It's Not Work, It's Gardening!: You're grounded!
It all started the previous weekend when I went to the winter garden talk at Schlafly Gardenworks, the large (compared to mine) urban garden that grows food for their restaurants. Jack (the head gardener) mentioned at the talk that they have a big pile of used coffee grounds, and that anybody is welcome to come and take some.
I got in contact with Jack during the week to talk about bamboo, and Jack asked what I fertilized my bamboos with, quickly following that question by asking if I wanted to try coffee grounds. Apparently there weren't many (any?) takers on his offer at the meeting.
So on a warm sunny afternoon I drove down there during my lunch break and picked up several bags of the grounds, then spent an hour or more just chatting with Jack while he worked in the garden. It was one of those "recharge" afternoons, if you know what I mean. Warm sunshine, talking about plants, shovel in the ground (or coffee grounds pile). Just wonderful!
I was concerned about the acidity of the grounds, but Jack explained that the brewing process removes most of the acidity, and that the spent grounds are only slightly acidic. I found some other references that confirmed this, and my own soil acidity meter showed they were essentially neutral. (Although my meter may not be working, as I tested it in some vinegar and it still read "neutral".) Note that I also found plenty of sites that indicate that coffee grounds are highly acidic (5.0 or even lower!) but those seemed to be based on old research, or were just repeating the same information. I trust this page about coffee grounds, from Oregon State University, and they say the grounds are neutral.
The grounds are a good source of Nitrogen, but it won't be immediately available to your plants -- it needs to be broken down first. The OSU page talks about using it in compost piles, but Jack uses it directly in his beds. Not only is it a great source of organic matter, but it apparently attracts earthworms, repels or kills slugs (the caffeine is toxic to them), and can also kill some soil-dwelling pests, and leaf-dwelling ones when used in a foliar spray.
So I added loads of it to each of my bamboos, along with the organic fertilizer I normally use (Milorganite). I pulled back some of the mulch in order to get the grounds into contact with the soil, but also just dumped grounds on top of mulch -- removing all of the mulch would have been a lot of work.
I had placed them there last year when I remade the bed adjacent to this box and liked the way they looked, so left them. I don't want them to interfere with any of the shoots this bamboo will be producing this spring though, so it's time to move them. This plant usually starts shooting mid-April, but this mild winter who knows? I suspect things will wake up earlier than usual, but we'll see.
So most of the bamboos got a few shovels full of used coffee grounds. I'll see if that makes any difference on growth, pest damage, or any other noticeable aspect. It may be difficult to tell this year, since mild winter should result in big growth increases in all of these bamboos -- I won't know if it was the coffee grounds that produced the results or some other factor.
Since I didn't have enough to use on all of my bamboos, I went back yesterday and got a few more bags which I haven't yet spread (garage smells great!). I'm planning on using it not only around the rest of the bamboos, but in the veggie beds too. I've seen Jack's results with his veggies, and he's definitely doing something right!
I have a feeling that coffee grounds will become one of my favorite soil amendments and bamboo mulches. It certainly makes my yard smell delicious!
Great post! I've been wanting to do the same. I did some research last year and came to the same conclusion you did, i.e. coffee grounds are essentially neutral or a touch acidic. That is great for us since our soil is alkaline. You've inspired me to ask the coffee shop up the street for some grounds!
I am so jealous! The one espresso shack I inquired of for grounds wanted more for their buckets of used tat than three times the volume purchased as top grade organic fertilizer at the local garden center. I sniffed at them and drove away. I haven't had the heart to ask other places. I'm going to be really interested to see what you discover about this experiment.
I wasn't really clear on how much grounds I actually got. Those bags in the first photo are 55 lb. grain sacks. I estimate each bag was about 35 lb. when filled, so that's 175 lb. of grounds. On the first trip. (I went back for more remember.) Fantastic resource!
If any of you live near a Starbucks (and really who doesn't? Except me, unfortunately) they bag up their grounds and give them away for free. Also I've heard that grounds can be toxic for your pets.
Coffee grounds are only mildly acidic after composting, prior to that the PH can be nuetral to very acidic depending on how they where brewed. Here in the south western New Jersey we are already acidic with PH values around 6.0 to 6.5 if near a house structure. I have personally killed plants in the past with coffee grounds mostly due to our already acidic soil. So has a fair warning if your soil is already borderline acidic, consider composting coffee grounds first.
M: Good info! Can you point me to some sources that indicate that some brewing methods can leave the grounds as quite acidic? I want to research this as thoroughly as possible. In my case it's nice to know that a local grower uses them with no issues -- it gives me more confidence to just dive in.
I thought that the foundations of buildings (if concrete) made the nearby soil more alkaline, not acidic?
I'm envious of your haul. A few years ago I would pick them up at Starbuck's but often they had the paper inside which was a devil to deal with. Maybe I'll drop by there again sometime. Thanks for reminding me.
hello,after those years,is good to use coffee grounds for bamboos?


Science Teachers Outreach held at Rau Secondary School in Moshi-Kilimanjaro by Josephh Clowry.
On the 23rd of May the director of YST, Dr. Kamugisha Gozibert visited Shyinyanga to meet some of YST's partner schools.
An opportunity to link up with students from Ireland and Tanzania!


text:
China sells 10-year debt
China's government sold 26 billion yuan ($3.8 billion) of 10-year bonds, following three failed auctions for shorter-dated debt this month, on speculation inflation will remain subdued.
The finance ministry sold the securities at an average yield of 3.48 percent and a highest of 3.51 percent, according to traders at China Citic Bank Co. and China Construction Bank Corp. in Beijing, who asked not to be identified. It attracted bids for 1.88 times the amount of debt on offer, compared with an average of 1.5 times at auctions this year, the traders said.
"Inflation expectations aren't that high at the moment so investors are still willing to buy long-term bonds," said Tang Guohui, a fixed income analyst at Industrial Securities Co. in Shanghai. "The short-term bond auctions failed because the central bank allowed money-market rates to rise."
The shortage of demand at previous sales reflected concern that the country's 4 trillion yuan economic stimulus package would cause stock and property market bubbles. The government said last week that consumer prices fell 1.7 percent in June from a year earlier, even as economic growth accelerated to 7.9 percent in the second quarter.
"We don't see any possibility the global economy will recover rapidly," said Lu Zhengwei, senior economist at Industrial Bank Co. in Shanghai. "China's consumer prices will climb at most 3 percent next year."
The average yield matched the 3.48 percent median estimate in a Bloomberg News survey. The government last sold similar- maturity debt in June at 3.09 percent.
Demand for government debt also rebounded because China State Construction Engineering Corp. moved closer to completing its initial public offering, reducing demand for funds in the financial system.
The Beijing-based company plans to raise as much as 50.2 billion yuan in the world's biggest IPO since March 2008. It started selling shares to institutional investors yesterday and to retail investors today.
The seven-day repurchase rate, which measures funding availability on the interbank market, fell 31 basis points, or 0.31 percentage point, to 1.96 percent as of 10:05 a.m. in Shanghai, according to the China Interbank Funding Center.
The yield on China's one-year central bank bills has risen 39 basis points this month to 1.73 percent as policy makers guided rates higher to rein in lending. Loans rose almost fivefold in June from a year earlier to 1.5 trillion yuan.
"It's still possible for government bill sales to miss their target in the future," said Huang Yanhong, an analyst at Bank of Nanjing Co., a Chinese lender partly owned by BNP Paribas SA. "It will happen if investors are short of funds amid new share subscriptions and also if the central bank bill yield rises fast.
Asian stocks rise on earnings speculations
Apple exceeds profit forecasts as iPhone sale rises

redpajama_set_name: RedPajamaCommonCrawl


The Italian Voice, a newspaper geared toward the Italian American community, will honor Joseph Leone, a Partner in the firm, in their December issue. Honorees are recognized for their success as Italian Americans in their fields and for their integrity, reliability and dedication to fair and ethical business practices. Joe will receive the Stivale D'Italia Award of Excellence Plaque in official recognition of the honor.


| Excellent Guardian Angel style movie!
I saw this movie several years ago on British TV which I believe is called "We're Fighting Back", and if so it's well worth watching!
The movie is about a group of a crime fighters who go out to protect the public. They particularly patrol the subways trains (like new york), no uniforms as such but they all wear t-shirt bearing a logo/motif on the front of a 'clenched fist against a flat hand, also wearing beret style caps (just like Guardian Angels).
It starts off with a neighbour-hood constantly being the brunt of hardened thugs terrorising the public, mugging, attacking and pretty much making the area an unsafe and no-go area.
One of the residents who has had enough of the crime escalating in his street decides to start a crime fighting alliance - several guys who will be willing to support his battle of the New York streets, and becoming part of his Guardian patrol group.
The 'Guardians' are soon to deal with the day to day battle of crime on the streets, and they turn to the Subway trains next which are constantly becoming a muggers paradise after dark.
As they operate on the subway trains, each member of the 'guardians' would patrol their section of the train carriage, and when the train got to a station they would step out to check each member of their 'angels' were present at designated carriage - but if they were not, this meant there was trouble, so all the other angels would go to assist.
Lots of drama, defence & crime fighting against hardened thugs. Well worth watching if you can ever get the chance - just wish the production companies would release this on DVD!


John F. Kennedy Elementary School PTA in Somerville has launched their fall fundraising season with Close Buy Catalog, a local goods school fundraising catalog featuring only made in New England products.
The Gronk in the Ville!
On Monday, New England Patriot Rob Gronkowski, aka the Gronk was in Somerville at Foss Park and he through his "Gronk Foundation" donated $31,000 in equipment to the Somerville Pop Warner.


Did you know that we manufacture custom Germantown Football Sublimated Shorts here at Lightning Wear.
Check out these very fresh Sublimated Statue of Liberty Pinnies we made for a gift shop up in Ellis Island.
Get started on making your Lacrosse Uniform Sets from Lightning Wear.
Take a sweet look at these Girls Sublimated Shorts we made for a huge group playing in this weekend girls lacrosse tournament in Baltimore Maryland.
Yes it is definitely that time to turn up the volume and break out your DJ Shorts from Lightning Wear Apparel.
Check out these Lake Forest Lacrosse Uniforms we made for our pals in Illinois.
You can never feel the vibrations fully until you have put on a custom pair of these Rasta Flag Lacrosse Shorts from Lightning Wear.


text:
Molecular dynamics simulation of reversible electroporation with Martini force field
Cheng Zhou1 &
Kefu Liu1
BioMedical Engineering OnLine volume 18, Article number: 123 (2019) Cite this article
After the discovery of membrane-reversible electroporation decades ago, the procedure has been used extensively in biology, biotechnology and medicine. The research on the basic mechanism has increasingly attracted attention. Although most research has focused on models that consider all atomic and molecular interactions and much atomic-level information can be obtained, the huge computational demand limits the models to simulations of only a few nanometers on the spatial scale and a few nanoseconds on the time scale. In order to more comprehensively study the reversible electroporation mechanism of phospholipid membrane on the nanoscale and at longer time intervals of up to 100 ns, we developed a dipalmitoylphosphatidylcholine (DPPC) phospholipid membrane model with the coarse-grained Martini force field. The model was tested by separately examining the morphology of the phospholipid membrane, the hydrophilic channel size, the distribution of the voltage potential on both sides of the membrane, and the movement of water molecules and ions during electroporation.
The results showed that the process went through several stages: (1) the formation of the pore with defects originating on the surface. (2) The maintenance of the pore. The defects expanded to large pores and the size remains unchanged for several nanoseconds. (3) Pore healing stage due to self-assembly. Phospholipid membrane shrunk and the pore size decreased until completely closed. The pores were not circular in cross-section for most of the time and the potential difference across the membrane decreased dramatically after the pores formed, with almost no restoration of membrane integrity even when the pores started to close.
The mechanism of the reversible electroporation process on the nanoscale level, including defects, expansion, stability, and pore closing stages on a longer time scale of up to 100 ns was demonstrated more comprehensively with the coarse-grained Martini force field, which took both the necessary molecular information and the calculation efficiency into account.
The discovery and utilization of cell membrane electroporation date from decades ago [1], and the research on the electroporation mechanism has increasingly attracted attention. Most of the current mechanistic studies are based on macroscopic and static models such as calculating the electric field distribution with the electric circuit model and the transmembrane voltage measured experimentally. However, as a non-equilibrium process on a molecular scale, the electroporation mechanism is determined by the movement of phospholipid molecules, water molecules and ions. Classical static and macro-models cannot describe unbalanced microscopic processes, such as the kinetics of pore formation, dynamics behavior of pores, as well as the transport of small ions across the pores in membrane. Therefore, in this paper, in order to explore the relationship between the movement of molecules and the reversible electroporation process, the molecular dynamics simulation software GROMACS was used to simulate the electroporation dynamics to reveal the basic mechanism on a molecular scale.
In the earliest experiments, it was found that the permeability of cell membranes could be enhanced by exposing cells to a suitable electric field [1]. The transient pores that formed in the membrane allowed the intracellular delivery of drugs, DNA, and other molecules. Chang et al. were the first to use cryo-electron microscopy to observe the formation of nanoscale (20–120 nm) pores on the surface of the cell membrane [2]. They showed that the electroporation process was inconsistent with electrical breakdown, because electrical breakdown involved chemical reactions.
The theory that first explained the electroporation process treated the phospholipid membrane as a flexible surface [3] that was squeezed under the influence of an electric field. If the electric field strength was sufficiently large to increase the compression force above a critical value, the phospholipid membrane could no longer withstand it and broke. This model did not take the molecular structure of the phospholipid membrane into account. New models proposed in recent years have added information about pore structure on the surface of phospholipid membranes [4, 5]. Some researchers considered electroporation as a phase transition since the structure of the phospholipid bilayer was metastable and the formation of pores could be considered the nucleation process of the other phase [6]. The electric field influences the nucleation rate and determines whether it will develop into a 'crystallization' process with irreversible electroporation. In the transient hydrophilic pore model, the phospholipid molecule itself was assumed to randomly generate some penetrating channels due to fluctuations in its thermal motion [4, 7]. When placed in a suitable electric field, the polar head of a phospholipid molecule is pushed toward the interior of a randomly generated tunnel, causing it to increase in length.
None of the above theoretical models took into account the effects of atomic motion. The method of molecular dynamics simulation is considered a powerful tool for elucidating the electroporation process. Molecular dynamics simulation has been used for decades as a research method, and has become a basic research tool in fields such as nanomaterials, bioengineering, and biochemistry. Tieleman et al. were the first to use all-atom molecular dynamics to simulate the electroporation process of phospholipid membranes [8]. They found that 3.04 ns after electric field application, the membrane underwent intense deformation and water molecules entered the membrane from both sides. When the water molecules from both sides met, pore channels were formed and the water molecules assumed a strong orientation within them. Tarek also used an all-atomic molecular dynamics simulation to study the electroporation process [9], and his simulations showed that under an electric field, water molecules on each side of the phospholipid membrane moved to the inside and when they met in the middle, hydrophilic pores were formed, and at the same time, the polar head of the phospholipid molecules tilted towards the hydrophilic pores. Bockmann et al. [10] reported that pore formation proceeded quickly within 0.5 ns once the process started and suggested a four-state pore formation model. Pore radius of 0.5 nm was then determined with 10 lipid headgroups tilting into the hydrophobic core forming a hydrophilic pore, which was in agreement with the experiment results. Polak et al. [11] found that electroporation threshold differed substantially, which depended not only on the "electrical" properties of the membrane, i.e., its dipole potential, but also on the properties of its component hydrophobic tails. Kotnik et al. [12] reported that without the strong electric field, the pore formation rate was generally too slow to be observable in such simulations, which typically covered a submicrosecond time span, but in sufficiently strong electric fields, the pore formation rate increases dramatically, and pore initiation was well discernible on a nanosecond time scale. Delemotte et al. [13] showed that at the molecular level, the external electric field and charge imbalance produced similar effects: provided the transmembrane voltage are higher than a certain threshold, hydrophilic pores stabilized by the membrane lipid headgroups form within the nanosecond time scale across the lipid core and both methods induced similar electric field distributions within the membrane core.
A dipalmitoylphosphatidylcholine (DPPC) phospholipid bilayer was established with ion concentration gradient across the membrane, and then the simulation was performed using the coarse-grained Martini force field. The model was tested by separately examining the pore morphology, the hydrophilic channel size, the distribution of the voltage potential across the membrane, and the movement of particles during electroporation.
Morphology of the phospholipid membrane during electroporation
Figure 1 and the video (see Additional file 1) show the changes in the phospholipid membrane resulting from application of the electroporation field. Due to the presence of the ion concentration gradient, the phospholipid membrane was subjected to large surface tension and expanded in the initial stage (within 1 ns). As the phospholipid membrane's volume was constant, the membrane inevitably thinned, which promoted pore formation. Several visible defects on the membrane surface appeared early, e.g., at 0.08 ns, and then gradually evolved into larger pores concomitant with the expansion of the phospholipid membrane. When the expansion process of the phospholipid membrane was complete (at about 1 ns), the pore size tended to be stable for several nanoseconds. After 4 ns, the phospholipid membrane shrank, and the pore size gradually decreased until they completely closed. Reformation of the whole complete membrane took around 30 ns.
Morphological changes of phospholipids during electroporation within 32 ns. (1) 0–1 ns, initial stage with defects. (2) 1–4 ns, defects evolved into larger pores, and (3) 4–32 ns, pore healing stage
Size of hydrophilic pores
Representative results are shown in Fig. 2. The dimensions of the pores in the x–y direction were measured to reflect their size and shape, since the pores were not ideal cylinders. From 0 to 1 ns, the pores increased rapidly from 0 to 6 nm (y direction) or 5 nm (x direction). Then, from 1 ns to 4 ns, the pore size remained relatively constant without much change. After 4 ns, the pore size in both directions gradually decreased, as well as the difference in diameter in the x and y directions, which implied that pores tended to become more circular. At 30 ns, the pores were all closed (0 nm).
Sizes (nm) of hydrophilic pores. a Pore size in the x and y direction over time. b Molecule figure indicates the x and y direction. 0–1 ns, pores increased rapidly. 1–4 ns, pore size remained relatively constant. 4–30 ns pore size gradually decreased. After 30 ns pores all closed
Microscopic process of pore formation
In order to further study the microscopic process of pore formation in the initial electroporation stage, here a pore was selected to observe the structure and molecular distribution near the pore. The results are illustrated in Fig. 3.
Microscopic process of pore formation (color coding as in Fig. 8). 0.04–0.08 ns, the hydrophobic region gradually exposed; 0.08–0.12 ns, the water chain formed with no obvious connectivity; 0.12–0.6 ns, water-chain pores completely formed
Due to the ion concentration gradients (namely electric potential difference) existing across the membrane, the hydrophobic region of the phospholipid was gradually exposed as the phospholipid membrane became thinner, and the probability of water molecules and ions entering the phospholipid hydrophobic region from both sides of the membrane gradually increased (0.04 to 0.08 ns). At 0.12 ns, water molecules from each side of the membrane met to form a chain of water molecules that almost penetrated the membrane. However, at this time the water chain was still incomplete with no obvious connectivity. At the same time, the hydrophilic heads of nearby phospholipid molecules tilted toward the water chain, which contributed to the energy required for the water molecule to contact the hydrophobic tail. The number of water molecules entering the hydrophobic region of the membrane was further increased and a more pronounced water chain was formed. In addition, ions also entered the phospholipid membrane through the water-chain pores, and ultimately passed through the membrane.
Electrical potential distribution across the membrane
The difference in electrical potential across the membrane reflected the stress imbalance on both sides, and therefore played a key role in the electroporation process. The distribution of the potential in the direction perpendicular to the phospholipid membrane (z direction) in the simulation box was calculated (Fig. 4a). Initially, the transmembrane potential difference was about 260 V, and the change in potential was located mainly in the phospholipid membrane and solution region, as the potential difference was caused by the distribution of ions. The potential difference then decreased over time. At 0.08 ns, the potential difference was reduced by more than half to around 100 V. The potential gradient at this time also decreased dramatically, indicating that the electric field force and the stress imbalance were much reduced. Afterwards, the potential difference and the slope of the potential decreased gradually, hence the electric field force was also greatly diminished. As can be seen in Fig. 4, during the initial stage of the electroporation simulation (0 to 0.5 ns), the potential difference rapidly decreased from 260 V to about 10 V. In subsequent simulations, the potential difference across the membrane fluctuated around this value but did not change much.
Distribution of electrical potential during the simulation. a Distribution of potential at different time in z direction; b transmembrane potential difference over time
Movement of water molecules and ions during electroporation
The ion gradient causes the difference in membrane potential. The number of ions and water molecules in the upper compartment of the simulation box was calculated (Fig. 5) to determine the direct relationship between ion concentration and phospholipid morphology.
Number of particles over time in the upper compartment of the simulation box. a Sodium ions and chloride ions; b water molecules
Initially, there were no Cl− ions, 192 Na+ ions, and 10,434 water molecules in the upper compartment. In the initial stage (0 to 1 ns), the number of Cl− ions increased rapidly while the number of Na+ ions decreased drastically, with the fastest ion migration speed. Because at this stage the pores were not yet formed completely, the transmembrane resistance for the ions was still high. However, as Fig. 4 shows, the potential difference across the membrane was also maximal during this stage. The extremely high electric field force during this time easily overcame the transmembrane resistance and caused the rapid migration of ions. In addition, the number of water molecules gradually increased, indicating that the direction of movement of the water molecules was consistent with that of the Cl− ions.
In the second stage (1 to 4 ns), pores with a stable size formed. However, the ion migration speed was not higher than that in the first stage because the concentration difference across the phospholipids was already small. The potential difference across the membrane was likewise small, and the electric field force for driving ion diffusion was not significant (Fig. 4). This indicates that in the second stage, the migration of ions mainly depended on their concentration difference as they passed through the channels formed in the phospholipid membrane. In addition, the movement of the water molecules was not the same as in the first stage, as they returned to the lower compartment of the simulation box.
In the final stage, along with the gradual closure of the pores (4 ns), the ion migration speed was reduced to zero and these events are considered the most important features of this part of the process. At the same time, the diffusion rate of water molecules also decreased until it reached a steady state. At steady-state conditions, the number of positive and negative ions in the aqueous solution was not the same, which implied an unbalanced charge distribution across the membrane. However, as the imbalance in the ion distribution decreased, along with the difference in potential (Fig. 4), the driving force for ion transport became insufficient to overcome the transmembrane resistance of the closed or restored phospholipid membrane.
This MD simulation is aimed at investigating reversible electroporation in a long time-scale and providing a more comprehensive understanding of the mechanism, especially the healing stage. By using the coarse-grained Martini force field, we witnessed the process of (1) defects origination in the membrane within 1 ns, (2) water chain and pore channel formation with defects evolution, which lasts and keeps steady for around 3 ns, and (3) pore healing due to self-assembly until completely closed in the hydrophobic domain when subjected to an electrical field of maximum 10.5 V/nm. The whole process took about 30 ns. The overall results here presented are believed to capture the essence of the reversible electroporation in lipid bilayer. However, there are several limitations in this work should be noted:
We have a hypothesis here that applying an external electric field (Eext) can lead to an ion concentration difference. In the equilibrium state, there is no ion concentration difference across the phospholipid membrane. When an external electric field is applied, the equilibrium distribution is destroyed and the ions are unevenly distributed, which will form another electric field (Eion) on the membrane and affect electroporation process accordingly. Once the ion concentration difference is formed, Eion will exist continually until the ion concentration difference disappears, no matter the external electric field (Eext) is removed or not. What we focused on in this simulation was the influence of the uneven ion concentration caused by Eext, namely an indirect method to deal with the effect of electric field.
It is noteworthy that the electric potential difference or the electric field strength across the membrane was calculated based on the ion concentration, namely a double integral of the molecular charge density distribution. Although the electric field strength (10.5 V/nm) here is much higher than that commonly used in the experiment, it is essential in the MD simulation, because the increased ion concentration will not lead to significant distortion of electroporation process, and only by artificially increasing the field strength can speed up the electroporation process, which enables the whole process to be observed in a short and acceptable simulation time. Since the electric field Eion is also much higher than Eext, the influence of the external electric field can be even neglected.
As this work is based on coarse-grained (not atomistically detailed) force field, it is preferred to analyze or understand the results in a qualitative or semiquantitative way rather than comparing specific values with other results. Nevertheless, results including pore formation time and pore size are even comparable with previous simulations [8,9,10,11] which adopted different models and initial conditions.
In this investigation, the simulation of reversible electroporation mechanism was performed with a non-reactive Martini force field, but in reality, the system involves chemical reactions [14,15,16] that cannot be simulated using this force field. In order to achieve this, a more accurate model is needed, including: (a) adopting all-atom force field directly with reactive force field. It is the most complex and accurate at the cost of computation efficiency. (b) In principle, introducing reaction field to Martini force field is possible and it is not likely to affect the molecule behavior very much. (c) Multi-scale simulation of a mixture of the all-atom and the coarse-grained model to accelerate the computation. Under the coarse-grained framework, higher precision simulations that reflect more molecular details can be implemented in the area we care about. However, the general theory and algorithms of multi-scale simulation have not yet been established at present. The simulation involving reaction needs to be extending by further study.
The electroporation process in phospholipid bilayers was studied by coarse-grained molecular dynamics. Due to the application of an electric field, the equilibrium in the ion distribution across both sides of the membrane was destroyed. The ion concentration difference on each side caused a difference in electrical potential across the membrane. We simulated the electroporation process caused by this uneven ion distribution and divided the process into three stages:
Pore formation stage When the phospholipid membrane was exposed to a difference in ion concentration, it first spread and the thickness decreased. At the same time, many defects appeared on the surface of the membrane, and these defects evolved into larger pores as the membrane extended. Under the setup conditions of the simulation in this study, the process occurred within the first nanosecond of electroporation. At this stage, water molecules entered the hydrophobic region of the phospholipid membrane from both sides and formed a water chain that communicated through the membrane's pores. The ion migration was also faster, and the difference in ion concentration on each side of the phospholipid membrane decreased rapidly.
Pore maintenance stage This stage occurred after the formation of the channels, at about 1 ns. The size of the pores and the area and thickness of the phospholipid membrane remained stable, and this stage can last for several nanoseconds. Under the analog setup conditions of this study, the stage lasted approximately 3 ns. During this time, the ions could still undergo transmembrane migration, but the speed rapidly decreased.
Pore healing stage When the difference in ion concentration between the two sides of the phospholipid membrane was reduced, the area of the phospholipid membrane was also reduced. At the same time, the size of the pores gradually decreased until the pores were completely closed, and the phospholipid membrane was completely healed. This process can last for tens of nanoseconds. Under our simulation conditions, the process lasted from 3 to 30 ns. During this time, the transmembrane ion migration decreased until it stopped completely.
The overall results here presented are believed to provide a comprehensive mechanism of reversible electroporation. The adoption of coarse-grained Martini force field enabled the simulation to run in a long time-scale with relatively low computation cost. For chemical reaction involved, the model should be extended to be more accurate with reactive force field or the mixture of all-atom and coarse-grained force field. The findings and the results can provide the basis for the biomedical applications including cell apoptosis, drug and gene delivery.
Molecular dynamics simulations
Previous molecular dynamics simulations of electroporation were based on all-atom models. Although the all-atom model can yield atomic-level information about the process, the computational demand limits the simulation to only a few nanometers in distance and a few nanoseconds in time. In order to study the microscopic process of reversible electroporation more comprehensively, we conducted our study using a coarse-grained molecular dynamics simulation method. Before the molecular dynamics simulation was run, the energy of the system was minimized to ensure that the force on each atom was within an appropriate range to avoid the instability inherent in numerical integration. The algorithm implemented the steepest descent method, and the convergence criterion was that the force per particle was < 100.0 kJ mol−1 nm−1. The energy-minimized structure was used as the input for the simulation with time steps of 20 fs and periodic boundary conditions were applied. According to the standard treatment of the Martini force field, the Lennard-Jones effect was truncated at 1.2 nm and translated from 0.9 nm to 1.2 nm, ensuring force continuity. To calculate the static electric field, the particle mesh Ewald (PME) method was used [17] with a cutoff radius of 1.4 nm. The temperature was controlled at about 310 K by the Berendsen method [18] and the coupling constant was 1.0 ps. The Parrinello–Rahman method was adapted to control the horizontal and vertical pressure on the membrane at 1 bar, and the coupling constants were 10.0 ps, which gave a phospholipid membrane surface tension of 0 dyn/cm. The simulation was performed in an NVT ensemble.
All the force field parameters in this study were from the Martini force field [19, 20], which is a coarse-grained (CG) force field that can take both the molecular information needed and the calculation efficiency into account. As it is not an atomistically detailed force field neglecting some atom degrees of freedom, sampling the energy landscape is considered as effectively as possible rather than as accurately as possible, which makes the Martini force field available to simulate a more complicated system and at longer time scale compared with all-atom force field. It has been widely used to study various structures and transformation process of phospholipid.
In the Martini force field, on average, an interaction center (namely a particle) represents four heavy atoms and associated hydrogen atoms. The interaction between particles is divided into two types: bonded and non-bonded interactions. Bonded interactions exist between particles within the same molecule, whereas non-bonded interactions between all particles. The systematical building-process of Martini force field combines "up-to-bottom" and "bottom-to-up" strategies. For the non-bonded interactions, the parameters are obtained by the reproduction of experimental partitioning free energies between polar and apolar solvents of a large number of chemical compounds (the "up-to-bottom" strategy); while the bonded interactions are derived from reference all-atom simulations (the "bottom-to-up" strategy).
The non-bonded interactions include van der Waals interactions and electrostatic interactions:
$$U_{\text{nonbonded}} = 4\varepsilon_{ij} \left[ {\left( {\frac{{\sigma_{ij} }}{{r_{ij} }}} \right)^{12} - \left( {\frac{{\sigma_{ij} }}{{r_{ij} }}} \right)^{6} } \right] + \frac{{q_{i} \,q_{j} }}{{4\pi \varepsilon_{0} r_{ij} }},$$
where \(U_{\text{nonbonded}}\) is the non-bonded energy between two particles (i ,j), εij and σij are the intensity and distance of van der Waals interaction, rij is the distance of the particle pair, qi is the charge of particle i, ε0 = 8.8542 × 10−12 C2 N−1m−2 is the dielectric constant in vacuum. In order to keep the model simple, only four main types of non-bonded interactions are defined: polar, non-polar, apolar, and charged. Each type has corresponding van der Waals interaction parameters.
The bonded interactions include the stretching, bending, and torsional terms, which are described by the following set of functions:
$$U_{\text{stretching}} = \frac{ 1}{ 2}k_{r} \left( {r_{ij} - r_{ij}^{0} } \right)^{2} ,$$
$$U_{\text{bending}} = \frac{ 1}{ 2}k_{\theta } \left( {\theta_{ijk} - \theta_{ijk}^{0} } \right)^{2} ,$$
$$U_{\text{torsional}} = \sum\limits_{n = 0}^{5} {C_{n} } \cos^{n} \left( {\phi_{ijkl} - 180^\circ } \right),$$
where \(k_{r}\), \(k_{\theta }\) and \(C_{n}\) are the force constants, representing the intensity of each term; \(r_{ij}\), \(\theta_{ijk}\), \(\phi_{ijkl}\) are the bond length, bond angle and dihedral angle between the atoms, respectively; \(r_{ij}^{0}\) and \(\theta_{ijk}^{0}\) are the equilibrium bond length and angle when the molecule is in the energy-minimized state.
The phospholipid membrane was composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (Fig. 6). Every coarsely granulated DPPC molecule contained 12 Martini particles, each with a mass of 72 atomic mass unit (amu). The sn-1 and sn-2 alkyl chains consisted of four hydrophobic particles, labeled C1B–C4B and C1A–C4A, respectively. The polar head of DPPC consisted of two particles: a negatively charged PO4 particle representing a phosphate group, and a positively charged NC3 particle representing a choline group. The glycerol backbone was represented by two neutral particles (GL1 and GL2). GL1 linked the phosphate group and the sn-2 alkyl chain. GL2 linked GL1 and the sn-1 alkyl chain. A model with polarizable water molecules was adopted, which has proven to be suitable for studying electrostatic forces [21], including the electroporation process in this study.
Molecular structure and coarse-grained model of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). NC3: choline group, PO4: phosphate group. GL1 and GL2: glycerol skeleton. sn-1 (C1B–C4B) and sn-2 (C1A–C4A): alkyl chains
Establishing the model
In order to construct the membrane, 512 phospholipid molecules and 10,477 water molecules were placed randomly and they formed a phospholipid bilayer due to self-assembly (Fig. 7). Then a 100-ns simulation was performed.
Diagram showing formation of the DPPC lipid bilayer. a Top view; b side view. In the figure, the NC3 particles ('choline') in the phospholipid molecule are shown in blue, the phosphate group particles (PO4) are yellow-brown, the neutral head of the phospholipid molecule (GL1, GL2) is pink and the hydrophobic tail particles are silver. The water molecules are blue-green
After forming the phospholipid membrane, a simulation box was constructed (Fig. 8). The entire simulation system consisted of two phospholipid membranes, each containing 512 phospholipid molecules. These two phospholipid membranes divided the aqueous solution into two regions, each contained 178 Na+ ions (corresponding to a physiological concentration of 0.15 M), but only the lower side contained 356 Cl− ions. The entire simulation box was electrically neutral, but the solution had a net charge due to ion separation. This imbalanced ion distribution was formed by placing the phospholipid membrane in an electric field. The distribution of the electric potential in the vertical direction, perpendicular to the phospholipid membrane, is shown in Fig. 8b. It can be seen that the potential difference across the entire simulated box was about 260 V (the origin of the z axis was the lowest point of the box in Fig. 8a) and the electric field strength was approximately 10.5 V/nm. Since the periodic boundary condition (PBC) was adopted, the solution was connected with only one phospholipid bilayer in the simulation box. Therefore, our entire simulation system contained two phospholipid bilayers in order to obtain two unconnected solution regions.
Setup for simulation of the electroporation process. a Side view of the simulation box; b distribution of the electric potential in the vertical direction. In a, the choline (NC3) particles in the phospholipid molecule are blue, the PO4 group particle is yellow-brown, the neutral head (GL1, GL2) is pink, and the hydrophobic tail particles are silver. The sodium ions are purple, the chloride ions green-yellow, and the water is blue-green
Structure of the self-assembled phospholipid bilayer
The phospholipid molecule will self-assemble to form a phospholipid bilayer structure once dissolved in aqueous solution, as the molecule is amphiphilic: the head is hydrophilic and the tail is hydrophobic. Figure 7 shows a typical bilayer structure in which the phospholipid molecules assembled into two layers. The molecules in one layer had the same orientation, while those in the other layer were oppositely oriented. This allowed the hydrophobic tail groups to associate with each other and avoid contact with water. The hydrophilic heads of the phospholipid molecules were distributed between the water and the hydrophobic tail region, in direct contact with water.
Figure 9 shows the density distribution of different phospholipid components in the vertical direction, perpendicular to the bilayer. The hydrophobic tails of the phospholipid molecules were distributed between −2 and 2 nm, and the hydrophilic heads from −3 to −1 nm and from 1 to 3 nm. It can be seen that the thickness of the phospholipid membrane was 4–5 nm in the absence of external force. The density of water and ions gradually increased starting 1 nm from the middle of the phospholipid layer. Beyond 3 nm, water density reached the bulk maximum, while in the hydrophobic region, water density was zero, which means no water molecules entered the interior of the intact phospholipid bilayer. The energy barrier across the membrane was much too high for polar molecules such as water to overcome without the help of an additional force. Thus, electroporation of a phospholipid membrane can be regarded as a breaking down of the hydrophobic region by means of an electric field so that water or ions can easily cross the membrane.
Density distribution of the self-assembled phospholipid structure. a Particle density in z direction. b Molecule figure indicates the z direction. The density distribution in the figure is translated so that in the middle of the phospholipid bilayer membrane, z = 0
Hardware condition for simulation
The simulation process was mainly carried out on a Dell C6100 server with the open-source software, GROMACS 4.5.5 [22, 23]. The operating system was Red Hat Enterprise Linux Server release 5.6 (Tikanga). The main configuration of the computer node was 2*Intel(R) Xeon(R) CPU E5645@2.40 GHz 6-core processors, and 24 GB DDR3 1333 MHz memory.
All data generated or analyzed during this study are included in this published article, and the datasets used during the current study are available from the corresponding author on reasonable request.
DPPC:
dipalmitoylphosphatidylcholine
PME:
particle mesh Ewald
amu:
atomic mass unit
PBC:
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Tieleman DP, Leontiadou H, Mark AE, Marrink SJ. Simulation of pore formation in lipid bilayers by mechanical stress and electric fields. J Am Chem Soc. 2003;125(21):6382–3.
Tarek M. Membrane electroporation: a molecular dynamics simulation. Biophys J. 2005;88(6):4045–53.
Böckmann RA, De Groot BL, Kakorin S, Neumann E, Grubmüller H. Kinetics, statistics, and energetics of lipid membrane electroporation studied by molecular dynamics simulations. Biophys J. 2008;95(4):1837–50.
Polak A, Bonhenry D, Dehez F, Kramar P, Miklavčič D, Tarek M. On the electroporation thresholds of lipid bilayers: molecular dynamics simulation investigations. J Membr Biol. 2013;246(11):843–50.
Kotnik T, Rems L, Tarek M, Miklavčič D. Membrane electroporation and electropermeabilization: mechanisms and models. Annual review of biophysics. 2019;6(48):63–91.
Delemotte L, Tarek M. Molecular dynamics simulations of lipid membrane electroporation. J Membr Biol. 2012;245(9):531–43.
Akbarian D, Hamedi H, Damirchi B, Yilmaz DE, Penrod K, Woodward WH, Moore J, Lanagan MT, van Duin AC. Atomistic-scale insights into the crosslinking of polyethylene induced by peroxides. Polymer. 2019;21(183):121901.
Akbarian D, Yilmaz DE, Cao Y, Ganesh P, Dabo I, Munro J, Van Ginhoven R, van Duin AC. Understanding the influence of defects and surface chemistry on ferroelectric switching: a ReaxFF investigation of BaTiO 3. Phys Chem Chem Phys. 2019;21(33):18240–9.
Kowalik M, Ashraf C, Damirchi B, Akbarian D, Rajabpour S, van Duin AC. Atomistic scale analysis of the carbonization process for C/H/O/N-based polymers with the ReaxFF reactive force field. J Phys Chem B. 2019;123(25):5357–67.
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Berendsen HJ, Postma JV, van Gunsteren WF, DiNola AR, Haak JR. Molecular dynamics with coupling to an external bath. J Chem Phys. 1984;81(8):3684–90.
Marrink SJ, De Vries AH, Mark AE. Coarse grained model for semiquantitative lipid simulations. J Phys Chem B. 2004;108(2):750–60.
Marrink SJ, Risselada HJ, Yefimov S, Tieleman DP, De Vries AH. The MARTINI force field: coarse grained model for biomolecular simulations. J Phys Chem B. 2007;111(27):7812–24.
Yesylevskyy SO, Schäfer LV, Sengupta D, Marrink SJ. Polarizable water model for the coarse-grained MARTINI force field. PLoS Comput Biol. 2010;6(6):e1000810.
Berendsen HJ, van der Spoel D, van Drunen R. GROMACS: a message-passing parallel molecular dynamics implementation. Comput Phys Commun. 1995;91(1–3):43–56.
Pronk S, Páll S, Schulz R, Larsson P, Bjelkmar P, Apostolov R, Shirts MR, Smith JC, Kasson PM, van der Spoel D, Hess B. GROMACS 45: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics. 2013;29(7):845–54.
The authors would like to thank the National Natural Science Foundation of China (No. 51877046), Jihua Laboratory Projects of Guangdong Province (No. 18002U100) and Pioneering Project of Academy for Engineering and Technology of Fudan University (No. gyy2018-002) for their supports.
This work is supported by the National Natural Science Foundation of China (No. 51877046), Jihua Laboratory Projects of Guangdong Province (No. 18002U100) and Pioneering Project of Academy for Engineering and Technology of Fudan University (No. gyy2018-002).
Department of Light Sources & Illuminating Engineering, Fudan University, 220 Handan Road, Yangpu District, Shanghai, 200433, China
Cheng Zhou
& Kefu Liu
Search for Cheng Zhou in:
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CZ did most of the work , KL checked and revised the manuscript. Both authors read and approved the final manuscript.
Correspondence to Kefu Liu.
Both authors give their consent for publication.
Additional file 1. Morphological changes of phospholipids during the whole reversible electroporation process.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Zhou, C., Liu, K. Molecular dynamics simulation of reversible electroporation with Martini force field. BioMed Eng OnLine 18, 123 (2019) doi:10.1186/s12938-019-0743-1
Martini force field
Reversible electroporation
Phospholipid membrane


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March 28, 2017 / 1:52 AM
Cincinnati Reds 2017 season preview: Time to speed up rebuilding
Jeff Wallner, The Sports Xchange
Cincinnati Reds' Eugenio Suarez (L) high fives teammate Billy Hamilton after Hamilton hit a solo home run during the first inning against the Chicago Cubs of the Cubs home opener at Wrigley Field in Chicago on April 11, 2016. Photo by Brian Kersey/UPI | License Photo
Cincinnati Reds Joey Votto is examined by trainers after being injured at second base in the eighth inning against the St. Louis Cardinals at Busch Stadium in St. Louis on September 29, 2016. Votto came out of the gam as St. Louis won the game 4-3. Photo by Bill Greenblatt/UPI | License Photo
Cincinnati Reds Joey Votto runs to second base after hitting a double in the ninth inning against the St. Louis Cardinals at Busch Stadium in St. Louis on July 28, 2015. Votto also hit a three run home run in the sixth inning as Cincinnati won the game 4-0. Photo by Bill Greenblatt/UPI | License Photo
CINCINNATI -- How much longer?
That's the question on the minds of many Cincinnati Reds fans going into the 2017 season. While most pundits agree the Reds still are a year or two away from being reasonably close to wrapping up their rebuilding process, there are a few pieces of the puzzle that should fit better this summer.
For one thing, the everyday lineup is pretty much set now that the club dealt Brandon Phillips to the Atlanta Braves and handed the second base job to talented prospect Jose Peraza. It's still possible that shortstop Zack Cozart could be traded, which would open up the position for 23-year old Dilson Herrera. There is not much suspense left in the batting order.
Center fielder Billy Hamilton is coming off a breakout year offensively. Left fielder Adam Duvall was a National League All-Star who hit 33 home runs and drove in 103 runs in his first full season in Cincinnati. And, Joey Votto was, well, Joey Votto, hitting .408 after the All-Star break.
RELATED Los Angeles Dodgers LHP Scott Kazmir to open season on DL
The Reds' rebuild focuses mostly on young pitching, and if you trust the scouting reports, they have an abundance of quality arms.
Over the past two seasons, rookies started 166 of 324 games for Cincinnati, including a club-record 110 in 2015. And, there are several young arms that could crack the rotation this season, including left-handers Amir Garrett and Cody Reed, who's coming off a rough major league debut last year but had a strong spring.
Heading into the final week of spring training, as many as seven pitchers were vying for three spots in the rotation, a testament to the Reds' pitching depth and how they performed in the spring.
RELATED Season tickets for Los Angeles Chargers sold out
"I'm not looking at 2017 to be a replay of 2016, because we say it's a rebuild," manager Bryan Price told MLB.com. "I'm looking for 2017 to be a significant improvement over 2016. In order to do that, you have to have pitching depth in the rotation and bullpen. I think we've satisfied some of those issues we had last year. Now we have to stay healthy."
That's been an issue. Cincinnati's retooling process might be further along if it hadn't been for injuries. In the past three seasons, 36 different players have made 57 separate appearances on the disabled list. That streak of bad luck will extend into this season.
Right-hander Homer Bailey, who signed a six-year, $105 million contract in 2014, will begin the year on the DL again after undergoing forearm and elbow surgeries last year. Raisel Iglesias who could be the club's future closer, came down with back issues and a sore elbow late in spring training. Anthony DeSclafani, the projected Opening Day starter, will begin the season on the DL and catcher Devin Mesoraco is being brought along slowly after hip and shoulder injuries limited him to just 16 games in 2016.
RELATED Pittsburgh Pirates 2017 season preview: Andrew McCutchen switch, Jung Ho Kang status keys
Due to injuries, 34-year old veteran right-hander Scott Feldman will get the start on Opening Day, a move more out of necessity than one intended to be part of the grand plan. That said, Feldman has made two prior Opening Day starts: in 2010 for Texas and 2014 for Houston.
"His experience and the way he commands the strike zone, I think really makes him the best candidate," Price told the Cincinnati Enquirer. "He's exactly the guy we were looking for and targeted -- a veteran guy with presence that commands the zone and has a veteran savvy that will serve us well. That will work well in an Opening Day environment."
If the pitching holds up and the club can get healthy, the Reds could surprise some folks by improving on last year's 94-loss performance. It won't be easy.
Perhaps the biggest obstacle confronting Cincinnati in its quest for contention is the NL Central itself. The defending world champion Chicago Cubs have a core of young players locked up for the foreseeable future. The St. Louis Cardinals are perennial contenders.
The Pittsburgh Pirates and Milwaukee Brewers are trying to beat Cincinnati to the finish line with rebuilds of their own, but the Reds still have 19 games each against the Cubs and the rival Cardinals. Not an easy task.
Price and the entire coaching staff were brought back for 2017, but beyond that is anyone's guess. Dick Williams, who assumed the general manager's chair in taking over for Walt Jocketty, will want to see improvement in his first season at the helm.
"The last couple of years, we were preaching that we would have to make tough decisions, trade players, restock the system," Williams told MLB.com. "I think people are starting to see the fruits of that labor. We're starting to get some of these exciting young players opportunities. This year, we'll learn about what those guys can do."
Scott Feldman
Adam Duvall
Lions officially hire Saints' Dan Campbell as next head coach
Chiefs' Patrick Mahomes practices, remains in concussion protocol
Arians warns Buccaneers about thinking ahead to home-field Super Bowl
Elite quarterbacks headline AFC, NFC Championship games
Ted Thompson, ex-Packers GM, dies at 68
Texas A&M defeats North Carolina at Orange Bowl


No need for a sympathetic head tilt with these delightfully off-kilter initial cards. To the point and minimal, with a bright palette that highlights creativity, fun and a strong sense of identity in the palm of your hand.
This was inspired by a classic 3-letter monogram, but updated to look more modern. The rotated letters keep it feeling a bit more playful and it means you can rotate your card any way when you hand it out.


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MUNDIAL GT - OPTIMUM MOTORSPORT
Sexta, 07 Setembro 2018 18:16 | Actualizado em Segunda, 13 Janeiro 2020 05:12
Optimum Motorsport wins British GT Snetterton appeal
Optimum Motorsport is pleased to announce today (4 September) that it has won its appeal against the stewards' decision taken during the British GT Championship opening race at Snetterton in May.
The team immediately appealed the 30-second time penalty applied to the result, one that dropped them from fourth position at the chequered flag to ninth.
Having won the appeal, the team gain an additional ten points in the championship and lead the drivers' standings with 149.5 points ahead of the season finale at Donington.
"I'm very happy with this decision," stated Team Principal Shaun Goff. "We never accepted this incident was anything other than a racing accident. We're delighted with the outcome and now we can look forward to a strong showing at Donington where we stand a good chance of taking the championship honours."


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Why a Natural Shoreline on Your Property?
What's in Your Water? Should You Be Testing?


Thank you for the invite yesterday and it was great to catch up and meet new colleagues last night.
Amazing turnout and event – and had a really good time.
Thank you for your kind invitation to the new office opening last night, it was good to catch up with so many people. The office looks great place to work, I wish you all continued success.
Just wanted to say thanks very much to you and to all your colleagues at SGP for your excellent hospitality last evening at the official opening of your new offices – which do your practice proud.
It was an excellent turn out, very friendly and I really enjoyed catching up with so many people and making a few new connections.
A special thank you to the team at the VR suite who finally persuaded me to try it and go on a virtual tour: very surreal when I found myself hovering above desks and to have people talking to me when all I could see is an empty room, but frivolity aside - clearly a very powerful and useful design tool.
Thank you also for the presents on leaving – I especially like the book with images of a selection of the firm's work, which is beautifully produced and really showcases well the range and quality of the work SGP is doing.
Thank you very much for your kind hospitality and congratulations on putting on such a well-attended event!
Just wanted to drop you both a quick note to thank you and your colleagues for the kind hospitality at last night's reception, which was very much appreciated.
The new offices are really impressive and what a great location and outlook.
I hope that the evening was a great success and look forward to catching up with you both in the near future.
Thank you for the invitation and hospitality.
Great to meet you both last night. Fabulous office and great turnout!
Thank you for the invite yesterday and it was great to catch up and meet new colleagues last night. Amazing turnout and event – and had a really good time.


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Home / Places to stay / Glascoed Farm Cottages
Glascoed Farm Cottages
Glascoed Farm, Henllan Amgoed, Whitland, Pembrokeshire, SA34 0SE
About Glascoed Farm Cottages
Award-winning cottages in peaceful location. Romantic four-poster, wood-burners. Lovely gardens and walks. Stunning location, central for all of the Coastal and Countryside attractions of West Wales..
The Old Farmhouse is a five-star graded cottage sleeping up to seven plus cot in four bedrooms with four bathrooms (2 en-suite). Small private garden with gate to larger water gardens and farm walks.
The Coach House is an intimate four-star graded cottage for two featuring a king-size four-poster bed. A perfect hideaway in a stunning countryside location, and perfect for touring all of West Wales.
Both cottages have access to the hot tub spa and the sauna suite and both cottages are served with solar power as part of out commitment to low-carbon living.
Well-behaved dogs are welcome (see our terms & conditions) and visitors horses can be accommodated too.
£32.00 - £32.00 B&B per person per night
£395.00 - £825.00 per unit per week (largest)
£220.00 - £375.00 per unit per week (smallest)
Rates: Per unit per week (smallest) £220 – £375
Rates: Per unit per week (largest) £395 – £825
Disabled access statement available
Euros accepted
DISABILITY ACCESS STATEMENT
GLASCOED FARM HOLIDAY COTTAGES
The buildings are historic. The first written conveyance available (in Latin) is dated 1540 (Library of Wales, Aberystwyth). We have converted the buildings, which comprise the original main farmhouse and the coach house, as sympathetically as possible to provide good access in general terms: but in buildings of this age it must be appreciated there is limited scope for further adaptation for wheel chairs or other disabled aids.
The Old Farmhouse has a ground-floor double bedroom suite, which includes a large bathroom accessed by two steps down (with handrail). The bedroom is on the same level as the main living accommodation, which is very helpful for those with more limited mobility: and precludes the need to use the main staircase to the first floor.
We remain committed to assisting those who are less physically able and will be pleased to answer questions about the suitability of the buildings for any potential client.
Clive Mortlock


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Culver City Weather ForecastCulver City Weather Forecast
Culver City (California) forecast issued: 9 pm Mon 18 Jan 2021 local time
Culver City Weather Forecast. Providing a local hourly Culver City weather forecast of rain, sun, wind, humidity and temperature.
The Long-range 12 day forecast also includes detail for Culver City weather today. Live weather reports from Culver City weather stations and weather warnings that include risk of thunder, high UV index and forecast gales. See the links below the 12-day Culver City weather forecast table for other cities and towns nearby along with weather conditions for local outdoor activities.
Culver City is 29 m above sea level and located at 34.02° N 118.40° W. Culver City has a population of 39717. Local time in Culver City is PST.
Culver City Weather Today
Mostly dry. Warm (max 21°C on Wed afternoon, min 14°C on Mon night). Mainly strong winds.
Culver City Weather (4–7 days)
Light rain (total 4mm), mostly falling on Fri night. Very mild (max 15°C on Thu night, min 10°C on Fri night). Wind will be generally light.
10 Day Culver City Weather
Mostly dry. Very mild (max 16°C on Wed morning, min 8°C on Mon night). Winds decreasing (fresh winds from the WNW on Mon morning, calm by Tue afternoon).
Culver City Weather Next Week
Moderate rain (total 15mm), heaviest on Fri afternoon. Very mild (max 18°C on Thu morning, min 11°C on Wed night). Winds increasing (calm on Thu morning, fresh winds from the SSE by Fri morning).
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14 16 17 15 18 18 15 18 16 13 14 13 10 12 12 10 13 12 9 12 11 8 10 11 10 13 13 11 15 14 12 14 12 13 14 14
14 15 15 14 17 18 15 18 16 13 14 12 8 11 11 10 12 11 6 9 10 6 10 11 9 11 13 9 14 14 12 14 10 11 13 14
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18 17 16 14 14 15 18 17 15 14 14 14 14 17 21 21 18 17 16 15 15 18 19 18 16 15 14 13 13 14 14 12 12 11 9 8 8 11 12 12 11 11 11 10 10 12 13 12 11 10 10 8 6 9 11
None None None None None Low Low Low None None None None None Low Low Low None None None None None Low Mod Low None None None None None Low Low Low None None None None - - - - - - - - - - - - - - - - - - -
None None None None None None None None None None None None None None None Low Low Low Low Low Low Low Low Low None None None None None None None
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Culver City Weather
Live weather stations near Culver City
Culver City Live Weather Today: Recent reports from weather stations near Culver City
Santa Monica Airport
Mist dry and cloudy.
Light winds from the N.(Wind varies from 005 to 009 degs)
BUOY-ICAC1
5 km WSW
Light winds from the ESE
Hawthorne Airport
Wind obs. (9 kph from 110 degs) was rejected
Haze dry and cloudy.
Wind obs. (7 kph from 1 degs) was rejected
* NOTE: not all weather stations near Culver City update at the same time and we only show reports from the nearest stations that are deemed current. Weather reports submitted by any ship (SYNOP) that is close to Culver City within an acceptable time window are also included.Read More
Culver City Weather Photos
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Culver City Location map
Weather forecast for places near Culver City:
4 km Mostly dry. Moderate rain (total 14mm), heaviest on Fri night. Mostly dry. Moderate rain (total 16mm), heaviest on Fri night.


You don't have to be brilliant to do well in the stock market, just don't be stupid!
Robert C. Merton is perhaps one of the most brilliant financial theorists in the world. He received the Alfred Nobel Memorial Prize in Economic Sciences in 1997 for a new method to determine the value of derivatives, Merton's research focuses on finance theory, including life-cycle finance, optimal intertemporal portfolio selection, capital asset pricing, pricing of options, risky corporate debt, loan guarantees, and other complex derivative securities. No doubt, he is an incredibly brilliant man, but at the same time, he was the principal of Long-Term Capital Management, a speculative hedge fund that collapsed in the late 1990s after losing $4.6 billion. Not only was he extremely smart, but all 15 of his partners were super smart too.
Long-Term Capital Management did business with nearly everyone important on Wall Street. Indeed, much of LTCM's capital was composed of funds from the same financial professionals it traded with. After its collapse on September 23, 1998, Goldman Sachs, AIG, and Berkshire Hathaway offered to buy out the fund's partners for $250 million, to inject $3.75 billion, and to operate LTCM within Goldman's own trading division. The offer was stunningly low to LTCM's partners, because at the start of the year their firm had been worth $4.7 billion. Warren Buffett gave Meriwether less than one hour to accept the deal; the time period lapsed before a deal could be worked out.
Unfortunately similar to the case of Robert C. Merton and LTCM, the stock market is full of smart people and companies doing stupid things… the list is long: Jeff Skilling of Enron, Bernard Madoff, AIG, Lehman Brothers, Fannie Mae, Freddie Mac, and so on.
As Warren Buffett mentioned in this great speech–what is important are the qualitative factors of a person, because everyone has the intelligence to do just the right thing.
Control (a .344 lifetime average) that no player had ever done before.
He had the intelligence of a lead-off hitter and the brawn of a power hitter; the patience of a bench warmer, and the bat control of a single hitter.
His contribution to the game was to reduce the waiting time to hit a perfect pitch; he said that a good hitter can hit a pitch that is over the plate three times better than a great hitter with a questionable ball in a tough spot. Williams knew, for example, that a high and inside strike pitted his weakness against the pitcher's strength. If he consistently swung at those pitches, his batting average would suffer. A low and outside pitch produced the same results – a success rate far below Williams' lifetime batting average. However if Williams received a pitch in his optimum strength zone, he put all his muscle into it, knowing that he could consistently produce a higher batting average. In constructing a template for success, Williams outlined a pattern of patience. He realized that it was often better to take a pitch on the fringe of the strike zone rather than swing for a low average. A called strike was better than making an out.
In this video, one of the most successful investors in the world: Carl Icahn speaks about the great danger we are facing ahead. Carl Icahn speaks about the carried interest loophole, the problem of repatriation and inversions and how these problems don't get fixed in Washington.
He also speaks about companies engineering their finances. Companies today artificially inflate their earnings through mergers and acquisitions and stock buybacks. Finally, he speaks about the two biggest problems of all: Introduction of permanent low-interest rates by the Fed, and oversell of high bond yields to the public, especially by BlackRock.
Unquestionably, Expedia Stock is a great business and for that reason one of the top positions in my portfolio. Lately however, I have been seeing that the company is spending too much too fast on marketing and acquisitions, betting heavily on a future that looks bright on paper, but has yet to be proven.
While Expedia may be right, in some instances organic growth is more important that growth through acquisitions and over spending on marketing When this kind of rapid growth happens, companies can accumulate dangerous amounts of debt too quickly; the smallest tailwind can cause serious trouble and instability.
In November 2014, Expedia completed its acquisition of Australian-based Wotif Group for $658 million. Wotif Group is a prominent player in the Asia Pacific market with a host of travel brands under its umbrella, including Wotif.com, lastminute.com.au, travel.com.au, Asia Web Direct, LateStays.com, GoDo.com.au, and Arnold Travel Technology. Wotif's portfolio focuses on hotel and air, offering consumers more than 29,000 bookable properties across the globe. The group currently operates from Australia, China, Indonesia, Malaysia, New Zealand, Singapore, Thailand, UK, and Vietnam.
Expedia acquired French car rental company, Auto Escape, in June 2014 for $85 million. The acquisition increased Expedia's exposure to the $36.9 billion global car rental industry, which is expected to grow at a compounded rate of 13.6% to reach $79.5 billion by 2019, according to Transparency Market Research.
Auto Escape offers car rental services from over 300 car rental suppliers in 125 countries, and has a fleet of over 800,000 vehicles. It is estimated that Auto Escape's revenues increased fivefold in the last five years to €120 million ($160 million). Auto Escape became a part of the CarRentals.com brand, a business unit managed by Expedia's Hotwire Group.
In January 2015, Expedia acquired online travel agency Travelocity from technology company Sabre Corp for $280 million in cash. The deal follows a 2013 marketing agreement in which Expedia Inc's technology powered platforms for Travelocity's U.S. and Canadian websites, while Travelocity drove additional web traffic to Expedia.
Expedia is currently trying to seal a $1.34 billion takeover of Orbitz. Expedia's blockbuster purchase of Orbitz may not significantly alter the competitive landscape for consumers, but it still represents a dramatic move in the larger, ongoing battle for travelers' clicks, dollars and long-term loyalty. The newly enlarged Expedia will essentially control 75 percent of the U.S. online travel agency (OTA) market, based on 2013 figures. However, OTAs handle just 16 percent of total bookings, meaning that the company's total is closer to 12 percent, hardly a monopolistic scenario – at least in the current market.
In 2013, Expedia agreed to take a majority stake in leading German travel metasearch site Trivago in a cash and stock deal worth $632 million.
Founded in 2005, Dusseldorf, Germany based Trivago has a strong presence in hotel metasearch in Germany, Spain, the UK, Italy, France, and has recently entered the U.S. market.
Expedia has signed a definitive agreement to take a 62% stake in Trivago, which expects to take in $132 million in net revenue in 2012.
This year, Expedia made a $270 million investment in Decolar/Despegar, getting close to a 20% stake of the largest online travel agency (OTA) in Latin America. Expedia is known for making these strategic investments before fully acquiring companies, and if history repeats itself, this may be the first step in a future acquisition, two or three years down the road. That was the model they used with Travelocity – they began investing back in 2013, and finally acquired the company two years later.
When taken together, these acquisitions add up $3.2 billion since 2013. For a company that is generating a yearly net income of ~$300 million putting their debt to equity ratio at 1.05, double that of Priceline at 0.45. This means Expedia is highly leveraging its acquisitions through debt. To a certain point this strategy makes sense, but things may not materialize as quickly as they would like or need. For instance, the company is losing tons of money on its venture with elong, one of the biggest OTAs in China, where Expedia is betting bi. Last quarter the company lost $33 million on this deal, and they expect to continue losing money at the expense of positioning and expansion in the Chinese market. This investment is an example of a big bet that may or may not produce the expected results. There is fierce competition in China including Alibaba and Ctrip, and the market is not as mature or proven as those of the United States and Europe.
For Q1 2015, Expedia reported an operating loss of $51 million including China, compared to a $3 million operating loss last year. This led to an adjusted loss per share of $0.03, while analysts were expecting a profit of $0.09 per share. Excluding China, the company delivered an operating loss of $10.9 million. In the short terms this may not be a problem, but spending and acquiring companies like is 1999 won't be sustainable in the long run. Since 2010, shareholder equity has decreased -36%, while Priceline's shareholder equity is up 518%.
In order to maintain growth and market share Expedia is spending heavily on advertising. Total sales & marketing are up nearly 70% of core OTA revenue (22% Y/Y). Meanwhile, SG&A expenses rose 17% Y/Y. Overall, spending is outpacing revenue growth, which is expected to continue as competitive headwinds remain.
If this trend of massive acquisitions continues at the expense of shareholders value and debt, I may sell my position in the coming months. I already have the company under close observation, and will be monitoring all future developments. While I think acquisitions can be quite valuable when they make good strategic and business sense, they also have the potential to be very dangerous if they are leveraged and made on assumptions that may never be fully realized.
I have found that through history there is nothing more true than that statement. People, governments, and companies have all come and go with these cycles. Sometimes this happens slowly and sometimes quickly, but they all are born, grow, diversify, expand, decline, and often eventually disappear.
… Take Google, for example, and ask yourself: why is it so successful, on so many levels? Because they've been able to carve out a unique category that has put them years ahead of their competition; they are not competing like crazy (the way a restaurant in San Francisco would, for example).
This statement articulates the main reason I will be keeping Google (GOOG) in my portfolio for years to come. Google right now is a very successful company, and every single technology company on the planet wants to operate just like them. The model is perfect for our time, and I expect the company to keep growing and diversifying at least for 10 more years. However, this doesn't mean that the Google model will be the perfect model for the future. At some point Google will stop growing, it will slow down, and eventually will start to decline.
As companies grow they become more difficult to operate and responsibilities and goals inevitably decentralized. While Larry Page and Sergey Brin may have hired their own employees, I assume that's no longer the case now that they have more than 40,000 employees worldwide. The original Google employees from the 80's and 90's are now married, have kids and priorities in their lives may have changed. Over time this shifting in employee focus and tenure can result in a slow down in efficiency and innovation, change frequently becomes difficult, and new smart people that want to move up the ladder face a lot of internal barriers from "originals" employees more interested in protecting their jobs before retirement than in coming up with the next great idea (let alone investing the energy required to realize it).
If a life cycle can be applied to a company, Google is still in the expansion phase, and it may stay in this phase for as many as 10 more years. At the same time, Google's innovation and diversification has slowed. Google maps was launched in 2004, and Youtube in 2006 – both from acquisitions, in the case of Google maps from Keyhole, and YouTube from former PayPal employees that created the company in 2005. Since 2006, Google has not launched any significant product or service as big or with the potential of Google Maps or YouTube. However, despite the lack of recent innovation, these two products, plus Google Fiber and Google AdWords offer Google a lot of room for growth. The focus of the company has changed from innovation to expansion and distribution. That is what it makes Google so attractive it is a company that, with its current lineup of products, has the potential for growth every single year over the next 10 year at a reasonably healthy rate. It is true that the company may have its ups and downs like any other company, but a company in this phase has a very good chance of outperforming the S&P 500 over the next 10 years.
Personally, I look for businesses that have a competitive advantage, a big barrier to entry for competitors, and are expanding, just as Google is right now. As long as technology trends don't change, Google would be a reasonably safe bet. 20 years from now, Google may not be that relevant anymore (even if that is hard to imagine), but today they can easily sustain their growth and the expansion and continue to be a company that will do well for years to come.
Why I increased my investment in Energy?
The future of Intel is not computers, it is wearables, servers and the Internet of things.


I was privileged to have known and worked with Cesar the last few years of his life.
He and my Dad, Lalo Guerrero, had known each other for many, many years going back to the 1950s. But, I didn't meet Cesar until he guested on a TV talk show I produced in the early 1990s, El Show de Paul Rodriguez.
Dad used to travel with his band in the 1950s and that included through California's Central Valley. A young Cesar used to go to Dad's dances wherever he played in the area and they became friends. Cesar used to give Dad advice about where and when to take the band. "Lalo, Tulare in July. Grapes." Or "September, the Central Coast, lettuce." This was long before the UFW, but Cesar knew that world. He knew where and when the campesinos would be working and wanting to have a good time after a long, hard week in the fields. So, Dad used to book his tours according to Cesar. Dad used to say, "Well, I guess you could say Cesar was my booking agent through fruits and vegetables." Dad tells that story in the documentary I produced with Nancy De Los Santos, "Lalo Guerrero:The Original Chicano." A treasure to have it on video.
It took me two years to get Cesar on El Show de Paul Rodriguez. Guess he wasn't into talk shows. I finally mentioned it to Jocelyn Sherman and bingo. At last. Cesar agreed to do the show. He wanted to give away VHS tapes of "No Grapes. No Uvas." I asked if he was sure since we had such a big television audience and that could be costly, the mailing alone. "No, I want it to be available to all," he said. And he said they were prepared with brand new telephone lines and all kinds of stuff. He called me after the airing to say the response had been incredible and, I recall, blew out a lot of the new telephone lines because of the large number of callers. He was suddenly into talk shows and asked if I could get him on some others. I called friends at some of the other talk shows on the English language networks and went to a couple of appearances with him. I was very glad to return that long ago favor when he helped Dad with bookings.
When Cesar passed so suddenly in 1993, I was asked to get Latino Hollywood up for the funeral. I got a school bus donated and off we all went at dawn: Eddie Olmos, Cheech Marin, Paul Rodriguez, Dad and so many others. A difficult but amazingly beautiful experience. I was always in awe of Cesar. Proud to know him.


A month after his emergency heart-bypass operation, Detroit Symphony Orchestra Music Director Leonard Slatkin talks.
While boarding a plane at Metro Airport a couple of months ago, Leonard Slatkin suddenly felt his heart go into overdrive, racing like crazy.
"But it felt like one of those things I knew would pass," said the 73-year-old Detroit Symphony Orchestra music director in an exclusive phone interview Tuesday, his first since his May 22 heart-bypass operation.
Over 45 minutes, Slatkin, who will be honored June 23 at the DSO's Heroes Gala fundraiser, discussed his health, his upcoming move to St. Louis, and his excitement over next year's American Music Festival at the DSO.
Of his recovery, "I'm getting better," Slatkin said. "It wasn't fun, but at least I'm not under house arrest anymore and can drive and walk around."
As it happens, the racing pulse on that flight to Amsterdam did indeed pass. But on Slatkin's return to Detroit, doctors suggested he wear a heart monitor for two weeks.
"Sure enough," he said, "there were incidents when my pulse was up to 189" -- once when he was conducting, he said, and the other while asleep.
"The odd thing," Slatkin added, "is I didn't notice it."
The initial suspect was atrial fibrillation, when the heart can beat wildly out of control. But subsequent tests found clogged arteries, and doctors recommended a bypass.
"It just seemed like the only decision that made sense," said Slatkin, who had a heart attack in 2009.
He admitted that bowing out of conducting the last three concerts of this year's classical season was a blow -- not least because they were meant to be his last three as music director before transitioning to music director laureate for the 2018-19 season.
"It was sad not be able to do them," Slatkin said. "But I realized -- if I didn't take care of this, they could well be my final three concerts."
After his week at the University of Michigan Medical Center, Slatkin began implementing life changes consistent with boosting heart health: physical therapy, exercise and reforming what had been a gourmand's diet.
"It's low salt, low fat, and sometimes no flavor," he said. "But it's been a lot of fun experimenting with things, too, and seeing what one can do."
Alas, that hasn't included grilling, one of his favorite summer pastimes. For the first month after surgery, the recovering patient wasn't supposed to lift more than 10 pounds.
"And the lid of our grill," Slatkin said drily, "weighs more than 10 pounds."
The grill, which is packed, will have to wait till after the June 24 move from Bloomfield Hills to Clayton, Missouri, where Slatkin and his wife, composer Cindy McTee, have just built a house.
"We're relocating to St. Louis, my old stomping grounds," he said, "and where my grandfather settled when he came over from Russia."
Slatkin was music director at the St. Louis Symphony from 1979-96.
The couple originally thought of staying in Detroit or relocating to the West Coast or the Arizona desert. "But ultimately," he said, "I have some really deep, long friendships in St. Louis."
While guest-conducting in the city a year ago, Slatkin said he encouraged McTee, who didn't know the city, to drive around: "And she really found it was to her taste at this point in her life."
Moving to St. Louis, of course, will have no impact on Slatkin's commitment for the next three seasons to conduct the DSO four weeks a year.
In 2018-19, he's actually stretched that to five weeks. Slatkin will open the classical season in October and close it in June, as well as conduct the three-week American Music Festival in February.
Slatkin noted that whoever is chosen as the new music director will decide whether to continue the February music festivals.
"It's not my place to say. But they have been very successful," he said, "and I said it would feel strange if I didn't do an American Music Festival."
As for the search currently under way to find his successor, Slatkin said he has no inside information.
"I asked to really not be involved at all," he said.
"Sometimes the conductor is, but when I left previous positions, I said, 'You have to find the person you think is the right mix for the orchestra and audience.'"
Given what Slatkin calls his bent for American music, he said he thinks there's a fair chance the board might pick somebody with different interests.
"They might look at a younger and more Euro-centric conductor," he suggested, "or not."
Meantime, Slatkin's keeping to his regimen, watching what he eats and building up his strength.
He tips his hat to McTee who, he said, really took responsibility for his recovery.
"I go into curmudgeon mode when I'm not feeling well," he said. "But she kept pushing me when I didn't want to exercise. She'd say, 'You are going to exercise.'"


text:
Q: Change a text file in random access I have a text file and want to change it in some places, for example in byte range 4030 to 4060, in this way:

*

*if there is a character 'C' or 'c' followed by 'G' or 'g' ,must be changed in 'B' character

The input file is a text file and I want to get a changed text output file.  There is no random access in text file and so I must open file in binary form and make changes, but the output file will be binary and I don't have any idea to get a text output. The code is below:
int main()
{
    string str, cstr;
    ReadTextFile("in", 4030, 4060);
    return 0;
}

string ReadTextFile(string path, int from, int to)
{
    fstream fp(path.c_str(), ios::in|ios::out|ios::binary);
    char *target;
    string res, str;
    target = new char[to - from + 1];
    if (!target)
    {
        cout << "Cannot allocate memory." << endl;
        return "";
    }
    fp.seekg(from);
    fp.read(target, to - from);
    target[to - from] = 0;
    res = target;
    str = changestring(res);
    fp.seekg(from);
    fp.write((char *)&str, to-from);
    return res;
}

string changestring(string str)
{
    int l = str.length();
    l = l-1;
    for (int i = 0; i <= l; i++)
    {
        if (str[i] == 'C' ||  str[i] == 'c')
        {
            int j = i+1;
            if (str[j] == 'G' || str[j] == 'g')
                str[i] = 'B';
        }
    }
    return str;
}

A: You misunderstand text and binary. It's not true that if you open a file in binary mode then the output will be binary. All binary mode does in practice is stop \n being translated to \r\n on Windows systems. In other words it affects the line endings in a text file, nothing else.
You are getting binary in your output because you are writing the pointers internal to a string to your output, write characters to your file instead.
Change
fp.write((char *)&str, to-from);

to
fp.write(str.c_str(), to-from);

This would also work
fp << str;

To get text output, write text. It doesn't matter whether you do that with write or with <<, text is just text. Stop thinking in terms of two modes of output, binary and text, it's not accurate, its what you write not how you write it that determines whether the output is text or binary.

redpajama_set_name: RedPajamaStackExchange


25/06/2016 in Amateur Radio, Austria, SOTA, Summits on the Air.
20/06/2016 in Amateur Radio, Austria, SOTA, Summits on the Air.
01/06/2016 in Amateur Radio, Austria, SOTA, Summits on the Air.
01/06/2016 in Austria, SOTA, Summits on the Air.
31/05/2016 in Austria, SOTA, Summits on the Air.


also a scrapmatts printer tray that I am showing you today.
I have used graphic 45 by the sea collection for my back ground papers and this gave me a perfect colour palette for the remainder of the project.
created using kraft card stock and scrapmatts chip board pieces and assorted other materials.
I also used embossing powders and distress inks.
I think this project demonstrates you can create your own unique embellishments with a few resources and simple inking and embossing techniques.
I love to create detail if I can to give my creation dimension and interest.
I am teaching this project locally at the end of April, so I had to use my signature detailed style whilst trying to keep a simple construction technique.
Your comments will be received with appreciation and I hope you will return to see my final round projects for my graphic 45 DT submission.


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