding pathways, and propose a three-stage mechanism of ESCRT-driven budding: (i) membrane-bound ESCRT-I-II complexes form clusters, or domains, and induce a line tension on the domain boundaries through local segregation of lipids; (ii) as the domain boundary energy exceeds a threshold level, the membrane patch sequestered by the ESCRT assemblies buckles and forms a bud; (iii) the ESCRT-I-II complexes scaffold the bud neck and thus stabilize a neck diameter optimized for ESCRT-III protein binding and bud scission. To validate the model, we compare its predictions for bud shapes, sizes, and formation kinetics to experiment [13], [20]. We also relate our model to recent experiments probing ESCRT-induced lipid segregation in membranes [21].
In the framework of membrane elasticity theory, the energy of membrane deformations consists of the mean curvature term(1)and the Gaussian curvature term(2)where and are the mean and Gaussian bending rigidity moduli, respectively; and are the principal local curvatures; is the spontaneous curvature; and the integrals are performed over the membrane surface [22]. For uniform membranes with fixed topology, the energy term does not depend on membrane shape. Consistent with in vitro experiments [13], we will consider symmetric lipid bilayers with no spontaneous curvature, .
Eq. (1) implies that the energetic cost of forming a vesicle bud [23]–[27] is approximately , which exceeds for typical membrane bending rigidities . This large energetic cost effectively eliminates random thermal fluctuations as a main factor. Instead, the energetic cost of the nascent vesicle bud has to be offset by the molecular machinery that drives the budding process. In ESCRT-induced budding, the source of this offset energy is provided by the binding of the ESCRT proteins to the membrane. But unlike BAR domains or clathrin proteins, the ESCRT machinery does not seem to use the scaffold mechanism as the primary mode of action. Indeed, the scaffold mechanism cannot easily account for the significantly different bud radii in vitro (m [13]) and in vivo ( [20]), and appears to be contradicted by the experimental observation that the ESCRT proteins colocalize in the neck of buds.
How does the ESCRT machinery deform a flat membrane patch into a bud [28]? There are two relevant observations that help answer this question. Firstly, there is increasing evidence that the insertion of amphipathic, cationic peptide segments into the lipid bilayer can induce negative Gaussian curvature [29]–[31], which is a characteristic of the bud necks where the ESCRT proteins localize. The Vps37 subunit of ESCRT-I has such a cationic helix at its N-terminus [32], as does the Vps22 subunit of ESCRT-II [33]. It is therefore possible that the ESCRT-I-II proteins generate negative Gaussian curvature by inserting amphipathic and cationic segments into the membrane. In the lowest order of approximation, the resulting curvature-dependent binding energy,(3)is proportional to the membrane Gaussian curvature integrated over an ESCRT-rich membrane patch , where is a coupling constant. Remarkably, we also arrive at an energy term that is formally equivalent to Eq. (3) on an entirely different route. If we assume that by binding to the membrane, the ESCRT-I-II proteins locally perturb its Gaussian bending modulus according to Eq. (2), then the parameter can be understood as the Gaussian bending modulus contrast . The energy term in Eq. (3) thus accounts for both the preferential binding to saddle-shaped features of the membrane and for changes in the Gaussian bending modulus in response to binding, with different interpretations of the constant .
The second relevant observation is that late endosomes contain raft-like domains that are rich in cholesterol and sphingomyelin [34]–[36]. The in vitro experiments [13] were performed on vesicles containing cholesterol, raft-favoring lipids and PI3P, which is consistent with the possibility that ESCRTs could use lipid rafts or domains to promote membrane budding [17], [18]. The ESCRT proteins could bind to pre-existing rafts or induce lipid domain formation. In fact, proteins with multiple lipid binding sites can induce lipid segregation in membranes close to the demixing point [37]–[40]. Lipid segregation has been suggested as a factor driving scission in endocytosis [41]. The enhancement of lipid segregation upon protein binding is primarily an entropic effect. Lipid segregation has to overcome an entropic penalty to move away from a perfectly mixed state. This penalty is reduced by concentrating certain lipids through preferential binding because the entropic cost of rearranging the resulting clusters of protein-bound lipids is smaller than that of rearranging individual lipids.
We consider a single lipid-segregated domain . A line tension acts on the boundary of this domain, where the corresponding energy is proportional to the length of the boundary line(4)To quantify the preferential binding of ESCRTs to lipid rafts or domains, we introduce an additional energy term in the spirit of mean field theory(5)Here, is an effective ESCRT binding free energy per unit area, and denotes the intersection of the lipid-segregated domain and the ESCRT-rich domain on the membrane, where we assume that the latter is a subset of the former, . In the following, we assume the shapes of and to be circular and ring-shaped, respectively.
As we show below, a minimal model of ESCRT proteins inducing or enhancing lipid segregation can explain central experimental results, in particular the narrow yet different bud-size distributions in vivo and in vitro. Moreover, the line tension on the boundaries of the lipid-segregated membrane domains [42]–[44] can also provide the large energetic driving force for vesicle budding [45]–[48].
Our model is defined by the energy functional(6)which combines the energy terms in Eqs. (1), (3), (4), and (5). The four corresponding parameters , , , and depend on membrane lipid composition, on external conditions such as temperature, and on ESCRT molecular properties, which are not entirely understood yet. Therefore, the model parameters can take different values depending on the molecular details of the ESCRT-membrane system. We thus use the spherical cap approximation [45], [49] to derive analytical solutions that allow us to map out the entire parameter space of the model. We then minimize the energy functional Eq. (6) with respect to the membrane shape and the ESCRT cluster location, and investigate the membrane states, or morphologies, that are stable in different parameter regimes. Interestingly, we find a range of physically meaningful parameters in which the minimum energy states closely resemble the fluorescence images in Ref. [13]. We also investigate possible budding pathways that lead without activation barriers from a flat membrane state to the coatless membrane bud with ESCRTs localized in the neck.
In the following, we first develop an approximate analytic treatment that accurately captures the energetics. We then report the results of numerical calculations that allow us to test the analytical solutions and to provide detailed information about the bud shapes. Finally, we analyze possible budding pathways.
Energetics and morphology diagrams
To make our model analytically tractable, we applied the spherical cap approximation, which is known to capture the energetics of unassisted membrane budding [45], [49]. In the spherical cap approximation, the energy functional Eq. (6) becomes a simple function whose minima can be found analytically (see Methods). The phase diagram that results from the energy minimization for displays three regimes which are shown in Fig. 2. For small line tensions (left side of Fig. 2), the membrane resists bending and remains flat. The lipid-segregated domain and the ESCRT-rich domain colocalize entirely. For large line tensions and large binding energies (top right corner of Fig. 2), the membrane buckles and forms an ESCRT-covered bud. Again and colocalize. For large and small (bottom right corner of Fig. 2), the minimum energy configurations are coatless buds with ESCRTs localized in the bud neck.
Results are shown for a Gaussian bending modulus fixed at . The green squares represent the flat membrane state. The magenta triangles correspond to ESCRT-coated membrane buds. The orange circles represent bare lipid buds with ESCRTs localized in the bud neck. The black solid lines are the coexistence lines, and the black dotted line represents the spinodal at which the energetic barrier to bud formation vanishes. Exemplary membrane shapes are shown for indicated points. The red and blue lines represent lipid domains and ESCRT-rich domains, respectively.
Membrane shape
To test the spherical cap approximation and to calculate bud shapes, we minimized the energy functional Eq. (6) numerically with respect to the membrane shape and the location of the ESCRT cluster (see Methods). To distinguish different morphologies of the minimum energy configurations, we introduced the area fraction , where and denote the areas of the lipid segregated domain and the ESCRT-rich domain , respectively. We grouped all the minimum energy configurations found numerically into three distinct categories: (i) flat membrane domains with zero bending energy and area fraction , (ii) ESCRT-coated buds with bending energy and area fraction , and (iii) coatless buds with , small area fraction , and the ESCRT cluster located primarily in the bud neck region. The resulting morphology diagram agrees very well with the diagram based on the spherical cap approximation (Fig. 2).
The spherical cap model predicts discontinuous transitions between the different morphological phases, with a kink in the minimum energy at the transition lines. In the complete model, however, the kink is rounded at the transition line due to the finite-size of the bud neck (Fig. 3A).
(A) Minimum energy rescaled by the membrane bending rigidity as a function of the ESCRT binding energy rescaled by the area of the lipid-segregated domain. (B) Minimum energy as a function of the cosine of the membrane tangent angle along the lines of longitude at the boundary of the ESCRT-coated domain. (C) as a function of the binding energy rescaled by the area of the lipid-segregated domain . (D) as a function of the area fraction , where is the area of the ESCRT-rich domain .
To additionally quantify the bud morphologies, we determined the tangent angle (see Methods) along the longitude at the boundary of the ESCRT-coated domain. The angle as well as the area fraction exhibit an abrupt change at the transition line , as shown in Fig. 3, which further justifies the distinction between ESCRT-coated buds and uncoated buds.
It is instructive to have a closer look at the membrane buds shown in the bottom right corner of Fig. 2. The principal part of these buds is coatless membrane, and the ESCRT cluster is localized only in the bud neck. These coatless buds assume the shape of a sphere. The geometry of the bud neck follows that of a catenoid, a minimal surface with no mean curvature, , and thus zero bending energy [24]–[27]. As a result, the bending energy of the buds approaches the bending energy of a spherical vesicle, . We note that our calculated bud shapes, as shown in the top right corner of Fig. 2, resemble the tomographic images of early endosomes [20] and the fluorescence images of GUV buds observed in vitro [13].
Budding mechanisms
Within the framework of the spherical cap approximation, we also analyzed possible budding pathways. We focused in particular on activation-less pathways along which the buds grow without having to cross significant energy barriers (see Methods). This analysis revealed a three-stage mechanism of ESCRT-mediated membrane budding. In stage (i) the ESCRT-rich domain and the lipid-segregated domain colocalize entirely on the flat membrane if . As the domain radius increases beyond , the energy of the flat domain becomes larger than the energy of an ESCRT-covered bud. As the domain radius increases to , the barrier between these membrane states vanishes entirely and the system enters into stage (ii) in which the flat membrane domain buckles and forms an ESCRT-covered bud. The resulting bud has a radius(7)which, interestingly, is determined by membrane material parameters. In stage (iii) the ESCRTs coalesce into the bud neck with no energy barriers if . The third stage is driven by the energy of the ESCRT preferential binding to membrane regions with negative Gaussian curvature. Importantly, in stage (iii), the bud is cleared of ESCRTs for possible recycling.
If , the mechanism of ESCRT-mediated membrane budding is different. The flat membrane with ESCRTs bound is then unstable and the system evolves spontaneously to a transient state, in which ESCRT-free buds with radius are formed. The mini-buds, induced by a strong binding preference to saddle-shaped membrane structures, are next squeezed bigger by line tension if the radius of the ESCRT-coated membrane patch is smaller than . The latter condition puts an upper bound on the ESCRT-free bud radius . Interestingly, on this budding pathway the ESCRTs never coat the bud. We note, however, that this pathway is somewhat problematic since the linear approximation in Eq. (3) may not be applicable for large parameters . In addition, effects of finite membrane thickness cannot be ignored in this regime.
The detailed physical mechanisms used by the ESCRT machinery to control membrane budding are currently unknown. This lack of knowledge has motivated us to consider a simple, phenomenological model for ESCRT-induced membrane budding. Our model is based on well-established physical principles to ensure robustness. Following earlier studies of membrane tubulation [16] and fission [19] driven by ESCRT-III proteins, we have cast our model in the framework of membrane elasticity theory. The key assumption in our model is that ESCRTs induce or enhance the formation of raft-like domains in lipid membranes. Indeed, recent total internal reflection fluorescence (TIRF) images show that the ESCRT-II proteins induce lipid phase separation in model membranes with endosome-like composition [21]. These direct experimental observations thus support a central element of our theoretical model. However, to test and further quantitate our model will require additional studies of ESCRT-membrane interactions. In particular, line tension measurements would provide critical input in building a fully quantitative description of the budding process. In addition, studies of the different components of the ESCRT system, including modifications such as Vps20 myristoylation, should provide further guidance at which stage and to what extent lipid phase separation is induced. The TIRF experiments [21] indicate interesting variations, with the yeast ESCRT-II protein complex forming somewhat smaller and more numerous clusters than the human ESCRT-II protein complex under the same conditions. Ultimately, it will be important to determine if membrane curvature is induced in a way consistent with our theoretical analysis. Such measurements would provide critical tests to what extent our model captures ESCRT-induced budding, and would help in its quantitation and refinement.
The energetic analysis of our model suggests a three-stage mechanism of vesicle budding induced by the ESCRT proteins. In the first stage, the ESCRT-I-II binding induces lipid phase segregation, consistent with the TIRF experiments [21]. We speculate that in this stage, ESCRT-I-II complexes loosely assemble on the membrane to form clusters or domains. The ESCRT-membrane interactions enhance lipid segregation in the membrane and induce a line tension on the domain boundaries. Once the lipid-segregated membrane domain exceeds a critical size, as determined by membrane material parameters according to our model, the process enters the second stage. Beyond a size threshold set by the ratio , the energy barrier to membrane shape deformation disappears, and the membrane patch sequestered by ESCRTs buckles and forms a bud. In the third stage, according to our energy function, the ESCRT-I-II complexes concentrate in the neck of the newly formed bud. This final step is driven the ESCRT binding energy to the membrane portions with negative Gaussian curvature. While our model lacks molecular detail of this curvature-dependent binding, recent experiments [29]–[31] suggest possible mechanisms. In particular, it could be caused by the insertion of amphipathic and cationic segments of the membrane-bound ESCRT proteins into the membrane, or by the formation of an assembly whose shape is complementary to that of the bud neck [50]. The formation of neck-coating assemblies by the ESCRT-I-II complexes not only would help recruit ESCRT-III proteins for scission, but could also clear the ESCRT-I-II proteins from the bud lumen and set them up for recycling.
The third stage of the budding process described above is dynamically feasible within the overall time scale of the process. The diffusion coefficient of membrane proteins is of order [51]. Provided a gradient to the bud neck region, the process of clearing in vitro buds with radius [13] would be completed within . The clearing process would be two orders of magnitude faster for the in vivo buds with radius [20].
One immediate consequence of the three-stage mechanism is that the complete buds have a radius that is determined by membrane material parameters, namely membrane rigidity and line tension . Therefore, the bud radius attains a particular value for a vesicle at given conditions, which explains the narrow distribution of bud sizes observed both in vitro [20] and in vivo [13]. We note, however, that both the line tension and the bending rigidity may be affected by details of the ESCRT-membrane interactions, which could explain small ILV size variations in response to mutations [52].
Despite the 40-times smaller radius , the in vivo buds have the same bending energies and shapes as the buds observed in vitro. This conclusion is a direct consequence of the scale invariance of the membrane bending energy Eq. (1). However, the line tensions necessary to induce vesicle budding, , are different. For in vivo buds with radius and bending rigidity [53], we estimate ; for in vitro buds with , we get assuming the same membrane rigidity . Line tensions in this range have been measured in GUVs [42], [47].
We note that the bending rigidity of early endosomes [53] is an order of magnitude lower than the bending rigidity of lipid membranes in the liquid-ordered phase [47], [54]. This difference in mechanical properties may be caused by variations in the composition and by the presence and activity of proteins in the membrane of endosomes. The membrane-sculpting protein Sar1 of the COPII complex, for example, has been shown to lower the membrane rigidity below [55]. The rigidity of fluid membranes has also been shown to decrease gradually with increasing concentration of the HIV-1 fusion peptide in the bilayer [56].
The three-stage budding process discussed above occurs if the Gaussian bending modulus contrast is limited to a window . This relation puts an upper bound on the binding energy . For typical bending rigidities and line tensions , the required energy is of order per or smaller.
If the affinity of ESCRTs to regions with negative Gaussian curvature is high, , our model predicts a different budding pathway. In this case, flat ESCRT-coated membrane domains are unstable, and small ESCRT-free buds form spontaneously. The mini-buds induced by a strong binding preference to saddle-like membrane shapes can next be squeezed bigger by line tension . The latter transition occurs if the radius of the ESCRT-coated domain does not exceed . The resulting ESCRT-free buds have a radius . For and as above, we get the bud radius smaller than approximately , which is consistent with the size of late endosome buds, [20].
In contrast to the three-stage mechanism, in the pathway starting with mini-buds the ESCRTs never enter the bud lumen. It is thus tempting to picture this alternative budding route as follows: ESCRT-0 and -I bind to the membrane with and the membrane remains flat. When ESCRT-II proteins bind to ESCRT-I and to the membrane, becomes larger than and the ESCRT-free membrane buds are formed spontaneously. The condition for forming a finite-size, ESCRT-free bud is that the radius of the ESCRT patch at the ESCRT-II arrival can not exceed . We notice, however, that this pathway might be an artifact of our model since the linear approximation in Eq. (3) could be inapplicable for large . Nevertheless, we cannot entirely exclude this alternative pathway.
Overall, we favor the three-stage mechanism as a possible route for ESCRT-induced budding. It concisely explains the uniform bud sizes seen in experiment, and makes quantitative and testable predictions for their dependence on physical parameters. In particular, according to Eq. (7) the vesicle bud radius should increase linearly as a function of the ratio . Since the line tension depends sensitively on temperature [42], it should be possible to test this theoretical prediction in future experiments on GUVs.
A key element of the model is the Gaussian bending energy . Because of experimental difficulties, our understanding of Gaussian curvature effects is rather limited. However, factors affecting the Gaussian bending modulus [57], [58] or enhancing the binding to membranes with negative Gaussian curvature [29]–[31] have been established. As detailed structural models of the full-length ESCRT protein supercomplexes emerge [50], [59], it will be important to identify the interactions responsible for the preferential association of the ESCRT proteins to saddle-shaped bud-neck membrane regions. It may also be possible to study binding of ESCRT proteins or protein fragments to model systems with negatively curved membranes [29]–[31]. In this way, one could gain a microscopic quantification of the parameter , putting it on similar footing as the other membrane-bending terms.
We consider only axisymmetric vesicle buds as the lowest energy shape by symmetry. Their surface is described in terms of two parameters, the angle of rotation around the axis of symmetry, and the arc length along longitudes. The vesicle bud surface then has Cartesian coordinates , and , where and and is the distance from the -axis, which is the axis of symmetry of the bud. One can introduce a tangent angle such that and . In this parametrization, the principal local curvatures are and [60], [61]. Therefore, in this parametrization, Eq. (1) reads(8)The lower limit of integration, , corresponds to the "south pole" of the vesicle bud at which . With these boundary conditions we have and . The upper limit of integration in Eq. (8), , corresponds to the bud rim, where and to connect the bud smoothly to a flat membrane at a distance from the axis.
The boundaries of the ESCRT-occupied membrane patch are located at and with . Eq. (3) takes the following form in the arc-length parametrization,(9)which simplifies to(10)In this parameterization, the energy terms in Eqs. (4) and (5) become(11)and(12)The total energy is .
Numerical calculations
It is convenient to use dimensionless variables in numerical calculations. Here we introduce(13)and with . With these dimensionless variables, the boundaries of the ESCRT-occupied membrane patch are described by parameters and , where . The energy functional(14)depends then on the bud shape, which is described by(15)on the domain location as given by and ; and on three dimensionless parameters(16)(17)and(18)In deriving the equations above we used a relation between the contour length and the membrane area , namely(19)The membrane energy (14) is minimized numerically with respect to the bud shape and the domain boundaries and using a simulated annealing method. To this end, the function is assumed to be smooth at and approximated by a Fourier series(20)that fulfills the boundary conditions and . Here, is the number of Fourier amplitudes . In simulated annealing Monte Carlo moves, the amplitudes and the boundary positions and are varied. We performed the numerical calculations with up to Fourier modes as in [62], [63]. For any set of parameters , , and , numerical calculations were repeated twice with different initial configurations to ensure convergence to the global minimum.
Spherical cap approximation
To make our model analytically tractable, we approximate the nascent membrane bud by a spherical cap of radius with a budding angle . A convenient budding coordinate is . We also define the area ratio where and denote the areas of the lipid-segregated domain and the ESCRT-rich domain , respectively. The domain of the two dimensionless parameters and is the unit square. In the following, we write the energy of the bud in terms of and , considering two cases:
Case I: The whole lipid-segregated domain forms a spherical cap and thus the membrane surface parametrization is given by for with . Then and the energy functional Eq. (6) becomes a two-variable function(21)
Case II: Only the ESCRT-free membrane patch forms a bud and therefore the membrane surface parametrization is given by for and for with . Then and(22)
In both cases I and II, the determinant of the Hessian matrix is always negative within the unit square , , which implies that the local minima of can be found only at the square boundaries. A simple analysis of Eqs. (21) and (22) shows that the local minima of the energy function are located only at the corners of the unit square in the − plane. First, . Second, , and . Third, . We compare these energies to find the global minimum of the energy function and, in this way, construct the phase diagram in Fig. 2.
Point corresponds to the flat membrane state, which is obviously the starting point of the budding process. Point on energy surface corresponds to an ESCRT-coated bud, whereas point on energy surface corresponds to coatless buds with radius . Point corresponds to a coatless membrane bud with finite radius and ESCRTs localized in the neck. Fluorescence microscopy shows that this state is the end point of the ESCRT-induced budding.
The transition from the initial, flat membrane state at point to the end point at occurs spontaneously with no energy barriers if either (i) and , or (ii) and . In case (i), the transition occurs on energy surface . In case (ii), the system evolves on energy surface . In both cases (i) and (ii) the transitions occur from point to along the line and next from point to along the line. We checked that for some parameter combinations, a saddle point can emerge within the unit square, separating local minima at and . However, if the saddle point is in the unit square, it is higher than . We also checked that for positive parameters and , the saddle point cannot merge with point . So the barrier-free path should indeed go from point to to .
In the case , the system stays on energy surface . It is then instructive to analyze the energy landscape as the circular domain grows. As the domain radius increases beyond , the minimum at becomes higher than the energy at . As the domain radius increases to , the barrier between and disappears entirely. However, if , the point is not a local minimum, with an energy already higher than the minimum at . So if budding is induced at the radius , then the ESCRTs either stay on the bud, if , or they coalesce into the neck immediately after bud formation if .
Another budding scenario is possible if and . The system evolves then on energy surface from point to with no energy barriers, which means that flat, ESCRT-coated membrane domains are unstable and small, ESCRT-free buds with radius form spontaneously. The mini-buds induced by a strong binding preference to membrane portions with negative Gaussian curvature are next squeezed bigger by line tension if . The system evolves then from point to . The resulting ESCRT-free buds have a radius . In contrast to the three-stage mechanism, in this budding pathway the ESCRTs never enter the bud lumen.
Author Contributions
Conceived and designed the experiments: BR EB JHH GH. Performed the experiments: BR. Analyzed the data: BR EB JHH GH. Contributed reagents/materials/analysis tools: BR GH. Wrote the paper: BR EB JHH GH.
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Norwegian retailers will face complex challenges as they seek to innovate in an increasingly cashless future—rapidly evolving cybercrime threats, new European privacy regulations and an increasingly competitive, global market. In this post, we briefly explore current areas of concern for managing cyber risk in the retail sector.
Only 6% of Norwegians use cash regularly and our large financial institutions are lobbying to eliminate the cash economy by 2020. Whether this deadline is met or not, it's clear that Norwegian retailers will demonstrate to the world both the opportunity and the risk that come with an increasingly cashless future. At the same time, retailers must learn to navigate new European privacy regulations while continuing to develop personalized, omnichannel customer experiences in an increasingly competitive, global market.
In addition to the obvious benefits, adoption of new technology also brings risk—risk that is often not so easy to see at the outset. The risk associated with new technology becomes understood over time and is constantly changing, but like all risk, it can be assessed and managed. In this blog post, we briefly explore current areas of concern for managing cyber risk in the retail sector.
Criminals continue to target online stores to obtain account information, extort ransom or execute fraudulent transactions. In the last two years, 150,000 Norwegians were victims of identity theft. One of the ways this personal information is often stolen and later used is via online retail sites. Criminals who may have previously engaged in traditional on-site robbery and fraud are increasingly moving online. Europol's latest annual cybercrime report found that remote cybercrime is becoming easier to execute and offers a low risk, high reward alternative for existing criminal organizations.
The 2013 breach of US Target Corporation that affected 110 million customers began with attackers obtaining legitimate network access credentials that Target had provided to one of their vendors. There have been many similar incidents in the Scandinavian region—businesses have seen thousands of customers impacted because vendors' security was compromised. In 2012, many Norwegian online stores had their user accounts and passwords breached through the weak security of their hosting and content management provider, Demo Store AS. In 2013, the Coop HotellKupp reward program experienced a breach of 96,000 customer records when their service provider Loyaltybuild was compromised.
Although online attacks are on the rise, on-site fraud and breaches still occur via electronic POS terminals. The use of fraudulent cards copied via skimming or real cards obtained under stolen identities is an ongoing problem in Norway. POS malware is also becoming increasingly sophisticated and effective at stealing card information directly from vulnerable retail POS systems. Sophisticated attacks may combine remote hacking with on-site activities such as installation of malware via exposed USB ports or even swapping out entire devices with malicious look-alikes.
Datatilsynet's report "The Great Data Race" explains several common commercial uses of personal information for online profiling and tracking that may violate Norway's existing privacy laws—and more rules are coming. Tougher European data protection rules will be applicable in 2018, including possible fines of up to 4% of retailers' global revenue for non-compliance.
Norwegian retailers that store, process or transmit cardholder data in the course of business are responsible for maintaining PCI DSS compliance. Although the means of measuring and reporting compliance differs based on the number of transactions a retailer processes annually, this compliance requirement applies to retailers of all sizes. However, as the Target breach and others have demonstrated, compliance audits are an observation at a point in time and a successful audit does not guarantee that a merchant will be able to effectively prevent or detect a future breach.
A successful approach to cyber risk management starts with business leaders becoming aware of strategic threats and the business's current capabilities to prevent, detect and respond to those threats. Even in the event of significant breaches, customers have shown a surprising capacity to forgive and forget. However, retailers should expect cyber threats and consumer awareness of these issues to continue increasing into the future. This means gaining and keeping customer trust will increasingly depend on retailers not only taking reasonable steps to prevent breaches, but also having strong detection capabilities and a tested response plan in place.
It's not enough to achieve compliance with standards at a point in time, or even to maintain it over time. Today's retail threat landscape is changing faster than the smorgasbord of available standards can be updated to address it. Compliance with relevant standards can be a great starting point for effective risk management, but it's not the finish line.
Cyber risk management is best undertaken as a continuous process that includes threat awareness, the establishment of business-specific performance indicators and ongoing measurement to track the effectiveness of controls against current threats. From this state of awareness, business leaders can support the integration of appropriate security and privacy measures as part of normal operations using an adaptive approach. By shifting resources over time to continually address the most relevant threats and vulnerabilities for their business, retailers can effectively protect their most valuable assets—their customers' loyalty and trust.
Be om tilbud

subset: commoncrawl


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On this segment of Ignite Your Life LIVE! Carolin works with Entrepreneur Coach Lisa Steadman and Social Media Coach Kerianne Mellott to move past some issues that were preventing her from doing well at her new job. Was she able to focus, or did her fears get the better of her?
The Relationship Readiness Rejuvenation Retreat
June 3rd-5th!
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This review is a bit out of the ordinary (and a tad long to boot, scroll down to the bottom if you want some screenshots rather than just large blocks of text), and so is the product at hand, which is also why the contents of the info box on the left is a bit different from the usual photo/video stuff. 3D Sex Villa is a virtual sex game or simulator, placing… Read More
Titanime Review
Very large collection of downloadable hentai movies, everything from Angel Blade, Bible Black and Discipline to Marine A GoGo, Taboo Charming Mother and many others. The collection is even more impressive when you factor in the two additional specialized hentai movie sites (lesbian and bondage) also included in the price (although some movies do appear on at least two sites, probably because they cover several themes) HentaiKey is a pretty mean competitor… Read More
Comixxx Archive Review
Another quick review update of Comixxx Archive before my most recent membership expires. I already mentioned the redesigned interface last year, so this time I really only have two things to bring up – the first is that the collection of adult comics has, as you would expect, grown since last time, now weighing in at nearly 3,400 comics, or more than 121,000 pages. The second is that the site network you… Read More
Bondanime Review
If you're after the more extreme and bizarre (in some cases very bizarre) hentai videos, the third site in this small hentai DVD network (well, small in terms of sites, they certainly aren't lacking in content) should provide you with many hours of video entertainment. This is the stuff that sometimes (but entirely unjustified in my opinion, this is strictly fantasy afterall) gives hentai a bad name. Extreme bondage, tentacle porn, violence,… Read More
Where Titanime covers a broad range of hentai subcategories and Bondanime delivers the most extreme and rough hentai videos, Lestai gives you nearly 150 hentai DVDs with one or more lesbian scenes. There is some crossover between the three sites, probably because you'll be hard pressed to find a hentai DVD covering solely lesbian toon sex, but based on my sample comparisons (didn't feel like cross checking for more than a hundred… Read More
With a huge amount of downloadable hentai DVDs, scanned hentai and Western style (US/Europe) erotic comic books with a reported total of no less than 3,000 Gigabyte of content, there's little doubt that HentaiKey is pretty much paradise for fans of hentai and toon porn. There's enough content to keep visitors entertained for months on end – 24/7. Given the nature of an AEN network there are many inconsistensies in design and… Read More


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We strive to develop long term partnerships with our clients, many of whom have been with us for over a decade. Our client retention rate is over 98%.
Since 2003, we have provided outsourcing services to over 400 clients including many of the top 100 UK insurance companies, FTSE Eurotop 100 and Fortune Global 500 companies.
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Braai Week Part 4, Steak
I'll come right out and say it. I don't like Steak. Yes, once again, I expect to be thrown with rotten tomatoes. I seriously would rather eat a juicy lamb chop or a nice fatty pork chop than eat steak. The only steak I don't mind, is fillet. So for me, the most important part of steak is the sauce. I usually top a palm-sized steak with about 2 ladles of sauce. But then again, I am a bit of a sauce slut. If I could have creamy mushroom or pepper sauce with vegetables I would..actually, I think I have!
So for me, it's less about the marinade but more about the sauce. Our butcher is really excellent and they have fabulous marinated steaks which I buy sometimes, but other times a drizzle of olive oil and soy with some cracked black pepper is more than enough. I like using Soy sauce with steaks because it just enhances the meatiness of the beef so well. It's almost the same as when you use Bovril in a beef stew, it just works!
I'm also a bit of a Steakhouse person when it comes to steak. I don't like fancy schmancy sauces over my steak. I like creamy mushroom and pepper. That's it. But when I make it myself it's never as gloopy and floury as at a steakhouse. It's light, flavourful and delicious. When we were in Malaysia I had a great Bearnaise sauce with my steak and it was nice, but not as nice as my pepper sauce.
And you won't find a simpler sauce recipe than these two. One day I might venture further into red wine sauces etc. but for now, I'm very happy with my 'bad taste in sauces' as I've been told!
Creamy mushroom sauce:
250ml cream
150g sliced mushrooms
2 garlic cloves, crushed
1tbsn cornflour mixed with 2tbsn milk
drizzle of lemon juice
Salt & pepper to taste
- In a small saucepan, fry the garlic and mushrooms in a tiny bit of butter or oil for about a minute. Add the cream and turn down the heat. Allow to simmer for 7-10 minutes until the mushrooms are cooked then add the cornflour mix and allow to thicken. It mustn't be incredibly thick. Just thick enough to coat your mouth pleasantly.
- Add some lemon juice and seasoning and serve.
Creamy Pepper sauce
1 tin Evaporated milk
2tbsn cornflour mixed with 100ml milk
2-3tbsn freshly cracked pepper / 2tbsn green peppercorns
Salt to taste
- Combine the evaporated milk and cornflour mix in a small saucepan. Allow to come to a simmer and wait for it to thicken. Add the pepper and salt and serve.
I hope everyone has a fabulous braai day filled with smokey flavours and lots of raucous laughter. Be safe and DON'T DRINK AND DRIVE!!
I tried the sauce with red wine on the steak it was divine. I love my steak but i still need to find the best way to make it. I know you say your not a fan but for some of us who use your blog to become kitchen goddesses ( I wish) please make an effort on how to grill or fry the perfect steak. I get it wrong.
Have you tried the Italian salad, bed of rocket with slices of med cooked steak (any cut will do)
drizzled with olive oil and balsamic vinegar, topped with parmesan shavings? Oh, now I'm drooling!!
For the mushroom sauce replace the cornflour mix with a packet cream of mushroom soup. This just heightens the mushroom flavour and quite delicious
By the way both sauces are delicious. So many unfortunate people say that they only eat fillet. Don't get me wrong fillet is a great piece of meat and definitely has its place, but as far as I am concerned its nowhere near the braai. While fillet is extremely tender it is also seriously lacking in flavor. Perhaps this is why so many people prefer to have sauce with their braai meat. While sirloin/porterhouse or rump are well known for their flavor especially on a braai, why not try a ribeye? This can be prepared by your butcher, either with bone or without, or even with a bit of fat or without. This cut is extremely tender, like fillet, but also has a good flavor like sirloin or rump. An extremely underrated cut of steak. It is also still priced fairly well. But watch this cut eventually take the same route as chicken wings and ox tail, once considered offal now considered almost a delicacy.
Also consider using dried porcini mushrooms in addition to the fresh mushrooms; they bring a lot of mushroomy flavour to the sauce. Use the water from rehydrating the mushrooms as stock in soups, stews or even your mushroom sauce.
The cream sauce can be used as a base for various other sauce options, even the black pepper one. Add smoked paprika to take the flavour into a different direction. Add a knob of butter for extra smoothness (you've already used cream so stop complaining about your weight/heart/colesterol). A handfull of grated cheese makes it cheese sauce but don't always be stuck with plain old cheddar, unless it is a properly matured one. Think Parmesan, Brie, Blue, etc. You can even try a Mustard version. I usually fry up half an onion as part of any sauce recipe and liquidise it with the sauce. If you want a few chunky bits of mushroom, simply keep a few mushrooms aside for that purpose.
Here is another trick: use Hoisin sauce (you can funk it up a bit with various additions like some chopped garlic, Teriyaki or Soy sauce, even Worcestershire sauce or HP Sauce) as marinade, preferably overnight. By the time you are done no one will ever know what you had used but you'll get compliments aplenty.
Roll the steak (works particulalry well with a whole fillet of game) in a mix of ground corriander (liberal quantities), ground cumin, black pepper, ground sea salt and chopped herbs (I go for sage, rosemary, mint, organo or a mix of these).
Make sure the coals are scorchingly hot. Even better: take the cast iron grid from your gas grill and put it on top of the charcoal grill over the fire and heat the cast iron grill up. Best of both worlds. Now throw the steak on there, cook to your desired state; anything more than medium and your red meat privileges are revoked. Use any of the cream based sauces to serve. For game, which is particularly low in fat, you can add olive oil in the marinade.
You do not need the spice/herb mix to make an excellent steak, or even the marinade for that matter. Mature, good quality steak on very hot coals plus salt are the minimum requirement.
love the new site! Have a super braai weekend 🙂
I also only eat fillet… the mushroom sauce sounds sooo yummy. My husband will adore it so will have to make it for him soon


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Finding: CONFIRMED
Explanation: It's probably no surprise that the same chemical compound called thiols that's found in skunks' noxious spray is also present in rotting flesh and feces. And what better team to tackle those malodorous molecules than MythBusters Jamie Hyneman and Adam Savage?
To start, after coaxing three separate skunks to spray, the pair finally got one to release its torrent of thiols. Then they scoured their skunk-soaked clothes and bathroom in a variety of home remedies, followed by a sniff test to determine whether each potential solution was confirmed, busted or plausible.
1) Beer — busted. Still skunky and not like bad beer.
2) Tomato paste — plausible. Eau de skunk still snuck through the acidic sauce.
3) Feminine hygiene product — busted. Douche doesn't double as a skunk scrub.
4) Commercial skunk-scent remover — plausible. Orangey with a hint of skunk.
5) Hydrogen peroxide, baking soda and dish soap — confirmed. From skunky stench to squeaky clean.
Why did hydrogen peroxide work? The oxygen molecules in the soapy solution neutralized the skunk scent by bonding to those terrible thiols. By altering that chemical makeup, this stink-fighting antidote made the smell quickly disappear.
With this confirmed skunk home remedy, the MythBusters proved it might actually be harder to entice the animal to spray on command than get rid of its stench!


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Clinton Corners, NY 12514
Contact Us
About Metro Builders Corp.
Metro Builders Corp. has been building homes, both on contract and for sale, throughout Dutchess, Orange & Ulster counties since 1996 - in 2006, we began building homes in Saratoga county. We have built Colonials, Craftsman style houses, Raised Ranches & custom planned houses. All of our work is done under the close supervision of Carl J. Diesing, Vice President. The company has established a solid reputation for quality workmanship and commitment to customer satisfaction. In 2002, the company also began developing homes in the Arts and Crafts style, which are designed to blend into their natural settings. We consider ourselves to be a mainstream-to-custom builder.
New Construction
We will build a house to your exact specifications. You can also select from one of our spec homes already in progress, or completed.
Metro Builders Corp. is committed to building quality subdivisions with presentation and quality as our top priorities.
Home Warranties
For Sale
In Process of Being Built
Asking price: $474,900
Agent: Carol Raike
Address: 7 Oakwood Court Milton, NY
County: Saratoga
Total Sqft: 2,820
Bedrooms: 4
Baths: 2 Full, 1 Half Bath
Lot Size: 1.953 Acres
Style: Colonial
The BEST LOT at Barrett Creek Estates is waiting for you!! Custom kitchen designed by Curtis Lumber! Still time to select your own cabinetry, granite counter tops, 5 foot tile shower in master bath with large linen closet. 1st floor with hardwood, tile in laundry area, foyer & baths. Mudroom makes for a clean & organized living, Full front porch, gutters and pressure treated deck also included at this price! Front of house with custom stone this home will shows like a model! Customize NOW!


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Actor Tommy Lee Jones has just listed the 50-acre polo farm he owns in Wellington, Fla. for $26.75 million, according to a South Florida gossip blog (via Zillow).
Jones bought the property a decade ago and built the equestrian center himself. The Lincoln star is a known polo lover who also owns a ranch in Texas.
In addition to recreational facilities and miles of horse trails, there's a four-bedroom, two-bathroom house on the property.


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While researching medical laboratory services in Australia I came across a significant error that I had seen before. The error was in a report prepared by Price-Waterhouse-Cooper for the Australian government on international laboratory services. The report stated that half of Canada' medical laboratory services are delivered by the private sector. This was an incorrect figure that appeared in the Carter Commission, a report to the Ministry of Health in the United Kingdom, which was the source cited by Price-Waterhouse. The correct figure is approximately 25% delivered by for-profit corporations.
It is not clear how Carter arrived at this figure. He refers to a couple of sources from Canada who provided information on Calgary's Laboratory Services (CLS) and services in Oshawa, Ontario. But the figure, fifty per cent private, to my knowledge is not published anywhere nor is it likely that either of these sources would have said it. My guess is that Carter confused Ontario, where 50% of lab services are delivered by for-profit corporations, with Canada. An easy mistake considering that many people in Ontario regularly confuse the two.
A second point highlighted by Price Waterhouse is that there is a national cap on pathology expenditures in Canada. This also is incorrect. There is no national system of spending on specific clinical services. The federal government transfers money to the provinces albeit with some strings attached but none that directly relate to pathology services. The provinces make the clinical allocation decisions and the federal money only accounts for about 25% of health care spending. Only four of ten provinces in Canada use for-profit laboratory corporations and only two of these have a hard cap on private laboratory expenses. Once again the province of Ontario may have been conflated with Canada.
But when using other national examples to help make informed decisions, especially when the information is provided by professional researchers and high-priced consultants, mistakes like this are shoddy and unescusable: an "F" for a first year health policy paper. They are also mistakes which overstate the role of the private sector, encouraging further privatization.
Carter also made a couple of other errors. In his report Calgary Laboratory Services was identified as an independent service provider: which is a British term for a for-profit provider. Even when the CLS had private sector partners it was always 50% controlled by the public sector and since 2006 it has been wholly within the public sector. This misinformation was also carried over into the Price-Waterhouse report. The real story from Canada on public-private laboratory partnerships is that all of them have ended and the work reverted back to the public sector.
Price-Waterhouse also create a false impression of the low-cost of Australian laboratory services. In their per capita comparison table they use an expense figure for Australia which is only for community, or outpatient tests (maybe 60% of Australia's laboratory tests) and compare it to expenditures for Canada and Britain which are total expenditures on all laboratory services: inpatient, outpatient and community. It is little wonder that Australia's per capita costs come out lower.
The surprising fact is that in the written description Price-Waterhouse's consultants acknowledge that Australia's costs do not include inpatient care and the figure they use for Britain is similar to the one in the Carter Commission, a report they're familiar with. Granted the data is confusing because the OECD figures are misleading, but a small bit of critical thinking would have exposed this glaring inconsistency. Rather than making a weak statement like, the national systems may not be comparable, some thought may have inspired a bit more intellectual rigour with a statement like, Australia's numbers are apples compared to Canada's and the United Kingdom's oranges, and the per capita costs as calculated are not comparable. Better yet, this table should have just been left out.
There are other less significant errors in both reports which systematically paint a rosier picture of for-profit corporations delivering public health care than is deserved. Price-Warehouse's data, specifically the per capita cost comparisons, is used in subsequent government documents to support polices favoring private delivery.
This is the paper trail to ill-informed policy in Australia. The Carter Commission in Britain does a poor job of researching Canada's laboratory system, probably using a hired consultant, publishes its incorrect findings, which a private consultant in Australia reads and uses, apparently uncritically, in a report which is then used to formulate government policy. It is a tangled ill-informed web that these consultants have woven: but it is very lucrative.


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From vegetables and fruit to locally sourced meats and treats, farmers markets around Atlanta have as much selection as your local grocery store. Summertime means a visit to your local farmers market for the freshest ingredients of the season.
Keeping that fresh summer spirit in mind, which farmers market were named the Best in Atlanta?
Marietta Square Farmers Market wins the No. 1 spot for good reason. Market visitors have offerings from over 65 vendors, ranging from fruits and vegetables to home goods and kitchen staples. Arrive empty handed and leave with everything you need to make from-scratch meals at home. You can even dress your dinner table with freshly cut local flowers.
You'll feel refreshed and inspired by visiting our second runner up, the Grant Park Farmers Market. Let free weekly celebrity chef demonstrations featuring tops chefs from the south spark ideas for your own at-home menu.
Your third place pick is none other than Freedom Farmers Market, now open year-round. This farmers market features live music, a kid's tent and a local chef with hot prepared foods. They also hold monthly events featuring the fruit or vegetable of the season.
Looking for a farmers market closer to your own community? Check out our other official nominees for a variety of farmers market experiences: Decatur Farmers Market (DeKalb), East Atlanta Village Farmers Market (Intown), Snellville Farmers Market (Gwinnett) and Farmers Market at Sweet Apple Village (North Fulton).
Farmers markets can be found all around Atlanta. Here are some that could be near you or right in your neighborhood.
If visualizing straight-from-the-farm veggies are just what you need to inspire a healthy summertime meal, walk through this photo guide to one of metro Atlanta's most revered and longstanding farmers markets.
A visit to your local farmers market is free. Here are a few more wallet-friendly things to do around Atlanta.
Photos: Top Atlanta Farmers Markets
Read about our other Best of Atlanta winners and nominees here.
Support real journalism. Support local journalism. Subscribe to The Atlanta Journal-Constitution today. See offers.
Your subscription to the Atlanta Journal-Constitution funds in-depth reporting and investigations that keep you informed. Thank you for supporting real journalism.


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Dirty Works are a brand that are available in Sainsbury's in the UK. I've tried lots of their products in the past, like their pedicure accessories, Banish The Bags Eye Cream and Lift and Firm Super Skin Serum and their Blemish Buster 2 in 1 scrub and mask. From what I've tried of the range so far, I can honestly say that the quality is far superior to other brands I've tried that are typically sold in supermarkets. They're high quality, great value for money and actually do what they say they're going to do. For me, you can't ask any more than that. Over the last few weeks I've been trying out more of a cosmetic product from them. Something that isn't going to change your skin in the long run but something that's designed to make it look the best it can for that special occasion. If you like adding a little something extra to complete your look, making your body appear flawless then you have to try this!
Bling It On! Shimmer Lotion
Dirty Works
The Dirty Works Bling It On! Shimmer Lotion is a light but bright gold shimmery lotion. Not to be mistaken with a body butter, cream or lotion; I wouldn't recommend this to be used solely as a moisturiser for your body as that isn't its purpose- use this as the last step in your routine, to beautiful and perfect your skin. It can be used all over the body, wherever you want to add a bit of shimmer. Light, thin and almost gel like in formula, it is very quick and easy to apply and dries quickly on the skin which is perfect if, like me, you're always rushing around before going on a night out! It's also lightly fragranced, like a soft, fresh perfume.
The shimmer in it is pretty subtle, it isn't like chunky glitter so it wont make you look like a disco ball! I personally like to wear this on a night out as I find that it gives that your legs but better look, very polished and well presented. I'd recommend applying it to your legs as it adds a gorgeous sheen, particularly down your shins. It looks gorgeous applied to your arms, shoulders and decollete too. Whilst I favour wearing this for a night out, it would also look fantastic worn during the day, perhaps with a pretty summer dress for afternoon cocktails with the girls, or even on holiday as shimmer lotions look gorgeous on sun drenched skin.
|Im wearing the shimmer lotion here…
Plus a sneek peek of a WhatLauraWore outfit post coming up!
Blight It On! is photo friendly too. I'm wearing it in the photo above and there's no scary flash back created so you'll still look good in photos when wearing it which is something that's pretty important to me as I'm photographed quite a bit before I go out for my blog and when I'm out if I'm at an event. In photos, I find that it gives that your skin but better look and in real life you can see the shimmer more, it makes your skin look flawless. The Bling It On! Shimmer Lotion makes your skin look great whether you have a tan or not as it has a slight tint to it so it blurs imperfections and gives a slight golden tint to your skin. In the photo above I'm not actually wearing any false tan and I still felt confident going out as I had my shimmer lotion on! I've also tried using this over false and gradual tan and it looks amazing. I can't wait to have my birthday night out when I'm tanned to the max and wearing this- gorgeous!
The Dirty Works Bling It On! Shimmer Lotion is available from Sainsbury's for £4.99 for a 200ml tube which will last you AGES! Sainsbury's often have offers on the Dirty Works products too, if you're quick you can save 33% on the Shimmer Lotion and lots of the other products in the range- what better excuse for a haul?! You can also check out the full Dirty Works range on their website here.


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Delta Goodrem releases the video to the second single of her upcoming untitled album.
'Dear Life' is a beautiful song about life's struggles and joys and reflects Delta's own struggles along the way. I am loving her music this era so far and I can't wait for the release of her new album, hopefully in the Summer/Fall of this year!
Check out the video below:


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A 2013 Monroe County budget that keeps property taxes at 21.25 mills won unanimous approval Thursday from the three commissioners.
A 2013 Monroe County budget that keeps property taxes at 21.25 mills won unanimous approval Thursday from the three commissioners.
The $93.9 million spending plan reduces overall spending by about $5 million from the current year.
Health insurance costs to taxpayers have been reduced by $872,000 — to $9.29 million — by raising deductibles, co-pays and out-of-pocket premiums paid by workers.
Unionized workers at the Pleasant Valley Manor, the county nursing home, agreed to $500,000 in wage and benefit concessions, though the county will provide a $1 million subsidy to the home.
The county's annual employee retirement plan contribution will go up by $486,330, to $3.88 million.
The county eliminated 12 positions through layoffs or not filling vacancies in the planning, purchasing, maintenance, parking deck and register and recorder departments.
But 12 corrections officers were added at the Snydersville prison — eight full-time and four part-time workers.
A 50 percent private donation to the county's $28,000 child safety seat program helped preserve that program for another year, Commissioners Chairman John Moyer said.
The vector control program, which tests for the presence of the West Nile Virus, will benefit from an expected one-third contribution from the 10 municipalities where the virus is commonly found. State funding accounts for another one-third of its $85,171 budget.
Children and Youth Services — with a $15.3 million budget — has the largest expenditure among Monroe County departments, though $11.45 million of it is covered by state grants.
The Monroe County Correctional Facility has a $13.5 million budget next year, all but $256,657 covered by local taxpayers.
Various judicial functions — including the courts, public defender and district attorney offices, and sheriff's office — are expected to accumulate $18.1 million in expenses. About $7.5 million of the total will be covered by grants.


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Pyro Pet Cat Candle
A cute cat shaped candle reveals the wild beast inside her, a grinning metallic skeleton with fire blazing in her eyes! Kisa is available in three colors: pink, light grey and black. The dimensions of a Kisa candle are: 6.7 x 2.9 x 4.5 inches (17 x 7.5 x 11.5 cm). Each candle should have a burning time of approximately 20 hours. Materials: Paraffin wax, cotton wick and aluminium.
Additional information
|Choose Color
Grey, Black


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The Trump administration has put a hold on $647 million in federal funding for a proposed electrification of the Caltrain commuter rail system in the San Francisco Bay peninsula. This is the main passenger rail line serving Silicon Valley. The US DOT did this at the request of California's Republican Congressmen, who see it as a further subsidy to California's high speed rail line, which would apparently use the tracks.
I haven't ridden Caltrain, but it appears to be a diesel engine system with bi-level coaches similar to Chicago's Metra or Boston's MBTA commuter system. Again, this isn't a line I've studied in detail, but at first blush electrification seems like a good thing. A $647 million federal subsidy isn't gigantic as these things go.
A couple things jump out to me however.
The first is that "Resistance" has consequences. When mayors and other urban leaders dedicate themselves to the unconditional undermining of the President, don't expect him to do them any favors. As I noted on New York PBS, it may be difficult for Trump to pull existing funding for sanctuary cities, but there is plenty of new funding they want that is at risk.
San Francisco and Silicon Valley have been, with rare exceptions like Peter Thiel, implacably hostile to Trump. They can't expect him to treat them nicely in return.
Secondly, Silicon Valley is America's most economically prosperous area. It's a bit odd that the project is in jeopardy because of $647 million in federal funds. Apple alone as over $200 billion in cash. Silicon Valley is awash in funds and needs its workers to be able to get around. The Silicon Valley Leadership Group should pony up the money themselves if it's really such a great project. If a rail project in Silicon Valley fails for want of far less than a billion dollars, it must not have delivered that much value.
Thirdly, from a progressive point of view, I'm not sure why the feds should prioritize money into one of America's prosperous regions. Federal aid should be directed into more struggling areas that need transit investment, like Detroit and Baltimore. Rich regions should pay their own way, particularly since these projects yield big economic returns for them. If they can't muster the political will to do it, that's not Washington's problem.
Urban leaders have a choice to play at Resistance or play ball. If they choose the former, they need to be prepared to start self-funding a lot more projects. This would also be aligned with a more progressive approach to urban regional funding, in which the feds prioritize struggling, poorer cities over prospering ones.


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While corporate participation in social media is increasing, many companies still do not have a strategic plan for its use. IT Business Edge's Ann All has written several times about research that supports this. For instance, a Burson-Marsteller study released in early 2010 found many companies risked confusing customers and/or diluting their brands by creating multiple Twitter and Facebook accounts, some of which lapse into disuse.
A new survey of 100 companies by search and social media consulting company Digital Brand Expressions (DBE) similarly found that while 78 percent of respondents were using social media, just 41 percent said they have a strategic plan. Click through for results of the survey.
Eight Tips for Creating a Social Media Policy
The key to a good social-media policy is finding the right balance for your company.
Social Media Authors Go In-Depth on New Communication Tools
Tap into the communication power provided by social networking and Web 2.0 technologies
Seven Truths About Social Media
'Social' doesn't mean 'free' -- you need to make a plan of attack and then stick to it
Related Topics : Cox Communications, Business Development, Contact Center Solutions, CRM Solutions, Database Acceleration
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Predictive analytics uses past data to forecast outcomes and target the right prospects – a move that's redefining the use of data and insights in marketing. ... More >>
Five steps CIOs and CMOs can take to patch the IT/marketing disconnect and create a cohesive, efficient working environment. ... More >>


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I hate anyone who makes a profit by incarcerating human beings.
Monday, December 12, 2011
Private Prisons Must Comply with Public Records Laws
Well at least in Tennessee and Florida, they do. In most jurisdictions, private prisons, as private companies, are not subject to public records or FOIA laws, which has presented a tremendous obstacle to members of the public who wish to exert some semblance of oversight on the system. But in a recent ruling in Tennessee, it was held that CCA must comply with public records requests as must any agency performing an inherently governmental function. This is a tremendous victory for advocates like myself, who recognize that the industry is in dire need of effective oversight and that it has skirted the bounds of accountability for decades by continually avoiding turning over records related to its operations. CCA will almost undoubtedly appeal the decision, but for the time being, I want to savor this victory.
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